Research in Veterinary Science 1991, 51, 72-77
Interleukin-l-like activity in synovial fluids and sera of horses with arthritis W. H. A L W A N * t , S. D. CARTER*t, J. B. DIXON*, D. BENNETT't, S. A. M A Y t , G. B. EDWARDS-t,
Departments of Veterinary Pathology*, and Veterinary Clinical Sciencet, University of Liverpool, PO Box 147, Liverpool L69 3BX
Synovial fluid samples of horses with osteoarthritis were investigated to detect interleukin-1 0L-0 activity which could contribute to the disease pathogenesis. Of the 32 samples tested, 12 (37.5 per cent) showed an augmented phytohaemagglutinin induced proliferation of C3H/HeJ mouse thymocytes. Positive results were also seen in horses with infected arthritis, osteochondritis, traumatic arthritis and undefined synovial effusions. Normal synovial fluid and sera from all groups failed to show any detectable IL-1 activity. Fractionation of synovial fluid showed that the IL-1 activity was in the 15 to 20 Kd fractions. In the absence of mitogen, synovial fluid failed to stimulate thymocytes and did not stimulate the growth of an interleukin-2 (IL-2) dependent CTLL cell line, but synovial fluid stimulated IL-2 release by mouse spleen cells incubated with suboptimal doses of lectin. Evidence of an IL-1 inhibitor in synovial fluid from osteoarthritic horses was provided by ultrafiltration experiments and by the inhibitory activity of synovial fluid at particular dilutions in the thymocyte assay. The presence of IL-l-like activity could be relevant in the pathogenesis of arthritis in horses.
INTERLEUKIN-1 (IL-1) is a monokine produced by macrophages with a molecular weight of about 15,000 Daltons (Mizel et al 1978) which has been implicated in the pathogenesis of rheumatoid arthritis (Nouri et al 1984) and osteoarthritis (Alwan et al 1988) in man. In recent years it has become clear that IL-1 is not only produced by macrophages, but can be synthesised and secreted from a variety of connective tissues and cells of human and animal origin in culture (Dingle et al 1979, Rath et a11988). In particular, synovial ceils can secrete IL-1 (Wood et al 1985) and IL-1 is thought to have many properties of relevance to rheumatic diseases in man and animals. These include the activation of hepatocytes with synthesis and release of acute phase reactants (Sztein et al 1981), stimulation
of synovial adherent cells to release prostaglandin E 2 and collagenase (Miller and Dinarello 1987), action on chondrocytes (as catabolin) to cause cartilage degradation (Jasin and Dingle 1981) and to induce bone resorption (Heath et al 1985). Furthermore, IL-I has been demonstrated in the synovial fluids of human patients with a variety of joint diseases (Nouri et al 1984, Bendtzen et al 1985, Miossec et al 1986). The degenerative joint disease seen in the horse has many similarities with the human disorder, both clinically and pathologically. In addition, equine osteoarthritis is characterised by degeneration of cartilage macromolecules, particularly proteoglycans (Alwan et al 1990, 1991) and antibodies against these molecules and other debris may lead to immune complex formation within the joint (Niebauer et al 1988) where they could have a stimulatory effect on IL-1 production. Equine IL-1 has recently been characterised and shown to be active in assays routinely used for measuring human IL-1 (May et al 1990). Therefore, a study was undertaken to find out whether synovial fluids from horses with osteoarthritis and other types of arthritides contained IL-1 activity. Materials and methods
Patients and samples Synovial fluid (SF) aspirates were collected from horses under sterile conditions, centrifuged at 10,000 g for 20 minutes to remove cells and debris and stored in aliquots at - 7 0 ° C until assayed. Blood from horses was also collected, and sera were separated by centrifugation and stored in 0.5 ml aliquots. The animals investigated comprised cases of osteoarthritis (32 joints), septic arthritis (six joints), osteochondritis (five joints), traumatic arthritis (four joints), synovial effusion of unknown cause (four joints) and 34 normal joints. Cases o f osteoarthritis were referred to the university Large A n i m a l H o s p i t a l as lameness problems for a second opinion. The diagnosis of
Reprint requests to Dr S. D. Carter, Department of Veterinary Pathology, University of Liverpool, PO Box 147, Liverpool L69 3BX
72
lnterleukin-1 in horses with arthritis osteoarthritis, osteochondritis and traumatic arthritis was based on the history, clinical examination and radiological assessment. Most cases involved the carpal and fetlock joints. Septic arthritis was diagnosed by clinical and radiological examination and by cytological examination of the SF (high polymorphonuclear cell counts) and in some cases by a positive bacterial culture from the SF. Horses with normal joints were selected from clinical cases presented at the Large Animal Hospital with no history of locomotor disease; most were colic cases. The absence of joint disease was confirmed by clinical examination, SF analyses and, in some cases, radiography.
IL-1 a s s a y
The IL-1activity of SF was determined by measuring the augmenting effect on phytohaemagglutinin (PHA)-stimulated thymocytes (Gery et al 1972). Thymuses from four- to six-week-old C3H/HeJ mice were finely minced in sterile phosphate buffered saline (PBS) and dispersed by repeated pipetting and passage through a stainless steel screen, washed three times and the final cell preparation adjusted to 2 × 107 m l - ~in RPMI 1640 medium supplemented with 50 iu m l - l penicillin, 50/~g m l - 1 streptomycin, 5 per cent newborn calf serum, 3 × 10 -5 M 2-mercaptoethanol and 1 per cent L-glutamine (Flow Laboratory). Aliquots of cell suspension containing 1-5x 106 cells well -~ were dispersed into 96 well plates. Test samples were diluted with culture medium and added to cultures in quadruplicate at a final dilution of 1 in 10 either with or without 1 /xg m l - l PHA (Wellcome). Cells were also cultured in medium alone or medium plus PHA. Cultures were incubated for 72 hours at 37°C in a 5 per cent carbon dioxide incubator. The rate of DNA synthesis was determined by measuring the incorporation of 3H-thymidine, 0.5 /~Ci well - l (Amersham, TRA 360, 2 Ci mmol -l) after a 16 hour incubation. The effect of SF on the PHA response of thymocytes was compared with the background proliferative response of cells cultured with PHA only. On each occasion the PHA-induced proliferation was less than 500 counts per minute (cpm) (mean + 2 SD = 890 cpm). Before testing SF for IL-l-like activity, the fluids were diluted 1:5, passed through two filters (0.45/zm and 0" 20 tzm) and applied to a micro-partition system (MPS-1 with YM Diaglo 10 membrane; Amicon) and centrifuged at 3000 g for 10 minutes at 4°C to remove low molecular weight materials ( < 10,000 Kd). sF were tested in the assay both before and after filtration. In addition some SF samples were also tested at a range of concentrations. In each experiment, a standard IL-1 preparation was added to aliquots of cells. The
73
standard was prepared by culturing adherent cells from peripheral blood mononuclear leucocyte preparations, isolated from the blood of a normal horse by Percoll (sp gr 1-075) separation, with 10 #g ml-1 lipopolysaccharide (Difco) and 1 Izg m l - l indomethacin (Sigma). Two days later, culture supernatants (SN) were collected, centrifuged, dialysed, filtered and used in the IL-1 assay at 5 per cent. The proliferation produced by this preparation was large (5000 to 6000 cpm) and consistent.
IL-2 a s s a y
IL-2 activity was assayed by an IL-2 dependent mouse CTLL cell line (kindly provided by Dr Stuart Marshall-Clarke, University of Liverpool) according to the method of Leibson et al (1981). The cells were cultured at 1 × 104 well-1 together with filtered SF samples diluted in culture medium at a final dilution of 1 in 10, for 48 hours. The incorporation of 3Hthymidine was determined over a 12 hour period. In each experiment, standard horse IL-2 was cultured with aliquots of CTLL cell line at a final dilution of 5 per cent. The It-2 standard was prepared by culturing horse blood lymphocytes, which were separated by Ficoll-Hypaque density sedimentation, at 5× 106 ml-1 with 5/zg ml-1 concanavalin A (Con A) for 48 hours, the cells were removed by centrifugation and the supernatant dialysed, filtered and used in the IL-2 assay. Gel filtration chromatography SF samples were treated with sheep testis hyaluronidase (100 iu ml-1) to reduce viscosity, diluted 1:2 in Pns and passed through a 0.45 /~m filter. Six SF samples from horses with osteoarthritis and two samples from infected arthritis cases (which showed the highest IL-1 activity) were separated by gel filtration on a 2.6 × 100 cm column of ACA 34 Ultrogel and eluted with PBs pH 7-2. Four ml fractions were collected, the OD was read at 280 nm in a spectrophotometer and four peaks were collected, concentrated, filter sterilised and assayed for ILq activity at 1:10 dilution.
Production o f mouse IL-2 In experiments performed to monitor IL-2 production by C3H/HeJ mouse spleen cells upon stimulation with sv or standard horse ILq, 5× 106 spleen cells were incubated in RPMI 1640 with other supplements (as detailed above) and Con A at 1 t~g ml-1 or 5 #g m l - l . The cultures treated with a suboptimal dose of Con A (1 #g ml -~) were supplemented with either 5 per cent of the standard horse
W. H. Alwan, S. D. Carter, J. B. Dixon, D. Bennett, S. A. May, G. B. Edwards
74
IL-I or 1:10 dilution of the filtered SF from horses with arthritis. At the end of the culture period (48 hours), the cells were removed by centrifugation and the supernatants were assayed for IL-2 activity.
assay and added at 10 per cent. Cell proliferation was measured after three days by the incorporation of 3H-thymidine. Results
D.IO.G4.1 IL.1 assay
IL-1 in synovial fluids
This assay, which detects [L-1 but not IL-6, was performed on 19 osteoarthritis SF samples. The D.10.G4.1 mouse cell line was maintained in tissue culture. SF samples were treated as for the thymocyte
9000-
8000--
7000-
6000--
=~5000-E 4000-
In experiments designed to test the effect of SF on the PHA-induced proliferative response of C3H/HeJ thymocytes, the results were expressed as cpm and compared with the background proliferative response of cells and PHA only. Any sample with cpm more than double the background response was considered to have IL-l-like activity. Using the equine IL-1 standard in the assay, a 10- to 20-fold increase of the background PHA response was observed. Normal synovial fluids had consistently low levels of lEd; all were below the line defined by the background PnAstimulated uptake of 3H-thymidine by the thymocytes in the absence of synovial fluids (Fig 1). Comparisons with the synovial fluids from horses with joint diseases showed that some had increased levels and that some, particularly in infective arthropathies, were very high. Statistical analysis (Student's t test/Mann-Whitney) showed that all four patient groups had significantly greater levels of SF IL-1 than the normal SF samples (in all groups P
Interleukin-l-like activity in synovial fluids and sera of horses with arthritis W. H. A L W A N * t , S. D. CARTER*t, J. B. DIXON*, D. BENNETT't, S. A. M A Y t , G. B. EDWARDS-t,
Departments of Veterinary Pathology*, and Veterinary Clinical Sciencet, University of Liverpool, PO Box 147, Liverpool L69 3BX
Synovial fluid samples of horses with osteoarthritis were investigated to detect interleukin-1 0L-0 activity which could contribute to the disease pathogenesis. Of the 32 samples tested, 12 (37.5 per cent) showed an augmented phytohaemagglutinin induced proliferation of C3H/HeJ mouse thymocytes. Positive results were also seen in horses with infected arthritis, osteochondritis, traumatic arthritis and undefined synovial effusions. Normal synovial fluid and sera from all groups failed to show any detectable IL-1 activity. Fractionation of synovial fluid showed that the IL-1 activity was in the 15 to 20 Kd fractions. In the absence of mitogen, synovial fluid failed to stimulate thymocytes and did not stimulate the growth of an interleukin-2 (IL-2) dependent CTLL cell line, but synovial fluid stimulated IL-2 release by mouse spleen cells incubated with suboptimal doses of lectin. Evidence of an IL-1 inhibitor in synovial fluid from osteoarthritic horses was provided by ultrafiltration experiments and by the inhibitory activity of synovial fluid at particular dilutions in the thymocyte assay. The presence of IL-l-like activity could be relevant in the pathogenesis of arthritis in horses.
INTERLEUKIN-1 (IL-1) is a monokine produced by macrophages with a molecular weight of about 15,000 Daltons (Mizel et al 1978) which has been implicated in the pathogenesis of rheumatoid arthritis (Nouri et al 1984) and osteoarthritis (Alwan et al 1988) in man. In recent years it has become clear that IL-1 is not only produced by macrophages, but can be synthesised and secreted from a variety of connective tissues and cells of human and animal origin in culture (Dingle et al 1979, Rath et a11988). In particular, synovial ceils can secrete IL-1 (Wood et al 1985) and IL-1 is thought to have many properties of relevance to rheumatic diseases in man and animals. These include the activation of hepatocytes with synthesis and release of acute phase reactants (Sztein et al 1981), stimulation
of synovial adherent cells to release prostaglandin E 2 and collagenase (Miller and Dinarello 1987), action on chondrocytes (as catabolin) to cause cartilage degradation (Jasin and Dingle 1981) and to induce bone resorption (Heath et al 1985). Furthermore, IL-I has been demonstrated in the synovial fluids of human patients with a variety of joint diseases (Nouri et al 1984, Bendtzen et al 1985, Miossec et al 1986). The degenerative joint disease seen in the horse has many similarities with the human disorder, both clinically and pathologically. In addition, equine osteoarthritis is characterised by degeneration of cartilage macromolecules, particularly proteoglycans (Alwan et al 1990, 1991) and antibodies against these molecules and other debris may lead to immune complex formation within the joint (Niebauer et al 1988) where they could have a stimulatory effect on IL-1 production. Equine IL-1 has recently been characterised and shown to be active in assays routinely used for measuring human IL-1 (May et al 1990). Therefore, a study was undertaken to find out whether synovial fluids from horses with osteoarthritis and other types of arthritides contained IL-1 activity. Materials and methods
Patients and samples Synovial fluid (SF) aspirates were collected from horses under sterile conditions, centrifuged at 10,000 g for 20 minutes to remove cells and debris and stored in aliquots at - 7 0 ° C until assayed. Blood from horses was also collected, and sera were separated by centrifugation and stored in 0.5 ml aliquots. The animals investigated comprised cases of osteoarthritis (32 joints), septic arthritis (six joints), osteochondritis (five joints), traumatic arthritis (four joints), synovial effusion of unknown cause (four joints) and 34 normal joints. Cases o f osteoarthritis were referred to the university Large A n i m a l H o s p i t a l as lameness problems for a second opinion. The diagnosis of
Reprint requests to Dr S. D. Carter, Department of Veterinary Pathology, University of Liverpool, PO Box 147, Liverpool L69 3BX
72
lnterleukin-1 in horses with arthritis osteoarthritis, osteochondritis and traumatic arthritis was based on the history, clinical examination and radiological assessment. Most cases involved the carpal and fetlock joints. Septic arthritis was diagnosed by clinical and radiological examination and by cytological examination of the SF (high polymorphonuclear cell counts) and in some cases by a positive bacterial culture from the SF. Horses with normal joints were selected from clinical cases presented at the Large Animal Hospital with no history of locomotor disease; most were colic cases. The absence of joint disease was confirmed by clinical examination, SF analyses and, in some cases, radiography.
IL-1 a s s a y
The IL-1activity of SF was determined by measuring the augmenting effect on phytohaemagglutinin (PHA)-stimulated thymocytes (Gery et al 1972). Thymuses from four- to six-week-old C3H/HeJ mice were finely minced in sterile phosphate buffered saline (PBS) and dispersed by repeated pipetting and passage through a stainless steel screen, washed three times and the final cell preparation adjusted to 2 × 107 m l - ~in RPMI 1640 medium supplemented with 50 iu m l - l penicillin, 50/~g m l - 1 streptomycin, 5 per cent newborn calf serum, 3 × 10 -5 M 2-mercaptoethanol and 1 per cent L-glutamine (Flow Laboratory). Aliquots of cell suspension containing 1-5x 106 cells well -~ were dispersed into 96 well plates. Test samples were diluted with culture medium and added to cultures in quadruplicate at a final dilution of 1 in 10 either with or without 1 /xg m l - l PHA (Wellcome). Cells were also cultured in medium alone or medium plus PHA. Cultures were incubated for 72 hours at 37°C in a 5 per cent carbon dioxide incubator. The rate of DNA synthesis was determined by measuring the incorporation of 3H-thymidine, 0.5 /~Ci well - l (Amersham, TRA 360, 2 Ci mmol -l) after a 16 hour incubation. The effect of SF on the PHA response of thymocytes was compared with the background proliferative response of cells cultured with PHA only. On each occasion the PHA-induced proliferation was less than 500 counts per minute (cpm) (mean + 2 SD = 890 cpm). Before testing SF for IL-l-like activity, the fluids were diluted 1:5, passed through two filters (0.45/zm and 0" 20 tzm) and applied to a micro-partition system (MPS-1 with YM Diaglo 10 membrane; Amicon) and centrifuged at 3000 g for 10 minutes at 4°C to remove low molecular weight materials ( < 10,000 Kd). sF were tested in the assay both before and after filtration. In addition some SF samples were also tested at a range of concentrations. In each experiment, a standard IL-1 preparation was added to aliquots of cells. The
73
standard was prepared by culturing adherent cells from peripheral blood mononuclear leucocyte preparations, isolated from the blood of a normal horse by Percoll (sp gr 1-075) separation, with 10 #g ml-1 lipopolysaccharide (Difco) and 1 Izg m l - l indomethacin (Sigma). Two days later, culture supernatants (SN) were collected, centrifuged, dialysed, filtered and used in the IL-1 assay at 5 per cent. The proliferation produced by this preparation was large (5000 to 6000 cpm) and consistent.
IL-2 a s s a y
IL-2 activity was assayed by an IL-2 dependent mouse CTLL cell line (kindly provided by Dr Stuart Marshall-Clarke, University of Liverpool) according to the method of Leibson et al (1981). The cells were cultured at 1 × 104 well-1 together with filtered SF samples diluted in culture medium at a final dilution of 1 in 10, for 48 hours. The incorporation of 3Hthymidine was determined over a 12 hour period. In each experiment, standard horse IL-2 was cultured with aliquots of CTLL cell line at a final dilution of 5 per cent. The It-2 standard was prepared by culturing horse blood lymphocytes, which were separated by Ficoll-Hypaque density sedimentation, at 5× 106 ml-1 with 5/zg ml-1 concanavalin A (Con A) for 48 hours, the cells were removed by centrifugation and the supernatant dialysed, filtered and used in the IL-2 assay. Gel filtration chromatography SF samples were treated with sheep testis hyaluronidase (100 iu ml-1) to reduce viscosity, diluted 1:2 in Pns and passed through a 0.45 /~m filter. Six SF samples from horses with osteoarthritis and two samples from infected arthritis cases (which showed the highest IL-1 activity) were separated by gel filtration on a 2.6 × 100 cm column of ACA 34 Ultrogel and eluted with PBs pH 7-2. Four ml fractions were collected, the OD was read at 280 nm in a spectrophotometer and four peaks were collected, concentrated, filter sterilised and assayed for ILq activity at 1:10 dilution.
Production o f mouse IL-2 In experiments performed to monitor IL-2 production by C3H/HeJ mouse spleen cells upon stimulation with sv or standard horse ILq, 5× 106 spleen cells were incubated in RPMI 1640 with other supplements (as detailed above) and Con A at 1 t~g ml-1 or 5 #g m l - l . The cultures treated with a suboptimal dose of Con A (1 #g ml -~) were supplemented with either 5 per cent of the standard horse
W. H. Alwan, S. D. Carter, J. B. Dixon, D. Bennett, S. A. May, G. B. Edwards
74
IL-I or 1:10 dilution of the filtered SF from horses with arthritis. At the end of the culture period (48 hours), the cells were removed by centrifugation and the supernatants were assayed for IL-2 activity.
assay and added at 10 per cent. Cell proliferation was measured after three days by the incorporation of 3H-thymidine. Results
D.IO.G4.1 IL.1 assay
IL-1 in synovial fluids
This assay, which detects [L-1 but not IL-6, was performed on 19 osteoarthritis SF samples. The D.10.G4.1 mouse cell line was maintained in tissue culture. SF samples were treated as for the thymocyte
9000-
8000--
7000-
6000--
=~5000-E 4000-
In experiments designed to test the effect of SF on the PHA-induced proliferative response of C3H/HeJ thymocytes, the results were expressed as cpm and compared with the background proliferative response of cells and PHA only. Any sample with cpm more than double the background response was considered to have IL-l-like activity. Using the equine IL-1 standard in the assay, a 10- to 20-fold increase of the background PHA response was observed. Normal synovial fluids had consistently low levels of lEd; all were below the line defined by the background PnAstimulated uptake of 3H-thymidine by the thymocytes in the absence of synovial fluids (Fig 1). Comparisons with the synovial fluids from horses with joint diseases showed that some had increased levels and that some, particularly in infective arthropathies, were very high. Statistical analysis (Student's t test/Mann-Whitney) showed that all four patient groups had significantly greater levels of SF IL-1 than the normal SF samples (in all groups P
