International Immunology, Vol. 3, No. 3, pp.
273-278
©1991 Oxford University Press 0953-8178/91 $3.00
Age-associated increase in interleukin 6 in MRL/lpr mice Bo Tang, Tadashi Matsuda1, Shizuo Akira, Norikazu Nagata2, Susumu Ikehara2, Toshio Hirano1, and Tadamitsu Kishimoto The Institute for Molecular and Cellular Biology, Osaka University, Suita Osaka 565, Japan 'Division of Molecular Oncology, Biomedical Research Center, Osaka University Medical School, 4-3-57, Nakanoshima, Kita-ku, Osaka 530, Japan 2 Department of Pathology, Kansai Medical College, Moriguchi, Osaka 570, Japan
Abstract It has been demonstrated that abnormal expression of Interleukin 6 (IL-6) may be involved In the pathogenesls of a variety of autoimmune diseases and glomerulonephrttls. In this study, we demonstrate an age-associated Increase In IL-6 in the sera of MRL/lpr mice but not in control congenic MRL/+ mice, normal strains of BALB/c, C3H/HeN mice, and other autoimmune prone mice, such as (NZB x NZW)F1, (NZB x BXSB)F1 mice. IL-6 activity was detected in the sera of MRL/lpr mice by 3 weeks of age and it was neutralized by anti-mouse IL-6 antibody. The serum IL-6 level was gradually Increased with age and reached up to 410 pg/ml around 30 weeks of age. The expression of IL-6 mRNA was detected in the spleen and lymph nodes from 8 weeks of age. Expression of IL-6 mRNA was also detected In C57BL/6-lpr mice, Indicating the possible linkage of abnormal expression of the IL-6 gene and the Ipr gene. Southern blot analysis demonstrated that both the promoter region and 3' untranslated region of the IL-6 gene were apparently normal, suggesting that deregulated production of IL-6 may be due to abnormal transcriptional control. The data suggest that abnormal expression of the IL-6 gene may play a key role In the development of polyclonal B cell activation and glomerulonephritis in MRL/lpr mice.
Introduction Upon stimulation with antigen, B cells proliferate and differentiate into antibody-forming plasma cells under the control of a variety of interleukins (1). It is conceivable that deregulated expression of these interleukins and/or their receptors could induce abnormal B cell proliferation and/or differentiation. We first suggested that abnormal interleukin 6 (IL-6) production might be involved in polyclonal plasmacytosis, causing autoimmune conditions in patients with cardiac myxoma (2). Since then much evidence has been accumulated supporting this notion (3). Abnormal IL-6 production has been demonstrated in patients with rheumatoid arthritis (RA) (4,5), Castleman's disease (6), AIDS (7,8), and alcoholic liver cirrhosis (9) where polyclonal plasmacytosis is observed. Furthermore, IL-6 was found to be a growth factor for myeloma/plasmacytoma and may play a critical role in the generation of plasma cell neoplasias (10-12). Moreover, it was demonstrated that IL-6 is a growth factor for renal mesangial cells, suggesting the involvement of IL-6 in the pathogenesis of mesangial proliferative glomerulonephritis (13). In accordance
Correspondence to: 8. Tang Transmitting editor: K. Okumura
with these suggestive data, a massive plasmacytosis and mesangial proliferative glomerulonephritis were generated in IL-6 transgenic mice (14). MRL/Mp-lpr/Mp-lpr (MRL/lpr) mice spontaneously develop a massive lymphadenopathy with hypergammaglobulinemia, presence of autoantibodies, high levels of acute phase proteins, plasmacytosis, arthritis and proliferative mesangial glomerulonephritis (15). An abnormal proliferative T cell population was reported to produce B cell differentiation factor (I-BCDF) which was suggested as causing a polyclonal B cell activation in MRL/lpr mice (16). Furthermore, these T cells were found to express mRNA transcripts for IFN7 and TNFa (17). However, the pathological significance of these cytokines in MRL/lpr mice has not yet been established. In this report, we demonstrate an age-associated increase in IL-6 in the sera of MRL/lpr mice and discuss a possible involvement of deregulated expression of the IL-6 gene in the pathological manifestation in MRL/lpr mice.
Received 26 November 1990; accepted 10 December 1990
Downloaded from http://intimm.oxfordjournals.org/ at Cornell University Library on July 12, 2015
Key words: IL-6 gene expression, autoimmune disease, B cell activation, lymphadenopathy
274
Interieukin 6 in MRL/lpr mice
C 3 H/HeN, BALB/c (NZB x NZW)F1, (NZB x BXSBJF,, MRL/MP-lpr/MP-lpr and MRL/Mp+/Mp + (MRL/+) mice of either sex were purchased from Clea Japan Inc. (Tokyo, Japan). C57BL/6-lpr/lpr(C57BL/6-lpr) mice were purchased from Charles River Japan, Inc. (Japan).
buffer was replaced with a fresh one containing ^P-labeled probes. The probes used in this study were the 1.8 kb of BamH\IXba\ digested fragment (probe A) and the 1.0 kb of EcoRI digested fragment (probe B) of the mouse IL-6 genomic DNA (20) as indicated in Fig. 5A. The membranes were incubated at 65°C for a further 16 h, washed with 2 x SSC twice at room temperature, twice with 0.5 x SSC, 1 % SDS at 65°C and twice with 0.1 x SSC at room temperature.
Preparation of polyclonal rabbit anti-mouse IL-6
Results
Methods Animals
Rabbits were immunized with 50 ng of recombinant mouse IL-6 in complete Freund's adjuvant at 2 week intervals for 2 months. The IgG fraction of antiserum was prepared by an ion-exchange chromatography. This antibody could neutralize murine IL-6 but not human IL-6 activity.
The assay for IL-6 activity was performed by utilizing an IL-6-dependent murine hybridoma clone, MH60-BSF2, as described previously (18). Briefly, MH60-BSF2 cells were seeded in 96-well flat-bottom micro-plates in 200 /il of RPMI1640 supplemented with 10% FCS, 5 x 10~5 M 2-ME and antibiotics (100U/ml of penicillin and 100/ig/ml of streptomycin) and cultured at a density of 5 x 104/ml in the presence of serially diluted sera obtained from different strains of mice for 48 h. The cells were pulsed with 1 /iCi of pHJthymidine (5 Ci/mmol) during the final 8 h of the culture and harvested on glass filter paper by an automatic cell harvester. The radioactivity was counted by Beckman liquid scintillation counter. IL-6 activity was indicated as the concentration of recombinant human IL-6 which can exert the same activity as the test sample.
IL-6 activity in the sera of MRL/lpr mice was measured utilizing an IL-6-dependent murine hybridoma, MH60-BSF2. Fig. 1A shows representative data on the serum obtained from a 20-week-old MRL/lpr mouse. As shown in Fig. 1A, proliferation of MH60-BSF2 was induced by the addition of serum from the MRL/lpr mouse in a dose-dependent manner. As shown in Fig. 1B, the growth-inducing activity of the serum was almost completely neutralized by rabbit anti-mouse IL-6 antibody but not by anti-human IL-6 antibody, indicating that the activity was due to mouse IL-6. Age-associated increase in serum IL-6 level It is well known that serological and immunological abnormalities in MRL/lpr mice develop at an early age, and disease severity is related to increasing age. To elucidate the relationship between serum IL-6 and MRL/lpr mouse age, the sera from 82 MRL/lpr mice of either sex with a variety of ages were measured for IL-6 activity. IL-6 activity in the sera of MRU + mice, other autoimmune prone mice such as (NZB x NZW)F1 and (NZB x BXSB)F1, and normal mouse strains, BALB/c and CsH/HeN was also
Extraction of RNA and Northern blot analysis Total cellular RNA was isolated by the guanidinium isothiocyanate/cesium chloride method. Poly(A)+ RNA was prepared utilizing an oligo-dT column as described previously (19). Po)y(A)+ RNA (2 HQ) was denatured at 60°C for 15 min, isolated on a 1 % agarose/formamid gel and transferred onto GeneScreen plus (NEN, Boston). Membranes were baked under vacuum at 80°C for 2 h and prehybridized with 50% of formamid, 1 % SDS, 1 M NaCI, 10% dextran sulfate and 20/ig/ml of denatured salmon sperm DNA at 42°C for 12 - 1 6 h. A 637 bp EcoRI/H/ndlll fragment of the cDNA encoding mouse IL-6(20) was labeled with fj^PjCTP by Multiprime DNA labeling systems (Amersham) and used as a probe. Hybridization was performed at42°C for 16 h. The membranes were washed twice with 2 x SSC at room temperature, twice with 0.2 x SSC, 1 % SDS at 60°C and twice with 0.1 x SSC at room temperature. The membranes were dried at room temperature and autoradiography was performed by a standard technique. 1/160
Preparation of high molecular weight DNA and Southern blot analysis High molecular weight DNA from both BALB/c and MRL/lpr mice was prepared by the phenol/proteinase K method (21). DNAs (10/tg/lane) were digested by restriction enzymes, electrophoresed through 1 % agarose gel, and transferred onto GeneScreen plus (NEN, Boston). After drying at room temperature for 1 h, membranes were pre-hybridized with 1 M NaCI, 1 % SDS, 0.1 M Tris - HCI for 12 - 1 6 h at 65°C, then the
1/80 1/40 M u t t o n of mnmt
1/20
23 SO 100 Concentration of Ab (jig/raT)
Fig. 1. IL-6 activity in the serum of MRL/lpr mouse and its inhibition by anti-mouse IL-6 antibody. (A) IL-6 activity in the serum from a 20-weekotd MRL/lpr mouse was measured using an IL-6 dependent murine hybridoma. MH60-BSF2 as described in Methods. Data represent the mean of triplicate cultures. Standard error was within 10% of the mean. (B) MH60 BSF-2 eels were cultured with 20-fold diluted serum obtained from the same mouse as in (A) in the presence of various concentrations of rabbit anti-mouse IL-6 antibody ( • ) or rabbit anti-human IL-6 antibody (•)•
Downloaded from http://intimm.oxfordjournals.org/ at Cornell University Library on July 12, 2015
Assay for IL-6 activity
IL-6 activity in the serum of MRL/lpr mice
Interleukin 6 in MRL/lpr mice 275 strains did not show any age-associated increase in serum IL-6 activity up to 40 weeks of age (MRL/+ mice: r = -0.303 P > 0.132; BALB/c mice: r = 0.189 P > 0.425; C3H/HeN mice: r = 0.225 P > 0.46). In addition, no significant increase in IL-6 activity was observed in (NZB x NZW)F1 mice (73 ± 54 pg/ml, 2 8 - 5 0 weeks of age) and in (BXSB x NZB)F1 mice (180 ± 24 pg/ml, 2 8 - 3 2 weeks of age).
measured. As shown in Fig. 2, IL-6 activity was detectable in the sera of MRL/lpr mice by 3 - 6 weeks of age (170 ± 97 pg/ml, equivalent to human recombinant IL-6). The serum IL-6 levels of MRL/lpr mice gradually increased during 7 - 1 2 weeks of age (230 ± 150 pg/ml), while fymphadenopathy was absent or barely detectable. The serum IL-6 level was elevated during 1 3 - 2 0 weeks of age (320 ± 180 pg/ml) when the mice had significant cervical lymphadenopathy and reached up to 410 ± 260 pg/ml during 21 - 3 0 weeks of age when the mice had other systemic features such as skin lesions and proteinuria. Premature death resulted. Statistical analysis suggested that there was a significant relationship between age and serum IL-6 levels in MRL/lpr mice {r = 0.441, P < 0.001). However, MRL/+ mice and normal
The expression of the IL-6 gene in vivo We further examined the expression of the IL-6 gene in MRL/lpr mice of different ages. Poly A + RNAs isolated from the spleens of 2 - 3 mice of different ages from MRL/lpr, MRL/+ and normal BALB/c mice were analyzed by Northern blot utilizing mouse IL-6
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273-278
©1991 Oxford University Press 0953-8178/91 $3.00
Age-associated increase in interleukin 6 in MRL/lpr mice Bo Tang, Tadashi Matsuda1, Shizuo Akira, Norikazu Nagata2, Susumu Ikehara2, Toshio Hirano1, and Tadamitsu Kishimoto The Institute for Molecular and Cellular Biology, Osaka University, Suita Osaka 565, Japan 'Division of Molecular Oncology, Biomedical Research Center, Osaka University Medical School, 4-3-57, Nakanoshima, Kita-ku, Osaka 530, Japan 2 Department of Pathology, Kansai Medical College, Moriguchi, Osaka 570, Japan
Abstract It has been demonstrated that abnormal expression of Interleukin 6 (IL-6) may be involved In the pathogenesls of a variety of autoimmune diseases and glomerulonephrttls. In this study, we demonstrate an age-associated Increase In IL-6 in the sera of MRL/lpr mice but not in control congenic MRL/+ mice, normal strains of BALB/c, C3H/HeN mice, and other autoimmune prone mice, such as (NZB x NZW)F1, (NZB x BXSB)F1 mice. IL-6 activity was detected in the sera of MRL/lpr mice by 3 weeks of age and it was neutralized by anti-mouse IL-6 antibody. The serum IL-6 level was gradually Increased with age and reached up to 410 pg/ml around 30 weeks of age. The expression of IL-6 mRNA was detected in the spleen and lymph nodes from 8 weeks of age. Expression of IL-6 mRNA was also detected In C57BL/6-lpr mice, Indicating the possible linkage of abnormal expression of the IL-6 gene and the Ipr gene. Southern blot analysis demonstrated that both the promoter region and 3' untranslated region of the IL-6 gene were apparently normal, suggesting that deregulated production of IL-6 may be due to abnormal transcriptional control. The data suggest that abnormal expression of the IL-6 gene may play a key role In the development of polyclonal B cell activation and glomerulonephritis in MRL/lpr mice.
Introduction Upon stimulation with antigen, B cells proliferate and differentiate into antibody-forming plasma cells under the control of a variety of interleukins (1). It is conceivable that deregulated expression of these interleukins and/or their receptors could induce abnormal B cell proliferation and/or differentiation. We first suggested that abnormal interleukin 6 (IL-6) production might be involved in polyclonal plasmacytosis, causing autoimmune conditions in patients with cardiac myxoma (2). Since then much evidence has been accumulated supporting this notion (3). Abnormal IL-6 production has been demonstrated in patients with rheumatoid arthritis (RA) (4,5), Castleman's disease (6), AIDS (7,8), and alcoholic liver cirrhosis (9) where polyclonal plasmacytosis is observed. Furthermore, IL-6 was found to be a growth factor for myeloma/plasmacytoma and may play a critical role in the generation of plasma cell neoplasias (10-12). Moreover, it was demonstrated that IL-6 is a growth factor for renal mesangial cells, suggesting the involvement of IL-6 in the pathogenesis of mesangial proliferative glomerulonephritis (13). In accordance
Correspondence to: 8. Tang Transmitting editor: K. Okumura
with these suggestive data, a massive plasmacytosis and mesangial proliferative glomerulonephritis were generated in IL-6 transgenic mice (14). MRL/Mp-lpr/Mp-lpr (MRL/lpr) mice spontaneously develop a massive lymphadenopathy with hypergammaglobulinemia, presence of autoantibodies, high levels of acute phase proteins, plasmacytosis, arthritis and proliferative mesangial glomerulonephritis (15). An abnormal proliferative T cell population was reported to produce B cell differentiation factor (I-BCDF) which was suggested as causing a polyclonal B cell activation in MRL/lpr mice (16). Furthermore, these T cells were found to express mRNA transcripts for IFN7 and TNFa (17). However, the pathological significance of these cytokines in MRL/lpr mice has not yet been established. In this report, we demonstrate an age-associated increase in IL-6 in the sera of MRL/lpr mice and discuss a possible involvement of deregulated expression of the IL-6 gene in the pathological manifestation in MRL/lpr mice.
Received 26 November 1990; accepted 10 December 1990
Downloaded from http://intimm.oxfordjournals.org/ at Cornell University Library on July 12, 2015
Key words: IL-6 gene expression, autoimmune disease, B cell activation, lymphadenopathy
274
Interieukin 6 in MRL/lpr mice
C 3 H/HeN, BALB/c (NZB x NZW)F1, (NZB x BXSBJF,, MRL/MP-lpr/MP-lpr and MRL/Mp+/Mp + (MRL/+) mice of either sex were purchased from Clea Japan Inc. (Tokyo, Japan). C57BL/6-lpr/lpr(C57BL/6-lpr) mice were purchased from Charles River Japan, Inc. (Japan).
buffer was replaced with a fresh one containing ^P-labeled probes. The probes used in this study were the 1.8 kb of BamH\IXba\ digested fragment (probe A) and the 1.0 kb of EcoRI digested fragment (probe B) of the mouse IL-6 genomic DNA (20) as indicated in Fig. 5A. The membranes were incubated at 65°C for a further 16 h, washed with 2 x SSC twice at room temperature, twice with 0.5 x SSC, 1 % SDS at 65°C and twice with 0.1 x SSC at room temperature.
Preparation of polyclonal rabbit anti-mouse IL-6
Results
Methods Animals
Rabbits were immunized with 50 ng of recombinant mouse IL-6 in complete Freund's adjuvant at 2 week intervals for 2 months. The IgG fraction of antiserum was prepared by an ion-exchange chromatography. This antibody could neutralize murine IL-6 but not human IL-6 activity.
The assay for IL-6 activity was performed by utilizing an IL-6-dependent murine hybridoma clone, MH60-BSF2, as described previously (18). Briefly, MH60-BSF2 cells were seeded in 96-well flat-bottom micro-plates in 200 /il of RPMI1640 supplemented with 10% FCS, 5 x 10~5 M 2-ME and antibiotics (100U/ml of penicillin and 100/ig/ml of streptomycin) and cultured at a density of 5 x 104/ml in the presence of serially diluted sera obtained from different strains of mice for 48 h. The cells were pulsed with 1 /iCi of pHJthymidine (5 Ci/mmol) during the final 8 h of the culture and harvested on glass filter paper by an automatic cell harvester. The radioactivity was counted by Beckman liquid scintillation counter. IL-6 activity was indicated as the concentration of recombinant human IL-6 which can exert the same activity as the test sample.
IL-6 activity in the sera of MRL/lpr mice was measured utilizing an IL-6-dependent murine hybridoma, MH60-BSF2. Fig. 1A shows representative data on the serum obtained from a 20-week-old MRL/lpr mouse. As shown in Fig. 1A, proliferation of MH60-BSF2 was induced by the addition of serum from the MRL/lpr mouse in a dose-dependent manner. As shown in Fig. 1B, the growth-inducing activity of the serum was almost completely neutralized by rabbit anti-mouse IL-6 antibody but not by anti-human IL-6 antibody, indicating that the activity was due to mouse IL-6. Age-associated increase in serum IL-6 level It is well known that serological and immunological abnormalities in MRL/lpr mice develop at an early age, and disease severity is related to increasing age. To elucidate the relationship between serum IL-6 and MRL/lpr mouse age, the sera from 82 MRL/lpr mice of either sex with a variety of ages were measured for IL-6 activity. IL-6 activity in the sera of MRU + mice, other autoimmune prone mice such as (NZB x NZW)F1 and (NZB x BXSB)F1, and normal mouse strains, BALB/c and CsH/HeN was also
Extraction of RNA and Northern blot analysis Total cellular RNA was isolated by the guanidinium isothiocyanate/cesium chloride method. Poly(A)+ RNA was prepared utilizing an oligo-dT column as described previously (19). Po)y(A)+ RNA (2 HQ) was denatured at 60°C for 15 min, isolated on a 1 % agarose/formamid gel and transferred onto GeneScreen plus (NEN, Boston). Membranes were baked under vacuum at 80°C for 2 h and prehybridized with 50% of formamid, 1 % SDS, 1 M NaCI, 10% dextran sulfate and 20/ig/ml of denatured salmon sperm DNA at 42°C for 12 - 1 6 h. A 637 bp EcoRI/H/ndlll fragment of the cDNA encoding mouse IL-6(20) was labeled with fj^PjCTP by Multiprime DNA labeling systems (Amersham) and used as a probe. Hybridization was performed at42°C for 16 h. The membranes were washed twice with 2 x SSC at room temperature, twice with 0.2 x SSC, 1 % SDS at 60°C and twice with 0.1 x SSC at room temperature. The membranes were dried at room temperature and autoradiography was performed by a standard technique. 1/160
Preparation of high molecular weight DNA and Southern blot analysis High molecular weight DNA from both BALB/c and MRL/lpr mice was prepared by the phenol/proteinase K method (21). DNAs (10/tg/lane) were digested by restriction enzymes, electrophoresed through 1 % agarose gel, and transferred onto GeneScreen plus (NEN, Boston). After drying at room temperature for 1 h, membranes were pre-hybridized with 1 M NaCI, 1 % SDS, 0.1 M Tris - HCI for 12 - 1 6 h at 65°C, then the
1/80 1/40 M u t t o n of mnmt
1/20
23 SO 100 Concentration of Ab (jig/raT)
Fig. 1. IL-6 activity in the serum of MRL/lpr mouse and its inhibition by anti-mouse IL-6 antibody. (A) IL-6 activity in the serum from a 20-weekotd MRL/lpr mouse was measured using an IL-6 dependent murine hybridoma. MH60-BSF2 as described in Methods. Data represent the mean of triplicate cultures. Standard error was within 10% of the mean. (B) MH60 BSF-2 eels were cultured with 20-fold diluted serum obtained from the same mouse as in (A) in the presence of various concentrations of rabbit anti-mouse IL-6 antibody ( • ) or rabbit anti-human IL-6 antibody (•)•
Downloaded from http://intimm.oxfordjournals.org/ at Cornell University Library on July 12, 2015
Assay for IL-6 activity
IL-6 activity in the serum of MRL/lpr mice
Interleukin 6 in MRL/lpr mice 275 strains did not show any age-associated increase in serum IL-6 activity up to 40 weeks of age (MRL/+ mice: r = -0.303 P > 0.132; BALB/c mice: r = 0.189 P > 0.425; C3H/HeN mice: r = 0.225 P > 0.46). In addition, no significant increase in IL-6 activity was observed in (NZB x NZW)F1 mice (73 ± 54 pg/ml, 2 8 - 5 0 weeks of age) and in (BXSB x NZB)F1 mice (180 ± 24 pg/ml, 2 8 - 3 2 weeks of age).
measured. As shown in Fig. 2, IL-6 activity was detectable in the sera of MRL/lpr mice by 3 - 6 weeks of age (170 ± 97 pg/ml, equivalent to human recombinant IL-6). The serum IL-6 levels of MRL/lpr mice gradually increased during 7 - 1 2 weeks of age (230 ± 150 pg/ml), while fymphadenopathy was absent or barely detectable. The serum IL-6 level was elevated during 1 3 - 2 0 weeks of age (320 ± 180 pg/ml) when the mice had significant cervical lymphadenopathy and reached up to 410 ± 260 pg/ml during 21 - 3 0 weeks of age when the mice had other systemic features such as skin lesions and proteinuria. Premature death resulted. Statistical analysis suggested that there was a significant relationship between age and serum IL-6 levels in MRL/lpr mice {r = 0.441, P < 0.001). However, MRL/+ mice and normal
The expression of the IL-6 gene in vivo We further examined the expression of the IL-6 gene in MRL/lpr mice of different ages. Poly A + RNAs isolated from the spleens of 2 - 3 mice of different ages from MRL/lpr, MRL/+ and normal BALB/c mice were analyzed by Northern blot utilizing mouse IL-6
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