IL6

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IL-6 Tadashi Matsuda1 and Toshio Hirano2,* 1

Department of Immunology, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama, 930-0194, Japan 2 Division of Molecular Oncology, Biomedical Research Center, Osaka University Graduate School of Medicine, 2-2 Yamada-oka. Suita, Osaka, 565-0871, Japan * corresponding author tel: 81-6-6879-3880, fax: 81-6-6879-3889, e-mail: [email protected] DOI: 10.1006/rwcy.2000.06001.

SUMMARY Interleukin 6 (IL-6) is a pleiotropic cytokine that is produced by many different cell types and plays a role in a wide range of responses, such as immune responses, acute-phase reactions, and hematopoiesis (Sehgal et al., 1989; Heinrich et al., 1990; Hirano and Kishimoto, 1990; Van Snick, 1990; Hirano, 1992, 1998).

BACKGROUND

Discovery In early 1980, Muraguchi and Kishimoto and their colleagues (Muraguchi et al., 1981), and Teranishi and Hirano and their colleagues (Teranishi et al., 1982), independently showed that the culture supernatant fractions of stimulated human peripheral blood mononuclear cells and tonsillar mononuclear cells respectively induce immunoglobulin (Ig) production in Epstein±Barr virus (EBV)-transformed B lymphoblastoid cell lines. Kishimoto's group called this active factor TRF or BCDF (Muraguchi et al., 1981; Okada et al., 1983), and Hirano's group termed it TRF-like factor or BCDFII (Teranishi et al., 1982; Hirano et al., 1984a,b). Teranishi et al. (1982) partially purified the TRFlike factor and showed that it is recovered in the fractions corresponding to molecular weights of 22 kDa and 36 kDa, by gel filtration, and that its isoelectric point is 5±6. This active factor was further purified to homogeneity, renamed BSF-2 (Hirano

et al., 1985), and molecularly cloned in 1986 (Hirano et al., 1986). In 1980, an inducible mRNA species of about 13S coding for a novel human fibroblast-type IFN, named IFN- 2, was reported (Weissenbach et al., 1980). The 26 kDa protein without any antivirus activity, which is induced in fibroblasts upon stimulation with IL-1, was also reported (Content et al., 1985). The nucleotide sequences of the cDNAs encoding human IFN- 2 and 26 kDa protein were determined in 1986 (Haegeman et al., 1986; Zilberstein et al., 1986). A growth factor for murine hybridomas was found in the supernatant of human endothelial cells (Astaldi et al., 1980) and human monocytes (Aarden et al., 1985). A hybridoma growth factor (Van Snick et al., 1987) and plasmacytoma growth factor (Nordan et al., 1987) were purified, and their partial N-terminal amino acid sequences were determined. It was found that all these molecules were identical, and it was proposed that this molecule be referred to as interleukin 6 (IL-6) at the conference entitled `Regulation of the Acute Phase and Immune Responses: A New Cytokine', held in New York City on 12±14 December 1988 (Sehgal et al., 1989).

Alternative names IL-6 has many alternative names, such as interferon 2 (IFN- 2) (Weissenbach et al., 1980; May et al., 1986; Zilberstein et al., 1986), T cell replacing factorlike factor (Teranishi et al., 1982), B cell differentiation factor (BCDF) (Okada et al., 1983), BCDFII (Hirano et al., 1984a,b), 26 kDa protein (Haegeman

538 Tadashi Matsuda and Toshio Hirano Figure 1 Homology among human, mouse, and rat, and viral IL-6 proteins.

et al., 1986), B cell stimulatory factor 2 (BSF-2) (Hirano et al., 1985, 1986), hybridoma plasmacytoma growth factor (HPGF) (Aarden et al., 1985; Nordan and Potter, 1986; Van Damme et al., 1987a; Van Snick et al., 1988), hepatocyte stimulating factor (HSF) (Andus et al., 1987; Gauldie et al., 1987), and monocyte-granulocyte inducer type 2 (MGI-2) (Shabo et al., 1988).

Structure Human IL-6 is a protein of 186 amino acids glycosylated at positions 73 and 172 (Hirano et al., 1986; May et al., 1988; Santhanam et al., 1989). It is synthesized as a precursor protein of 212 amino acids. At least five different molecular forms of IL-6 with molecular masses from 21 to 28 kDa are expressed in monocytes. This difference is derived from posttranslational alterations such as glycosylation and phosphorylation (May et al., 1988). Human IL-6 shows homology with IL-6 from the mouse and rat to a value of 65% and 68% at the DNA level and 42% and 58% at the protein level respectively (Van Snick et al., 1988; Northemann et al., 1989). The mouse and rat protein sequences are 93% identical. The open reading frame K2 in the genome of human herpesvirus 8 (HHV-8) encodes a structural homolog of IL-6, termed viral IL-6 (vIL-6). vIL-6 protein sequence shows 62% similarity to that of human IL-6 (Neipel et al., 1997) (Figure 1).

GENE AND GENE REGULATION

Accession numbers GenBank: Human gene, 5-flanking region: M22111 Mouse gene: M20572 Rat gene: M26745 Human cDNA: X04602 Mouse cDNA: X06203 Rat cDNA: M26744 HHV-8 cDNA: U75698 Viral IL-6: AAC57089 CDS for viral IL-6: 17261±17875.

Sequence See Figure 2.

Chromosome location The human IL-6 gene has a length of approximately 5 kb and contains five exons and four introns (Yasukawa et al., 1987; Tanabe et al., 1988). It maps to human chromosome 7p21 between the markers D7S135 and D7S370 (Sehgal et al., 1986; Bowcock et al., 1988). The murine gene maps to chromosome 5 (Mock et al., 1989). The sequence similarity in the coding region of the human and mouse IL-6 genes is about 60%, where the 30

IL-6 539 untranslated region and the first 300 bp of 50 flanking region are highly (80%) conserved (Tanabe et al., 1988). The nucleotide sequences of IL-6 and G-CSF genes resemble each other in a way, suggesting a possible evolutionary relationship.

Regulatory sites and corresponding transcription factors The production of IL-6 is regulated by a variety of stimuli. IL-6 production is induced in T cells or T cell clones by T cell mitogens or antigenic stimulation (Van Snick et al., 1987; Hodgkin et al., 1988; Horii et al., 1988; Espevik et al., 1990). Lipopolysaccharide (LPS) enhances IL-6 production in monocytes and fibroblasts, whereas glucocorticoids inhibit it (Helfgott et al., 1987; Sehgal, 1992). Various viruses induce IL-6 production in fibroblasts (Sehgal et al., 1988; Van Damme et al., 1989) or in the CNS (Frei et al., 1988). HIV also induces IL-6 production (Nakajima et al., 1989; Breen et al., 1990; Emilie et al., 1990). A variety of peptide factors, such as IL-1, TNF, IL-2, IFN , and PDGF (Content et al., 1985; May et al., 1986; Wong and Goeddel, 1986; Zilberstein et al., 1986; Kohase et al., 1987; Van Damme et al., 1987a,b; Kasid et al., 1989), PKC (Sehgal et al., 1987), calcium ionophore (Sehgal et al., 1987), and various agents causing an increase in intracellular cyclic AMP level (Zhang et al., 1988a,b), also induce IL-6 production. Conversely, IL-4 and IL-13 inhibit IL-6 production in monocytes (Gibbons et al., 1990; Velde et al., 1990; Minty et al., 1993).

Several potential transcriptional control elements, such as glucocorticoid-responsive element, an activating protein 1 (AP-1)-binding site, a c-fos serum-responsive element (SRE) homology, c-fos retinoblastoma control element homology, a cAMPresponsive element and an NFB-binding site, have been identified within the conserved region of the IL6 promoter (Ray et al., 1988; Ray et al., 1989; Sehgal, 1992). Among them, c-fos SRE and AP-1-like elements appear to contain the major cis-acting regulatory elements that confer responsiveness to several reagents (including serum, forskolin, and phorbol ester) upon the heterologous herpesvirus thymidine kinase (TK) promoter. The 23 base pair oligonucleotide designated as a multiple responsive element (MRE) within the IL-6 enhancer region (173±151), which contains a CGTCA motif, binds nuclear proteins. A single copy of MRE inserted upstream of the herpesvirus TK promoter renders heterologous promoter inducible by IL-1, TNF, and serum, as well as by activators of protein kinase A (forskolin) and protein kinase C (phorbol ester). The IL-1responsive element was also mapped within the region from 180 to 111 base pairs of the IL-6 gene, and a nuclear factor, NF-IL6 CCAAT/enhancer-binding protein (C/EBP ), was identified that binds specifically to a 14 base pair palindrome (Isshiki et al., 1990; Shimizu et al., 1990; Akira et al., 1994). NF-IL6 is a transcription factor of 32 kDa belonging to a family of DNA-binding proteins characterized by a leucine zipper that mediates dimerization and a basic DNA-binding domain. NF-IL6 is also known as C/EBP (CCAAT/enhancer-binding protein ),

Figure 2 Gene and cDNA sequences for human, mouse, and rat IL-6. Human 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141

IL-6 gene sequence GGATCCTCCT GCAAGAGACA AATAAGAAAT TCTTGGGTGC GTCCGTAGTT TCCTTCTAGC CGATAAACAC AAACTCTGCA GGTGAGTAGT AATCTCCCCC TCCAGGAGAC CTGGGCATGC GGCAGTGGGG AGAGCACTCG TGGAGGATTC CCAAGGGTCA GCTGAAGCAG GTGAAGAAAT CACCTGGAGA CGCCTTGAAG CAGCCGCCTC ACAGGGAGAG TTCTTCATAA TCCCAGGCTT ATCTTTGGTT TTTACAATAC CACTGAAAAA AAAAAAATTT TCTCTTTGTA AAACTTCGTG TGAGTCACTA ATAAAGAAAA CTCAATGACG ACCTAAGCTG TCATTGCACA ATCTTAATAA CCTCCAACAA AGATTTATCA ACCCCCAATA AATATAGGAC

CCATCCTGAG CGACGGGGAC TTCTTTTTGA AGATGCCACA TTTCTGCCCT AGGTGCCGAT CAGCACAGGC CTTGGGAGAG GGCAGAAGAC TAACTGCACG CCAGAACACA GGGGGGCTGC AAATTAACTG TTTTTTTTTC CATGACTTCA AAGAAGTAAA CACTTTTCCC GGTTTCCAAT AATGTGGGAT TGGAGATGTC

GGGAAGAGGG AGCAGATTCA TTTCAAATCA AGGTCCTCCT GAACCAAGTG GAAACAGTGG AAACCTCTGG GGCAGGCAGC GCGGTGGTGG AAATTTGAGG GCAAGAACTC GATGGAGTCA GAACGCTAAA AAAAAACATA GCTTTACTCT GGAAGAGTGG CCTAGTTGTG CAGCCCCACC TTTCCCATGA TCTGAGGCTC

CTTCTGAACC GAGCCTAGAG AGACTTACAG TTGACATCCC GCTTCAGTAA TGAAGAGACT CACAAGAGCA AGCCAACCTC CAAAAAGGAG GTGGCCAGGC AGATGACTGG GAGGAAACTC TTCTAGCCTG GCTTTAGCTT TGTCAAGACA TTCTGCTTCT TCTTGCGATG CGCTCTGGCC GTCTCAATAT ATTCTGCCCT

AGCTTGACCC CCGTGCCTGC GGAGAGGGAG CAACAAAGAA GTTTCAGGGC CAGTGGCAGT AAGTCCTCAC CTCTAAGTGG TCACACACTC AGTTCTACAA TAGTATTACC AGTTCAGAAC TTAATCTGGT ATTTTTTTTT TGCCAAGTGC TAGCGCTAGC CTAAAGGACG CCACCCTCAC TAGAGTCTCA CGAG

Figure 2 (Continued ) Mouse IL-6 gene 1 GGATCCTGAG 61 GCCTGGAATC 121 ACTAGTCTGA 181 GAAGACTTGA 241 CTGTCATCCA 301 TTCGATATCT 361 CAACAGACCT 421 GCAGCAGTGG 481 CTAAGAAGAT 541 CCAAGATTGC 601 GCAACTCCTG 661 TCCTGACAAG 721 GGTGCTGGGG 781 AACATCTGTA 841 GTGTGTGTGT 901 GCGCGTGCCT 961 TGAATTTCAG 1021 AAAAGAAGAG 1081 CCTTCCTAGT 1141 TTTCCAATCA 1201 AATGTGGGAT 1261 TGGGGATGTC 1321 AACCGCTATG 1381 GTATTGAGAC 1441 GCATCAGCTA 1501 CCACGCAGGA 1561 CTTCCCTACT 1621 TGTCTATACC 1681 AATGAGAAAA 1741 TTCTCTTTGC 1801 CTAAAGGTCA 1861 TTGGGAAGGG 1921 TCACAGTGGG 1981 CCCTCATTAT 2041 ACCAGAAAGT 2101 TCTAGGGTCA 2161 CACACACACA 2221 CAGCACTTGG 2281 TTGAGTTCCA 2341 AAAAAAAAAC 2401 TACCTAGATT 2461 AAATCACAAA 2521 GGAAAATCCT 2581 TGGCACAGAC 2641 CATCAAAATT 2701 ATCCTTGAGA 2761 TTAGGAGAGT 2821 CTTAGTAGGG 2881 GACTTGGCCC 2941 TTGCAGTTGT 3001 AATCTGAAAC 3061 GTAGAAACTT 3121 CTGCATTTGT 3181 TGTAACTGGC 3241 TAGGGTGAGG 3301 GGAGCTAAAT 3361 AAAAAACATT 3421 GTCATGAGGA 3481 CCCACTATGC 3541 GTTCATGAGC 3601 GAGAACACTC 3661 GACAAGTCAG 3721 AGGGTCAGAA 3781 GTGCCTCTCT 3841 TAGAAGATAC 3901 TACATTTCAA 3961 TCCCCGCTCC 4021 ATATAAAGTT 4081 CTGCTACATA 4141 TCCAACCTAT 4201 TGGGGGCCCT 4261 CTAGCCTCAC 4321 AATACTGTCG 4381 ATCTTCCATC 4441 TTGTTCCCCA 4501 TTCTTGTGTT

sequence AGTGTGTTTT ATTCTGAATG AAAAGAAACT GCATTGGAGG GTAGAAGGGA TTATCTTCCA TCAAGCCTCC GATCAGCACT AGCCAAGAGA TTGACAACAG GAAACAACTG ACACAGGAAA GTGGGAGAGG GATCCTTACA GTGTGTGTGT GCGTTTAAAT TTTTCTTTCC TGCTCATGCT TGTGATTCTT GCCCCACCCA TTTCCCATGA TGTAGCTCAT AAGTTCCTCT TGTGAGAGAG GCAGCAGGTC GACTTCCATC TCACAAGTCC ACTTCACAAG GAGGTGGGTA TCTTGAATTA GACTAGACGT GTTCCTTTCC CCATTCTCTG TCCTTTTTCC GCTTTTTGGC GCCCAGATAA CACACACACA GCAGCACGCA GGATATCCAA AACAACAACA AAAATATCTC CAAGATTAAT CAACTATTTA CCTTCCAGAT TCTGTCCTTT CAAATGTATA AACAAGCTTA TTCTAAGAAG TCTAGGGTGC GCAATGGCAA TTCCAGAGAT GTCACGTTTA ACAGTCCCAG CTGCAGTAGC GAAAGTTTAC GTTTAAAGTC TTTTTTGTCT AAGTAACTTA TGGGATTCTT CATCTCTTCT TTCACACCCC ACAAGAACAA GTGGATTCCC CTACTTGTCC TGAACAGCAG ATGCTATCCC CTTACCCACC TGCAAGACCA TGCAGCTAGA AGGGTTGCAG GTGTTCCATC ACGAGACAGC TGGTTTGGTG CTTTTGTCTT TTCTAAGGAG TTGGAAATTG

GTAAATGGTT CTAGCTAGAT AACCAAAGGG GGTTATTCAG GCTTCAAACA TATACCATGA TTGCATGACC AACAGATAAG CCACTGGGGA ACAGAAGATA CACAAAATTT AACAAGCAAT GAGTGTGTGT GACATACAAA GTGTGTGTGT AACATCAGCT CATCAAGACA TCTTAGGGCT TCGATGCTAA CTCTGGCCCC GTCTCAAAAT TCTGCTCTGG CTGCAAGTAA GAGTGTGAGG CAACTGTGCT CAGTTGCCTT GGAGAGGAGA TCGGAGGCTT GGCTGTGAAA GAAATTCTCT GTTCTCTCTT TGTCTGGAAG TTCCAATTTA TTATCTCTTT TGAATGTAGT GATGCATATT CACAACCTAG GGAAGATCTC GACTACATTG AACAAACAAA CATCTTTAAA AGCCTAGGTC GTACTCTTTA GGCAATATCC ACCAAGTTCC AGAGGGACAT AGGGACTGAG GTATAGCTTC TTGTTGTAAG TTCTGATTGT ACAAAGAAAT CTTTCAACAA TCAGGCAACA TTGAATACAA CAACCAGTGC TCTATTCTCA AAAAAAGTGG CAGATTTAAT CACCACTGTT CTGGCTGCCC TCTCCTTCCA GAGTACAGGA ACTGCCACAC AGATAATTCA GAATCTTTTT CACAGTCCCC AACTCCCACT AGGGGCCTCT GATATGAGCT ACCCCTTCAG CAATAGAAGA TATATCAGGG GCTGATTATG AGCTCCAAAC GAATGAAGTA TACCTTGGGT

TTGGATTTTA ATCTGGAGAC AAGAAGTCTG AGTGAGACGT CAAGCTAGCT ATCAAAGAAA TGGAAATTGG GGCAACTCTC GAATGCAGAG TTTCTGTACT GGAGGTGAAC ATGCAACATT CTTTGTATGA AGAATCCTAG GTGTGTGTAT TTACGTTCTC TGCTCAAGTG AGCCTCAAGG ACGACGTCAC ACCCCCACCC TAGAGAGTTG AGCCCACCAA GTGAAGGCAG CAGAGAGCCA ATCTGCTCAC CTTGGGACTG CTTCACAGAG AATTACACAT CTGATGAAGA GCTGGGATCT TCTCACCTCT ATACAGAATG GCTATTGCTT GCCTTCATTT AAGTCCTGTG GTATAGAGCC ATTAGGCTGG TGTGAGTTAG AGAGATCCTT CAAACAAGAA TGAAAGTATT TGGGTGTGTA TCTGCATAAT TGTGACTTAA TACTCTATGC CAATAATTCT CCTAAGGGTG CCTTTCTGTG AGGTGTTTAA ATGAACAACG GATGGATGCT TTCTTCAAGG AAGATGGGGG AAGGAGATTG TATAAATGTT TCTTGGCTTT AAGAGTAGAT TAAAAAGCAG AAATGTTAGA CTGGAAGGAT GCCGGTGAGA AGGGCACTGC TCTGGGGAGG GGAGCTGATG TTCAATTTTT TATCCCTATG TCTTGGCCCT ATTCCCAATG CTGGGGGTAC CTCCTTGGGT CTGTGAGCAT TCCTTTCAGC GGATGGATCC TTTGTCTCTG TCCACATGTT ATTCTAAGTT

TGTACAGAGC AGGTGGACAG TTTAAGTTTG ACCACCTTCA AAGATACAAT CTTCAACAAC GTGTTTTGGG ACAGAGACTA AATAGGCTTG TCACCCACTT AAACCATTAG ACTGTCTGTT TCTGAAAAAA CCTCTTATTC GTGTGTGTCG TTTCTCCTTA CTGAGTCACT ATGACTTAAG ATTGTGCAAT TCCAACAAAG ACTCCTAATA GAACGATAGT TTCCTTGCCC GCATTGTGGG TTGCCGGTTT ATGCTGGTGA GATACCACTC GTTCTCTGGG CCCAGTGTGG AGGGCCCTTA TTGCTGGTTT TGACTGCATT CTTAGGTGGG ATCCCTTGAA TGTGTGTGAA CAATAAAGTG GCATGGTGGT AGGCCAGCTT CTCAAAAAAA AAAAAAAACC TGACTCTGCT GAACCAACTT TATGGAGTTA ATTTATAAGC TGGGCAGTTT TTATGAAACA CATTTTTATT TCCTGGCTAT GTGATTGCTC ATGATGCACT ACCAAACTGG TTCTCTCTTG GATACATCAA AGAGGTACCT CACTCAATCT GAGGTGTTAG ATAAAACATG TTTATTTAAA AGTCATAGCA CCAGGCCTAC TAGTCTACAT AGGATAGAAG GGGTGGGGGG CTGCCTGTTG TATTAGATAT CCCCTATACC GGCATTCCCT ATGGCTGACT TGGTTAGTTC ACTTTCTCTA CCACTTTTTG AAAATCTTGC CTAGGTGGGG TAACTCCTTC GGTCTTCCTT TCTGGGCTAA

CTACTTTCAA AAAACCAGGA ACCCAGCCTA GATTCAAATC GAGGTCCTTC ATGAGGACTG GTGTCGGGCA AAGGTCTTAA GACTTGGAAG TACCCACCTG AAACAACTGG GTCCAGGTTG CTCAGGTCAG ATGTGTGTGT TCTGTCATGC TAAAACATTG TTTAAAGAAA CACACTTTTC CTTAATAAGG ATTTTTATCA AATATGAGAC CAATTCCAGA TCTGGCGGAC TTGGCCAGCA TTCCTTTTCT CAACCACGGC CCAACAGACC AAATCGTGGA CGTCCATTCA GGATTTGAAG TGAGTGGAGG TCTAGAAAAT GATTCCTTTT GTCTACAAGG ATCAGGATGC CTAACACACA GGATGCCTCC GGTCTACATA AAAAAAAAAA CAACCAACCC GACAGAAGAA TGTAATCTTG AAACAGATAA CCCAAGATAT TTATCGTTGA TAGGAAATAC TATTGGGCTG GAAAGAGCAT TGGTAATCTT TGCAGAAAAC ATATAATCAG CACGTGATAC TACCACTTGA CAGCAGCTGC ATGTCCATCA ATGATTAAAA TTATGTAAAC CAGGTAAAGG AGTTTAGCTA ACTCCCTACT CAGGTCTCAG GGAAGGAGTG ATATTAAGTG TCTAATTCTG TTTCTTCATT CTCCCCCCCC GTACTGGGGC AGGCCATCTT ATATTGTTGT GCTCCTCCAT CCAGCATTGC TGGTATATGC TAGTCTCTGG CATGGGTATT CTTCTTGATT TAATATCCAC

IL-6 541 Figure 2 (Continued ) 4561 4621 4681 4741 4801 4861 4921 4981 5041 5101 5161 5221 5281 5341 5401 5461 5521 5581 5641 5701 5761 5821 5881 5941 6001 6061 6121 6181 6241 6301 6361 6421 6481 6541 6601 6661 6721 6781 6841 6901 6961 7021 7081 7141 7201 7261 7321 7381 7441 7501 7561 7621 7681 7741 7801 7861 7921 7981 8041 8101 8161 8221

TTATCAGTGA TACTCTCCAG AATAGTACTC CTGGGTTCTT ATGTCCTTAT TTCCGGTAGT TACAAGCTTG GCATCTGCTG CAGGGTTGTT CTTCTCAGCC TAATGGGGTT TGGATATTAG GCCTTTTTGT TCCATTTGTC CCCCTGTTCC CTCTGGTTTT GAATGGATCA AGAAGTCCTG GATGTCCTTT AAAACTAGTT AACCACCAGT TTAGCTCCCT ATTGTGTTCC CAATTGCTCT ATTCCTAGAA TTTCCTCTGG ATAACAAGAA TCAACCAAGA CCTCAGGGTA GGCCTTGCTT TACCTGCTTT GACAGCCATA ATCCTTGGCA CTAAAATGTC TAGATATGAG TGGAATGAAA GAGAGTTTTT TCCCAATTTA AGGTCTAGGA ATATAGTATT TGGGCCCATG AGAGTCTGAA ACTGATGGGA AGACAAATAA GTAAGGTTAG GAGTTGTTAG GTGAGTCAAA CAGCCCAGAA TTCCTCCTTT CTCCTAACAG ATCTTGAAAT TGCGTTATGC TCCTGTTGTC GGGACACTAT ATTTATGATT TTTGAAATGA TTGGAATGTA TGTGATGTAT AAACCAGTGA CCTAAGTTAC AAAGTTAAAT CTGGACTGAG

GTGCATATCA ATACATCCAT CATTTGTAAA TCCAGCTTCT TACCAGTTGG ACTATGTCTA CAATCCCAAC TCACCTGAAT TTGATTTGCA ATTCAGTATT ATTTGAATTT TCCCCTATCA CTTTATTGAC GATTCTCGAT CATATCTACG ATGTGGAGTT ATTCGGGCTG AGTTCAAATC TCTGGTGTGT ATTTAAAAAA TGAGCCCGCA TGTCAAAGAT ATTGACTACC GTAGTTTTTT AGAACTGACT TCTTCTGGAG AGACAAAGCC GGTGAGTGCT GCGGCACTTT TGTTTTACTT AAACTGTATT CATAGTCACC AGTGACATTT AGGGAAGGAG ATAATGACTC CCCTCTTGCT GCTAAGGCTG ATGTGCACAG AGGGAACTAA CAGGCTCCTA GAAGGGTGTT TGGAAACCAC GCTTCTGTTT GATGGTGGTG AATTCTGTTG GCATGGGTCT GCAGATGGAC CACGCCACAA AGGTAAAAGA ATAAGCTGGA CACTTGAAGA CCTAAGCATA AGGTATCTGA TTTAATTATT GATATTTATT TAACCTAAAA TAAGTTACCT TTTTATAATG CTGAAAGACG ATCCAAACAT AAGAAGTGAA AGTAAGGGAT

AGTGACTTAT TTGACCAAGA TGTACCACAT GGCTATTATG AACATTCTGC ATTTTTTGAG AGCAATGGAG TTTTGATCTT TTTCCCTGAT CCTCAGTTGA GAATTTCTGG GATTTAGGAT AGTGTCTTTT TTTACTGTAC AGGGTTTTTT CTTTGATCCA GAGAGATGGC CCAGCAACAT CTGAAGACAG AAAAAAAAAG CCAATTGTTG CAAGTGACCA TGTCTGTTGC TTTTTTTTGA TCCTTTTCCA TACCATAGCT AGAGTCCTTC TCCCCATCTC TTCCAGACAG TGGGGTTTTG AATAGAATGT CATTATGAGA TTGTAACCAG GGATCAGGGC AGAGTGTGGG AAGGCTGCTT CTTTTGCCAC GAACCACTTA GATTCATTTT AGTAGTGGGC TCCAGACTTC GAAGGAACAC TCCAGTAGAT CTGACCTCTG CTATTAAAAA CTCTCGAGTA TTAGCTCGTC GAAAAAAAAA TTTACATAAA GTCACAGAAG ATTTCTAAAA TCAGTTTGTG CTTATGTTGT TTTAATTTAT ATTTTTATGA ATCTATTTGA CAATGAATTG TTTAGACTGT CATCTCAGCT CCTCCCCCAA AGCTGCACAT CTAACTAAGC

TTTGTGATTG ATTTCATAAA TTTCTGTATC AATAAGGCTG GTATATGCCC GAACAGCCAG GAGTGTTCCT AGCCATTCTG GATTAAGGAT GAATTCTTTG AGTTCAGCTT TGGTAAAAAT CCCTTACAGA AAGCCATTGC TCCCACTTTC CTTAGACTTG TCAGTGGTTA GGTGGTTCAT CTACAGTGTA AATGGATCAA AAAATGCTCT TAAGTGTGTG TGTATCAATA CAGCAGGAAT TTTACTTATA ACCTGGAGTA AGAGAGATAC TCATGCAGTG CTGCTCAGAA TTTGAGGTTC TACTAATTGT GCACAGAGAC AGTTCTAATG TTCTGGCTAC CGAACAAAGC TTGCCACTTG TTGTAGTTTC GGGTCTTGTT TGTAAACAGC AAGCCTTCCA TTCATGCTAC ATTTGTTTTA ACAGTATGTT GACGCTTACT ACTAATAATT AGCTTGGAAC TCATTCATTC AATGTGCAAT ATAGTCCTTC GAGTGGCTAA GTCACTTTGA GACATTCCTC TCTCTACGAA TGATAATTTA AGTGTCACTT TATAAATATT CTAATTTAAA CTTCAAACAA GGTAAAGTTC ATCAATAATT TAGTTAATTT CGCCTTTG

GGTTACCTCA TCCATTGTTT CATTCCTCTG CTATGAACAT AGAAGAGGTA ACTGATTTCC CTTTCTCCAC ACTGGTGTGA GTTGAACATT TTTAGCTTTG CTTGAGCTCT CCTTTCCCAA AGCTTTGCAA TGTTCTGTTC TCCTCTATAA AGCTTTGTAC AGAGCACTGA AACCATCTAT CTTACATGTA TTCACACTCT CTTTTTCCCA GGTTCATTTC CCATGCAGTT CTTATTCTCA GGAAATTTGC CATGAAGAAC AGAAACTCTA TGGGAAAGAG GGGAGAGAGT TCTTTTGCAA GTAAGAGGTA AAAAGTGACT CAGAGAAGTT ATTAGCCAGA CAGATGCAAT TAGTTTCTTG TTGCCTTAAA CCAGCAGGGT TGCTTGTTTA GTTAGTCTTC CACACTAAGA GATTCCTCTG AGCATGGATG CTCTAGTGGC AATCACCTTG AAAGCTTCTC TAAATTAGAA ATTTAACCAG CTACCCCAAT GGACCAAGAC GATCTACTCG ACTGTGGTCA GAACTGACAA AATAAGTAAA GAAATGTTAT CTGTTACTAG TATGTTTTTA ATAAATTATA TTACCCAACA AAGCACTTTT CAGGTCTTGT

CTAAGGATCA TTAATAGCTG TTGAGGACAT AGTGGAGCAT TTGCTGGATC AGAGTGGTTA ATCCTTACCA GGTGGAATCT TTTTCAAGTG TACACATTTT TTGTATATAT TCTGTTGGTG TTTTATGAGG AGGAATTTTT ATTTCAGTGT AAGGAGATAA CTGCTCTTTC AATAAGATCT ATAAATAAAT TCTACATGAT CTGGATGGTT TGTGTCTTCA TTTTTTATCA AATTGAATCT CTATTGAAAA AACTTAAAAG ATTCATATCT ACACCCGGCA CTGAACAACA AGAACATCAA TGAAAACTAT TTAATATTTA TAGCCAAAAG AGAAGAATGG AAGAAGGGCC CCTTAAACCA CCAGAGAGTT CTTACTTAGG TGACCCTGCT CCCATCGCAG GACTCTCAAC TGCCACCTTT CTTGGATAAC AGACAGAACA AAAAAGAATG CCTGGCTTGG CTTCTTCCCA TCTTTGTTTT TTCCAATGCT CATCCAATTC GCAAACCTAG GAAAATATAT TATGAATGTT CTTTAAGTTA ATGTTATAGT CAGATGGTTC AAGAAATCTT TTATATTTAA TGAGCAAGGT TATGACATGT ACATTCTTTT

Exon 1: 1328–1346, Exon 2: 1509–1693, Exon 3: 2947–3060, Exon 4: 6042–6191, Exon 5: 7453–7620

TCF-5 (transcription factor 5), LAP (liver activator protein), SF-B (silencer factor B), and IL-6D-BP (IL6-dependent DNA-binding protein). The expression of NF-IL6 is induced by IL-6. NF-IL6-deficient mice

are highly susceptible to infection by Listeria monocytogenes, with macrophages containing a large number of pathogens in the cytoplasm. The tumor cytotoxicity of macrophages is severely impaired.

Figure 2 (Continued ) Rat IL-6 gene sequence 1 AATTCTTCAA ATGGGAAACC 61 GTTTAGAGTT TAGAGTTTAG 121 CTCCCATAAA CAAATGCCAA 181 TACATACAAA GGAACAAGAC 241 AAGCTCTGGG GTTGTCAAGG 301 GATGATATGT ATAGACTGTT 361 AAGTCTAGGA GTAAAGCACA 421 CTTTGTCTGC GTGTGTGAAT 481 GGCATCAGAT ACCCAGGAAC 541 ACCCACATCC TCTTGAAAGA 601 CTCATAATGA ATTTTCAATA 661 AGACTAACTT CATCCTTGGT 721 GGTCCCAGGT GAACTGATAG 781 TAAAAATAGT AGAGTTGGTG 841 TGACCTAGTA TTATTTTCCT 901 TTTAGCCACT AATAAACAGC 961 TCAAAAGGCT ATCGTCTCCA 1021 TATAGACATC ATCCAGACCA 1081 AGAGAGATAT ATGTGTGAAA 1141 GATGAAGCTG ACAAGAGTTC 1201 TTACTTTCCT CTGTTGTCTT 1261 GAGCATTTTT TTAAAAATTG 1321 GCTTATTGAG TCATTTAAAA 1381 TGAGAGAACA CATGGCAGGG 1441 AGCTGCCATT TTAAGGTCCA 1501 TTTCCATTTT CAACTACAAT 1561 AAGAATGGGA AACCTCACTT 1621 TCCACCCATA TTTTAACTGT 1681 GCAAAGTGAA GAAACTGATT 1741 TATACAGAGC CTACTTTCAG 1801 GGTGGACAGA AAACCAGGGA 1861 TTAAGCTTGA CCCAGCCTAG 1921 CCACCTTTAG ATTCAAATCC 1981 TACAACGAGG TCCTTCTTTG 2041 CAAGTCTCCT TGCATGACCT 2101 ACTAACAAAT AAGGCAACTC 2161 AGACCACTAG GGAGAAATCA 2221 ATGACAGAAG ATATTTCTGT 2281 AACTGCACAA AATTTGGAGG 2341 GGAAGAACAA GCAATATGCA 2401 TGGGGGTGGA GTGGGAGTGG 2461 AACATCTTTA GATTCTCACA 2521 AATTCATGAG TGTTTGTGTG 2581 TTCTCCTTAT AAAACATTGT 2641 GTCACTTTTA AAGAAAGAAA 2701 TTAAACACAC TTTCCCCCTC 2761 GCAATCTTAA TAAGGTTTCC 2821 CAAAGATTTT TATCAAATGT 2881 CAATAAATAT GAGACTGGGG 2941 AAGTCAACTC CATCTGCCCT 3001 GGCAGTTTCT CGCCCTCTGG 3061 CCAGCCAAGT GGGTTGGCTA 3121 TCTCATTTGC TTCTTTCCCT 3181 GACTGATGTT GTTGACAGCC 3241 CAGAGGATAC CACCCACAAC 3301 CATATGTTCT CAGGGAGATC 3361 GACGGCCCAA TGTGGGCATC 3421 GGTTCTAGAG CCCTTTGGAT 3481 TCTTCTCTTT GCTGGTTTTG 3541 GATACAGAAT GTGAACTGCA 3601 TAGTTGTTGT TTCTTGGGTA 3661 CTTCATTTGT CCTTTGAACT 3721 GCTGTGTGTG TGTGTGTGTG 3781 GGATGCTCCA GGGTCAGCCC 3841 CATAAAAAAA AATAGATAAG 3901 ACAAGCAGGA AGATTTTTGT 3961 TACCCAGGAC TACATTGGGA 4021 ACCCAAAACC AACCAACCAA 4081 CATCTTTAAA TGCAAAGTAT 4141 AGATTAATAG CCTATTCTCT 4201 ATTCCCAATT TATTTAGTAC 4261 GATGGAATAT TCTCTGACTT 4321 TTACTAAGCT CCTACTCTAT 4381 AAGAGGGACT TCAATAAGTC 4441 CTTGTAGTCA AAAGAGAGCC 4501 CTTAGTAAGG TTCTAAGAAG

TGTCAAGATA AGTTACTGTG TCAAAGGCAG AGAATCACCC CCATTTATGC AATTCTCCTA GCTGGCTAAT GTGCGTCACG AAGAGTTACA GCAATAGATG GCACCACCAA ACAGTAAGCT TTATGCCAGA CTTACTCTTT GTTAAAGAGT TAGCAAATGG CATGTACCCT TTAACATACA TGTATATGTA CACCGAAACT GTTTGAACAT AAAACAGAAA ATTGTTAAGT AGTAGGGGAA AGGGCACTAA CCCCTAGAAC CTTCAATAGG CCCAGCAACA TCGATGCTAA CCTGTAATCA CTAATCTGTA AAGTCTCGGA TGTCACCCAG CTGTAAACCC GGAAATGTTT TATTACAGAA GAAAGTGGAC TCTTCACTCC TGAACAAACC GCAGGCATCA GAGTGTGTGT GACATGAAAA TGCGCACATG GCATTTCAGT AAGAGTGATC CTAGCTGTGA AATCAGCCCC GGGATTTTCC ATGTCTGTAG TCAGGAACAG CGGAGCTATT GCAGCCAGCA TTCCTCCACG ACTGCCTTCC AGACCAGTAT TTGGAAATGA CATTCATTCT TTGAAGCTAA GGTGGGAGTT TTTCTAGAAA GGGATTCCTT CTACAAGGAC TGTGTGTGTG AGCTAAGATG GCTGGGCATA GAGTTAGAGG GATCCTTTCT CCAACCAACC TTTGACTCCG AGGTCTGGGT TCTAATTATG AAATTTATTT GCTGGGCAGT TTTATTTAAA TGGGACTGAG GTGTAGCTTC

CAGAGAAAAG GTAATATTTT TGGTGCTAAT TGCCTTCCAA TTGAGAATTA TAAAAAAAAC TAGTGAGTAA TATATTCAGT GATGATTTAG ACCTTAACGT TCTCATTTCT GGTTGTTCTC AAAGCTCCCT CTTAGTTATC AAATAAACAA TGGCATTGGA GCCCCCACCC GTGTGTATCT TAAATATATG ACTGGGGTTT TTTATCATGG TTTGAATGTT GGAGAAAAGA GTAGAGACAT AAGTAAAACC CTATGTGAGG CAGTTTCTCG GTCATTGCTT GAGTCTGATT TTTTGAGTGC AAATAAATTG CATTAGAAGG TTAAAGGGAG TGAATAAACG TGGGGATGTT ACTAAAAGTC TTGGACTTGA ATAGTAGCCC ATTAGAAGCA CTACTGTCTG CTTTGTATGA AATTAGAAGA TGTTTAAATA TTTTCCCCCT AGGCTTCTTA TTCTTTGGAT ACCCACTCTG CATGAGTCTC CTCATTCTGT CTATGAAGTT GAGACTGTGA GCCAGCATCA CAGGAGACTT CTACTTCACA ATACCACTTC GAAAAGAGGT CTTTGCTCCT AGATCAGACT GTGTGTGTGT ATTCACAGTG TTTCTTTATC CAGAAAGTGC TGTGTGTGTG CATATTGTAT ATGGTGGATA AAAGCTCAGT CAAAAACAAA AACCAACCCT CTTGACAGAA GTGTAGAATC GAGTTAGAGA GCCCCAAAAT TTTATCATCG CATAGGCAAT CCTAAGGGAC CCTTTTCTTA

AATGTATAAT GGTTTAAGAA AAAGATCCAT ACCTTGAATA TGCTCAGAAA TTAGCAGAAT GGGACCTAGT GCCAACTGAG AACCACGATG CCAAGTCATC TTAGGGATAA CATAGTAACT GAGGGAGAAG TTTACGGCAG GAACACAGAT CTTCTAACCC CCCACCTCCA CTATGTATAA AGCTGAGAAA AGGCACCACT ATGCATGCCT GTAGTTAGTC AACTCTATAC ACTGAGTTCC ATCCTGCTTT ACTAGTGCCT TTTATGTACA TCAGAGTCTT TGTAAATAGT TATCTAGGTA AACAAAGGGA GTCATTCTGA CTTCAAACAT TAAGGACTGC CTGGCAGCAG TTACTAGTAA GAGGAAACCA ACCTGGCAAC GCTGGTCCTG TGTGACTCCA TCTGAAGAAA GTAAATCCTG ACATCAGCTT ATCAAGTGCT AGGATAGCCT GCTAAATGAC GCCCCACCCC AAAAGTAGAG CTCGAGCCCA TCTCTCCGCA GAGGAGTGTG GGCGCCCAGC CCAGCCAGTT AGTCCGGAGA ACAAGTCGGA GGGTAGGCTG GAATTGGGAA AGACTTGTAT GGTTCCTTCC GACCATTCTC AAAGAGTAAG TTTTTGGCTG TGTGTGTGTG AGAGCCTAAT CCTCCCAGCA CTACATATTG ACAAAATAAG ACCTAGATTA AGAAAAAAAA AACTTTTTAA TGATGGCACA ATCATCACAA AATCGTTGAA ACCTAAGTAA CCTTTTTATT ATGTGTCCTG

GGTAGATAGA GAGTGTTTTT GCTGGTGACA CTAGAAACTT CTTGAAGGAT ACTTTGGGAA TTGAGTATGG GCTATAAGAG TAGAAACTGA TGTTCAGCTC AATGGTTCAG TCACAAGCAA TTCCAGCTCT CTCTGTATGA CTTGGGCTAA AAATTATTAC TGAAAAGCGA ACATGCATTT ACCTCTTCCA TGTAGGACCA CCTACTTAAA TAACAGTCAA CTTAGAGACC AGGCTATTTC CTCTCCCCCA CACACAGTGA CAAACAAGAG AAGGCAATCT TTTGGGTTTA TCTGGAGACA AGAAGTCTGT GTGAGACATG CTAGCTACGA AACAAACCTT TGGGATCAGC GATGGCCAAG GGAGTGCTTG TCCTGGAAAC ATAACATGCA GGTCAGGTGC CTTAGGTCAG GCCTCTTATT TAGCTTCACT CAAGTGCTGA CAAGGATGAC GTCACATTGT CACCCTCCAA AGTCGACTCC CCAGGAACGA AGTAAGTGAA AGGCAGGGAG TGGGCTATTT GCCTTCTTGG GGAGACTTCA GGCTTAATTA TGAAAGTGAT TTCTCTGCTG TCTCTCTCTC CTGTCTGGAA AGTTCCAATT ATCTCTTGGC AATGTAGTAA TGTGTAATCA AAAGTACTAA CTTGGGAGGC AGTTCCAGGA AAAACAAAAA AAATATCTCA AACACAGTCA TCTTGGGAAG GACCCTTCCA TTTCTGTCCT ATAAATGTAT GAGTACAAAG TATTGGGCTG GCTATGGAAG

Figure 2 (Continued ) 4561 4621 4681 4741 4801 4861 4921 4981 5041 5101 5161 5221 5281 5341 5401 5461 5521 5581 5641 5701 5761 5821 5881 5941 6001 6061 6121 6181 6241 6301 6361 6421 6481 6541 6601 6661 6721 6781 6841 6901 6961 7021 7081 7141 7201 7261 7321 7381 7441 7501 7561 7621 7681 7741 7801 7861 7921 7981 8041 8101 8161 8221 8281 8341 8401 8461 8521 8581 8641 8701 8761 8821 8881 8941 9001 9061 9121 9181

AGCATGACTT ATCTTTTGCA AAAACAATCT ACCAGGTAGG ATTGATACCT ACCACTTGGA CAGTGGCTGC ATGTTTCAAG TTTTCTCTAA GTAACTTACA TTAAATGTTT TTCTCTGGTT CCCCTCTCCT CTCAGGTCAG GAGTCGGGTC TGGTGCCTTT CAATTCTGTA GAAAGAACTG CTGGTCTTCT AGAAAGACAA AAGAGGTGAG CCCAGCATCT CAACAGTTCT TCTTTTGCAA CTTAAGAGAT CCATTATGAG TTTTGTTACC GGGATCAAGG TCAGAGTGTG TAAGGCTGCT GTGCACAGAA GGAACTGAGA CTCCTAAGTC TGTTTCCATC AGGTGAAGGA TTAACATGGA TGGCAGACAG TTGAAAAAAA AAGTACCTAC ATTCTAAATT TTAACCAGTC ACCCCAACTT ACCAAGACCA TCTACTCGGC GTGGTCAGAA TGGCAATATG AGTAAACTAT TATTATGTTA CCTAGCCAGA TTTTAAAAAA TTATCCCATT CCCACCCTCT TCTCCACCCT CCTCCCATTG ATCCCTGTGT TGATATTGTG ACTCCTCCAT ATTTGTAAAG ACTTCTTTCA GGGGCAGTCT TTTCCTCCTG TCCTTCTTCT GCTAATATCT TGGGACAGTG AGTTTCACCT GTGACTATCA ACAGGTCACA CATAGCCAAT CCCACTCTGA CTTGAAACCA CTTTGCCTAC GCAACTGCCA ACCAGCTGCA CCCACTCCAT AGAACTTGAT TCTGGATCCC GCAGCAGCAT CGCTGTGGTG

GGCCCTCTAG GTTGTGCAAT GAAACTTCCA AACTTGTCAC GCATATATAC TGTAACTGGC TAGGGTGAGG TCTTTGTTCT AAAAATGAAA GCTTTAATTA AGCAAGTTTA GCCCCTGGAA TCCAGCCAGT CCCAGATAAG AGAAGTAGAT CTCTACTTGT GAAAATGCTG ATATCTCCTT GGAGTTCCGT AGCCAGAGTC TGCGTCCCCA CAGGGTAGCA TTGACTGTAA AGACTGTCAA AAGAAAGCCT AACATAGAGC ACAGACCTAA GCTTCTGGCT GATGAGTCAA TCTGCCACTT ACCACCCAGG TTCATTTTTG ATGGGGCAAG ATTCTTCATG ATGCATTTGA TATTTGGATA AACAGCAAAG AAGAATGGAG CTCCTCTAAC AGAACTTCCC TTTCTTTTTT CCAATGCTCT TCCAACTCAT AAACCTAGTG AATATATCCT AATGTTGAAA AAGTTAATTT TAGTTTTGAA TGGTTTCTTG AAATCTTTGT TCTCGATTTC CACTCCCCCC GACATTTCCC ATGTGGACAA TCTCTTGGGA TTCTTCTTAT TGGAGACCCT CTTTGCAGAG TCTGCGATAT CTGGATGGCC TGAGTATTTT TGAGCTTCAT GCTTACCAGT AAAGACATAC GACTCCCAGG GTCACATAAG TAGGCTCAAG GAATTTCCCT GAAGTGGGAT TGGACTGTCT AGTGCCACCA AGCTCAAGCA GTGTCCCCTC TCTCAGCTCT GACCCTGGCC AGACTGTGCT GGGGTAAGTG GAGCTGATTC

TGGTGTTTGT GGCAATTCTG GAAATACAAA ATTTACTTCC AGTCTCAGTC CTACAGGAGC GAAAGTTTAC CCTCTTGGCT GAGTAGTTAT AAAAGCAATT GCTAGTTCAT GGATTCAGGC GATATAATCT AACAAGAGTA TCTCACTATC CCAGATAATT AACAGCAGGA CCATTTTACT TTCTACCTGG ATTCAGAGCA TCTCTCATGC GCACTTTTTC GGCCTTGCTT TACTTGCTTT TAACAGACAA ACAAAAGTGA TGCAGAGAAG ACCACTAGCC GCCAGATGCG GTAGTTTCTT GTCTTGTTCC TAAACAGCTG CCTTCCTGTT CTACCACACC TTTAGATCCC ACAGATGAAT TTAGGATTCT TTGTTAAGCA TTGGGTGAGT ATAGCCCAGA CCTCCTTTAG CCTAATGGAG CTTGAAAGCA TGCTATGCCT GTCGATGGGT CACTATTTTA ATGATTGATA AAGATAATAT CAATATATAA GATGTATTTT CCATCCAGAA TAACCCCCCC TGTACTGGGG GGCCATCCTC TAGTGGTTTA GGGGTTGCAA GCTCTCAGTT CCTCTCAGGA TGTTTGGGTT TTTCAGTTTT TTTCCCCCTT GTGGTTTGTG TACTGCATAC TGGTTCCCAG AAACCAGGGT GGCAACACCC ACAGATATCC TCCCAGACAC ATGGTTTTAT CTTTTCATCG TCTAATCTCC TACTGCTGAA TTTCTCCCTC TCCTCTGCAT TCCTTGCAGG TCCCCACCTC CAGTCAAACC AGGGAGCCCC

TGTAGGAGTA ATTGTATGAA GAAATGATGG AGCAATTCTT AGGTAACAAA TTGCATACAA ATCACTCAAT TTGAGGTGTT AAAAAATGTT TCTTTGAACA GTCTAGTACA CTACTCTCCC GCATGGGTTT CAGGAAGTGC ATACTCTGGG AAAGGAGGTC ATCTTATTCT TATAGGAAAT AGTTTGTGAA ATACTGAAAC AGTGTAGGGA CAGATAGCTG TGTTTTACTT AAACTGTATT AAGGAAGCAT CTTTAATATT TAGCCAAAAG ATTAAATAGT ATAAGAGGGC GCCTCAAACC AGCAGAGTCT CTTGTTTATG AGTCCCCATG AAGAAACTCT TCTTCTGTTA AAGATGGTGC GTTGCTATTG TGGGTTTCTC CAAAGCAGAT GCACTCCACA ATAAAAGACT AAGTTAGAGT CTTGAAGAAT AAGCATATCA ATCTAAATTA ATTATTTTTA TTTATACTTT AAAAATCTAT GTTTACCTCA TTAAATTCTG ACCTGATATC TCCGCTTCTA CATCGAGCCT TGCTACATAT GCTCTGGGAG ATCCTTTCAG CAATGGTTGG GACAGCTATA TGGTGGCTGT TCAGTCTCTG CTAAGGTTTG AATTGTGTTT CATGTATGTT TTGCGGAGGC GGTCACTAAC CAAGCCCCTT GTTATAATGA TCCTCCCTGC TCATCCACTT GGATCTGACA CAAAGAAAGC CTGCTGACCA AGCACTGGGC CCTAGTGGTT CCCAGGGATG CTGGAACATT TCCTTTATCC ACAATTAATT

CTTAAGTGCT CAGCGATGAT ATGCTTCCAA CAGCCAATGT GATGGGGAAG AAGGAGATTG CTACGTCCAT AGATGATTAA ATGTAAATGT GGTAAAGTCC GTAGTTTATG TACTGAGGAC CTTATTTCTT ACTGGTGGGT ACCCAGGGGG AGCTGATGCT CAAATTGAGT TTGCCTATTG GAACAACTTA CCTAGTTCAT AGAGGTGCTA CTCAAAAGGG TGGGGTTTTG AAATAGAATG ATCCAACCTT TAATCCCTGG CTAAAATGTC AGATATGGAG CTGGAATAGA AGGGCATTTT GACTTAGAAG GCCCTGCTAT CCAGTGGGTC CAACAAAGTC TCTGGTAGCA TTTGGATGCT GAAAACTAAT TCACGGTAAG TGACCTAGTT AGGAAAAAAT CATATAAAAT CACAGAAGGA TTCTAAAGGT GTTTGTGGAC TGTTGTTCTC ATTTATTGAT TTATGAAGTG TTGATATGAA ATGAATTGCT TTTTATTTAC CCAACCTCAT CCCACCCACT TGACAGGACC GCAGATGAAG CTCTGGGCTG CTACTTCAGT CTACGAGCAT TCAGGCTCCT ATATGGGATG TTCCACACTT ATGCATCCAC TGGATACTAC CTTTTGTGAT TTGAATCCTG CCATAGCCCT CACAGGTAGA GGTACATAAA AAAAGGTATT TCTGCCATGA TGGGGAGTTA CTCCTGCGCT GCCAAGGCTA TAGATCCACT CCCAGATGCC GTGAGTCAGC CCCACAGCCT TGCCTCCCCT CTACAGGTTA

CATTCTGGTA GCACTGTCAG ACTGGATATA TCTCTCTTGC TACATCCAAC AGAGGCACCT CAGGAGCCAA AAAAGATTTT CTTGAGGAAA CACTACACTG AGCCATTCTC ACTCTTCATA AAAAGCAGGT AGAAGGCAAG AAATATTAAG GCCTATTGCC CTATCTCCTA AAAATCTGCT CAAGATAACA ATCTTCAAAC GGAGAAAACA AGAGTCTGAG TTTGGGCCTC TTATTAATTG ACACAGTCAT CAAGTGACAT AGGAAAGGAA GACTGATGGC ACCCTCATGC CCAGTTTAAT GTCTAGGAAG TTTATTCAGG CACGGTAGAG TGAATGGAAA ATACTGTATG TATTCTCTAG ACTAATCTCC CTTGGAGCAA AATCTCATTC TGTGCAATAC AGTCCTTCCT GTGGCTAAGG CACTATGAGG ATTCCTCACT TACGAAGAAC AATTTAAATA TCACTTGAAA TATTCTCTTA AATTTAAATT ATTTTAAATG CCCCTAGTCC CCCTTCCACC AAAGGCCTCT CCATAGGTCC TCTAGTTGGT CGTTTCTCTA CTGCCTTTGT GTCAGCATGC AATCCCATGT TGTCTCTATA ATTTTGGTCT AAGCTTTTGG TGGGTTACTA GAGACCCTGA CCATAGATTT GGACACGACC GGCCTGGAGT TAATCTCAGG CAATAAACAC CAAAGAAGGC CTCAGCAGCA CCCCAGTGGG TCCACAGCCC CAAGAGCCTG TCTGGCTATG GACACCCCCA GCCTCCAGAA TCTCACTGAG

544 Tadashi Matsuda and Toshio Hirano Figure 2 (Continued ) 9241 AATGATATCT TCCATCTATT TGCCTAAGAA TTTCATGAAG TCATTGTTTT 9301 TTTTTATTTA CATTTCAAAT GTTATCCCCT TTCCCCCACC CAACCATCCA 9361 CCTCCCCACT CTGACATTCC CCTACATTGG ATGGGGGTCA GCCTTGGCAG 9421 TTCTCCTCCC ATTGGTGCCC AACAAGGCCA TCCTCAGCTA CATATGCAGC 9481 GGTCTGTCCA TGTGTACTCT TTGGATGATA GTTTAGTCAC TGGGAGCTCT 9541 TATTGTTGTT CTTAGGGGTT GCAAGCCCCT TCAGCTTCTT CATTCCTTTC 9601 CCATTGGGAA CCCCATTCTT AGTTCAATAG TTGTCTGTGA GCCTCTGTAC 9661 CTGCAGAGCC TCTCAGGAAA CAGATATATC GGGCTCCTGT CAGCATGTAC 9721 CAGCAATATT GTCTGAATTT GGTGACTGTA TGTATATATG CTGGATCCCC 9781 GGCTGTGAAT GGCCATTCCT TCAGCCTCTG CTCCAAACTT TGTCTCCATA 9841 TGAATATTTT TGTTCCCCCA TCTAAGAGGA CTGAAGCATC CACACTTTAG 9901 TCTTGAACTT CATGTGGTCT GTGGATTGTA TCTTGGGTAA TCTGAGCTTT 9961 TCCACTTATC AGTGAGTTCA TACCATGTGT GCTTTTTTGT GATTGGGTTA 10021 GATGATATTT TCTACTTTCA TCCACTTGCC TATGAATTTC ATGAATT

TTTATTAGAT ACCCTTCCTG GATCAAGGGC TGGAGCCATA GGTTGGTTGG TCTAGCTCTG TTGTCATGCT TTTTTGGCAT AGGTGGAGCA TATACTCCTA TCATCCTTCT TGGGCTATTT CCTTGCTCAG

Exon 1: 2973–2991, Exon 2: 3154–3338, Exon 3: 4632–4745, Exon 4: 5616–5765, Exon 5: 6991–7158

TNF and IFN , which are known to be involved in macrophage activation, are induced normally. Nitric oxide formation is also induced normally. However, the induction of G-CSF in macrophages and fibroblasts does not occur in the absence of NFIL6. The results indicate that NF-IL6 has a crucial role in the bactericidal and tumoricidal activities of macrophages, as well as the existence of an NOindependent mechanism for these activities (Akira et al., 1995). The NFB-binding motif, located between 73 and 63 base pairs relative to the mRNA cap site, is required for the IL-1/TNF-induced expression of the IL-6 gene (Libermann and Baltimore, 1990; Zhang et al., 1990). The antisense oligonucleotide of NFB inhibits the expression of IL-6 mRNA in tumor cells derived from HTLV-I tax transgenic mice (Kitajima et al., 1992). Consistent with this, p40tax induces IL-6 mRNA through the NFB-binding site, concomitantly inducing NFB-binding protein (Muraoka et al., 1993). The involvement of NFB is also implicated in IL-6 gene induction by nonstructural regulatory protein 1 (NS-1), the nonstructural regulatory protein of human parvovirus B19 (Moffatt et al., 1996). In monocytic cell lines, the NFB site is crucial for LPS-induced IL-6 gene expression (Dendorfer et al., 1994; Sanceau et al., 1995). The synergistic induction of the IL-6 gene by IFN and TNF, in monocytic cells, involves cooperation between the interferon regulatory factor 1 (IRF-1) and NFB p65 homodimers, with a concomitant removal of the negative effect of the retinoblastoma control element (Sanceau et al., 1995). NFB is also involved in CD40-mediated IL-6 gene expression (Hess et al., 1995). Although the NFB site functions as a potent IL-1/TNF-responsive element in nonlymphoid cells, its activity is repressed in lymphoid cells and NFB-binding factor containing c-Rel seems to act as a repressor in lymphoid cells

(Nakayama et al., 1992). p53 and retinoblastoma also repress the IL-6 gene promoter, although the biological significance of this remains to be evaluated (Sehgal, 1992).

PROTEIN

Accession numbers SwissProt: Human: P05231 Mouse: P08505 Rat: P20607 Viral IL-6: AAC57089

Sequence See Figure 3.

Description of protein Early crystallographic studies of growth hormone (GH) and IL-2 led to the prediction that most cytokines would have a left-handed up-up-downdown four-helix bundle structure with two long and one short loop connections (Abdel-Megid et al., 1987; Brandhuber et al., 1987). In human IL-6, the four main helices are: Thr20±Lys46 (helix A), Glu80± Asn103 (helix B), Glu109±Lys129 (helix C), and Glu156±Met184 (helix D). In addition, Pro141± Glu152 make up a small helix C±D loop (Xu et al., 1996). Subsequent X-ray diffraction and NMR studies confirmed this prediction about other cytokines including IL-6, prolactin, erythropoietin, IL-4, G-CSF, GM-CSF, LIF, oncostatin M (OSM), CNTF, and cardiotropin 1 (CT-1). The evidence suggests an

IL-6 545 Figure 2 (Continued ) Human IL-6 cDNA 1 GAGAAGCTCT 61 GGTCCAGTTG 121 GTACCCCCAG 181 TCAGAACGAA 241 GAGACATGTA 301 CTGAACCTTC 361 ACTTGCCTGG 421 CAGAACAGAT 481 CTGATCCAGT 541 ACCACAAATG 601 ACAACTCATC 661 CGGCAAATGT 721 CAGAAACCTG 781 AGCGTTAGGA 841 GTTAATTTAT 901 TAGTTTTGAA 961 AGATCATTTC 1021 TAAAGAAATA 1081 CATTTTAAAA

sequence ATCTCCCCTC CCTTCTCCCT GAGAAGATTC TTGACAAACA ACAAGAGTAA CAAAGATGGC TGAAAATCAT TTGAGAGTAG TCCTGCAGAA CCAGCCTGCT TCATTCTGCG AGCATGGGCA TCCACTGGGC CACTATTTTA GTAAGTCATA ATAATAATGG TTGGAAAGTG TTTATATTGT AATTCAGC

CAGGAGCCCA GGGGCTGCTC CAAAGATGTA AATTCGGTAC CATGTGTGAA TGAAAAAGAT CACTGGTCTT TGAGGAACAA AAAGGCAAAG GACGAAGCTG CAGCTTTAAG CCTCAGATTG ACAGAACTTA ATTATTTTTA TTTATATTTT AAAGTGGCTA TAGGCTTACC ATTTATATAA

GCTATGAACT CTGGTGTTGC GCCGCCCCAC ATCCTCGACG AGCAGCAAAG GGATGCTTCC TTGGAGTTTG GCCAGAGCTG AATCTAGATG CAGGCACAGA GAGTTCCTGC TTGTTGTTAA TGTTGTTCTC ATTTATTAAT AAGAAGTACC TGCAGTTTGA TCAAATAAAT TGTATAAATG

CCTTCTCCAC CTGCTGCCTT ACAGACAGCC GCATCTCAGC AGGCACTGGC AATCTGGATT AGGTATACCT TGCAGATGAG CAATAACCAC ACCAGTGGCT AGTCCAGCCT TGGGCATTCC TATGGAGAAC ATTTAAATAT ACTTGAAACA ATATCCTTTG GGCTAACTTA GTTTTTATAC

AAGCGCCTTC CCCTGCCCCA ACTCACCTCT CCTGAGAAAG AGAAAACAAC CAATGAGGAG AGAGTACCTC TACAAAAGTC CCCTGACCCA GCAGGACATG GAGGGCTCTT TTCTTCTGGT TAAAAGTATG GTGAAGCTGA TTTTATGTAT TTTCAGAGCC TACATATTTT CAATAAATGG

sequence GATAGTCAAT CCTTCTTGGG GAGACTTCAC GCTTAATTAC ATTCTGATTG TACAAAGAAA TTTCCTCTGG ATAACAAGAA TCAACCAAGA TCCTAACAGA TCTTGAAATC GCGTTATGCC TGTTGTCAGG ACACTATTTT TATGATTGAT GAAATGATAA TTGGAATGTA TTGTGATGTA

TCCAGAAACC ACTGATGCTG AGAGGATACC ACATGTTCTC TATGAACAAC TGATGGATGC TCTTCTGGAG AGACAAAGCC GGTAAAAGAT TAAGCTGGAG ACTTGAAGAA TAAGCATATC TATCTGACTT AATTATTTTT ATTTATTATT CCTAAAAATC TAAGTTTACC TTTTTATAAT

GCTATGAAGT GTGACAACCA ACTCCCAACA TGGGAAATCG GATGATGCAC TACCAAACTG TACCATAGCT AGAGTCCTTC TTACATAAAA TCACAGAAGG TTTCTAAAAG AGTTTGTGGA ATGTTGTTCT AATTTATTGA TTTATGAAGT TATTTGATAT TCAATGAATT GTTTAGACTG

TCCTCTCTGC CGGCCTTCCC GACCTGTCTA TGGAAATGAG TTGCAGAAAA GATATAATCA ACCTGGAGTA AGAGAGATAC TAGTCCTTCC AGTGGCTAAG TCACTTTGAG CATTCCTCAC CTACGAAGAA TAATTTAAAT GTCACTTGAA AAATATTCTG GCTAATTTAA TCTTCAAACA

AAGAGACTTC TACTTCACAA TACCACTTCA AAAAGAGTTG CAATCTGAAA GGAAATTTGC CATGAAGAAC AGAAACTCTA TACCCCAATT GACCAAGACC ATCTACTCGG TGTGGTCAGA CTGACAATAT AAGTAAACTT ATGTTATATG TTACCTAGCC ATATGTTTTT AATAAATTAT

Rat IL-6 cDNA sequence 1 AGCTCATTCT GTCTCGAGCC 61 AGCTATGAAG TTTCTCTCCG 121 GTTGACAGCC ACTGCCTTCC 181 CACCCACAAC AGACCAGTAT 241 CAGGGAGATC TTGGAAATGA 301 CGATGATGCA CTGTCAGAAA 361 CTTCCAAACT GGATATAACC 421 GTTCCGTTTC TACCTGGAGT 481 CAGAGTCATT CAGAGCAATA 541 CTCATATAAA ATAGTCCTTC 601 GTCACAGAAG GAGTGGCTAA 661 ATTTCTAAAG GTCACTATGA 721 CAGTTTGTGG ACATTCCTCA 781 TATGTTGTTC TCTACGAAGA 841 TAATTTATTG ATAATTTAAA 901 TTTTATGAAG TGTCACTTGA 961 ATTTGATATG AATATTCTCT 1021 CAATGAATTG CTAATTTAAA

CACCAGGAAC CAAGAGACTT CTACTTCACA ATACCACTTC GAAAAGAGTT ACAATCTGAA AGGAAATTTG TTGTGAAGAA CTGAAACCCT CTACCCCAAC GGACCAAGAC GGTCTACTCG CTGTGGTCAG ACTGGCAATA TAAGTAAACT AATATTATGT TACCTAGCCA TTTTTT

GAAAGTCAAC CCAGCCAGTT AGTCCGGAGA ACAAGTCGGA GTGCAATGGC ACTTCCAGAA CCTATTGAAA CAACTTACAA AGTTCATATC TTCCAATGCT CATCCAACTC GCAAACCTAG AAAATATATC TGAATGTTGA ATAAGTTAAT TATAGTTTTG GATGGTTTCT

TCCATCTGCC GCCTTCTTGG GGAGACTTCA GGCTTAATTA AATTCTGATT ATACAAAGAA ATCTGCTCTG GATAACAAGA TTCAAACAAG CTCCTAATGG ATCTTGAAAG TGTGCTATGC CTGTCGATGG AACACTATTT TTATGATTGA AAAAGATAAT TGCAATATAT

CTTCAGGAAC GACTGATGTT CAGAGGATAC CATATGTTCT GTATGAACAG ATGATGGATG GTCTTCTGGA AAGACAAAGC AGATAAAAGA AGAAGTTAGA CACTTGAAGA CTAAGCATAT GTATCTAAAT TAATTATTTT TATTTATACT ATAAAAATCT AAGTTTACCT

CDS: 34-672 Mouse IL-6 cDNA 1 CACCAAGAAC 61 CATCCAGTTG 121 GTCCGGAGAG 181 CAAGTCGGAG 241 TGCAATGGCA 301 CTTCCAGAGA 361 CTATTGAAAA 421 AACTTAAAAG 481 ATTCATATCT 541 TCCAATGCTC 601 ATCCAATTCA 661 CAAACCTAGT 721 AAATATATCC 781 GAATGTTGGG 841 TAAGTTAATT 901 TTATAGTTTT 961 AGATGGTTTC 1021 AAAGAAATCT 1081 ATTATATTT CDS: 34-662

CDS: 65-700

evolutionary relationship among these molecules acting on the immune, hematopoietic, endocrine, and nerve systems (Bazan, 1990, 1992; Sprang and Bazan, 1993). However, unlike the other cytokines

such as GH, G-CSF, and LIF, no AB-loop helix was reported for IL-6 (Xu et al., 1996). Recently, a 1.9 AÊ crystal structure of human IL-6 has been reported, and this prediction has been

546 Tadashi Matsuda and Toshio Hirano Figure 3 Amino acid sequences for human, mouse, rat, and viral IL-6 proteins.

confirmed (Somers et al., 1997). The X-ray structure of IL-6 is composed of loops and an additional minihelix. Out of 185 residues, 157 are well defined in the final structure, with 18 N-terminal and 8 A±B loop amino acids displaying no interpretable electron density. Mutagenesis combined with epitope mapping studies using IL-6-specific neutralizing monoclonal antibodies showed the importance of four functionally distinct regions in IL-6. The first class of IL-6 mutant, called site1 mutant, shows reduced binding to the IL-6 receptor (Savino et al., 1993). Two additional distinct classes of IL-6 mutant, site 2 and site 3 mutants, bind to the IL-6 receptor but fail to generate signals (Brakenhoff et al., 1994; Ehlers et al., 1994). An IL-6 mutant with both site 2 and site 3 mutations not only fails to generate signals, but also functions as an antagonist in an IL-6-dependent proliferation assay. The fourth binding site on IL-6 is predicted as an IL-6/IL-6 interaction, which may be necessary for the sequential assembly of a functional hexameric IL6 receptor complex (Somers et al., 1997): IL-6 binds to the soluble IL-6 receptor to form a heterodimer. Moreover, in the presence of the soluble gp130, a hexameric complex is formed that is composed of IL-6, soluble IL-6 receptor and soluble gp130 in a 2 : 2 : 2 stoichiometry (Simpson et al., 1997). These mutational and structural analyses concerning the molecular basis for the actions of cytokines have also shown the possibility of engineering homologs of cytokines with enhanced or altered biologic properties. Some of the mutated recombinant

proteins are superior to the parent protein and useful in clinical applications. A series of artificial cytokines called Herlequin, made on the basis of sequences of human IL-6 and G-CSF, were produced (Grazi Cusi and Ferrero, 1997). Some of these chimeric molecules maintain the activity of either IL-6 or G-CSF, and at least one, Herlequin 11 has both biologic activities. Another example is the construction of a fusion protein, based on the `receptor conversion', which is composed of IL-6 and a soluble IL-6 receptor  in which the two moieties are linked by a flexible peptide chain (Fischer et al., 1997). This fusion is fully active at an 100- to 1000-fold lower concentration than the combination of unlinked IL-6 and soluble IL-6 receptor on gp130-expressing cells and is used to expand human hematopoietic progenitor cells efficiently.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce IL-6 is produced by a variety of cell types. The main sources are macrophages, fibroblasts, and endothelial cells. Other cell type sources are: T cells (Van Snick et al., 1987; Hodgkin et al., 1988; Horii et al., 1988; Espevik et al., 1990). B cells (Tanaka et al., 1988)

IL-6 547 Monocytes/macrophages (Helfgott et al., 1987; Horii et al., 1988; Sehgal, 1992). Eosinophils (Hamid et al., 1992) Mast cells (Burd et al., 1989) Chondrocytes (Guerne et al., 1990) Osteoblasts (Ishimi et al., 1990) Glial cells (Yasukawa et al., 1987) Astrocytes (Yasukawa et al., 1987) Trophoblasts (Kameda et al., 1990) Keratinocytes (Grossman et al., 1989) Fibroblasts (Sehgal et al., 1988; Van Damme et al., 1989) Endothelial cells (Astaldi et al., 1980) Smooth muscle cells (Loppnow and Libby, 1990) Mesangial cells (Horii et al., 1989; Ruef et al., 1990) Islet cells (Campbell and Harrison, 1990) Thyroid cells (Bendtzen et al., 1989)

RECEPTOR UTILIZATION The IL-6 receptor consists of two subunits: the  chain (IL-6R), an 80 kDa transmembrane glycoprotein which binds IL-6 with low affinity, and the chain (gp130), a 130 kDa transmembrane glycoprotein which binds to the IL-6±IL-6R heterodimer to form the high-affinity signal transducing complex. (see the chapter on IL-6 Ligand and Receptor Family).

IN VITRO ACTIVITIES

In vitro findings IL-6 and Immune Responses After antigen stimulation, B cells proliferate and differentiate to antibody-producing cells under the control of many cytokines produced by T cells and macrophages (Kishimoto and Hirano, 1988). IL-6 was identified as one of the factors acting on B cells in the culture of supernatants of PHA- or antigenstimulated peripheral mononuclear cells that induce immunoglobulin production in Epstein±Barr virus (EBV)-transformed B cell lines (Muraguchi et al., 1981; Teranishi et al., 1982). Furthermore, it was demonstrated that IL-6 functions in the late phase of SAC stimulation of normal B cells (Hirano et al., 1984a; Teranishi et al., 1984) or leukemic B cells (Yoshizaki et al., 1982), inducing immunoglobulin production when other cytokines, such as IL-2, are available. IL-6 acts on B cell lines at the mRNA level and induces the biosynthesis of secretory type Ig (Kikutani et al., 1985). Transcriptional activation is

the primary mechanism for the quantitative increase of secretary Ig mRNAs (Raynal et al., 1989). Furthermore, IL-6 activates the immunoglobulin heavy chain enhancer (E) in large but not unstimulated small B cells obtained from transgenic mice carrying the E and  light chain promoter-driving chloramphenicol acetyltransferase (CAT) gene (Miller et al., 1992). IL-6 acts on B cells activated with SAC or PWM to induce immunoglobulin production, but not on resting B cells (Muraguchi et al., 1988). Anti-IL-6 antibody inhibits PWM-induced Ig production, indicating that IL-6 is essential for PWM-induced Ig production (Muraguchi et al., 1988). Roles of IL-6 were also demonstrated in IL-4-dependent IgE (Vercelli et al., 1989) synthesis and in polysaccharide-specific antibody production in human B cells, as well as in the influenza A virus-specific primary response in murine B cells (Hilbert et al., 1989). AntiIL-6 antibody inhibits IL-4-driven IgE production, suggesting that endogenous IL-6 plays an obligatory role in the IL-4-dependent induction of IgE (Vercelli et al., 1989). Indeed, IL-4 induces IL-6 production in normal human B cells (Smeland et al., 1989). An obligatory role for IL-6 in antibody production is shown in IL-2-induced immunoglobulin production in SAC-stimulated B cells (Xia et al., 1989). In this case, IL-2 does not induce IL-6 production but may induce the IL-6 responsiveness in SAC-stimulated B cells, which produce IL-6 spontaneously. The dependence on IL-2 of the action of IL-6 in B cells was previously demonstrated using partly purified IL6 (Teranishi et al., 1984), and this was confirmed with recombinant IL-6 (Splawski et al., 1990), indicating that, as well as antigenic stimulation, additional signals provided by growth factors such as IL-2 are required for B cells to acquire IL-6 responsiveness. IL-6 is required differently for antigen-specific antibody production by primary and secondary murine B cells. The former response is dependent on IL-6, but the latter is not (Hilbert et al., 1989). IL-6 and IL-1 synergistically stimulate the growth and differentiation of murine B cells activated with anti-Ig or dextran sulfate (Vink et al., 1988). In addition, IL6 increases IgA production in murine Peyer's patch B cells (Beagley et al., 1989; Kunimoto et al., 1989) or human appendix B cells that express the IL-6 receptor (Fujihashi et al., 1991). This effect of IL-6 is not the result of isotype switching, as membrane-bound IgAnegative B cells were not induced to secrete IgA by IL-6 (Beagley et al., 1989). These facts indicate that IL-6 plays a role in the mucosal immune response (Fujihashi et al., 1992). IL-6 is also reported to augment the in vivo production of anti-sheep red cell (SRBC) antibodies

548 Tadashi Matsuda and Toshio Hirano in mice (Takatsuki et al., 1988). Consistent with these results, transgenic mice or mice bearing a retrovirus expressing IL-6 show massive plasmacytosis and hypergammaglobulinemia (Suematsu et al., 1989; Brandt et al., 1990). Similarly, transferring pre-B cells derived from IL-6 transgenic mice into RAG2deficient or SCID mice results in significantly more IgG and IgA production than is seen with wild-type preB cells (Oka et al., 1989). All these results show that IL-6 plays roles in Ig production in vivo. However, IL-6 may not be essential for immunoglobulin production and could be compensated for by other factors in vivo. IL-6 is involved in T cell activation, growth, and differentiation (Van Snick, 1990; Houssiau and Van Snick, 1992). IL-6 induces CD25, IL-2 receptor  chain, expression in one T cell line (Noma et al., 1987) and in thymocytes (Le et al., 1988), and functions as a second signal for IL-2 production by T cells (Garman et al., 1987). IL-6 promotes the growth of human T cells stimulated with PHA (Houssiau et al., 1988; Lotz et al., 1988) or mouse peripheral T cells (Uyttenhove et al., 1988). It also acts on murine thymocytes to induce proliferation (Helle et al., 1988; Le et al., 1988; Uyttenhove et al., 1988). The effects of IL-6 are synergistic with those of IL-1 and TNF (Le et al., 1988). IL-6 enhances the proliferation response of thymocytes to IL-4 and phorbol myristate acetate (Hodgkin et al., 1988). As IL-6 stimulates thymocyte proliferation and IL-1 can induce IL-6 production in thymocytes (Helle et al., 1989), the effect of IL-1 on thymocyte proliferation may be mediated by induced IL-6. After the removal of thymocytes with a low buoyant density that are capable of producing IL-6 following stimulation with IL-1, IL-1 cannot induce cell proliferation but IL-6 or IL-2 is still co-mitogenic; the IL-1 induced proliferation of thymocytes thus seems to be dependent on endogenous IL-6 production. A part of the effect of IL-6 on T cell growth is mediated by endogenously produced IL-2. Anti-CD25 antibody generally inhibits IL-6induced T cell proliferation (Garman et al., 1987; Le et al., 1988; Helle et al., 1989; Kawakami et al., 1989; Tosato et al., 1990). IL-1 and IL-6 synergistically induce IL-2 production (Holsti and Raulet, 1989; Houssiau et al., 1989) and CD25 expression in T cells (Houssiau et al., 1989). IL-6 also induces the differentiation of cytotoxic T lymphocytes (CTLs) in the presence of IL-2 from murine as well as human thymocytes and splenic T cells (Okada et al., 1988; Takai et al., 1988; Uyttenhove et al., 1988). Using purified murine T cells, both IL-1 and IL-6 were demonstrated to be required for the generation of CTLs and the induction of CD25 and IL-2 (Renauld

et al., 1989). IL-6 also induces serine esterase and perforin, required for mediating target cell lysis in the granules of CTLs (Takai et al., 1988; Liu et al., 1990), suggesting a role in the differentiation and expression of CTL function. IL-6 and Hematopoiesis IL-6 and IL-3 synergistically induce the proliferation of murine pluripotent hematopoietic progenitors in vitro (Ikebuchi et al., 1987). The combination of IL-6 and IL-3 acts on blast cell colony-forming cells to cause them to leave G0 earlier. IL-6 appears to trigger the entry into the cell cycle of the dormant progenitor cells, whereas IL-3 can support the continued proliferation of progenitors after they exit from the G0 phase (Ogawa, 1992). The colonyforming units in the spleen (CFU-S) were increased by culturing bone marrow cells in the presence of both IL-6 and IL-3 (Bodine et al., 1989; Okano et al., 1989b). Bone marrow cells cultured with IL-3 and IL6 for 6 days had a much higher capacity to rescue lethally irradiated mice than did cells cultured with IL-3 alone. These data indicate that the combination of IL-6 and IL-3 stimulates hematopoietic stem cells in vitro and could therefore be applied in bone marrow transplantation. IL-6 synergizes with M-CSF in the colony-forming unit-macrophage (CFU-M) with respect to both the number and size of macrophage colonies (Bot et al., 1989). IL-6 has also been to found to act synergistically with GM-CSF. Colony-forming units in culture (CFU-C) in the spleen and femur of mice that had been exposed to 750 rads and reconstituted with bone marrow cells were increased when IL-6 was injected (Okano et al., 1989a). Furthermore, the survival rate of lethally irradiated mice transplanted with 5  104 bone marrow cells was increased by IL-6 treatment from 20% to 75% on day 21 (Suzuki et al., 1989). One of the interesting reports on IL-6 and the hematopoietic system is that the defect in differentiation of hematopoiesis in Fanconi anemia may be caused by a deficiency in IL-6 production (Rosselli et al., 1992). Consistent with the possible role of IL-6 in hematopoietic stem cells, IL-6-deficient mice showed a decrease in the absolute number of CFUSd12 and pre-CFU-S progenitors and a reduced functionality of long-term repopulating stem cells. Primitive clonal progenitors in the bone marrow and in spleen are markedly reduced compared with controls, and the function of the long-term repopulating stem cell compartment was compromised. In vitro megakaryopoiesis is supported by several hematopoietic CSFs. IL-6 was found to induce the

IL-6 549 maturation of megakaryocytes synergistically with IL-3 (Ishibashi et al., 1989b); IL-6 promoted marked increments in megakaryocyte size and acetyl cholinesterase activity. Furthermore, IL-6 induced a significant shift towards higher ploidy classes. These effects of IL-6 on megakaryocytes have subsequently been confirmed (Lotem et al., 1989; Williams et al., 1990). The role of IL-6 in megakaryocyte development is further demonstrated by the fact that antimouse IL-6 monoclonal antibody inhibits megakaryocyte development in mouse bone marrow cultures in both the absence and the presence of IL-3 (Lotem et al., 1989). Human megakaryocytes were demonstrated to express IL-6 receptor and produce IL-6, suggesting that IL-6 regulates the terminal maturation of megakaryocytes in an autocrine manner (Hegyi et al., 1990). The number of mature megakaryocytes in the bone marrow was increased in IL-6 transgenic mice (Suematsu et al., 1989). Moreover, it was found that the administration of IL-6 increased platelet number in both mice (Ishibashi et al., 1989a) and monkeys (Asano et al., 1990). The additive or synergistic effect of IL-3 and IL-6 on megakaryocytopoiesis was further demonstrated in mice (Carrington et al., 1991) and monkeys (Geissler et al., 1992). The in vivo effect of IL-6 was consistent with the results obtained in IL-6-deficient mice, which showed the reduction of megakaryocyte progenitors (Bernad et al., 1994). Human and mouse myeloid leukemic cell lines, such as human histiocytic U937 cells and mouse myeloid M1 cells, can be induced to differentiate into macrophages and granulocytes in vitro by several synthetic and natural products. Several factors have been identified that can induce the differentiation of leukemic cells, such as G-CSF (Nicola et al., 1983), macrophage/granulocyte inducer type 2 (MGI-2) (Sachs, 1987), which was found to be identical to IL-6 (Shabo et al., 1988), D-factor (Tomida et al., 1984), and LIF (Gearing et al., 1987). IL-6 actually induces the growth inhibition and macrophage differentiation of several human and murine myeloid leukemic cells lines, suggesting a role for IL-6 in the final maturation of cells of the granulocyte±macrophage lineage (Miyaura et al., 1988; Shabo et al., 1988; Onozaki et al., 1989; Oritani et al., 1992; Revel, 1992). Consistent with this, the predominant cell types of GM-CFU colonies were monoblasts and macrophages in IL-6-deficient mice and wild-type mice respectively (Bernad et al., 1994). As for the involvement of IL-6 in granulopoiesis, the administration of IL-6 stimulates granulopoiesis in vivo in the absence of G-CSF receptor signals. GCSF receptor-deficient mice have a severe quantitative defect in granulopoiesis, although phenotypically

normal neutrophils are detected. In IL-6 and G-CSF receptor doubly deficient mice, the neutropenia was significantly worsened compared with that in G-CSF receptor-deficient mice. Almost normal numbers of myeloid progenitors were detected in the bone marrow of IL-6 and G-CSF receptor-deficient mice, and the terminal differentiation into mature neutrophils was also observed. These results demonstrate that IL-6 is an independent regulator of granulopoiesis in vivo (Liu et al., 1997). The IL-6 and soluble IL6 receptor complex in vitro is able to expand early CD34‡ progenitors that are IL-6 receptor negative and gp130 low in conjunction with stem cell factor (Sui et al., 1995). This evidence was confirmed in double transgenic mice expressing both human IL-6 and soluble IL-6. In contrast to single transgenics, the double transgenic mice develop a dramatic extramedullary hematopoiesis in the liver and spleen, but not in the bone marrow (Peters et al., 1997). IL-6 and Acute-Phase Reaction and Liver Regeneration The biosynthesis of acute-phase proteins by hepatocytes is regulated by several factors, including IL-1, TNF, and hepatocyte-stimulating factor (HSF). It was found that recombinant IL-6 can function as HSF (Gauldie et al., 1987). In addition, the activity of crude HSF can be neutralized by anti-IL-6 (Andus et al., 1987), indicating that HSF activity is exerted by IL-6 itself (Heinrich et al., 1990; Gauldie et al., 1992). IL-6 can induce a variety of acute-phase proteins, such as fibrinogen, 1-antichymotrypsin, 1-acid glycoprotein, and haptoglobulin, in the human hepatoma cell line HepG2. In addition to these proteins, it induces serum amyloid A, C-reactive protein (CRP), and 1-antitrypsin in human primary hepatocytes (Castell et al., 1988). The proteins induced in rats by IL-6 are fibrinogen, cysteine proteinase inhibitor, 2-macroglobulin, and 1-acid glycoprotein. The in vivo administration of IL-6 in rats induces typical acute-phase reactions similar to those induced by terpentine (Geiger et al., 1988), and the IL-6-induced expression of mRNAs for acutephase proteins is more rapid than that induced by terpentine. These results confirm the in vivo effect of IL-6 in the acute-phase reaction. It has been reported that serum levels of IL-6 correlate well with those of CRP and with fever in patients with severe burns (Nijsten et al., 1987), and an increase in serum IL-6 concentration has been observed before an increase in serum CRP level in those undergoing surgical operations (Nishimoto et al., 1989; Shenkin et al., 1989), supporting a causal role of IL-6 in the acute-phase response. In fact,

550 Tadashi Matsuda and Toshio Hirano IL-6-deficient mice are severely defective in the inflammatory acute-phase response after tissue damage or infection (Kopf et al., 1994). It is also likely that different patterns of cytokines are involved in systemic and localized tissue damage: IL-6 is an essential mediator of the inflammatory response to localized inflammation, such as in the terpentine-induced response, but not to a systemic response induced by lipopolysaccharide (Fattori et al., 1994b). IL-6 and TNF are important components of the early signaling pathways leading to regeneration in the liver (Michalopoulos and DeFrances, 1997). At a very early stage of the regenerative response in liver, TNF induces IL-6 production and the rapid activation of a set of transcription factors such as STAT3, NFB, AP-1, and C/EBP (Diehl et al., 1994). In both TNF type I receptor- and IL-6-deficient mice, DNA synthesis after partial hepatectomy is severely impaired and the activation of NFB and STAT3 does not take place (Cressman et al., 1996; Yamada et al., 1997). The administration of sufficient amounts of IL-6 can repair the defect of DNA synthesis, hepatocyte regeneration, and liver damage. Without hepatectomy, IL-6 does not stimulate hepatocyte proliferation since IL-6 transgenic mice do not show any morphological liver alternation. Thus, IL-6 is crucial to liver regeneration and cannot be substituted by other endogenously produced cytokines. Double transgenic mice expressing both human IL-6 and soluble IL-6 receptor in liver parenchyma were generated (Maione et al., 1998). In contrast to single transgenic mice, the double transgenic mice developed progressive extramedullary hematopoiesis in the liver and spleen. In hepatic periportal areas, elevated levels of both transgenes induce hepatocytes to proliferate and cause the early development of hyperplastic nodules that closely mimic human nodular regenerative hyperplasia of liver. The disorder is characterized by diffuse micronodular transformation of the hepatic parenchyma and the late formation of large liver adenomas. These findings support the role of IL6 as a major cytokine, responsible for both physiologic and pathologic proliferation of the hepatic parenchyma. IL-6 and CNS The IL-1 stimulation of glioblastoma or astrocytoma cells induces the expression of IL-6 mRNA (Yasukawa et al., 1987). Both virus-infected microglial cells and astrocytes also produce IL-6, indicating the possible involvement of IL-6 in nerve cell functions. IL-1 and TNF are important inducers of IL-6

in astrocytes. Neurotransmitters including substance P, norepinephrine, VIP, adenosine, and histamine have been reported to induce IL-6 production in astrocytes. LPS is one of the important inducers of IL-6 in microglia. In fact, IL-6 induces the neurite outgrowth of PC12 cells into neural cells (Satoh et al., 1988; Ihara et al., 1996; Wu and Bradshaw, 1996) (also see chapter on IL-6 receptor). Furthermore, IL-6 can support the survival of cultured cholinergic neurons. IL-6 can also alter the neuronal response to NMDA receptor activation (Hama et al., 1989). Several studies using the reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization techniques have shown that CNS cells express both IL-6 and IL-6 receptors. IL-6 and IL-6 receptor mRNAs are detected in several brain regions, including the hippocampus, striatum, hypothalamus, neocortex, cerebellum, and brainstem. Their mRNA levels tend to be higher in forebrain structures than in more caudal regions. In the hippocampus, IL-6 mRNA expression levels decrease during development, whereas IL-6 receptor mRNA levels increase, suggesting that IL-6 and IL-6 receptor expression are regulated differentially in this CNS region. The developmental timing of the expression of IL-6 receptor in the hypothalamus seems to be linked to the maturation of the hypothalamic-pituitary-adrenal axis (HPA), consistent with a role of IL-6 in the regulation of hormone release. IL-6 stimulates the secretion of adrenocorticotropic hormone either through the corticotropinreleasing hormone (Naitoh et al., 1988) or directly (Fukata et al., 1989). IL-6 also stimulates the release of a variety of anterior pituitary hormones such as prolactin, growth hormone, and luteinizing hormone (Spangelo et al., 1989). Anterior pituitary cells produce IL-6 spontaneously (Spangelo et al., 1990). IL-6 is released in the CNS during various pathologic conditions, including AIDS±dementia complex, Alzheimer's disease, multiple sclerosis, systemic lupus erythematosus, CNS trauma, and viral and bacterial meningitis (Gruol and Nelson, 1997).

Bioassays used The biologic activity of IL-6 is determined by a number of bioassays: the proliferation of murine hybridoma/plasmacytoma cells 7TD1, B9 and MH60.BSF2, the differentiation of murine leukemia M1 cells and the antibody production of the human B cell lines CESS and SKW6-Cl4. Both human and murine IL-6 are equally active on human and murine cells, but murine IL-6 is inactive on human cells.

IL-6 551

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS Osteoporosis is a major cause of morbidity in older people. A variety of compounds, including hormones, nutrients, and cytokines, modulate bone remodeling. IL-6 increases with age and menopause. Additionally, murine models suggest that IL-6 plays a central role in bone resorption (Ershler et al., 1997). Estrogen deficiency causes a marked bone loss by stimulating osteoclastic bone resorption. The concentrations of IL-1, IL-6, IL-6 receptor, and prostaglandins detected in the bone marrow of ovariectomized mice are insufficient to account for the increase in bone resorption caused by estrogen withdrawal. The increase in bone resorption induced by ovariectomy can be explained by a cumulative effect of these cytokines (Pacifici, 1996). IL-6 alone cannot induce osteoclast formation in co-culture of mouse bone marrow and osteoblastic cells. Soluble IL-6 receptor strikingly stimulates osteoclast formation induced by IL-6. In the presence of dexamethasone, IL-6 alone could induce osteoclast formation. The treatment of osteoblastic cells with dexamethasone induced a marked increase in the expression of IL-6 receptor (Udagawa et al., 1995). In IL-6-deficient mice, an analysis of bone metabolism revealed a specific bone phenotype (Poli et al., 1994). IL-6-deficient female mice have a normal amount of trabecular bone but higher rates of bone turnover than control littermates. Estrogen deficiency induced by ovariectomy causes a significant loss of bone mass together with an increase in bone turnover rates in wild-type mice. In contrast, in IL-6-deficient mice, ovariectomy does not induce any change in either bone mass or bone turnover rate. These results indicate that IL-6 plays an important role in the local regulation of bone turnover and, at least in mice, appears to be essential for the bone loss caused by estrogen deficiency. In accordance with this, the enhanced osteoclast development in ovariectomized mice is prevented by the administration of anti-IL-6 antibody (Jilka et al., 1992), and estrogen inhibits the IL-1- and TNF-induced production of IL-6 (Girasole et al., 1992). Trophoblast produces IL-6 in vivo, although the biological significance of IL-6 in the placenta is unknown (Kameda et al., 1990). Because IL-6 stimulates hepatic lipogenesis in mice, and IL-6 is induced by TNF, the lipogenic effects of TNF may be in part mediated by IL-6 (Grunfeld et al., 1990). IL-6 is produced by vascular smooth muscle cells (Loppnow and Libby, 1990) and may be involved in their cell growth (Nabata et al., 1990), suggesting the

possible involvement of IL-6 in arteriosclerosis. The intraperitoneal injection of either LPS or IL-1 does not cause a fever response in IL-6-deficient mice, and the fever response is recovered by the intracerebroventricular injection of recombinant IL-6, but not IL1 and LPS (Chai et al., 1996). IL-6 is a growth factor for various cells, including plasmacytoma, myeloma, and hybridoma. On the other hand, IL-6 acts as a growth inhibitor for a number of carcinoma and leukemia cell lines, including breast carcinoma, ovarian carcinoma, and myeloleukemic cell lines (Revel, 1992).

Knockout mouse phenotypes In IL-6-deficient mice, the numbers of thymocytes and peripheral T cells are consistently reduced by 20± 40% compared with controls (Kopf et al., 1994). Spleen cells from IL-6-deficient mice and control mice show similar levels and kinetics of IL-2 receptor expression after stimulation with Con A. The numbers of B cells in the bone marrow and spleen are normal, and their expression of B220, IgM, IgD, and CD23 is within the normal range. IL-6-deficient mice show a reduced IgG response but no reduction in IgM response to both a soluble protein antigen and vesicular stomatitis virus (VSV) antigen (Kopf et al., 1994). A striking effect is observed in the mucosal IgA antibody response in IL-6-deficient mice: the number of IgA-producing cells was greatly reduced (Ramsay et al., 1994). This reduced IgA response was completely restored after intranasal infection with recombinant IL-6 vaccinia viruses. Furthermore, IL6-deficient mice infected intranasally with recombinant vaccinia virus expressing the hemagglutinin (HA) glycoprotein of influenza virus have strongly reduced numbers of HA-specific IgA and IgG antibodyproducing cells. Likewise, the intraduodenal injection of ovalbumin resulted in an impaired IgA response. In contrast, in IL-6-deficient mice after either inoculation with Helicobacter felis or repeated peroral immunization with soluble protein (ovalbumin) in the presence of cholera toxin, normal mucosal IgA and IgG responses were observed (Bromander et al., 1996). Such conflicting results may be explained by the fact that the former responses probably reflect complement dependence, but the latter does not require complement: C3 complement component plays an important role in both antibody production and germinal center development. The local production of C3 and germinal center formation are impaired in IL-6-deficient mice (Kopf et al., 1998). Germinal centers from IL-6- and from C3-deficient mice have a

552 Tadashi Matsuda and Toshio Hirano comparable defect in IgG2a and IgG2b antibody production (Kopf et al., 1998). Therefore, IL-6induced C3 production seems to play an important role to generate high-affinity antibodies within the germinal centers. In IL-6-deficient mice, T cells show a normal pattern of expression of TCR , , , and  chains and CD4, CD8, CD44, and CD24 markers. The generation of CTLs and their lytic activity were reduced 3- to 10-fold in IL-6-deficient mice infected with VV-WR, although CTL function against LCMV or VV was not reduced (Kopf et al., 1994). T cell activation and T helper cell subset differentiation were not significantly altered in IL-6-deficient mice. An inability to clear Listeria monocytogenes is observed in IL-6-deficient mice (Kopf et al., 1994; Dalrymple et al., 1995). This inability is most likely due to the inability of neutrophils to function in IL-6deficient mice, suggesting that IL-6 plays a critical role in listeriosis by stimulating neutrophils (Dalrymple et al., 1995). IL-6-deficient mice show an increased susceptibility to Escherichia coli infection and are unable to induce neutrophilia following challenge with E. coli (Dalrymple et al., 1996). Furthermore, IL-6-deficient mice are more susceptible than wild-type mice to virulent Candida albicans. Impairment of macrophage, neutrophil, and T helper type 1 (TH1)-associated protective immunity is observed in IL-6-deficient mice (Romani et al., 1996). These findings indicate a role in the function of macrophages and neutrophils in vivo.

Transgenic overexpression To show the in vivo function of IL-6 in the CNS, two types of IL-6 transgenic mouse were produced (Campbell et al., 1993; Fattori et al., 1995). NSEIL-6 mice were made by expressing human IL-6 in the CNS neurons under the control of the rat neuronspecific enolase promoter (Fattori et al., 1995). GFAP-IL-6 mice were produced by expressing the murine IL-6 in astrocytes under the control of the murine glial fibrillary-acidic protein promoter (Campbell et al., 1993). In NSE-IL-6 mice, reactive gliosis is found throughout the CNS, as shown by the increased size and number of GFAP-positive astrocytes and ramified microglia. The GFAP-IL-6 transgenic mice also showed reactive gliosis throughout the CNS. In contrast to the NSE-IL-6 mice, the GFAP-IL-6 mice show several neuronal and vascular pathologic and behavioral abnormalities. The GFAPIL-6 mice also show significant neurodegeneration, including the loss and damage of neurons in the hippocampus formation and cerebellum. A number of

motor problems, such as motor uncoordination, ataxia, and tremor are also present in GFAP-IL-6 mice (Campbell et al., 1993). After injury in the hypoglossary nerve in adult mice, the expression of IL-6 increased in Schwann cells at the lesional site as well as in the cells of hypoglossal neurons in the brainstem. The regeneration of axotomized hypoglossal nerve in vivo was significantly retarded by the administration of anti-IL-6 receptor antibodies. In double transgenic mice expressing both human IL-6 and soluble IL-6 receptor, regeneration of the axotomized nerve was strikingly accelerated compared with nontransgenic controls (Hirota et al., 1996). The IL-6 signal may play an important role in nerve regeneration after trauma in vivo. See the chapter on the IL-6 Receptor.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Role in experiments of nature and disease states A possible involvement of deregulated expression of the IL-6 gene in polyclonal B cell abnormalities was first suggested in patients with cardiac myxoma (Hirano et al., 1987). Since then, much evidence has been accumulated indicating that the deregulated production of IL-6 could be involved in a variety of diseases and malignancies. Considering the possible involvement of IL-6 in such diseases, it might be worth noting that IL-6 was identified as virus-induced IFN- 2 (Weissenbach et al., 1980) or found in the culture supernatants of cells infiltrating the pleural effusion of patients with tuberculous pleurisy (Teranishi et al., 1982), which also contained factors capable of inducing immunoglobulin production in activated human B cells (Hirano et al., 1981). The consequence of a deregulated expression of IL-6 has been evaluated in either transgenic mice overexpressing the IL-6 gene or mice generated by targeted disruption of the IL-6 gene. IL-6 and Autoimmune Diseases Patients with cardiac myxoma show a variety of autoimmune symptoms, such as hypergammaglobulinemia, the presence of autoantibodies and an

IL-6 553 increase in acute-phase proteins. The production of IL-6 by cardiac myxoma cells first suggested the possible involvement of IL-6 in B cell abnormality and autoimmune diseases (Hirano et al., 1987; Jourdan et al., 1990). Before this finding, it was demonstrated that the pleural effusion cells of patients with pulmonary tuberculosis, when stimulated with purified protein derivative, produce a large amount of factors capable of inducing Ig production in activated normal B cells (Hirano et al., 1981). One of these factors was identified as IL-6 (Teranishi et al., 1982; Hirano et al., 1984a,b). It is noteworthy that patients with pulmonary tuberculosis often have a wide range of autoantibodies (Shoenfeld and Isenberg, 1988), and in certain cases a significant diffuse hypergammaglobulinemia is reported (Sela et al., 1987). IL-6 was also found to be produced by islet beta and thyroid cells (Bendtzen et al., 1989; Campbell and Harrison, 1990), suggesting that IL-6 may be involved in type I diabetes (Campbell et al., 1991). The observation that anti-IL-6 antibody inhibits the development of insulin-dependent diabetes in NOD/WEHI mice may support the role of IL-6 in autoimmune disease (Campbell et al., 1991). The expression of IL-6 by beta cells does not cause insulitis or diabetes in C57BL/6  CBA mice, but the interaction of IL-6 and diabetes susceptibility genes causes insulitis in NOD/F1 mice (DiCosmo et al., 1994). IL-6 production is also observed in patients with rheumatoid arthritis (Hirano et al., 1988; Bhardwaj et al., 1989), the type II collagen-induced murine model of arthritis (Takai et al., 1989), and MRL/lpr mice (Tang et al., 1991), which develop autoimmune disease with proliferative glomerulonephritis and arthritis. Other interesting evidence is that a striking increased prevalence of agalactosyl IgG has been observed in a variety of autoimmune and/or IL-6related diseases, such as pulmonary tuberculosis, rheumatoid arthritis, Crohn's disease, sarcoidosis, leprosy, Castleman's disease, Takayasu's arthritis, multiple myeloma, and pristane-induced arthritis (Nakao et al., 1991; Rook et al., 1991; Rook and Stanford, 1992). In accordance with these facts, IL-6 transgenic mice showed an increase in agalactosyl IgG activity (Rook et al., 1991), further strengthening the intimate relationship between IL-6 and certain autoimmune diseases. Taken as a whole, the evidence suggest that IL-6 plays a role in autoimmune disease, although IL-6 alone is not sufficient for its generation (Hirano, 1992). The requirement for IL-6 in the process of autoimmune disease has been shown in mouse models of arthritis and experimental autoimmune encephalomyelitis (EAE). C57BL/6 mice develop antigen-induced

arthritis (AIA). C57BL/6 crossed with an IL-6null mutation gave rise to only mild arthritis. In IL-6-deficient mice, the articular cartilage was well preserved. The lymph node cells of IL-6-deficient mice produced much more TH2 cytokine than those of wild-type mice with either antigen-specific or nonspecific stimulation in vitro culture (Ohshima et al., 1998). Collagen-induced arthritis (CIA) in DBA/ 1J mice and the inflammatory polyarthritis of TNF transgenic mice were investigated in relation to the requirement of IL-6 for disease onset. Crossing an IL6-null mutation into both arthritis-susceptible genetic backgrounds ablated IL-6. DBA/1J, IL-6-deficient mice were completely protected from CIA, accompanied by a reduced antibody response to type II collagen and the absence of inflammatory cells and tissue damage in knee joints, while the arthritis in TNF transgenic mice was not affected by inactivation of the IL-6 gene. These results suggest that IL-6 is critical for CIA (Alonzi et al., 1998). EAE provoked by myelin oligodendrocyte glycoprotein (MOG) is referred to as the animal model of multiple sclerosis (MS). IL-6-deficient mice were resistant to MOGinduced EAE (Okuda et al., 1998). No infiltration of inflammatory cells was observed in the central nervous system of IL-6-deficient mice. IL-6 and Chronic Inflammatory Proliferative Disease Chronic inflammatory proliferative disease (CIPD) may be categorized as a disease in which chronic inflammation and immunological reactions, with prolonged cell proliferation in lesions, are operating and underlying the manifestation of a variety of symptoms. Mesangial cell proliferative glomerulonephritis, psoriasis, and Kaposi's sarcoma may be categorized as CIPD. From this point of view, rheumatoid arthritis is also considered in this category, because one of its prominent features is chronic expansion of synovial cells. Glomerulonephritis is accompanied by a variety of autoimmune diseases, and several growth factors have been suggested as candidates that induce the pathologic growth of mesangial cells (Horii et al., 1989; Ruef et al., 1990). IL-6 is a possible autocrine growth factor for rat mesangial cells. It is produced by renal mesangial cells in patients with mesangial cell proliferative glomerulonephritis (Horii et al., 1989). IL-6 is detected in urine samples from patients with mesangial proliferative glomerulonephritis but not from those with other types of glomerulonephritis. There is a correlation between the level of IL-6 in urine and the progressive stage of mesangial proliferative glomerulonephritis. MT-I/IL-6 transgenic

554 Tadashi Matsuda and Toshio Hirano mice, in which the IL-6 gene is driven by the mouse metallothionein I gene promoter, show a progressive kidney pathology in which the initial membranous glomerulonephritis is followed by focal glomerulosclerosis and finally by extensive tubular damage. This are similar to the damage observed in patients in the terminal stages of multiple myeloma (myeloma kidney) (Fattori et al., 1994a). Other CIPDs that may be related to IL-6 are psoriasis (Grossman et al., 1989) and Kaposi's sarcoma (Miles et al., 1990), in which IL-6 is considered to be one of the growth factors for keratinocytes and Kaposi's sarcoma cells. IL-6 is synthesized and released by normal human epidermis (Paquet and Pierard, 1996). Keratinocytes express both IL-6 and IL-6 receptors. IL-6 has been shown to stimulate keratinocyte proliferation in vitro. An overproduction of IL-6 is observed in the supernatant of cell cultures of psoriatic keratinocytes (Grossman et al., 1989). In psoriatic skin, IL-6 is present in the cytoplasm of keratinocytes and in corneocytes. Furthermore, increased susceptibility to the growth-stimulating effect of IL-6 is shown in psoriatic keratinocytes. The cells of AIDS-related Kaposi's sarcoma (AIDSKS) synthesize and release large amounts of IL-6 both in vitro and in vivo. These cells also possess IL-6 receptor and proliferate in response to IL-6. IL-6 acts on AIDS-KS cells as an autocrine growth factor (Miles et al., 1990). Most intriguing finding is that HHV-8 or Kaposi's sarcoma-associated herpesvirus (KSHV), which is identified with high frequency in all epidemiological forms of Kaposi's sarcoma, encodes a structural homolog of IL-6, termed viral IL-6 (vIL-6) (Moore et al., 1996; Neipel et al., 1997; Nicholas et al., 1997a,b). Plasma Cell Neoplasia IL-6 is a potent growth factor for murine plasmacytoma cells (Aarden et al., 1985; Van Damme et al., 1987a,b; Van Snick et al., 1988) and human myeloma cells (Kawano et al., 1988), suggesting the possible involvement of IL-6 in the generation of plasmacytoma or myeloma (Hirano, 1991). There is a significant association between the occurrence of plasma cell neoplasia and chronic inflammation (Isobe and Osserman, 1971; Isomaki et al., 1978). Plasmacytoma can be induced in Balb/c mice by mineral oil or pristane, both of which are potent inducers of chronic inflammation and IL-6 (Potter and Boyce, 1962; Nordan and Potter, 1986). Constitutive IL-6 production in a murine plasmacytoma cell line caused by the insertion of an IAP retrotransposon in the IL-6 gene has been reported (Blankenstein et al., 1990). Expression of the IL-6

gene in an IL-6-dependent murine plasmacytoma cell line caused the cells to proliferate in an autocrine manner (Tohyama et al., 1990; Vink et al., 1990). These cells showed greatly enhanced tumorigenicity, and monoclonal antibodies capable of blocking the binding of IL-6 to its receptor inhibited their growth in vivo (Vink et al., 1990). IL-6 was also demonstrated to be an autocrine growth factor for EBV-transformed B cell lines (Yokoi et al., 1990), and the expression of an exogenous IL-6 gene in these B cell lines conferred a growth advantage and in vivo tumorigenecity (Scala et al., 1990). However, it is controversial whether all myeloma cells produce IL-6, because only some myeloma cell lines have been found to produce it (Kawano et al., 1988; Klein et al., 1989; Shimizu et al., 1989; Hata et al., 1990), and bone marrow adherent cells rather than bone marrow nonadherent cell populations containing myeloma cells have been demonstrated to be major producers of IL-6 (Klein et al., 1989). In any case, evidence indicates that IL-6 plays an important role in the in vitro growth of myeloma cells and generation of plasma cell neoplasia. This is further supported by the following findings. The in vitro IL-6 responsiveness of myeloma cells obtained from patients with multiple myeloma correlates directly with the in vivo labeling index of these tumors (Zhang et al., 1989), and increased serum IL-6 levels correlate well with disease severity in multiple myeloma and plasma cell leukemia (Bataille et al., 1989). The administration of anti-IL-6 antibodies suppresses myeloma cell growth (Klein et al., 1991). IL-6 prevents apoptosis and induces the expression of Bcl-xL in murine myeloma cells (Schwarze and Hawley, 1995). Furthermore, STAT3, which is a major signaling molecule activated through the IL-6 receptor and involved in the expression of IL-6 biologic activities, is constitutively activated in bone marrow mononuclear cells from patients with multiple myeloma and in human myeloma cell line U266, which expresses Bcl-xL (Catlett-Falcone et al., 1999). These findings provide evidence that IL-6 contributes to the pathogenesis of multiple myeloma. IL-6 alone may not, however, be sufficient for the generation of plasmacytoma. Plasma cells generated in the IL-6 transgenic mice were not transplantable to syngenic mice, indicating that additional factors may be required for malignant transformation. An interesting finding is that C57BL/6 IL-6 transgenic mice, when backcrossed with Balb/c mice, showed a progression from polyclonal plasmacytosis to fully transformed monoclonal plasmacytoma that contained chromosomal translocation with c-myc gene rearrangement (Suematsu et al., 1992). The evidence strongly supported the hypothesis that the

IL-6 555 deregulated expression of the IL-6 gene can trigger polyclonal plasmacytosis, resulting in the generation of malignant monoclonal plasmacytoma (Hirano, 1991). In fact, in IL-6-deficient mice, pristane cannot induce plasmacytoma (Hilbert et al., 1995). Interestingly, no plasmacytomas were shown in Balb/c IL-6-deficient mice treated with a myc/raf-expressing retrovirus (J3V1) in combination with pristane oil, while the frequency of myeloid tumors in IL-6deficient mice was comparable to that of control mice (Lattanzio et al., 1997). An intriguing finding is that HHV-8 (KSHV) is detected in the bone marrow dendritic cells of multiple myeloma patients but not those of normal individuals or patients with other malignancies. Interestingly, two out of eight patients with monoclonal gammopathy of undetermined significance (MGUS), a precursor to myeloma, were also positive for HHV-8 (Rettig et al., 1997). Since the genome of HHV-8 encodes vIL-6, which is functional in B9 proliferation assays (Moore et al., 1996), HHV-8 may play a role in the transformation of MGUS to myeloma. In relation to this issue, the germinal centers of the hyperplastic lymph nodes of patients with Castleman's disease produce large quantities of IL-6 (Yoshizaki et al., 1989). Castleman's disease is a rare B cell lymphoproliferative disorder consisting of giant lymph node hyperplasia with plasma cell infiltration, fever, anemia, hypergammaglobulinemia, and an increase in the plasma level of acute phase proteins. The presence of HHV-8 DNA is also associated with multicentric Castleman's disease (Neipel et al., 1997; Parravinci et al., 1997). All these findings provide evidence that the constitutive production of IL-6 as a result of virus infection, chronic inflammation, and/or prolonged bacterial infection underlies the molecular basis of the generation of plasmacytoma and myeloma and the manifestation of clinical symptoms (Hirano, 1991, 1998).

IN THERAPY

Preclinical ± How does it affect disease models in animals? It has been shown that the administration of IL-6 with IL-3 increases platelet number in both mice (Ishibashi et al., 1989a) and monkeys (Asano et al., 1990). A combination of these factors may be valuable for the effective control of thrombocytopenia and anemia. A variety of inhibitors of IL-6, such as anti-IL-6 monoclonal antibody and mutated IL-6,

would be used to treat patients with multiple myeloma, Castleman's disease, and rheumatoid arthritis (Klein et al., 1991; Bataille et al., 1995; Elliott and Maini, 1995; Matsuno et al., 1998). The fusion protein composed of IL-6 and a soluble IL-6 receptor would be used to stimulate cells expressing gp130, for example, to expand hematopoietic stem cells (Fischer et al., 1997) and accelerate the regeneration of liver in patients with hepatitis.

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