Expression of Mhc class I and II mRNA in a macrophage-like cell line (SHK-1) derived from Atlantic salmon, Salmo salar L., head kidney

Fish & Shellfish Immunology (1999) 9, 473–489 Article No. fsim.1999.0213 Available online at http://www.idealibrary.com on

Expression of Mhc class I and II mRNA in a macrophage-like cell line (SHK-1) derived from Atlantic salmon, Salmo salar L., head kidney E. O. KOPPANG1*, B. H. DANNEVIG2, Ø. LIE3, K. RØNNINGEN1, AND C. McL. PRESS1 1

Department of Morphology, Genetics and Aquatic Biology, Norwegian School of Veterinary Science, N-0033 Oslo, Norway; 2National Veterinary Institute, and 3BioSoft, Oslo Research Park, Oslo, Norway (Received 7 October 1998, accepted 11 January 1999) A cell-line derived from Atlantic salmon (Salmo salar L.) head kidney (SHK-1) consisting of macrophage-like cells was investigated for major histocompatibility complex (Mhc) class I and class II messenger ribonucleic acid (mRNA) expression following stimulation with lipopolysaccaride (LPS), human recombinant insulin-like growth factor-I (IGF-I), and virus infection. A reverse transcriptase-polymerase chain reaction (RT-PCR) was used to provide a semi-quantitative comparison of specific mRNA levels. Incubation with LPS from Escherichia coli (100 ìg ml
1) resulted in a decrease in the expression of Mhc class I heavy chain mRNA after 24 h followed by a weak increase or slightly reduced expression. Incubation with LPS from Aeromonas salmonicida subsp. salmonicida (10 ìg ml
1 and 100 ìg ml
1) induced a more marked reduction in expression after 24 h than LPS from E. coli, and a greater increase in expression after 72 h. Similar observations were also recorded with respect to Mhc class II mRNA expression. Following infection with two di#erent viruses that are pathogenic for Atlantic salmon (infectious salmon anaemia (ISA) virus, and infectious pancreatic necrosis (IPN) virus), the level of expressed mRNA for both Mhc classes increased at 24 h post-infection, whereas the level was markedly reduced or absent 72 h post-infection. IGF-I (10 ìg ml
1 and 100 ìg ml
1) induced either a reduction or absence of Mhc transcripts following 3 h and 48 h incubation. The present study shows that a cell-line consisting of macrophage-like cells expresses Mhc class I and class II, and that the expression can be modified in response to bacterial components, infective viruses, and hormonal stimulation. The study demonstrates a link in expression between the two Mhc classes, indicating common regulatory mechanisms.  1999 Academic Press Key words:

Major histocompatibility complex, Mhc, macrophage, lipopolysaccaride, LPS, IPNV, ISAV, virus, insulin-like growth factor-I, IGF-I, Atlantic salmon

I. Introduction The diagnosis of viral diseases and the investigation of viral pathogenesis are greatly facilitated by an ability to grow virus in cell culture. Following the *Corresponding author. Present address: Biosoft, Oslo Research Park, Gaustadalleen 21,0349 Oslo, Norway. E-mail: [email protected] 1050–4648/99/060473+17 $30.00/0

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establishment of the cell line SHK-1 (salmon head kidney-1), Dannevig et al. (1995) isolated and identified the causative viral agent of the infectious salmon anaemia (ISA). The SHK-1 cell line has been central to the characterisation of the virus (Mjaaland et al., 1997; Falk et al., 1997), and to the diagnosis of and control measures for the disease (K. Falk, National Veterinary Institute, Oslo, pers. comm.). The SHK-1 cell line was derived from the head kidney of Atlantic salmon (Salmo salar L.), and the characterisation studies of the SHK-1 cell line (Dannevig et al., 1997) showed that the cells were able to phagocytose bacteria, but did not appear to be able to kill them. On the basis of this and other phenotypic and functional findings, the SHK-1 cells were described as macrophage-like cells. An important property of macrophages is the ability to present antigens to T-cells, which is central for the propagation of a specific immune response. Macrophages possess cell-surface molecules encoded by genes of the major histocompatability complex (Mhc) that are able to bind short peptides derived from bacteria, viruses or other foreign substances. In mammals, along with all nucleated cells, macrophages express genes of Mhc class I to produce an integral membrane subunit that is noncovalently associated with â2 microglobulin, and is primarily involved in the presentation of endogenous antigens. However, macrophages belong to a more restricted range of cells that also express genes of Mhc class II to form a heterodimer of two transmembrane subunits, one á and one â chain, which are predominantly associated with the presentation of exogenous antigens. While multiple cell types of epithelial or mesenchymal origin may be induced to express Mhc class II (Glimcher & Kara, 1992; Ferrante et al., 1996), dendritic cells, B-cells and macrophages constitutively express significant levels of Mhc class II on their surface. In mammalian tissues, the tight regulation of Mhc expression is predominantly at the level of transcription (Rosa & Fellous, 1988; Mach et al., 1996), and the level of cell surface expression of Mhc molecules closely correlates with the level of intracellular Mhc messenger ribonucleic acid (mRNA) (David-Watine et al., 1990; Glimcher & Kara, 1992). In teleost fish, the genes of Mhc class I and II have been cloned and sequenced in a number of species including carp (Cyprinus carpio) (Hashimoto et al., 1990), rainbow trout (Oncorhynchus mykiss) (Juul-Madsen et al., 1992; Hansen et al., 1996) and Atlantic salmon (Grimholt et al., 1993; Hordvik et al., 1993). The demonstration of mixed leucocyte reactions and graft rejection (Stet & Egberts, 1991), and the investigation of the tissue distribution of Mhc messenger ribonucleic acid (mRNA) expression and the changes in those levels following vaccination (Koppang et al., 1998a, b) argue for the presence of functional Mhc genes in teleost fish. Furthermore, macrophages have been shown to be necessary for immune responses in vitro (Clem et al., 1985), and antigen presentation has been demonstrated using long-lived monocyte-like cell lines from channel catfish (Ictalurus punctatus) (Vallejo et al., 1991). Recently, it has been demonstrated that the expression of MHC class II in macrophages isolated from the head kidney of rainbow trout can be modulated by the synergistic action of cytokines and bacterial products (Knight et al., 1998).

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The aim of the study was to investigate the regulation of Mhc genes in macrophage-like cells from the head kidney of Atlantic salmon. The SHK-1 cells represent a defined cell population with macrophage-like properties, and as such are well suited for studies of gene regulation and function in teleost immune cells. The levels of Mhc class I and class II mRNA expression in the SHK-1 cell line was investigated at various times after exposure to bacterial components and a hormonal growth factor, and after infection with pathogenic fish viruses. The level of Mhc specific mRNA was estimated using a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) procedure previously described by Koppang et al. (1998a, b). II. Materials and methods CELLS

SHK-1 cells were grown at 20 C in Leibovitz’s L-15 cell culture medium supplemented with foetal calf serum (FCS, 5% v/v), L-glutamine (4 mM), 2-mercaptoethanol (2-ME, 40 ìM) and gentamicin (50 ìg ml
1). The cells were subcultured by standard procedure using 0·05% trypsin and 0·02% EDTA in 0·01 M PBS. All cell culture reagents, except 2-ME which was obtained from Gibco BRL, Uxbridge, U.K., were from BIO-Whittaker Inc., Walkersville, MD, U.S.A. Cell cultures of 30–60 passages were used in this study. STIMULANTS AND VIRUS

Lipopolysaccharide (LPS) from Escherichia coli (E. coli LPS) was obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. LPS from Aeromonas salmonicida subsp. salmonicida was kindly provided by the National Veterinary Institute, Oslo, Norway (Thuvander et al., 1993; Reitan & Secombes, 1997). Infectious salmon anaemia (ISA) virus, strain Glesvaer/2/90 (Mjaaland et al., 1997), which had been passaged four times in SHK-1 cells, and infectious pancreatic necrosis (IPN) virus, strain no. 44 of serotype Sp (Melby & Falk, 1995) were used. Virus titre (50% tissue culture infective dose, TCID50) was estimated by end point dilution according to Falk et al. (1997) and Taksdal et al. (1998). Recombinant (E. coli) human insulin-like growth factor-I (IGF-I) was supplied by Boehringer-Mannheim, Mannheim, Germany. EXPERIMENTS

SHK-1 cells grown for 3 days at 20 C in 75 cm2 cell culture flasks (Costar, Cambridge, MA, U.S.A.) with 20 ml fully supplemented medium were used. After removal of the growth medium, the cells were washed twice with L-15 medium without FCS in all experiments. In experiments with LPS and IGF-I, fresh medium with FCS and supplemented with the indicated stimulants was added in a volume of 10 ml. Final concentrations of stimulants were 100 ìg ml
1 of E. coli LPS, 10 and 100 ìg ml
1 of Aeromonas salmonicida subsp. salmonicida LPS, and 10 and 100 ng ml
1 of IGF-I. Control cells were treated

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identically but received no stimulants. The cells were incubated at 20 C until harvesting at 24 h and 72 h subsequent to stimulation with LPS and at 3 h and 48 h for cells stimulated with IGF-I. Infection of SHK-1 cells with ISA virus or IPN virus was performed by incubating the cells with 3 ml of virus supernatant diluted in L-15 without FCS for 4 h at 15 C. Thereafter the inoculate was removed and replaced with 10 ml L-15 medium with FCS. The infective doses during inoculation were 105 TCID50ml
1 for ISA virus and 10 and 102 TCID50ml
1 for IPN virus. Control cells were treated identically, but without virus. The infected and non-infected cell cultures were incubated at 15 C until harvesting at 24 h and 72 h post infection. To verify that inoculation of SHK-1 cells with ISA virus and IPN virus resulted in a successful infection and virus replication, parallel cell cultures inoculated with the respective viruses were incubated for a longer time period for observation of cytopathogenic e#ects. At the indicated times, single cell suspensions were obtained by trypsination of the adherent cell layer after washing the cells twice with PBS. The cell suspension was washed and concentrated by centrifugation (5 min at 1200 rpm and 4 C, Beckman GS-6R Centrifuge) and resuspended in 1 ml of L-15 medium without FCS. The cell number was estimated using a haemocytometer. The cell suspension was kept on ice from immediately after harvesting. Results from single experiments are presented (see Figs 1–3). One experiment consisted of the addition of a specific stimulant or virus to one cell culture flask with SHK-1 cells and the corresponding control flask. The results presented in each figure are from experiments conducted at di#erent days using cell cultures of di#erent passages. The number of experiments performed with the various stimulants and viruses varied between two and eight. However, when only two experiments were conducted, two di#erent concentrations of the stimulants at each time point were used.

mRNA ISOLATION

From each flask, total mRNA was isolated. QuickPrep Micro mRNA Purification Kit (Pharmacia Biotech AB, Uppsala, Sweden) was used according to the manufacturer’s protocol. The extracted mRNA was precipitated in 95% ethanol together with glycogen solution and potassium acetate solution (provided in the kit and according to standard protocol) after centrifugation, and a barely visible pellet was redissolved in 200 ìl diethyl pyrocarbonate (DEPC)-treated water. The concentration of isolated mRNA was measured with a spectrophotometer (GeneQuant II RNA/DNA Calculator, Pharmacia Biotech, Norway), and the total mRNA content in each sample was adjusted to 5 ìg ml
1 total mRNA.

REVERSE TRANSCRIPTASE (RT) REACTION

The reverse transcriptase reaction was performed at 37 C for 1 h with Ready To Go T-Primed First Strand Kit (Pharmacia Biotech AB, Uppsala,

Mhc EXPRESSION IN SALMON MACROPHAGE-LIKE CELL LINE

Expression relative to unstimulated control

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72 h

24 h E. coli LPS

24 h 72 h A. s. salmonicida LPS

Fig. 1. SHK-1 cells were incubated for 24 h and 72 h with either E. coli LPS (100 ìg ml
1) or A.s. salmonicida LPS (10 ìg ml
1 and 100 ìg ml
1). The level of expression of Mhc class I heavy chain (a) and Mhc class II â chain (b) mRNA was determined by RT-PCR as detailed in the Materials and Methods section and expressed relative to the unstimulated control. The level of expression equals (experiment intensity value/control intensity value) minus 1. Di#erent bars indicate individual experiments * mRNA was not detected in the control or stimulated flasks. § level of mRNA expression was the same as in the control flask. 10–10 ìg ml
1; 100–100 ìg ml
1

Sweden), using 33 ìl of the 200 ìl mRNA elution according to the manufacturer’s protocol. The RT reaction was run with an excess of Not I d(T) 18 primer, allowing all mRNA in the solution to be converted to complementary deoxyribonucleic acid (cDNA). Rabbit globulin RNA, which was provided in the kit, was used in each run as an external control to verify the procedure (data not

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3.0

(a)

Expression relative to unstimulated control

2.5

n=4

n=4

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§ * 100

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n=4

n=4

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Expression relative to unstimulated control

2.0 1.5 1.0 0.5 10

100

0

100

10

–0.5 –1.0 72 h

24 h ISA virus

24 h

72 h IPN virus

Fig. 2. SHK-1 cells were incubated for 24 h and 72 h after inoculation with either ISA virus (105 TCID50ml
1) or IPN virus (10 and 102TCID50ml
1). The level of expression of Mhc class I heavy chain (a) and Mhc class II â chain (b) mRNA was determined and expressed relative to the unstimulated control. The level of expression equals (experiment intensity value/control intensity value) minus 1. Di#erent bars indicate individual experiments. * mRNA was not detected in the control or stimulated flasks. § level of mRNA expression was estimated as
1%. 10–10 TCID50 ml
1; 100–102 TCID50 ml
1

shown). An additional control was also run by using synthesized RNA provided with a poly-T tail (pAW 109 RNA, Perkin Elmer, Applied Biosystems Division, Foster City, CA, USA); 400,000 copies of the pAW 109 RNA were added to 33 ìl of the collected tissue solutions and subjected to the RT reaction together with the extracted mRNA.

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1.0

Expression relative to unstimulated control

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48 h Mhc class I

3h

48 h Mhc class II

Fig. 3. SHK-1 cells were incubated for 3 h and 48 h with recombinant (E. coli) human IGF-I (10 ng ml
1 and 100 ng ml
1). The level of expression of Mhc class I heavy chain and Mhc class II â chain mRNA was determined and expressed relative to the unstimulated control. The level of expression equals (experiment intensity value/ control intensity value) minus 1. Di#erent bars indicate individual experiments 10–10 ng ml
1; 100–100 ng ml
1

PCR PRIMERS

Mhc class I heavy chain A sense primer was selected from the Mhc-Sasa class I á2 domain, whereas the corresponding antisense primer was located in the transmembrane/ cytoplasmic domain region. Sense primer :5 -cat tac ctc acc cag acc tgc att g-3 . Corresponding antisense primer: 5 -tga cga ccc aac aac aac agc aac-3 . Both the sense and antisense primers were selected from clone p 23 (GenBank Accession number L07605; Grimholt et al., 1993). The PCR was expected to yield a 455 basepair (bp) product from amplified cDNA. Lack of amplification or amplification of a PCR product larger than 455 bp would indicate genomic DNA contamination as an intron is present between the á2 domain and á3 domain and between the á3 domain and the transmembrane domain. Mhc class II â chain A sense primer was selected from the Mhc-Sasa class II â1 domain, whereas the corresponding antisense primer was located in the â2 domain. Sense primer: 5 -cag att caa cag cag tgt ggg gaa g-3 . Corresponding antisense primer: 5 -tca cat cag act tca cct cac gtc c-3 . Both sense and antisense primers were selected from clone Sasa c144 (GenBank Accession number X70165; Hordvik et al., 1993). The PCR was expected to yield a 313 bp product from amplified cDNA. Lack of amplification or amplification of a PCR product larger than 313 bp would indicate genomic DNA contamination as an intron is present between the â1 and â2 domains.

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Controls Primers for the external control (rabbit globulin mRNA) were provided with the RT-kit (Pharmacia) and for the additional control, Perkin Elmer provided primers DM 151 and DM 152 for pAW 109 RNA amplification. POLYMERASE CHAIN REACTION

The PCR was run in a Perkin-Elmer thermo-cycler 9600. For each cell culture sample, 20, 29, 30 and 35 cycles of PCR (94 C 1 min, 61 C 30 sec and 72 C 30 sec) were run using 2·5 units of Taq polymerase (Perkin-Elmer/Cetus, Emeryville, California, U.S.A.), 5 ìl of the resulting RT solution containing cDNA, 0·05 nmol of each primer, 10 ìl 2nM dNTP solution, 10 ìl PCR bu#er solution and ultrapure autoclaved water, to give a total reaction volume of 100 ìl for both Mhc class I and Mhc class II transcript amplification. The PCR products from the SHK-1 cells were run on a 1·5% agarose gel together with one standard molecular weight ladder (123 DNA Ladder, Gibco BRL, Life Technologies, U.S.A.). Two products from Mhc class I as well as Mhc class II amplification were also run on a polyacrylamide gel to determine the exact size. The external RT reaction controls were run according to the manufacturer’s protocol. The additional RT reaction controls were run taking 5 ìl of the RT solution and subjecting it to 24 cycles of PCR (94 C 1 min, 55 C 30 sec and 72 C 30 sec). Negative controls included using distilled water instead of the tissue RT solution, and omitting either the Taq polymerase, the sense primer, the anti-sense primer or both primers from the PCR protocol. SEQUENCING AND SEQUENCE ANALYSIS

The nucleotide sequence of the PCR products was determined using Sequenase PCR Product Sequencing Kit (Amersham Life Science, Cleveland, OH, U.S.A.) according to the manufacturer’s protocol. Five ìl of the PCR products from two samples of Mhc class I products and two samples of Mhc class II products were used in this reaction. COMPUTER-ASSISTED MORPHOMETRIC ANALYSIS

The gels were placed on a UV table and scanned with a CCD video camera (Model XC-77CE, Sony, Japan and with camera adaptor CMA-D2, Sony, Japan). The digital images were analysed using the computer software, Gel-Pro Analyzer (Version 2·0, Media Cybernetics, Silver Spring, MA, U.S.A.). The Lane Profile Analysis option allowed the intensity value of the resulting bands in each lane to be estimated (Anonymous, 1995). As the total mRNA extracted from each individual flask was standardised to 5 ìg ml
1 prior to the RT step, the resulting PCR product is an estimate of the proportion of specific Mhc class I or II mRNA in the total mRNA from all cells in a single flask. The level of expression of both Mhc class I heavy chain and Mhc class II â chain detected at a specific time point varied between flasks of SHK-1 cells. To standardise the presentation of the results, the intensity value determined for each flask of SHK-1 cells following various treatments is

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presented relative to the intensity value determined for the unstimulated control flask, i.e. for Figs 1, 2 and 3, the level of expression equals (experiment intensity value/control intensity value) – 1. III. Results Isolated mRNA from SHK-1 cells was evaluated at various PCR cycle numbers ranging from 20 to 35. After 20 cycles of PCR, weak bands of expected length for Mhc class I heavy chain (455 bp) and Mhc class II â chain (313 bp) were detected in a very limited number of the samples (data not shown). After 35 cycles of PCR most samples gave a high amount of PCR product, indicating a plateau e#ect (Anonymous, 1991). However, all samples from IGF-I stimulated SHK-1 cells that did not produce a Mhc class I or II product after 30 cycles of PCR, also failed to give detectable products after 35 cycles. To facilitate the di#erentiation of gene expression in the various experiments, 30 cycles of PCR were used as this gave a low but detectable level of PCR products for most samples (Figs 1, 2 and 3). As a control, two PCR products from the Mhc class I amplification were sequenced and found to correspond to the expected expressed gene fragment of clone p 23 (Grimholt et al., 1993), and two PCR products from the Mhc class II amplification were sequenced and found to correspond to the expected expressed gene fragment in the Mhc-Sasa class II â chain sequence (Hordvik et al., 1993). The external control for the RT reaction, rabbit globulin mRNA, which was provided in the kit, gave in each reaction a band of the expected 550 bp. The addition of synthesized RNA to the cell mRNA solution produced a band of 308 bp of equal intensity. The negative controls did not produce any PCR product. LPS

The addition of LPS, regardless of origin or dose, resulted in a decrease in the expression of Mhc class I heavy chain by SHK-1 cells compared to the level of expression in the controls after 24 h incubation (Fig. 1a). In one experiment with E. coli LPS (100 ìg ml
1), mRNA could not be detected. After 72 h incubation, LPS from E. coli induced a weak increase in expression in four experiments, while in two experiments there was a slightly lower expression compared to controls. The higher concentration (100 ìg ml
1) of LPS from Aeromonas salmonicida subsp. salmonicida seemed to induce a more marked reduction in expression of Mhc class I heavy chain than the lower concentration (10 ìg ml
1) after 24 h incubation. Exposure of SHK-1 cells to Aeromonas salmonicida subsp. salmonicida LPS for 72 h, resulted in a substantially higher level of expression compared to the controls for both concentrations (Fig. 1a). The level of expression of Mhc class II â chain by SHK-1 cells followed a similar pattern to the expression of Mhc class I heavy chain after stimulation with E. coli and Aeromonas salmonicida subsp. salmonicida LPS (Fig. 1b). Following stimulation with E. coli LPS for 24 h, mRNA could not be detected in one experiment. Following stimulation with Aeromonas salmonicida

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subsp. salmonicida LPS for 24 h, no obvious di#erence in levels of expression of Mhc class II â chain (Fig. 1b) could be observed for the two concentrations used (10 and 100 ìg ml
1), as was observed for the expression of Mhc class I (Fig. 1a). VIRUS

Following infection with ISA virus (105 TCID50 ml
1) and IPN virus (10 and 10 TCID
1), an increase in the expression of Mhc class I heavy chain by SHK-1 cells was observed at 24 h post-infection (Fig. 2a). At this time point, Mhc class I mRNA could not be detected in one experiment with ISA virus and in two experiments with IPN virus. At 72 h post-infection, both viruses caused a reduction in or had no e#ect on the level of expression of Mhc class I heavy chain except in one experiment in which IPN virus-infected SHK-1 cells (infective dose 10 TCID50 ml
1) showed a considerably higher level of expression than corresponding control cells. The level of Mhc class II â chain expression by virus-infected SHK-1 cells also showed a time-dependent variation. (Fig. 2b). For cells infected with ISA virus, the level of expression was clearly higher than in the controls (four of four experiments) at 24 h post-infection, while at 72 h, the level of expression was lower compared to the controls in three of the four experiments performed and higher than the control in one experiment. SHK-1 cells inoculated with the higher dose of IPN virus (102TCID50 ml
1) showed a higher level of expression of Mhc class II â chain compared to controls in three of three experiments performed at 24 h post-infection, while the level of expression cells inoculated with the lower dose (10 TCID50 ml
1) was slightly higher than in controls in two of the three experiments performed and lower in one experiment. At 72 h post-infection, the IPN virus-infected cells showed generally a lower level of expression of Mhc class II â chain compared to controls with one exception (Fig. 2b). In this experiment with the lower infective dose of IPN virus, the level of expression of both Mhc class I heavy chain and Mhc class II â chain was considerably higher compared to controls. Cytopathogenic e#ects were observed in parallel cultures inoculated with the respective viruses. 2

IGF-I

Exposure of SHK-1 cells for IGF-1 resulted in a reduction in the level of expression of both Mhc class I heavy chain and Mhc class II â chain compared to controls (Fig. 3). The reduction in expression was obvious after 3 h and uniform at 48 h for both concentrations used (10 and 100 ng ml
1). IV. Discussion The present study shows that SHK-1 cells express Mhc class I heavy chain and Mhc class II â chain mRNA. The detection of both Mhc class I heavy chain and Mhc class II â chain mRNA in the SHK-1 cell line is consistent with observations in mammalian macrophage cell lines (Chang & Lee, 1986) and

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with the recent description of Mhc class II â chain transcription in macrophages isolated from the head kidney of rainbow trout (Knight et al., 1998). The primers used for the detection of Mhc transcripts and the RT-PCR procedure have previously been used to estimate the level of Mhc class I and II mRNA in tissues of non-vaccinated and vaccinated Atlantic salmon (Koppang et al., 1998a, b). As in these tissues studies, a number of PCR cycles was determined to facilitate the di#erentiation of levels of mRNA expression. However in contrast to the tissue studies, the present study was standardised to a given amount of isolated total mRNA (5 ìg ml
1) rather than a given amount of tissue (0.1 g). The present approach, which gave an estimate of the proportion of specific Mhc mRNA in the total isolated mRNA, was justified as the SHK-1 cell line represents a defined cell population (Dannevig et al., 1997). While variation existed between the amounts of total mRNA isolated from flasks of stimulated and unstimulated cells, changes in proportions of specific Mhc mRNA would provide an indication of the e#ect of the stimulus given to the SHK-1 cells. An association between the amounts of total mRNA obtained and the passage of the cell line used was not observed (data not shown), however enzyme histochemical studies of the SHK-1 cells have shown phenotypic diversity within the cell line (Dannevig et al., 1997). Whether the obtained variability in the mRNA levels was a consequence of this diversity is unknown, but the possible presence of cells at di#erent stages of development should be considered in further studies of gene expression in the SHK-1 cell line. The SHK-1 cell line was derived from the head kidney of Atlantic salmon and, based on morphological, phenotypic and functional characteristics, has been described as a macrophage-like cell (Dannevig et al., 1997). However, the apparent inability of this cell line to kill bacteria raised questions as to whether this cell line lacked or had lost other features of macrophages such as the ability to regulate the expression of Mhc genes. An important property of macrophages is the ability to regulate expression in response to various stimuli such that a specific and appropriate immune response is initiated against an antigen. The regulation of Mhc gene expression in SHK-1 cells proved to be under close regulation as evidenced by changes in levels of expression following the stimuli administered in the present study. LPS from E. coli and Aeromonas salmonicida subsp. salmonicida caused a downregulation of both Mhc class I and class II expression after 24 h incubation followed by an upregulation after 72 h incubation. Koerner et al. (1987) investigated Mhc II expression in murine peritoneal macrophages and found that LPS exerted an initial dose and time dependent suppressive e#ect on the accumulation of mRNA. These investigators and others have shown that bacterial LPS can also increase Mhc class I and class II expression in vivo and/or in vitro, an e#ect that is probably related to the known ability of LPS to mediate release of IFN-ã, TNF-á and other cytokines from non-T cells including macrophages (Maehorea & Ho, 1977; Havell & Spitalny, 1983; Blanchard et al., 1986; Jephthah-Ochola et al., 1988; Cockfield et al., 1990; Nash et al., 1992). TNF-á and IFN-ã upregulate Mhc class I and class II transcription resulting in an increased level of cytoplasmic mRNA, although posttranscriptional regulation is also important, and these cytokines probably

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function through di#erent pathways (David-Watine et al., 1990; Freund et al., 1990; Glimcher & Kara, 1992). Knight et al. (1998) investigated the e#ect of human recombinant TNF-á and E. coli LPS on the expression of Mhc class II â chain in macrophages isolated from the head kidney of rainbow trout. Using Northern blot analysis, TNF-á was found to have no e#ect on Mhc class II mRNA levels in isolated macrophages and E. coli LPS only a small stimulatory e#ect up to 8 h after stimulation. A synergistic e#ect of these two substances was demonstrated at 4 h post-stimulation and the percentage of macrophages reacting with a polyclonal anti-Mhc class II â chain serum increased with time (72 h). In the present study, the more sensitive method of RT-PCR was used to detect changes in Mhc mRNA levels 24–72 h after stimulation with LPS from two bacterial sources. Interestingly, Aeromonas salmonicida subsp. salmonicida LPS was found to have a stronger inductive e#ect on Mhc class I and class II expression after 72 h incubation compared with the level of expression induced by E. coli LPS (Fig. 1). Whether this observation represents an e#ect of the source of the bacterial LPS and possible structural di#erences between the bacteria (Rietschel et al., 1996), deserves further investigation. The reaction to both virus and IGF-I administered in the present study showed that the fish macrophage-like cell line responded in a manner similar to mammalian macrophage cell lines. Viral infection often entails enhanced transcription and expression of Mhc genes while viral transformation leads to decreased expression (Brown et al., 1991). Accordingly, the present study found that both IPN, an aquatic birnavirus and ISA, a virus suggested to belong to the Orthomyxoviridae family (Falk et al., 1997; Mjaaland et al., 1997), showed an initial increase in the levels of mRNA expression followed by a decline in expression. Dannevig et al. (1995) observed cytopathogenic e#ects in SHK-1 cells four to seven days after inoculation with ISA virus propagated in cell culture. However, the results of the present study would suggest that significant disturbances of cellular function are in e#ect as early as 72 h post-inoculation. A reduction in protein synthesis has been observed in ISA infected SHK-1 cells at 72 h post-infection using 35S methionine in vivo labelling (Dannevig et al., 1995). The e#ect of IGF-I on the immune system has been documented in mammals (Murphy, 1996; Clarc, 1997), but the actions of this highly conserved hormone (Duguay et al., 1992) on the immune system of teleosts have not been investigated, although Secombes et al. (1996) suggested that IGF-I could be regarded as a cytokine in fish, and a number of studies have shown that human and fish IGF-I are equally potent in mammalian and fish bioassay systems (Duan, 1998). In the present study, recombinant (E. coli) human IGF-I induced a dramatic decline in both Mhc class I and class II mRNA expression in SHK-1 cells after only 3 h exposure, and expression was not detected after 48 h. As specific IGF-I receptor transcripts have been identified in teleosts (Elies et al., 1996) and observations in mammalian cells indicate that a rapid specific response in Mhc class I occurs following incubation with IGF-I (Saji et al., 1992) or suppression of IGF-I (Shevelev et al., 1997; Trojan et al., 1996), shorter time intervals from exposure to harvesting (3 h and 48 h) were undertaken to detect the response to incubation with IGF-I. Interestingly,

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Sakai et al. (1995, 1996) found that the administration of growth hormone (GH) activated phagocytic activity and superoxide anion production in isolated rainbow trout head kidney macrophages in vitro and enhanced resistance to Vibrio anguillarum infection in vivo (Sakai et al., 1997). IGF-I is a mediator of the growth e#ect and osmoregulatory action of GH in salmonids (Sakamoto & Hirano, 1993; Sakamoto et al., 1993). Furthermore, the administration of GH to salmonids has been shown to increase IGF-I production in the liver, which is the main site of production (Duan et al., 1994). Whether IGF-I and the down-regulation of Mhc gene expression are involved in the GH-induced activation of salmonid macrophages is not known. An important observation in the present study is the close link between Mhc class I and Mhc class II mRNA levels in the SHK-1 cell line following treatment with LPS, virus or IGF-I. Shared regulatory elements in the promoters of Mhc class I and class II genes have been identified and suggest a common regulation of transcription of class I and class II genes (van den Elsen et al., 1998). Conserved sequence elements within the promoter of Mhc class I genes have been found in the zebrafish, chicken, chimpanzee, gorilla and orang-utan (van den Elsen et al., 1998). Atlantic salmon class II promotors are currently isolated and two conserved regulatory elements have been identified (Lundin & Syed, Norwegian School of Veterinary Science, pers. comm.). While the autocrine induction of cytokines and their known e#ects on Mhc gene expression (discussed above) may mediate the common regulation of transcription of class I and class II genes, the recent evidence that endogenous antigens can be presented by Mhc class II (Guéguen & Long, 1996; Malnati et al., 1992) and that exogenous antigens can be presented by Mhc class I (Kovacsovics-Bankowski & Rock, 1995; Norbury et al., 1995; Sousa & Germain, 1995) may provide opportunities for the future development of vaccines against fish diseases. The production of e#ective vaccines particularly against viral diseases is an important objective for the aquaculture industry. The specific induction of helper and cytotoxic T cell populations is a field of intense investigation in fish (Stuge et al., 1997) and an understanding of the phagosome to cytosol pathway for exogenous antigens would have clear implications for vaccine design and strategies. Knowledge of the regulation of Mhc genes gained in a salmonid macrophage-like cell line that can support the growth of pathologically significant viruses, may contribute to the development of these future vaccines. The authors thank Inger Austrheim and Monika Jankowska at the National Veterinary Institute, Oslo for skilled technical assistance.

References Anonymous. (1991). Quantitative RT-PCR. Methods and applications, Book 3. Clontech Laboratories, California, USA. Anonymous. (1995). Gel-Pro Analyzer Version 2·0 Reference Guide. Media Cybernetics, Silver Spring, MD. Blanchard, D. K., Djeu, J. Y., Klein, T. W., Friedman, H. & Stewart, W. E. (1986). Interferon-ã induction by lipopolysaccharide: dependence on interleukin 2 and macrophages. Journal of Immunology 136, 963–970.

486

E. O. KOPPANG ET AL.

Brown, C. D., Pampeno, C. & Meruelo, D. (1991). Virus-induced modulation of H-2 class I gene expression. In Immunogenetics of the major histocompatibility complex (R. Srivastava, B. P. Ram & P. Tyle, eds.) pp. 268–308. New York: VCH Publishers. Chang, R. J. & Lee, S. H. (1986). E#ects of interferon-ã and tumor necrosis factor-á on the expression of an Ia antigen on a murine macrophage cell line. Journal of Immunology 9, 2853–2856. Clarc, R. (1997). The somatogenic hormones and insulin-like growth factor-1: stimulators of clymphopoiesis and immune function. Endocrine Reviews 2, 157–179. Clem, L. W., Sizemore, R. C., Ellsaesser, C. F. & Miller, N. W. (1985). Monocytes as accessory cells in fish immune responses. Developmental and Comparative Immunology 9, 803–809. Cockfield, S. M., Urmson, J., Pleasants, J. R. & Halloran, P. F. (1990). The regulation of expression of MHC products in mice. Factors determining the level of expression in kidneys of normal mice. Journal of Immunology 8, 2967–2974. Dannevig, B. H., Brudeseth, B. E., Gjøen, T., Rode, M., Wergeland, H. I., Evensen, Ø. & Press, C. McL. (1997). Characterisation of a long-term cell line (SHK-1) developed from the head kidney of Atlantic salmon (Salmo salar L.). Fish & Shellfish Immunology 7, 213–266. Dannevig, B. H., Falk, K. & Namork, E. (1995). Isolation of the causal virus of infectious salmon anaemia (ISA) in a long-term cell line from Atlantic salmon head kidney. Journal of General Virology 76, 1353–1359. David-Watine, B., Israël, A. & Kourilsky, P. (1990). The regulation and expression of MHC class I genes. Immunology Today 11, 286–292. Duan, C. (1998). Nutritional and developmental regulation of insulin-like growth factors in fish. Journal of Nutrition 128, 306–314. Duan, C., Duguay, S. J., Swanson, P., Dickho#, W. W. & Plisetskaya, E. M. (1994). Tissue-specific expression of insulin-like growth factor I ribonucleic acids in salmonids: developmental, hormonal and nutritional regulation. In Perspectives in Comparative Endocrinology (K. G. Davey, S. S. Tobe & D. E. Peter, eds) pp. 365–372. Toronto, Canada: National Research Council of Canada. Duguay, S. J., Park, L. K., Samadpour, M. & Dickho#, W. W. (1992). Nucleotide sequence and tissue distribution of three insulin-like growth factor I prohormones in salmon. Molecular Endocrinology 6, 1202–1210. Elies, G., Groigno, L., Wol#, J., Boeuf, G. & Boujard, D. (1996). Characterization of the insulin-like growth factor type 1 receptor messenger in two teleost species. Molecular and Cellular Endocrinology 124, 131–140. Elsen, P. J. van den, Gobin, S. J. P., Eggermond, M. C. van & Peijnenburg, A. (1998). Regulation of MHC class I and II gene transcription: di#erences and similarities. Immunogenetics 48, 208–221. Falk, K., Namork, E., Rimstad, E., Mjaaland, S. & Dannevig, B. H. (1997). Characterization of infectious salmon anemia virus, an Orthomyxo-like virus isolated from Atlantic salmon (Salmo salar L.). Journal of Virology 12, 9016–9023. Ferrante, P., Apollonio, I., DelNero, A., Petonzelli, F. & Mazzilli, M. C. (1996). Regulation of HLA gene expression. Minerva Biotecnologica 8, 32–42. Freund, Y. R., Dedrick, R. L. & Jones, P. P. (1990). Cic-acting sequences required for class II gene regulation by interferon ã and tumor necrosis factor á in a murine macrophage cell line. Journal of Experimental Medicine 171, 1283–1299. Glimcher, L. H. & Kara, C. J. (1992). Sequences and factors: a guide to MHC class-II transcription. Annual Review of Immunology 10, 13–49. Grimholt, U., Hordvik, I., Fosse, V. M., Olsaker, I., Endresen, C. & Lie, Ø. (1993). Molecular cloning of major histocompatibility complex class I cDNA from Atlantic salmon (Salmo salar). Immunogenetics 37, 469–473. Guéguen, M. & Long, E. O. (1996). Presentation of a cytosolic antigen by major histocompatibility complex class II molecules requires a long-lived form of the antigen. Proceedings of the National Academy of Sciences USA 93, 14692–14697. Hansen, J. D., Strassburger, P. & Du Pasquier, L. (1996). Conservation of an alpha 2 domain within the teleostean world, MHC class I from the rainbow

Mhc EXPRESSION IN SALMON MACROPHAGE-LIKE CELL LINE

487

trout Oncorhynchus mykiss. Developmental and Comparative Immunology 6, 417–425. Hashimoto, K., Nakanishi, T. & Kurosowa, Y. (1990). Isolation of carp genes encoding major histocompatibility complex antigens. Proceedings of the National Academy of Sciences USA 87, 6863–6867. Havell, E. A. & Spitalny, G. L. (1983). Endotoxin-induced interferon synthesis in macrophage cultures. Journal of the Reticuloendothelial Society 33, 369–380. Hordvik, I., Grimholt, U., Fosse, V. M., Lie, Ø. & Endresen, C. (1993). Cloning and sequence analysis of cDNAs encoding the MHC class II B chain in Atlantic salmon (Salmo salar). Immunogenetics 37, 437–441. Jephthah-Ochola, J., Urmson, J., Farkas, S. & Halloran, P. F. (1988). Regulation of MHC expression in vivo. Bacterial lipopolysaccharide induces class I and II MHC products in mouse tissues by a T cell-independent, cyclosporine-sensitive mechanism. Journal of Immunology 2, 792–800. Juul-Madsen, H. R., Glamann, J., Madsen, H. O. & Simonsen, M. (1992). MHC class II beta-chain expression in the rainbow trout. Scandinavian Journal of Immunology 35, 687–694. Knight, J., Stet, R. J. & Secombes, C. J. (1998). Modulation of MHC class II expression in rainbow trout Oncorhynchus mykiss macrophages by TNFá and LPS. Fish & Shellfish Immunology 8, 545–553. Koerner, T. J., Hamilton, T. A. & Adams, D. O. (1987). Suppressed expression of surface Ia on macrophages by lipopolysaccharide: evidence for regulation at the level of accumulation of mRNA. Journal of Immunology 1, 239–243. Koppang, E. O., Lundin, M., Press, C. McL., Rønningen, K. & Lie, Ø (1998a). Di#ering levels of Mhc class II â chain expression in a range of tissues from vaccinated and non-vaccinated Atlantic salmon (Salmo salar L.). Fish & Shellfish Immunology 8, 183–196. Koppang, E. O., Press, C. McL., Rønningen, K. & Lie, Ø (1998b). Expression of Mhc class I mRNA in tissues from vaccinated and non-vaccinated Atlantic salmon (Salmo salar L.). Fish & Shellfish Immunology 8, 577–587. Kovacsovics-Bankowski, M. & Rock, K. L. (1995). A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules. Science 267, 243–246. Mach, B., Steimle, V., Martinez-Soria, E. & Reith, W. (1996). Regulation of MHC class II genes: lessons from a disease. Annual Review of Immunology 14, 301–331. Maehora, N. & Ho, M. (1977). Cellular origin of interferon induced by bacterial lipopolysaccharide. Infection and Immunity 15, 78. Malnati, M. S., Marti, M., LaVaute, T., Jaraquemada, D., Biddison, W., DeMars, R. & Long, E. O. (1992). Processing pathways for presentation of cytosolic antigen to MHC class II-restricted T cells. Nature 357, 702–704. Melby, H. P. & Falk, K. (1995). Study of the interaction between a persistent infectious pancreatic necrosis virus (IPNV) infection and experimental infectious salmon anaemia (ISA) in Atlantic salmon, Salmo salar L. Journal of Fish Diseases 18, 579–586. Mjaaland, S., Rimstad, E., Falk, K. & Dannevig, B. H. (1997). Genomic characterization of the virus causing infectious salmon anemia in Atlantic salmon (Salmo salar L.): an orthomyxo-like virus in a teleost. Journal of Virology 10, 7681–7686. Murphy, W. J. (1996). E#ects of growth hormone, insulin-like growth factor-I, and somatostatin on immune function and development. In The Physiology of Immunity (J. A. Marsh & M. D. Kendall, eds) pp. 197–209. New York: CRC Press. Nash, A. D., Barcham, G. J., Andrews, A. E. & Brandon, M. R. (1992). Characterisation of ovine alveolar macrophages: regulation of surface antigen expression and cytokine production. Veterinary Immunology and Immunopathology 31, 77–94. Norbury, C. C., Hewlett, L. J., Prescott, A. R., Shastri, N. & Watts, C. (1995). Class I MHC presentation of exogenous soluble antigen via macropinocytosis in bone marrow macrophages. Immunity 3, 783–791.

488

E. O. KOPPANG ET AL.

Reitan, L. J. & Secombes, C. J. (1997). In vitro methods for vaccine evaluation. In Fish Vaccinology (R. Gudding, A. Lillehaug, P. J. Midtlyng & F. Brown, eds) pp. 293–301. Developments of Biological Standardization, Karger, Basel, Switzerland. Rietschel, E. T., Brade, H., Holst, O., Brade, L., Muller-Loennies, S., Mamat, U., Zahringer, U., Beckmann, F., Seydel, U., Brandenburg, K., Ulmer, A. J., Mattern, T., Heine, H., Schletter, J., Loppnow, H., Schonbeck, U., Flad, H. D., Hauschildt, S., Schade, U. F., Di Padova, F., Kusumoto, S. & Schumann, R. R. (1996). Bacterial endotoxin: Chemical constitution, biological recognition, host response, and immunological detoxification. Current Topics in Microbiology and Immunology 216, 39–81. Rock, K. L. (1996). A new foreign policy: MHC class I molecules monitor the outside world. Immunology Today 17, 131–137. Rosa, F. M. & Fellous, M. (1988). Regulation of HLA-DR gene by IFN-gamma: transcriptional and post-transcriptional control. Journal of Immunology 140, 1660–1664. Saji, M., Moriarty, J., Ban, T., Singer, D. S. & Kohn, D. L. (1992). Major histocompatibility complex class I gene expression in rat thyroid cells is regulated by hormones, methimazole, and iodide as well as interferon. Journal of Clinical Endocrinology and Metabolism 75, 871–878. Sakai, M. & Kajita, Y., Kobayashi, M. & Kawauchi, H. (1997). Immunostimulating e#ect of growth: In-vivo administration of growth hormone in rainbow trout enhances resistance to Vibrio anguillarum infection. Veterinary Immunology and Immunopathology 57, 147–152. Sakai, M., Kobayashi, M. & Kawauchi, H. (1995). Enhancement of chemiluminescent responses of phagocytic cells from rainbow trout, Oncorhynchus mykiss, by injection of growth hormone. Fish & Shellfish Immunology 5, 375–379. Sakai, M., Kobayashi, M. & Kawauchi, H. (1996). In vitro activation of fish phagocytic cells by GH, prolactin and somatolactin. Journal of Endocrinology 151, 113–118. Sakamoto, T. & Hirano, T. (1993). Expression of insulin-like growth factor I gene in osmoregulatory organs during sea water adaption of the salmonid fish: possible mode of osmoregulatory action of growth hormone. Proceedings of National Academy of Sciences USA 90, 1912–1916. Sakamoto, T., McCormick, S. D. & Hirano, T. (1993). Osmoregulatory actions of growth hormone and its mode of action in salmonids: a review. Fish Physiology and Biochemistry 11, 155–164. Secombes, C. J., Hardie, L. J. & Daniels, G. (1996). Cytokines in fish: an update. Fish & Shellfish Immunology 6, 291–304. Shevelev, A., Burfeind, P., Schulze, E., Rininsland, F., Johnson, T. R., Trojan, J., Chernicky, C. L., Hélène, C., Ilan, J. & Ilan, J. (1997). Potential triple helix-mediated inhibition of IGF-I gene expression significantly reduces tumorigenicity of glioblastoma in an animal model. Cancer Gene Therapy 2, 105–112. Sousa, C. R. & Germain, R. N. (1995). Major histocompatibility complex class I presentation of peptides derived from soluble exogenous antigen by a subset of cells engaged in phagosytosis. Journal of Experimental Medicine 182, 841–851. Stet, R. J. M. & Egberts, E. (1991). The histocompatibility system in teleostean fishes: from multiple histocompatibility loci to a major histocompatibility complex. Fish & Shellfish Immunology 1, 1–16. Stuge, T. B., Yoshida, S. H., Chinchar, V. G., Miller, N. W. & Clem, W. (1997). Cytotoxic activity generated from channel catfish peripheral blood leukocytes in mixed leukocyte cultures. Cellular Immunology 177, 154–161. Taksdal, T., Ramstad, A., Stangeland, K. & Dannevig, B. H. (1998). Induction of infectious pancreatic necrosis (IPN) in covertly infected Atlantic salmon, Salmo salar L., post-smolts by stress exposure, by injection of IPN virus (IPNV) and by cohabitation. Journal of Fish Diseases 21, 193–204.

Mhc EXPRESSION IN SALMON MACROPHAGE-LIKE CELL LINE

489

Thuvander, A., Wichardt, U. P. & Reitan, L. J. (1993). Humoral antibody response of brown trout Salmo trutta vaccinated against furunculosis. Diseases of Aquatic Organisms 17, 17–23. Trojan, J., Duc, H. T., Upegui-Gonzalez, L. C., Hor, F., Guo, Y., Anthony, D. & Iljan, J. (1996). Presence of MHC-I and B-7 molecules in rat and human glioma cells expressing antisense IGF-I mRNA. Neuroscience Letters 212, 9–12. Vallejo, A. N., Ellsaesser, C. F., Miller, N. W. & Clem, L. W. (1991). Spontaneous development of functionally active long-term monocyte-like cell lines from channel catfish. In Vitro Cell Developmental Biology 4, 279–286.