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The Journal of Immunology

IFN-␥ Stimulates the Expression of a Novel Secretoglobin That Regulates Chemotactic Cell Migration and Invasion Moonsuk S. Choi, Rabindranath Ray, Zhongjian Zhang, and Anil B. Mukherjee1 IFNs are a family of cytokines that alert the immune system against viral infections of host cells. The IFNs (IFN-␣, IFN-␤, and IFN-␥) interact with specific cellular receptors and stimulate the production of second messengers, leading to the expression of antiviral and immunomodulatory proteins. We report in this study that IFN-␥ stimulates the expression of a novel gene that encodes a protein with 30% amino acid sequence identity with uteroglobin, the founding member of the newly formed Secretoglobin (SCGB) superfamily. We named this protein IFN-␥-inducible SCGB (IIS), because its expression in lymphoblast cells is augmented by IFN-␥ treatment. IIS is expressed in virtually all tissues, and the highest level of expression is detectable in lymph nodes, tonsil, cultured lymphoblasts, and the ovary. Interestingly, although the expression of IIS mRNA is not significantly different in resting lymphoid cells, it is markedly elevated in activated CD8ⴙ and CD19ⴙ cells. Furthermore, treatment of lymphoblast cells with IIS antisense phosphorothioate (S)-oligonucleotides prevents chemotactic migration and invasion. Taken together, these results raise the possibility that this novel SCGB has immunological functions. The Journal of Immunology, 2004, 172: 4245– 4252. potent cytokine, IFN-␥ mediates a wide range of immunological activities, primarily in response to viral infections or to inflammatory stimuli (1– 4). In addition, IFNs (i.e., IFN-␣, IFN-␤, and IFN-␥) may regulate the amplification of Ag presentation to specific T cells and are expressed constitutively by most cells. The physiological functions of IFNs are mediated through both autocrine and paracrine mechanisms. The type I (␣ and ␤) and type II (␥) IFNs regulate overlapping sets of several hundred genes at the transcriptional level (3, 5, 6) (supplemental data for Ref. 3 at http://arjournals.annualreviews.org/ doi/suppl/10.1146/annurev.immunol.15.1.749). IFN-␥ also stimulates the expression of uteroglobin (UG),2 the founding member of the newly formed Secretoglobin (SCGB) superfamily of proteins (7) that manifests potent anti-inflammatory and immunomodulatory properties (reviewed in Ref. 8). Blastokinin (9) or UG (10), first isolated from the uterus of rabbits during early pregnancy, is a homodimeric protein in which the identical 70-aa subunits are connected in antiparallel orientation by an N-terminal and a C-terminal disulfide bond forming a central hydrophobic cavity. Although the functional significance of this central cavity is unclear, it is suggested that it may sequester hydrophobic ligands such as progesterone, retinol, and polychlorinated biphenyls (11–13). During the past two decades, the isolation and characterization of cDNAs encoding UG from the mouse (14), pig (15), Syrian hamster (16), horse (17), and human (18, 19) have been reported.

A

Section on Developmental Genetics, Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 Received for publication October 27, 2003. Accepted for publication January 22, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Anil B. Mukherjee, Section on Developmental Genetics, Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 9S241, Bethesda, MD 20892-1830. E-mail address: [email protected] 2 Abbreviations used in this paper: UG, uteroglobin; SCGB, secretoglobin; MGB, mammaglobin; LIP, lipophilin; IIS, IFN-␥-inducible SCGB; SSCP, single-strand conformation polymorphism; SNP, single nucleotide polymorphism; STRP, short tandem repeat polymorphism; IP-10, IFN-␥-inducible protein-10; S, phosphorothioate.

Copyright © 2004 by The American Association of Immunologists, Inc.

Moreover, several paralogous proteins in different mammalian species have also been described. These paralogous proteins include the rat prostatic binding protein (20) also known as prostatein (21) or prostate-␣ protein (22). Prostatein forms oligomers among its subunits C1/C3 and C2/C3, and is reported to inhibit microtubule assembly (23). Several homologous proteins to rat prostatein have been described in other species. The rat prostatein C3 shows 34 and 41% sequence identity with human lacryglobin (24) and mammaglobin (MGB)1 (25), respectively. The protein sequence of lacryglobin has been reported to be identical with that of MGB2 (26) and lipophilin (LIP)-C (27). Although the biological functions of most proteins in this superfamily remain unclear, UG has been recognized as a multifunctional protein with potent antiinflammatory, antichemotactic, and tumor suppressor-like activities (8). MGB1 is reported to be expressed at high levels in mammary tumors (28). Interestingly, among the proteins of the SCGB superfamily, the expression of only the UG gene has been reported to be stimulated by IFN-␥ (14, 29, 30). Thus, it is of interest to determine whether the expression of other members of this superfamily of genes are inducible by cytokines. In this study, we report the characterization of the cDNA and the gene encoding a novel SCGB, the expression of which in lymphoblasts is stimulated by IFN-␥, and we named this protein IFN-␥inducible SCGB (IIS). This protein bears 30% amino acid sequence identity with UG, a multifunctional protein with potent anti-inflammatory/immunomodulatory properties. We also found that IIS is expressed in virtually all tissues, and although the expression of IIS mRNA is not significantly different in resting lymphoid cells, it is markedly elevated in activated CD8⫹ T cells and CD19⫹ B cells. Moreover, treatment of the lymphoblast cells with IIS antisense phosphorothioate (S)-oligonucleotide markedly inhibited chemotactic migration and invasion, raising the possibility that IIS may have immunological functions.

Materials and Methods Materials Polyclonal Ab of IIS was raised in the rabbit (Covance Laboratories, Vienna, VA) against a synthetic oligopeptide (NH2-SFKKRLSLKKSWWKCOOH) corresponding to the amino acid sequence of IIS (residues 70 – 83) (Peptide Technologies, Gaithersburg, MD). Specificity of this antiserum 0022-1767/04/$02.00

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IFN-␥-INDUCIBLE SCGB

toward IIS was also tested before and after adsorption with the oligopeptide that was used as the immunogen for raising this Ab. The results showed that, although unadsorbed antiserum readily recognized IIS protein band in Western blots, the postadsorbed antiserum was ineffective in recognizing this protein. Human recombinant cytokines (IFN-␥, IL-4, and IL-13) and TNF-␣ were obtained from Sigma-Aldrich (St. Louis, MO) and Endogen (Woburn, MA), respectively. Genomic DNAs of 34 unrelated individuals were obtained from the DNA Polymorphism Discovery resources of Coriell Cell Repositories (Camden, NJ).

TGA TTT ATT AAA GC-3⬘, annealed at 58°C. PCR products were subcloned into TOPO TA vector (Invitrogen). The nucleotide sequence was determined by using a Beckman Coulter (Fullerton, CA) CEQ 2000 following the manufacturer’s protocol. To investigate the tissue-specific expression, PCR was also performed with first-strand cDNAs from various human tissues as supplied in MTC panel II (Clontech) using the same primers and conditions mentioned above. PCR products were resolved by electrophoresis using 1.2% agarose gels. The amplification of GAPDH was used to normalize cDNA loading into each lane.

Cell culture

Characterization of RT-PCR products by hybridization with IIS cDNA probe

Immortalized normal human lymphoblast cells were obtained from Dr. K. Wisniewski (New York State Institute for Basic Research, Staten Island, N.Y.). These cells were grown in RPMI 1640 (Invitrogen, San Diego, CA) supplemented with 16% heat-inactivated FBS (Invitrogen), 1 mM L-glutamine, and penicillin (100 U/ml)-streptomycin (100 ␮g/ml), and incubated in a humidified incubator in an atmosphere of 5% CO2 and 95% air at 37°C. Human colon carcinoma (T-84; ATCC no. CCL248; American Type Culture Collection, Manassas, VA) and the NIH 3T3 cells were grown in Dulbecco’s modified minimal essential medium (Invitrogen) supplemented with 10% heat-inactivated FBS (Invitrogen), 1 mM L-glutamine, and penicillin (100 U/ml)-streptomycin (100 ␮g/ml), and incubated in a humidified atmosphere with 5% CO2 at 37°C.

Complimentary DNA from various human tissues (MTC panel II; Clontech) were subjected to PCR as described above, and the products were resolved by electrophoresis using 1% agarose gels, and cDNA bands were transferred on to Hybond-N⫹ nylon membrane (Amersham Biosciences, Buckinghamshire, U.K.). The blot was hybridized with [␣-32P]dCTP-labeled IIS cDNA probe at 42°C overnight using DIG Easy Hyb (Boehringer Mannheim, Mannheim, Germany). After prehybridization for 2 h, the blot was washed three times with SSC (2⫻)/0.1% SDS solution for 15 min each and then three times with SSC (0.1⫻)/0.1% SDS solution for 15 min each. Autoradiographs were prepared using BioMax x-ray film (Kodak).

Single-strand conformation polymorphism (SSCP)

Expression of IIS protein in COS-1 cells

To detect mutations (polymorphism), we carried out SSCP. Five fragments of IIS gene were amplified from genomic DNAs of normal individuals (n ⫽ 34) by PCR solution added [␣-32P]dCTP. The fragment (382 bp) of promoter region 1 was amplified using 5⬘-GTG GCT ACA CAT CAC AGA AAG-3⬘ (forward) and 5⬘-CAC AGG TGA ATT ATG GCT TC-3⬘ (reverse) primers at annealing temperature of 64°C. The fragment (398 bp) of promoter region 2 was amplified using 5⬘-GAG AAC ACA GCC TTC CAG C-3⬘ (forward) and 5⬘-CAA TGA GTG ATT TGG ATT CG-3⬘ (reverse) primers at annealing temperature of 63°C. The fragment (160 bp) of exon 1 was amplified using 5⬘-CTC CAT GAC TAC ACA GGC TC-3⬘ (forward) and 5⬘-GCT GGA CTC ATG ACT GAT G-3⬘ (reverse) primers at annealing temperature of 65°C. The fragment (311 bp) of exon 2 was amplified using 5⬘-CTG TCT GGT GTA ACC TCA GG-3⬘ (forward) and 5⬘-GCT GAG TTG AAT TCT GCC TC-3⬘ (reverse) primers at annealing temperature of 65°C. The fragment (210 bp) of exon 3 was amplified using 5⬘-CAG CAG CAG CAT GAC TGA C-3⬘ (forward) and 5⬘-GAC CAG TGG AGA TGT GCA G-3⬘ (reverse) primers at annealing temperature of 66°C. The amplified fragments were resolved on 0.5⫻ MDE gel (BioWhittaker Molecular Applications, Rockland, ME) with 0.6⫻ Tris-borateEDTA (TBE) running buffer at constant 3 W for 12–16 h, and autoradiographs were obtained. Thermo sequenase radiolabeled terminator cycle sequencing kit (USB, Cleveland, OH) was used for the sequencing following the manufacturer’s protocol. The samples were resolved by electrophoresis using 6% sequencing gel, and autoradiographs were prepared by using BioMax x-ray film (Kodak, Rochester, NY).

cDNA containing the entire coding sequence of IIS was amplified using 5⬘-ATG AGG CTG TCA GTG TGT CTC C-3⬘ (forward) and 5⬘-TCA CTA TTT CCA CCA GGA CT-3⬘ (reverse) primers, and cDNA encoding the mature IIS (without the signal peptide sequence) without signal peptide sequences was amplified using 5⬘-CTT GTC TGC CCA GCT GTT GCT TC-3⬘ (forward) and 5⬘-TCA CTA TTT CCA CCA GGA CT-3⬘ (reverse) primers and subcloned into pcDNA4/HisMax-TOPO TA expression vector (Invitrogen). The recombinant plasmids were linearized with BglII and electroporated into COS-1 cells. The transfected cells were culture for 48 h in Dulbecco’s modified minimal essential medium containing 10% FBS, 0.8 mM L-glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin.

RT-PCR Total RNAs were extracted from human lymphoblast and human colon carcinoma cells using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. Total RNAs were reverse-transcribed using ThermoScript RT-PCR system (Invitrogen). Semiquantitative PCR was performed to amplify target fragments of cDNA. Five microliters of the RT-PCR product was used for the PCR. The PCR conditions are as follows: denaturation at 94°C for 1 min, followed by 35 cycles of amplification at 94°C for 30 s and at different annealing temperature for 2 min, and a final incubation at 68°C for 5 min using AdvanTaq Plus DNA polymerase (Clontech, Palo Alto, CA). The primers that amplify LIP-B and an unknown cDNA fragment are 5⬘-CTG CTG CTA CCA GGC CAA TG-3⬘ (forward) and 5⬘-GTC ACA CAC TAC ATT TCT TC-3⬘ (reverse), and the primers that amplify only LIP-B are 5⬘-CCT CTG TTC AAG TTA AGT C-3⬘ (forward) and 5⬘-CCG CAA TGA GGC TTC GTT TGG-3⬘ (reverse). The annealing temperature is 60°C. The primers used to amplify MGB1 were 5⬘-GAC AAT GCC ACT ACA AAT GCC-3⬘ (forward) and 5⬘-CAT TGC TCA GAG TTT CAT CCG-3⬘ (reverse) with annealing temperature of 66°C. The primers used for the amplification of other cDNAs are as follows: MGB2, forward, 5⬘-CTC CTG GAG GAC ATG GTT G-3⬘, reverse, 5⬘-CTA TGT GAC TGG TTG AGG-3⬘, annealing temperature 66°C; LIP-A, forward, 5⬘-CAG TGG TCT GCC AAG CTC TTG G-3⬘, reverse, 5⬘-CAT AGG CCA TCG TAT CCA CGC-3⬘, annealed at 66°C; UG, forward, 5⬘-CAG AGA TCT GCC CGA GCT TTC-3⬘, reverse, 5⬘-GCT TAA TGA TGC AAA CAC TGG-3⬘, annealed at 58°C; IIS, forward, 5⬘-CTC ACA GCC GAA TAA GCC ACC-3⬘, reverse, 5⬘-GTG CAG GGC AAG

Expression of rIIS protein in Escherichia coli IIS cDNA containing the entire coding region was amplified using 5⬘-ATG AGG CTG TCA GTG TGT CTC C-3⬘ (forward) and 5⬘-CAT TTT TTC ACT ATT TCC ACC AGG ACT-3⬘ (reverse) primers and subcloned into PshI/EcoRV site of pET-42a(⫹) vector (Novagen, Madison, WI), which includes an integrated T7 promoter. E. coli strain, BL21 (DE3) was transfected with the cDNA construct, and the transformed cells were inoculated into Luria-Bertani broth with kanamycin and grown until it reached an OD reading of 1.0 at 600 nm. Expression of the IIS protein was induced with 0.4 mM isopropyl-␤-D-thiogalactopyranoside for 2 h.

Purification of rIIS protein from the transfected E. coli culture IIS protein from the transfected E. coli lysates was purified using BugBuster Ni-NTA His 䡠 Bind purification kit and His 䡠 Bind columns (Novagen). The transfected E. coli cells (1.0 g of cell paste) were harvested and added with 5 ml of BugBuster reagent and 5 ␮l of Benzonase (Novagen) followed by incubation for 20 min on a shaking platform. The solution was centrifuged, and the supernatant was passed through a 0.45-␮m syringeend filter and then loaded onto His 䡠 Bind column precharged with Ni2⫹. The column was washed with 10 vol of 1⫻ binding buffer and 6 vol of 1⫻ washing buffer. The bound protein was eluted with 6 vol of 1⫻ elution buffer.

Immunoprecipitation and immunoblotting Human lymphoblasts were harvested and lysed with lysis buffer containing protease inhibitor mixture and immunoprecipitated using protein A immunoprecipitation kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD) according to the supplier’s instructions. The cell lysate was precleared using 50% resin slurry. The IIS Ab was added to the precleared sample followed by an addition of 50 ␮l of 50% resin slurry and incubated overnight at 4°C with gentle agitation. The resin was pelleted and washed three times with lysis buffer and resuspended in 40 ␮l of 1⫻ SDS-PAGE sample buffer. Proteins were resolved by electrophoresis on 18% Tris-glycine gels (Invitrogen). Total proteins from transfected COS-1 cells and transfected E. coli cells were extracted using T-PER mammalian and bacterial protein extraction buffers (Pierce, Rockford, IL), respectively, according to the manufacturer’s instructions. The concentration of total proteins was determined using Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). One hundred micrograms of total protein from the transfected COS-1 cells

The Journal of Immunology were resolved by electrophoresis using 18% Tris-glycine gel (Invitrogen) followed by electrotransferring onto Immobilon-P transfer membrane (Millipore, Bedford, MA). The blots were incubated in blocking solution containing 4% BSA at 4°C. They were washed with TBST solution (10 mM Tris-HCl (pH 8.0), 0.15 M NaCl, and 0.1% Tween 20) and incubated with anti-Xpress Ab (diluted 1/5000; Invitrogen) for 1 h followed by incubation with HRP-conjugated rabbit anti-mouse Ab. The blots were washed with TBST solution and developed using ECL chemiluminescence detection kit (ECL kit; Amersham Biosciences) according to manufacturer’s instructions. The purified proteins from the transfected E. coli culture were resolved on 10% Tris-glycine gel followed by transferring onto Immobilon-P transfer membrane. The membrane was incubated in blocking solution containing 1% gelatin at 4°C. The blot was washed and incubated with alkaline phosphatase-conjugated S 䡠 Tag Ab (diluted 1/5000; Novagen) for 15 min. The blots were washed with TBST solution and developed using S 䡠 Tag AP Western blot kit (Novagen) following the manufacturer’s instruction. For detecting the IIS protein bands, the blots were incubated with anti-IIS Ab (diluted 1/1000) for 1 h, washed with TBST solution, and incubated with HRP-conjugated goat anti-rabbit IgG (diluted 1/1000; Amersham Biosciences) for 1 h, washed five times (10 min each), and detected by ECL chemiluminescence detection. The specificity of the Ab was determined by preabsorption of the antiserum with the synthetic oligopeptide Ag, and this postadsorbed antiserum failed to recognize the IIS protein band in Western blot. The prestained protein marker (Bio-Rad Laboratories) was used as a molecular mass standard.

Quantitative real-time RT-PCR Cultured human lymphoblast cells were treated with the following cytokines: TNF-␣ (10 ng/ml), IFN-␥ (10 ng/ml), IL-4 (0.5 ng/ml), or IL-13 (10 ng/ml) for 1, 3, and 6 h, respectively. The cells were also treated with different concentrations of IFN-␥ (0.1, 1, or 10 ng/ml) for 1 h to obtain a dose-response curve. Total RNA was isolated from the treated cells using TRIzol (Invitrogen) and an RNeasy mini-kit (Qiagen, Valencia, CA), followed by DNase treatment to eliminate genomic DNA contamination. Quantitative real-time RT-PCR was performed using Smart Cycler system (Cepheid, Sunnyvale, CA). First-strand cDNA was synthesized from 1 ␮g of total RNA using Superscript III first-strand synthesis system (Invitrogen) following the manufacturer’s protocol. Real-time PCR was performed with 2.5 ␮l of cDNA and primers (forward, 5⬘-CTC ACA GCC GAA TAA GCC ACC-3⬘; reverse, 5⬘-GTG CAG GGC AAG TGA TTT ATT AAA GC-3⬘) using QuantiTect SYBR Green (Qiagen) following the manufacturer’s protocol under the following conditions: denaturation at 94°C for 15 min followed by 50 cycles of amplification at 94°C for 15 s, 58°C for 30 s, and 72°C for 30 s. Real-time PCR was also performed with first-strand cDNAs from resting and activated blood cells using Human Blood Fractions MTC panel (Clontech). The data from each PCR run was analyzed using Cepheid Smart Cycler software program (Cepheid) with FAM as the reference dye. The final data were normalized to ␤-actin and are presented as fold induction. Quantitation was performed using at least three separate total RNA samples for each treatment group.

4247 using the cell invasion assay kit as indicated above. The migrated and invaded cells were counted using a hemocytometer.

Statistical analysis The statistical significance was analyzed using Student’s t test. Data are presented as the mean ⫾ SD. The results were considered significant at p ⬍ 0.05.

Results IIS is a novel member of the SCGB family To delineate the levels of expression of various members of the SCGB family in human lymphoid cells, we performed RT-PCR using total RNA from a lymphoblast cell line, using primers for UG, LIP-A, LIP-B, MGB1, and MGB2 (Fig. 1A). We performed DNA sequencing of the PCR products to confirm the identity of each of these genes. Unexpectedly, we found that the amplified products for putative LIP-B contained a DNA sequence that is unrelated to that of LIP-B. LIP-B cDNA and the unidentified RTPCR product were independently amplified by PCR using more specific primers for LIP-B and a separate set of primers for the unique sequence. The PCR products were analyzed by DNA sequencing. The results of repeated experiments showed that not only all members of the SCGB gene family but also the putative new gene is also expressed in human lymphoblasts (Fig. 1A). To further delineate the identity of this unique RT-PCR product, we performed a BLAST search of GenBank database and uncovered that the sequence of the unidentified RT-PCR product is identical with that of the expressed sequence tag 378247, MAGE sequences, and MAGI Homo sapiens cDNA sequence (GenBank accession no. AW966174). Interestingly, we were also able to amplify this cDNA by RT-PCR using total RNA from human colon carcinoma cells. Further analysis of the full-length cDNA sequence contained a single open reading frame of 252 nt. Because this unique sequence has not been previously described and because the expression of this gene is stimulated by IFN-␥, we named this gene IIS. The cDNA sequence of IIS has been submitted to GenBank (accession no. AY236538). The IIS cDNA encodes a protein of 83 aa, which has a calculated molecular mass of 9.2 kDa and a calculated isoelectric point of 8.9.

Antisense S-oligonucleotide treatment A phosphorothioate IIS antisense oligonucleotide (5⬘-TTT GGC AAC TTG GAG GTT TA-3⬘) was used to inhibit IIS protein expression. The corresponding IIS sense oligonucleotide (5⬘-TAA ACC TCC AAG TTG CCA AA-3⬘) was used as one of the controls. When the lymphoblast cells reached 30 – 40% confluence, they were rinsed once with serum-free OptiMEM I (Invitrogen). The S-oligonucleotides were then delivered to the cells by Oligofectamine (Invitrogen) according to the manufacturer’s instructions. Treatment of the cells with Oligofectamine alone was also used as a control. The cells were incubated for 60 h at 37°C before using for migration and invasion assays.

Cell migration and invasion assay The cell migration assay was performed using QCM 96-well 5-␮m cell migration plates (Chemicon, Temecula, CA). The cells were collected after transfection with IIS antisense or sense S-oligonucleotides and incubated for 60 h. Following incubation, the cells were rinsed with serum-free RPMI 1640 medium and resuspended in the same serum-free medium. One hundred fifty microliters of the conditioned medium from NIH 3T3 cell culture was placed into 96-well feeder tray, and 1 ⫻ 105 cells in 100 ␮l of serumfree RPMI 1640 medium was placed into cell migration chamber plate and then incubated for 24 h at 37°C in a CO2 incubator. The cell invasion assay was performed using QCM 96-well cell invasion plate (Chemicon). The transfected cells were rinsed with serum-free RPMI 1640 medium and then resuspended in serum-free RPMI 1640 medium. The conditions for the invasion assay are identical with those of the migration assay conducted

FIGURE 1. The mRNA expression of SCGB family members in human lymphoblast cells, the gene structure of IIS, and exon/intron boundaries. A, The mRNA expression levels of SCGB family members in human lymphoblast cells by semiquantitative RT-PCR. Lane 1, UG; lane 2, LIP-A; lane 3, MGB1; lane 4, MGB2; lane 5, LIP-B; lane 6, IIS. B, Schematic structure of the human IIS gene with the exons shown by boxes and the introns shown by horizontal lines. The coding region is shown by solid boxes. There is an STRP in intron 2 and an SNP in the 3⬘ region of the gene. C, Exon-intron boundaries of the human IIS gene. The consensus nucleotides of exon-intron boundaries are in bold.

4248 BLAST search against the human genome database using the IIS cDNA coding sequence enabled us to identify the genomic sequence containing the IIS gene. We found that the entire IIS gene sequence was contained in human chromosome 11, clones RP11703H8 (GenBank accession no. AP003306), pDJ741n15 (accession no. AC004127), and CTD-253 D15 (accession no. AP003064). To determine IIS gene structure, we further analyzed these clone sequences and found that the entire IIS gene spanned ⬃3 kb of genomic DNA and a total of three exons and two introns (Fig. 1B). The sequences adjacent to the splice sites were in good conformance with the consensus splice rule (Fig. 1C). The relative sizes of the exons and introns are closely conserved in SCGB family members except for exon 3 of IIS, which encodes the shortest amino acid sequences among the human SCGB members. Subsequent BLAST analysis demonstrated that cDNA sequence of IIS also showed high homology to those of other SCGB family members, especially with LIP-B cDNA sequences showing a 72.6% homology. The IIS gene promoter sequence was analyzed using NSITE DB program to determine the transcription factors that potentially regulate this gene. Several transcription factor-binding consensus sequences including those of NF-␬B, hepatocyte NF-1A, -1B, -1C, SP1, IFN-stimulated response elements, and ␥-IFN-activated sites are present within ⫺1500 bp of the IIS gene promoter region. Polymorphisms in the IIS gene Because single nucleotide polymorphisms (SNP) have been reported in the UG gene (19), we sought to determine whether SNPs are also detectable in the IIS gene. IIS genomic DNAs from 34 normal healthy individuals were analyzed for polymorphism by SSCP and DNA sequencing. There were five different SSCP patterns identified in amplicons of the exon 3 region (Fig. 2A). We found an SNP (G3 A substitution) in the 3⬘-flanking region near exon 3 (Fig. 2B). The frequency of occurrence of this SNP was 0.971 for the G allele and 0.029 for the A allele (n ⫽ 34 individuals). Short tandem repeat polymorphisms (STRP) such as C(T)8,

FIGURE 2. Polymorphism of IIS gene. A, SSCP analysis of IIS genomic DNA. Each lane shows different SSCP pattern from normal individuals. B, SNP (G3 A) of IIS gene in 3⬘ region of the gene shows three different genotypes: GG (left panel), GA (middle panel), and AA (right panel). Arrows indicate SNP sites. C, There is STRP (C(T)8, C(T)9, or (CTTT)2(CTTTT)) of IIS gene in intron 2. Panels show C(T)8/C(T)8 (left), C(T)9/C(T)9 (middle), and (CTTT)2(CTTTT)/C(T)8 (right) genotypes.

IFN-␥-INDUCIBLE SCGB C(T)9, or (CTTT)2(CTTTT) in intron 2 were detected, and this polymorphism appears to occur at frequencies of 0.662 for C(T)8, 0.309 for C(T)9, and 0.029 for (CTTT)2(CTTTT) in these individuals (Fig. 2C). IIS mRNA expression in various tissues We performed Northern hybridizations using poly(A)⫹ RNA blot of various tissues to see the level of IIS mRNA expression. However, this approach was unsuccessful, because the signal from these tissues was extremely low (data not shown). Therefore, we determined the expression of IIS mRNA by semiquantitative RTPCR. The identities of the RT-PCR products were confirmed by Southern hybridization using IIS cDNA probe as well as by DNA sequencing. The results show that the expression of IIS mRNA is the highest in the ovary, lymph node, and tonsil, and in a lymphoblast cell line. Moderate expression levels were detected in the small intestine, colon, bone marrow, and fetal liver, and in a colon carcinoma cell line, T-84, and the lowest expression levels were detected in the spleen, thymus, prostate, and the testis (Fig. 3). IIS protein expression in bacterial and mammalian cells IIS cDNA was subcloned into PshI/EcoRV site of pET-42a(⫹) vector, and the expression constructs were electroporated into E. coli strain BL21 (DE3). After induction with isopropyl-␤-D-thiogalactopyranoside, IIS protein was purified using BugBuster NiNTA His 䡠 Bind purification kit and His 䡠 Bind columns. The eluted protein (molecular mass, 35 kDa) was detected with S 䡠 Tag Ab and with IIS Ab (Fig. 4A). These results show that IIS mRNA is translated to IIS protein in the bacteria, and that it is stable in the bacteria. We also expressed the IIS cDNA in COS-1 cells. The IIS cDNA was subcloned into pcDNA4/His 䡠 Max-TOPO vector and was electroporated into COS-1 cells. The lysates of the transfected cells were resolved by SDS-PAGE, and protein bands were analyzed by Western blot. The results show that IIS protein was stably expressed in COS-1 cells both without (Fig. 4B, lane 2) and with the signal peptide (lane 3). No change in the apparent molecular mass of this protein was observed when we resolved the protein by SDS-PAGE under both reducing and nonreducing conditions (Fig. 4B), suggesting the absence of subunit structure involving interchain disulfide bonds. Using IIS Ab and immunoprecipitation, we further confirmed that a cultured human lymphoblast cell line expresses IIS protein (Fig. 4C, upper panel). The specificity of the IIS Ab was determined by adsorbing the total antiserum with the synthetic oligopeptide corresponding to the IIS amino acid sequence (residues 70 – 83), which failed to recognize the protein band (Fig. 4C, lower panel).

FIGURE 3. The mRNA expression levels of IIS in various human tissues and cell lines. The mRNA expression levels were detected by RT-PCR and then Southern hybridization. Note the highest level of expression of IIS mRNA in the ovary, lymph node, and tonsil, and in lymphoblasts. The amplification of GAPDH was used to normalize the total RNA added to each RT-PCR.

The Journal of Immunology

FIGURE 4. Expression of IIS protein. A, Immunoblot analysis of rIIS expressed in E. coli. IIS protein was detected with S 䡠 Tag Ab (left panel) and IIS Ab (right panel) following the purification using Ni2⫹ resin. Lane 1, Lysate from the cells transfected with vector alone; lane 2, lysate from the cells transfected with vector containing IIS coding sequence. B, Immunoblot analysis of IIS expression in COS-1 cells. Cell lysates from the culture of COS-1 cells expressing His-tagged IIS protein were resolved by SDS-PAGE under reducing (R) and nonreducing (NR) conditions, respectively, and then immunoblotted with anti-Xpress Ab. Lane 1, Lysate of cells transfected with vector only; lane 2, lysate of cells transfected with plasmid containing IIS cDNA encoding the mature polypeptide sequence 9 without the leader peptide; lane 3, the lysate from cells transfected with plasmid containing full-length IIS cDNA cloning sequence. C, Immunoblot analysis of IIS protein expression in lymphoblasts. Cell lysates from lymphoblasts were immunoprecipitated with IIS Ab and resolved by SDSPAGE under reducing conditions, and immunoblots were prepared using IIS Ab. Note that the preadsorption of the antiserum with the synthetic oligopeptide, used as the Ag for generating IIS Ab, failed to recognize the IIS protein band.

IIS is inducible by IFN-␥

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FIGURE 5. The induction of IIS mRNA expression in cultured lymphoblast cells. A, The induction of IIS expression by cytokines (TNF-␣, IFN-␥, IL-4, and IL-13) in the indicated treatment time. B, The induction of IIS expression by varying doses of IFN-␥. The levels of expression are represented as fold induction compared with the expression level in control cells. The experiments are repeated at least three times, and the results are expressed as the mean of fold induction ⫾ the SD of the mean. ⴱ, Significant differences (p ⬍ 0.05).

mRNA expression was markedly stimulated in activated CD8⫹ cells compared with that in resting cells (Fig. 6B). Most interestingly, activated CD19⫹ B cells expressed significantly higher levels of IIS mRNA compared with that in resting cells (Fig. 6B).

Because it has been reported previously that the expression of UG is inducible by IFN-␥, we sought to determine whether the expression of other members of this family of genes are also induced by IFN-␥. Accordingly, we determined the IIS mRNA levels by quantitative real-time PCR of total cellular RNA extracted from human lymphoblast cells that were treated with IFN-␥ (10 ng/ml), TNF-␣ (10 ng/ml), IL-4 (0.5 ng/ml), and IL-13 (10 ng/ml) for up to 6 h, and compared the results with those from untreated controls. When lymphoblast cells were treated with IFN-␥ for 1 h, the expression levels of IIS mRNA were significantly increased (Fig. 5A). However, the cells treated with cytokines other than IFN-␥ failed to show any stimulation of IIS mRNA expression (Fig. 5A). To determine a dose-related effect of IFN-␥ on the changes in IIS mRNA expression, the cells were incubated with 0.1–10 ng/ml IFN-␥ for 1 h. The expression levels of IIS mRNA were significantly increased in a dose-dependent manner up to 11.68-fold of nonstimulated cells (Fig. 5B). This results show that IIS expression is inducible by IFN-␥.

Because it has been reported that IFN-␥-inducible protein-10 (IP10) induces chemotaxis (31), we sought to determine whether IIS has any effect on cellular migration and invasion. Accordingly, lymphoblast cells were transfected with IIS antisense oligonucleotides and used for chemotaxis assay. Conditioned medium from fibroblast cell culture was used as the chemoattractant, and cell migration and invasion assays were conducted as previously reported (32, 33). The results show that chemotactic migration of antisense oligo-transfected cells was markedly inhibited compared with that of the control and sense oligonucleotide-transfected cells (Fig. 7A). Similarly, invasion of the antisense oligo-transfected cells was also significantly lower than that of control and sense oligonucleotide-transfected cells (Fig. 7B). These results suggest that inhibition of IIS expression suppresses migration and invasion in lymphoblast cells.

Stimulation of IIS expression in activated white blood cells

Discussion

To delineate which blood cells are specific for the expression of IIS, we investigated its expression in different cell types of blood cells by quantitative real-time PCR. IIS mRNA expression in various types of resting WBCs was not significantly different from that of lymphoblasts (Fig. 6A). IIS mRNA expression in CD4⫹ T cells was not induced in activated cells (Fig. 6B). However, IIS

In this study, we characterized IIS. We assigned IIS to a novel member of SCGB family based upon the following: 1) cDNA and amino acid sequences of IIS showed a high homology to those of SCGB family members, especially with LIP-B cDNA sequences showing a 72.6% homology; 2) IIS gene is mapped to human chromosome 11, the region in which SCGB gene cluster is localized;

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IFN-␥-INDUCIBLE SCGB

FIGURE 6. The expression of IIS mRNA in different types of white blood cells. A, The expression level of resting cells from each cell type is represented in fold increase compared with that of lymphoblasts. B, A comparison of the IIS expression levels in each type of activated WBCs (f) with that in the resting counterparts (u). The results are expressed as the mean of three independent experiments ⫾ SD of the mean. ⴱ, Significant differences (p ⬍ 0.05).

FIGURE 7. The effect of IIS antisense S-oligonucleotide treatment of lymphoblasts on cellular migration and invasion. The results of cell migration assay are presented in A, and cell invasion assay in B. The results are expressed as the total number of migrated or invaded cells per 0.005 mm3. The results are expressed as the mean of three independent experiments ⫾ SDs of the means. ⴱ, Significant differences (p ⬍ 0.05).

3) IIS gene structure is virtually identical with all genes in the SCGB family; and 4) IIS protein contains an N-terminal and a central cysteine residue that are identical with those of LIP-A and -B, MGB-1 and -2, RYD-5, UGRP-1, and YGB, which are members of the SCGB family. The proteins in the SCGB family are divided into four groups according to the number of conserved cysteines. As indicated above, UG has two conserved cysteines at ⫹3 and ⫹69 positions (Fig. 8A). Other members, such as LIP-A, LIP-B, MGB1, MGB2, and YGB (34), not only have the two cysteines at ⫹3 and ⫹69 but also have an additional cysteine at ⫹44 position. In contrast, three members of this family, RYD5, UGRP1, and UGRP2, have only one cysteine at the center of the molecule (35, 36). IIS is the only member described so far that has two cysteines: one at the Nterminal residue 3, and one at the center of the C terminus of the molecule (residue 44). A comparison of the amino acid sequences between IIS and YGB shows that amino acid residues 1–77 in these two proteins are identical. However, the third cysteine residue at position 78 of YGB is lacking in IIS, and from here the amino acid residues are not identical. It is likely that a deletion of 1 nt in the YGB cDNA sequence may have occurred at codon ⫹78, resulting in a frame shift that led to the origin of this new member. The phylogenetic analysis also suggests that IIS and YGB may have evolved from the same ancestral gene (Fig. 8B). We have detected both SNP and STRP in the IIS gene using genomic DNA samples from 34 apparently normal subjects. Although the significance of these polymorphisms is unclear at this time, the occurrence of a (G3 A) SNP in the UG gene (19) has been suggested to predispose individuals to asthma (19, 37, 38)

and found to be associated with a rapid progression of IgA nephropathy (39 – 41). Recently, it has been reported that UG gene polymorphisms affect the progression of IgA nephropathy by modulating the level of expression of UG (40), which protects against abnormal renal glomerular deposition of IgA and fibronectin (42), characteristic of IgA nephropathy. Whether the SNP and STRP in the IIS gene uncovered in the present study are predisposing factors for any human disease needs to be investigated. The amino acid sequence deduced from IIS cDNA sequence suggests that, like most SCGB family of proteins, this protein also contains a leader peptide. The founding member of this protein family, UG, which is induced by IFN-␥, is also a secreted protein, and recently, we have demonstrated that UG binds to as-yet-unidentified cell surface binding protein(s) with high affinity and specificity and regulates cellular migration and invasion (32, 33, 43). Consistent with these findings, we find that IIS is also induced by IFN-␥, and the suppression of IIS expression by antisense oligonucleotide treatment of the cells inhibits chemotactic migration and invasion. It has been reported that an IFN-␥ response element is present in the 5⬘ promoter region of the UG gene (29). We also detected a sequence in the promoter region of the IIS gene that is similar to the IFN-␥ response element in the UG promoter. Another IFN-␥-inducible protein, IP-10, which belongs to the superfamily of chemokines, stimulates activation and recruitment of leukocytes as well as nonhemopoietic cells (44, 45). Furthermore, IFN-␥-inducible T cell ␣-chemoattractant has been reported to stimulate transendothelial migration of normal blood T lymphocytes (46). Interestingly, the deduced amino acid sequence of mature IIS polypeptide bears 25% sequence identity with that of

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FIGURE 8. Amino acid sequences of the human SCGB family of proteins and the SCGB phylogenetic tree. A, Alignment of amino acid sequences of SCGB family members. Accession numbers for the sequences were as follows: IIS, AY236538; UG, NM_003357; YGB (Ref. 34); LIP-A, NM_006552; LIP-B, NM_006551; MGB1, NM_002411; MGB2, NM_002407; RYD5*, BK 000201; UGRP2, NM_052863; UGRP1, NM_054023. Asterisk indicates that the accession number is for nucleotide sequences that were used for predicting the gene products. The consensus cysteines are represented in bold within boxes. B, Phylogenetic tree of the human SCGB family. This phylogenetic tree was generated after the alignment of the sequences using CLUSTAL W.

IP-10 for which receptor-mediated functions have been identified (reviewed in Ref. 47). It is tempting to speculate that IIS may also manifest similar properties as those of IP-10 and this SCGB, like IP-10, may have a receptor-mediated function. The facts that the expression of this protein is augmented in activated CD8⫹ and CD19⫹ cells, that its expression in lymphoblast cells is stimulated by IFN-␥, and that IIS antisense S-oligonucleotide treatment inhibits chemotactic migration and invasion, suggest that this protein may have immunological functions. Future studies may delineate the molecular mechanism(s) by which IIS contributes to orchestrate the IFN-␥-mediated immune response.

Acknowledgments We thank Dr. Krystyna Wisniewski for the generous gift of lymphoblast cells used in this study. We also thank Drs. Ida Owens and Janice Y. Chou for critical review of this manuscript and for helpful suggestions.

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