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Identification of an alternatively spliced isoform of the common cytokine receptor γ chain in chickens
BBRC Biochemical and Biophysical Research Communications 299 (2002) 321–327 www.academicpress.com
Identification of an alternatively spliced isoform of the common cytokine receptor c chain in chickens Wongi Min, Hyun Soon Lillehoj,* and Raymond Hugh Fetterer U.S. Department of Agriculture, Parasite Biology, Epidemiology, and Systematics Laboratory, Animal and Natural Resources Institute, Building 1040, BARC-East, Beltsville, MD 20705, USA Received 6 October 2002
Abstract The common cytokine receptor c ðcc Þ chain is shared by at least six cytokine receptors and plays a critical role in the regulation of immune responses. In this study, we discovered that, unlike mammals, chickens possess two different cc gene transcripts, chcc -a and chcc -b. Sequence comparisons between the cDNAs and a cc genomic clone isolated by PCR revealed that chcc -b contained an in-frame 78 bp insertion between Gly-222 and Val-223 of the chcc -a sequence. This insertion most likely resulted from alternative splicing such that the fifth intron was not removed from the chcc -b transcript. Furthermore, while chcc -a and chcc -b transcripts were expressed equally in the spleen, thymus, bursa, and cecal tonsils, they were differentially expressed during the time course of Con A stimulation of splenic T lymphocytes. Western blot analysis of normal spleen lymphocytes identified 45, 53, and 64 kDa immunoreactive bands whereas only 64 kDa band was detected in Con A-activated splenic lymphocytes. COS-7 cells transfected with chcc -b secreted 42 kDa proteins. Taken together, our results document that chickens express an alternative spliced cc receptor which is larger than the conventional transcript and this novel isoform generates soluble receptors in the transfected COS-7 cells. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Chicken; Common cytokine receptor c chain; Isoform; Gene regulation
The cc chain is a shared component of the receptors for IL-2, -4, -7, -9, -15, and -21 [1,2] and is a member of the cytokine receptor superfamily displaying a characteristic spacing of four conserved cysteine residues and a WSXWS motif [3,4]. The cc chain is expressed as a transmembrane glycoprotein on CD4þ and CD8þ T cells, B cells, NK cells, monocytes/macrophages, neutrophils, and granulocytes [4,5], where it forms heterodimeric or heterotrimeric complexes with specific interleukin receptors thereby augmenting ligand affinity, receptor internalization, and signal transduction [4,6]. The cytoplasmic region of cc contains two Src homology region 2 (SH2) domains [7] that contribute to intracellular signal transduction through interaction with phosphotyrosine residues of various effector molecules [1,8].
* Corresponding author. Fax: 1-301-504-5103. E-mail address: [email protected] (H.S. Lillehoj).
The critical role of the cc chain in regulating lymphocyte development and proliferation is illustrated by the discovery that some forms of X-linked SCID (XSCID) were caused by mutations in the human [9–11], murine [12–14], or canine [15–17] cc genes. XSCID is characterized by profound developmental impairment of T and NK cells. A review of the literature related to cc gene defects suggests that cc chains of different species may have different functions. Thus, while B cell levels were unaffected or, in some cases, slightly elevated in XSCID [1,18], c= knockout mice showed a marked c decrease in B cell numbers [12,13]. Furthermore, mice with a targeted deletion in the cc gene and dogs with a naturally occurring form of XSCID exhibited a somewhat different phenotype compared with human XSCID [19,20]. In light of the facts that cc Õs from different species may be functionally diverse, we became interested in elucidating the role of two chicken cc genes cloned from an expressed sequence tag (EST) cDNA. Sequence comparisons between the cDNAs and a cc genomic
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clone indicated that one cDNA represents a conventional form of cc whereas the other cDNA identifies a novel spliced transcript which contains an in-frame 78 bp insertion. Furthermore, differential expression of two forms of chicken cc chains was seen in Con A-activated splenic T cells and transiently transfected COS-7 cells using Northern and Western blot analyses.
Materials and methods Chickens. Fertilized eggs of White Leghorn SC chickens were obtained from Hyline International Production Center (Dallas Center, IA) and hatched at the Animal and Natural Resources Institute (Beltsville, MD). Chickens were kept in wire cages and were provided with water and food ad libitum. Isolation of chicken cc cDNA and genomic clones. The 50 region of the partial cc cDNA EST clone pat.pk0020.e7 [21] was extended by rapid amplification of cDNA ends (RACE, version 2.0, Life Technologies, Gaithersburg, MD). Total RNA was isolated from splenic lymphocytes of 5- or 6-week-old chickens using the Trizol (Life Technologies), first strand cDNA was synthesized from 5.0 lg RNA with the cc specific primer 50 -GCAGCAGCAGGCAGGAGGCGAT using reverse transcriptase (Superscript II RT, Life Technologies), and RNA template was removed with RNase (Life Technologies). The first strand product was purified with a GlassMax DNA isolation spin cartridge (Life Technologies), a homopolymeric tail added to the 30 end using dCTP and TdT, and the tailed cDNA was amplified by PCR with a 50 -RACE abridged anchor primer and the cc specific primer 50 -TCAGCACCGTGTGGATCCAGA. PCR conditions were 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, for 35 cycles using Platinum Taq DNA polymerase (Promega, Madison, WI). Two PCR clones were isolated following agarose gel electrophoresis and the amplified cDNAs were cloned into the pCR3.1 vector (Invitrogen, San Diego, CA) and sequenced. A genomic cc clone was isolated from a chicken cosmid library (Clontech, Palo Alto, CA) that originated from a male adult Leghorn liver by PCR using the cc specific primers 50 -GCGCAGCAAGATCAACAACTA and 50 -CCCACGTAGCTGC TCTTGTA and the PCR conditions described above. The sequence data were submitted with GenBank Accession Nos. AJ419897 for chcc -a, AJ419896 for chcc -b, and AJ419898 for genomic sequence. Northern blot analysis. PolyðAÞþ RNA was isolated using the PolyA Tract mRNA Isolation System IV (Promega) from total RNA of various tissues, subjected to electrophoresis through 1% agarose (Northern Max-Gly Kit, Ambion, Austin, TX), and transferred to a positively charged nylon membrane (Boehringer–Mannheim, Mannheim, Germany). The blot was hybridized with a digoxigenin-labeled chicken cc cDNA probe in DIG Easy Hyb solution (Boehringer– Mannheim) at 55 °C overnight. The probe was generated by PCR using the chicken cc specific primers 50 -TCAGCACCGTGTGGATC CAGA and 50 -AAGGTAGGCCGCGCACTGAGA. The blot was washed twice in 2 SSC and 0.1% SDS for 15 min at room temperature followed twice by 0.1 SSC and 0.1% SDS for 15 min at 55 °C. Detection was carried out with the DIG DNA Labeling and Detection Kit (Boehringer–Mannheim) according to manufacturerÕs instruction. As a control, the chicken b actin gene was amplified by PCR with the primers 50 -TCTGGTGGTACCACAATGTACCCT and 50 -CCAGT AATTGGTACCGGCTCCTC. Cell culture. Splenic lymphocytes were isolated by Histopaque-1077 (Sigma, St. Louis, MO) centrifugation, resuspended at 1 107 /ml in IMDM (Life Technologies) supplemented with 10% FCS (HyClone, Logan, UT), and stimulated with 12.5 lg/ml Con A (Sigma) at 41 °C in 5% CO2 . Immunomagnetic purification of B cells and flow cytometry. Chicken B cells were purified from splenic lymphocytes using the Magnetic Cell
Sorting System (autoMACS, Miltenyi Biotec, Cologne, Germany) according to manufacturerÕs protocol. Briefly, splenic cells were sequentially incubated with the B cell specific Bu mAb [22] and goat anti-mouse IgG microbeads, and B cells enriched by the autoMACS separator. B cell purity was verified by flow cytometry with phycoerythrin-conjugated Bu or CD8 mAbs specific for B or T cells as described [23] using the EPICS-XL flow cytometer (Coulter, Hialeah, FL). B cells prepared in this manner were routinely >96% pure. Expression of recombinant chicken cc and development of anti-cc mAbs. For recombinant cc production, the extracellular region of the chcc -a gene was amplified by PCR using the following primers: 50 -GATCGGATCCATGGCGGTGCCCGGGGCTTCG, and 50 -TC AGCACCGTGTGGATCCAGA (Fig. 1). The recombinant plasmid in pMal-c2 vector (New England Biolabs, Beverly, MA) was transformed into competent Escherichia coli DH5a and the transformant was selected on ampicillin agar plates at 37 °C and induced with 1.0 mM isopropyl-b-D -thiogalactopyranoside for 3 h. The recombinant protein fused with maltose binding proteins was purified on amylose affinity column (New Englind Biolabs) according to manufacturerÕs instructions. For hybridoma production, female BALB/c mice of 5–6 weeks of age were immunized subcutaneously with 50 lg recombinant chcc -a in FreundÕs complete adjuvant and boosted two times biweekly with 50 lg antigen in incomplete FreundÕs adjuvant. Three days prior to fusion, 10 lg of the recombinant antigen in PBS was intravenously injected. Splenocytes were fused with SP2/0 myeloma cells (ATCC, Manassas, VA) in 50% polyethylene glycol and hybridoma cells were selected in IMDM containing 20% FCS, hypoxanthine, aminopterin, and thymidine (Sigma, St. Louis, MO). Hybridomas producing antigen reactive mAbs were identified by ELISA. Briefly, flat-bottomed 96-well microtiter plates (Coster, Corning, NY) were coated with 100 ll E. coliexpressed affinity purified protein (5 lg/ml) in 0.1 M carbonate buffer, pH 9.6 at 4 °C overnight and washed three times with PBS containing 0.05% Tween 20 (PBS-T). Wells were blocked with 200 ll PBS containing 1% BSA (PBS-BSA) for 1 h at room temperature and washed with PBS-T and 100 ll hybridoma supernatant was added for 2 h at room temperature. After washing with PBS-T, bound antibodies were detected at OD450 with horseradish peroxidase-conjugated goat antimouse IgG antibody (Sigma) and 0.01% tetramethylbenzidine (Sigma) in 0.05 M phosphate-citrate buffer, pH 5.0. Positive hybridomas were cloned by limiting dilution using mouse thymus feeder cells and ascites were produced by intraperitoneal injection of 5 106 cells into BALB/c mice. One mAb (cM1-11) was selected from several positive clones for further study and its ascites were purified on the Hi-Trap Protein G column (Amersham Pharmacia Biotech, Piscataway, NJ) using fast protein liquid chromatography (FPLC) for Western blot analysis. Construction of chicken cc expression plasmids and Western blot analysis. The full-length chcc -a and chcc -b cDNAs were amplified by PCR from single stranded cDNAs of splenic lymphocytes using the following primers: forward primer (HindIII cloning site is underlined), 50 -AGGCAAGCTTCGCACTGAGAGTTGCGCCATG; reverse primer (EcoRI cloning site is underlined and 6 copy histidine tag is indicated by subscript), 50 -ATCCGAATTCTCAðATGÞ6 CGCTCCCA CGTAGCTGCT. The PCR products (chcc -a-His and chcc -b-His) were digested with HindIII and EcoRI and cloned into the corresponding restriction endonuclease sites of pcDNA3 (Invitrogen). COS-7 cells were transiently transfected with 10 lg constructs using Lipofectamine (Life Technologies), incubated 48 h at 37 °C in IMDM without 10% FCS, and harvested. Cell-free culture supernatants from transfected COS-7 cells were concentrated up to 10-fold using the Amicon Centriplus (YM-10) (Millipore, Bedford, MA). Cells were extracted with SDS–PAGE sample buffer (0.125 M Tris–HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.004% bromophenol blue), heated at 94 °C for 4 min, and resolved on 10% SDS–PAGE gels. Proteins were electrophoretically transferred to nitrocellulose (Immobilon-P, Millipore, Bedford, MA), blocked with 1% nonfat dried milk in DulbeccoÕs PBS at 4 °C overnight, and incubated with FPLC-puri-
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Fig. 1. Molecular features of the chcc -a and chcc -b cDNAs. (A) Schematic representation of chcc -a and chcc -b. SS, predicated signal sequence; WSXWS, WSXWS motif; TM, putative transmembrane region. (B) Nucleotide and deduced amino acid sequences of the chcc -a cDNA. The predicted signal sequence is indicated by the single underline. The WSXWS motif is indicated by the double underline. The putative transmembrane region is indicated by the dashed underline. The four conserved cysteine residues are boxed. Five potential N-linked glycosylation sites are in bold. (C) Nucleotide and deduced amino acid sequences of the chcc -b cDNA in the region containing the 26 amino acid insertion (underlined). (D) Hydrophobicity plots of chcc -a and chcc -b. Hydrophobicity plots were generated using the ExPASy TMpred program. Region A indicates the change due to the 26 amino acid insertion in the chcc -b cDNA.
fied mAb cM1–11 for 2 h followed by HRP conjugated rabbit antimouse IgG antibody (Sigma) for 40 min at room temperature. The membrane was washed five times with PBS and five times with distilled water, and developed with Sigma Fast DAB peroxidase substrate (Sigma). For Western blot analysis with normal and Con A-activated splenic lymphocytes using chemiluminescent substrates (Pierce, Rockford, IL), membranes were blocked overnight in blocking buffer (SuperBlock, Peirce) containing 0.05% Tween 20. Membranes were then washed with washing buffer and incubated for 2 h with mAb cM1–11. The membrane was washed six times with wash buffer, incubated with HRP conjugated rabbit anti-mouse IgG antibody (Sigma) for 40 min at room temperature, and washed six times with wash buffer. The membrane was incubated in luminol solution (Super Signal Dura-extended, Pierce) and light emitting from membrane was imaged with a cooled ccd digital video camera and imaging software (UVP, Upland, CA).
Results and discussion Isolation of chcc -a and chcc -b cDNAs. Two full-length cDNA clones (chcc -a and chcc -b) were isolated following 50 -RACE extension of the chicken pat.pk0020.e7 cc EST cDNA [21]. Sequence analysis confirmed that both cDNAs were derived from the chicken cc gene. The chcc a cDNA was approximately 1.4 kb in length and contained a 1044 bp open reading frame predicted to encode a protein of 348 amino acids and a molecular mass of 39.7 kDa. By hydrophobicity analysis, a predicted signal sequence (residues 1–21) and transmembrane domain (236–254) were identified and the remaining regions
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were inferred to comprise the extracellular domain of the mature protein (22–235) and the cytoplasmic domain (236–348) (Fig. 1A). Compared with mammalian cc sequences, chcc -a shared 36% sequence identity with dog and cow cc Õs, 35% identity with mouse cc , 34% identity with human cc , and 21% identity with rainbow trout cc [3,15,24–26]. As shown in Fig. 1B, chcc -a contained the four conserved cysteines and the WSXWS motif, both hallmarks of the cytokine receptor superfamily [4]. The translated chcc -a sequence also contained five potential N-linked glycosylation sites (Asn-X-Ser/ Thr) in the extracellular domain and a region in its cytoplasmic domain (255–299) with limited homology to SH2 domains [7]. A leucine zipper-like domain [3] present in the cc chains of human and cow, but absent from dog and mouse cc , was not evident in the chicken sequence. As shown in Figs. 1A and C, the predicted amino acid sequence of the chcc -b cDNA was identical to chcc -a with the exception of an in-frame 78 bp (26 amino acids) insertion at a position between the codons encoding Gly-222 and Val-223. Correspondingly, the chcc -b cDNA was predicted to encode a protein of 374 amino acids and a molecular mass of 42.5 kDa. Hydrophobicity analysis indicated that the 26 amino acid insertion did not alter the hydrophobic profile of the putative transmembrane region of the chcc -b chain (Fig. 1D). Considering only a single cc transcript that has been detected in mammals, different mechanisms of immune regulation involving the cc chain may exist between mammals and avians. Genomic organization of the chicken cc chain. A genomic cc clone was isolated by PCR from 6 104 chicken cosmid clones using chicken cc specific primers. Sequence analysis revealed that this genomic clone consisted of eight exons spanning approximately 2.9 kb (Fig. 2). The exon/intron organization of the chicken cc
gene was remarkably similar its mammalian counterparts and all exon/intron boundaries contained consensus splice donor and acceptor sites. Sequence comparisons between the genomic and cDNA clones revealed that the extracellular region of the molecule was encoded by exons 1–6, the transmembrane domain by exon 6, and the cytoplasmic region by exons 6–8. The four consensus cysteine residues were located in exons 2 and 3, two in each exon, and the WSXWS motif resided in exon 5. Interestingly, when compared to the structures of the human, mouse, and cow cc genes, minor differences in the sizes of all exons were noted, with the exception of exon 5; this exon was identical in size between all four sequences compared. It remains to be determined if this trend will continue when cc genes from other species are sequenced. When comparing intron sizes, it was noted that the first intron of chicken cc was markedly larger than its mammalian equivalents while the other six were shorter. Expression of chcc -a and chcc -b transcripts. Sequence comparisons between the cDNAs and a cc genomic clone revealed that chcc -b is a novel isoform which resulted from an alternative splicing harboring the fifth intron (Fig. 3A). Because of the critical role of the cc chain in regulating mammalian lymphocyte development and proliferation [9–11], we next compared expression levels of chcc -a and chcc -b transcripts by Northern blot analysis of chicken tissues and mitogenactivated lymphocytes. The expected size of the chcc -a transcript was approximately 1.4 kb and that of the chcc -b transcript slightly larger as a result of insertion of the intron between exons 5 and 6 (Fig. 3A). As shown in Fig. 3B, both transcripts were expressed abundantly in the spleen, thymus, bursa, and cecal tonsils with the chcc -a mRNA consistently expressed at higher levels than chcc -b. Low expression of both transcripts was detected also in kidney, heart, and muscle. In view of the
Fig. 2. Schematic comparison of the structures of chicken and mammalian cc genes. Exons (boxes) are numbered at the top in Roman numerals. The numbers indicate lengths in base pairs encoded by each exon and intron. WSXWS, WSXWS motif; TM, putative transmembrane region; black shaded boxes, untranslated regions; light shaded boxes, translated regions. The four arrows indicate conserved cysteine residues. Human cc , GenBank Accession No. L19546; mouse cc , Accession No. U21795; cow cc , Accession No. U33748.
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Fig. 3. Schematic representation of the splicing pattern and Northern blot analyses of the chcc -a and chcc -b transcripts. (A) The chcc -b transcript contains a 78 bp insertion between exons 5 and 6 of chcc -a, most likely due to alternative splicing allowing the intron to remain in the mature transcript. (B) Expression of chcc -a and chcc -b transcripts in various tissues. PolyðAÞþ RNA was isolated from 10-week-old chickens and analyzed by Northern blotting for expression of chcc -a and chcc -b transcripts (arrows a and b). (C) Flow cytometric analysis of B cells purified by immunomagnetic separation with Bu mAb. Buþ ; Bu , and unseparated splenic lymphocytes were stained with Bu and CD8 mAbs (upper panel). Expression of chcc -a and chcc -b transcripts in Buþ ; Bu , and unseparated splenic lymphocytes (lower panel). (D) Expression of chcc -a and chcc -b transcripts in normal and Con A-activated splenic lymphocytes. PolyðAÞþ RNA was isolated from 1-, 2-, 3-, or 4-week-old chickens following nonactivation or activation with 12.5 lg/ml Con A for 48 h and analyzed by Northern blotting for expression of chcc -a and chcc -b transcripts (arrows a and b). (E) Time course of Con A activation for expression of chcc -a and chcc -b transcripts. Splenic lymphocytes were isolated from 5-week-old chickens, activated with 12.5 lg/ml of Con A for the indicated times, and analyzed by Northern blotting for expression of chcc -a and chcc -b transcripts (arrows a and b).
fact that the chicken cc transcripts were expressed at high levels in lymphoid organs as well as previous studies demonstrating mammalian cc expression in splenic T and B cells [4,27], next we assessed chcc -a and chcc -b mRNAs in chicken T and B cells. B cells were isolated from splenic lymphocytes by immunomagnetic separation using the Bu mAb [22]. B cell purity was confirmed by flow cytometry using the Bu mAb to detect B cells and the CTLA3 CD8 mAb [23] to stain contaminating T cells (Fig. 3C, upper panel). When Buþ ; Bu , and unseparated splenocytes were examined for cc transcripts, all three populations contained both chcc -a and chcc -b mRNAs (Fig. 3C, lower panel). To confirm that these bands represented the two different transcripts, they were excised from the gel and amplified by RT-PCR, and the presence of both cc forms was confirmed by DNA sequence analysis (data not shown). To explore further the expression of chicken cc transcripts in T cells, splenic lymphocytes were stimu-
lated with the T cell mitogen Con A and analyzed by Northern blotting. As shown in Fig. 3D, nonactivated splenocytes from chickens of 1, 2, 3, or 4 weeks of age expressed both chcc -a and chcc -b transcripts. In contrast, 48 h Con A-stimulated T cells from the same age groups contained only the chcc -a transcript. In an experiment to examine the kinetics of chicken cc transcript expression, splenic T cells were stimulated with Con A for 0, 4, 24, or 48 h and chcc -a and chcc -b mRNAs were identified by Northern blotting (Fig. 3E). While both mRNAs were detected at 0 and 4 h, only the chcc -a transcript was seen at the later two time points. Expression of chicken cc forms. To better characterize the proteins encoded by chcc genes, we attempted to develop mouse mAbs specific for chcc -a and chcc -b forms. For the production of mAbs detecting chcc -a and chcc -b forms, E. coli-expressed recombinant chcc -a protein and 26 peptides (SSGPERPRSPRRPRSVLTVS PRLVAG) conjugated with keyhole limpet hemocyanin,
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Fig. 4. Detection of chicken cc forms by Western blot analysis. Normal (A) and Con A-activated splenic lymphocytes (B) were immunoblotted with mAb cM1–11 using chemiluminescence. The arrows on the left indicate the positions of the chcc proteins. (C) Whole-cell lysates (lanes 2 and 4) and cell-free supernatants (lanes 3 and 5) were collected from COS-7 cells transiently transfected with the chcc -a-His plasmid or chcc -b-His plasmid 48 h after transfection. The blot was developed using peroxidase conjugated anti-mouse IgG antibody and Sigma Fast DAB peroxidase substrate. Lane 1 indicates molecular weight marker. Lanes 2 and 3 were transfected with chcc -b-His plasmid. Lanes 4 and 5 were transfected with chcc -a-His plasmid. The box indicates the positions of the chcc proteins. All samples were resolved by 10% SDS–PAGE and Western blot analysis was performed as described in Materials and methods.
respectively, were used. Several mAbs specifically detecting both chcc -a and chcc -b forms were generated but no antibody specific for the 26 peptides was produced presumably due to weak immunogenicity of this peptide in mice. One mAb (cM1–11) detecting both chcc -a and chcc -b proteins was chosen to analyze the proteins expressed from chcc genes. Using chemilumisol detection system, 45, 53, and 64 kDa protein bands were identified in normal splenic lymphocytes (Fig. 4A) whereas only the 64 kDa band was detected in Con A-activated splenic lymphocytes (Fig. 4B). To further characterize gene products encoded by the two cc transcripts, the chcc -a and chcc -b cDNAs were subcloned into the pcDNA3 eucaryotic expression vector with C-terminal histidine tags and transiently transfected into COS-7 cells. As shown in Fig. 4C, mAb (cM1–11) identified 44 kDa band in the chcc -b-Histransfected cells (lane 2) whereas two immunoreactive bands of 41 and 43 kDa were detected in the chcc -a-Histransfected cells (lane 4). Expression of both chcc -a and chcc -b proteins in COS-7 cells clearly suggests that both forms are present in chicken cells. The same mAb also detected two immunoreactive bands of 42 and 44 kDa proteins in the concentrated cell-free supernatant from the chcc -b-His-transfected COS-7 cells (Fig. 4C, lane 3) but not from the chcc -a-His-transfected cells (Fig. 4C, lane 5). Using an anti-polyhistidine mAb, the same size bands were detected in the cell lysates of chcc -a-His and chcc -b-His-transfected COS-7 cells, but none in the cellfree supernatants from them (unpublished observation).
This result suggests a possibility that the chcc -b transcript may produce soluble forms. In mammals, soluble forms of cc could not be detected in the supernatants from normal and activated PBL, serum from healthy individuals or in patients with different disorders [28]. In some disorders, however, soluble cc forms have been detected including serum from patients with inflammatory bowel disease [29], synovial fluid from rheumatoid joints [30], and sera of certain inbred mice [31]. In conclusion, this report clearly documents the existence of a novel cc isoform whose expression is downregulated after a mitogenic activation in the avian system. The relevance of this finding to the biological function of cc receptors in chickens remains to be investigated. However, our findings suggest a possibility of species difference involving cc chain-mediated immunoregulation between mammals and avians.
Acknowledgment We thank Dr. Erik P. Lillehoj for editing and critical review of the manuscript.
References [1] K. Sugamura, H. Asao, M. Kondo, N. Tanaka, N. Ishii, M. Nakamura, T. Takeshita, The common c-chain for multiple cytokine receptors, Adv. Immunol. 59 (1995) 225–277.
W. Min et al. / Biochemical and Biophysical Research Communications 299 (2002) 321–327 [2] H. Asao, C. Okuyama, S. Kumaki, N. Ishii, S. Tsuchiya, D. Foster, K. Sugamura, The common c-chain is an indispensable subunit of IL-21 receptor complex, J. Immunol. 167 (2001) 1–5. [3] T. Takeshita, H. Asao, K. Ohtani, N. Ishii, S. Kumaki, N. Tanaka, H. Munakata, M. Nakamura, K. Sugamura, Cloning of the c chain of the human IL-2 receptor, Science 257 (1992) 379– 382. [4] B.H. Nelson, D.M. Willerford, Biology of the interleukin-2 receptor, Adv. Immunol. 70 (1998) 1–81. [5] N. Ishii, T. Takeshita, Y. Kimura, K. Tada, M. Kondo, M. Nakamura, K. Sugamura, Expression of the IL-2 receptor c chain on various populations in human peripheral blood, Int. Immunol. 6 (1994) 1273–1277. [6] J.P. DiSanto, R. Kuhn, W. Muller, Common cytokine receptor c chain ðcc Þ-dependent cytokines: understanding in vivo functions by gene targeting, Immunol. Rev. 148 (1995) 19–34. [7] C.A. Koch, D. Anderson, M.F. Moran, C. Ellis, T. Pawson, SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins, Science 252 (1991) 668–674. [8] K. Sugamura, H. Asao, M. Kondo, N. Tanaka, N. Ishii, K. Ohbo, M. Nakamura, T. Takeshita, The interleukin-2 receptor c chain: its role in the multiple cytokine receptor complexes and T cell development in XSCID, Annu. Rev. Immunol. 14 (1996) 179–205. [9] M. Noguchi, H. Yi, H.M. Rosenblatt, A.H. Filipovich, S. Adelstein, W.S. Modi, O.W. McBride, W.J. Leonard, Interleukin-2 receptor c chain mutation results in X-linked severe combined immunodeficiency in humans, Cell 73 (1993) 147–157. [10] J.M. Puck, S.M. Deschenes, J.C. Porter, A.S. Dutra, C.J. Brown, H.F. Willard, P.S. Henthorn, The interleukin-2 receptor c chain maps to Xq13.1 and is mutated in X-linked severe combined immunodeficiency, SCIDX1, Hum. Mol. Genet. 2 (1993) 1099– 1104. [11] J.P. DiSanto, S. Certain, A. Wilson, H.R. MacDonald, P. Avner, A. Fischer, G. de Sain Basile, The murine interleukin-2 receptor c chain gene: organization, chromosomal localization and expression in the adult thymus, Eur. J. Immunol. 24 (1994) 3014– 3018. [12] X. Cao, E.W. Shores, J. Hu-Li, M.R. Anver, B.L. Kelsall, S.M. Russell, J. Drago, M. Noguchi, A. Grinberg, E.T. Bloom, W.E. Paul, S.I. Katz, P.E. Love, W.J. Leonard, Defective lymphoid development in mice lacking expression of the common cytokine receptor c chain, Immunity 2 (1995) 223–238. [13] J.P. DiSanto, W. Muller, D. Guy-Grand, A. Fischer, K. Rajewsky, Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor c chain, Proc. Natl. Acad. Sci. USA 92 (1995) 377–381. [14] W.J. Leonard, E.W. Shores, P.E. Love, Role of the common cytokine receptor c chain in cytokine signaling and lymphoid development, Immunol. Rev. 148 (1995) 97–114. [15] P.S. Henthorn, R.L. Somberg, V.M. Fimiani, J.M. Puck, D.F. Patterson, P.J. Felsburg, IL-2Rc gene microdeletion demonstrates that canine X-linked severe combined immunodeficiency is a homologue of the human disease, Genomics 23 (1994) 69–74. [16] R.L. Somberg, R.P. Pullen, M.L. Casal, D.F. Patterson, P.J. Felsburg, P.S. Henthorn, A single nucleotide insertion in the canine interleukin-2 receptor c chain results in X-linked severe
[17]
[18] [19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
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combined immunodeficiency disease, Vet. Immunol. Immunopathol. 47 (1995) 203–213. P.J. Felsburg, B.J. Hartnett, P.S. Henthorn, P.F. Moore, S. Krakowka, H.D. Ochs, Canine X-linked severe combined immunodeficiency, Vet. Immunol. Immunopathol. 69 (1999) 127–135. M.E. Conley, Molecular approaches to analysis of X-linked immunodeficiencies, Annu. Rev. Immunol. 10 (1992) 215–238. S. Kumaki, M. Kondo, T. Takeshita, H. Asao, M. Nakamura, K. Sugamura, Cloning of the mouse interleukin 2 receptor c chain: demonstration of functional differences between the mouse and human receptors, Biochem. Biophys. Res. Commun. 193 (1993) 356–363. L.I. Sharara, C.A. Vosshenrich, W. Muller, J.P. DiSanto, On the role of the common cytokine receptor c chain in B-cell vs. T-cell development, Res. Immunol. 148 (1997) 449–453. V.G. Tirunagaru, L. Sofer, J. Cui, J. Burnside, An expressed sequence tag database of T-cell-enriched activated chicken splenocytes: sequence analysis of 5251 clones, Genomics 66 (2000) 144–151. T. Veromaa, O. Vainio, E. Eerola, P. Toivanen, Monoclonal antibodies against chicken Bu-1a and Bu-1b alloantigens, Hybridoma 7 (1988) 41–48. H.S. Lillehoj, E.P. Lillehoj, D. Weinstock, K.A. Schat, Functional and biochemical characterizations of avian T lymphocyte antigens identified by monoclonal antibodies, Eur. J. Immunol. 18 (1988) 2059–2065. X. Cao, C.A. Kozak, Y.J. Liu, M. Noguchi, E. OÕConnell, W.J. Leonard, Characterization of cDNAs encoding the murine interleukin 2 receptor (IL-2R) c chain: chromosomal mapping and tissue specificity of IL-2R c chain expression, Proc. Natl. Acad. Sci. USA 90 (1993) 8464–8468. J. Yoo, R.T. Stone, S. Solinas-Toldo, R. Fries, C.W. Beattie, Cloning and chromosomal mapping of bovine interleukin-2 receptor c gene, DNA Cell Biol. 15 (1996) 453–459. T. Wang, C.J. Secombes, Cloning and expression of a putative common cytokine receptor c chain (cC) gene in rainbow trout (Oncorhynchus mykiss), Fish Shellfish Immunol. 11 (2001) 233– 244. M. Kondo, Y. Ohashi, K. Tada, M. Nakamura, K. Sugamura, Expression of the mouse interleukin-2 receptor c chain in various cell populations of the thymus and spleen, Eur. J. Immunol. 24 (1994) 2026–2030. K. Lundin, A.M. Tuukkanen, C. Jansson, T. Nordstrom, C. Lindqvist, No soluble common cytokine receptor c chain (cc ) in activated human lymphocyte cultures-comparison with soluble IL-2Ra, Immunol. Lett. 82 (2002) 235–240. O.H. Nielsen, I. Kirman, K. Johnson, M. Giedlin, T. Ciardelli, The circulating common c chain (CD132) in inflammatory bowel disease, Am. J. Gastroenterol. 93 (1998) 323–328. J. Nishio, H. Kohsaka, T. Shimamura, J. Hamuro, N. Miyasaka, Abundant expression of common cytokine receptor c chain (CD132) in rheumatoid joints, J. Rheumatol. 28 (2001) 240–244. U. Meissner, H. Blum, M. Schnare, M. Rollinghoff, A. Gessner, A soluble form of the murine common c chain is present at high concentrations in vivo and suppresses cytokine signaling, Blood 97 (2001) 183–191.
Identification of an alternatively spliced isoform of the common cytokine receptor c chain in chickens Wongi Min, Hyun Soon Lillehoj,* and Raymond Hugh Fetterer U.S. Department of Agriculture, Parasite Biology, Epidemiology, and Systematics Laboratory, Animal and Natural Resources Institute, Building 1040, BARC-East, Beltsville, MD 20705, USA Received 6 October 2002
Abstract The common cytokine receptor c ðcc Þ chain is shared by at least six cytokine receptors and plays a critical role in the regulation of immune responses. In this study, we discovered that, unlike mammals, chickens possess two different cc gene transcripts, chcc -a and chcc -b. Sequence comparisons between the cDNAs and a cc genomic clone isolated by PCR revealed that chcc -b contained an in-frame 78 bp insertion between Gly-222 and Val-223 of the chcc -a sequence. This insertion most likely resulted from alternative splicing such that the fifth intron was not removed from the chcc -b transcript. Furthermore, while chcc -a and chcc -b transcripts were expressed equally in the spleen, thymus, bursa, and cecal tonsils, they were differentially expressed during the time course of Con A stimulation of splenic T lymphocytes. Western blot analysis of normal spleen lymphocytes identified 45, 53, and 64 kDa immunoreactive bands whereas only 64 kDa band was detected in Con A-activated splenic lymphocytes. COS-7 cells transfected with chcc -b secreted 42 kDa proteins. Taken together, our results document that chickens express an alternative spliced cc receptor which is larger than the conventional transcript and this novel isoform generates soluble receptors in the transfected COS-7 cells. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Chicken; Common cytokine receptor c chain; Isoform; Gene regulation
The cc chain is a shared component of the receptors for IL-2, -4, -7, -9, -15, and -21 [1,2] and is a member of the cytokine receptor superfamily displaying a characteristic spacing of four conserved cysteine residues and a WSXWS motif [3,4]. The cc chain is expressed as a transmembrane glycoprotein on CD4þ and CD8þ T cells, B cells, NK cells, monocytes/macrophages, neutrophils, and granulocytes [4,5], where it forms heterodimeric or heterotrimeric complexes with specific interleukin receptors thereby augmenting ligand affinity, receptor internalization, and signal transduction [4,6]. The cytoplasmic region of cc contains two Src homology region 2 (SH2) domains [7] that contribute to intracellular signal transduction through interaction with phosphotyrosine residues of various effector molecules [1,8].
* Corresponding author. Fax: 1-301-504-5103. E-mail address: [email protected] (H.S. Lillehoj).
The critical role of the cc chain in regulating lymphocyte development and proliferation is illustrated by the discovery that some forms of X-linked SCID (XSCID) were caused by mutations in the human [9–11], murine [12–14], or canine [15–17] cc genes. XSCID is characterized by profound developmental impairment of T and NK cells. A review of the literature related to cc gene defects suggests that cc chains of different species may have different functions. Thus, while B cell levels were unaffected or, in some cases, slightly elevated in XSCID [1,18], c= knockout mice showed a marked c decrease in B cell numbers [12,13]. Furthermore, mice with a targeted deletion in the cc gene and dogs with a naturally occurring form of XSCID exhibited a somewhat different phenotype compared with human XSCID [19,20]. In light of the facts that cc Õs from different species may be functionally diverse, we became interested in elucidating the role of two chicken cc genes cloned from an expressed sequence tag (EST) cDNA. Sequence comparisons between the cDNAs and a cc genomic
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clone indicated that one cDNA represents a conventional form of cc whereas the other cDNA identifies a novel spliced transcript which contains an in-frame 78 bp insertion. Furthermore, differential expression of two forms of chicken cc chains was seen in Con A-activated splenic T cells and transiently transfected COS-7 cells using Northern and Western blot analyses.
Materials and methods Chickens. Fertilized eggs of White Leghorn SC chickens were obtained from Hyline International Production Center (Dallas Center, IA) and hatched at the Animal and Natural Resources Institute (Beltsville, MD). Chickens were kept in wire cages and were provided with water and food ad libitum. Isolation of chicken cc cDNA and genomic clones. The 50 region of the partial cc cDNA EST clone pat.pk0020.e7 [21] was extended by rapid amplification of cDNA ends (RACE, version 2.0, Life Technologies, Gaithersburg, MD). Total RNA was isolated from splenic lymphocytes of 5- or 6-week-old chickens using the Trizol (Life Technologies), first strand cDNA was synthesized from 5.0 lg RNA with the cc specific primer 50 -GCAGCAGCAGGCAGGAGGCGAT using reverse transcriptase (Superscript II RT, Life Technologies), and RNA template was removed with RNase (Life Technologies). The first strand product was purified with a GlassMax DNA isolation spin cartridge (Life Technologies), a homopolymeric tail added to the 30 end using dCTP and TdT, and the tailed cDNA was amplified by PCR with a 50 -RACE abridged anchor primer and the cc specific primer 50 -TCAGCACCGTGTGGATCCAGA. PCR conditions were 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, for 35 cycles using Platinum Taq DNA polymerase (Promega, Madison, WI). Two PCR clones were isolated following agarose gel electrophoresis and the amplified cDNAs were cloned into the pCR3.1 vector (Invitrogen, San Diego, CA) and sequenced. A genomic cc clone was isolated from a chicken cosmid library (Clontech, Palo Alto, CA) that originated from a male adult Leghorn liver by PCR using the cc specific primers 50 -GCGCAGCAAGATCAACAACTA and 50 -CCCACGTAGCTGC TCTTGTA and the PCR conditions described above. The sequence data were submitted with GenBank Accession Nos. AJ419897 for chcc -a, AJ419896 for chcc -b, and AJ419898 for genomic sequence. Northern blot analysis. PolyðAÞþ RNA was isolated using the PolyA Tract mRNA Isolation System IV (Promega) from total RNA of various tissues, subjected to electrophoresis through 1% agarose (Northern Max-Gly Kit, Ambion, Austin, TX), and transferred to a positively charged nylon membrane (Boehringer–Mannheim, Mannheim, Germany). The blot was hybridized with a digoxigenin-labeled chicken cc cDNA probe in DIG Easy Hyb solution (Boehringer– Mannheim) at 55 °C overnight. The probe was generated by PCR using the chicken cc specific primers 50 -TCAGCACCGTGTGGATC CAGA and 50 -AAGGTAGGCCGCGCACTGAGA. The blot was washed twice in 2 SSC and 0.1% SDS for 15 min at room temperature followed twice by 0.1 SSC and 0.1% SDS for 15 min at 55 °C. Detection was carried out with the DIG DNA Labeling and Detection Kit (Boehringer–Mannheim) according to manufacturerÕs instruction. As a control, the chicken b actin gene was amplified by PCR with the primers 50 -TCTGGTGGTACCACAATGTACCCT and 50 -CCAGT AATTGGTACCGGCTCCTC. Cell culture. Splenic lymphocytes were isolated by Histopaque-1077 (Sigma, St. Louis, MO) centrifugation, resuspended at 1 107 /ml in IMDM (Life Technologies) supplemented with 10% FCS (HyClone, Logan, UT), and stimulated with 12.5 lg/ml Con A (Sigma) at 41 °C in 5% CO2 . Immunomagnetic purification of B cells and flow cytometry. Chicken B cells were purified from splenic lymphocytes using the Magnetic Cell
Sorting System (autoMACS, Miltenyi Biotec, Cologne, Germany) according to manufacturerÕs protocol. Briefly, splenic cells were sequentially incubated with the B cell specific Bu mAb [22] and goat anti-mouse IgG microbeads, and B cells enriched by the autoMACS separator. B cell purity was verified by flow cytometry with phycoerythrin-conjugated Bu or CD8 mAbs specific for B or T cells as described [23] using the EPICS-XL flow cytometer (Coulter, Hialeah, FL). B cells prepared in this manner were routinely >96% pure. Expression of recombinant chicken cc and development of anti-cc mAbs. For recombinant cc production, the extracellular region of the chcc -a gene was amplified by PCR using the following primers: 50 -GATCGGATCCATGGCGGTGCCCGGGGCTTCG, and 50 -TC AGCACCGTGTGGATCCAGA (Fig. 1). The recombinant plasmid in pMal-c2 vector (New England Biolabs, Beverly, MA) was transformed into competent Escherichia coli DH5a and the transformant was selected on ampicillin agar plates at 37 °C and induced with 1.0 mM isopropyl-b-D -thiogalactopyranoside for 3 h. The recombinant protein fused with maltose binding proteins was purified on amylose affinity column (New Englind Biolabs) according to manufacturerÕs instructions. For hybridoma production, female BALB/c mice of 5–6 weeks of age were immunized subcutaneously with 50 lg recombinant chcc -a in FreundÕs complete adjuvant and boosted two times biweekly with 50 lg antigen in incomplete FreundÕs adjuvant. Three days prior to fusion, 10 lg of the recombinant antigen in PBS was intravenously injected. Splenocytes were fused with SP2/0 myeloma cells (ATCC, Manassas, VA) in 50% polyethylene glycol and hybridoma cells were selected in IMDM containing 20% FCS, hypoxanthine, aminopterin, and thymidine (Sigma, St. Louis, MO). Hybridomas producing antigen reactive mAbs were identified by ELISA. Briefly, flat-bottomed 96-well microtiter plates (Coster, Corning, NY) were coated with 100 ll E. coliexpressed affinity purified protein (5 lg/ml) in 0.1 M carbonate buffer, pH 9.6 at 4 °C overnight and washed three times with PBS containing 0.05% Tween 20 (PBS-T). Wells were blocked with 200 ll PBS containing 1% BSA (PBS-BSA) for 1 h at room temperature and washed with PBS-T and 100 ll hybridoma supernatant was added for 2 h at room temperature. After washing with PBS-T, bound antibodies were detected at OD450 with horseradish peroxidase-conjugated goat antimouse IgG antibody (Sigma) and 0.01% tetramethylbenzidine (Sigma) in 0.05 M phosphate-citrate buffer, pH 5.0. Positive hybridomas were cloned by limiting dilution using mouse thymus feeder cells and ascites were produced by intraperitoneal injection of 5 106 cells into BALB/c mice. One mAb (cM1-11) was selected from several positive clones for further study and its ascites were purified on the Hi-Trap Protein G column (Amersham Pharmacia Biotech, Piscataway, NJ) using fast protein liquid chromatography (FPLC) for Western blot analysis. Construction of chicken cc expression plasmids and Western blot analysis. The full-length chcc -a and chcc -b cDNAs were amplified by PCR from single stranded cDNAs of splenic lymphocytes using the following primers: forward primer (HindIII cloning site is underlined), 50 -AGGCAAGCTTCGCACTGAGAGTTGCGCCATG; reverse primer (EcoRI cloning site is underlined and 6 copy histidine tag is indicated by subscript), 50 -ATCCGAATTCTCAðATGÞ6 CGCTCCCA CGTAGCTGCT. The PCR products (chcc -a-His and chcc -b-His) were digested with HindIII and EcoRI and cloned into the corresponding restriction endonuclease sites of pcDNA3 (Invitrogen). COS-7 cells were transiently transfected with 10 lg constructs using Lipofectamine (Life Technologies), incubated 48 h at 37 °C in IMDM without 10% FCS, and harvested. Cell-free culture supernatants from transfected COS-7 cells were concentrated up to 10-fold using the Amicon Centriplus (YM-10) (Millipore, Bedford, MA). Cells were extracted with SDS–PAGE sample buffer (0.125 M Tris–HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.004% bromophenol blue), heated at 94 °C for 4 min, and resolved on 10% SDS–PAGE gels. Proteins were electrophoretically transferred to nitrocellulose (Immobilon-P, Millipore, Bedford, MA), blocked with 1% nonfat dried milk in DulbeccoÕs PBS at 4 °C overnight, and incubated with FPLC-puri-
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Fig. 1. Molecular features of the chcc -a and chcc -b cDNAs. (A) Schematic representation of chcc -a and chcc -b. SS, predicated signal sequence; WSXWS, WSXWS motif; TM, putative transmembrane region. (B) Nucleotide and deduced amino acid sequences of the chcc -a cDNA. The predicted signal sequence is indicated by the single underline. The WSXWS motif is indicated by the double underline. The putative transmembrane region is indicated by the dashed underline. The four conserved cysteine residues are boxed. Five potential N-linked glycosylation sites are in bold. (C) Nucleotide and deduced amino acid sequences of the chcc -b cDNA in the region containing the 26 amino acid insertion (underlined). (D) Hydrophobicity plots of chcc -a and chcc -b. Hydrophobicity plots were generated using the ExPASy TMpred program. Region A indicates the change due to the 26 amino acid insertion in the chcc -b cDNA.
fied mAb cM1–11 for 2 h followed by HRP conjugated rabbit antimouse IgG antibody (Sigma) for 40 min at room temperature. The membrane was washed five times with PBS and five times with distilled water, and developed with Sigma Fast DAB peroxidase substrate (Sigma). For Western blot analysis with normal and Con A-activated splenic lymphocytes using chemiluminescent substrates (Pierce, Rockford, IL), membranes were blocked overnight in blocking buffer (SuperBlock, Peirce) containing 0.05% Tween 20. Membranes were then washed with washing buffer and incubated for 2 h with mAb cM1–11. The membrane was washed six times with wash buffer, incubated with HRP conjugated rabbit anti-mouse IgG antibody (Sigma) for 40 min at room temperature, and washed six times with wash buffer. The membrane was incubated in luminol solution (Super Signal Dura-extended, Pierce) and light emitting from membrane was imaged with a cooled ccd digital video camera and imaging software (UVP, Upland, CA).
Results and discussion Isolation of chcc -a and chcc -b cDNAs. Two full-length cDNA clones (chcc -a and chcc -b) were isolated following 50 -RACE extension of the chicken pat.pk0020.e7 cc EST cDNA [21]. Sequence analysis confirmed that both cDNAs were derived from the chicken cc gene. The chcc a cDNA was approximately 1.4 kb in length and contained a 1044 bp open reading frame predicted to encode a protein of 348 amino acids and a molecular mass of 39.7 kDa. By hydrophobicity analysis, a predicted signal sequence (residues 1–21) and transmembrane domain (236–254) were identified and the remaining regions
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were inferred to comprise the extracellular domain of the mature protein (22–235) and the cytoplasmic domain (236–348) (Fig. 1A). Compared with mammalian cc sequences, chcc -a shared 36% sequence identity with dog and cow cc Õs, 35% identity with mouse cc , 34% identity with human cc , and 21% identity with rainbow trout cc [3,15,24–26]. As shown in Fig. 1B, chcc -a contained the four conserved cysteines and the WSXWS motif, both hallmarks of the cytokine receptor superfamily [4]. The translated chcc -a sequence also contained five potential N-linked glycosylation sites (Asn-X-Ser/ Thr) in the extracellular domain and a region in its cytoplasmic domain (255–299) with limited homology to SH2 domains [7]. A leucine zipper-like domain [3] present in the cc chains of human and cow, but absent from dog and mouse cc , was not evident in the chicken sequence. As shown in Figs. 1A and C, the predicted amino acid sequence of the chcc -b cDNA was identical to chcc -a with the exception of an in-frame 78 bp (26 amino acids) insertion at a position between the codons encoding Gly-222 and Val-223. Correspondingly, the chcc -b cDNA was predicted to encode a protein of 374 amino acids and a molecular mass of 42.5 kDa. Hydrophobicity analysis indicated that the 26 amino acid insertion did not alter the hydrophobic profile of the putative transmembrane region of the chcc -b chain (Fig. 1D). Considering only a single cc transcript that has been detected in mammals, different mechanisms of immune regulation involving the cc chain may exist between mammals and avians. Genomic organization of the chicken cc chain. A genomic cc clone was isolated by PCR from 6 104 chicken cosmid clones using chicken cc specific primers. Sequence analysis revealed that this genomic clone consisted of eight exons spanning approximately 2.9 kb (Fig. 2). The exon/intron organization of the chicken cc
gene was remarkably similar its mammalian counterparts and all exon/intron boundaries contained consensus splice donor and acceptor sites. Sequence comparisons between the genomic and cDNA clones revealed that the extracellular region of the molecule was encoded by exons 1–6, the transmembrane domain by exon 6, and the cytoplasmic region by exons 6–8. The four consensus cysteine residues were located in exons 2 and 3, two in each exon, and the WSXWS motif resided in exon 5. Interestingly, when compared to the structures of the human, mouse, and cow cc genes, minor differences in the sizes of all exons were noted, with the exception of exon 5; this exon was identical in size between all four sequences compared. It remains to be determined if this trend will continue when cc genes from other species are sequenced. When comparing intron sizes, it was noted that the first intron of chicken cc was markedly larger than its mammalian equivalents while the other six were shorter. Expression of chcc -a and chcc -b transcripts. Sequence comparisons between the cDNAs and a cc genomic clone revealed that chcc -b is a novel isoform which resulted from an alternative splicing harboring the fifth intron (Fig. 3A). Because of the critical role of the cc chain in regulating mammalian lymphocyte development and proliferation [9–11], we next compared expression levels of chcc -a and chcc -b transcripts by Northern blot analysis of chicken tissues and mitogenactivated lymphocytes. The expected size of the chcc -a transcript was approximately 1.4 kb and that of the chcc -b transcript slightly larger as a result of insertion of the intron between exons 5 and 6 (Fig. 3A). As shown in Fig. 3B, both transcripts were expressed abundantly in the spleen, thymus, bursa, and cecal tonsils with the chcc -a mRNA consistently expressed at higher levels than chcc -b. Low expression of both transcripts was detected also in kidney, heart, and muscle. In view of the
Fig. 2. Schematic comparison of the structures of chicken and mammalian cc genes. Exons (boxes) are numbered at the top in Roman numerals. The numbers indicate lengths in base pairs encoded by each exon and intron. WSXWS, WSXWS motif; TM, putative transmembrane region; black shaded boxes, untranslated regions; light shaded boxes, translated regions. The four arrows indicate conserved cysteine residues. Human cc , GenBank Accession No. L19546; mouse cc , Accession No. U21795; cow cc , Accession No. U33748.
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Fig. 3. Schematic representation of the splicing pattern and Northern blot analyses of the chcc -a and chcc -b transcripts. (A) The chcc -b transcript contains a 78 bp insertion between exons 5 and 6 of chcc -a, most likely due to alternative splicing allowing the intron to remain in the mature transcript. (B) Expression of chcc -a and chcc -b transcripts in various tissues. PolyðAÞþ RNA was isolated from 10-week-old chickens and analyzed by Northern blotting for expression of chcc -a and chcc -b transcripts (arrows a and b). (C) Flow cytometric analysis of B cells purified by immunomagnetic separation with Bu mAb. Buþ ; Bu , and unseparated splenic lymphocytes were stained with Bu and CD8 mAbs (upper panel). Expression of chcc -a and chcc -b transcripts in Buþ ; Bu , and unseparated splenic lymphocytes (lower panel). (D) Expression of chcc -a and chcc -b transcripts in normal and Con A-activated splenic lymphocytes. PolyðAÞþ RNA was isolated from 1-, 2-, 3-, or 4-week-old chickens following nonactivation or activation with 12.5 lg/ml Con A for 48 h and analyzed by Northern blotting for expression of chcc -a and chcc -b transcripts (arrows a and b). (E) Time course of Con A activation for expression of chcc -a and chcc -b transcripts. Splenic lymphocytes were isolated from 5-week-old chickens, activated with 12.5 lg/ml of Con A for the indicated times, and analyzed by Northern blotting for expression of chcc -a and chcc -b transcripts (arrows a and b).
fact that the chicken cc transcripts were expressed at high levels in lymphoid organs as well as previous studies demonstrating mammalian cc expression in splenic T and B cells [4,27], next we assessed chcc -a and chcc -b mRNAs in chicken T and B cells. B cells were isolated from splenic lymphocytes by immunomagnetic separation using the Bu mAb [22]. B cell purity was confirmed by flow cytometry using the Bu mAb to detect B cells and the CTLA3 CD8 mAb [23] to stain contaminating T cells (Fig. 3C, upper panel). When Buþ ; Bu , and unseparated splenocytes were examined for cc transcripts, all three populations contained both chcc -a and chcc -b mRNAs (Fig. 3C, lower panel). To confirm that these bands represented the two different transcripts, they were excised from the gel and amplified by RT-PCR, and the presence of both cc forms was confirmed by DNA sequence analysis (data not shown). To explore further the expression of chicken cc transcripts in T cells, splenic lymphocytes were stimu-
lated with the T cell mitogen Con A and analyzed by Northern blotting. As shown in Fig. 3D, nonactivated splenocytes from chickens of 1, 2, 3, or 4 weeks of age expressed both chcc -a and chcc -b transcripts. In contrast, 48 h Con A-stimulated T cells from the same age groups contained only the chcc -a transcript. In an experiment to examine the kinetics of chicken cc transcript expression, splenic T cells were stimulated with Con A for 0, 4, 24, or 48 h and chcc -a and chcc -b mRNAs were identified by Northern blotting (Fig. 3E). While both mRNAs were detected at 0 and 4 h, only the chcc -a transcript was seen at the later two time points. Expression of chicken cc forms. To better characterize the proteins encoded by chcc genes, we attempted to develop mouse mAbs specific for chcc -a and chcc -b forms. For the production of mAbs detecting chcc -a and chcc -b forms, E. coli-expressed recombinant chcc -a protein and 26 peptides (SSGPERPRSPRRPRSVLTVS PRLVAG) conjugated with keyhole limpet hemocyanin,
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Fig. 4. Detection of chicken cc forms by Western blot analysis. Normal (A) and Con A-activated splenic lymphocytes (B) were immunoblotted with mAb cM1–11 using chemiluminescence. The arrows on the left indicate the positions of the chcc proteins. (C) Whole-cell lysates (lanes 2 and 4) and cell-free supernatants (lanes 3 and 5) were collected from COS-7 cells transiently transfected with the chcc -a-His plasmid or chcc -b-His plasmid 48 h after transfection. The blot was developed using peroxidase conjugated anti-mouse IgG antibody and Sigma Fast DAB peroxidase substrate. Lane 1 indicates molecular weight marker. Lanes 2 and 3 were transfected with chcc -b-His plasmid. Lanes 4 and 5 were transfected with chcc -a-His plasmid. The box indicates the positions of the chcc proteins. All samples were resolved by 10% SDS–PAGE and Western blot analysis was performed as described in Materials and methods.
respectively, were used. Several mAbs specifically detecting both chcc -a and chcc -b forms were generated but no antibody specific for the 26 peptides was produced presumably due to weak immunogenicity of this peptide in mice. One mAb (cM1–11) detecting both chcc -a and chcc -b proteins was chosen to analyze the proteins expressed from chcc genes. Using chemilumisol detection system, 45, 53, and 64 kDa protein bands were identified in normal splenic lymphocytes (Fig. 4A) whereas only the 64 kDa band was detected in Con A-activated splenic lymphocytes (Fig. 4B). To further characterize gene products encoded by the two cc transcripts, the chcc -a and chcc -b cDNAs were subcloned into the pcDNA3 eucaryotic expression vector with C-terminal histidine tags and transiently transfected into COS-7 cells. As shown in Fig. 4C, mAb (cM1–11) identified 44 kDa band in the chcc -b-Histransfected cells (lane 2) whereas two immunoreactive bands of 41 and 43 kDa were detected in the chcc -a-Histransfected cells (lane 4). Expression of both chcc -a and chcc -b proteins in COS-7 cells clearly suggests that both forms are present in chicken cells. The same mAb also detected two immunoreactive bands of 42 and 44 kDa proteins in the concentrated cell-free supernatant from the chcc -b-His-transfected COS-7 cells (Fig. 4C, lane 3) but not from the chcc -a-His-transfected cells (Fig. 4C, lane 5). Using an anti-polyhistidine mAb, the same size bands were detected in the cell lysates of chcc -a-His and chcc -b-His-transfected COS-7 cells, but none in the cellfree supernatants from them (unpublished observation).
This result suggests a possibility that the chcc -b transcript may produce soluble forms. In mammals, soluble forms of cc could not be detected in the supernatants from normal and activated PBL, serum from healthy individuals or in patients with different disorders [28]. In some disorders, however, soluble cc forms have been detected including serum from patients with inflammatory bowel disease [29], synovial fluid from rheumatoid joints [30], and sera of certain inbred mice [31]. In conclusion, this report clearly documents the existence of a novel cc isoform whose expression is downregulated after a mitogenic activation in the avian system. The relevance of this finding to the biological function of cc receptors in chickens remains to be investigated. However, our findings suggest a possibility of species difference involving cc chain-mediated immunoregulation between mammals and avians.
Acknowledgment We thank Dr. Erik P. Lillehoj for editing and critical review of the manuscript.
References [1] K. Sugamura, H. Asao, M. Kondo, N. Tanaka, N. Ishii, M. Nakamura, T. Takeshita, The common c-chain for multiple cytokine receptors, Adv. Immunol. 59 (1995) 225–277.
W. Min et al. / Biochemical and Biophysical Research Communications 299 (2002) 321–327 [2] H. Asao, C. Okuyama, S. Kumaki, N. Ishii, S. Tsuchiya, D. Foster, K. Sugamura, The common c-chain is an indispensable subunit of IL-21 receptor complex, J. Immunol. 167 (2001) 1–5. [3] T. Takeshita, H. Asao, K. Ohtani, N. Ishii, S. Kumaki, N. Tanaka, H. Munakata, M. Nakamura, K. Sugamura, Cloning of the c chain of the human IL-2 receptor, Science 257 (1992) 379– 382. [4] B.H. Nelson, D.M. Willerford, Biology of the interleukin-2 receptor, Adv. Immunol. 70 (1998) 1–81. [5] N. Ishii, T. Takeshita, Y. Kimura, K. Tada, M. Kondo, M. Nakamura, K. Sugamura, Expression of the IL-2 receptor c chain on various populations in human peripheral blood, Int. Immunol. 6 (1994) 1273–1277. [6] J.P. DiSanto, R. Kuhn, W. Muller, Common cytokine receptor c chain ðcc Þ-dependent cytokines: understanding in vivo functions by gene targeting, Immunol. Rev. 148 (1995) 19–34. [7] C.A. Koch, D. Anderson, M.F. Moran, C. Ellis, T. Pawson, SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins, Science 252 (1991) 668–674. [8] K. Sugamura, H. Asao, M. Kondo, N. Tanaka, N. Ishii, K. Ohbo, M. Nakamura, T. Takeshita, The interleukin-2 receptor c chain: its role in the multiple cytokine receptor complexes and T cell development in XSCID, Annu. Rev. Immunol. 14 (1996) 179–205. [9] M. Noguchi, H. Yi, H.M. Rosenblatt, A.H. Filipovich, S. Adelstein, W.S. Modi, O.W. McBride, W.J. Leonard, Interleukin-2 receptor c chain mutation results in X-linked severe combined immunodeficiency in humans, Cell 73 (1993) 147–157. [10] J.M. Puck, S.M. Deschenes, J.C. Porter, A.S. Dutra, C.J. Brown, H.F. Willard, P.S. Henthorn, The interleukin-2 receptor c chain maps to Xq13.1 and is mutated in X-linked severe combined immunodeficiency, SCIDX1, Hum. Mol. Genet. 2 (1993) 1099– 1104. [11] J.P. DiSanto, S. Certain, A. Wilson, H.R. MacDonald, P. Avner, A. Fischer, G. de Sain Basile, The murine interleukin-2 receptor c chain gene: organization, chromosomal localization and expression in the adult thymus, Eur. J. Immunol. 24 (1994) 3014– 3018. [12] X. Cao, E.W. Shores, J. Hu-Li, M.R. Anver, B.L. Kelsall, S.M. Russell, J. Drago, M. Noguchi, A. Grinberg, E.T. Bloom, W.E. Paul, S.I. Katz, P.E. Love, W.J. Leonard, Defective lymphoid development in mice lacking expression of the common cytokine receptor c chain, Immunity 2 (1995) 223–238. [13] J.P. DiSanto, W. Muller, D. Guy-Grand, A. Fischer, K. Rajewsky, Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor c chain, Proc. Natl. Acad. Sci. USA 92 (1995) 377–381. [14] W.J. Leonard, E.W. Shores, P.E. Love, Role of the common cytokine receptor c chain in cytokine signaling and lymphoid development, Immunol. Rev. 148 (1995) 97–114. [15] P.S. Henthorn, R.L. Somberg, V.M. Fimiani, J.M. Puck, D.F. Patterson, P.J. Felsburg, IL-2Rc gene microdeletion demonstrates that canine X-linked severe combined immunodeficiency is a homologue of the human disease, Genomics 23 (1994) 69–74. [16] R.L. Somberg, R.P. Pullen, M.L. Casal, D.F. Patterson, P.J. Felsburg, P.S. Henthorn, A single nucleotide insertion in the canine interleukin-2 receptor c chain results in X-linked severe
[17]
[18] [19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
327
combined immunodeficiency disease, Vet. Immunol. Immunopathol. 47 (1995) 203–213. P.J. Felsburg, B.J. Hartnett, P.S. Henthorn, P.F. Moore, S. Krakowka, H.D. Ochs, Canine X-linked severe combined immunodeficiency, Vet. Immunol. Immunopathol. 69 (1999) 127–135. M.E. Conley, Molecular approaches to analysis of X-linked immunodeficiencies, Annu. Rev. Immunol. 10 (1992) 215–238. S. Kumaki, M. Kondo, T. Takeshita, H. Asao, M. Nakamura, K. Sugamura, Cloning of the mouse interleukin 2 receptor c chain: demonstration of functional differences between the mouse and human receptors, Biochem. Biophys. Res. Commun. 193 (1993) 356–363. L.I. Sharara, C.A. Vosshenrich, W. Muller, J.P. DiSanto, On the role of the common cytokine receptor c chain in B-cell vs. T-cell development, Res. Immunol. 148 (1997) 449–453. V.G. Tirunagaru, L. Sofer, J. Cui, J. Burnside, An expressed sequence tag database of T-cell-enriched activated chicken splenocytes: sequence analysis of 5251 clones, Genomics 66 (2000) 144–151. T. Veromaa, O. Vainio, E. Eerola, P. Toivanen, Monoclonal antibodies against chicken Bu-1a and Bu-1b alloantigens, Hybridoma 7 (1988) 41–48. H.S. Lillehoj, E.P. Lillehoj, D. Weinstock, K.A. Schat, Functional and biochemical characterizations of avian T lymphocyte antigens identified by monoclonal antibodies, Eur. J. Immunol. 18 (1988) 2059–2065. X. Cao, C.A. Kozak, Y.J. Liu, M. Noguchi, E. OÕConnell, W.J. Leonard, Characterization of cDNAs encoding the murine interleukin 2 receptor (IL-2R) c chain: chromosomal mapping and tissue specificity of IL-2R c chain expression, Proc. Natl. Acad. Sci. USA 90 (1993) 8464–8468. J. Yoo, R.T. Stone, S. Solinas-Toldo, R. Fries, C.W. Beattie, Cloning and chromosomal mapping of bovine interleukin-2 receptor c gene, DNA Cell Biol. 15 (1996) 453–459. T. Wang, C.J. Secombes, Cloning and expression of a putative common cytokine receptor c chain (cC) gene in rainbow trout (Oncorhynchus mykiss), Fish Shellfish Immunol. 11 (2001) 233– 244. M. Kondo, Y. Ohashi, K. Tada, M. Nakamura, K. Sugamura, Expression of the mouse interleukin-2 receptor c chain in various cell populations of the thymus and spleen, Eur. J. Immunol. 24 (1994) 2026–2030. K. Lundin, A.M. Tuukkanen, C. Jansson, T. Nordstrom, C. Lindqvist, No soluble common cytokine receptor c chain (cc ) in activated human lymphocyte cultures-comparison with soluble IL-2Ra, Immunol. Lett. 82 (2002) 235–240. O.H. Nielsen, I. Kirman, K. Johnson, M. Giedlin, T. Ciardelli, The circulating common c chain (CD132) in inflammatory bowel disease, Am. J. Gastroenterol. 93 (1998) 323–328. J. Nishio, H. Kohsaka, T. Shimamura, J. Hamuro, N. Miyasaka, Abundant expression of common cytokine receptor c chain (CD132) in rheumatoid joints, J. Rheumatol. 28 (2001) 240–244. U. Meissner, H. Blum, M. Schnare, M. Rollinghoff, A. Gessner, A soluble form of the murine common c chain is present at high concentrations in vivo and suppresses cytokine signaling, Blood 97 (2001) 183–191.