Tissue-Specific mrna Expression Profiles of Human Toll-Like Receptors and Related Genes

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1 886 Biol. Pharm. Bull. 28(5) (2005) Vol. 28, No. 5 Tissue-Specific mrna Expression Profiles of Human Toll-Like Receptors and Related Genes Masuhiro NISHIMURA* and Shinsaku NAITO Division of Pharmacology, Drug Safety and Metabolism, Otsuka Pharmaceutical Factory, Inc.; 115 Tateiwa, Muya-cho, Naruto, Tokushima , Japan. Received December 27, 2004; accepted March 3, 2005: published online March 8, 2005 The mrna expression levels of 10 toll-like receptors (TLRs) and 21 related genes in total RNA from pooled specimens of fetal human tissues (brain and liver), from single and pooled specimens of various adult human tissues (adrenal gland, brain, heart, kidney, liver, lung, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, testis, thymus, thyroid gland, trachea, and uterus), and from two cell lines (Hep G2 and HeLa) were analyzed by real-time reverse transcription PCR. The mrna expression of the 10 human TLRs was successfully detected in all of the tissues and in HeLa cells. TLR2, TLR3, TLR6, and TLR9 were consistently expressed in Hep G2 cells, but TLR1, TLR4, TLR5, TLR7, TLR8, and TLR10 showed no or very weak expression in these cells. The mrna expression of many TLR-related genes (ICAM1, CD14, MyD88, LY96, TRIF, TICAM2, TIRAP, CD83, SOCS1, TNFAIP3, TOLLIP, IRAK1, IRAK2, IRAK4, and TRAF6) was successfully detected in all of the tissues and cell lines. The mrna expression of CD80, CD86, IRAK3, and CCL2 was successfully detected in all of the tissues and cell lines except for Hep G2 cells. The mrna expression of CCL5 was successfully detected in all of the tissues and cell lines except for fetal brain. The mrna expression of CXCL10 was successfully detected in all of the tissues except for fetal and adult brain. These results provide valuable information for studies concerning the regulation of TLR-related genes. Key words toll-like receptor; mrna expression; tissue distribution Toll-like receptors (TLRs), which have pathogen-associated molecular patterns, play important roles in innate immunity in mammals. 1) Recently, at least 10 members of the TLR family have been identified, and several ligands recognized by TLRs have been reported. 2,3) Triacylated bacterial lipopeptides are recognized by the cooperation of TLR2 with TLR1. 4) It has been reported that TLR1 coexpression inhibits the TLR2-mediated response to phenol-soluble modulin, a factor secreted by Staphylococcus epidermidis, while coexpression with TLR6 enhances the TLR2 response. 5) TLR3 recognizes the double-stranded RNA produced by most viruses during replication. 6) TLR4 is an essential receptor that transduces the signals of lipopolysaccharide (LPS). 7) The innate immune response to bacterial flagellin is mediated by TLR5. 8) TLR7 recognizes the single-stranded RNA (ssrna) viruses, vesicular stomatitis virus and influenza virus. 9) Heil et al. 10) reported that ssrna represents a physiological ligand for murine TLR7 and human TLR8. TLR9 recognizes unmethylated CpG DNA found in bacteria. 11) The specific ligands and functions of TLR10 are currently obscure, although it is known to be expressed in the spleen and lung. 12) It has been reported that the TLRs collaborate with many molecules such as MyD88, MD-2, TICAM, TIRAP, IRAK, TRAF6, etc. 1 3) The nomenclature for these TLRs and related genes is summarized in Table 1. The tissue distributions of many TLRs 6,12 15) and related genes such as MyD88 16) and TICAM2 17) have been reported. Information concerning gene expression in various tissues and cell lines may be important in assessing the feasibility of measuring the mrna expression level of target genes in target tissues and cell lines. However, the tissue distribution of the mrna expression of large numbers of human TLRs and related genes has not been evaluated under the same experimental conditions. The present study was therefore undertaken to investigate the mrna expression levels of 10 human TLRs and 21 related genes (ICAM1, CD14, MyD88, LY96, TRIF, TICAM2, TIRAP, CD80, CD83, CD86, SOCS1, TNFAIP3, TOLLIP, IRAK1, IRAK2, IRAK3, IRAK4, TRAF6, CCL2, CCL5, and CXCL10) using high-sensitivity real-time reverse transcription PCR (RT-PCR) in total RNA from pooled specimens of 2 fetal human tissues, from single and pooled specimens of 18 adult human tissues, and from 2 cell lines. MATERIALS AND METHODS Materials Total RNA prepared from pooled specimens of fetal human tissues (brain and liver), from single and pooled specimens of adult human tissues (adrenal gland, brain, heart, kidney, liver, lung, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, testis, thymus, thyroid gland, trachea, and uterus), and from two cell lines (Hep G2 human cell line and HeLa cell line ATCC#: CCL-2) was purchased from CLONTECH Laboratories, Inc. (Hilden, Palo Alto, CA, U.S.A.). The total RNA source information for these human tissues is shown in Table 2. Yeast trna was purchased from Life Technologies, Inc. (Rockville, MD, U.S.A.), and the TaqMan One-Step RT-PCR Master mix reagents kit was purchased from Applied Biosystems (Foster City, CA, U.S.A.). All other chemicals used in this study were of reagent grade. Oligonucleotides The pairs of primers and the TaqMan probes for the target mrnas were designed from the human mrna sequence (Table 3) using Primer Express software (Applied Biosystems). The GenBank accession number and position from the initiation codon are also shown in Table 3. Each primer and/or probe was homology searched by an NCBI BLAST search to ensure that it was specific for the target mrna transcript. The primers and TaqMan probes were synthesized by QIAGEN (Tokyo, Japan) and SIGMA- ALDRICH JAPAN K.K. Genosys Division (Ishikari, To whom correspondence should be addressed. nisimums@otsukakj.co.jp 2005 Pharmaceutical Society of Japan

2 May Table 1. Nomenclature for Target Genes Name Housekeeping genes b-actin b-2-microglobulin Glyceraldehyde 3-phosphate dehydrogenase Glucuronidase, beta Hypoxanthine phosphoribosyltransferase 1 Peptidylprolyl isomerase A Transferrin receptor Toll-like receptors Toll-like receptor 1 Toll-like receptor 2 Toll-like receptor 3 Toll-like receptor 4 Toll-like receptor 5 Toll-like receptor 6 Toll-like receptor 7 Toll-like receptor 8 Toll-like receptor 9 Toll-like receptor 10 Toll-like receptor-related genes Intercellular adhesion molecule 1 CD14 antigen Myeloid differentiation primary response gene (88) Lymphocyte antigen 96 TIR domain containing adaptor inducing interferon-beta Toll-like receptor adaptor molecule 2 Toll-interleukin 1 receptor (TIR) domain containing adaptor protein CD80 antigen CD83 antigen CD86 antigen Suppressor of cytokine signaling 1 Tumor necrosis factor, alpha-induced protein 3 Toll interacting protein Interleukin-1 receptor-associated kinase 1 Interleukin-1 receptor-associated kinase 2 Interleukin-1 receptor-associated kinase 3 Interleukin-1 receptor-associated kinase 4 TNF receptor-associated factor 6 Chemokine (C-C motif) ligand 2 Chemokine (C-C motif) ligand 5 Chemokine (X-X-C motif) ligand 10 Abbreviation, gene name and/or trivial name B2M GAPDH GUSB HPRT1 PPIA TFRC TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR7 TLR8 TLR9 TLR10 ICAM1, CD54 CD14 MyD88 LY96, MD-2, ESOP-1 TRIF, TICAM-1 TICAM2 TIRAP CD80 CD83 CD86 SOCS1 TNFAIP3, A20 TOLLIP IRAK1 IRAK2 IRAK3, IRAKM IRAK4 TRAF6 CCL2, MCP-1 CCL5, RANTES CXCL10, IP-10 Table 2. Total RNA Source Information for Various Tissues Tissue Pool size Age Sex Race Brain 21 fetuses weeks Female, male Caucasian Liver 63 fetuses weeks Female, male Caucasian Adrenal gland years Female, male Caucasian Brain 1 43 years Male Caucasian Heart years Female, male Caucasian Kidney 1 75 years Female unknown Liver 1 51 years Male Caucasian Lung years Female, male Caucasian Placenta years Female Caucasian Prostate years Male Caucasian Salivary gland years Female, male Caucasian Skeletal muscle years Female, male Caucasian Spleen years Female, male Caucasian Testis years Male Caucasian Thymus years Male Caucasian Thyroid gland years Female, male Caucasian Trachea years Female, male Caucasian Uterus years Female Caucasian Small intestine years Female, male Caucasian Spinal cord years Female, male Caucasian, no data available.

3 888 Vol. 28, No. 5 Table 3. Primers and Probes Used for RT-PCR Analysis mrna (GenBank No.) Forward primer (Position) Reverse primer (Position) Probe (Position) Housekeeping genes b-actin (NM_001101) 5 -AGATGGCCACGGCTGCT-3 ( ) 5 -AACCGCTCATTGCCAATGG-3 ( ) 5 -AGCTCCTCCCTGGAGAAGAGCTACGAGCT-3 ( ) B2M a) (NM_004048) 5 -TGCTCGCGCTACTCTCTCTTT-3 (26 46) 5 -ATGTCGGATGGATGAAACCC-3 ( ) 5 -CCTGGAGGCTATCCAGCGTACTCCAAAGAT-3 (51 80) GAPDH (NM_002046) 5 -CATGGGTGTGAACCATGAGAA-3 ( ) 5 -GGTCATGAGTCCTTCCACGAT-3 ( ) 5 -AACAGCCTCAAGATCATCAGCAATGCCT-3 ( ) GUSB a) (NM_000181) 5 -TCTGTCAAGGGCAGTAACCTG-3 ( ) 5 -CGCCACGACTTTGTTTTCTG-3 ( ) 5 -TCAAGTTGGAAGTGCGTCTTTTGGATGC-3 ( ) HPRT1 a) (NM_000194) 5 -GAACGTCTTGCTCGAGATGTG-3 ( ) 5 -CCAGCAGGTCAGCAAAGAATT-3 ( ) 5 -AGGCCATCACATTGTAGCCCTCTGTG-3 ( ) PPIA a) (NM_021130) TFRC a) (NM_003234) 5 -TCCTGGCATCTTGTCCATG-3 ( ) 5 -CCATCCAACCACTCAGTCTTG-3 ( ) 5 -CAAATGCTGGACCCAACACAAATGG-3 ( ) 5 -TTGGACATGCTCATCTGGG-3 ( ) 5 -ATGACCGAGATGGTGGAAACT-3 ( ) 5 -ACCCTTACACACCTGGATTCCCTTCCTTC-3 ( ) Toll-like receptors TLR1 (NM_003263) 5 -AACCCATTCCGCAGTACTCCA-3 ( ) 5 -AAGGCCACGTTTGCTCTTTTC-3 ( ) 5 -AGCAGTTATCACAAGCTCAAAAGTCTCATGGCC-3 ( ) TLR2 (NM_003264) 5 -CAATGATGCTGCCATTCTCAT-3 ( ) 5 -ATTATCTTCCGCAGCTTGCA-3 ( ) 5 -CATTGAGAAAAAAGCCATTCCCCAGCG-3 ( ) TLR3 (NM_003265) 5 -ACAACTTAGCACGGCTCTGGA-3 ( ) 5 -ACCTCAACTGGGATCTCGTCA-3 ( ) 5 -ACGCAAACCCTGGTGGTCCCATTTAT-3 ( ) TLR4 (NM_138554) 5 -AGTTTCCTGCAATGGATCAAGG-3 ( ) 5 -CTGCTTATCTGAAGGTGTTGCAC-3 ( ) 5 -AGGCAGCTCTTGGTGGAAGTTGAACGA-3 ( ) TLR5 (NM_003268) 5 -GGCTTAATCACACCAATGTCACTAT-3 ( ) 5 -GAAACCCCAGAGAACGAGTCAG-3 ( ) 5 -CTGGGCCTCCTGCAGACATATATTGTGTG-3 ( ) TLR6 (NM_006068) 5 -CCCATTCCACAGAACAGCAT-3 ( ) 5 -ATAAGTCCGCTGCGTCATGA-3 ( ) 5 -CCCAACAAGTACCACAAGCTGAAGGCT-3 ( ) TLR7 (NM_016562) 5 -TGGAAATTGCCCTCGTTGTT-3 ( ) 5 -GTCAGCGCATCAAAAGCATT-3 ( ) 5 -CCCATTTCCTTGTGCGCCGTGTAA-3 ( ) TLR8 (NM_016610) 5 -CTTCGATACCTAAACCTCTCTAGCAC-3 ( ) 5 -AAGATCCAGCACCTTCAGATGA-3 ( ) 5 -CCCTCAGGAAGATTAATGCTGCCTGGTT-3 ( ) TLR9 (NM_017442) 5 -AGTCAATGGCTCCCAGTTCCT-3 ( ) 5 -CGTGAATGAGTGCTCGTGGTA-3 ( ) 5 -TCTGCAGGTGCTAGACCTGTCCCACAATA-3 ( ) TLR10 (NM_030956) 5 -TATGACAGCAGAGGGTGATGC-3 (42 62) 5 -TGCGGGAACCTTTCTTAGAGA-3 ( ) 5 -CTGCCAGAAGAAAGGGAACTGATGACCA-3 (70 97) Toll-like receptor related genes ICAM1 (NM_000201) 5 -GCAGTCAACAGCTAAAACCTTCC-3 ( ) 5 -TAAGGTTCTTGCCCACTGGCT-3 ( ) 5 -TGTACTGGACTCCAGAACGGGTGGAACT-3 ( ) CD14 (NM_000591) 5 -TGTGAGCTGGACGATGAAGAT-3 (73 93) 5 -CAGACACACACTGGAAGGCTT-3 ( ) 5 -TGCGTCTGCAACTTCTCCGAACCTCA-3 ( ) MyD88 (NM_002468) 5 -CATCACCACACTTGATGACCC-3 ( ) 5 -TGCACAAACTGGATGTCGC-3 ( ) 5 -CATATGCCTGAGCGTTTCGATGCC-3 ( ) LY96 (NM_015364) 5 -ACTGTCAACACCATGAATCTTCC-3 ( ) 5 -CTGCAAAAAGAGTAATCGTCATCA-3 ( ) 5 -AAGCGCAAAGAAGTTATTTGCCGAGGA-3 ( ) TRIF (NM_182919) 5 -AGCGCCTTCGACATTCTAGGT-3 (28 48) 5 -AGAACCATGGCATGCAGGA-3 ( ) 5 -AAGCTCTTGTATCTGAAGCACAAACTGAAGACCCC-3 (64 98) TICAM2 (NM_021649) 5 -AAACCCGGAATAATCTTTGCTG-3 ( ) 5 -TAAGATTGTCCATGCAGACCCA-3 ( ) 5 -CCATGTGGCAGACAGCATTTACAGAATTTAGATG-3 ( ) TIRAP (NM_052887) 5 -CTGGCTCTCGGCCTAAGAA-3 (29 47) 5 -CATCGCTGGAGGTGCTTTC-3 ( ) 5 -CTCTAGGCAAGATGGCTGACTGGTTCAG-3 (50 77) CD80 (NM_005191) 5 -TGTTATCCACGTGACCAAGGA-3 ( ) 5 -TGTGCCAGCTCTTCAACAGAAA-3 ( ) 5 -TGAAAGAAGTGGCAACGCTGTCCTG-3 ( ) CD83 (NM_004233) 5 -CCCCAATGAAAGGCCCTATT-3 ( ) 5 -TTAGGTTTCTCTGCCCATCCG-3 ( ) 5 -CTGAAGATCCGAAACACTACCAGCTGCAACTC-3 ( ) CD86 (NM_175862) 5 -TATGGGCCGCACAAGTTTT-3 ( ) 5 -TCCTGTGGGCTTTTTGTGAT-3 ( ) 5 -ATTCGGACAGTTGGACCCTGAGACTTCA-3 ( ) SOCS1 (NM_003745) 5 -ACTGCTTTTTCGCCCTTAGC-3 ( ) 5 -AGCAGCTCGAAGAGGCAGTC-3 ( ) 5 -AAGATGGCCTCGGGACCCACGA-3 ( ) TNFAIP3 (NM_006290) 5 -TGCCCAGGAATGCTACAGAT-3 ( ) 5 -ACAAGTGGAACAGCTCGGATT-3 ( ) 5 -CCCATTGTTCTCGGCTATGACAGCCA-3 ( ) TOLLIP (NM_019009) 5 -ATGGACGACCGCATTGC-3 ( ) 5 -ACTTGTCCTCCACCTTGCCC-3 ( ) 5 -ACATCACCATCCCGGAGTCCCTGAG-3 ( ) IRAK1 (NM_001569) 5 -CCTCTGTGAGATTTCCCGGG-3 ( ) 5 -ACAGCATACACCGTGTTCCTCAT-3 ( ) 5 -ACCCACAACTTCTCGGAGGAGCTCAAGATC-3 ( ) IRAK2 (NM_001570) 5 -TCCCTGCAATGGATAACAACC-3 ( ) 5 -GCGGTGCTGCTTGGAATAT-3 ( ) 5 -AGCCCGGTTTACCTGAAGGACTTACTCCT-3 ( ) IRAK3 (NM_007199) 5 -TTGGTCCTGGGCACAGAAA-3 ( ) 5 -AATAGCTCGACGATGTCCCAT-3 ( ) 5 -AGACCATCGGTGACCTTTTACAGGTCCTC-3 ( ) IRAK4 (NM_016123) 5 -CAGACTCTCTTGCTTGGATGGT-3 ( ) 5 -AGCTGCACCCTGAGCAATCTT-3 ( ) 5 -CTCCACCACTTTCTTGGCACATGAGATG-3 ( ) TRAF6 (NM_145803) 5 -GGTCCGGAATTTCCAGGAAA-3 ( ) 5 -CATTTTAGCAGTCAGCTCCCG-3 ( ) 5 -ATTCACCAGTTAGAGGGTCGCCTTGTAAGA-3 ( ) CCL2 (NM_002982) 5 -TTGTGGCCAAGGAGATCTGT-3 ( ) 5 -CGGAGTTTGGGTTTGCTTGT-3 ( ) 5 -CCAAGCAGAAGTGGGTTCAGGATTCC-3 ( ) CCL5 (NM_002985) 5 -TCTACACCAGTGGCAAGTGCT-3 ( ) 5 -TCCCGAACCCATTTCTTCTC-3 ( ) 5 -CCAGCAGTCGTCTTTGTCACCCGAAA-3 ( ) CXCL10 (NM_001565) 5 -GGCATTCAAGGAGTACCTCTCTC-3 (52 74) 5 -GACAAAATTGGCTTGCAGGA-3 ( ) 5 -AGAACCGTACGCTGTACCTGCATCAGCA-3 (76 103) a) Indicates primers and probes that were sequences employed in our previous study. 18) The positions of the primers and probes were counted from the position of the initiation codon.

4 May Hokkaido, Japan). The TaqMan probes contained 6-carboxyfluorescein (FAM) at the 5 end and 6-carboxytetramethylrhodamine (TAMRA) at the 3 end and were designed to hybridize to a sequence located between the PCR primers. TaqMan RT-PCR Conditions The total RNAs obtained from adult human tissues and cell lines were diluted under the same conditions as in our previous studies. 18) The RT- PCR assay was performed using the ABI PRISM 7700 Sequence Detector system (Applied Biosystems) under the same conditions as in our previous studies. 18) Data Analysis Samples were deemed positive at any given cycle when the value of the emitted fluorescence was greater than the threshold value calculated by the instrument s software (Sequence Detector Ver ). The threshold cycle (Ct), which is defined as the cycle at which PCR amplification reaches a significant value (i.e., usually 15 times greater than the standard deviation of the baseline), is given as the mean value. The relative expression of each mrna was calculated by the DCt method (where DCt is the value obtained by subtracting the Ct value of PPIA mrna from the Ct value of the target mrna), as employed in previous studies ) Specifically, the amount of target mrna relative to PPIA mrna is expressed as 2 (DCt). Data are expressed as the ratio of target mrna to PPIA mrna. RESULTS AND DISCUSSION Table 4. Calibration Data and Limit of Quantification for Each mrna in the Total RNA Studied Limit of No. of Calibration curve Correlation quantification mrna data coefficient (pg total RNA/50 ml points Slope Intercept r reaction mixture) Toll-like receptor mrnas TLR TLR TLR TLR TLR TLR TLR TLR TLR TLR Toll-like receptor related gene mrnas ICAM CD MyD LY TRIF TICAM TIRAP CD CD CD SOCS TNFAIP TOLLIP IRAK IRAK IRAK IRAK TRAF CCL CCL CXCL The calibration curves show the threshold cycle (Ct) for analysis of total RNA per 50 ml of reaction mixture. The source of the total RNA was the adult human lung. In this study, we investigated the mrna expression levels of 10 human TLRs and 21 related genes in fetal human tissues (brain and liver), in adult human tissues (adrenal gland, brain, heart, kidney, liver, lung, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, testis, thymus, thyroid gland, trachea, and uterus), and in two cell lines (Hep G2 and HeLa). The sequence of each primer and/or probe for the 10 human TLRs was designed using a part of the mrna sequence that exhibited sufficient structural differences among the different members of the family. Furthermore, each primer and/or probe for the 10 human TLRs and 21 related genes was homology searched by an NCBI BLAST search to ensure that it was specific for the target mrna transcript. Analysis was conducted by RT-PCR using the ABI PRISM 7700 Sequence Detector System in the presence of the Taq- Man probe. Table 4 shows the calibration data and limit of quantification for each mrna in the total RNA studied. The source of the total RNA was pooled adult human lung. To prepare the calibration curve, various amounts ranging from 1.28 to pg total RNA were used. The lower limit of quantification for each mrna ranged between 1.28 pg and 800 pg of total RNA per 50 ml of reaction mixture. The upper limit of quantification for each mrna was higher than pg of total RNA per 50 ml of reaction mixture. The relative amounts of housekeeping gene mrnas in each tissue and cell line used in this study are shown in Table 5. Data are expressed as the ratio to the lowest value for each housekeeping gene. For example, the expression of b-actin mrna was 22-fold higher in Hep G2 cells than in skeletal muscle, and the expression of GAPDH mrna was 30-fold higher in skeletal muscle than in the spleen. Consistent with the findings of our previous study, 18) the expression of PPIA mrna showed the lowest variability among the 7 housekeeping genes studied. Therefore, in the present study, we considered that PPIA would be suitable as an endogenous control for the measurement of nuclear receptor mrnas. The process used to select the best housekeeping gene has been described in detail in our previous studies. 18,21) The mrna expression values for 10 human TLRs in various tissues and cell lines are shown in Table 6. TLR1 mrna was expressed at high levels in the kidney, lung, and spleen. Consistent with the findings of another study in the mouse, 13) TLR2 mrna was expressed at high levels in the lung and spleen. Furthermore, TLR2 was expressed ubiquitously, and Rock et al. 14) have also reported that TLR2 mrna is expressed in the brain, heart, and muscle. Consistent with the findings of another study, 14) TLR3 mrna was expressed at the highest levels in the placenta. Furthermore, TLR3 mrna was expressed ubiquitously, and Alexopoulou et al. 6) have also reported that TLR3 mrna is expressed in the lung, brain, and kidney in the mouse. TLR4 mrna was expressed at the highest levels in the spleen. TLR5 and TLR6 mrna were expressed ubiquitously. Similar to the findings of another study, 15) TLR7 mrna was predominantly expressed in the lung, placenta, spinal cord, and spleen, and TLR8 mrna was expressed at high levels in the lung and spleen. TLR9

5 890 Vol. 28, No. 5 Table 5. Relative Amounts of Human Housekeeping Gene mrnas in Various Tissues and Cell Lines Tissue b-actin B2M GAPDH GUSB HPRT1 PPIA TFRC Brain Liver Adrenal gland Brain Heart Kidney Liver Lung Placenta Prostate Salivary gland Skeletal muscle Small intestine Spinal cord Spleen Testis Thymus Thyroid gland Trachea Uterus Hep G HeLa Data are expressed as the ratio to the lowest value for each housekeeping gene. Total RNA at pg per 50 ml of the reaction mixture was used for each tissue. Experiments were performed in duplicate. The highest values of Ct for b-actin, B2M, GAPDH, GUSB, HPRT1, PPIA, and TFRC in various tissues and cell lines were 17.45, 20.26, 18.84, 24.26, 27.23, 21.65, and 26.31, respectively. Table 6. Expression of Human Toll-Like Receptor mrnas in Various Tissues and Cell Lines Tissue TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR7 TLR8 TLR9 TLR10 Brain Liver Adrenal gland Brain Heart Kidney Liver Lung Placenta Prostate Salivary gland Skeletal muscle Small intestine Spinal cord Spleen Testis Thymus Thyroid gland Trachea Uterus Hep G BLQ BLQ HeLa BLQ, below the limit of quantification. Data are expressed as the ratio of target mrna to PPIA mrna. Experiments were performed in duplicate. The highest values among the various tissues and cell lines are shown in boxes. mrna was expressed at high levels in skeletal muscle and the spleen, 15) and it has also been reported that TLR9 mrna is expressed in the spleen. Consistent with the findings of another study, 12) TLR10 mrna was most highly expressed in lymphoid tissues such as the spleen and thymus. Similar to the findings of another study, 22) TLR2, TLR3, TLR6, and TLR9 were consistently expressed in Hep G2 cells, but TLR1, TLR4, TLR5, TLR7, TLR8, and TLR10 showed no or

6 May Table 7. Expression of Human Toll-Like Receptor Related Gene mrnas in Various Tissues and Cell Lines Tissue ICAM1 CD14 MyD88 LY96 TRIF TICAM2 TIRAP CD80 CD83 CD86 Brain Liver Adrenal gland Brain Heart Kidney Liver Lung Placenta Prostate Salivary gland Skeletal muscle Small intestine Spinal cord Spleen Testis Thymus Thyroid gland Trachea Uterus Hep G BLQ BLQ HeLa Tissue SOCS1 TNFAIP3 TOLLIP IRAK1 IRAK2 IRAK3 IRAK4 TRAF6 CCL2 CCL5 CXCL10 Brain BLQ BLQ Liver Adrenal gland Brain BLQ Heart Kidney Liver Lung Placenta Prostate Salivary gland Skeletal muscle Small intestine Spinal cord Spleen Testis Thymus Thyroid gland Trachea Uterus Hep G BLQ BLQ BLQ HeLa BLQ BLQ, below the limit of quantification. Data are expressed as the ratio of target mrna to PPIA mrna. Experiments were performed in duplicate. The highest values among various tissues and cell lines are shown in boxes. very weak expression in these cells. The mrna expression values for 21 human TLR-related genes in various tissues and cell lines are shown in Table 7. ICAM1 mrna was widely expressed in the tissues and cell lines, and particularly high levels of ICAM1 mrna were detected in the lung. CD14, MyD88, LY96, TRIF, TICAM2, TIRAP, SOCS1, TNFAIP3, TOLLIP, and TRAF6 mrna were expressed ubiquitously. Hardiman et al. 16) and Oshiumi et al. 17) have reported that MyD88 and TICAM2 mrna are expressed in many human tissues, respectively. Kato et al. 23) have also reported that LY96 mrna is expressed in many mouse tissues. CD80, CD83, CD86, CCL2, CCL5, and CXCL10 mrna were expressed at high levels in the lung and spleen. IRAK1 mrna was expressed at high levels in the adrenal gland and skeletal muscle, with very weak expression in the other tissues and cell lines. IRAK2, IRAK3, and IRAK4 mrna were expressed ubiquitously, except for no detectable expression of IRAK3 in Hep G2 cells. The results of this study provide information concerning the tissue- and cell line-specific profiles of mrna expression of 10 human TLRs and 21 human TLR-related genes. These results provide valuable information for studies concerning

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