Human B cells immortalized with Epstein-Barr virus upregulate CCR6 and CCR10 and downregulate CXCR4 and CXCR5 - PubMed (original) (raw)

FIG. 1.

RT-PCR analysis for expression of various chemokine receptors. CD19+ B cells were purified from peripheral blood mononuclear cells by positive selection with magnetically activated cell sorting (Miltenyi Biotech, Bergisch Gladbach, Germany) after labeling with anti-CD19 microbeads. The purity of CD19+ B cells as examined by flow cytometry after staining with fluorescein isothiocyanate-labeled anti-CD19+ was >95%. By using Trizol reagent (GIBCO-BRL, Gaithersburg, Md.), total RNA was prepared from peripheral blood mononuclear cells treated with phytohemagglutinin (PHA) for 3 days, fresh peripheral blood CD19+ B cells, EBV-immortalized polyclonal B-cell lines (BCL-NU, BCL-SH, BCL-SM, and BCL-TOS), EBV-positive BL cell lines (AKATA, Daudi, Jijoye, Raji, and AG876), an EBV-negative BL cell line (Ramos), and a human non-BL B-cell line (BJAB). RNA was further purified using RNeasy (Qiagen, Hilden, Germany). Total RNA (1 μg) was reverse transcribed using oligo(dT)18 primer and SuperScript II reverse transcriptase (GIBCO-BRL). Resulting first-strand DNA (20 ng of total RNA equivalent) and original total RNA (20 ng) were amplified in a final volume of 20 μl containing 10 pmol of each primer and 1 U of Ex-Taq polymerase (Takara Shuzo, Kyoto, Japan). Amplification conditions were denaturation at 94°C for 30 s (5 min for the first cycle), annealing at 60°C for 30 s, and extension at 72°C for 30 s (5 min for the last cycle) for 33 cycles for all chemokine receptors and 27 cycles for glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Amplification products (10 μl each) were separated by electrophoresis on 2% agarose and stained with ethidium bromide. The primers used were as follows: +5′-GGCTGCTGGGGACTGTCTATGAAT-3′ and −5′-GCCCGGCCGATGTTGTTG-3′ for CXCR1, +5′-CCGCCCCATGTGAACCAGAA-3′ and −5′-AGGGCCAGGAGCAAGGACAGAC-3′ for CXCR2, +5′-CAACGCCACCCACTGCCAATACAA-3′ and −5′-CAGGCGCAAGAGCAGCATCCACA-3′ for CXCR3, +5′-ATCTTCCTGCCCACCATCTACTCCATCATC-3′ and −5′-ATCCAGACGCCAACATAGACCACCTTTTCA-3′ for CXCR4, +5′-AACTACCCGCTAACGCTGGAAATGGAC-3′ and −5′-CACGGCAAAGGGCAAGATGAAGACC-3′ for CXCR5, +5′-ATGGCAATGTCTTTAATCTCGACAA-3′ and −5′-TGAAAGCTGGTCATGGCATAGTATT-3′ for CXCR6, +5′-CAACTCCGTGCCAGAAGGTGAA-3′ and −5′-GCCAGGGCCCAAATGATGAT-3′ for CCR1, +5′-CCAACGAGAGCGGTGAAGAAGTC-3′ and −5′-TCCGCCAAAATAACCGATGTGAT-3′ for CCR2, +5′-GAGCCCGGACTGTCACTTTTG-3′ and −5′-CAGATGCTTGCTCCGCTCACAG-3′ for CCR3, +5′-AAGAAGAACAAGGCGGTGAAGATG-3′ and −5′-AGGCCCCTGCAGGTTTTGAAG-3′ for CCR4, +5′-CTGGCCATCTCTGACCTGTTTTTC-3′ and −5′-CAGCCCTGTGCCTCTTCTTCTCAT-3′ for CCR5, +5′-CCTGGGGAATATTCTGGTGGTGA-3′ and −5′-CATCGCTGCCTTGGGTGTTGTAT-3′ for CCR6, +5′-GTGCCCGCGTCCTTCTCATCAG-3′ and −5′-GGCCAGGACCACCCCATTGTAG-3′ for CCR7, +5′-GGCCCTGTCTGACCTGCTTTTT-3′ and −5′-ATGGCCTTGGTCTTGTTGTGGTT-3′ for CCR8, +5′-CACTGTCCTGACCGTCTTTGTCT-3′ and −5′-CTTCAAGCTTCCCTCTCTCCTTG-3′ for CCR9, +5′-TGCTGGATACTGCCGATCTACTG-3′ and −5′-TCTAGATTCGCAGCCCTAGTTGTC-3′ for CCR10, +5′-TGACCATCCACCGCTACC-3′ and −5′-ATCTGGGTCCGAAACAGC-3′ for XCR1, +5′-TGGCCTTGTCTGATCTGCTGTTTG-3′ and −5′-ATGGCTTTGGCTTTCTTGTGGTTC-3′ for CX3CR1, +5′-GCCAAGGTCATCCATGACAACTTTGG-3′ and −5′-GCCTGCTTCACCACCTTCTTGATGTC-3′ for G3PDH. Representative results from three independent experiments are shown. The lower panel shows the signal intensity ratio between each chemokine and G3PDH.