Involvement of IL-6, Apart from Its Role in Immunity, in Mediating a Chronic Response during Experimental Arthritis
Identifieur interne : 000464 ( Main/Corpus ); précédent : 000463; suivant : 000465Involvement of IL-6, Apart from Its Role in Immunity, in Mediating a Chronic Response during Experimental Arthritis
Auteurs : Alfons S. K. De Hooge ; Fons A. J. Van De Loo ; Onno J. Arntz ; Wim B. Van Den BergSource :
- The American Journal of Pathology [ 0002-9440 ] ; 2000.
Abstract
Interleukin-6 (IL-6) is highly produced during arthritis but its exact function is still unknown. In this study we examined if IL-6, apart from its role in immunity, was involved in the local inflammatory response in experimental arthritis. IL-6 deficient (IL-6/) and wild-type mice were first compared in the antigen-induced arthritis model. IL-6 deficiency resulted in a mild, transient inflammation whereas wild-type mice developed a chronic, destructive synovitis. Wild-type mice immunized with one-tenth of the normal antigen dose still developed chronic arthritis despite low antibody levels, excluding reduced humoral immunity in IL-6/ mice as a crucial phenomenon. In addition, passive immune-complex-induced arthritis did not differ between wild-type and IL-6/ mice. Another option is reduced levels of Th1 cells in IL-6/ mice. However, transfer of antigen-specific wild-type lymph node cells to IL-6/ mice enhanced acute joint inflammation and increased cartilage damage but still could not sustain chronic inflammation, suggesting involvement of nonimmune elements of IL-6 activity in chronicity. In line with this, nonimmunologically mediated zymosan-induced arthritis developed similarly in the first week, but only wild-type mice developed chronic synovitis. These results indicate an important role for IL-6 in propagation of joint inflammation, potentially independent of its role in immunity.
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DOI: 10.1016/S0002-9440(10)64846-8
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>Involvement of IL-6, Apart from Its Role in Immunity, in Mediating a Chronic Response during Experimental Arthritis</title><author><name sortKey="De Hooge, Alfons S K" sort="De Hooge, Alfons S K" uniqKey="De Hooge A" first="Alfons S. K." last="De Hooge">Alfons S. K. De Hooge</name><affiliation><mods:affiliation>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: A.deHoogereuma.azn.nl</mods:affiliation></affiliation></author><author><name sortKey="Van De Loo, Fons A J" sort="Van De Loo, Fons A J" uniqKey="Van De Loo F" first="Fons A. J." last="Van De Loo">Fons A. J. Van De Loo</name><affiliation><mods:affiliation>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</mods:affiliation></affiliation></author><author><name sortKey="Arntz, Onno J" sort="Arntz, Onno J" uniqKey="Arntz O" first="Onno J." last="Arntz">Onno J. Arntz</name><affiliation><mods:affiliation>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</mods:affiliation></affiliation></author><author><name sortKey="Van Den Berg, Wim B" sort="Van Den Berg, Wim B" uniqKey="Van Den Berg W" first="Wim B." last="Van Den Berg">Wim B. Van Den Berg</name><affiliation><mods:affiliation>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:3FFA4817B217FC3EC96B07D190272F56761F0188</idno><date when="2000" year="2000">2000</date><idno type="doi">10.1016/S0002-9440(10)64846-8</idno><idno type="url">https://api.istex.fr/document/3FFA4817B217FC3EC96B07D190272F56761F0188/fulltext/pdf</idno><idno type="wicri:Area/Main/Corpus">000464</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a">Involvement of IL-6, Apart from Its Role in Immunity, in Mediating a Chronic Response during Experimental Arthritis</title><author><name sortKey="De Hooge, Alfons S K" sort="De Hooge, Alfons S K" uniqKey="De Hooge A" first="Alfons S. K." last="De Hooge">Alfons S. K. De Hooge</name><affiliation><mods:affiliation>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: A.deHoogereuma.azn.nl</mods:affiliation></affiliation></author><author><name sortKey="Van De Loo, Fons A J" sort="Van De Loo, Fons A J" uniqKey="Van De Loo F" first="Fons A. J." last="Van De Loo">Fons A. J. Van De Loo</name><affiliation><mods:affiliation>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</mods:affiliation></affiliation></author><author><name sortKey="Arntz, Onno J" sort="Arntz, Onno J" uniqKey="Arntz O" first="Onno J." last="Arntz">Onno J. Arntz</name><affiliation><mods:affiliation>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</mods:affiliation></affiliation></author><author><name sortKey="Van Den Berg, Wim B" sort="Van Den Berg, Wim B" uniqKey="Van Den Berg W" first="Wim B." last="Van Den Berg">Wim B. Van Den Berg</name><affiliation><mods:affiliation>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">The American Journal of Pathology</title><title level="j" type="abbrev">AJPA</title><idno type="ISSN">0002-9440</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000">2000</date><biblScope unit="volume">157</biblScope><biblScope unit="issue">6</biblScope><biblScope unit="page" from="2081">2081</biblScope><biblScope unit="page" to="2091">2091</biblScope></imprint><idno type="ISSN">0002-9440</idno></series><idno type="istex">3FFA4817B217FC3EC96B07D190272F56761F0188</idno><idno type="DOI">10.1016/S0002-9440(10)64846-8</idno><idno type="PII">S0002-9440(10)64846-8</idno></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0002-9440</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract">Interleukin-6 (IL-6) is highly produced during arthritis but its exact function is still unknown. In this study we examined if IL-6, apart from its role in immunity, was involved in the local inflammatory response in experimental arthritis. IL-6 deficient (IL-6/) and wild-type mice were first compared in the antigen-induced arthritis model. IL-6 deficiency resulted in a mild, transient inflammation whereas wild-type mice developed a chronic, destructive synovitis. Wild-type mice immunized with one-tenth of the normal antigen dose still developed chronic arthritis despite low antibody levels, excluding reduced humoral immunity in IL-6/ mice as a crucial phenomenon. In addition, passive immune-complex-induced arthritis did not differ between wild-type and IL-6/ mice. Another option is reduced levels of Th1 cells in IL-6/ mice. However, transfer of antigen-specific wild-type lymph node cells to IL-6/ mice enhanced acute joint inflammation and increased cartilage damage but still could not sustain chronic inflammation, suggesting involvement of nonimmune elements of IL-6 activity in chronicity. In line with this, nonimmunologically mediated zymosan-induced arthritis developed similarly in the first week, but only wild-type mice developed chronic synovitis. These results indicate an important role for IL-6 in propagation of joint inflammation, potentially independent of its role in immunity.</div></front></TEI><istex><corpusName>elsevier</corpusName><author><json:item><name>Alfons S.K. de Hooge</name><affiliations><json:string>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</json:string><json:string>E-mail: A.deHoogereuma.azn.nl</json:string></affiliations></json:item><json:item><name>Fons A.J. van de Loo</name><affiliations><json:string>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</json:string></affiliations></json:item><json:item><name>Onno J. Arntz</name><affiliations><json:string>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</json:string></affiliations></json:item><json:item><name>Wim B. van den Berg</name><affiliations><json:string>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>IL-6 and Chronic Synovitis in Experimental RA</value></json:item></subject><language><json:string>eng</json:string></language><abstract>Interleukin-6 (IL-6) is highly produced during arthritis but its exact function is still unknown. In this study we examined if IL-6, apart from its role in immunity, was involved in the local inflammatory response in experimental arthritis. IL-6 deficient (IL-6/) and wild-type mice were first compared in the antigen-induced arthritis model. IL-6 deficiency resulted in a mild, transient inflammation whereas wild-type mice developed a chronic, destructive synovitis. Wild-type mice immunized with one-tenth of the normal antigen dose still developed chronic arthritis despite low antibody levels, excluding reduced humoral immunity in IL-6/ mice as a crucial phenomenon. In addition, passive immune-complex-induced arthritis did not differ between wild-type and IL-6/ mice. Another option is reduced levels of Th1 cells in IL-6/ mice. However, transfer of antigen-specific wild-type lymph node cells to IL-6/ mice enhanced acute joint inflammation and increased cartilage damage but still could not sustain chronic inflammation, suggesting involvement of nonimmune elements of IL-6 activity in chronicity. In line with this, nonimmunologically mediated zymosan-induced arthritis developed similarly in the first week, but only wild-type mice developed chronic synovitis. These results indicate an important role for IL-6 in propagation of joint inflammation, potentially independent of its role in immunity.</abstract><qualityIndicators><score>9.364</score><pdfVersion>1.7</pdfVersion><pdfPageSize>585 x 783 pts</pdfPageSize><refBibsNative>true</refBibsNative><keywordCount>1</keywordCount><abstractCharCount>1409</abstractCharCount><pdfWordCount>7380</pdfWordCount><pdfCharCount>44386</pdfCharCount><pdfPageCount>11</pdfPageCount><abstractWordCount>197</abstractWordCount></qualityIndicators><title>Involvement of IL-6, Apart from Its Role in Immunity, in Mediating a Chronic Response during Experimental Arthritis</title><pii><json:string>S0002-9440(10)64846-8</json:string></pii><genre><json:string>research-article</json:string></genre><host><volume>157</volume><pii><json:string>S0002-9440(10)X6128-4</json:string></pii><pages><last>2091</last><first>2081</first></pages><issn><json:string>0002-9440</json:string></issn><issue>6</issue><genre></genre><language><json:string>unknown</json:string></language><title>The American Journal of Pathology</title><publicationDate>2000</publicationDate></host><categories><wos><json:string>PATHOLOGY</json:string><json:string>CELL BIOLOGY</json:string></wos></categories><publicationDate>2000</publicationDate><copyrightDate>2000</copyrightDate><doi><json:string>10.1016/S0002-9440(10)64846-8</json:string></doi><id>3FFA4817B217FC3EC96B07D190272F56761F0188</id><fulltext><json:item><original>true</original><mimetype>application/pdf</mimetype><extension>pdf</extension><uri>https://api.istex.fr/document/3FFA4817B217FC3EC96B07D190272F56761F0188/fulltext/pdf</uri></json:item><json:item><original>false</original><mimetype>application/zip</mimetype><extension>zip</extension><uri>https://api.istex.fr/document/3FFA4817B217FC3EC96B07D190272F56761F0188/fulltext/zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/document/3FFA4817B217FC3EC96B07D190272F56761F0188/fulltext/tei"><teiHeader><fileDesc><titleStmt><title level="a">Involvement of IL-6, Apart from Its Role in Immunity, in Mediating a Chronic Response during Experimental Arthritis</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>ELSEVIER</publisher><availability><p>ELSEVIER</p></availability><date>2000</date></publicationStmt><notesStmt><note>Supported by a grant from the Dutch League against Rheumatism (NR97-2-402).</note><note type="content">Section title: Regular Articles</note><note type="content">Figure 1: Joint swelling at day 1 and 2 after mBSA injection. Immunized and naive mice were injected with 60 g of mBSA. Joint swelling was measured as the ratio of 99mTc uptake in the arthritic knee (right) over the nonarthritic knee (left). , IL-6/; , wild type (n = 7; , P < 0.05; , P < 0.005, Student's t-test).</note><note type="content">Figure 2: Inflammation and cartilage damage in wild-type (A and B) and IL-6/ (C and D) mice on day 2 (A and C) and 7 (B and D) of the AIA. Sections were stained with safranin-O and cartilage damage was observed as loss of red staining. E: Position of patella (P), femur (F), synovium (S), growth plate (GP), and cartilage (C) is shown. Original magnification, 200.</note><note type="content">Figure 3: mRNA expression in synovia from wild-type and IL-6/ mice on day 1 (A) and 2 (B) of the AIA. The number of PCR cycles needed to first detect the specific band on an agarose gel was compared between synovia from arthritic and contralateral nonarthritic knee joints. Increased mRNA expression for the investigated gene in arthritic synovia results in a reduced number of PCR cycles to first detect the specific band. Four mice per group were used and equal amounts of cDNA were used as assessed by PCR for glyceraldehyde-3-phosphate dehydrogenase (all appeared at cycle 14). Basal expression in the nonarthritic synovia did not differ between wild-type and IL-6/ mice. No signal for IL-6 was detected at the end point of the PCR with cDNA from IL-6/ mice. , IL-6/; , wild type.</note><note type="content">Figure 4: A: mBSA-specific antibody subclasses before onset of AIA. Titers as depicted are the dilution of sera needed to give half the maximal optical density at 450 nm as described in Materials and Methods. , IL-6/; , wild type (n = 7). B: mBSA-specific IgG2a titers in mice immunized and boostered with 100 g of mBSA (IL-6/. or with 10 or 100 g of mBSA (wild type). Each symbol represents one mouse (P = 0.22, IL-6/ (100 g) versus wild type, (10 g) Student's t-test). Total IgG titers also did not differ (IL-6/: 1/720 194, wild type: 1/734 180). C: Joint swelling in wild-type mice immunized with 10, 30, or 100 g of mBSA (n = 8). Joint swelling was measured as the ratio of 99mTc uptake in the arthritic knee (right) over the nonarthritic knee (left). , IL-6/; , wild type (A and C: , P < 0.05; , P < 0.005, Student's t-test). One of three experiments is shown.</note><note type="content">Figure 5: A: mBSA-specific increase in cpm (cpm mBSA minus cpm medium). Lymph nodes from seven mice per group were pooled and enriched for T cells as described in Materials and Methods. Lymph node cells (2106. together with 2107 irradiated antigen-presenting cells were incubated for 72 hours with mBSA at 25 g/ml. Cells were plated in sixfold. Cultures were labeled with 3H for the last 16 hours. Antigen-presenting cells were used from naive mice of the same strain. IL-6/ and wild-type mice did not differ in the response to concanavalin A at 1 g/ml (IL-6/, 28,002 7,291 cpm; wild type, 25,436 1,216 cpm. One of four experiments is shown. B: Th1/Th2 multiplex RT-PCR of IL-6/ (100 g mBSA) or wild-type (10 g mBSA) T cells stimulated for 24 hours with conA (1 g/ml). C = positive control.</note><note type="content">Figure 6: Joint swelling on day 2 of AIA in IL-6/ mice after transfer of 4107 lymph node cells derived from mBSA (wild-type/mBSA) or ovalbumin (wild-type/OVA. immunized wild-type mice (A) or of mBSA (IL-6//mBSA. immunized IL-6/ mice (B). NT = normal AIA in wild-type mice immunized with 10 g of mBSA. Joint swelling was measured as the ratio of 99mTc uptake in the arthritic knee (right) over the nonarthritic knee (left) (n = 6; , P < 0.05, Student's t-test, n.s. = not significant). , IL-6/, , wild type. One of three experiments is shown.</note><note type="content">Table 1: Development of Joint Inflammation and Cartilage Damage during AIA</note><note type="content">Table 2: Joint Inflammation and Cartilage Damage at Day 7 of AIA after Transfer of Wild-Type Lymph Node Cells</note><note type="content">Table 3: Synovial Infiltrate on Day 7 and 21 of Zymosan-Induced Arthritis</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a">Involvement of IL-6, Apart from Its Role in Immunity, in Mediating a Chronic Response during Experimental Arthritis</title><author><persName><forename type="first">Alfons S.K.</forename><surname>de Hooge</surname></persName><email>A.deHoogereuma.azn.nl</email><affiliation>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</affiliation></author><author><persName><forename type="first">Fons A.J.</forename><surname>van de Loo</surname></persName><affiliation>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</affiliation></author><author><persName><forename type="first">Onno J.</forename><surname>Arntz</surname></persName><affiliation>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</affiliation></author><author><persName><forename type="first">Wim B.</forename><surname>van den Berg</surname></persName><affiliation>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</affiliation></author></analytic><monogr><title level="j">The American Journal of Pathology</title><title level="j" type="abbrev">AJPA</title><idno type="pISSN">0002-9440</idno><idno type="PII">S0002-9440(10)X6128-4</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000"></date><biblScope unit="volume">157</biblScope><biblScope unit="issue">6</biblScope><biblScope unit="page" from="2081">2081</biblScope><biblScope unit="page" to="2091">2091</biblScope></imprint></monogr><idno type="istex">3FFA4817B217FC3EC96B07D190272F56761F0188</idno><idno type="DOI">10.1016/S0002-9440(10)64846-8</idno><idno type="PII">S0002-9440(10)64846-8</idno></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2000</date></creation><langUsage><language ident="en">en</language></langUsage><abstract><p>Interleukin-6 (IL-6) is highly produced during arthritis but its exact function is still unknown. In this study we examined if IL-6, apart from its role in immunity, was involved in the local inflammatory response in experimental arthritis. IL-6 deficient (IL-6/) and wild-type mice were first compared in the antigen-induced arthritis model. IL-6 deficiency resulted in a mild, transient inflammation whereas wild-type mice developed a chronic, destructive synovitis. Wild-type mice immunized with one-tenth of the normal antigen dose still developed chronic arthritis despite low antibody levels, excluding reduced humoral immunity in IL-6/ mice as a crucial phenomenon. In addition, passive immune-complex-induced arthritis did not differ between wild-type and IL-6/ mice. Another option is reduced levels of Th1 cells in IL-6/ mice. However, transfer of antigen-specific wild-type lymph node cells to IL-6/ mice enhanced acute joint inflammation and increased cartilage damage but still could not sustain chronic inflammation, suggesting involvement of nonimmune elements of IL-6 activity in chronicity. In line with this, nonimmunologically mediated zymosan-induced arthritis developed similarly in the first week, but only wild-type mice developed chronic synovitis. These results indicate an important role for IL-6 in propagation of joint inflammation, potentially independent of its role in immunity.</p></abstract><textClass><keywords scheme="keyword"><list><head>Article category</head><item><term>IL-6 and Chronic Synovitis in Experimental RA</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2000-08-25">Registration</change><change when="2000">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><original>false</original><mimetype>text/plain</mimetype><extension>txt</extension><uri>https://api.istex.fr/document/3FFA4817B217FC3EC96B07D190272F56761F0188/fulltext/txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier doc found" wicri:toSee="Elsevier, no converted or simple article"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 5.1.0//EN//XML" URI="art510.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="gr3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="gr4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="gr5"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="gr6"></istex:entity></istex:docType><istex:document><article docsubtype="fla"> <item-info> <jid>AJPA</jid> <aid>64846</aid> <ce:pii>S0002-9440(10)64846-8</ce:pii> <ce:doi>10.1016/S0002-9440(10)64846-8</ce:doi> <ce:copyright type="society" year="2000">American Society for Investigative Pathology</ce:copyright> <ce:doctopics> <ce:doctopic> <ce:text>IL-6 and Chronic Synovitis in Experimental RA</ce:text> </ce:doctopic> </ce:doctopics> </item-info> <ce:floats> <ce:figure id="fig1"> <ce:label>Figure 1</ce:label> <ce:caption> <ce:simple-para id="spara10">Joint swelling at day 1 and 2 after mBSA injection. Immunized and naive mice were injected with 60 μg of mBSA. Joint swelling was measured as the ratio of <ce:sup>99m</ce:sup>Tc uptake in the arthritic knee (<ce:bold>right</ce:bold>) over the nonarthritic knee (<ce:bold>left</ce:bold>). ▪, IL-6<ce:sup>−/−</ce:sup>; □, wild type (<ce:italic>n</ce:italic> = 7; *, <ce:italic>P</ce:italic> < 0.05; **, <ce:italic>P</ce:italic> < 0.005, Student's <ce:italic>t</ce:italic>-test).</ce:simple-para> </ce:caption> <ce:link locator="gr1"></ce:link> </ce:figure> <ce:figure id="fig2"> <ce:label>Figure 2</ce:label> <ce:caption> <ce:simple-para id="spara20">Inflammation and cartilage damage in wild-type (<ce:bold>A</ce:bold> and <ce:bold>B</ce:bold>) and IL-6<ce:sup>−/−</ce:sup> (<ce:bold>C</ce:bold> and <ce:bold>D</ce:bold>) mice on day 2 (<ce:bold>A</ce:bold> and <ce:bold>C</ce:bold>) and 7 (<ce:bold>B</ce:bold> and <ce:bold>D</ce:bold>) of the AIA. Sections were stained with safranin-O and cartilage damage was observed as loss of red staining. <ce:bold>E:</ce:bold> Position of patella (P), femur (F), synovium (S), growth plate (GP), and cartilage (C) is shown. Original magnification, ×200.</ce:simple-para> </ce:caption> <ce:link locator="gr2"></ce:link> </ce:figure> <ce:figure id="fig3"> <ce:label>Figure 3</ce:label> <ce:caption> <ce:simple-para id="spara30">mRNA expression in synovia from wild-type and IL-6<ce:sup>−/−</ce:sup> mice on day 1 (<ce:bold>A</ce:bold>) and 2 (<ce:bold>B</ce:bold>) of the AIA. The number of PCR cycles needed to first detect the specific band on an agarose gel was compared between synovia from arthritic and contralateral nonarthritic knee joints. Increased mRNA expression for the investigated gene in arthritic synovia results in a reduced number of PCR cycles to first detect the specific band. Four mice per group were used and equal amounts of cDNA were used as assessed by PCR for glyceraldehyde-3-phosphate dehydrogenase (all appeared at cycle 14). Basal expression in the nonarthritic synovia did not differ between wild-type and IL-6<ce:sup>−/−</ce:sup> mice. No signal for IL-6 was detected at the end point of the PCR with cDNA from IL-6<ce:sup>−/−</ce:sup> mice. ▪, IL-6<ce:sup>−/−</ce:sup>; □, wild type.</ce:simple-para> </ce:caption> <ce:link locator="gr3"></ce:link> </ce:figure> <ce:figure id="fig4"> <ce:label>Figure 4</ce:label> <ce:caption> <ce:simple-para id="spara40"><ce:bold>A:</ce:bold> mBSA-specific antibody subclasses before onset of AIA. Titers as depicted are the dilution of sera needed to give half the maximal optical density at 450 nm as described in Materials and Methods. ▪, IL-6<ce:sup>−/−</ce:sup>; □, wild type (<ce:italic>n</ce:italic> = 7). <ce:bold>B:</ce:bold> mBSA-specific IgG2a titers in mice immunized and boostered with 100 μg of mBSA (IL-6<ce:sup>−/−</ce:sup>. or with 10 or 100 μg of mBSA (wild type). Each symbol represents one mouse (<ce:italic>P</ce:italic> = 0.22, IL-6<ce:sup>−/−</ce:sup> (100 μg) <ce:italic>versus</ce:italic> wild type, (10 μg) Student's <ce:italic>t</ce:italic>-test). Total IgG titers also did not differ (IL-6<ce:sup>−/−</ce:sup>: 1/720 ± 194, wild type: 1/734 ± 180). <ce:bold>C:</ce:bold> Joint swelling in wild-type mice immunized with 10, 30, or 100 μg of mBSA (<ce:italic>n</ce:italic> = 8). Joint swelling was measured as the ratio of <ce:sup>99m</ce:sup>Tc uptake in the arthritic knee (<ce:bold>right</ce:bold>) over the nonarthritic knee (<ce:bold>left</ce:bold>). ▪, IL-6<ce:sup>−/−</ce:sup>; □, wild type (<ce:bold>A</ce:bold> and <ce:bold>C:</ce:bold> *, <ce:italic>P</ce:italic> < 0.05; **, <ce:italic>P</ce:italic> < 0.005, Student's <ce:italic>t</ce:italic>-test). One of three experiments is shown.</ce:simple-para> </ce:caption> <ce:link locator="gr4"></ce:link> </ce:figure> <ce:figure id="fig5"> <ce:label>Figure 5</ce:label> <ce:caption> <ce:simple-para id="spara50"><ce:bold>A:</ce:bold> mBSA-specific increase in cpm (cpm mBSA minus cpm medium). Lymph nodes from seven mice per group were pooled and enriched for T cells as described in Materials and Methods. Lymph node cells (2×10<ce:sup>6</ce:sup>. together with 2×10<ce:cross-ref refid="bib7"><ce:sup>7</ce:sup></ce:cross-ref> irradiated antigen-presenting cells were incubated for 72 hours with mBSA at 25 μg/ml. Cells were plated in sixfold. Cultures were labeled with <ce:sup>3</ce:sup>H for the last 16 hours. Antigen-presenting cells were used from naive mice of the same strain. IL-6<ce:sup>−/−</ce:sup> and wild-type mice did not differ in the response to concanavalin A at 1 μg/ml (IL-6<ce:sup>−/−</ce:sup>, 28,002 ± 7,291 cpm; wild type, 25,436 ± 1,216 cpm. One of four experiments is shown. <ce:bold>B:</ce:bold> Th1/Th2 multiplex RT-PCR of IL-6<ce:sup>−/−</ce:sup> (100 μg mBSA) or wild-type (10 μg mBSA) T cells stimulated for 24 hours with conA (1 μg/ml). C = positive control.</ce:simple-para> </ce:caption> <ce:link locator="gr5"></ce:link> </ce:figure> <ce:figure id="fig6"> <ce:label>Figure 6</ce:label> <ce:caption> <ce:simple-para id="spara60">Joint swelling on day 2 of AIA in IL-6<ce:sup>−/−</ce:sup> mice after transfer of 4×10<ce:cross-ref refid="bib7"><ce:sup>7</ce:sup></ce:cross-ref> lymph node cells derived from mBSA (wild-type/mBSA) or ovalbumin (wild-type/OVA. immunized wild-type mice (<ce:bold>A</ce:bold>) or of mBSA (IL-6<ce:sup>−/−</ce:sup>/mBSA. immunized IL-6<ce:sup>−/−</ce:sup> mice (<ce:bold>B</ce:bold>). NT = normal AIA in wild-type mice immunized with 10 μg of mBSA. Joint swelling was measured as the ratio of <ce:sup>99m</ce:sup>Tc uptake in the arthritic knee (<ce:bold>right</ce:bold>) over the nonarthritic knee (<ce:bold>left</ce:bold>) (<ce:italic>n</ce:italic> = 6; *, <ce:italic>P</ce:italic> < 0.05, Student's <ce:italic>t</ce:italic>-test, n.s. = not significant). ▪, IL-6<ce:sup>−/−</ce:sup>, □, wild type. One of three experiments is shown.</ce:simple-para> </ce:caption> <ce:link locator="gr6"></ce:link> </ce:figure> <ce:table id="tbl1" frame="topbot" rowsep="0" colsep="0"> <ce:label>Table 1</ce:label> <ce:caption> <ce:simple-para id="spara70">Development of Joint Inflammation and Cartilage Damage during AIA</ce:simple-para> </ce:caption> <tgroup cols="9" altimg="si1.gif"> <colspec colnum="1" colname="col1"></colspec> <colspec colnum="2" colname="col2"></colspec> <colspec colnum="3" colname="col3"></colspec> <colspec colnum="4" colname="col4"></colspec> <colspec colnum="5" colname="col5"></colspec> <colspec colnum="6" colname="col6"></colspec> <colspec colnum="7" colname="col7"></colspec> <colspec colnum="8" colname="col8"></colspec> <colspec colnum="9" colname="col9"></colspec> <thead> <row valign="top"> <entry></entry> <entry namest="col2" nameend="col3" align="center" rowsep="1">DAY 1 AIA</entry> <entry namest="col4" nameend="col5" align="center" rowsep="1">DAY 2 AIA</entry> <entry namest="col6" nameend="col7" align="center" rowsep="1">DAY 4 AIA</entry> <entry namest="col8" nameend="col9" align="center" rowsep="1">DAY 7 AIA</entry> </row> <row valign="top" rowsep="1"> <entry></entry> <entry align="center">IL-6−/− (<ce:italic>n</ce:italic> = 6)</entry> <entry align="center">WT (<ce:italic>n</ce:italic> = 10)</entry> <entry align="center">IL-6−/− (<ce:italic>n</ce:italic> = 9)</entry> <entry align="center">WT (<ce:italic>n</ce:italic> = 10)</entry> <entry align="center">IL-6−/− (<ce:italic>n</ce:italic> = 8)</entry> <entry align="center">WT (<ce:italic>n</ce:italic> = 13)</entry> <entry align="center">IL-6−/− (<ce:italic>n</ce:italic> = 8)</entry> <entry align="center">WT (<ce:italic>n</ce:italic> = 10)</entry> </row> </thead> <tbody> <row valign="top"> <entry align="left">Infiltrate</entry> <entry align="center">0.5 ± 0.3</entry> <entry align="center">0.7 ± 0.3</entry> <entry align="center">0.8 ± 0.4</entry> <entry align="center">1.6 ± 0.4<ce:cross-ref refid="tbl1fn2"><ce:sup>†</ce:sup></ce:cross-ref></entry> <entry align="center">0.4 ± 0.3</entry> <entry align="center">2.3 ± 0.8<ce:cross-ref refid="tbl1fn2"><ce:sup>†</ce:sup></ce:cross-ref></entry> <entry align="center">0.3 ± 0.2</entry> <entry align="center">2.8 ± 0.6<ce:cross-ref refid="tbl1fn2"><ce:sup>†</ce:sup></ce:cross-ref></entry> </row> <row valign="top"> <entry align="left">Exudate</entry> <entry align="center">1.5 ± 0.4</entry> <entry align="center">2.1 ± 0.5<ce:cross-ref refid="tbl1fn1"><ce:sup>*</ce:sup></ce:cross-ref></entry> <entry align="center">0.7 ± 0.4</entry> <entry align="center">1.9 ± 1.0*</entry> <entry align="center">0.1 ± 0.2</entry> <entry align="center">0.6 ± 0.5*</entry> <entry align="center">0.1 ± 0.1</entry> <entry align="center">1.5 ± 0.9<ce:cross-ref refid="tbl1fn2"><ce:sup>†</ce:sup></ce:cross-ref></entry> </row> <row valign="top"> <entry align="left">Patella</entry> <entry align="center">0.2 ± 0.2</entry> <entry align="center">0.4 ± 0.4</entry> <entry align="center">1.7 ± 0.6</entry> <entry align="center">2.5 ± 0.5*</entry> <entry align="center">1.7 ± 0.6</entry> <entry align="center">2.6 ± 0.7*</entry> <entry align="center">0.5 ± 0.7</entry> <entry align="center">3.0 ± 0.0<ce:cross-ref refid="tbl1fn2"><ce:sup>†</ce:sup></ce:cross-ref></entry> </row> <row valign="top"> <entry align="left">Femur</entry> <entry align="center">0.2 ± 0.3</entry> <entry align="center">0.7 ± 0.7</entry> <entry align="center">1.6 ± 0.9</entry> <entry align="center">2.3 ± 0.7</entry> <entry align="center">1.6 ± 0.6</entry> <entry align="center">2.7 ± 0.7<ce:cross-ref refid="tbl1fn2"><ce:sup>†</ce:sup></ce:cross-ref></entry> <entry align="center">0.8 ± 0.8</entry> <entry align="center">3.0 ± 0.0<ce:cross-ref refid="tbl1fn2"><ce:sup>†</ce:sup></ce:cross-ref></entry> </row> <row valign="top"> <entry align="left">Medial tibia</entry> <entry align="center">nd</entry> <entry align="center">nd</entry> <entry align="center">1.1 ± 0.6</entry> <entry align="center">2.6 ± 0.5<ce:cross-ref refid="tbl1fn2"><ce:sup>†</ce:sup></ce:cross-ref></entry> <entry align="center">1.9 ± 0.7</entry> <entry align="center">2.8 ± 0.4<ce:cross-ref refid="tbl1fn2"><ce:sup>†</ce:sup></ce:cross-ref></entry> <entry align="center">1.3 ± 0.9</entry> <entry align="center">2.6 ± 0.7<ce:cross-ref refid="tbl1fn2"><ce:sup>†</ce:sup></ce:cross-ref></entry> </row> </tbody> </tgroup> <ce:legend> <ce:simple-para id="spara80">Cellular infiltrate, exudate, and cartilage damage as observed in safranin-O-stained sections. Cartilage damage was assessed as loss of red staining observed in patella, femur, or medial tibia. Histology was scored by two independent observers and ranged from 0 (no infiltrate/exudate or no depletion) to 3 (maximal infiltrate/exudate or maximal depletion). Statistical differences between IL-6<ce:sup>−/−</ce:sup> and wild-type mice are indicated (nd, not determined;</ce:simple-para> </ce:legend> <ce:table-footnote id="tbl1fn1"> <ce:label>*</ce:label> <ce:note-para><ce:italic>P</ce:italic> <0.05</ce:note-para> </ce:table-footnote> <ce:table-footnote id="tbl1fn2"> <ce:label>†</ce:label> <ce:note-para><ce:italic>P</ce:italic> <0.005; Student's t-test). WT, wild type.</ce:note-para> </ce:table-footnote> </ce:table> <ce:table id="tbl2" frame="topbot" rowsep="0" colsep="0"> <ce:label>Table 2</ce:label> <ce:caption> <ce:simple-para id="spara90">Joint Inflammation and Cartilage Damage at Day 7 of AIA after Transfer of Wild-Type Lymph Node Cells</ce:simple-para> </ce:caption> <tgroup cols="4" altimg="si2.gif"> <colspec colnum="1" colname="col1"></colspec> <colspec colnum="2" colname="col2"></colspec> <colspec colnum="3" colname="col3"></colspec> <colspec colnum="4" colname="col4"></colspec> <thead> <row valign="top" rowsep="1"> <entry align="left">Acceptor</entry> <entry align="center">IL-6<ce:sup>−/−</ce:sup> (<ce:italic>n</ce:italic> = 6)</entry> <entry align="center">IL-6<ce:sup>−/−</ce:sup> (<ce:italic>n</ce:italic> = 11)</entry> <entry align="center">WT (<ce:italic>n</ce:italic> = 11)</entry> </row> </thead> <tbody> <row valign="top"> <entry align="left">Donor</entry> <entry align="center">WT/OVA</entry> <entry align="center">WT/mBSA</entry> <entry align="center">No transfer</entry> </row> <row valign="top"> <entry align="left">Infiltrate</entry> <entry align="char" char=".">0.2 ± 0.1</entry> <entry align="char" char=".">0.8 ± 0.7</entry> <entry align="char" char=".">2.7 ± 0.5<ce:cross-ref refid="tbl2fn2"><ce:sup>†</ce:sup></ce:cross-ref></entry> </row> <row valign="top"> <entry align="left">Exudate</entry> <entry align="char" char=".">0.0 ± 0.0<ce:cross-ref refid="tbl2fn1"><ce:sup>*</ce:sup></ce:cross-ref></entry> <entry align="char" char=".">0.6 ± 0.6</entry> <entry align="char" char=".">1.5 ± 0.6<ce:cross-ref refid="tbl2fn2"><ce:sup>†</ce:sup></ce:cross-ref></entry> </row> <row valign="top"> <entry align="left">Patella</entry> <entry align="char" char=".">0.3 ± 0.6*</entry> <entry align="char" char=".">1.4 ± 1.1</entry> <entry align="char" char=".">3.0 ± 0.0<ce:cross-ref refid="tbl2fn2"><ce:sup>†</ce:sup></ce:cross-ref></entry> </row> <row valign="top"> <entry align="left">Femur</entry> <entry align="char" char=".">0.8 ± 0.9<ce:cross-ref refid="tbl2fn2"><ce:sup>†</ce:sup></ce:cross-ref></entry> <entry align="char" char=".">2.0 ± 0.7</entry> <entry align="char" char=".">3.0 ± 0.0<ce:cross-ref refid="tbl2fn2"><ce:sup>†</ce:sup></ce:cross-ref></entry> </row> <row valign="top"> <entry align="left">Medial tibia</entry> <entry align="char" char=".">0.8 ± 0.8*</entry> <entry align="char" char=".">2.1 ± 1.0</entry> <entry align="char" char=".">2.9 ± 0.3*</entry> </row> </tbody> </tgroup> <ce:legend> <ce:simple-para id="spara100">Cellular infiltrate, exudate, and cartilage damage at day 7 after cell transfer and induction of AIA was scored as described in <ce:cross-ref refid="tbl1">Table 1</ce:cross-ref>. WT/OVA, transfer of lymph node cells from ovalbumin immunized wild-type mice. WT, wild-type mice immunized with 10 μg of mBSA. Statistical differences relative to IL-6<ce:sup>−/−</ce:sup> mice plus WT/mBSA lymph node cells are indicated</ce:simple-para> </ce:legend> <ce:table-footnote id="tbl2fn1"> <ce:label>*</ce:label> <ce:note-para><ce:italic>P</ce:italic> <0.05;</ce:note-para> </ce:table-footnote> <ce:table-footnote id="tbl2fn2"> <ce:label>†</ce:label> <ce:note-para><ce:italic>P</ce:italic> <0.005; Student's<ce:italic>t</ce:italic>-test). WT/mBSA, transfer of lymph node cells from mBSA-immunized wild-type mice.</ce:note-para> </ce:table-footnote> </ce:table> <ce:table id="tbl3" frame="topbot" rowsep="0" colsep="0"> <ce:label>Table 3</ce:label> <ce:caption> <ce:simple-para id="spara110">Synovial Infiltrate on Day 7 and 21 of Zymosan-Induced Arthritis</ce:simple-para> </ce:caption> <tgroup cols="3" altimg="si3.gif"> <colspec colnum="1" colname="col1"></colspec> <colspec colnum="2" colname="col2"></colspec> <colspec colnum="3" colname="col3"></colspec> <thead> <row valign="top" rowsep="1"> <entry align="left">Infiltrate</entry> <entry align="center">IL-6<ce:sup>−/−</ce:sup></entry> <entry align="center">Wild-type</entry> </row> </thead> <tbody> <row valign="top"> <entry align="left">Day 7 ZIA</entry> <entry align="center">1.4 ± 0.6</entry> <entry align="center">1.9 ± 1.0 ns</entry> </row> <row valign="top"> <entry align="left">Day 21 ZIA</entry> <entry align="center">0.3 ± 0.3</entry> <entry align="center">2.2 ± 0.7*</entry> </row> </tbody> </tgroup> <ce:legend> <ce:simple-para id="spara120">Cellular infiltrate as observed in safranin-O-stained sections. Histology was scored as described in <ce:cross-ref refid="tbl1">Table 1</ce:cross-ref> (n = 7 per group). One of three experiments is shown. (*, <ce:italic>P</ce:italic> <0.005, Student's t-test; ns = not significant).</ce:simple-para> </ce:legend> </ce:table> </ce:floats> <head> <ce:article-footnote> <ce:note-para>Supported by a grant from the <ce:grant-sponsor id="GS1">Dutch League against Rheumatism</ce:grant-sponsor> (<ce:grant-number refid="GS1">NR97-2-402</ce:grant-number>).</ce:note-para> </ce:article-footnote> <ce:dochead> <ce:textfn>Regular Articles</ce:textfn> </ce:dochead> <ce:title>Involvement of IL-6, Apart from Its Role in Immunity, in Mediating a Chronic Response during Experimental Arthritis</ce:title> <ce:author-group> <ce:author> <ce:given-name>Alfons S.K.</ce:given-name> <ce:surname>de Hooge</ce:surname><ce:cross-ref refid="cor1"><ce:sup>*</ce:sup></ce:cross-ref> <ce:e-address type="email">A.deHooge@reuma.azn.nl</ce:e-address> </ce:author> <ce:author> <ce:given-name>Fons A.J.</ce:given-name> <ce:surname>van de Loo</ce:surname> </ce:author> <ce:author> <ce:given-name>Onno J.</ce:given-name> <ce:surname>Arntz</ce:surname> </ce:author> <ce:author> <ce:given-name>Wim B.</ce:given-name> <ce:surname>van den Berg</ce:surname> </ce:author> <ce:affiliation> <ce:textfn>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</ce:textfn> </ce:affiliation> <ce:correspondence id="cor1"> <ce:label>*</ce:label> <ce:text>Address reprint requests to Alfons S. K. de Hooge, Rheumatology Research Laboratory, University Medical Center Nijmegen, Geert Grooteplein Zuid 8, 6525 GA Nijmegen, The Netherlands. E-mail:</ce:text> </ce:correspondence> </ce:author-group> <ce:date-accepted day="25" month="8" year="2000"></ce:date-accepted> <ce:abstract> <ce:abstract-sec> <ce:simple-para id="spara130">Interleukin-6 (IL-6) is highly produced during arthritis but its exact function is still unknown. In this study we examined if IL-6, apart from its role in immunity, was involved in the local inflammatory response in experimental arthritis. IL-6 deficient (IL-6<ce:sup>−/−</ce:sup>) and wild-type mice were first compared in the antigen-induced arthritis model. IL-6 deficiency resulted in a mild, transient inflammation whereas wild-type mice developed a chronic, destructive synovitis. Wild-type mice immunized with one-tenth of the normal antigen dose still developed chronic arthritis despite low antibody levels, excluding reduced humoral immunity in IL-6<ce:sup>−/−</ce:sup> mice as a crucial phenomenon. In addition, passive immune-complex-induced arthritis did not differ between wild-type and IL-6<ce:sup>−/−</ce:sup> mice. Another option is reduced levels of Th1 cells in IL-6<ce:sup>−/−</ce:sup> mice. However, transfer of antigen-specific wild-type lymph node cells to IL-6<ce:sup>−/−</ce:sup> mice enhanced acute joint inflammation and increased cartilage damage but still could not sustain chronic inflammation, suggesting involvement of nonimmune elements of IL-6 activity in chronicity. In line with this, nonimmunologically mediated zymosan-induced arthritis developed similarly in the first week, but only wild-type mice developed chronic synovitis. These results indicate an important role for IL-6 in propagation of joint inflammation, potentially independent of its role in immunity.</ce:simple-para> </ce:abstract-sec> </ce:abstract> </head> <body> <ce:sections> <ce:para id="para10">Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by a chronic inflammation of the joints. This inflammation finally leads to tissue destruction that disables the patient. Although the exact cause of RA is not yet known pro- and anti-inflammatory cytokines seem to play an important role in the pathology of the disease.<ce:cross-ref refid="bib1"><ce:sup>1</ce:sup></ce:cross-ref> Interleukin-6 (IL-6) is a member of the IL-6 family to which leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and IL-11 also belong.<ce:cross-refs refid="bib2 bib3"><ce:sup>2,3</ce:sup></ce:cross-refs> Both IL-6 and the agonistic soluble IL-6 receptor are found in large quantities in synovial fluid and serum of RA patients.<ce:cross-ref refid="bib4"><ce:sup>4</ce:sup></ce:cross-ref> The main producers of IL-6 in the inflamed joint are articular chondrocytes and synovial fibroblasts.<ce:cross-refs refid="bib5 bib6"><ce:sup>5,6</ce:sup></ce:cross-refs> Studies on the relation of disease activity and IL-6 concentration have yielded conflicting results.<ce:cross-refs refid="bib7 bib8 bib9"><ce:sup>7–9</ce:sup></ce:cross-refs> Anti-IL-6 monoclonal antibodies showed transitory clinical improvement in RA patients.<ce:cross-ref refid="bib10"><ce:sup>10</ce:sup></ce:cross-ref> Surprisingly, this effect was accompanied by an increase in IL-6 serum levels, which makes it unclear what caused the improvement.</ce:para> <ce:para id="para20">Both pro- and anti-inflammatory properties have been ascribed to IL-6, complicating the establishment of its role in RA. IL-6 plays an important role in the maturation of B cells into antibody-secreting plasma cells,<ce:cross-ref refid="bib11"><ce:sup>11</ce:sup></ce:cross-ref> differentiation of osteoclasts<ce:cross-ref refid="bib12"><ce:sup>12</ce:sup></ce:cross-ref> and macrophages,<ce:cross-ref refid="bib13"><ce:sup>13</ce:sup></ce:cross-ref> generation of an acute-phase response in the liver,<ce:cross-refs refid="bib14 bib15 bib16"><ce:sup>14–16</ce:sup></ce:cross-refs> and has a co-stimulatory role in T cell activation.<ce:cross-refs refid="bib17 bib18"><ce:sup>17,18</ce:sup></ce:cross-refs> On the other hand, IL-6 can induce expression of IL-1 receptor antagonist, soluble tumor necrosis factor (TNF) receptor, and tissue inhibitor of metalloproteinases,<ce:cross-refs refid="bib19 bib20"><ce:sup>19,20</ce:sup></ce:cross-refs> which could down-regulate inflammation and reduce connective tissue damage in the inflamed joint. IL-6 also can reduce TNF production.<ce:cross-ref refid="bib21"><ce:sup>21</ce:sup></ce:cross-ref></ce:para> <ce:para id="para30">The dual face of IL-6 as a pro- and anti-inflammatory protein is also reflected by studies in IL-6 gene knockout (IL-6<ce:sup>−/−</ce:sup>) mice. The local inflammatory response against turpentine was impaired in IL-6<ce:sup>−/−</ce:sup> mice whereas systemic inflammatory reactions on lipopolysaccharide were not.<ce:cross-ref refid="bib22"><ce:sup>22</ce:sup></ce:cross-ref> The inflammatory response against <ce:italic>Candida albicans</ce:italic> was also impaired in IL-6<ce:sup>−/−</ce:sup> mice.<ce:cross-ref refid="bib23"><ce:sup>23</ce:sup></ce:cross-ref> Xing et al<ce:cross-ref refid="bib24"><ce:sup>24</ce:sup></ce:cross-ref> in contrast found increased inflammatory reactions in endotoxic lung or during endotoxemia in IL-6<ce:sup>−/−</ce:sup> mice and proposed an anti-inflammatory role of IL-6 during acute infection. IL-6<ce:sup>−/−</ce:sup> mice also had a higher incidence of arthritis after infection with <ce:italic>Borrelia burgdorferi</ce:italic><ce:cross-ref refid="bib25"><ce:sup>25</ce:sup></ce:cross-ref> demonstrating an anti-inflammatory role of IL-6. In a previous study we looked into the role of IL-6 in zymosan-induced arthritis (ZIA),<ce:cross-ref refid="bib26"><ce:sup>26</ce:sup></ce:cross-ref> a nonimmunologically mediated irritant-induced joint inflammation.<ce:cross-ref refid="bib27"><ce:sup>27</ce:sup></ce:cross-ref> During the first week of ZIA the inflammation developed synchronically in IL-6<ce:sup>−/−</ce:sup> and wild-type mice. Intriguingly, cartilage damage was increased in the IL-6<ce:sup>−/−</ce:sup> mice, pointing at a cartilage protective role for IL-6. A recent study by Ohshima et al<ce:cross-ref refid="bib28"><ce:sup>28</ce:sup></ce:cross-ref> showed the importance of IL-6 for development of antigen-induced arthritis (AIA), an immunologically mediated model with features of RA such as synovial hyperplasia, influx of inflammatory cells, and cartilage damage.<ce:cross-ref refid="bib29"><ce:sup>29</ce:sup></ce:cross-ref> Their study focused at the outcome of arthritis at day 14 and differences in the antigen-specific immunity. It remains unclear what caused amelioration of the disease in IL-6<ce:sup>−/−</ce:sup> mice: the developed, but impaired, antigen-specific immune response or the absence of IL-6 during the inflammation. In the present study we wanted to examine if IL-6, independent of its role in immunity was involved in the inflammatory response in different experimental arthritis models. In these models wild-type and IL-6<ce:sup>−/−</ce:sup> mice were compared. We confirmed that initial inflammation in IL-6<ce:sup>−/−</ce:sup> mice did not develop into a chronic inflammatory infiltrate during AIA. Differences in cellular but not humoral immunity had major influence on the onset of AIA. However, transfer of wild-type lymph node cells enhanced the mild inflammatory response in IL-6<ce:sup>−/−</ce:sup> mice but still did not lead to a chronic infiltrate. In the nonimmunologically mediated ZIA we also found that the acute inflammation of the first week did not develop into a chronic synovial infiltrate in IL-6<ce:sup>−/−</ce:sup> mice. These results suggest that in both immunologically and nonimmunologically mediated experimental arthritis, there is an important role for IL-6 in propagation of the inflammatory infiltrate.</ce:para> <ce:section id="cesec10"> <ce:section-title>Materials and Methods</ce:section-title> <ce:section id="cesec20"> <ce:section-title>IL-6<ce:sup>−/−</ce:sup> and Wild-Type Mice</ce:section-title> <ce:para id="para40">Homozygous IL-6<ce:sup>−/−</ce:sup> and wild-type (C57Bl/6×129/Sv) F2 mice<ce:cross-ref refid="bib30"><ce:sup>30</ce:sup></ce:cross-ref> and IL-6<ce:sup>−/−</ce:sup> mice back-crossed into C57Bl/6 for eight generations (N8) were obtained from Dr. M. Kopf (Basel, Switzerland) and bred in our SPF animal facilities. IL-6 deficiency was routinely checked by polymerase chain reaction (PCR) of genomic DNA. A standard diet and acidified tap water were provided <ce:italic>ad libitum</ce:italic>. At the age of 11 to 13 weeks the animals were used in the experiments. Experiments were performed according to national and institutional regulations for animal use.</ce:para> </ce:section> <ce:section id="cesec30"> <ce:section-title>AIA</ce:section-title> <ce:para id="para50">Mice were immunized with 100 μg of methylated bovine serum albumin (mBSA; Sigma, St. Louis, MO) in Freund's complete adjuvant (Difco, Detroit, MI) divided over the front paws and both flanks. They also received an intraperitoneal injection of 2×10<ce:cross-ref refid="bib9"><ce:sup>9</ce:sup></ce:cross-ref> heat-killed <ce:italic>Bordetella pertussis</ce:italic> bacteria (National Institute of Public Health, Bilthoven, The Netherlands) in 1 ml of saline. Seven days later, mice were boostered with 100 μg of mBSA in Freund's complete adjuvant divided over two places in the neck region. Three weeks after the booster 60 μg of mBSA in saline (6 μl total volume) was injected in the knee joint cavity of the right hind leg to induce arthritis.</ce:para> </ce:section> <ce:section id="cesec40"> <ce:section-title>Immune-Complex-Induced Arthritis</ce:section-title> <ce:para id="para60">An immune-complex-induced arthritis was elicited in naive mice as described by van Lent et al.<ce:cross-ref refid="bib31"><ce:sup>31</ce:sup></ce:cross-ref> Mice were injected intravenously with 0.2 ml of a polyclonal rabbit anti-lysozyme serum of which the complement had been heat-inactivated. Arthritis was induced 16 hours later by injecting 6 μl of poly-<ce:small-caps>l</ce:small-caps>-lysine-coupled lysozyme (3 μg; Sigma) in the joint.</ce:para> </ce:section> <ce:section id="cesec50"> <ce:section-title>ZIA</ce:section-title> <ce:para id="para70">ZIA was elicited by intra-articular injection of 180 μg of zymosan A (<ce:italic>Saccharomyces cerevisiae</ce:italic>) as described before.<ce:cross-ref refid="bib26"><ce:sup>26</ce:sup></ce:cross-ref></ce:para> </ce:section> <ce:section id="cesec60"> <ce:section-title>Assessment of Joint Swelling</ce:section-title> <ce:para id="para80">Mice were injected subcutaneously in the neck with 20 μCi of <ce:sup>99m</ce:sup>Technetium pertechnetate (<ce:sup>99m</ce:sup>Tc) in 0.2 ml of physiological saline. After 15 minutes, mice were sedated by intraperitoneal injection of 4.5. chloral hydrate (0.1 ml/10 mg of body weight; Merck, Darmstadt, Germany). Accumulation of the isotope because of increased blood flow and edema in the knee was determined in duplicate by external γ counting using a NaI crystal. The ratio of <ce:sup>99m</ce:sup>Tc uptake in the inflamed over the contralateral knee joint was determined and a ratio higher than 1.1 indicated joint swelling.</ce:para> </ce:section> <ce:section id="cesec70"> <ce:section-title>Histological Evaluation of Knee Joints</ce:section-title> <ce:para id="para90">Knee joints were dissected, fixed in formalin, decalcified, dehydrated, and embedded in paraffin. Standard frontal sections of 7 μm were prepared. For assessing cartilage damage, sections were stained with safranin-O and counterstained with fast green. Serial sections were scored in a blind-folded manner by two independent observers. Cartilage depletion was scored from 0 (normal safranin-O staining, no depletion) up to 3 (complete loss of safranin-O staining, complete depletion). Also cellular infiltrate and exudate were scored in a scale from 0 up to 3.</ce:para> </ce:section> <ce:section id="cesec80"> <ce:section-title>Semiquantitative Reverse Transcriptase (RT)-PCR on Synovial mRNA</ce:section-title> <ce:para id="para100">Synovial mRNA was isolated and quantitated as described by van Meurs et al.<ce:cross-ref refid="bib32"><ce:sup>32</ce:sup></ce:cross-ref> Patellae were isolated from knee joints and two pieces of tissue adjacent to the patella were punched out with a 3-mm biopsy punch (Stiefel Laboratorium GMbH, Offenbach am Main, Germany). The tissue was immediately frozen in liquid nitrogen. Tissue samples were homogenized in a freeze mill, thawed in 1 ml of TRIzol reagent, and further processed according to the manufacturers protocol. All reagents for RNA isolation and RT-PCR were from Life Technologies (Breda, The Netherlands). Isolated RNA was treated with DNase before being reverse-transcribed into cDNA with Moloney murine leukemia virus reverse transcriptase. After increasing numbers of PCR cycles samples were taken and run on an agarose gel. The cycle number at which the PCR product was first detected on the gel was taken as a measure for the amount of specific mRNA originally present in the isolated synovial RNA. PCR for glyceraldehyde-3-phosphate dehydrogenase was performed to verify that equal amounts of cDNA were used. Primers, annealing temperature, and MgCl<ce:inf>2</ce:inf> concentration for murine VCAM-1,<ce:cross-ref refid="bib33"><ce:sup>33</ce:sup></ce:cross-ref> ICAM-1, E- and P-selectin,<ce:cross-ref refid="bib34"><ce:sup>34</ce:sup></ce:cross-ref> MCP-1, Mip-1α and Mip-2 (Blom et al, submitted), TNF-α, IL-1β, and glyceraldehyde-3-phosphate dehydrogenase<ce:cross-ref refid="bib35"><ce:sup>35</ce:sup></ce:cross-ref> were as described.</ce:para> </ce:section> <ce:section id="cesec90"> <ce:section-title>Assessment of mBSA-Specific Antibody Titers</ce:section-title> <ce:para id="para110">mBSA-specific antibody titers in sera were determined by enzyme-linked immunosorbent assay. Ninety-six-well flat-bottom plates (Costar, Corning, NY) were coated overnight with mBSA (0.1 mg/ml. Sigma), blocked the next day with gelatin (1% in phosphate-buffered saline) and incubated with serial dilutions of the sera. Horseradish peroxidase-labeled secondary antibodies were from Southern Biotechnologies Associates (goat anti-mouse IgG1 or anti-mouse IgG2b. Birmingham, AL) or from Nordic (rat anti-mouse IgG or anti-mouse IgG2a. Tilburg, The Netherlands). Color development after adding substrate (5-aminosalicylzuur, 0.8 mg/ml, 0.8 μl 30. H<ce:inf>2</ce:inf>O<ce:inf>2</ce:inf>/ml in 50 mmol/L of sodium-phosphate, pH 6.0) was monitored in an enzyme-linked immunosorbent assay reader at 450 nm. Dilution curves were plotted and the dilution at optical density 50% was determined. A standard serum of mBSA immunized C57Bl/6 mice was included for intra-assay variation.</ce:para> </ce:section> <ce:section id="cesec100"> <ce:section-title>Lymphocyte Stimulation Test</ce:section-title> <ce:para id="para120">Antigen-specific T cell immunity was determined by the proliferative response of T cells to mBSA. Axillary and inguinal lymph nodes were isolated under sterile conditions and disrupted. The suspension was enriched for T cells by nylon wool adherence for 30 minutes. Nonadherent cells were washed from the column and adjusted to 2×10<ce:cross-ref refid="bib6"><ce:sup>6</ce:sup></ce:cross-ref> cells/ml in medium (RPMI/penicillin 100 U/ml and streptomycin 100 μg/ml/1 mmol/L pyruvate/5% fetal calf serum; Life technologies). Cells were plated in a 96-well round-bottom plate (Costar) and antigen-presenting cells were added at 2×10<ce:sup>7</ce:sup>/ml. As antigen-presenting cells irradiated spleen cells from naive mice were used after disruption and lysis of erythrocytes (in 17 mmol/L Tris/144 mmol/L ammonium chloride, pH 7.2). Cells were incubated in three- or sixfold with medium, medium with twofold serial dilutions of mBSA starting at 25 μg/ml (data shown for 25 μg/ml) or with concanavalin A (Flow laboratories, Irvine UK) at 1 μg/ml as an aspecific stimulus. The plates were incubated for 3 days at 37°C and 5% CO<ce:inf>2</ce:inf>. For the last 16 hours the cells were labeled with 0.25 μCi <ce:sup>3</ce:sup>H-thymidine. After harvesting, incorporation of <ce:sup>3</ce:sup>H-thymidine was measured in a β-plate reader and the increase in counts per minute (cpm) caused by stimulation with mBSA was determined.</ce:para> </ce:section> <ce:section id="cesec110"> <ce:section-title>Multiplex PCR for T Cell Subsets</ce:section-title> <ce:para id="para130">T cells were isolated and stimulated as described above. After 1 day RNA was isolated as described above. For RT-PCR analysis of T cell subsets the mouse Th1/Th2 CytoXpress kit was used according to the manufacturers protocol (BioSource, Camarillo, Ca). Products were run on a 2% agarose gel and analyzed with the multianalyst system (BioRad, Richmond, CA).</ce:para> </ce:section> <ce:section id="cesec120"> <ce:section-title>Cell Transfer Experiments</ce:section-title> <ce:para id="para140">For cell transfer experiments single-cell preparations of lymph nodes (axillary and inguinal) were made from immunized wild-type or IL-6<ce:sup>−/−</ce:sup> mice. To prevent a possible graft-<ce:italic>versus</ce:italic>-host reaction C57Bl/6 mice were used as donors and C57Bl/6 IL-6<ce:sup>−/−</ce:sup> (N8) mice as recipients. Arthritis development did not differ between these mice and the C57Bl/6×129/Sv IL-6<ce:sup>−/−</ce:sup> mice (data not shown). Lymph node cells (4×10<ce:sup>7</ce:sup>) in 200 μl of RPMI medium were injected in the tail vein of immunized IL-6<ce:sup>−/−</ce:sup> mice before induction of arthritis in the knee joint. As controls IL-6<ce:sup>−/−</ce:sup> mice were injected intravenously with lymph node cells from ovalbumin (Sigma. immunized wild-type mice.</ce:para> </ce:section> <ce:section id="cesec130"> <ce:section-title>IL-6 Measurement</ce:section-title> <ce:para id="para150">For measuring local IL-6 production in the joint the patella was isolated with surrounding soft tissue (tendon and synovium). The patella was incubated in 200 μl of serum-free RPMI 1640 for 1 hour. The concentration of IL-6 in these patellar wash-outs and sera was determined by a B9 bioassay as described before.<ce:cross-ref refid="bib26"><ce:sup>26</ce:sup></ce:cross-ref></ce:para> </ce:section> <ce:section id="cesec140"> <ce:section-title>Statistical Analysis</ce:section-title> <ce:para id="para160">Statistical comparison between groups was performed with Student's <ce:italic>t</ce:italic>-test. Values of <ce:italic>P</ce:italic> < 0.05 were considered significant.</ce:para> </ce:section> </ce:section> <ce:section id="cesec150"> <ce:section-title>Results</ce:section-title> <ce:section id="cesec160"> <ce:section-title>Joint Inflammation Subsided after Day 1 in. IL-6<ce:sup>−/−</ce:sup> Mice</ce:section-title> <ce:para id="para170">After immunization a mono-articular arthritis was induced by injecting 60 μg of mBSA in the knee joint cavity of wild-type and IL-6<ce:sup>−/−</ce:sup> mice. Joints of both strains became highly swollen at day 1 after injection (<ce:cross-ref refid="fig1">Figure 1</ce:cross-ref><ce:float-anchor refid="fig1"></ce:float-anchor>) but thereafter swelling disappeared rapidly in IL-6<ce:sup>−/−</ce:sup> mice, whereas it subsided more gradually and remained significantly higher in wild-type mice. Histological evaluation of the arthritic knee joints showed onset of inflammation in IL-6<ce:sup>−/−</ce:sup> mice but the inflammatory infiltrate disappeared rapidly within the first week (<ce:cross-ref refid="tbl1">Table 1</ce:cross-ref><ce:float-anchor refid="tbl1"></ce:float-anchor>). In both strains the first cells entering the joint were predominantly polymorphonuclear cells. At day 2 a synovial infiltrate became apparent although smaller in knee joint cavities of IL-6<ce:sup>−/−</ce:sup> mice (<ce:cross-ref refid="fig2">Figure 2, A and B</ce:cross-ref><ce:float-anchor refid="fig2"></ce:float-anchor>). By day 7 joints from wild-type mice were highly inflamed whereas the cellular infiltrate and the cartilage proteoglycan depletion had almost disappeared in the IL-6<ce:sup>−/−</ce:sup> mice (<ce:cross-ref refid="fig2">Figure 2, C and D</ce:cross-ref>; <ce:cross-ref refid="tbl1">Table 1</ce:cross-ref>). In wild-type mice the inflammation persisted for weeks. Flare-up of the inflammation in wild-type mice by low doses of mBSA, that normally do not induce arthritis, further illustrates reactivity of the chronic synovitis (joint swelling 24 hours after injection on day 21 of AIA, intra-articular saline 1.15 ± 0.09 or intra-articular 2 μg mBSA 1.58 ± 0.24, <ce:italic>n</ce:italic> = 7, one of three experiments).</ce:para> </ce:section> <ce:section id="cesec170"> <ce:section-title>Expression of Adhesion Molecules and Chemokines during Onset of AIA</ce:section-title> <ce:para id="para180">IL-6 together with its soluble receptor has been shown to stimulate gene expression mediating cellular influx<ce:cross-refs refid="bib36 bib37"><ce:sup>36,37</ce:sup></ce:cross-refs> and this could be impaired in joints of IL-6<ce:sup>−/−</ce:sup> mice during onset of AIA. We therefore compared synovial gene expression in the arthritic joint with basal expression in the contralateral uninjected joint by RT-PCR.<ce:cross-ref refid="bib32"><ce:sup>32</ce:sup></ce:cross-ref> Both IL-6<ce:sup>−/−</ce:sup> and wild-type mice showed increased mRNA expression for the investigated adhesion molecules (ICAM-1, VCAM-1, E-selectin, and P-selectin), chemokines (MCP-1, Mip-1α, and Mip-2) and cytokines (TNF-α and IL-1β) in the arthritic joint at 24 (<ce:cross-ref refid="fig3">Figure 3A</ce:cross-ref><ce:float-anchor refid="fig3"></ce:float-anchor>) and 48 hours (<ce:cross-ref refid="fig3">Figure 3B</ce:cross-ref>) after injection. IL-6 mRNA expression was also highly increased in wild-type mice but undetectable in IL-6<ce:sup>−/−</ce:sup> mice. For this set of genes IL-6<ce:sup>−/−</ce:sup> mice responded similarly to wild-type mice at the mRNA level. This suggests that joints of IL-6<ce:sup>−/−</ce:sup> mice could support a normal influx of inflammatory cells during onset of AIA.</ce:para> </ce:section> <ce:section id="cesec180"> <ce:section-title>Complement Activation in IL-6<ce:sup>−/−</ce:sup> Mice</ce:section-title> <ce:para id="para190">A reduced activation of complement in IL-6<ce:sup>−/−</ce:sup> mice could contribute to the short-lasting inflammation. To test this possibility we induced an immune-complex-induced-arthritis in both strains by administrating antigen and antibodies to naive mice. Previously this model was shown to depend on complement activation.<ce:cross-ref refid="bib31"><ce:sup>31</ce:sup></ce:cross-ref> During immune-complex arthritis joint swelling on day 2 did not differ between both strains (IL-6<ce:sup>−/−</ce:sup>,1.37 ± 0.16. wild-type, 1.34 ± 0.12; one of two experiments, <ce:italic>n</ce:italic> = 6). Also the inflammatory infiltrate at day 7 did not differ between IL-6<ce:sup>−/−</ce:sup> and wild-type mice (IL-6<ce:sup>−/−</ce:sup>, 1.5 ± 1.0; wild-type, 1.2 ± 0.6; one of two experiments, <ce:italic>n</ce:italic> = 6). These results indicate that IL-6<ce:sup>−/−</ce:sup> mice can support a normal complement-mediated joint inflammation.</ce:para> </ce:section> <ce:section id="cesec190"> <ce:section-title>Differences in Antigen-Specific Antibody Subclasses before Induction of AIA</ce:section-title> <ce:para id="para200">On the day of arthritis induction comparable mBSA-specific IgG1 and slightly lower IgM levels were found in IL-6<ce:sup>−/−</ce:sup> mice. In contrast, levels of the complement binding IgG2a and IgG2b subclasses were greatly reduced in the IL-6<ce:sup>−/−</ce:sup> mice (<ce:cross-ref refid="fig4">Figure 4A</ce:cross-ref><ce:float-anchor refid="fig4"></ce:float-anchor>).</ce:para> <ce:para id="para210">Next, we used an immunization protocol to lower the IgG2a levels in wild-type mice to elucidate their role during onset of inflammation. Wild-type mice were immunized and boostered with 10, 30, or the normal 100 μg of mBSA. Wild-type mice immunized with 10 μg of mBSA developed IgG2a titers comparable to normally immunized IL-6<ce:sup>−/−</ce:sup> mice (<ce:cross-ref refid="fig4">Figure 4B</ce:cross-ref>), but they still showed significantly higher joint swelling on day 2 (<ce:cross-ref refid="fig4">Figure 4C</ce:cross-ref>). Although there was a dosage effect on joint swelling at day 2, development of arthritis at day 7 was not reduced after immunization with less antigen (infiltrate at day 7 was 2.7 ± 0.4, 2.6 ± 0.6, and 2.8 ± 0.3, respectively, for wild-type mice in the 10-, 30-, and 100-μg groups). These results suggest that lower antibody titers were not primarily responsible for a mild inflammatory response as observed in IL-6<ce:sup>−/−</ce:sup> mice.</ce:para> </ce:section> <ce:section id="cesec200"> <ce:section-title>T Cell Immunity in IL-6<ce:sup>−/−</ce:sup> Mice</ce:section-title> <ce:para id="para220">Because even the wild-type mice immunized with the lowest amount of mBSA (10 μg) did develop a normal arthritis despite reduced antibody levels we chose this group to evaluate T cell immunity. T cells were isolated from lymph nodes of wild-type (10 μg mBSA)- or IL-6<ce:sup>−/−</ce:sup> (100 μg mBSA)-immunized mice and stimulated with mBSA in the presence of irradiated spleens from naive mice as antigen-presenting cells. IL-6<ce:sup>−/−</ce:sup> T cells responded to the antigen in the same way as wild-type T cells (<ce:cross-ref refid="fig5">Figure 5A</ce:cross-ref><ce:float-anchor refid="fig5"></ce:float-anchor>). The ratio of mBSA-specific proliferation of IL-6<ce:sup>−/−</ce:sup> over wild-type T cells (0.90 ± 0.14 from four different experiments) showed that IL-6<ce:sup>−/−</ce:sup> T cells responded normally to the antigen <ce:italic>in vitro</ce:italic>. Multiplex RT-PCR analysis showed higher IL-4 and IL-5 expression after ConA stimulation of IL-6<ce:sup>−/−</ce:sup> T cells (+20.0% and + 11.4%, respectively; <ce:cross-ref refid="fig5">Figure 5B</ce:cross-ref>) suggesting a minor shift toward the Th2 type in IL-6<ce:sup>−/−</ce:sup> mice after immunization.</ce:para> </ce:section> <ce:section id="cesec210"> <ce:section-title>Transfer of Wild-Type Lymphocytes Restored Joint Swelling on Day 2 in IL-6<ce:sup>−/−</ce:sup> Mice</ce:section-title> <ce:para id="para230">To adapt for possible differences in cellular immunity we transferred lymph node cells from immunized wild-type mice (10 μg mBSA) to immunized IL-6<ce:sup>−/−</ce:sup> mice (100 μg mBSA. at onset of arthritis. Again immunizations yielding comparable IgG2a titers were used.</ce:para> <ce:para id="para240">Transfer of mBSA-specific wild-type lymph node cells completely restored joint swelling in IL-6<ce:sup>−/−</ce:sup> mice on day 2 of AIA (<ce:cross-ref refid="fig6">Figure 6A</ce:cross-ref><ce:float-anchor refid="fig6"></ce:float-anchor>). Transfer of ovalbumin-specific wild-type or mBSA-specific IL-6<ce:sup>−/−</ce:sup> lymph node cells in contrast did not restore joint swelling in IL-6<ce:sup>−/−</ce:sup> mice (<ce:cross-ref refid="fig6">Figure 6, A and B</ce:cross-ref>). The reverse experiment with transfer of mBSA-specific IL-6<ce:sup>−/−</ce:sup> lymph node cells to wild-type mice did not influence joint swelling in wild-type mice (<ce:cross-ref refid="fig6">Figure 6B</ce:cross-ref>).</ce:para> <ce:para id="para250">Although transfer of wild-type cells restored joint swelling on day 2 and led to an increase in cartilage damage it did, however, not sustain joint inflammation at day 7 in IL-6<ce:sup>−/−</ce:sup> mice (<ce:cross-ref refid="tbl2">Table 2</ce:cross-ref><ce:float-anchor refid="tbl2"></ce:float-anchor>). At 14 days after transfer of wild-type cells and induction of arthritis, joint inflammation was absent in IL-6<ce:sup>−/−</ce:sup> mice (data not shown).</ce:para> </ce:section> <ce:section id="cesec220"> <ce:section-title>ZIA Does Not Become Chronic in IL-6<ce:sup>−/−</ce:sup> Mice</ce:section-title> <ce:para id="para260">The results of the cell transfer pointed at an important role for IL-6 in developing a chronic infiltrate. This was confirmed in the nonimmunologically mediated ZIA model. During the first week of ZIA, inflammation did not differ between both strains. By week 3, however, inflammation persisted in wild-type mice whereas it had disappeared in IL-6<ce:sup>−/−</ce:sup> mice (<ce:cross-ref refid="tbl3">Table 3</ce:cross-ref><ce:float-anchor refid="tbl3"></ce:float-anchor>). Although the time scale was different in ZIA and AIA, we found in both models that IL-6 was important in either maintaining or turning the acute inflammation into a chronic synovitis.</ce:para> </ce:section> </ce:section> <ce:section id="cesec230"> <ce:section-title>Discussion</ce:section-title> <ce:para id="para270">IL-6 is a protein that is highly expressed in joints and serum of arthritic patients, but its exact role during arthritis is not yet known. The generation of IL-6 knockout mice<ce:cross-ref refid="bib30"><ce:sup>30</ce:sup></ce:cross-ref> made it possible to assess the role of IL-6 in murine arthritis models. In most of these models the role of IL-6 in immune development and the possible role in joint inflammation itself will be intermingled. In our study on AIA we found influence of IL-6 on both humoral and cellular immunity. During onset of arthritis the T cell plays a major role, because transfer of wild-type lymph node cells restored early joint swelling in IL-6<ce:sup>−/−</ce:sup> mice. The cell transfer, however, also showed that there are two phases in the AIA model in which IL-6 plays an important role. First, IL-6 is necessary for developing a good immune response before induction of arthritis. Second, IL-6 is important for developing and maintaining the inflammatory infiltrate.</ce:para> <ce:para id="para280">In a study by Ohshima et al,<ce:cross-ref refid="bib28"><ce:sup>28</ce:sup></ce:cross-ref> on the immunologically mediated AIA, IL-6<ce:sup>−/−</ce:sup> mice hardly showed joint inflammation on day 14 of AIA. During the first days of the AIA we observed that IL-6<ce:sup>−/−</ce:sup> mice do develop an inflammatory infiltrate and show cartilage damage. Joint inflammation, however, decreased rapidly and by day 7 almost no inflammatory cells were seen. Another striking observation was the sharp decrease in joint swelling in IL-6<ce:sup>−/−</ce:sup> mice after day 1. These findings pointed at an important role for IL-6 during the first days of the AIA in development of an inflammatory infiltrate.</ce:para> <ce:para id="para290">The decrease in joint swelling after day 1 could be caused by reduced expression of genes facilitating cellular influx. IL-6 has been reported to stimulate cellular influx.<ce:cross-refs refid="bib36 bib37"><ce:sup>36,37</ce:sup></ce:cross-refs> However, when we compared mRNA expression for chemokines, adhesion molecules, and pro-inflammatory cytokines we found small or no differences for the investigated set of genes between wild-type and IL-6<ce:sup>−/−</ce:sup> mice on day 1 and 2 of the AIA. This indicates that synovia of IL-6<ce:sup>−/−</ce:sup> mice show a normal pro-inflammatory reaction and it seems that the reduced joint swelling and cellular influx cannot be explained by reduced pro-inflammatory gene expression. This is in line with equal synovial mRNA expression for TNF-α and IL-1β in IL-6<ce:sup>−/−</ce:sup> and wild-type mice on day 4 of the AIA.<ce:cross-ref refid="bib28"><ce:sup>28</ce:sup></ce:cross-ref></ce:para> <ce:para id="para300">One of the first cells that enter a site of inflammation is the neutrophil. Romani et al<ce:cross-ref refid="bib23"><ce:sup>23</ce:sup></ce:cross-ref> had found that IL-6<ce:sup>−/−</ce:sup> mice could not mount a peripheral blood neutrophilia in response to infection with <ce:italic>C. albicans</ce:italic>. In our model the number of neutrophils in blood smears did not differ between immunized wild-type and IL-6<ce:sup>−/−</ce:sup> mice on day 0 and 7 of the arthritis (data not shown). Neutrophils were also the predominant type of cells in the infiltrate during onset of arthritis in both IL-6<ce:sup>−/−</ce:sup> and wild-type mice, although the total infiltrate in IL-6<ce:sup>−/−</ce:sup> mice was reduced. Polymorphonuclear cells and macrophages derived from IL-6<ce:sup>−/−</ce:sup> mice did not exhibit differences in <ce:italic>in vitro</ce:italic> chemotaxis as compared to wild-type cells.<ce:cross-ref refid="bib37"><ce:sup>37</ce:sup></ce:cross-ref> This finding, the results from our RT-PCR analysis, and equal cell influx during the immune-complex-induced arthritis and the first week of ZIA<ce:cross-ref refid="bib26"><ce:sup>26</ce:sup></ce:cross-ref> strongly support that neutrophils could enter the arthritic joint in IL-6<ce:sup>−/−</ce:sup> mice.</ce:para> <ce:para id="para310">Because the developed mBSA-specific immunity is important for inducing arthritis in the mBSA-injected knee joint we compared the immune status of wild-type and IL-6<ce:sup>−/−</ce:sup> mice. IL-6 plays a role in B cell maturation into antibody-secreting plasma cells.<ce:cross-ref refid="bib11"><ce:sup>11</ce:sup></ce:cross-ref> Lower mBSA- or collagen-specific total IgG titers have been reported for IL-6<ce:sup>−/−</ce:sup> mice.<ce:cross-refs refid="bib28 bib38 bib39"><ce:sup>28,38,39</ce:sup></ce:cross-refs> When we looked into IgG subclasses we found strongly reduced IgG2a and IgG2b titers in IL-6<ce:sup>−/−</ce:sup> mice, whereas IgG1 titers were not affected. The effect of IL-6 deficiency on antibody subclasses seems to depend in part on the antigen or the type of adjuvant used for immunization.<ce:cross-ref refid="bib40"><ce:sup>40</ce:sup></ce:cross-ref> This seems also true for experimental arthritis because during Lyme arthritis, in which no previous immunization takes place, IL-6<ce:sup>−/−</ce:sup> mice developed wild-type levels of IgG1 and IgG2a.<ce:cross-ref refid="bib25"><ce:sup>25</ce:sup></ce:cross-ref> The reduction in IgG2a and IgG2b subclasses we found in the IL-6<ce:sup>−/−</ce:sup> mice during AIA seemed not to have a great influence on the primary inflammation. Wild-type mice immunized to generate low IgG2a titers still showed joint swelling at day 2 and developed normal joint inflammation.</ce:para> <ce:para id="para320">Besides antibody levels the activation of complement or complement levels itself could also differ between IL-6<ce:sup>−/−</ce:sup> and wild-type mice. Kopf et al,<ce:cross-ref refid="bib41"><ce:sup>41</ce:sup></ce:cross-ref> had found equal basal levels for complement C3, but IL-6<ce:sup>−/−</ce:sup> mice failed to increase C3 after immunization. When we compared IL-6<ce:sup>−/−</ce:sup> and wild-type mice in the passive immune-complex-induced arthritis we did not find differences in joint swelling. Immune-complex arthritis is a passive immunization model that depends on complement activation in response to immune complexes.<ce:cross-ref refid="bib31"><ce:sup>31</ce:sup></ce:cross-ref> Our results showed that complement activation was not impaired in IL-6<ce:sup>−/−</ce:sup> mice. The above findings showed that the humoral immunity was probably not the main determining factor during onset of AIA. IL-6 also has a role in cellular immunity and a different T cell response against mBSA could occur in IL-6<ce:sup>−/−</ce:sup> mice during AIA.</ce:para> <ce:para id="para330">Antigen-specific cellular immunity in IL-6<ce:sup>−/−</ce:sup> mice was compared with that in wild-type mice immunized with one-tenth of the normal amount of mBSA. These wild-type mice still developed AIA although their humoral immunity was reduced to that found in IL-6<ce:sup>−/−</ce:sup> mice. T cells from wild-type and IL-6<ce:sup>−/−</ce:sup> mice proliferated to the same extent in response to mBSA. Higher mRNA expression of IL-4 and IL-5 after conA stimulation of IL-6<ce:sup>−/−</ce:sup> T cells, however, suggested a small shift toward the Th2 type in IL-6<ce:sup>−/−</ce:sup> mice. <ce:italic>In vitro</ce:italic> results of Ohshima et al,<ce:cross-ref refid="bib28"><ce:sup>28</ce:sup></ce:cross-ref> also suggested a shift toward the Th2 type in IL-6<ce:sup>−/−</ce:sup> mice.</ce:para> <ce:para id="para340">The difference in T cell subtypes could influence arthritis development in IL-6<ce:sup>−/−</ce:sup> mice. To investigate the importance of the T cell type in more detail, we transferred lymph node cells from wild-type to IL-6<ce:sup>−/−</ce:sup> mice. Previous experiments with C57Bl6 mice had shown that the T cell fraction in lymph-node cell preparations could transfer AIA.<ce:cross-ref refid="bib42"><ce:sup>42</ce:sup></ce:cross-ref> For these experiments wild-type mice immunized with 10 μg instead of 100 μg of mBSA were used as donors because they had mBSA-specific IgG2a titers comparable to IL-6<ce:sup>−/−</ce:sup> mice and still showed higher joint swelling and developed a chronic arthritis. Transfer of lymph node cells from mBSA-immunized but not from ovalbumin-immunized wild-type mice restored joint swelling on day 2 in IL-6<ce:sup>−/−</ce:sup> mice to wild-type levels. mBSA-specific antibody titers, as assessed by IgG2a, did not increase between day 1 and 2 after transfer and induction of arthritis (data not shown). This suggests an important involvement of the antigen-specific cellular response in joint swelling during onset of arthritis. Transfer of lymph node cells from immunized IL-6<ce:sup>−/−</ce:sup> mice to immunized IL-6<ce:sup>−/−</ce:sup> mice did not restore joint swelling. This shows that the restored joint swelling after transfer of mBSA-specific wild-type cells is not caused by increased T cell numbers but instead suggests influence of the T cell subtype on joint swelling.</ce:para> <ce:para id="para350">Despite equal joint swelling, IL-6<ce:sup>−/−</ce:sup> mice receiving mBSA-immunized wild-type lymph node cells did not develop arthritis as severe as that found in wild-type mice. In synovial washouts on day 1 or 2 after cell transfer no locally produced IL-6 was measured in a B9 bioassay. In joints of wild-type mice, IL-6 is expressed at high levels during AIA.<ce:cross-ref refid="bib43"><ce:sup>43</ce:sup></ce:cross-ref> The inability to develop a chronic infiltrate by IL-6<ce:sup>−/−</ce:sup> mice even after transfer of wild-type lymph node cells could be caused by a reduced local immune development. In human rheumatoid synovium, the development of germinal centers has been described.<ce:cross-ref refid="bib44"><ce:sup>44</ce:sup></ce:cross-ref> Interestingly, IL-6<ce:sup>−/−</ce:sup> mice develop smaller germinal centers compared to wild-type mice<ce:cross-ref refid="bib41"><ce:sup>41</ce:sup></ce:cross-ref> and this could also be the case in inflamed synovia.</ce:para> <ce:para id="para360">Transfer of the wild-type lymph node cells 3 days before injection of mBSA showed that they could survive in IL-6<ce:sup>−/−</ce:sup> mice and remained capable of restoring joint swelling (day 2: 1.68 ± 0.30). Their survival in the inflamed joint, however, could be impaired, as an anti-apoptotic function of IL-6 has been described.<ce:cross-refs refid="bib45 bib46"><ce:sup>45,46</ce:sup></ce:cross-refs> IL-6 has been shown to enhance <ce:italic>in vitro</ce:italic> the survival of T cells by inducing the anti-apoptotic protein Bcl-2.<ce:cross-ref refid="bib47"><ce:sup>47</ce:sup></ce:cross-ref> A very recent report showed increased apoptosis of mucosal T cells after inhibition of IL-6 signaling in Crohn's disease.<ce:cross-ref refid="bib48"><ce:sup>48</ce:sup></ce:cross-ref> This further supports an anti-apoptotic role of IL-6 in chronic inflammatory diseases. Increased apoptosis in the joints of IL-6<ce:sup>−/−</ce:sup> mice could inhibit formation of a normal infiltrate and influence local immune development despite the presence of wild-type T cells. An anti-apoptotic role of IL-6 would also influence survival of other synovial cells such as macrophages and fibroblasts. We currently have started experiments on this issue. A recent report showing development of collagen-induced arthritis in mice lacking functional T and B cells<ce:cross-ref refid="bib49"><ce:sup>49</ce:sup></ce:cross-ref> further stresses the importance of nonimmunological mechanisms in experimental arthritis.</ce:para> <ce:para id="para370">During the nonimmunologically mediated ZIA, inflammation developed normally in IL-6<ce:sup>−/−</ce:sup> mice during the first week but declined thereafter. Preliminary results showed up-regulation of suppressors of cytokine signaling (SOCS) expression in the inflamed synovia during ZIA. For SOCS3 there was a small increase in expression in both strains. The increase of SOCS1 mRNA expression, however, was much higher for IL-6<ce:sup>−/−</ce:sup> mice than for wild-type mice at day 14 of the ZIA. This suggests a causal relationship with an early relapse of arthritis in IL-6<ce:sup>−/−</ce:sup> mice.</ce:para> <ce:para id="para380">SOCS1 belongs to the SOCS family of proteins,<ce:cross-refs refid="bib50 bib51"><ce:sup>50,51</ce:sup></ce:cross-refs> an important group of inhibitors of cytokine signaling by the JAK-STAT pathway. Differences in expression of SOCS proteins could affect the inflamed synovium in several ways. First, SOCS proteins inhibit signaling by different pro-inflammatory cytokines such as IL-2,<ce:cross-ref refid="bib52"><ce:sup>52</ce:sup></ce:cross-ref> interferon-γ,<ce:cross-ref refid="bib50"><ce:sup>50</ce:sup></ce:cross-ref> and members of the IL-6 family.<ce:cross-refs refid="bib50 bib53"><ce:sup>50,53</ce:sup></ce:cross-refs> Second, there is evidence that SOCS proteins could influence other pathways besides the JAK-STAT pathway. SOCS1 for example has been shown to inhibit the proliferative signaling of the Kit receptor in hematopoietic and fibroblast cells.<ce:cross-ref refid="bib54"><ce:sup>54</ce:sup></ce:cross-ref> Third, SOCS proteins might inhibit signaling by binding to activated signaling proteins and targeting them for degradation by the proteasome.<ce:cross-refs refid="bib55 bib56"><ce:sup>55,56</ce:sup></ce:cross-refs></ce:para> <ce:para id="para390">Although SOCS proteins function in a negative feedback loop it has also been suggested that they can be expressed independent of cytokine signaling.<ce:cross-refs refid="bib51 bib57 bib58"><ce:sup>51,57,58</ce:sup></ce:cross-refs> Future research will have to address the regulation of cytokine signaling in inflamed synovia and the role of SOCS proteins in chronicity of arthritis.</ce:para> <ce:para id="para400">In conclusion, we have found in the immunologically mediated AIA and also in the nonimmunologically mediated ZIA that IL-6 plays an important role in progression of initial joint inflammation into a chronic infiltrate. 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Arthritis</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>Involvement of IL-6, Apart from Its Role in Immunity, in Mediating a Chronic Response during Experimental Arthritis</title></titleInfo><name type="personal"><namePart type="given">Alfons S.K.</namePart><namePart type="family">de Hooge</namePart><affiliation>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</affiliation><affiliation>E-mail: A.deHoogereuma.azn.nl</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Fons A.J.</namePart><namePart type="family">van de Loo</namePart><affiliation>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Onno J.</namePart><namePart type="family">Arntz</namePart><affiliation>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Wim B.</namePart><namePart type="family">van den Berg</namePart><affiliation>Rheumatology Research Laboratory, University Medical Center Nijmegen, Nijmegen, The Netherlands</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article"></genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2000</dateIssued><dateValid encoding="w3cdtf">2000-08-25</dateValid><copyrightDate encoding="w3cdtf">2000</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><physicalDescription><internetMediaType>text/html</internetMediaType></physicalDescription><abstract>Interleukin-6 (IL-6) is highly produced during arthritis but its exact function is still unknown. In this study we examined if IL-6, apart from its role in immunity, was involved in the local inflammatory response in experimental arthritis. IL-6 deficient (IL-6/) and wild-type mice were first compared in the antigen-induced arthritis model. IL-6 deficiency resulted in a mild, transient inflammation whereas wild-type mice developed a chronic, destructive synovitis. Wild-type mice immunized with one-tenth of the normal antigen dose still developed chronic arthritis despite low antibody levels, excluding reduced humoral immunity in IL-6/ mice as a crucial phenomenon. In addition, passive immune-complex-induced arthritis did not differ between wild-type and IL-6/ mice. Another option is reduced levels of Th1 cells in IL-6/ mice. However, transfer of antigen-specific wild-type lymph node cells to IL-6/ mice enhanced acute joint inflammation and increased cartilage damage but still could not sustain chronic inflammation, suggesting involvement of nonimmune elements of IL-6 activity in chronicity. In line with this, nonimmunologically mediated zymosan-induced arthritis developed similarly in the first week, but only wild-type mice developed chronic synovitis. These results indicate an important role for IL-6 in propagation of joint inflammation, potentially independent of its role in immunity.</abstract><note>Supported by a grant from the Dutch League against Rheumatism (NR97-2-402).</note><note type="content">Section title: Regular Articles</note><note type="content">Figure 1: Joint swelling at day 1 and 2 after mBSA injection. Immunized and naive mice were injected with 60 g of mBSA. Joint swelling was measured as the ratio of 99mTc uptake in the arthritic knee (right) over the nonarthritic knee (left). , IL-6/; , wild type (n = 7; , P < 0.05; , P < 0.005, Student's t-test).</note><note type="content">Figure 2: Inflammation and cartilage damage in wild-type (A and B) and IL-6/ (C and D) mice on day 2 (A and C) and 7 (B and D) of the AIA. Sections were stained with safranin-O and cartilage damage was observed as loss of red staining. E: Position of patella (P), femur (F), synovium (S), growth plate (GP), and cartilage (C) is shown. Original magnification, 200.</note><note type="content">Figure 3: mRNA expression in synovia from wild-type and IL-6/ mice on day 1 (A) and 2 (B) of the AIA. The number of PCR cycles needed to first detect the specific band on an agarose gel was compared between synovia from arthritic and contralateral nonarthritic knee joints. Increased mRNA expression for the investigated gene in arthritic synovia results in a reduced number of PCR cycles to first detect the specific band. Four mice per group were used and equal amounts of cDNA were used as assessed by PCR for glyceraldehyde-3-phosphate dehydrogenase (all appeared at cycle 14). Basal expression in the nonarthritic synovia did not differ between wild-type and IL-6/ mice. No signal for IL-6 was detected at the end point of the PCR with cDNA from IL-6/ mice. , IL-6/; , wild type.</note><note type="content">Figure 4: A: mBSA-specific antibody subclasses before onset of AIA. Titers as depicted are the dilution of sera needed to give half the maximal optical density at 450 nm as described in Materials and Methods. , IL-6/; , wild type (n = 7). B: mBSA-specific IgG2a titers in mice immunized and boostered with 100 g of mBSA (IL-6/. or with 10 or 100 g of mBSA (wild type). Each symbol represents one mouse (P = 0.22, IL-6/ (100 g) versus wild type, (10 g) Student's t-test). Total IgG titers also did not differ (IL-6/: 1/720 194, wild type: 1/734 180). C: Joint swelling in wild-type mice immunized with 10, 30, or 100 g of mBSA (n = 8). Joint swelling was measured as the ratio of 99mTc uptake in the arthritic knee (right) over the nonarthritic knee (left). , IL-6/; , wild type (A and C: , P < 0.05; , P < 0.005, Student's t-test). One of three experiments is shown.</note><note type="content">Figure 5: A: mBSA-specific increase in cpm (cpm mBSA minus cpm medium). Lymph nodes from seven mice per group were pooled and enriched for T cells as described in Materials and Methods. Lymph node cells (2106. together with 2107 irradiated antigen-presenting cells were incubated for 72 hours with mBSA at 25 g/ml. Cells were plated in sixfold. Cultures were labeled with 3H for the last 16 hours. Antigen-presenting cells were used from naive mice of the same strain. IL-6/ and wild-type mice did not differ in the response to concanavalin A at 1 g/ml (IL-6/, 28,002 7,291 cpm; wild type, 25,436 1,216 cpm. One of four experiments is shown. B: Th1/Th2 multiplex RT-PCR of IL-6/ (100 g mBSA) or wild-type (10 g mBSA) T cells stimulated for 24 hours with conA (1 g/ml). C = positive control.</note><note type="content">Figure 6: Joint swelling on day 2 of AIA in IL-6/ mice after transfer of 4107 lymph node cells derived from mBSA (wild-type/mBSA) or ovalbumin (wild-type/OVA. immunized wild-type mice (A) or of mBSA (IL-6//mBSA. immunized IL-6/ mice (B). NT = normal AIA in wild-type mice immunized with 10 g of mBSA. Joint swelling was measured as the ratio of 99mTc uptake in the arthritic knee (right) over the nonarthritic knee (left) (n = 6; , P < 0.05, Student's t-test, n.s. = not significant). , IL-6/, , wild type. One of three experiments is shown.</note><note type="content">Table 1: Development of Joint Inflammation and Cartilage Damage during AIA</note><note type="content">Table 2: Joint Inflammation and Cartilage Damage at Day 7 of AIA after Transfer of Wild-Type Lymph Node Cells</note><note type="content">Table 3: Synovial Infiltrate on Day 7 and 21 of Zymosan-Induced Arthritis</note><subject><genre>Article category</genre><topic>IL-6 and Chronic Synovitis in Experimental RA</topic></subject><relatedItem type="host"><titleInfo><title>The American Journal of Pathology</title></titleInfo><titleInfo type="abbreviated"><title>AJPA</title></titleInfo><genre type="Journal">journal</genre><originInfo><dateIssued encoding="w3cdtf">200012</dateIssued></originInfo><identifier type="ISSN">0002-9440</identifier><identifier type="PII">S0002-9440(10)X6128-4</identifier><part><detail type="volume"><number>157</number><caption>vol.</caption></detail><detail type="issue"><number>6</number><caption>no.</caption></detail><extent unit="issue pages"><start>1757</start><end>2161</end></extent><extent unit="pages"><start>2081</start><end>2091</end></extent></part></relatedItem><identifier type="istex">3FFA4817B217FC3EC96B07D190272F56761F0188</identifier><identifier type="DOI">10.1016/S0002-9440(10)64846-8</identifier><identifier type="PII">S0002-9440(10)64846-8</identifier><accessCondition type="use and reproduction" contentType="">© 2000American Society for Investigative Pathology</accessCondition><recordInfo><recordContentSource>ELSEVIER</recordContentSource><recordOrigin>American Society for Investigative Pathology, ©2000</recordOrigin></recordInfo></mods></metadata><enrichments><istex:catWosTEI uri="https://api.istex.fr/document/3FFA4817B217FC3EC96B07D190272F56761F0188/enrichments/catWos"><teiHeader><profileDesc><textClass><classCode scheme="WOS">PATHOLOGY</classCode><classCode scheme="WOS">CELL BIOLOGY</classCode></textClass></profileDesc></teiHeader></istex:catWosTEI></enrichments><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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