Increase of L-chain proteins in the sera of patients with systemic lupus erythematosus and the synovial fluids of patients with peripheral rheumatoid arthritis.код для вставкиСкачать
Increase of L-Chain Proteins in the Sera of Patients with Systemic Lupus Erythematosus and the Synovial Fluids of Patients with Peripheral Rheumatoid Arthritis By WALLACE V. EPSTEIN AND MARGARET TAN B specific for determinants of the light ( L ) polypeptide chains of the human immunoglobulins, it has become possible to measure free L-chains in the presence of normal or increased quantities of intact immunoglohulins.1,2 The quantities of L-chain proteins (Hence Jones proteins) found in the serum and urine of patients with multiple myeloma and macroglobulinemia have lieen established by both isolation technics3 and by direct immunologic methods2 The present report deals with the presence of increased quantities of L-chain proteins in the synovial fluids of patients with rheumatoid arthritis and the sera of those having systemic lupus erythematosus. using 2 per cent agar in 0.075M barbital buffer, pH 8.6. Serum was examined undiluted and synovial fluid was examined after the addition of hyaluronidase. Plates were inspected for preeipitin bands after 24 hours in a moist chamber at room temperature and again after staining. Concentration of the antigen was estimated by doubling dilutions of the antigen using separate pipettes at each dilution step. Gel filtration chromatography of synovial fluid was performed in 3.5 x 80 cm. columns of medium grade Sephadex (2 parts G200 to 1 part G100) in 0.1M NaCI, 0.01M PO,, pH 8 buffer. Chromatography was performed at room temperature. Y MEANS OF ANTISERUM MATEHIALSAND METHODS Antiserum to L-chain proteins was prepared by iirimnnizatioii of rabbits with L-chains prepared t)y the retliiction and alkylation of pooled human y - ( : glol~ulins.?’he antiserum has specificity for Lchain determinants relatively unavailable in intact g:imnia globulins. The antiserum was “absorbed” 11) the addition of y-G globulin until no precipitating ability for intact gamma globulin remained when tested by precipitation in agar. The details of these steps have been described previously.2 Antisrrnni specific for type K or type h deteriniiiants was prepared by absorption of this antiserinti with any type K or type h Bence Jones protein. The minimum concentration of L-chain protein that ciin be detected by these antisera is approximatel) 0.02 mg./ml. Precipitiii reactions in agar were performed F r i m the Rkeurnutic. Disease Group, Departirierit ( i f Mediczrke, Uniuersity of California School ot Mrdicitie, Sari Francisco, Calif. Supportc.d by u grartt fTo?rL the National Instih t e s of Health ( U S P H S 0229), Bethesda, M d . Presercted ‘n part at the Interim Meeting Of the Rheurnatisrn Association, December, Serum and Synovial Fluid All sera were from patients with clinically active systemic lupus erythematosns or peripheral rheumatoid arthritis. All of the former had positive LE cell preparations either at the time the specimen was obtained or no longer than 6 months earlier. All sera were examined for rheumatoid factor by the FII tanned cell hemagglutination procedure. Some serum specimens had been maintained in our serum reference collection at -20 C. without preservatives, for as long as 2 years. Synovial fluids were obtained by knee joint aspiration into syringes containing heparin, together with simultaneous venous blood specimens. Tests of synovial fluid for rheumatoid factor activity were performed after the addition of hyaluronidase and without the application of heat for the inactivation of complement. Our attempts to classify the renal status of the patients having systemic lupus erythematosus were fraught with difficulty. In several instances. no tests of renal function had been obtained at the same time as the serum specimen was obtained. It 1964, Washington, D. C . WALLACEv. EPSTEIN, M.D. : Associate Professor of Medicine; MARGARETTAN, B.A., M.S.: Research Biochemist; both of the Rheumatic Disease Group, Department of Medicine, Unicersity of California School of Medicine, Sun Francisco, Calif. Arriericari 713 ARTHRITISAND RHEUMATISM,VOL. 9, No. 5 (OCTOBER), 1966 714 EPSTEIN AND TAN Table 1.-Precipitin Test for Increased L-Chain Protein in Sera of Patimts with SLE Number Tested With renal failure Withont renal failure L-chain Positive Negative 10 9 1 20 19 1 appeared most reliable to limit classification to those with renal insufficiency at or within 3 months previous to the time the blood specimen was obtained. The serum creatinine and the creatinine clearance were the determinations most frequently found in the patients’ records and were used with any other available tests to group patients into those with and those without gross renal insufficiency. RESULTS except for one serum from a patient free of renal failure, in which only type K light chain protein was found. We have been unable to demonstrate a direct relationship between the amount of free L-chain protein in the serum and the presence or extent of renal insufficiency. In five cases urine specimens of patients having positive serum tests for increased free L-chain were examined with serial determinations over a 2-month period. In all instances, the free Lchain concentration (0.04 to 0.16 mg. per ml.) was equal or one doubling dilution greater in urine than in that patient’s serum. Again, no direct correlation was noted between the daily total proteinuria and the amount of free L-chain protein in the serum or urine. Systemic Lupus Erythematosus ( S L E ) Rheumatoid Arthritis The sera of ten patients having definite SLE associated with renal insufficiency and 20 without renal insufficiency were tested tor increased quantities of free L-chain protein. Tests of 47 normal sera using these same reagents revealed that they cmtained less than 0.02 mg./ml. of free L-chain proNine of ten patients with renal insufficiency due to SLE and 19 of 20 without renal insufficiency were found to conPain greater than 0.02 mg. per ml. (Table 1). Figure 1 shows the precipitin pattern of six different sera from patients with SLE arranged in a clockwise manner according to intensity of precipitin band produced by the whole serum. The center well contains antiserum to human L-chains absorbed with y-C: globulin. The double precipitin band of serum 1 and 2 was often noted in strongly positive sera. Antiserum specific for type A or type K light chain proteins was used to test the serial dilution of all positive sera. It revealed that type h light chain was present in amounts equal to or greater than type K Some 40 sera of patients with definite peripheral rheumatoid arthritis, having positive serum tests for rheumatoid factor and negative L. E. cell preparations, were examined. Two of these formed a precipitin band under the same conditions used to examine the SLE sera. Table 2 presents the results obtained for those sera and their corresponding synovial fluid specimens when tested against L-chain antiserum. The specimens are listed according to the FII hemagglutination titer of the serum. The quantities of free L-chain protein in synovial fluid ranged between 0.02 to 0.64 mg. per ml. with no correlation noted between the concentration of L-chain protein and the rheumatoid factor titer (Table 2). The specimen listed under Osteoarthritis that exhibits elevated rheumatoid factor levels and increased L-chain concentration in the synovial fluid had been obtained from a patient cared for in our clinic, and thought to have clinical osteoarthritis despite the known positive test for rheumatoid factor. No attempt was made to cor- LNCHEASE C F L-CHAIK P'HOTEINS IN SERA OF PATIENTS WITH SLE 715 Fig. 1.-Double diffusion in agar resulting in precipitin bands between six different serd from patients with SLE in peripheral wells and antiserum to human L-chains in center well. relate these results with drug therapy at the time the specimens were obtained. Fractionation of Synovial Fluids Three specimens of- synovial fluid were p'issed wparately through a column of a mixture of Sephadex G200 and G100. The protein distribution elution pattern as 5hown in Figure 2 is typical of the three 5pecimens. Rheumatoid factor activity was regularly found concentrated in peak I. The free L-chain activity was found only in peak 111 protein as shown in Figure 3. Specimens of Bence Jones proteins or of Lchain proteins of normal pooled 7-G globulin, when passed through these same columns, emerged in tubes corresponding to the peak I11 distribution. DrscussIoN A series of s t ~ i d i e s ~ have - ~ demonstrated that proteins having characteristics of Bence Tone5 proteins and the L-chains of human immunoglobulins are found in the serum and urine of normal individuals 716 EPSTEIN AND TAN Table 2.-Precipitin Test for Increased L-Chain Protein in Serum and Synovial Fluid Synovial Fluid Serum FII Titer Rheumatoid arthritis FII Titer L-chain (mg./ce.) Type K Type 0 0.08 0.08 0 0.08 0 0 0 0 0 0 0 0 0 0.02 0 0 0.02 224 1,280 112 28 7,000 14,000 7,000 56,000 3,584 14,000 112,000 7,000 14,000 0.08 0.08 0.08 0.16 0.08 0.32 0.08 0.02 0.08 0.02 0.02 0.02 0.08 0.08 0.16 0.64 0.16 <56 0.08 <56 0.02 0.04 <56 <56 <56 224 896 0 0 0 0 0 <56 <56 <58 56 224 0 0 0 0 0 0 0 0 0.02 0.02 <56 <56 <56 112 0 0 0 0 <56 <56 <56 <56 0 0 0 0 0 0 224 640 1,792 3.584 14,000 28,000 56,000 56,000 56,000 56,000 56,000 224,000 224,000 S.L.E. Osteoarthritis 'I'ramriatit arthriti5 L chain (mg./cc.) O0 0 0 0 0 0 ' 0 designates less than 0.02 mg./cc. The minimum concentration of L-chain protein to form precipitin line with the antiserum is 0.02 mg./cc. Using the antisera described in the present study, we have found that normal serum contains 0.005-0.008 mg./cc. of free Lchain.2 Normal urine contains approximately 3 mg. per day.? The sera of normal pregnant women contain approximately 0.016 mg./ml.* Interpretation of our findings of greater than normal quantities of L-chain proteins in the sera of patients having SLE and in the synovial fluids of patients having peripheral rheumatoid arthritis is made difficult by our lack of knowledge of the normal synthetic rate, metabolic fate, degradation and excretory pathways of L-chain proteins. It must be assumed that the individual polypeptide chains of the immunoglobulins, like 7-C globulin itself, must be synthesized and catabolized in connection with mechanisms that regulate the total intravascular mass of the protein; the serum concentration depends on the rate of synthesis, the plasma volume and the fractional catabolic rate. There may also be an endogenous catabolism of L-chain protein in the kidney itself3 as well as a normal urinary excretion of the protein. Our inability to correlate the magnitude of the increased L-chain concentration in the sera of patients having SLE with the degree of renal insufficiency does not eliminate the possibility that this serum abnormality reflects renal involvement in this disease in most, if not all, patients. Indeed, in the cases in which we have performed serial studies, L-chain proteinuria, but not total proteinuria, parallels or precedes the increase of the L-chain concentration in the 717 IlCCREASE OF L-CHAIN PROTEINS IN SERA OF PATIENTS WITH SLE o.D.280 R.A. S Y N O V / A L FLU/D 1.0- 1.6 - - 1.4 1.2 9 - - U I 5 Fig. 2.-GeI 1 1 I J 30 35 40 4 5 5 0 55 60 6 5 70 7 5 80 TUBE N U M B E R filtration chromatography of synovial fluid obtained from a patient 10 15 20 2 5 having peripheral rheumatoid arthritis. serum. Since decreased renal excretion alone could not explain such findings, one might postulate ;i decrease in the catabolic rate of I,-chain protein. Solomon et aL3 have demonstrated a decrease in the Bence Jones protein catabolic rates in patients with hlood urea nitrogen levels above 30 mg. per 100 ml. No catabolic or excretory organ other than the kidney has been described. It is also possible that the increased serum concentration of L-chain protein reflects the involvement of gamma globulins in the disease process with accelerated synthesis of the immunoglobulins and therefore of the I,-chain protein. Andersen and Jensen!' have found an increased rate of y-G globulin synthesis in 10 of 16 patients with collagen diseases and an increased fractional catabolic rate in 11. Vaughan and Barnettlo have recently reported an increase in serum L ~ h a i n sin the course of a serum sickness rewtion in it patient who had received horse serum. The transient increase of serum L-chains occurred at the time of the maximum symptomatic reaction and may have been related to both increased immunoglobulin synthesis and to peripheral catabolism in tissues. Both of the major antigenic types of L-chain protein appear represented in the serum increase found in SLE. The equal or at times increased quantity of type h to type K protein appears at variance with the reported normal excess of type K to type h in intact immunoglobu1ins.ll Whether increased type h protein is characteristic of the antibodies elaborated in this disorder will await completion of a wider ranging survey. It will be of interest to examine the possibility that decrease or loss of the catabolic role of the kidneys for L-chain proteins is a feature of the renal lesion of SLE. It appeared likely that the sera of patients with peripheral rheumatoid arthritis EPSTEIN AND TAN Fig. 3.-Precipitin band formation in agar using fractions shown in Figure 2 in concentrations of 1.0 mg./nil. Antiserum same as in Figure 1. would constitute the most pertinent control for the present observation of elevated Lchain in the serum of patients with SLE. Wc applicd criteria which tend to eliminate those cases in which one cannot easily decidc whether the patient has rheumatoid arthritis or SLE. The presence of a strongly positive test for rheumatoid factor and a negative LE cell preparation were used as criteria for the diagnosis of rheumatoid arthritis. It appears that the sera of patients with peripheral rheumatoid arthritis regularly lack increased concentrations of Lchain proteins. It was of considerable intercst to find increased concentration of Lchain protein in all but one of the synovial fluid samples of the RA patients (Table 2). One synovial fluid sample from a patient diagnosed as having osteoarthritis also showed detectable L-chain concentration. It should be noted that no adjustment was made for differences in the total protein concentration of the RA synovial fluids compared to those obtained from patients with osteoarthritis or traumatic arthritis. No correlation was found between the amount of L-chain protein in synovial fluid and its rheumatoid factor concentration. At the present time, we have no information bearing on whether L-chain is synthesized de nouo in the synovial membrane resulting from an immunologic event within the joint or is selectively concentrated from serum into synovial fluid. The difference in concentration between serum and synovial fluid indicates the presence of a concentration gradient maintained by the synovial membrane. The concentration of type A light chain in the synovial fluid is in most instances greater than that of type K . This suggests either preferential involvement of type A producing cells or preferential breakdown of type A immunoglobulins. ACKNOWLEDGMENTS The authors wish to express their appreciation to Mrs. Nancy IIelmers for her technical assistance. INCREASE OF L-CHAIN PROTEINS IN SERA OF PATIENTS WITH SLE 719 SUMMARY Greater than normal coricentrations of the free L-chains of human immuiioglobuliris were fcnnd in the sera of 28 out of 30 patients having systemic lupus erythematosus. No clear correlation between L-chain proteinemia a n d the presence or absence of azotemin w a s noted. Only t w o of 40 sera of patients with peripheral rheumatoid arthritis w a s found to have a n increased concentration of L-chain protein; 12 oiit of 1 :3 specinens of synovial fluids from patients with rheumatoid arthritis showed iricreased concentrations of L-chain proteins in contrast t o one of nine from patients with osteoarthritis or traumatic arthritis. The increased concentration of L-chain protein in t h e sera of patients with SLE, a n d the synovial fluid of patients with RA consisted of both of t h e two major antigenic types with type X in greater concentration than type K in some cases. SUMMARIO IN INTERLINCUA Plus-cii"-iiorniaI coriceiitrationes del libere catenas L d e immunoglobulinas human esseva trovate in l e sero d e 28 de 30 patientes con disseminate lupus erythematose. Nulle nette correlation esseva notate inter le proteinemia a catena L e le presentia o absentia de azotemia. Un augmentate concentration de proteina a catena L esseva constatate in solinente duo de 40 seros ah patientes con peripheric arthritis rheumatoide. Dece-duo de 13 speciriiens de liquido synovial ab patientes con arthritis rheumatoide moiistrava augmentate concentrationes de porteinas a catena L, sed iste mesme constatation esseva facite in solmente u n de novem patientes con osteoarthritis o arthritis traumatic. Le augmentate concentrationes de proteina a catena L in le sero d e patientes con d:sseminate Iup~iserythematose e in le liquido synovial de patientes con arthritis rheumatoide interessava a m b e major typos antigenic, sed in certe cases le typo L esseva representate in n n plus alte concentration q u e le typo K. REFERENCES 1. Epstein. W.; Tan, M.,and Gross, D. Blocked antigenic sites on the L-chain of human gamnra globillin. Nature 202: 1175, 1964. 9. Tan, M., and Epstein, W.: A direct immunologic assay of human sera for Bence Jones proteins (L-chains). J. Lab. Clin. Med. 66:344, 1965. ' 3. Solomon, A,, Waldmann, T. A., Fahey, J. L., mcl McFarlane, A. S.: Metabolism of Bence Jones proteins. J. Clin. Invest. 43:103, 1964. 4. Hcrgg'ird, I. On a y-globulin of low moleciilar weight in normal human plasma and lirinr. Clin. Chim. Acta 6:545, 1962. Fi. Webb, T.>Rose, B., and Sehon, A. I%.:Biocolloids in normal human urine. 11. Physicocheinical and iimnunochemical characteristics. Canad. J. Biochem. Physiol. 36:1167, 1958. 6. Hcrggard, I., and Edelman, G. M.: Normal counterparts to Bence-Jones proteins: free L polypeptide chains of human y-globulin. Proc. Nat. Acad. Sci. 49:330, 1963. 7. Stevenson, G. T.: Further studies of the 8. 9. 10. 11. gamma related proteins of normal urine. J. Clin. Invest. 41:1190, 1962. Epstein, W., Fong, S., and Tan, M.: Naturally occurring macroglohulin antibody of fetal origin in the normal human newborn. Immunology 10259, 1966. Andersen, S. B., and Jensen, K. B.: Metabolism of yG-globulin in collagen disease. Clin. Sci. 29 533, 1965. Vaughan, J. H., ancl Barnett, E. V.: Increased production of circulating L-chains in acute serum sickness. Arthritis Rheum. 8:476, 1965. Presented at the Anniial Meeting of the ARA, June 17-18, 1965, Philaclelphia, Pa. Fahey, J. L.: Two types of 6.6s gamma globulin, Beta ,,.,-globulins and 18s gamma,macroglobulins in normal sernm ancl gamma niic.roglobulins in normal iirine. J . Innniinol. 91:438. 1963.