0192-0561/92 $5.00 + .00 Pergamon Press Ltd. International Society for lmmunopharmacology.
lnt. J. lmmunopharmac., Vol. 14, No. 4, pp. 565-571, 1992.
Printed in Great Britain.
INTERLEUKIN-6 (IL-6) IN A D J U V A N T ARTHRITIS OF RATS AND ITS PHARMACOLOGICAL MODULATION P1A THEISEN-POPP,
HARTMUT
PAPE
and
REINER
MULLER-PEDDINGHAUS*
Troponwerke, Department of Antiphlogistic Research, Cologne, F.R.G. (Received 28 June 1991 and in final form 15 November 1991)
The levels of serum interleukin-6 (IL-6) were measured during the course of adjuvant arthritis (FAA) in male Lewis rats. In the course of the disease serum IL-6 levels increase with a clear correlation with morphologic disease signs. Additionally, the FAA-inducing antigen, heat-killed Mycobacterium tuberculosis, was found to be a strong inducer of 1L-6 production by spleen cells in vitro. The effects of the anti-rheumatic drugs indometacin, dexamethasone, cyclophosphamide and cyclosporin A (CsA) on IL-6 levels during FAA were determined. Complete normalization of serum IL-6 levels was observed with dexamethasone, cyclophosphamide and CsA whereas indometacin only partly reduced serum IL-6 levels.
Abstract --
Interleukin-6 (IL-6) exerts multiple actions on the growth, differentiation and function of lymphoid and non-lymphoid cells. It has recently been suggested that this cytokine plays a role in the pathogenesis of autoimmune disorders, in which the excessive production of IL-6 may lead to abnormal B-cell differentiation and antibody production. One example is cardiac myxoma, a condition often associated with autoimmune phenomena, such as hypergammaglobulinemia and autoantibodies (Sutton, Mercier, Giuliani & Lie, 1980). Myxoma cells in vitro produce significant amounts of IL-6 (Hirano et al., 1986, 1987). The autoimmune phenomena disappear after tumour removal. The question arose as to whether IL-6 might be involved in the pathogenesis of other autoimmune disorders, such as rheumatoid arthritis (RA). An elevation of IL-6 in the serum and synovial fluid of RA-patients was shown by different groups (Waage, Kaufmann, Espevik & Husby, 1989; Houssiau, Devogelaer, Van Damme, de Deuxchaisnes & Van Snick, 1988; Hirano et al., 1988). The adjuvant arthritis of rats (FAA), a T-cell mediated autoimmune disease, is one of the most important pharmacological models of rheumatoid arthritis (for review see Billingham, 1983). One aspect of our studies was to investigate the role of IL6 as a disease parameter of chronic inflammation.
Therefore, we measured IL-6 levels in the serum of FAA-rats during the course of the disease. Furthermore, the IL-6 production of spleen cells in response to in vitro stimulation with the arthritisinducing antigen, heat-killed Mycobacterium tuberculosis, was tested. A second aspect of this investigation was to examine the influence on serum IL-6 levels of drugs employed presently in the treatment of RA. Rats were treated orally with a non-steroidal anti-inflammatory drug (indometacin), dexamethasone, cyclophosphamide and cyclosporin
A (CsA). EXPERIMENTAL
PROCEDURES
Rats. Inbred male Lewis rats (120-150 g) were purchased from the Moellegaard Breeding Center (Skensved, Denmark). Animals were kept under standardized conditions on a standard diet (Altromin, Lage, F.R.G.) and water ad libitum. Induction and evaluation o f F A A . Heat-killed Mycobacterium tuberculosis (Difco, Detroit, MI) was suspended in paraffin oil (5 mg/ml, Merck, Darmstadt, F.R.G.) and injected intraplantarly into the footpad (50/al) of the right hind paw on day 0. In order to evaluate the progression of the disease two parameters were defined: (1) the swelling of the hind paws determined plethysmographically; and (2) the
*Author to whom correspondence should be addressed at: Department of Antiphlogistic Research, Troponwerke GmbH & Co. KG, Berliner Stra~e 156, 5000 Cologne 80, F.R.G. 565
566
P. THEISEN-PoPP el al.
arthritic score involving the quantification of seven arthritic sites: the three non-injected paws, ears, nose and tail. Parameters of evaluation were any sign of swelling and erythema of the involved sites. Drug treatment. Rats were treated from day 0 until day 12 or 18, and drug administration was done orally by gavage in a 1% traganth suspension with an application volume of 5 ml/kg. A shamtreated group served as the control. Indometacin 1.5 mg/kg/day, dexamethasone at 0.5 mg/kg/day, cyclosporin A at 20 mg/kg/day, whereas the cyclophosphamide dose was 20 mg/kg/day. Serum samples. Blood samples were obtained by cardiac puncture. Blood was centrifuged and the serum removed. Sera were stored at - 2 0 ° C until testing for IL-6 activity. Cell preparation and culture conditions. Spleens were removed from sacrificed animals under sterile conditions and teased with forceps in PBS. After brief sedimentation to remove coarse particles, the cell suspension was washed and subsequently resuspended in RPMI 1640 containing 100 U/ml penicillin, 100/~g/ml streptomycin, 2 mM L-glutamine, 50 mM 2-mercaptoethanol and 10% foetal calf serum (FCS; Seromed, Berlin, F.R.G.). Cell counts and viability testing by the dye-exclusion method (0.5% trypan blue) were performed in a Neubauer haemocytometer. To test the IL-6 release of the splenocytes, cells were cultured in sterile culture flasks (1.3 x 106/ml) without mitogen, with heat-killed M. tuberculosis (50 Mg/ml), lipopolysaccharide (LPS Escherichia coli 055:B5, Calbiochem, La Jolla, CA, 2/~g/ml) or concanavalin A (Con A; LKB, Uppsala, Sweden, 4/~g/ml) over a period of 6 4 h at 37°C in a humidified atmosphere. Supernatants of the cultures were kept at - 2 0 ° C until testing for IL-6 activity. IL-6 bioassay. To measure IL-6 activity in the serum and spleen cell supernatants, the murine IL-6dependent hybridoma cell line 7TDI was used (generously provied by Dr Van Snick, Ludwig Institute for Cancer Research, Brussels, Belgium). The culture conditions have been described elsewhere (Van Damme et al., 1987b). In brief, cells were cultured in culture flasks (1 x 104/ml) in DMEM medium supplemented with 100 U/ml penicillin 100/~g/ml streptomycin, 10% FCS, 1.5 mM L-glutamine, 0.24 mM L-asparagine, 0.55 mM L-arginine, 50 ~M 2-mercaptoethanol, 0.1 mM hypoxanthine and 16 MM thymidine. To test IL-6 activity, cells were cultured in 96-well flat, bottom microtitre plates (1 x 103/well) with serial dilutions of the sera and supernatants, for 3 days in a humidified atmosphere. Cell proliferation
was determined by MTT-Test (Mosmann, 1983). Briefly, 10 M1 MTT (3-[4,5-dimethylthiazol-2-yi]-2, 5-diphenyltetrazolium bromide; 5 mg/ml) was added to the wells for 4 h; then 100 ~1 SDS in 0.01 N HCI was added to incubate overnight. Extinction was measured in the EL310 microplate reader (Biotek Instruments, Vermont) at 570 nm (test)/630 nm (reference). The standard used in this assay was a human, recombinant IL-6 (Boehringer, Mannheim, F.R.G.). IL-6 activity in serum and supernatant is expressed in U/ml, with 1 U/ml defined as the concentration supporting half maximal proliferation of 7TD1 cells. One unit of IL-6 corresponds to 5 pg of the cytokine. The 7TDI cells do not respond to IL-1 a or /3, tumour necrosis factor a, IL-2, interferon a, [3, ), or colony stimulating factors (Van Damme, Cayphas, Opdenakker, Billiau & Van Snick, 1987a; Van Snick et al., 1986). Statistical analysis. Data are expressed as the arithmetic mean _+ standard deviation (S.D.) of triplicate cultures in the proliferation assay. Data of disease activity (paw swelling, arthritic index) are given as arithmetic mean _+ S.D. of the individual rats. The statistical significancce of the data was determined using Student's t-test or the M a n n Whitney U-test.
RESULTS
Course o f adjuvant arthritis o f Lewis rats. As shown in Fig. 1, in the conditions used the disease is characterized by two stages: an acute phase involving the swelling of the injected (primary) paw and [ml]
O - P r i m a r y paw swelling • - S e c o n d a r y paw swelling - Arthritic index /
A~
100
A
/
• 80
~
60
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,
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lnt. J. lmmunopharmac., Vol. 14, No. 4, pp. 565-571, 1992.
Printed in Great Britain.
INTERLEUKIN-6 (IL-6) IN A D J U V A N T ARTHRITIS OF RATS AND ITS PHARMACOLOGICAL MODULATION P1A THEISEN-POPP,
HARTMUT
PAPE
and
REINER
MULLER-PEDDINGHAUS*
Troponwerke, Department of Antiphlogistic Research, Cologne, F.R.G. (Received 28 June 1991 and in final form 15 November 1991)
The levels of serum interleukin-6 (IL-6) were measured during the course of adjuvant arthritis (FAA) in male Lewis rats. In the course of the disease serum IL-6 levels increase with a clear correlation with morphologic disease signs. Additionally, the FAA-inducing antigen, heat-killed Mycobacterium tuberculosis, was found to be a strong inducer of 1L-6 production by spleen cells in vitro. The effects of the anti-rheumatic drugs indometacin, dexamethasone, cyclophosphamide and cyclosporin A (CsA) on IL-6 levels during FAA were determined. Complete normalization of serum IL-6 levels was observed with dexamethasone, cyclophosphamide and CsA whereas indometacin only partly reduced serum IL-6 levels.
Abstract --
Interleukin-6 (IL-6) exerts multiple actions on the growth, differentiation and function of lymphoid and non-lymphoid cells. It has recently been suggested that this cytokine plays a role in the pathogenesis of autoimmune disorders, in which the excessive production of IL-6 may lead to abnormal B-cell differentiation and antibody production. One example is cardiac myxoma, a condition often associated with autoimmune phenomena, such as hypergammaglobulinemia and autoantibodies (Sutton, Mercier, Giuliani & Lie, 1980). Myxoma cells in vitro produce significant amounts of IL-6 (Hirano et al., 1986, 1987). The autoimmune phenomena disappear after tumour removal. The question arose as to whether IL-6 might be involved in the pathogenesis of other autoimmune disorders, such as rheumatoid arthritis (RA). An elevation of IL-6 in the serum and synovial fluid of RA-patients was shown by different groups (Waage, Kaufmann, Espevik & Husby, 1989; Houssiau, Devogelaer, Van Damme, de Deuxchaisnes & Van Snick, 1988; Hirano et al., 1988). The adjuvant arthritis of rats (FAA), a T-cell mediated autoimmune disease, is one of the most important pharmacological models of rheumatoid arthritis (for review see Billingham, 1983). One aspect of our studies was to investigate the role of IL6 as a disease parameter of chronic inflammation.
Therefore, we measured IL-6 levels in the serum of FAA-rats during the course of the disease. Furthermore, the IL-6 production of spleen cells in response to in vitro stimulation with the arthritisinducing antigen, heat-killed Mycobacterium tuberculosis, was tested. A second aspect of this investigation was to examine the influence on serum IL-6 levels of drugs employed presently in the treatment of RA. Rats were treated orally with a non-steroidal anti-inflammatory drug (indometacin), dexamethasone, cyclophosphamide and cyclosporin
A (CsA). EXPERIMENTAL
PROCEDURES
Rats. Inbred male Lewis rats (120-150 g) were purchased from the Moellegaard Breeding Center (Skensved, Denmark). Animals were kept under standardized conditions on a standard diet (Altromin, Lage, F.R.G.) and water ad libitum. Induction and evaluation o f F A A . Heat-killed Mycobacterium tuberculosis (Difco, Detroit, MI) was suspended in paraffin oil (5 mg/ml, Merck, Darmstadt, F.R.G.) and injected intraplantarly into the footpad (50/al) of the right hind paw on day 0. In order to evaluate the progression of the disease two parameters were defined: (1) the swelling of the hind paws determined plethysmographically; and (2) the
*Author to whom correspondence should be addressed at: Department of Antiphlogistic Research, Troponwerke GmbH & Co. KG, Berliner Stra~e 156, 5000 Cologne 80, F.R.G. 565
566
P. THEISEN-PoPP el al.
arthritic score involving the quantification of seven arthritic sites: the three non-injected paws, ears, nose and tail. Parameters of evaluation were any sign of swelling and erythema of the involved sites. Drug treatment. Rats were treated from day 0 until day 12 or 18, and drug administration was done orally by gavage in a 1% traganth suspension with an application volume of 5 ml/kg. A shamtreated group served as the control. Indometacin 1.5 mg/kg/day, dexamethasone at 0.5 mg/kg/day, cyclosporin A at 20 mg/kg/day, whereas the cyclophosphamide dose was 20 mg/kg/day. Serum samples. Blood samples were obtained by cardiac puncture. Blood was centrifuged and the serum removed. Sera were stored at - 2 0 ° C until testing for IL-6 activity. Cell preparation and culture conditions. Spleens were removed from sacrificed animals under sterile conditions and teased with forceps in PBS. After brief sedimentation to remove coarse particles, the cell suspension was washed and subsequently resuspended in RPMI 1640 containing 100 U/ml penicillin, 100/~g/ml streptomycin, 2 mM L-glutamine, 50 mM 2-mercaptoethanol and 10% foetal calf serum (FCS; Seromed, Berlin, F.R.G.). Cell counts and viability testing by the dye-exclusion method (0.5% trypan blue) were performed in a Neubauer haemocytometer. To test the IL-6 release of the splenocytes, cells were cultured in sterile culture flasks (1.3 x 106/ml) without mitogen, with heat-killed M. tuberculosis (50 Mg/ml), lipopolysaccharide (LPS Escherichia coli 055:B5, Calbiochem, La Jolla, CA, 2/~g/ml) or concanavalin A (Con A; LKB, Uppsala, Sweden, 4/~g/ml) over a period of 6 4 h at 37°C in a humidified atmosphere. Supernatants of the cultures were kept at - 2 0 ° C until testing for IL-6 activity. IL-6 bioassay. To measure IL-6 activity in the serum and spleen cell supernatants, the murine IL-6dependent hybridoma cell line 7TDI was used (generously provied by Dr Van Snick, Ludwig Institute for Cancer Research, Brussels, Belgium). The culture conditions have been described elsewhere (Van Damme et al., 1987b). In brief, cells were cultured in culture flasks (1 x 104/ml) in DMEM medium supplemented with 100 U/ml penicillin 100/~g/ml streptomycin, 10% FCS, 1.5 mM L-glutamine, 0.24 mM L-asparagine, 0.55 mM L-arginine, 50 ~M 2-mercaptoethanol, 0.1 mM hypoxanthine and 16 MM thymidine. To test IL-6 activity, cells were cultured in 96-well flat, bottom microtitre plates (1 x 103/well) with serial dilutions of the sera and supernatants, for 3 days in a humidified atmosphere. Cell proliferation
was determined by MTT-Test (Mosmann, 1983). Briefly, 10 M1 MTT (3-[4,5-dimethylthiazol-2-yi]-2, 5-diphenyltetrazolium bromide; 5 mg/ml) was added to the wells for 4 h; then 100 ~1 SDS in 0.01 N HCI was added to incubate overnight. Extinction was measured in the EL310 microplate reader (Biotek Instruments, Vermont) at 570 nm (test)/630 nm (reference). The standard used in this assay was a human, recombinant IL-6 (Boehringer, Mannheim, F.R.G.). IL-6 activity in serum and supernatant is expressed in U/ml, with 1 U/ml defined as the concentration supporting half maximal proliferation of 7TD1 cells. One unit of IL-6 corresponds to 5 pg of the cytokine. The 7TDI cells do not respond to IL-1 a or /3, tumour necrosis factor a, IL-2, interferon a, [3, ), or colony stimulating factors (Van Damme, Cayphas, Opdenakker, Billiau & Van Snick, 1987a; Van Snick et al., 1986). Statistical analysis. Data are expressed as the arithmetic mean _+ standard deviation (S.D.) of triplicate cultures in the proliferation assay. Data of disease activity (paw swelling, arthritic index) are given as arithmetic mean _+ S.D. of the individual rats. The statistical significancce of the data was determined using Student's t-test or the M a n n Whitney U-test.
RESULTS
Course o f adjuvant arthritis o f Lewis rats. As shown in Fig. 1, in the conditions used the disease is characterized by two stages: an acute phase involving the swelling of the injected (primary) paw and [ml]
O - P r i m a r y paw swelling • - S e c o n d a r y paw swelling - Arthritic index /
A~
100
A
/
• 80
~
60
; / Q'/-
,
A
o
,
0
T/'~
r//I
~~ 0 ~ 0 / ' ~/ i
5
,
E~]
.cu
,o
/,
