- Email: [email protected]
TRANSCRIPTION OF Fcγ RECEPTORS IN DIFFERENT RAT LIVER CELLS
Cell Biology International 2001, Vol. 25, No. 8, 821–824 doi:10.1006/cbir.2001.0761, available online at http://www.idealibrary.com on
SHORT COMMUNICATION
TRANSCRIPTION OF Fc RECEPTORS IN DIFFERENT RAT LIVER CELLS TORUNN LØVDAL and TROND BERG* Division of Molecular Cell Biology, Department of Biology, University of Oslo, P.O. Box 1050, Blindern, 0316 Oslo, Norway Received 8 February 2001; accepted 27 February 2001
Expression of Fc-receptors (FcRs) in different liver cells is poorly characterised. In the present study, transcription of FcRs in different rat liver cells was examined by means of Northern blot analysis and reverse transcriptase-polymerase chain reactions. We found that both Kupffer and liver endothelial cells produce mRNA for FcRIIB2, FcRIII and the -chain, whereas the level of FcRIIB1 mRNA is negligible. In contrast, parenchymal cells produce no FcR mRNA. 2001 Academic Press
K: Fc receptor, Kupffer cells, liver endothelial cells.
INTRODUCTION
MATERIALS AND METHODS
In addition to the neonatal Fc receptor (FcR), three different classes of FcRs that bind IgG have been defined in mouse and human: FcRI, FcRII, and FcRIII (reviewed by Gessner et al., 1998). Members of FcRII and FcRIII, but not of FcRI, have also been identified in rat (Zeger et al., 1990; Farber and Sears, 1991; Bocek and Pecht, 1993; Farber et al., 1993; Bocek, Jr et al., 1995). The liver is the main organ for uptake of IgGimmune complexes from the circulation (Johansson et al., 1996). Specific binding and uptake of small soluble IgG-immune complexes by Kupffer cells (KC) and liver endothelial cells (LEC), in absence of complement components (Muro et al., 1987; Johansson et al., 1996), indicate the presence of FcRs in these cells. Still, the expression of FcRs in different liver cells is poorly characterised. In the present study, transcription of FcRs in different rat liver cells was examined by means of Northern blot analysis and reverse transcriptase-polymerase chain reactions (RT-PCR).
Animals and cells Male Wistar rats (Møllegaard, Ejby, Denmark) were kept at a temperature of 213C, air humidity 40–60%, and a light–dark cycle of 12 and 12 h. They were given tap water, and fed with standard laboratory pellets (B&K Universal, Humberside, U.K.) ad libitum. Rats were anaesthetised by ether, and liver cells prepared by collagenase perfusion (Seglen, 1976). Suspensions of elutriated parenchymal cells (PC), and cultures of KC and LEC, were prepared as described earlier (Johansson et al., 1996; Lovdal et al., 2000). Monolayer cultures of the mouse peritoneal macrophage cell line J774A1, and normal rat kidney (NRK) cells, were grown in Dulbecco’s modified Eagle’s medium containing 4.4 g/l glucose, 2 m L-glutamine, 50– 100 U/ml penicillin, 50–100 g/ml streptomycin (Biowhittaker, Walkersville, MO, U.S.A.), and 10% fetal calf serum (Sigma-Aldrich, St Louis, MO, U.S.A.). RNA isolation and RT-PCR
*To whom correspondence should be addressed. Fax: +47 22 85 4605; E-mail: [email protected] 1065–6995/01/080821+04 $35.00/0
Poly A mRNA was isolated by means of magnetic oligo (dT) dynabeads (Dynal Biotech, Oslo, 2001 Academic Press
822
Norway), and cDNA prepared with AMV reverse transcriptase (Promega, Madison, WI, U.S.A.) in the absence of sodium pyrophosphate. We used primers specific for the rat B1-insert and -chain constructed by Bocek and co-workers (1995), and the following primers based on those constructed by Bocek and co-workers: rat FcRII sense primer 5 -ACGATTGTAGCTGCTGTCGC-3 and antisense primer 5 -GTAAGTTGATTAATTTTATT GGC-3 ; rat FcRIII sense primer 5 -AGCAA CSGCRTCCACCAGC-3 and antisense primer 5 CAGAGCATCATGTGTCCTGG-3 (in which S is C or G, and R is A or G; DNA Technology, Aarhus, Denmark). For human -actin we used the sense primer 5 -GAGCACGGCATTGTCACC AA-3 and antisense primer 5 -ATGTCACGTA CGATTTCCCG-3 (Medprobe, Oslo, Norway). PCR was performed in a Techne Genius temperature cycler (Cambridge, U.K.), using 0.1 U/l Taq DNA polymerase, 2 m magnesium chloride, and 0.2 m of each dNTP (Promega, Madison, WI, U.S.A.). Annealing of FcRII primers to cDNA took place at 56C. Touch-down PCR was performed for transcripts of the B1-insert and the -chain, with annealing temperature of 66–56C, whereas for transcripts of FcRIII it was 72–62C. The temperature was reduced 0.5C per cycle, followed by additional 20 cycles with annealing at 56C or 62C, respectively. All PCR routinely included negative control amplifications, in which no template was added. Cloning of PCR products The FcR PCR products of 644 bp (FcRIII), 457 bp (FcRIIB2), 573 bp (-chain), and 162 bp (B1-insert) were cloned into the pGEM-T vector, and transformed into competent JM109 cells (Promega, Madison, WI, U.S.A.). Recombinant clones were identified by colour-screening on indicator plates, and plasmid isolated following amplification in Luria-Bertani medium containing ampicillin (100 g/ml), under continuous agitation at 37C overnight. Sequences of all DNA products were verified by sequencing (GATC, Konstanz, Germany). Preparation of single-stranded cDNA probes Single-stranded cDNA probes were prepared as described by Espelund and co-workers (1990). Fulllength rat FcRIIIA and a human -actin fragment (422 bp), in pVZ1 and pBluescript SK, respectively, were used as templates. PCR was
Cell Biology International, Vol. 25, No. 8, 2001
performed using M13 primers (DNA Technology, Aarhus, Denmark), one of the two biotinylated, 0.2 U/l Taq DNA polymerase, 1.5 m MgCl2, and 0.2 m of each dNTP. Annealing of primers to cDNA took place at 65C. The single-stranded cDNA probes were labelled with digoxigenin (DIG; Roche, Basel, Switzerland) at 37C overnight, and kept at 20C. Northern blot analysis mRNA was separated by electrophoresis under denaturing conditions essentially as described by Galau and co-workers (1986). The formaldehyde gel-loading buffer contained 6.25% sucrose, 0.025% bromphenol blue, 25 m MOPS, 2.5 m EDTA (pH 8.0), 7.4% formaldehyde (pH>4), and 55% deionized formamide, and the gel and gel-running buffer 6% formaldehyde. Blotting of mRNA on to Hybond nylon membrane (Amersham Pharmacia Biotech, Uppsala, Sweden) took place by capillary-transfer, using 10SSC as transfer buffer (Sambrook et al., 1989). After blotting, mRNA was cross-linked to the moist membrane by UVirradiation (about 1.5 J per cm2) in a UV crosslinker (Hoefer Scientific Instruments, San Francisco, CA, U.S.A.). The membrane was treated at 68C for 20 min in 0.7SSC and 1SPEP, thereafter for 2–3 h in 0.7SSC, 1SPEP, 5Denhardt’s, and 100 g/ml salmon sperm DNA, before hybridization in 0.7SSC, 1SPEP, 1Denhardt’s, 10% dextran sulphate, and 100 g/ml salmon sperm DNA (Galau et al., 1986; Sambrook et al., 1989). Washing was performed at 68C for 320 min in 0.7SSC, 1SPEP, and 1Denhardt’s. Detection of DIGlabelled nucleic acids was performed using the chemiluminescent substrate CSPD (Roche, Basel, Switzerland). Before reprobing, membranes containing mRNA were stripped by boiling in 0.1% sodium dodecyl sulphate for 5 min. All images were processed for presentation using Adobe Photoshop (Adobe Systems, San Jose, CA, U.S.A.).
RESULTS AND DISCUSSION FcR transcription in different rat liver cells studied by Northern blot analysis The results obtained by Northern blot analysis, using the single-stranded full-length rat FcRIIIA cDNA probe, are presented in Figure 1. A smear of transcripts, ranging from 1.4–1.6 kb, was detected
Cell Biology International, Vol. 25, No. 8, 2001
STD
2.8 1.9 1.6
PC
LEC
KC
NRK
823
J774
FcgRIII
A
PC LEC KC AP NC
B
STD PC LEC KC NC
STD PC LEC KC NC
1 353 1 078 872 603 2.8 1.9 1.6
b-actin
Fig. 1. FcR transcription in different rat liver cells studied by Northern blot analysis. Poly A mRNA was isolated from cells by means of magnetic oligo (dT) dynabeads, separated by electrophoresis in a denaturing agarose gel (1.7%), and blotted on to a nylon membrane. The membrane was hybridised with single-stranded OIG-labelled full-length FcRIIIA cDNA from rat (upper panel), stripped, and reprobed with a singlestranded DIG-labelled -actin cDNA fragment (lower panel), which served as control for mRNA loading of the lanes. Origins of mRNA are marked above the lanes, and length of DIG-labelled RNA marker fragments is given in kb.
in KC, LEC and J774 cells (positive control). No transcripts of these sizes were demonstrated in PC or NRK cells (negative control). Bands were detected in all lanes following stripping and reprobing of the membrane with a -actin probe, confirming the presence of mRNA. Thus, both KC and LEC, but not PC seem to produce FcR mRNA. The appearance of the smear could be due to the high number of FcRIII transcripts found in rats. As many as eight different transcripts, ranging from 1.4 to 1.6 kb, have been characterised (Zeger et al., 1990; Farber and Sears, 1991; Farber et al., 1993). In agreement with the results obtained by Zeger and co-workers (1990), hybridisation of the full-length rat FcRIII probe not only to FcRIII transcripts (1.4 kb), but also to FcRIIB1 (1.6 kb) and FcRIIB2 (1.5 kb) transcripts, takes place in the J774 cells. It is therefore likely that crosshybridisation of this probe occurs in rat liver cells, as well. In addition, the full-length rat FcRIII probe seems to cross-hybridise to mRNA of about 1.9 kb. The smear of transcripts (range 1.4–1.6 kb) detected following reprobing with -actin cDNA, on the other hand, is probably due to incomplete stripping of the membrane.
(a)
(b)
(c)
(d)
1 353 1 078 872 603 310
Fig. 2. FcR transcription in different rat liver cells studied by RT-PCR amplification. Poly A mRNA was isolated from cells by means of magnetic oligo (dT) dynabeads, and cDNA prepared with AMV reverse transcriptase. In panel A, the cDNA quality was confirmed by PCR amplification of a -actin fragment (422 bp). pBluescript SK containing the -actin cDNA fragment was included as a positive control (actin plasmid, AP). In panel B, FcRIIB2 (a), FcRIII (b), FcRIIB1 (c), and -chain (d) fragments, of 457, 644, 162, and 573 bp, respectively, were amplified by PCR. FcRIIB2 and FcRIII PCR products were analysed in 2% agarose gels (a, b), whereas FcRIIB1 and -chain PCR products were analysed in 1% agarose gels (c, d). Origins of cDNA are marked above the lanes, no cDNA was added in the negative control (NC). Length of DNA marker fragments is given in bp.
FcR transcription in different rat liver cells studied by RT-PCR amplification To identify FcRs transcribed in different rat liver cells, primer sets for the unalike cytoplasmic domains of rat FcRII and FcRIIIA, as well as for the insert of the B1 isoform of FcRII, were used for RT-PCR amplification. Production of -actin PCR products in all cell types, confirmed the presence of cDNA (Fig. 2A). FcRIIB2 (Fig. 2Ba) and FcRIII (Fig. 2Bb) cDNA fragments, of 457 and 644 bp, respectively, were obtained by PCR in both KC and LEC, but not in PC. In addition, minor amounts of PCR products of similar sizes were obtained in both KC and LEC, probably representing alternative splice products. Because FcRIII is associated with -, - (only in
824
mast cells), or -chains (only in natural killer cells; reviewed by Gessner et al., 1998), we examined the presence of -chain mRNA in different rat liver cells. In accordance with transcription of the FcRIII gene, -chain transcripts were detected in KC and LEC, but not in PC (Fig. 2Bd). Variable results were obtained when primers for the B1insert were used. Occasionally, a cDNA fragment of 162 bp was obtained in all liver cell types examined (Fig. 2Bc). The B1-insert is located between the sequences recognised by the FcRII primers, which means that, if FcRIIB1 transcripts are produced in rat liver cells, FcRII primers should give rise to a 598 bp PCR product containing the B1-insert. However, a negligible amount of a PCR product of that size was detected in the KC and LEC using these primers. Thus, production of FcRIIB1 transcripts in rat liver cells is probably negligible and explains why the PCR results, using primers for the B1-insert, were not reproducible. The controls, in which no cDNA was added, were negative. The sequences of the PCR products of 162 bp (B1-insert), 457 bp (FcRIIB2), 644 bp (FcRIII), and 573 bp (the -chain) were verified by sequencing (data not shown). Our finding that FcR mRNA is present in the non-PC, but not in PC, is in agreement with the ability and inability of non-PC and PC, respectively, to endocytose small soluble IgG-immune complexes (Johansson et al., 1996). It is also in accordance with immunohistochemical staining of human and mouse liver sections, which has shown that both KC and LEC express low-affinity FcRs. FcRII, but not FcRIII was demonstrated in human LEC (Muro et al., 1993; Ahmed et al., 1995). We conclude that both KC and LEC produce mRNA for FcRIIB2, FcRIII and the -chain, whereas the level of FcRIIB1 mRNA is negligible. In contrast, PC do not produce FcR mRNA at all. ACKNOWLEDGEMENTS Thanks are due to Duane Sears for providing the full-length rat FcRIIIA cDNA cloned into the pVZ1 plasmid. Our sincere thanks also go to Gunnar Thoresen, Lene Malerød, Hanne Holla˚ s, and Lene K. Juvet for all methodological help. We also thank Marianne Frøystad for valuable
Cell Biology International, Vol. 25, No. 8, 2001
discussions. The study was supported by the Norwegian Cancer Society. REFERENCES A SS, M H, N M, K I, T Y, S H, 1995. Fc receptors in liver sinusoidal endothelial cells in NZB/W F1 lupus mice: a histological analysis using soluble immunoglobulin G-immune complexes and a monoclonal antibody (2.4G2). Hepatology 22: 316–324. B P, P I, 1993. Cloning and sequence of the cDNA coding for rat type II Fc gamma receptor of mast cells. FEBS Lett 331: 86–90. B P J, D L, D P, P I, 1995. Characterization of Fc gamma receptors on rat mucosal mast cells using a mutant Fc epsilon RI-deficient rat basophilic leukemia line. Eur J Immunol 25: 2948–2955. E M, S RAP, J KS, 1990. A simple method for generating single-stranded DNA probes labeled to high activities. Nucleic Acids Res 18: 6157–6158. F DL, G R, N MY, T M, K JP, S DW, 1993. Rat class III Fc gamma receptor isoforms differ in IgG subclass-binding specificity and fail to associate productively with rat CD3 zeta. J Immunol 150: 4364–4375. F DL, S DW, 1991. Rat CD16 is defined by a family of class III Fc gamma receptors requiring co-expression of heteroprotein subunits. J Immunol 146: 4352–4361. G GA, H DW, D L, 1986. Abscisic acid induction of cloned cotton late embryogenesis-abundant (Lea) mRNAs. Plant Mol Biol 7: 155–170. G JE, H H, T A, S RE, 1998. The IgC Fc receptor family. Ann Hematol 76: 231–248. J AG, L T, M KE, B T, S T, 1996. Liver cell uptake and degradation of soluble immunoglobulin C immune complexes in vivo and in vitro in rats. Hepatology 24: 169–175. L T, A E, B A, B T, 2000. Fc receptor mediated endocytosis of small soluble immunoglobulin G immune complexes in Kupffer and endothelial cells from rat liver. J Cell Sci 113: 3255–3266. M H, S H, K I, N S, 1993. Defect of Fc receptors and phenotypical changes in sinusoidal endothelial cells in human liver cirrhosis. Am J Pathol 143: 105–119. M H, S H, M N, N S, 1987. Fc receptors of liver sinusoidal endothelium in normal rats and humans. A histologic study with soluble immune complexes. Gastroenterology 93: 1078–1085. S J, F EF, M T, 1989. Molecular Cloning. A laboratory manual. Second Ed. New York, Cold Spring Harbor Laboratory Press. S PO, 1976. Preparation of isolated rat liver cells. Methods Cell Biol 13: 29–83. Z DL, H PM, S DW, 1990. Characterization and expression of an Fc gamma receptor cDNA cloned from rat natural killer cells. Proc Natl Acad Sci USA 87: 3425–3429.
SHORT COMMUNICATION
TRANSCRIPTION OF Fc RECEPTORS IN DIFFERENT RAT LIVER CELLS TORUNN LØVDAL and TROND BERG* Division of Molecular Cell Biology, Department of Biology, University of Oslo, P.O. Box 1050, Blindern, 0316 Oslo, Norway Received 8 February 2001; accepted 27 February 2001
Expression of Fc-receptors (FcRs) in different liver cells is poorly characterised. In the present study, transcription of FcRs in different rat liver cells was examined by means of Northern blot analysis and reverse transcriptase-polymerase chain reactions. We found that both Kupffer and liver endothelial cells produce mRNA for FcRIIB2, FcRIII and the -chain, whereas the level of FcRIIB1 mRNA is negligible. In contrast, parenchymal cells produce no FcR mRNA. 2001 Academic Press
K: Fc receptor, Kupffer cells, liver endothelial cells.
INTRODUCTION
MATERIALS AND METHODS
In addition to the neonatal Fc receptor (FcR), three different classes of FcRs that bind IgG have been defined in mouse and human: FcRI, FcRII, and FcRIII (reviewed by Gessner et al., 1998). Members of FcRII and FcRIII, but not of FcRI, have also been identified in rat (Zeger et al., 1990; Farber and Sears, 1991; Bocek and Pecht, 1993; Farber et al., 1993; Bocek, Jr et al., 1995). The liver is the main organ for uptake of IgGimmune complexes from the circulation (Johansson et al., 1996). Specific binding and uptake of small soluble IgG-immune complexes by Kupffer cells (KC) and liver endothelial cells (LEC), in absence of complement components (Muro et al., 1987; Johansson et al., 1996), indicate the presence of FcRs in these cells. Still, the expression of FcRs in different liver cells is poorly characterised. In the present study, transcription of FcRs in different rat liver cells was examined by means of Northern blot analysis and reverse transcriptase-polymerase chain reactions (RT-PCR).
Animals and cells Male Wistar rats (Møllegaard, Ejby, Denmark) were kept at a temperature of 213C, air humidity 40–60%, and a light–dark cycle of 12 and 12 h. They were given tap water, and fed with standard laboratory pellets (B&K Universal, Humberside, U.K.) ad libitum. Rats were anaesthetised by ether, and liver cells prepared by collagenase perfusion (Seglen, 1976). Suspensions of elutriated parenchymal cells (PC), and cultures of KC and LEC, were prepared as described earlier (Johansson et al., 1996; Lovdal et al., 2000). Monolayer cultures of the mouse peritoneal macrophage cell line J774A1, and normal rat kidney (NRK) cells, were grown in Dulbecco’s modified Eagle’s medium containing 4.4 g/l glucose, 2 m L-glutamine, 50– 100 U/ml penicillin, 50–100 g/ml streptomycin (Biowhittaker, Walkersville, MO, U.S.A.), and 10% fetal calf serum (Sigma-Aldrich, St Louis, MO, U.S.A.). RNA isolation and RT-PCR
*To whom correspondence should be addressed. Fax: +47 22 85 4605; E-mail: [email protected] 1065–6995/01/080821+04 $35.00/0
Poly A mRNA was isolated by means of magnetic oligo (dT) dynabeads (Dynal Biotech, Oslo, 2001 Academic Press
822
Norway), and cDNA prepared with AMV reverse transcriptase (Promega, Madison, WI, U.S.A.) in the absence of sodium pyrophosphate. We used primers specific for the rat B1-insert and -chain constructed by Bocek and co-workers (1995), and the following primers based on those constructed by Bocek and co-workers: rat FcRII sense primer 5 -ACGATTGTAGCTGCTGTCGC-3 and antisense primer 5 -GTAAGTTGATTAATTTTATT GGC-3 ; rat FcRIII sense primer 5 -AGCAA CSGCRTCCACCAGC-3 and antisense primer 5 CAGAGCATCATGTGTCCTGG-3 (in which S is C or G, and R is A or G; DNA Technology, Aarhus, Denmark). For human -actin we used the sense primer 5 -GAGCACGGCATTGTCACC AA-3 and antisense primer 5 -ATGTCACGTA CGATTTCCCG-3 (Medprobe, Oslo, Norway). PCR was performed in a Techne Genius temperature cycler (Cambridge, U.K.), using 0.1 U/l Taq DNA polymerase, 2 m magnesium chloride, and 0.2 m of each dNTP (Promega, Madison, WI, U.S.A.). Annealing of FcRII primers to cDNA took place at 56C. Touch-down PCR was performed for transcripts of the B1-insert and the -chain, with annealing temperature of 66–56C, whereas for transcripts of FcRIII it was 72–62C. The temperature was reduced 0.5C per cycle, followed by additional 20 cycles with annealing at 56C or 62C, respectively. All PCR routinely included negative control amplifications, in which no template was added. Cloning of PCR products The FcR PCR products of 644 bp (FcRIII), 457 bp (FcRIIB2), 573 bp (-chain), and 162 bp (B1-insert) were cloned into the pGEM-T vector, and transformed into competent JM109 cells (Promega, Madison, WI, U.S.A.). Recombinant clones were identified by colour-screening on indicator plates, and plasmid isolated following amplification in Luria-Bertani medium containing ampicillin (100 g/ml), under continuous agitation at 37C overnight. Sequences of all DNA products were verified by sequencing (GATC, Konstanz, Germany). Preparation of single-stranded cDNA probes Single-stranded cDNA probes were prepared as described by Espelund and co-workers (1990). Fulllength rat FcRIIIA and a human -actin fragment (422 bp), in pVZ1 and pBluescript SK, respectively, were used as templates. PCR was
Cell Biology International, Vol. 25, No. 8, 2001
performed using M13 primers (DNA Technology, Aarhus, Denmark), one of the two biotinylated, 0.2 U/l Taq DNA polymerase, 1.5 m MgCl2, and 0.2 m of each dNTP. Annealing of primers to cDNA took place at 65C. The single-stranded cDNA probes were labelled with digoxigenin (DIG; Roche, Basel, Switzerland) at 37C overnight, and kept at 20C. Northern blot analysis mRNA was separated by electrophoresis under denaturing conditions essentially as described by Galau and co-workers (1986). The formaldehyde gel-loading buffer contained 6.25% sucrose, 0.025% bromphenol blue, 25 m MOPS, 2.5 m EDTA (pH 8.0), 7.4% formaldehyde (pH>4), and 55% deionized formamide, and the gel and gel-running buffer 6% formaldehyde. Blotting of mRNA on to Hybond nylon membrane (Amersham Pharmacia Biotech, Uppsala, Sweden) took place by capillary-transfer, using 10SSC as transfer buffer (Sambrook et al., 1989). After blotting, mRNA was cross-linked to the moist membrane by UVirradiation (about 1.5 J per cm2) in a UV crosslinker (Hoefer Scientific Instruments, San Francisco, CA, U.S.A.). The membrane was treated at 68C for 20 min in 0.7SSC and 1SPEP, thereafter for 2–3 h in 0.7SSC, 1SPEP, 5Denhardt’s, and 100 g/ml salmon sperm DNA, before hybridization in 0.7SSC, 1SPEP, 1Denhardt’s, 10% dextran sulphate, and 100 g/ml salmon sperm DNA (Galau et al., 1986; Sambrook et al., 1989). Washing was performed at 68C for 320 min in 0.7SSC, 1SPEP, and 1Denhardt’s. Detection of DIGlabelled nucleic acids was performed using the chemiluminescent substrate CSPD (Roche, Basel, Switzerland). Before reprobing, membranes containing mRNA were stripped by boiling in 0.1% sodium dodecyl sulphate for 5 min. All images were processed for presentation using Adobe Photoshop (Adobe Systems, San Jose, CA, U.S.A.).
RESULTS AND DISCUSSION FcR transcription in different rat liver cells studied by Northern blot analysis The results obtained by Northern blot analysis, using the single-stranded full-length rat FcRIIIA cDNA probe, are presented in Figure 1. A smear of transcripts, ranging from 1.4–1.6 kb, was detected
Cell Biology International, Vol. 25, No. 8, 2001
STD
2.8 1.9 1.6
PC
LEC
KC
NRK
823
J774
FcgRIII
A
PC LEC KC AP NC
B
STD PC LEC KC NC
STD PC LEC KC NC
1 353 1 078 872 603 2.8 1.9 1.6
b-actin
Fig. 1. FcR transcription in different rat liver cells studied by Northern blot analysis. Poly A mRNA was isolated from cells by means of magnetic oligo (dT) dynabeads, separated by electrophoresis in a denaturing agarose gel (1.7%), and blotted on to a nylon membrane. The membrane was hybridised with single-stranded OIG-labelled full-length FcRIIIA cDNA from rat (upper panel), stripped, and reprobed with a singlestranded DIG-labelled -actin cDNA fragment (lower panel), which served as control for mRNA loading of the lanes. Origins of mRNA are marked above the lanes, and length of DIG-labelled RNA marker fragments is given in kb.
in KC, LEC and J774 cells (positive control). No transcripts of these sizes were demonstrated in PC or NRK cells (negative control). Bands were detected in all lanes following stripping and reprobing of the membrane with a -actin probe, confirming the presence of mRNA. Thus, both KC and LEC, but not PC seem to produce FcR mRNA. The appearance of the smear could be due to the high number of FcRIII transcripts found in rats. As many as eight different transcripts, ranging from 1.4 to 1.6 kb, have been characterised (Zeger et al., 1990; Farber and Sears, 1991; Farber et al., 1993). In agreement with the results obtained by Zeger and co-workers (1990), hybridisation of the full-length rat FcRIII probe not only to FcRIII transcripts (1.4 kb), but also to FcRIIB1 (1.6 kb) and FcRIIB2 (1.5 kb) transcripts, takes place in the J774 cells. It is therefore likely that crosshybridisation of this probe occurs in rat liver cells, as well. In addition, the full-length rat FcRIII probe seems to cross-hybridise to mRNA of about 1.9 kb. The smear of transcripts (range 1.4–1.6 kb) detected following reprobing with -actin cDNA, on the other hand, is probably due to incomplete stripping of the membrane.
(a)
(b)
(c)
(d)
1 353 1 078 872 603 310
Fig. 2. FcR transcription in different rat liver cells studied by RT-PCR amplification. Poly A mRNA was isolated from cells by means of magnetic oligo (dT) dynabeads, and cDNA prepared with AMV reverse transcriptase. In panel A, the cDNA quality was confirmed by PCR amplification of a -actin fragment (422 bp). pBluescript SK containing the -actin cDNA fragment was included as a positive control (actin plasmid, AP). In panel B, FcRIIB2 (a), FcRIII (b), FcRIIB1 (c), and -chain (d) fragments, of 457, 644, 162, and 573 bp, respectively, were amplified by PCR. FcRIIB2 and FcRIII PCR products were analysed in 2% agarose gels (a, b), whereas FcRIIB1 and -chain PCR products were analysed in 1% agarose gels (c, d). Origins of cDNA are marked above the lanes, no cDNA was added in the negative control (NC). Length of DNA marker fragments is given in bp.
FcR transcription in different rat liver cells studied by RT-PCR amplification To identify FcRs transcribed in different rat liver cells, primer sets for the unalike cytoplasmic domains of rat FcRII and FcRIIIA, as well as for the insert of the B1 isoform of FcRII, were used for RT-PCR amplification. Production of -actin PCR products in all cell types, confirmed the presence of cDNA (Fig. 2A). FcRIIB2 (Fig. 2Ba) and FcRIII (Fig. 2Bb) cDNA fragments, of 457 and 644 bp, respectively, were obtained by PCR in both KC and LEC, but not in PC. In addition, minor amounts of PCR products of similar sizes were obtained in both KC and LEC, probably representing alternative splice products. Because FcRIII is associated with -, - (only in
824
mast cells), or -chains (only in natural killer cells; reviewed by Gessner et al., 1998), we examined the presence of -chain mRNA in different rat liver cells. In accordance with transcription of the FcRIII gene, -chain transcripts were detected in KC and LEC, but not in PC (Fig. 2Bd). Variable results were obtained when primers for the B1insert were used. Occasionally, a cDNA fragment of 162 bp was obtained in all liver cell types examined (Fig. 2Bc). The B1-insert is located between the sequences recognised by the FcRII primers, which means that, if FcRIIB1 transcripts are produced in rat liver cells, FcRII primers should give rise to a 598 bp PCR product containing the B1-insert. However, a negligible amount of a PCR product of that size was detected in the KC and LEC using these primers. Thus, production of FcRIIB1 transcripts in rat liver cells is probably negligible and explains why the PCR results, using primers for the B1-insert, were not reproducible. The controls, in which no cDNA was added, were negative. The sequences of the PCR products of 162 bp (B1-insert), 457 bp (FcRIIB2), 644 bp (FcRIII), and 573 bp (the -chain) were verified by sequencing (data not shown). Our finding that FcR mRNA is present in the non-PC, but not in PC, is in agreement with the ability and inability of non-PC and PC, respectively, to endocytose small soluble IgG-immune complexes (Johansson et al., 1996). It is also in accordance with immunohistochemical staining of human and mouse liver sections, which has shown that both KC and LEC express low-affinity FcRs. FcRII, but not FcRIII was demonstrated in human LEC (Muro et al., 1993; Ahmed et al., 1995). We conclude that both KC and LEC produce mRNA for FcRIIB2, FcRIII and the -chain, whereas the level of FcRIIB1 mRNA is negligible. In contrast, PC do not produce FcR mRNA at all. ACKNOWLEDGEMENTS Thanks are due to Duane Sears for providing the full-length rat FcRIIIA cDNA cloned into the pVZ1 plasmid. Our sincere thanks also go to Gunnar Thoresen, Lene Malerød, Hanne Holla˚ s, and Lene K. Juvet for all methodological help. We also thank Marianne Frøystad for valuable
Cell Biology International, Vol. 25, No. 8, 2001
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