Studies on the Gm factorsComparison of the agglutinators in serum from patients with rheumatoid arthritis and in serum from healthy donors.код для вставкиСкачать
Arthritis and Rheumatism ~&ia~~rmrnUl of the gmerican Rheumatismassociation VOL. V, NO. 4 AUGUST, 1962 Studies on the Gm Factors: Comparison of the Agglutinators in Serum from Patients with Rheumatoid Arthritis and in Serum from Healthy Donors By ARTHURG. STEIXBERC With the technical assistance of lanet A. Wilson and Rachel Stauffer Titration and absorption studies of the agglutinators used to detect genetically determined human y-globulin groups showed that (a) the agglutinators in rheumatoid arthritis serum and in normal serum are probably the same, (b) the inhibition of R.A. sera by Cm (-) serum is due to a non-specific reaction, and (c) R.A. sera have at least two kinds of &M molecules, those with rheumatoid factor activity and those with both rheumatoid and gamma group activity. Studios de titration e absorption del agglutinatores usate pro deteger in human0 s gruppos de geneticamente determinate globulina gamma monstrava que (a) le agglutinatores in sero ab patientes con arthritis rheumatoide e in sero normal es probabilemente identic; (b) le inhibition de seros ab patientes con arthritis rheumatoide per le sero Cm-negative es attribuibile a un reao tion non-specific; e (c) seros ab patientes con arthritis rheumatoide ha a1 minus duo classes de moleculas B2M, i.e., illos con activitate attribuibile a factor rheumatic e illos con activitate tanto de factor rheumatoide como etiam del gruppo de globulina gamma. I T HAS BEEN DEMONSTRATED that human red blood cells (RBC) coated by selected incomplete anti-D sera can be caused to agglutinate by sera from some patients suffering from rheumatoid arthritis ( RA).6,27 Grubb and Laurell’ showed that the agglutinating action of the RA serum could be inhibited by pooled gamma globuin or by sera from certain individuals, and that the ability of these sera to inhibit the agglutination was genetically determined. Grubb and Laurel1 called inhibiting sera Gm( a+ ), the others Gm( a-). Different combinations of RA sera and incomplete anti-D sera have been used to identify a series of other genetically determined y-globulin factors [Gm(b),l0 Gm(x),” Gm-like,23and Gm(r)1.6]. From the Biobgical Lizboratoty, Western Reserve Unioersity, C h e l u n d , Ohio. This investigationwas suppotted in. part by PHS research grant RG-7214from the Dioision of General Medical Sciences, Public Health S h e . 331 ARTHRITIS& RHEUMATISM,VOL. 5, No. 4 (AUGUST),1962 332 ARTHUR G. STEINBERG Table 1.-Comparison of Ragg ~__ and SNagg Agglutinators Agglutinator Ragg SNagg Titer' against coated cells ~ _ _ ~ _ Inhibition titer of G m ( f ) sera vs. agglutinator Inhibition titer of Gm ( - ) sera vs. agglutinator Detect more than one Grn. factor? c64 > O , z8 Yes no _ _ 1024 c04 5 _ 5 . 128 0 'All titers are given as reciprocals. Although certain sera from patients with other diseases or from healthy individuals were found to cause sensitized RBC to agglutinate, these sera were not found to be inhibited by pooled gamma g l ~ b u l i n . ~ J ~Grubbb - ' ~ - ~ men~ tioned, as an addendum to a paper published in 1957, that he had found a serum from a healthy person which is inhibited by gamma globulin; however, he appears not to have tested it further. Ropartz and Lenoir'? confirmed Grubbs finding and demonstrated that sera from some blood donors, who were not demonstrably ill, could be used to detect Gm factors. Ropartz, Rousseau, and Rivat'" call such a serum "Serum Normal agglutinant" and abbreviate it SNagg. The term appeals to me and I suggest its retention as a generic term for Gm reagents from nonrheumatoid donors. Rheumatoid sera used as agglutinators in testing for y-globulin factors have been referred to as RA sera, anti-Gm reagin, or simply anti-Gm. I suggest that they be referred to as Rheumatoid agglutinators and abbreviated Ragg. Ragg and SNagg sera differ in their agglutinating titer, in the ease with which they are inhibited by Gm( ) and Gm( - ) sera and in the number of Data such as Gm factors which a given reagent can detect (table those summarized in table 1 have led Ropartz and his colleaguesZoto suggest that anti-Gm factors in Ragg and SNagg sera are different. They state that the action of SNagg sera is purely qualitative, while that of Ragg sera is both qualitative and quantitative. It should be noted that the agglutinators in Ragg and SNagg sera are associated with the BzM This is the same molecule which carries the Rheumatoid Agglutinating Factor ( R A F ) in RA sera.4a This paper is a report of comparisons of the agglutinators in Ragg and SNagg by means of titration, heating, and absorption experiments. + l).12J7-209z3 MATERIALS AND METHODS Detection of Crn Factor. The reagents used for Gm testing were: Group 0, R l R l red blood cells, an incomplete anti-D serum (Kim.) from a G m ( a + ) donor, a Ragg serum (Bowers) and two SNagg sera (Wils. and Scol.). Each of the agglutinating sera detects G m ( a ) with anti-D Kim. The Gm tests were performed as follows: One drop of washed, packed RBC was incubated with one drop of anti-D serum and eight drops of saline at 37" C. for 2 hours. Before use for Gm testing, the cells were washed four times in normal saline and resuspended in saline at a 0.3 per cent concentration. One drop of the diluted agglutinator was mixed on a slide with one drop of the diluted serum which was to be tested. This mixture was shaken for 5 minutes at room temperature. A drop of the cell suspension was then added, the mixture again shaken for about 5 minutes and then allowed to incubate for 45 minutes at room temperature. After the incubation STUMEG ON THE Gm 333 FACTORS Table B.-Agglutinatwn Titers of a Ragg Serum (Bowers) and a S N a g g Serum (Wh.) and Znhibitwn Titer8 of a Gm(a+) and a Gm(a-) Serum’ - Ragg titer against SRBC = 1/40962 Ragg 1/64 G m ( a + ) 1/16 no agglutination no agglutination Ragg 1/64 Gm( a-) 1/4 SNagg titer against SRBC = 1/32 no agglutination SNagg 1/8 G m ( a + ) 1/2-56 agglutination SNagg 1/8 Gm(a-) neat + + + + Unita in undiluted serum 4096 Agg.3 1024 Inhib.4 256 Inhib. 32 Agg. 1024 Inhib. 0 Inhib. ]See text for further explanation. 2SRBC = sensitized red blood cells. 3Agg. = agglutination units. 4Inhib. = inhibition units. the slide was again shaken and the results read under a dissecting microscope at 60X. Controls consisted of (1) known G m ( a + ) and Gm(a-) sera in the place of the serum to be tested; ( 2 ) the coated cells, plus saline, plus agglutinator; and (3) the coated cells, plus saline, plus the tested serum. Control ( 2 ) was used to check the activity of the agglutinator, control ( 3 ) was used to see if the tested serum was an agglutinator. If the cells failed to agglutinate, the serum being tested had inhibited the agglutinator and was said to be Gm( a+ 1. If the cells agglutinated, inhibition had not occurred and the serum was said to be Gm( a- ). Rheumatoid agglutinating factor (RAF) activity was detected by the FII latex test.21,22 Coated and uncoated latex particles were generously supplied by Hyland Laboratories. Gamma globulin was extracted from Gm( a- ) sera by Dr. Livia Blum. We are grateful to her for her assistance. THEDATA Agglutination and lnhibitiun Titers Table 2 shows the agglutination titers of Ragg Bowers and SNagg Wils., and the inhibition titers of a G m ( a + ) and a Gm(a-) serum against these agglutinators. The reciprocal of the maximum agglutination titer of the agglutinating serum was designated the number of agglutinating units in the undiluted serum. Thus Ragg Bowers undiluted is said to have 4096 agglutinating units. Ragg Bowers diluted 1/64 has 64 agglutinating units (i.e., 4096/64 = 64). At this dilution Ragg Bowers was inhibited by a G m ( a + ) serum diluted 1/16 but not by this serum diluted 1/32. The Cm( a+ ) serum diluted 1/16 is said to have 64 inhibition units and the undiluted serum 16 X 64 or 1024 inhibition units. The same reasoning is used for assigning units when the SNagg serum is used. Note that in this experiment the G m ( a + ) serum had the same number of inhibiting units (1024) against the Ragg serum as against the SNagg serum, and that while the Gm(a-) serum had 256 inhibiting units against the Ragg serum, it had no demonstrable activity against the SNagg serum (table 2). The above experiment was repeated with 19 Gm(a+) sera and nine Gm(a-) sera. The results are shown in table 3. The mean number of inhibiting units of the Gm( a + ) sera against Ragg and SNagg were 820.3 and 964.7, respectively, that is, less than one “tube” difference, since at this level the titer of the lower tube would be 512 and of the higher, 1024. Hence, the number of 334 ARTHUR G. STEINBERG inhibiting units in Gm( a+ ) sera against Ragg sera may be considered to be the same as those against SNagg sera. The mean number of inhibiting units of the Gm(a-) sera against Ragg was 241.8; but it was zero for these sera against the SNagg serum, because none of the sera used neat inhibited it. It will be shown in a later section that the inhibition of Ragg sera by Gm( - ) sera is probably due to non-Gm-specific 7-globulin (see page 335). Meutralization Experiments A 1:l mixture of a Gm( a+ ) serum diluted 1/64 and of Ragg Bowers diluted 1/64 when added to SRBC caused the cells to agglutinate. Hence, the inhibiting activity in the Gm( a+ ) serum had been neutralized by the agglutinator and the latter had retained sdicient activity to cause the SRBC to agglutinate. If the agglutinators in Ragg and SNagg sera are the same, one would not expect the above mixture of Gm( a+ ) and Ragg Bowers to inhibit the activity in a SNagg serum. If they are different, inhibition might occur. Seven G m ( a + ) sera were titrated against SNagg Wils. and SNagg Scol.; each SNagg being diluted '/8 for the test. When the inhibiting titers of the G m ( a + ) sera had been determined, each was diluted 1/64 and mixed with an equal volume of Ragg Bowers also diluted 1/64, hence, in the mixture, the concentration of each was 1/128. In each case the addition of the mixture PO SRBC led to agglutination. The mixture was submitted to a series of two-fold dilutions in saline. The concentration of each reagent in the final dilution was 1/8192. Each dilution was tested for its ability to inhibit each of the SNagg sera indicated above. Table 4 presents the protocols for the tests of two Gm(a+) sera against SNagg Wils. Apparently the addition of Ragg Bowers to these G m ( a + ) sera did not cause a reduction in the inhibition titer of either against SNagg Wils. A summary of the scores found for the seven sera tested against SNagg Wils. and SNagg Scol. is shown in table 5. There is no evidence of an effect of the Ragg on the titer of Gm( a+ ) sera against these two SNagg agglutinators. The data seem to indicate, in contradiction to the previous experiments, that the agglutinator in SNagg is not the same as that in Ragg. Titration Experiments with Heated Sera Steinberg and S t a ~ f f e have r ~ ~ reported the heating Gm(a-) sera at 63" C. rendered them Gm ( a + ). These tests were done against a Ragg agglutinator. The experiments have been repeated using one Ragg and two SNagg agglutinators. Twelve Gm(a-) and 13 G m ( a + ) sera were tested [six of the Gm(a+) were from Negroes]. The results are shown in table 6. Heated Gm( a- ) sera remained completely inactive against SNagg, while the titer of heated G m ( a + ) sera increased to about the same extent against SNagg as it did against Ragg, i.e., three to four tubes. Absorption Experiments The macroglobulin in serum from patients with rheumatoid arthritis will form precipitates with 7s gamma globulin preparation~.~-**~8 More important STUDIES ON THE Gm FACTORS 335 for our purposes is Fudenberg and Kunkel's 5b demonstration that the isolated 19s macroglobulin of RA sera will form 22s complexes with the 7-globulin in untreated normal sera regradless of the Gm type of the latter. It is assumed that the inhibition of Ragg sera by all undiluted or slightly diluted sera regardless of their Gm type is due to steric hindrance resulting from the combination of the 7-globulin in these sera with the RAF of the &M molecules in the Ragg serum. Support for this assumption is derived from experiments in which 7-globulin extracted from a Gm(a-) donor was titrated against Ragg Bowers. It was found that concentrations equal to or greater than about 300 mg. per cent inhibited the agglutinating activity of the Ragg Bowers serum diluted 1/32 (128 agglutinating units), while a concentration as great as 1600 mg. per cent failed to inhibit SNagg Wils. diluted l/s ( 4 units). If the assumption of steric hindrance is correct at least some of the P2M molecules in Ragg sera should have both Gm and RAF activity, while none of those in SNagg sera should show the combined activity. Absorption experiments with SRBC and with latex particles coated with Gm(a-) yglobulin were done to test this hypothesis. Two Ragg sera (Bowers and Bomb.) were absorbed repeatedly with packed RBC coated with a series of different anti-D's until all detectable activity against such cells was removed. (These experiments and others will be reported in detail elsewhere.) The absorbed sera were then titrated against latex particles coated with pooled gamma globulin. The titer of Ragg Bomb. against the latex particles was not changed by the absorption with the anti-D coated RBC, while Ragg Bowers showed a slight reduction in titer. These experiments indicate that not all the B2M molecules have both Gm and RAF activity. In another experiment Ragg and SNagg sera were absorbed with packed latex particles coated with pooled 7-globulin until the Ragg serum no longer agglutinated the particles (two absorptions). Both the Ragg and SNagg sera failed to agglutinate SRBC! The interpretation of these data is unclear because we have found (as suggested by Dr. Henry Kunkel) that saline or albumin used to wash latex particles coated with pooled 7-globulin becomes Gm( a+ ) . Apparently the 7-globulin is easily eluted from the latex particles. It is likely, therefore, that the inactivation of the Ragg and SNagg sera in the above experiments is not the result of absorption from them of macromolecules active in the Gm system, but rather the result of inhibition of the Gm agglutinqtors in them by the eluted G m ( a + ) 7-globulin which was present in the pooled 7-globulin used to coat the latex particles. The experiments were repeated using gamma globulin extracted from Gm( a- ) donors to coat the latex particles. Such particles, if the hvpothesis is correct, should absorb the rheumatoid factor and not inhibit the Gm activitv in a Ragg serum capable of detecting Gm(a), and, being coated with Gm( a- ) 7-globulin should have no effect on the SNagg serum. Ragg Bowers absorbed twice with latex particles coated with gamma globulin from a Gm( a- ) donor was inactive against particles coated with pooled 7-globulin and failed to cause SRBC to agglutinate, i.e., had no detectable anti-Gm ac- 336 ARTHUR G. S"BI%RG Table 3.-Mean Number of Znhibition Units in 19 Gm(a+) Sera and 9 Gm(a-) Sera vs. a Ragg and a SNagg Agglutinating Serum' Agglutinator SNacrcr Ram (Bowers) (Wils.) 820.3 24 1.8 964.7 0 19 Gm(a+) 9 Gm(a-) 'See text for further explanation. Table 4.-Titratwn of Gm(a+) Serum vs. SNagg Wils. Diluted 1 / 8 ( R I R 1 red blood cells were coated with 10 volumes of anti-D Kim. diluted 1/10.) ~Reciprocal of the dilution of the Gm(a+) serum Gm(a+) Donor R. KI. Good. '1 I Test reagent Gm(a+) G m ( a + ) 1/64 Rasg Bowera 1/64' + Gm(a+) Grn(a+) 1/64 Ragg Bowers 1/64' + 128 266 - - - - - - - 612 1024 2048 4096 ++ +++ ++++ ++++ ++ +++ ++++ ++++ + +++ ++++ ++++ +++ ++++ ++++ 8192 Score' ++++ 17 ++++ ++++ ++++ 17 16 15 ++ 'The mixture of G m ( a + ) 1/64: R a g Bowers 1/64: saline. l : l : l , gives a agglutination of the coated cells. In each of the tests indicated in the above table S N a g g Wils. 1/8 replaced the saline. ?The score equals the sun) of the plus signs which. as usual. indicate agglutination. Table 5.-Scores for 7 Gm(a+) Sera Tested in Two-fold Dilutions from 1/128 to 1 /8192 for lnhibition of SNaggI Wils. Scol. a b 87 12.4 82 11.7 ___---~_____ Total I X a 117 16.7 b 117 16.7 a. Untreated Gm( a + ) serum. b. An initial 1 : l mixture of Gm(a+) 1/64 plus Bowers 1/64 was titrated. 'See text for further explanation. tivity. A similarly treated SNagg serum (Wils.) retuined its ability to cause SRBC to agglutinate, i.e., retained its anti-Gm activity. DISCUSSION The indication, derived from the titration experiments summarized in tables 2 and 3, that the agglutinators in Ragg and SNagg sera are alike is apparently contradicted by the data of tables 4 and 5 which indicate that Gm( a+ ) serum inactivated by a Ragg serum remains active against a SNagg serum. The contradiction may be resolved with the aid of two observations: 1. A mixture of Ragg and G m ( a + ) sera which does not cause sensitized RBC to agglutinate will do so after dilution with ~ a l i n e . ~These J ~ observations suggest, as Grubb indicated, that dilution of the mixture leads to a separation of the agglutinator in the Ragg and the inhibitor in the Gm(a+ ) serum. The reaction may be summarized as follows: STUDIES ON THE Gm 337 FACTORS Table &--Mean Titers' (Expressed as Last 'Tube' of Serial Two-fold Dilutions Showing Inhibition) of Unheated (U)and of Heated (H)e Normal Sera e a i n s t Ragg and SNagg Agglutinators ~~ ~ ~ ~ SNazg (1/8) Rspz (1/64) ( Bowers ) Gm. U. No. - H .1 ~ 12 13 + 2.3 5.1 WilS. u. Scol. H.' U. H.' 0 0 10.7 13.7 ~~~ 6.4 9.7 0 9.8 13.4 0 'Each serum was made up in serial two-fold dilutions, the first tube being a 1/2 dilution. 'Heated at 63 C. until visible aggregates formed. The aggregates were not used for the agglutination inhibition tests. + ( a ) R(agg) I(nhibitor) RI dilution ( b ) RI---+ R + I 2. It was observed that, although Ragg 1/64 G m ( a + ) 1/64 saline (mixed 1:l:l) caused the sensitized cells to agglutinate [i.e., the Gm( a + ) was too dilute to inhibit the Ragg], Ragg 1/64 G m ( a + ) 1/64 SNugg % (mixed 1 : l : l ) did not cause the cells to agglutinate (table 4,first two columns of rows two and four). Paradoxically, the addition of more agglutinating reagent (the SNagg serum) led to an inhibition of agglutination. Recall that Gm( a- ) sera have approximateIy 250 inhibiting units against Hagg (tables 2 and 3 and text) and that this is assumed to be non-Gm-specific. If it is assumed ( a ) that dilution of the Ragg-Gm(a+) mixture frees the Gm inhibitor in the G m ( a + ) serum, and that the freed inhibitor inactivates the SNagg serum, and ( b ) that the postulated non-Gm-specific inhibitor in the SNagg serum and possibly in the Gm( a+ ) serum inhibit the Ragg serum, the data are explained. The reaction may be diagrammed as follows: + + + + a) ,/-9- "Diluted RI" t s-----3 w- n where R& and 3-I. -I indicate non-Gm-specific inhibition. If this reasoning is correct, the addition of SNagg at high concentrations (i.e., 7-globulin concentration greater than 300 mg. per cent), but not at low concentrations should inhibit the agglutination caused by the Ragg-Gm ( a+ ) mixture. The addition of Gm( a- ) sera in high concentrations should lead to the same result. The absorption of all detectable agglutinating activity from SNagg sera before their addition to the Ragg-Gm(a+) mixture should not influence the result, because the non-Gm-specific inhibitor is postulated to be 7s gamma globulin and the agglutinator is &M globulin. Finally, the mixture Ragg plus SNagg-unabsorbed should lead to agglutination (since both are agglutinators and SNagg is not inhibited by Gm(a-) sera, such as Ragg Bowers), but the mixture of Ragg plus SNagg-absorbed should not lead to agglutination, because the non-specific inhibitor in SNagg will inhibit Ragg. Table 7 shows the protocols of some of the experiments done to test this in- 338 ARTHUR C. STEINBERG Table 7.-Titratwn of SNagg W h . and Gm(a-) Sera (RIRI cells were coated with 10 volumes of anti-D Kim. dffuted 1/10.) Titer of serum Serum titrated 1/2 1/8 1/4 ( A . ) Against a mixture of Ragg Bowers 1/64 SNagg unabsorbed 0 0 SSagg absorbed1 0 1 Gm(a-) ( 2 samples) 0 0 Gm(a-) (1 sample) 0 0 + Gm( a + ) + ++ ( B . ) Against Ragg Bowers 1/04 SNagg unabsorbed SNagg absorbed1 Gm( a - ) ( 3 samples) ++++ 0 ++++ +++ 0 0 0 f ++++ ++++ +++ 1/16 1/64 +++ +++ +++ +++ ++++ ++++ ++++ 'Absorbed with cells coated with anti-D Kim. until no detectable agglutinating activity remained in the serum. terpretation. The experiments are clearly in agreement with the explanation offered above. The increased titer of heated Gm( a + ) sera against Ragg and SNagg agglutinators may be due to steric hindrance. It is assumed that the aggregates formed in heated Gm( a + ) sera are composed of 7-globulin with specific Gm activity and of other proteins. These larger molecules cover more sites, on the &M molecule, per active G m ( a i ) molecule than do the unaggregated r-globulin molecules, hence the higher titer. Evidence for this is derived from the demonstration, by electrophoresis, that Gm( a ) activity in heated serum extends from the region of the fast albumins to the y-glob~lins,'~~ while such activity is limited to the 7-globulins in unheated sera. The increased inhibitory activity of heated Gm(a-) sera against Ragg may be explained in essentially the same way. The aggregates react with RAF in Ragg and by steric hindrance inhibit the Gm activity of Ragg. The heated Gm(a-) sera do not react with the SNagg sera because SNagg sera have no RAF activity, and the Gm( a- ) sera have no Gm activity. The absorption experiments with latex particles coated with 7-globulin from Gm( a- ) donors indicate that in Ragg sera essentially all &M molecules with Gm activity also have RAF activity (since absorption removed both activities). The fact that the titer of the Ragg sera against the coated latex particles was either not reduced, or only slightly reduced, when all Gm agglutinating activity was absorbed from them indicates that only some of the &M molecules with R A F activity have Gm activity. In brief, we have demonstrated in Ragg sera two kinds of &M molecules with activity against human 7-globulin: ( a ) those with both RAF and Gm activity and ( b ) those with RAF activity but no Gm activity. The absence of RAF activity in the presence of the Gm activity in SNagg sera indicates a third type of B2M molecules, namely, those with Gm activity but no RAF activity. It will be recalled that the tests for R A F activity in sera from patients with rheumatoid arthritis are negative for SNagg sera. 'It is possible, however, that the RAF is present in SNagg sera but in too low concentration to be detected smm ON 339 THE cm FACTORS by the standard technics. SNagg Wils. concentrated five-fold by dialysis against 20 per cent PVP (polyvinylpyrrolidine) in saline, was still negative in the latex test. R a g Bowers similarly treated remained positive. Sera tested this way are 100 times as concentrated as they are in the usual latex test ( Hyland Laboratories ) . In the abstract submitted for the May, 1961, meeting of The American Society of Human Genetics, I indicated that I believe that the agglutinators in Ragg and SNagg were qualitatively different. The basis for this statement is the data in table 3, which are now interpreted differently, and I believe, correctly. SUMMARY The agglutinators in serum from patients with rheumatoid arthritis and in serum from healthy donors were compared by serologic methods. Titration and absorption experiments indicate that the agglutinators are probably the same and that sera from patients with rheumatoid arthritis, which can be used for Gm testing, have at least two kinds of &M macromolecules which react with human 7-globulin: ( a ) those with rheumatoid factor activity only and ( b ) those with both rheumatoid and Gm activity. The &M molecules in SNagg sera have Gm activity but no RAF activity and therefore represent a third type of molecule. REFERENCES 1. Brandtzaeg, Brita, Fudenberg, H., and Mohr, J.: The Gm(r) serum group. Acta genet. 11:170-177, 1961. 2. Christian, C. L.: Characterization of the “reactant” (gamma globulin fictor) in the F I1 precipitin reaction and the F I1 tanned sheep cell agglutination test. J. Exper. Med. 108: 139-157, 1958. 3. Epstein, W., Johnson, A., and Ragen, C.: Observations of a precipitin reaction between serum of patients with rheumatoid arthritis and a preparation (Cohn fraction 11) of human gamma globulin. Roc. Soc. 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