The pH dependence of the sensitized sheep cell reaction of sera from patients with rheumatoid arthritis.код для вставкиСкачать
The pH Dependence of the Seneitbed Sheep Cell Reaction of Sera from Patients with Rheumatoid arthritis By RALPHHEIMER,OLGAFEDERICO AND RICHARDH. FREYBEHG An effect of pH on the agglutinating activity of rheumatoid sera was demonstrated in the sensitized sheep cell test and the potentiated sensitized sheep cell test. Although the use of a b d e r i n g system did not change the incidence of positive tests, it did increase the titer of some sera. The studies suggest that inhibition of the agglutination system by some chemicals may be a function of pH, and that free amino groups may be essential for agglutination. Un effect0 de pH super le activitate agglutinante de seros rheumatoide esseva demonstrate in le test a sensibilisate cellulas ovin, simple e potentiate. Ben que le us0 de un systema de tamponage non alterava le frequentia de resultatos positive, ill0 augmentava nonobstante le titro de certe seros. Le studios suggere que le inhibition del systema agglutinatori per un substantia chimic es forsan un function de pH e que libere amino-gruppos es essential pro le agglutination. T HE SENSITIZED SHEEP CELL REACTION',' and the potentiated sheep erythrocyte agglutination reaction' have been widely used as a screening procedure for the diagnosis of rheumatoid arthritis. The basis of these tests is that serum samples of the majority of patients with active rheumatoid arthritis agglutinate sheep cells sensitized with nonagglutinating concentrations of rabbit (or other species) anti-sheep serum. When the effect of certain chemicals on the hemagglutination reaction was tested, certain inconsistent results led this laboratory to study the dependence of the titer on the pH of the agglutinating medium. The effect of certain chemicals believed to be inhibitors of the hemagglutination reaction was re-evaluated upon finding the reaction pH dependent. The reasons for the pH dependence of the hemagglutination titer were investigated, and a mechanism of interaction between the components of the system proposed. METHODS Agglutinution procedure. The sensitized sheep cell test was perfonned according to the method of Heller et a].'; the Potentiated sheep erythrocyte agglutination test was carried oiit as described by Heller et al." All serum samples were incubated at 56" C for 30 minutes in order to inactivate complement, and heterophilr antibodies were removed by absorption on equal volames of packed and washed sheep cells. Use of phosphate bufler in agglutinution procedure. Serum samples tested by the sensitized sheep cell agglutination reaction or by t ) potentiated ~ erythrocyte aggliitination reaction were serially diluted in isotonic saline or in a four per cent sheep seriirn solution ._ From the Loboruttwy for Research in Rheumlitif: Diseuses, The IIospitd for Special Surgery, New York 21, New York. Supported b y a grunt from the Nuticmu1 Institute of Arthritis c i n d Metaholic Diseases, United States Public Health Service, Bcthesdu, Manjland. We are indebted to Dr. H . E . Polluczek, Veterans Adrnini,stration Hospital, The Bronx, New York, for many helpful suggestions. 62 PH DEPENDENCE OF SHEEP CELL REACTION IN RHEUhlATOID ARTHRITIS 63 respectively, and 0.25 nil. aliquots of each dilution were pipetted in rows along the width of a 90 place test tube rack. Samples of 0.25 ml. of 0.2 M. phosphate buffer of varying pH were then added along the length of the test tube rack so that each long row constituted a series of dilutions at a different buffer range. By this procedure pipetting errors inherent in serial dilution were minimized. A series of phosphate buffers from p H 5.2 to pH 8 and 0.2 M. KC1-borate buffers above pH 8 were used. The d e c t of Uiumox, Sulfapyridine and Elkosin on the hemagglutinntion reaction. To each of 0.25 ml. serially diluted rheumatoid serum, 0.25 ml. of either Diamox (Lederle; diluted 1:27), Sulfapyridine (Lederle; diluted 1:13) or Elkosin (Ciba; diluted 1 : l O O ) was added. In control experiments, the rheumatoid serum, with and without added sulfonamides, was serially diluted in phosphate-saline buffer ( p H 7, 0.08 M.). Treatment of serum samples with iodoacetate, and amino group reagents. Serum samples wcre diluted fourteen-fold with saline and then dialyzed in the cold for 24 hours against 0.02 M. iodoacetic acid at pH 7. The samples were then redialyzed against excess phosphate buffer (0.1 M., p H 7 ) . One ml. serum samples were diluted fourteen-fold in bicarbonate buffer ( 1 M., pH 8 ) and stirred for 3 hours at 4 " C with slow addition of 0.0002 M. of acetic anhydride or carbobenzoxychloride or fluorodinitrobenzenc. The solutions were then dialyzed against frequently changed and excess phosphate buffer (0.1 M., pH 7 ) . The d e c t of acetate and phthalate buffers on the stability of the rheumatoid factor: Serum samples, diluted in saline, were dialyzed overnight in the cold against 0.1 M. acetate buffers ( p H range 3.7 to 5.6, in increments of .2 p H units) or against 0.1 M. phthalate buffers ( p H rangc 2.2 to 3.8 in increments of .2 pH units). The serum samples wcrc thcn rcdialyzed against excess phospbate-saline buffer ( p H 7, 0.08 M. ). RESITLTS The effect of pH on the sensitized sheep cell reaction and the potentiated sheep erythrocyte agglutination reaction was studied in serum samples of nine patients with active classical rheumatoid arthritis (table 1 ) . The titers were pH-dependent in all nine cases. In eight samples the sensitized sheep cell titers were well below control values when the reaction proceeded at pH 6.35, dropping to zero at pH 5 2 . The potentiated sheep erythrocyte agglutination titer of serum s,amples from patients with active classical rheumatoid arthritis also fell off at a similar p€I. Little potentiation of titers by sheep serum occurred below pH 5.85. The effect of pH on the titer of serum K from a patient, who six months subsequent to the testing died of metastatic carcinoma, was particularly interesting. A pH range was found ( p H 7.85 to 8.15) in which this serum sample had a strongly positive sensitized sheep cell titer (1:448), although at all other hydrogen ion concentrations the titer remained zero. The potentiated sheep erythrocyte agglutination titer cjf serum K showed a pronounced maximum between pH 7.85 and 8.15. The highest agglutination titers were obtained above pH 7. In some serwn samples maximal agglutination titers were found over a rather broad range (sera 104, 102, MM, W, 93 and 98) whereas in some others the optimum seemed more sharply defined (sera K and U ) . The optimal pH of the sensitized sheep cell reaction seemed to be around pH 8. Borate-saline buffer ( p H 8, 0.1 M.) was included in the sensitized sheep cell tests performed on a random sampling of test sera, obtained from forty patients treated at the rheumatic disease clinic, The results were compared with the ones obtained when no buffer system was used. Table 2 indicates that, whether a buffer was used or not, the sensitized sheep cell test gave essentially similar results. 5 5% 7% 6 4 7 5 6 8 5 4 8 5% 1 6.20 6.70 PH ti.90 i.00 i.20 7.50 -___-.7.65 6% 7 7% 7w 8 9 8 8 3 4 4 4 2 3 5 6357 7 7 7 7 5 5 6 5 % 5 5 M 6 6 7% 8 10 10 5% 6 6% 7 6% 8% 9% 8% 9% 10 5 % 7 6 % 8 7 9 7 8% 8% 9% 9% 9% 0 0 0 0 0 4 6 6% 7% 7% 9% 10% 8% 8% 9 12% 12 12 11% 12 4% 4% 5% 6 6% 7 7 7 9 9 8% 8% 10 10 7 9% 10 8 13% 13 10% 13 14 10 5 7% 1 6.36 7 9% 9% 14 11 6 10 8% 11 7 9% 7 6 4 7.X; 11 6 7 12 7 8 3 6% 8.45 11 10 12 10% 12 7 11 11 14% 8 9 8 10 10 5?40 9% 6% 6 7% 4 % 7 6 12 6 x 10 8.15 11 7 13% 6% 9% 0 3 6 6 10 6 8% h.85 Legend: The numerals in the columns indicate the number of tubes which contained aggliitinnted cells. ‘rhe relationship of the numbers to the titer of the serriin sample is given by the following: 1 2 3 4 5 etc. Number: 7 14 28 56 112 Titer 1: The term “Control” indicates the titer of the serum when tested without addition of hiiffer. 8 12% ssc pSEA 7 10 ssc pSEA 1 0 0 0 5 7 2 2 1 0 0 1 9% pSEA 13% 0 2 2 2 3 3 2 5 ssc ~~ 5 1 4 1 1 4 pSEA ‘ a 2 3 0 3 3 6.05 0 0 1 ssc 10 6 9 6% ~ 5.86 ___--I. tlrc IIemogglutiriation Titer Obtuinctl 6~ t l i c Setuitixd Slicep Cell 7 crt (SSC) and the Potentiuted Sheep Lrytltrcjcyte Test (13-SEA) 6.70 011 0 8% pSEA ssc pSEA ssc ssc 1% 1 1% 2 2 1 0 5 9 pSEA 9 pSEA ssc pSEA 3 8 6.50 pII 0 1 0 2 6.20 [I\ 0 0 0 0 ~ Control l.-’f%c E f f w t 7 ssc Test 83 h1hf 93 lo4 Serum No. ?‘AUL.E 2 3 r 4 m 6.5 P H DEPENDENCE OF SHEEP CELL REACTION IN RHEUMATOID ARTHRXTIS 8#) xQ 7.50 7M 650 6#)l I Dilution 1:14 1 128 I 1% I I I I I I I I 1412 1:W 1:448 14% 1:1792 13500 1:7T 1:14T SERIAL DILUTION OF SERUM FIG.1.-The effect of serial dilution of seriim on the pH of the agglutinating medium. As can be seen from figure 1, the pH of the agglutinating medium drops upon dilution of the serum sample. In the sensitized sheep cell test serum dilutions greater than 1 5 6 are run in pH ranges unfavorable for maximum agglutination." One would expect higher titers, therefore, in only those sera which are already positive, whereas serum samples with negative titers would remain unaffected, as they already fail to agglutinate near optimal pH conditions. Ten serum samples with titers in excess of 1:swere tested in the presence as well as absence of borate-saline buffer ( p H 8, 0.1 M . ) . One to two tube increases in titer occurred in five of the ten samples tested, while the titer of the other five remained unchanged. Increases in titer beyond two tubes were not encountered. When buffer systems other than borate were used, results essentially similar to those with phosphate or borate were obtained. The buffers and buffer ranges were: tris buffer (pH 7.1 to 8.6), barbital (pH 6.8 to 8.2) and glycine (pH 7.6 to 8.4). TABLE2.-A Summary of the Titers Obtained froin the Sera of Forty Patients Treated at the Rheumatic Disease Clinic, Using the Sensitized Sheep Cell Test with and without Borate Saline Bufier Titers: NO Buffer Borate Buffer (0.1 M., pH 8 ) 0 28 29 1:7 1:14 1:28 1:56 1:112 1:234 1:448 2 1 1 1 1 1 1 6 6 0 1 1 0 1 'The potentiated sheep erythrocyte agglutination test affords a more favorable pH medium for sera of higher titer owing to the buffering capacity of 2 per cent sheep serum. Some of the potentiation of the agglutination reaction by sheep serum is therefore due to a shift to a higher and more favorable pH. Sheep serum, however, does contain a component which i s responsible for potentiated agglutination over and above the pH effect. For instance, eledrophoretically obtained sheep serum albumin, ox serum iiiucoid fractions, crystalline lysozyme and the crystalline soy bean trypsin inhibitor failed to potentiate the sensitized sheep cell reaction. 66 RALPH HEIMER, OLGA FEDERICO AND RICHARD H. FREYBERC. TABLE3.-The Effect of Stclfonmmidos on ihe Sensitized Sheep Cell Test in Presence or Absence of p H 7 Phosphate Buffer Titer Control Elkohin Snlf npyridine Iliamox 1:448 1:28 1:224 1:s 3.4 8.3 5.9 No bitffer I PH 4 c, E5 Titer 1 :896 1 :896 1 :448 1 :448 PH 7 T 7 7 Phosphate rn Buffer TABLE 4.-The Effectof Glycylglycine (0.1 M ) on the Sensitized Sheep Cell Titer of Serum No. 103 Tested in Presence and Absence of (I Buffer System at p H 7 __- Titer Serum No. 103 in glycylglycine pH 5.4 Serum No. 103 in glycylglycinc pH 7 adjusted with PO, 1dFt.r 1:224 Serum No. 103 Control 1 :224 _____.__ The pH limitation of the sensitized sheep cell test and the potentiated sheep erythrocyte agglutination test, however, must be kept in mind when evaluating the effect of chemicals on the agglutination reaction. On reexamining the alleged inhibitory effect of certain sulionamides on the hemaggliitination reaction4 it was found that direct addition of dilute solutions or suspensions of commercial sulfonamide preparations ( the preparations were poorly soluble in saline) to the reacting medium resulted in inhibition of the hemagglutination test. Decreased titers, however, were directly related to the pH of the sulfonamide solution added. Upon adjusting the test suspensions to pH 7 no inhibition of the hemagglutination reaction occurred ( table 3 ) . Rheumatoid serum samples, treated directly with sulfonamide solutions or suspensions and then redialyzed against phosphate buffered saline ( p H 7, 0.08 M.), also had titers identical with untreated controls. These experiments clearly indicate that “sulfonamide inhibition” is in reality a pH effect. Similarly, glycylglycine, reported to be an inhibitor of the sensitized sheep cell reaction: depressed the hemagglutination titer because of low pH (5.4), while on adjusting the test suspension to pIf 7 no inhibition occurred (table 4). A number of other chemicals were tested for their effect on the hemagglutination reaction of sera from patients with rheumatoid arthritis. In all these experiments care was taken to test for agglutination at a comparable pH. Table 5 lists some common reagents, the structures affected by them, and their effect on the aggliitination reaction of rheumatoid sera. These tests PH 67 DEPENDENCE OF SHEEP CELL REACTION IN RHEUMATOID ARTHRITIS TABLE5.-Effect of Some Reagents on the Hemagglutination Reaction of Serum Samples from Individuals with Actbe Classical Rheumatoid Arthritis REAGENT GROUP AFFECTED DEGREE OF INHIBITION Saturated Periodate Iodoacwtate Carbobenzoxychloride Acetic Anhydride Fluorodinitrobenzene Certain Carbohydrates SH Amino Amino Amino None None ++++ +-t++ ++++ TABLE&-The pH Requirement for the Irreversible Inactivation of the Rheumatoid Factor Serum Control No. 81 101 102 87 122 ioa 83 96 82 U Titer 1:llZ 1:112 1:112 1:112 1 :224 i : m 1:448 1:448 1:896 1:3600 4.2 1:224 1:112 4.0 1:112 1:66 1:224 1 :896 1 :3600 Sensitized Sheep Cell Agglutination Titers at pH of acetate or phthalate buffer (0.1 M ) 8.8 8.13 8.4 3.2 8.0 2.8 1:112 1:llZ 1:112 l:l4 1:7 0 1:112 1:112 1:112 1:14 0 0 1:66 1:28 1:14 1:14 0 0 1:66 1:66 128 1:14 0 0 1:112 1:112 1:66 1:66 1:14 0 0 1:14 1:14 1:14 1:224 1:66 1:224 1:448 1:896 1:1792 1:448 1:448 1:224 1:1792 1:224 1:448 1:224 1:896 1:112 1:224 1224 1:448 1:66 1:14 1:112 1:224 1:14 0 1:28 1:66 2.6 2.4 0 1:14 0 1:14 1:66 1:14 indicate that the rheumatoid factor does not contain essential SH groups. The effectof amino group reagents may result either from blocking of the amino groups or from precipitation. The stability of the components of the hemagglutination reaction to acid conditions was also examined (table 6). Rheumatoid serum samples as well as rabbit anti-sheep serum were dialyzed in the cold against acid buffers, and then redialyzed against phosphate buffer ( p H 7., 0.08 M.), and the hydrogen ion concentration determined for irreversible inactivation of the rheumatoid factor and rabbit anti-sheep serum. In 0.1 M. acetate or phthalate buffers ( p H 5.0 to pH 2.2) a two tube loss of activity was observed upon exposing the rheumatoid serum samples to between pH 3.8 and 3.2. Mcst of the agglutinating activity of such serum samples disappeared between pH 2.8 and 3.4, depending on the sample tested. Some differences were found in the pH required for irreversible inactivation, although they were perhaps related to the initial titer. The decrease in the hemagglutination titer of rheumatoid sera when the test is carried out below pH 6.3 is, therefore, not due to acid sensitivity of any of the components of the reaction. Isoelectric precipitation of one of the participants in the hemagglutination reaction as a cause for the decreased titer also was considered. Serum samples and rabbit anti-sheep serum were stored in the cold for 18 hours in phosphate buffer ( p H 5.85, 0.1 M.) and then centrifuged at 2,000 x g for 20 minutes. The supernatant was redialyzed against phosphate buffer ( p H 7, 0.08 M.). The titer of the supernatant remained unchanged, ruling out isoelectric precipitation of any component of the reaction. DISCUSSION The pH of the test system is a determining factor for the magnitude of the 68 RALPH HEIMER, OLGA FEDERICO AND RICHARD H. FREYBERG agglutination between sensitized sheep cells and serum samples from individuals having classical rheumatoid arthritis. Maximal agglutination responses occur well above pH 7. A pH below 5.8 is unfavorable, while at pH 5.2 complete inhibition of agglutination occurs. Lacking in buffering capacity, the sensitized sheep cell test in many cases yields titers which are somewhat lower than those obtained when a proper buffer is added. Negative titers, however, remain unchanged, as the protein content of the test samples remains high enough to maintain a nearly optimal pH for agglutination. The addition of 2 per cent sheep serum as a diluent of all test samples (the potentiated sheep erythrocyte agglutination test) results in a pH which is more favorable to agglutination. The higher titers obtained with the potentiated sheep erythrocyte agglutination test are therefore partly a pH effect. However, increases in titer beyond two tubes are caused by a yet unknown constituent of sheep serum. Such potentiation has been shown to be a characteristic of serum samples from individuals with active rheumatoid arthriti~.~ Experimental data presented here suggest that coulombic forces rather than formation of covalent bonds are involved in this type of agglutination reaction. Neither isoelectric precipitation nor inactivation by acid of rheumatoid factors and rabbit anti-sheep serum are involved in the inhibition of agglutination below pH 5.8. Some serum samples, drawn from patients with collagen diseases other than classic rheumatoid arthritis, do agglutinate sensitized sheep cells even at pH 5.2," which demonstrates that interaction between rabbit anti-sheep serum and red cell does occur at such a pH. It is therefore likely that an alteration of electric charge on active sites of the rheumatoid factors is responsible for such inhibition. The pH range of the inhibition suggests that free amino groups are essential to agglutination, which may only proceed under relatively basic conditions with the amino group retaining its free electron pair. Inhibition of agglutination, occurring with weak acid, would be owing to formation of a center of positive charge on the amino group. The requirements for agglutination may be schematized as follows: -NHP: -NH,+ in acid does not agglutinate in base agglutinates Blocking of amino groups by acetylation, carbobenzoxylation, and dinitrophenylation, moreover, leads to loss of the ability of rheumatoid sera to agglutinate sensitized red cells. Whether such inactivation is due to blocking of free amino groups remains to be determined, as such treatment often leads to formation of insoluble proteins. SUMMARY Nine serum samples from individuals with active classical rheumatoid arthritis were examined for the effect of pH on the sensitized sheep cell agglutination titer and the potentiated sheep erythrocyte agglutination titer. Maximal agglutination occurred above pH 7, while inhibition was noted below pH 5.8. PH DEPENDENCE OF SHEEP CELL REACTION I N RHEUMATOID ARTHRlTIS 69 Routine screening of sera for agglutination activity is scarcely af€ected by the inclusion of a buffer system. Sera with titers in excess of 156 tend to exhibit higher titers when a buffer system is used, but negative sera remain unaffected because the pH of the test suspensions remains at all times near optimum. Inhibition of the hemagglutination reaction by addition of certain chemicals may be a consequence of an altered pH rather than a direct effect exerted by the chemical. This was shown to be the case for sulfonamides and glycylglycine. The pH dependence of the hemagglutination reaction is a manifestation of the electrostatic nature of the reaction. REFERENCES 1. Rose, H. M., Ragan, C., Pearce, E. and Lipman, M. 0.: Differential agglutination of normal and sensitized sheep erythrocytes by sera of patients with rheumatoid arthritis. Proc. SOC.Exper. Biol. & Med. 68:1, 1948. 2. Heller, G.,Jacobson, A. S. and Kolodny, M. H.: A modification of the hemagglutination test for rheumatoid arthritis, Proc. SOC.Erper. Biol. & Med. 72: 316, 1919. 3. -, -, - and Schuman, R. L.: The hemagglutination test for rheumatoid arthritis. I. An immunological analysis of the factors involved in the reaction. J. Immunology. 69:27, 1952. 4. Svartz, N. and Schlossmann, K.: Inhibition of the hemagglutination reaction in rheumatoid arthritis by means of sulfa compounds. Acta Med. Scand. 155131, 1950. 5. Williams, R. R., Stone, S. S., Jenkins, J., Evans, R. L. and Bunim, J. J.: Some characteristics of the inhibitor system for the sensitized sheep cell agglutination reaction. Ann. Rheumat. Dis. 15: 61, 1956. 6. Unpublished observations. Ralph Heimer, Ph.D., Hospital for Specinl Surgery, New York. Olga Federico, B.A., Hospital for Special Surgery, New York. Richard H . Freyberg, M.D., Hospital for Specinl Surgery, New York.