Transforming growth factor-β1 concentrations in equine synovial fluid

I-'quina Mutrition and Phyaiologg qoeletg Annual ~gmpo~ium Mau 2~'-~1, 1.097. Fort Worth, T~xa~.

TRANSFORMING GROWTH FACTOR-81 CONCENTRATIONS IN EQUINE SYNOVIAL FLUID L.D. Anderson; R.H. Raub; D.M. Grieger; J. Morris; J.D. Weber

SUMMARY A commercial assay for human TGF-g 1 was validated for use on equine synovial fluid and serum. This assay was then used to determine concentrations of TGF-gl in synovial fluid samples from 109 metacarpophalangealjoints of horses with varying age, gender, and joint condition. No difference in concentration was found between geldings and females. A difference (p =.04) was found between young horses (<48 months, 1101 pg/ml) and old horses (->120 months, 863 pg/ml), and between horses with visibly normal joints and those with severely diseased joints (885 versus 1002 pg/ml, p=0.01). These findings suggest concentrations of TGF-131 are elevated in joints from younger horses and joints with gross evidence of disease.

INTRODUCTION Transforming growth factor-beta (TGF-f3) has been shown to have multiple effects on joint related tissues in vitro and in vivo. In vivo studies have shown intra-articular TGF-B administration to induce osteoarthritis, t and intraarticular anti-TGF-g antibody administration to reverse synovial inflammation, z In addition, TGF-g 1 (a member of the TGF-g family of peptides) concentrations have been shown to be higher in synovial fluid of humans suffering from rheumatoid arthritis and seronegative spondyloarthropathies than in those with osteoarthritis, a However, systemic administration of TGF-13 was shown to suppress experimentally induced polyarthritis. 4 At the cartilage level, in vivo studies have shown a deficiency of TGF-g in osteochondritic areas of porcine and chick epiphyses, s as well as intra-articular TGF-g 1 administration causing articular cartilage hypertrophic zone changes, including loss of height, cell volume density, collagen volume density, and changes in cell morphology in the rat. ~ In vitro, TGF-13 has been shown to stimulate Authors' address: Kansas State University, Manhattan, KS 66506

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proteoglycan production in cartilage from healthy joints, 7 and osteoarthritic cartilage has been shown to be more sensitive to this stimulation, s This stimulatory action on cultured equine chondrocytes was confirmed for TGF-g 1, specifically, in serum free culture, but proteoglycan synthesis and accumulation was decreased by TGF-B1 when cultured in fetal bovine serum, a In addition, TGF-t31 expression as well as proteoglycan production in chondrocyte-like cell line culture has been shown to be decreased by greater than normal physiologic hydrostatic pressures.10 TGF-g 1 also has been shown to stimulate in vitro human chondrocyte mitogenesis] 1 and this stimulation is more pronounced in chondrocytes cultured from osteoarthritic joints, le TGF-gl also has been shown to modify the structure of glycosaminoglycans produced by porcine articular chondrocyte cultures.la Finally, TGF-f3 has been shown to increase the activity of transglutaminase, an enzyme involved in the normal and pathologic mineralization of cartilage, in cultured porcine articular chondrocytes. 14 Synovial tissue also is affected by TGF-fS. Human synovial tissue from patients with rheumatoid arthritis and seronegative spondyloarthropathy expressed higher concentrations of TGF-g than other growth factors or cytokines. 15 In addition, human rheumatoid synovial cells treated with TGF-13 proliferated more rapidly, 16 and synovial tissue of collagen-induced arthritic rats showed abundant and increasing expression of TGF-13 as well as TGF-g type I and II receptors during the development of disease] 7 The above evidence points to TGF-g, and particularly TGF-g 1, as an important factor in normal and pathologic conditions involving the joint. Systemic administration of TGF-131 or local administration of anti-TGF-131 may prove to have therapeutic value, and TGF-f31 concentrations may prove to be an early indicator of various joint diseases. Establishing normal values and the effects of age, diet, exercise, growth rate, and various joint diseases or conditions on these values may provide insight into T G F - g l ' s actions and potential areas for intervention to prevent or decrease the severity of disease. In this study, we validate a commercial human TGF-gl ELISA for use on equine

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serum and synovial fluid and do a preliminary study of the effects of age, gender and joint condition on TGF-f~I concentrations in equine synovial fluid.

MATERIALS AND METHODS

Sample Collection. Synovial fluid samples were obtained on two consecutive days at an equine slaughter house. Age was approximated by dental exam. 18 Age groups were divided into young (<48 months), middle (>48 months and <120 months), and old (>120 months). Sex of each horse was noted and each front foot was tagged for later identification. At a mean time of 5.75 hours after limb amputation during processing, individual synovial fluid samples were collected from left and right metacarpophalangeal joints. Samples were immediately placed in liquid nitrogen. Each joint sampled was then dissected open to assess visible abnormalities of joint surfaces. In addition, serum from blood was obtained from a live horse by left external jugular venipuncture and frozen for future assay validation procedures. ELISA Validation. Parallelism, recovery, inter- and intra-assay accuracy were evaluated to validate a commercial human TGF-B1 assay a for use on equine serum and synovial fluid. The manufacturer' s protocol provided with the assay kit was followed unless noted otherwise. The sandwich ELISA utilized a TGF-B receptor specific for TGF-B 1, -B2, and -133 in conjunction with an anti-TGF-131 antibody conjugated to horse radish peroxidase (HRP) to quantitatively detect TGF-131 using colorimetry. TGF-131 is secreted in vivo as a biologically inactive peptide noncovalently bound to a protein referred t o a s latency associated peptide (LAP). Transient acidification disassociates TGF-B from LAP rendering it biologically active. This activated form of TGF-131 is detectable in the assay. A preliminary ELISA run on three synovial fluid samples and one serum sample to compare activated and nonactivated samples yielded no detectable TGF-B 1 in the nonactivated samples. The manufacturer's protocol provided for activating cell culture supernatant was followed to activate equine synovial fluid samples. The activation protocol provided for human serum was used to activate equine serum samples. Synovial fluid samples were centrifuged for 10 minutes at 16,000 x g and the supernatant used in the assays. All unknown samples were tested in two replicates within one assay and their mean optical density (O.D.) compared to a standard curve for calculation of TGF-131 concentration. The standard curve was established for each assay according to the manufacturer's protocol using dilutions of recombinant human TGF-131 (rhTGF-131; provided in the kit) at concentrations of 0, 31.2, 62.5, 125,250, 500, 1000, and 2000 pg/ml. All assays were conducted in 98 aR and D Systems, 614 McKinley Place N.E., Minneapolis, MN 55413

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well plates provided in the kit and were analyzed on a Molecular Devices Vmax kinetic microplate plate reader at 450 and at 570 nM. Readings at 570 nM were subtracted from those at 450 nM to correct for optical imperfections in the plate as per manufacturer's protocol. Parallelism. Activated synovial fluid from two separate horses was initially assayed for parallelism at 50, 100 and 200 gl per well. A pool of 6 samples was made to assure an average representation and to provide an adequate sample volume for an additional test of parallelism. The pool was assayed at 6.25, 12.5, 25, 50, 100, and 200 ~1 per well. These values were plotted for comparison of parallelism to the standard curve within their respective assays. To test parallelism of equine serum, an activated sample was assayed at 50, 100, and 200 ~ per well and plotted against the standard curve. Recovery. Synovial fluid samples from 10 horses were pooled together to assure an average representation and to supply an adequate sample volume. The pool was activated and used for recovery testing. Fifty gl of the synovial fluid pool was added to each well. Since the samples were diluted during activation and the total final volume per well was 200 ~ , the final dilution of synovial fluid was 1:5,6. Recombinant human TGF-B 1 provided in the commercial assay kit were added to the synovial fluid pool to attain concentrations of 0, 50 and 100 pg/ml. All samples were run in triplicate and their mean optical density was used to calculate TGF-131 concentrations. Percent recovery was calculated as concentration of TGF-B1 in the samples to which rhTGF-B 1 was added divided by the sum of the TGF131 concentration in the sample to which no rhTGF-131 was added and the concentration of rhTGF-B 1 added. A single equine serum sample was activated and used to test recovery as well. To each well 100 gl of serum was added (final dilution of 1:60). Recombinant human TGF131 was added to serum to attain concentrations of 0, 50, and 100 pg/ml and calculation of percent recovery was done as described for synovial fluid recovery. Intra- and Inter-assay Accuracy. A pool created from ten synovial fluid samples was activated and assayed at 50 pl per well (final dilution of 1:5.6) in three assays. In one assay the sample was run in seven wells, and in the other two assays it was run in three wells. Their intra-assay mean, standard deviation and coefficient of variability percent (CV%) was calculated for each assay. The mean pool value for each assay was used to calculate the inter-assay mean, standard deviation, and coefficient of variation (CV). Synovial Fluid Samples. Assays of synovial fluid samples from 109 joints were performed using 50 gl of fluid per well (final dilution of 1:5.6). Of these, 57 samples came from geldings and 52 from females. Joint condition for each was classified as normal, as having moderate lesions, or as having severe lesions. Normal joints had no visible abnormalities. Moderate lesions included synovial adhesions, 2 to 3 mm articular cartilage defects, and mild JOURNAL OF EQUINE VETERINARY SCIENCE

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fibrillation of articular cartilage. Severe lesions included osteochondritic lesions, osteochondritis desicans lesions, chip fractures, severe fibrillation, and eburnation. Of the joints analyzed, 37 were normal, 35 had moderate lesions, 36 had severe lesions, and the joint condition was not determined for one horse. When samples were divided by age, 30 were 48 months old or less, 38 were greater than 48 months but less than 120 months, and 41 were 120 months or greater in age. Age, sex and joint condition effects were analyzed with the general linear models procedure of SAS.19

RESULTS ELISA Validation Parallelism. Increasing values of equine synovialfluid paralleled the standard curve within the range of 6.25 to 100 per well. However, parallelism was lost at a volume of 200 ~l per well (fig. 1). This loss of parallelism at 200 ~ of synovial fluid was consistent in all three assays. Assay values for 50, 100, and 200 ~ of equine serum per well also paralleled the standard curve (fig. 2). Recovery. Recovery of rhTGF-B 1 from synovial fluid samples was 95% at the 50 pg/ml concentration and 92% at the 100 pg/ml concentration. Recovery of rhTGF-B 1 from serum samples was 96% at the 50 pg/ml concentration and 87% at the 100 pg/ml concentration. Intra- and Inter-assay Accuracy. A common synovial fluid sample pool was measured in three assays to determine accuracy. One assay measured the pool in seven wells, had a mean TGF-61 concentration of 803 pg/ml, a standard deviation of 29.1 pg/ml, and a CV of 3.6%. A second assay measured the pool in triplicate, had a mean TGF-61 concentration of 876 pg/ml, a standard deviation of 35.0 pg/ml, and a CV of 4.0%. The third assay also measured the pool in triplicate, had a mean TGF-61 concentration of 816 pg/ml, a standard deviation of 39.1 pg/ml, and a CV of 4.8%. The

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Figure 2. Parallelism of TGF-81 in equine serum samples as compared to a standard curve. overall intra-assay CV was 4.1%. Using the means from the above three assays, assessment of inter-assay accuracy was made. Inter-assay mean was 831.5 pg/ml, standard deviation was 38.8 pg/ml, and CV was 4.7%.

Synovial Fluid Samples Analysis of variance showed no correlation between age and joint condition. Concentrations of TGF-61 in synovial fluid samples ranged from 406 to 2071 pg/ml, with a mean of 936 pg/ml. No difference was detected between mean synovial fluid concentrations in geldings (911 pg/ml) and females (963 pg/ml). Mean concentrations of TGF-B1 for the age groups were 1011,957, and 863 pg/ml for the young, middle, and old age groups, respectively. Least squares means analysis indicated a difference (p=0.04) between mean concentrations of the young and old age groups, but not between the middle and old age groups (p=0.19) or between the middle and young age groups (p--0.39). A good correlation for prediction was not found (R2=0.05). Least squares means analysis indicated a difference (p=0.01) between mean TGF-B 1 concentrations in healthy (885 pg/ml) versus severely diseased (1002 pg/ml)joints, but not between moderately diseased (898 pg/ml) and healthy joints (p=0.11) or between moderately and severely diseased joints (p=0.41). TGF-B1 concentration was different (p<.05) between healthy (805 pg/ml) and severely diseased (1104 pg/ml) joints within the moderate age group and within the old age group (p<.05; 729 vs 962 pg/ml). TGF-81 concentration was not different due to joint condition within the young age group.

DISCUSSION Parallelism demonstrated throughout a range of dilutions on three assays indicates that equine TGF-61 concen-

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trations are measured similarly to rhTGF-61 at these different concentrations. Loss of parallelism at the higher concentration of synovial fluid may indicate the presence of a substance that interferes with or competes for binding to receptors and/or antibody. Recoveries calculated indicate that the sample contents are not interfering with the assay's ability to detect rhTGF-B 1, and provide supporting evidence that the assay is consistently detecting equine TGF-131. Intra- and interassay accuracy results provide evidence that values are as repeatable for equine TGF-131 as for human TGF-61 within a single assay and between assays. Joint condition classification was based on the visual appearance of a general pathological progression of joint disease. Histochemical and biochemical analysis of joint condition would have been most beneficial to more accurately assess joint condition. However, limitations of the slaughter facility did not allow for such analysis. Statistical analysis of the data when categorized as healthy or diseased joints did not change the significance of the results. TGF-B1 secretion has been shown to be modulated during normal menstrual cycling humans, 2~ and 17-13estradiol treatment of osteoclasts in vitro has been shown to increase TGF-B accumulation. 21 These findings suggest different TGF-131 concentrations in synovial fluid between genders was likely to be seen. The findings of this study do not support this; however, all males were gelded and the state of estrous of the mares was not known. Future studies should include stallions and mares in known stages of estrous and gestation to study the effects of higher concentrations of sex hormones on TGF-131 concentrations in serum and synovial fluid. TGF-131 has been shown to stimulate cartilage mitogenesis.11 Cartilage cells in normally developing young growing horses may have a greater expression of TGF-61; this may be reflected in greater concentrations of TGF-131 in synovial fluid. The results of our study support this assumption, i.e., there was a difference in TGF-131 concentrations in synovial fluid between the youngest and oldest age groups of horses. Lack of a predictable correlation between age and synovial TGF-131 concentration may be due to the relatively few horses sampled in the young group that were in a significant growth phase; only seven of the 30 were 12 months or less in age. Also, uncontrolled factors such as genetics and nutrition may have affected any correlation with age. Previous research has shown that synovial fluid concentrations of TGF-131 were higher in patients suffering specific joint pathologies, and that the TGF-131 and its receptors were expressed in greater quantities in synovial tissue from arthritic joints. 17 Also, TGF-61's expression was decreased by increases in hydrostatic pressure] ~ as would occur with joint effusion. Evidence from previous studies in other species give mixed results for TGF-131 as a potential therapeutic agent in joint disease. Intra-articular 112

injection of TGF-131 appears to induce osteoarthritis, 1 while systemic administration may suppress polyarthritis. 4 In our study, the difference between concentrations in severely diseased and healthy joints supports previous reports 1,3,17,zz that suggest TGF-131 plays a role in joint disease and indicates that synovial fluid concentrations of TGF-131 may change in relation to some joint pathologies. Although, TGF-131 concentrations increased with severity of joint condition in the middle and old age groups this was not observed in the young age group. This may have been due to overall less severe and chronic joint pathologies in the young group, which visual categorization alone did not detect. Also, TGF-131 concentrations were higher in the younger group, this may have limited the ability to detect an increase in TGF-131 due to joint condition. In addition, the method used for determining joint condition may need to include histomorphologic and biochemical characterization of the joint.

CONCLUSIONS

A commercial human TGF-131 assay kit was validated for use on equine synovial fluid and serum. Differences were found between synovial fluid TGF-131 concentrations in metacarpophalangealjoints of young versus older horses, as well as between healthy joints and those with visibly severe joint disease. TGF-B1 is one of several factors that may affect the incidence of cartilage lesions in young, growing horses. Gaining a greater understanding of the role of TGF-131 in joint disease conditions may provide insight to improved management and therapy practices.

LITERATURE CITED 1. VandenBerg WB: Growth factors in experimental osteoarthritis: transforming growth factor beta pathogenic? J Rheumatol Suppl 1995;43:143-5. 2. Wahl SM, Allen JB, Costa GL, Wong HL, Dasch JR: Reversal of acute and chronic synovial inflammation by antitransforming growth factor beta. J Exp Med 1993; 177(1):225-30. 3. Schlaak JF, Pfers I, Meyer-Zum-Buschenfelde KH, Marker-Hermann E: Different cytokine profiles in the synovial fluid of patients with osteoarthritis, rheumatoid arthritis and seronegative spondyloarthropathies. Clin Exp Rheumatol 1996; 14(2): 155-62. 4. Brandes ME, Allen JB, Ogawa Y, Wahl SM: Transforming growth factor beta 1 suppresses acute and chronic arthritis in experimental animals. J Clin Invest 1991;87(3): 1108-13. 5. Thorp BH, Ekman S, Jakowlew SB, Goddard C: Porcine osteochondrosis: deficiencies in transforming growth factor-beta and insulin-like growth factor-I. Calcif Tissue Int 1995;56(5):37681. 6. Itayem R, Mengarelli-Widholm S, Hulth A, Reinholt TP: Ultra structural studies on the effect of transforming growth factorbeta 1 on rat articular cartilage. APMIS 1997;105(3):221-8. 7. Collier S, Ghosh P: Effects of transforming growth factor beta on proteoglycan synthesis by cell and explant cultures derived from the knee joint meniscus. Osteoarthritis Cartilage 1995;3(2):127-38.

JOURNAL OF EQUINE VETERINARY SCIENCE

8. Lafeber FP, VanderKraan PM, Huber-Bruning O, VandenBerg WB, Bijlsma JW: Osteoarthritic human cartilage is more sensitive to transforming growth factor beta than is normal cartilage. Br J Rheumatol 1993;32(4):281-6. 9. Fortier LA, Nixon A J, Mohammed HO, Lust G: Altered biological activity of equine chondrocytes cultured in a threedimensional fibrin matrix and supplemented with transforming growth factor beta-l. Am J Vet Res 1997;58(1 ):66-70. 10. Takahasi K, Kubo T, Kobayashi K, Imanishi J, Takigawa M, Arai Y, Hirasawa Y: Hydrostatic pressure influences mRNA expression of transforming growth factor-beta 1 and heat shock protein 70 in chondrocyte-like cell line. J Orthopedic Res 1997;15(1 ):150-8. 11. Guerne PA, Blanco F, Kaelin A, Desgeorges A, Lotz M: Growth factor responsiveness of human articular chondrocytes in aging and development. Arthritis Rheum 1995;38(7):960-8. 12. Jahng JS, Lee JW, Han CD, Kim S J, Yoo NC: Transforming growth factor-beta 1 responsiveness of human articular chondrocytes in vitro: normal versus osteoarthritis. Yonsei Med J 1997;38(1 ):40-51. 13. Zanni M, Tamburro A, Rotilio D: IL-1 beta and TGF-beta 1 modulate the sulphation grade of chondro-disaccharides in porcine articular cartilage: a capillary electrophoresis study. J Lipid Mediated Cell Signal 1995; 12(1 ):29-44. 14. Rosenthal AK, Derfus BA, Henry LA: Transglutaminase activity in aging articular chondrocytes and articular cartilage vesicles. Arthritis Rheumatol 1997;40(5):966-70.

15. Canete JD, Llena J, Collado A, Sanmarti R, Gaya A, Gratacos J, Blay M, Munoz-Gomez J: Comparative cytokine gene expression in synovial tissue of early rheumatoid arthritis and seronegative spondyloarthropathies. Brit J Rheumatol 1997;36(1 ):38-42. 16. Pronost S, Redini F, Vivien D, Galera P, Pujol JP: Human rheumatoid synovial cells (HRSC) in culture express TGF-beta receptors and are growth stimulated by the factor. Agents Actions 1993;Suppl 39:133-7. 17. Mussener A, Funa K, Kleinau S, Klareskog L: Dynamic expression of transforming growth factor-betas (TGF-beta) and their type I and type II receptors in the synovial tissue of arthritic rats. Clin Exp Immuno11997;107(1):112-9. 18. American Association of Equine Practitioners. Official Guide for Determining the Age of the Horse, fifth edition. 1988. 19. SAS: SAS/STATUser's Guide (4th Ed), Vol 2, Cary, NC: SAS Inc., 1989. 20. Polli V, Bulletti C, Galassi A, Borini A, Ciotti PM, Seracchioli R, Alfieri S, Flamingni C: Transforming growth factorbeta 1 in the human endometrium. Gynecol Endocrinol 1996;10(5):297-302. 21. Robinson, JA, Riggs BL, Spelsberg TC, Orsler MJ: Osteoclasts and transforming growth factor-beta: estrogenmediated isoform-specific regulation of production. 22. Jeffcott LB: Osteochondrosis in the horse-searching for the key to pathogenesis. Eq Vet J 1991 ;23(5):331-8.

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