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Specificity of rheumatoid factor with gamma globulin of different species.

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Chapter IV
Specificity of Rheumatoid Factor with Gamma
Globulin of Different Species
Chirmun: JOHN H. VAUGHAN,M.D.
DR.VAUGHAN:In discussing the reaction of rheumatoid factor with animal
r-globulins, I would first like to center your attention on a comparison of the
activity of rheumatoid factor with animal 7-globulins to that with human
7-globulin. The data reported by Dr. Butler1 on the reactivity of bisdiazotized
benzidine aggregates of 7-globulins with rheumatoid sera can illustrate this
point.
Figure 1 shows the results of tests of the rate of nitrogen absorption from
rheumatoid sera by animal and human 7-globulins made insoluble through
cross-linkage by bis-diamino-benzidine ( BDB ) . One finds that BDB-aggregated 7-globulins made from human 7-globulins absorb far more nitrogenous
material from rheumatoid serum than did BDB aggregates made from rabbit
or bovine 7-globulins. We like to consider these curves as a way of seeing
roughly what proportions of material in a rheumatoid serum are reactive with
the various types of 7-globulins.
All agglutinating activity was removed from this serum by absorption with
human 7-globulin aggregates a bit beyond the last point indicated on the
curve. Activity against rabbit, bovine, and equine 7-globulins was all removed
as well as activity against human 7-globulins.
By contrast, the same projected point on the rabbit or bovine 7-globulin
curves gave supernatants which were devoid of activity against rabbit or
bovine 7-globulin, respectively, but not against human 7-globulin and not
completely devoid of activity against the other types of 7-globulin in the case
of the bovine curve and in reverse.
Recovery of agglutinating activities from the insolubilized BDB 7-globulins
could be affected by exposing the washed insoluble material to a glycine buffer at pH 3.7. From each of the three types of 7-globulins used for absorption,
agglutinating activity against cells coated with human, rabbit, bovine, or
equine 7-globulins was recovered. This was true of all three of the types of
7-globulin used in the absorption. This suggested to Dr. Butler and me that
the relationship between these anti-7-globulin activities should be demonstrable in agar diffusion analyses.
In this experiment, soluble BDB aggregates of human 7-globulin and solnble BDB aggregates of rabbit 7-globulin formed precipitates with rheumatoid factor isolated from the bottom fractions of a sucrose density gradient
ultracentrifugation of the serum illustrated in figure 2. Fairly diffuse lines
were formed which did not cross each other. Although spurs were not seen,
as are characteristic of clinical cross-reacting systems, these data did indeed
suggest that the BDB aggregates of human and rabbit 7-globulins were interacting with common anti-7-globulin molecules in the rheumatoid factor.
446
SPECIFICITY OF RHEUMATOID FACTOR FOR OTHER GG
pg
w
447
CC-BDB
Fig. 1.- Adsorption of nitrogen to insoluble complexes.
Turning to the isospecih serum discussed earlier, we used the same technics
in analysis. With this serum, human 7-globulin regularly absorbs more nitrogen
from the serum than does either rabbit or bovine 7-globulin.
Doing the absorptions illustrated in figure 3, we were using very large
amounts of nitrogenous material and getting relatively small additions to the
insolubIe 7-globulin, so reproducing these curves exactly has been difficult.
In the absorption with human 7-globulin aggregates all the anti-Gm activity
[if one accepts the agglutination of Gm(a+) coated cells as evidence of
anti-Gm(a ) activity in this particular serum] was gone from the supernatant after 180 pg, N of BDB aggregate human y-globulin had been added to
the serum.
After exhaustive absorption (more than 2000 pg. N of BDB aggregate
human 7-globulin), all agglutinating activity was removed from this serum.
Exhaustive absorption with rabbit or bovine y-globulin could remove only
the agglutinating activity against the respective type of 7-globulin.
The autogenous 7-globulin prepared from the patient was made into insoluble 7-globulin by BDB and used for absorption. It can be noted that its
curve fell intermediately among the others. The fact that the autogenous
aggregated y-globulin curve was quantitatively less high than the RDB aggregate human 7-globulin (F-I1) curve was interesting and certainly must
have represented somehow the isospecificity of this serum.
We could not tell from this whether there was a difference because some
of the groupings in BDBlaggregated human YLglobulin were missing from
448
IMXfUNOLOGIC ASPECTS OF RHEUMATOID ARTHRITIS AND SLE
Fig. 2.-Precipitates formed in agar diffusion between ultracentrifugally isolated
rheumatoid factor and soluble y-globulin aggregates.
the autogenous BDB aggregates or whether they were just fewer in the autogenous aggregated material. We could not carry these curves out any further
with larger amounts of BDB 7-globulin because the errors of the experiment
became enormous in that area. When this serum was exhaustively absorbed
with repeated exposure to autogenous 7-globulin, essentially all the agglutinating activity was removed from the serum.
These data are consistent with the assumption that the apparent isospecificity that exists for anti-animal 7-globulin activities in the rheumatoid
factors in sera containing anti-7-globulin activities are part of a total immune
response to autogenous denatured 7-globulins.
D R . FUDENBERG:
Dr. Vaughan, in your experiments the specificity against
the patient’s own 7-globulin may have already been partially saturated. This
might explain why the autogenous 7-globulins absorbed less than others. It
would be perhaps wise to repeat these experiments using purely the 19s
rheumatoid factor antibodies.
DR.SINGER:In collaboration with Drs. Rafter, Plotz, Halberstam, and Schuller, we attempted to produce in experimental animals an antibody which would
have two main characteristics of rheumatoid factor: a large molecular size and
the ability to react with human 7-globulin.
Two experimental lines were followed. The first utilized heterologous
7-globulin and the second utilized autologous 7-globulin. Latex particles with
absorbed 7-globulin were used as particulate carriers. Our choice of this
method was based on the fact that human 7-globulin coated on latex particles
of 0.2 p served as a potent stimulator of antibody production. It also eliminated
the individual variation in antibody production in different animals of the
same species.
Our decision was justified by a study of goats immunized with latex particles
449
SPECIFICITY OF RHEUMATOID PACTOR FOR OTHER GG
75
ROC
ug. N
absorbed
25
500
)rg.
Fig. 3.-Adsorption
1000
1500
BDB-GC
of nitrogen to insoluble complexes.
alone, human 7-globulin alone, latex particles with absorbed human r-globulin,
and finally, human 7-globulin in Freund's adjuvant. When the goat serum
was tested for agglutinating antibodies, high titers were obtained both with
human 7-globulin coated on latex particles and with 7-globulin in Freund's
adjuvant. Most precipitating activity was in the system of 7-globulin in
Freund's adjuvant, less activity being exhibited by latex 7-globulins. Human
7-globulin showed low titers, low precipitation, and negative results were obtained with the latex particles alone, as expected.
Following these findings, latex particles were coated with human 7-globulin
and injected into cows, horses, sheep, goats, and rabbits. All primary and successive bleedings were studied by DEAE column chromatography, agglutinating activity, precipitation curves, sodium chloride density gradients, analytic
ultracentrifugation, double agar gel diffusion, and immunoelectrophoresis.
Prior to immunizations all animals showed a 19s component in their sera
as detected by analytic ultracentrifugation.
We found that all animals immunized with latex particles coated with
human 7-globulin produced two antibodies, one a 7s component and the
other a 19s macroglobulin. However, the pattern of distribution of antibody
of low and high molecular weight and the time of appearance of this antibody
varied among the different animals tested.
Figure 4' shows the chromatographic pattern of cow sera obtained at
varying intervals during the immunization periods. The location and titers of
450
IhlhlUn'OLOCIC ASPECTS OF HHEXJMATOID ARI'HRI'rIS AND SLE
FII
s.c
TITER
R
0
302
1
n
20
60
100
20
60
I00
Fig. 4.-Chromatography of cow sera (10 ml.) on diethylaminoethyl cellulose.
Cone sphere gradient 0.02M,pH8-0.3M, pH5-1M NaC1. #301-309.
serologically active fractions as determined by tanned sensitized sheep cells
are shown in relation to protein peaks. In cow sera, peak I of low molecular
weight coincided with the 7 2 region and peak I1 was located within the macroglobulins. The initial bleeding revealed serologic activity only within the
second, or macroglobulin, peak. After booster immunization activity was
noted in the y2 region, while at the end of 6 months the only serologically
active material again was a macroglobulin.
In horses, sheep and rabbits the distribution of antibodies appeared differently. For example, figure 5 illustrates the chromatographic pattern of
horse sera at varying intervals during the immunization period. Antibody
appears in both the y1 and 7 2 fractions and continues in both fractions throughout. The control percentage is from 2 to 5 per cent of the total y-globulin.
Figure 6 shows the precipitation curves obtained by adding human 7-globulin in varying amounts to aliquots of experimentally produced macroglobulin
obtained by column chromatography. The shape of the curve is similar to
that obtained with the precipitation of the rheumatoid factor and denatured
7-globulin.
The reactivity of various animal macroglobulins with various animal globnlins coated on tanned sheep cells was tested. As expected, in every case the
specificity was direct to human 7-globulin with less specificity shown to 7globulin of other species. The cow macroglobulin produced the greatest agglutination reaction with human 7-globulin and cross-reacted with rabbit,
horse and sheep but not cow sera. Similarly, sheep macroglobulin reacted
with human, rabbit and cow 7-globulins, but not with that of sheep. In every
instance there was no serologic activity with autologous 7-globulin.
451
SPECIFICITY OF RHEUMATOID FACTOR FOR 0Tm.R GG
FlI
S.C.,TITER
1
/05
I:","
20 60
20
60
loo
100
PER C€NT €LUAT€
Fig. 5.-Chromatography of horse sera ( 10 ml.) on diethylaminoethyl cellulose.
Cone sphere gradient 0.02M, pH8-0.3M, pH5-1M NaCl.
Following this line of investigation, rabbits and goats were immunized with
latex particles coated, respectively, with autologous rabbit and goat y-globulins. Animals immunized by this procedure produced agglutination titers not
only with the autologous 7-globulins but also with human 7-globulin. However, very low titers-1:32 and l:6P-were obtained. While these titers may
have been significant as being indicative of autoantibody production, they
were too low for quantitative data to be obtained. We are considering developing this technic further by utilizing autologous 7-globulin on latex particles
for the production of autoantibody.
In order to further characterize these antibodies we postulated that if two
major characteristics of rheumatoid factor were present, other similarities
with rheumatoid factor might exist. Table 1 compares rheumatoid factor with
the macroglobulin antibody to human 7-globulin produced in various animals.
They share the following characteristics: both are 19s macroglobulins which
react strongly in the tanned sheep cell and latex fixation tests with human
y-globulin; the agglutinating activity of both is destroyed by mercaptoethanol;
both exhibit cross-reactivitywith other species of 7-globulin; and they produce
similar precipitation curves. The only difkrence between them is that
rheumatoid factor reacts with autologous denatured y-globulin while none of
the animal macroglobulins so far produced shows such autologous activity.
In summary, the injection of heterologous y-globulin into animals produced
macroglobulin antibodies similar in most physical and immunochemical
452
IMMUNOLOGIC ASPECTS OF RHEUMATOID ARTHRITIS AND SLE
751
y601
PEAK Il 308
REACTION VOL. 4mf
$45
OV
540 ’ 7h0 960 ’ Idso’ I k O ’ d40
MICROGRAMS OF HUMAN GAMMA GLOBULIN N ADDED
lb0 ’ 3
b
Fig. 6.-Precipitation curve of a 19s macroglobulin cow serum immunized with
human y-globulin. (Vertical column = ppt. y N per ml.)
properties to rheumatoid factor. This strongly suggests that rheumatoid factor
is a true antibody. The most distinctive feature of rheumatoid factor is its ability to react with denatured autologous 7-globulin.
DR.MILGROM:
The data presented by Dr. Vaughan indicate that the rheumatoid factor may be considered an antibody primarily directed against human
y-globulin and that the reactions which are obtained with y-globulins of other
species origin should be considered as cross-reactions. On the basis of Dr.
Vaughan’s experiments as well as on the basis of experiments published by
one would have to conclude that there exist essentially
other
two factors: one which combines with human y-globulin only and a crossreacting factor which combines with both human and foreign species 7-globulin whose existence was rather well established by Dr. Vaughan’s experiments.
I will present the data only briefly. InasAs our findings were publi~hed,~
much as they were obtained by quite different technics, our results do not
contradict those of Dr. Vaughan.
We wished to explore whether in addition to these two factors there was
another factor directed only against foreign species 7-globulin (represented
by rabbit 7-globulin in our investigations). We confined our experiments to
the agglutination of sensitized red blood cells. Three indicator systems were
Iised. ( 1 ) A system elaborated by Cohen et ala5which consisted of alligator
red blood cells sensitized by subagglutinating doses of rabbit anti-alligator
erythrocyte serum. This system was designated Ar. (2) This was elaborated
in our laboratory6 on the basis of the discovery by Kellner and Hedal.’ It
consisted of rabbit blood group G red blood cells sensitized by incomplete
anti-G serum of rabbit origin. We will call this indicator system Rr. ( 3 ) A
system elaborated by Waller and Vaughan8 consisted of Rh-positive red
blood cells sensitized by incomplete anti-Rh serum. The anti-Rh serum “Ripley” used in this investigation is known to combine with most rheumatoid
sera. This was designated Hh.
453
SPECIFICITY OF RHEUMATOID FACTOR FOR OTHER GG
Table 1.-Comparison
between Animal 19s HGG Antibodies
and Rheumatoid Factors
/9 s
A m HGG.
/9 s
#EUM. FACTORS
+
In the experiments presented in figure 7, a mixture of Ar
Rr was added
to rheumatoid sera and examined for mixed agglutination. With all rheumatoid
sera tested, positive results were obtained and the clumps found on microscopic examination appeared as those presented in figure 8. However, when
Hh was added to rheumatoid sera, in most instances
the mixture of Ar
homologous agglutinates were recognized consisting of separate alligator
erythrocyte clumps and human erythrocyte clumps (fig. 9 ) . Only a minor
proportion of rheumatoid sera, about 1 out of 5, produced mixed agglutination
of the Ar
Hh mixture. These results already indicated the existence of a
separate factor for rabbit 7-globulin because if such a factor did not exist the
mixture of Ar
Hh should have always been agglutinated in mixed clumps as
was the case in the mixture of Ar Rr,
Further experiments were designed to compare the serologic properties of
mixed agglutinates composed of Ar
Rr and Ar
Hh (the latter were produced by exceptional rheumatoid sera capable of agglutinating Ar
Hh in
mixed clumps). The results of this experiment are presented in table 2.
As can be seen, the Ar Rr agglutinates were not affected by 1 per cent F-I1
of human serum but were completely dispersed by 1 per cent F-I1 of rabbit
serum. Mixed agglutinates composed of Ar
Hh were affected quite difhrently by F-I1 preparations. The addition of human F-I1 caused the dispersion of human red blood cells leaving the alligator red blood cells agglutinated, and the addition of rabbit F-I1 resulted in the dispersion of alligator
erythrccytes leaving human erythrocytes in clumps.
These experimental data can be interpreted by assuming the existence of
three types of rheumatoid factor: ( 1 ) one directed against human 7-globulin
only; ( 2 ) one capable of combining with both human and rabbit 7-globulin;
+
+
+
+
+
+
+
+
+
454
IMMUNOLOGIC ASPECTS OF RHEUMATOID ARTHRITIS AND SLE
I Appearance
Mixed Atglutination
I
Ar +Rr
P06.
I
0
0. o O O
Neq with mod
rheum.sera
&s with some
rheum.sera
I
AY+Ho
0 0
0 00 0
o0 0
@
::
Neq.
0
Fig. 7.-Ar
agglutination.
+
Rr mixture added to rheumatoid sera and examined for mixed
and ( 3 ) one directed against rabbit 7-globulin only. We believe that factors
1 and 3 appear in much larger quantities than factor 2. We could demonstrate
factor 2 only in 1 out of 5 rheumatoid sera (those which were capable of
Hh). Most probably in the remaining
producing mixed agglutination of Ar
sera the titer of the cross-reacting factor was not high enough to produce
positive mixed agglutination reactions.
+
SPECIFICITY OF RHEUMATOID FACTOR FOR OTHER GG
Fig. 8.-Clumps found upon microscopic examination of agglutination of Ar
455
+ Rr.
DR.VAUGHAN:Is this postulating a heteroligating type of antibody activity?
DR. MILGROM:Not many antibodies but many reactive sites. I think, according to Dr. Kunkel, there are seven reactive sites in rheumatoid factor.
DR.SINGER:Did you test with other than the rabbit 7-globulin?
DR.MILGROM:In this system bovine would not be active in the dispersion,
but I did not have an incomplete bovine antibody system which could be
applied for this type.
I think, as Dr. Milgrom indicated, there is no inconsistency
DR.MCDUFFIE:
between his findings and those of Dr. Vaughan. They certainly coincide with
the observations of Drs. Fudenberg and K ~ n k e l They
.~
showed there was no
relationship between the ability to agglutinate anti-D coated cells and the
ability to agglutinate sensitized sheep cells. Everything depends on the type
of coat being used.
We have been studying the reaction of rheumatoid factor with rabbit
y-globulin using different coats and an inhibition technic. Figure 10 illustrates
what happens when one tests the ability of human 7-globulin to inhibit the
agglutination of tanned sheep cells by rheumatoid serum. The first five lines
shew the results obtained with tanned cells coated with human 7s 7-globulin,
and the second five lines the results when cells coated with rabbit y-globulin
are used. Human u-globulin inhibits both systems equally well. Figure 11 shows
the reverse experiment. If rabbit y-globulin is used as an inhibitor, it does
not show any activity against cells coated with human 7-globulin. On the
other hand, it strongly inhibits the agglutination of cells coated with rabbit
7-globulin. The failure of rabbit 7-globulin to inhibit the reaction of cells
coated with human 7-globulin appears to be due to the absence of much antibody-like activity in most rheumatoid sera.
456
IMMUNOLOGIC ASPECTS OF RIIEUhlATOID ARTHRITIS AND SLE
Fig. 9.-Clumps found on microscopic examination of agglutination of Ar
+ Hh.
Next we investigated the effect of the denaturation and aggregation of the
reactants. The first part of figure 12 shows the results of inhibition tests against
tanned cells coated with 7s rabbit 7-globulin isolated by chromatography.Both
aggregated (20 minutes at 68 C.) and unaggregated rabbit 7-globulin appear
to be equally effective inhibitors. Somewhat different findings are observed
when aggregated rabbit 7-globulin is used to coat the cells, however. In the
second part of this figure it can be seen that unaggregated rabbit 7-globulin
is a rather poor inhibitor, whereas aggregated inhibits well. It may also be
noted that the absolute titer of the serum against aggregated coated cells is
slightly higher than against cells coated with unaggregated rabbit 7-globulin.
It does seem possible that the process of heating and aggregation has altered
the 7-globulin is some way so as to uncover or produce new antigenic groups.
We attempted by cross-absorption to see whether activity against the aggregated coated cells might remain after absorption with cells coated with
unaggregated rabbit 7-globulin. Unfortunately, using such cells to absorb the
sera we found that the coat comes off the cell and prozones occur when the
supernatants are tested. Thus the results were inconclusive on this point. We
are now using both aggregated and unaggregated anti-sheep cell antibodies
to coat the cells, but have no results to report as yet.
We have also compared the behavior of rheumatoid sera with that of a
sheep anti-rabbit 7-globulin serum (fig. 13); they are identical with those
obtained with rheumatoid serum. When unaggregated rabbit 7-globulin is
used to coat the cells, both aggregated and unaggregated rabbit 7-globulin
act as equally good inhibitors. If the cells are coated with aggregated rabbit
7-globulin, aggregated rabbit 7-globulin behaves as a much more potent inhibitor. We believe this phenomenon of enhanced reactivity following aggregation or denaturation may be characteristic of a number of antigen-antibody
systems.
457
SPECIFICITY OF RHEUMATOID FACTOR FOR OTHER GG
Table 2.-Dispersion of Mixed Agglutination: Effect of Fraction ZZ of Human
and Rabbit Origin on Mixed Agglutinates Composed of
Ar 4- RT and AT
Hh
+
Microscopic Appearance of
Ar + Rr agglutinates incubated with:
lr;r,FIIof
pooled human serum
______-__-____
Alligator
erythrocytes
Mammalian
erythrocytes
l%FIIof
pooled rabbit serum
saline
+ Hh agglutinatea incubated with:
-_
1% F I1 of
pooled human serum
1% F I1 of
pooled rabbit serum
Aaplutinated
Free
.._____.
._______
Free
Mixed
apglutinates
-_____-__-__
Ar
Mixed
agglutinates
Free
Free
Agglutinated
saline
- __
Mixed
agglutinates
____-_______.-_
Since the adsorption of 7-globulin onto the surface of a tanned cell h a y
produce some structural alteration in the molecule, the sensitized sheep cell
test was also employed. The results of agglutination and inhibition tests are
recorded in figure 14. Both 7 s and aggregated rabbit 7-globulin as well as
normal rabbit serum appear to be potent inhibitors; 7s human 7-globulin and
whole human serum were only slightly less reactive. Surprisingly, aggregated
human 7-globulin was much less reactive in the rheumatoid factor-rabbit y globulin system than was unaggregated human 7-globulin. The explanation
of this apparent paradox is not clear.
In summary, we believe that our results are most consistent with the hypothesis that the reacticn of rheumatoid factor with rabbit 7-globulin is analogous
to that of an immunologic cross-reaction. Although human 7-globulin contains
“antigenic” groups not present in rabbit 7-globulin, all the “antigenic” sites of
rabbit y-globulin are present in human 7-globulin. There may be quantitative
differences. The enhanced reactivity of rabbit 7-globulin after aggregation and
denaturation by heating may result from a change in antigenicity but this has
not yet been proved.
DR.VAUGHAN:
I would like to call your attention to the serum I discussed
in an earlier session in which autologous y-globulin from an individual had no
inhibitory activity for his own agglutinating activity, but acquired it after
the alkaline denaturation. This raises the major possibility that the. differences
between Dr.Milgrom’s data and ours are that we were dealing with partially
denatured 7-globulin in our BDB aggregates, whereas Dr. Milgrom was dealing
with a much more nearly native 7-globulin in his sensitized cells.
Dr. Vaughan, we have not tried your particular aggregate
DR. MCDUFFIE:
but have tested a number of insoluble conjugated y-globulins in an attempt
to measure rheumatoid factor quantitatively. We have had great difficulty
getting reliable quantitative data because of high blanks and nonspecific adsorption of serum proteins to the conjugates. I am a little surprised at the
large amounts of rheumatoid factor that appear to be present in rheumatoid
sera according to your data. I would also like to ask about the blanks with
normal sera. All our preparations have removed so much nitrogen from normal
sera containing no detectable rheumatoid factor that it has become a major
difficulty.
DR.VAUGHAN:
These sera, first of all, were selected for being especially high
458
TMMUKOLOGTC ASPECTS OF R3EUMATOID ARTHRITIS AND SLE
.
Coated
Conc
with
Inhibitor mg/ml
Human
I1
None
fiuman
I1
11
I1
11
II
11
It
None
Human
It
I1
II
I1
11
I1
Rabbit
0
Dilution (reciprocal) of RF pool 1 7
50 100 200 400 800 1600 3200 6400 12800 25600
- - * * * * *
- i-t - - - - * i+ * * *
0.05* * * * *
o . o 1 - * * * * u *
- - * * -+ t- i +- *- *
0.70 0.10 *
* - O . O 5 - + + + t t e 0.70
0.10
-I+
i+
0 . 0 1 - * + t i + * -
* * * i+
-* --- -- -* u u
-
---
---
--
. H i +
+
-
-
-
+
---
-
Fig. 10.-Inhibition of rheumatoid agglutination of tanned sheep cells coated
with human and rabbit y-globulin by human y-globulin.
Coated
with
Human
II
Conc
Inhibitor mg/ml
None
Rabbit
II
,I
I1
II
Rabbit
II
None
Rabbit
,I
,I
II
I,
0
50
Dilution (reciprocal) of RF pool #7
100 200 400 800 1600 3200 6400 12800 25600
- - * * * * * * *
*
+t
U
i+++
0 . 1 - * * * * * *
* * u
- * * * * * * * 4- 4 +1.0
- - - - .- - 0.5
- - - - - 0.1
- - - - - - - 1.0
i+
++ U I+ i+i+ ++
0 . 5 - i + i + U + f t + + +
ft
ft
*
*
*
i+
-
Fig. 11.-Inhibition of rheumatoid agglutination of tanned sheep cells coated
with human and rabbit y-globulin by rabbit y-globulin.
titer. They are not casual rheumatoid sera. Second, we have found it useful to
dilute these sera 1:6 or 1:lO and allow them to stand awhile. A spontaneous
precipitation occurs here, very small in quantity, but one which is quite
large in relation to the nitrogen added to the aggregates.
Hence what we are examining is essentially whole sera that have been
diluted up in buffer, allcwed to stand, exhaustively spun and then analyzed
with the inscjlubilized BDB aggregates. I think this circumvents the problem
you mention.
DR.KAPLAN: What kind of plot do you get if you graph the absorbed nitrogen to the amount of BDB-labeled F-II?Is this a changing proportion?
If you noticed, there was quite a difference in the amount of
DR.VAUGHAN:
nitrogen added per pg. of BDB-aggregated 7-globulin between the two sera
shown. We have yet to clarify in our minds whether this indeed represents
some difference in combining ratio for the BDB aggregates or whether this
is a difference in the way we prepare one bxtch of aggregates to another.
In terms of reproducibility, I would emphasize that these are orders of
differences and are reproducible in that way. Human y-globulin has consistently absorbed more than rabbit 7-globulin. But the absolute figures for quantities
absorbed vary by 10 to 20 fig. of nitrogen.
DR.WITEBSKY:
May I call attention to the fact that the method discussed
by Dr. Milgrom of dispersion cf agglutinates is a very fine quantitative procedvre. I feel I should recommend this method because it discloses many more
459
SPECIFICITY OF RHEUMATOID FACTOR FOR OTHER GC:
Rheumatoid serum pool 1 7
Sheep c e l i s coated with 7s RGC (0.8 mg/ml)
or aggregated RGG (0.8 mg/ml)
Coated
with
7s RGG
Inhibitor
AggRGG
0.8
0.5
0.1
0.8
0.5
0.1
None
75RcG
0.8
0.5
0.1
AggRGG
Reciprocal of d i l u t i o n s of rheumatoid serum
20 40
80 160 320 640 1,280 2,560 5,120
- * * t + * c c * *
None
7sRGG
Agg RGG
Conc.
mglml None
0.8
0.5
0.1
- ++ tF ft -- --- *
- *
- -*
*
- * * -
-
.
I
*
+
-
---
-
-
f
--
-
--
-
-
-- -- --
-
- * * * * + ) * **
*
-I+t+ +*
- + k + t . H + , U f . +
* * * * M i + . *
f
- ++
+
-- *++ + -- -- -- * * - - *
- -
--
10,240
--
-
*-
-
Fig. 12.-Inhibition of rheumatoid serum by 7s rabbit y-globulin and aggregated
rabbit y-globulin.
differences than does the absorption experiment with denatured material. I
believe the information gained this way is more accurate. Of course, the
material to be used in a dispersion experiment can be denatured also but I
believe one wants to get material as undenatured as possible.
Is there any relationship between the observation that the
DR. EPSTEIN:
precipitin reaction occurs best with dilution of the rheumatoid serum and the
fact that the prozone reaction, as seen in the Gm specific systems, will disappear as you dilute? Do you think that the failure to have as good a precipitin
reaction without dilution relates to the removal of a prozone reaction between
the 7s and 19s of the individual rheumatoid serum? Do you have an explanation for the observation made on the increase in precipitin reaction in large
volume?
DR.VAUGHAN:
Our thought was, as you indicated, that this was a matter of
diluting out inhibitor 7s more rapidly than the agglutinating or precipitating
activity, for whatever physicochemical reason this would be.
I think the major problem with accepting this unreservedly is Dr. Harboe’s
finding, confirmed by Dr. Marion Waller and others, that some of the sera
with prozones in the anti-Rh (Gm) system actually exhibit isospecificity. Thi;
would say that the y-globulin which should be there to inhibit and thereby
to induce the prozone is not there, or is not there in very large quantity.
DR.EPSTEIN:
The material that Dr. Vaughan spins off after dilution of the
serum theoretically should contain on dissociation the two constituents in
small quantity. Have you looked at the material spun off, dissociated and seen
its specificity?
DR.VAUGHAN:
We have not actually looked at them. We assumed that we
460
IMMUNOLOGIC ASPECTS OF RHEUMATOID ARTE-IRITIS AND SLE
(0.8 q h l )
Sheep c e l l s coated with IS I&%
or aggregated RGC (0.8 mglml)
Coated
with
Inhibitor
kCiDrOCa1
Conc.
lllglml
Sheep a n t i PCO-S-1228596
None
20
Of
dilutions of Anti BcG
5,120
80 160 320 640 1,280 2.560
40
10,240
~
7s RGG
None
7SRGG
- + u - I + c + t + * * * *
0.8
0.5
0.1
AgsRcG
Agg RGG
0.8
0.5
0.1
None
7SRGG
Ag*m
0.8
0.5
0.1
0.8
0.5
0.1
--- ** *
*
*
- - -
+6
-
-
+ + + + *
*
*
*
+
f
.
--
*
*
- + k + + i + * * * * *
*
--
*
*
-- *
++
*
-*
++
*
-
*
-
*
-
+t
++
+t
f+
--
-
-- ++
+c + + + + * f f + + f + + + i + + b
- * * * * * * *
+c ++ i+i+
- * i + * + * i + i + + t
20,480
-~
-
+t
* * * * * ++
*
-- -- -- -- - - - - -
Fig. 13.--Inhibition of sheep anC-rabbit-y-globuliii by 7s rabbit 7-globulin and
aggregated rabbit y-globulin.
would find, much as you did, that there was agglutinating activity there. This
is something we should do with the various types of specificities.
DR.WITEBSKY:
We always refer to an antibody against a protein of a foreign
species as “heteroantibody.”So, we would call the antibcdy effective against
rabbit 7-globulin a heteroantibody and we would use the word isoantibody to
denote an antibody against human 7-globulin.
DR. VAUCHAN:
This is a very difficult problem of terminology. Indeed, is
this an antibody to denatured autogenous 7-globulin?
DR.WITEBSKY:
From our point of view and I believe from the general immunologist’s point of view, an antibody found in human serum against rabbit
protein is a “heteroantibody”while an antibody against protein from different
human individuals, but not from the antibody-producer, is an isoantibody.
Only the antibody active against the protein of the individual himself would
be called autoantibody. Let us at least agree on that basic concept of terminology; otherwise we will never understand each other.
DR. VAUGHAN:
This is acceptable except for one possibility. Did you mean
by heteroantibody antibody that is evolved from being immunized with a
heterologous antigen? This is a problem. If we have antibody activity against
rabbit 7-globulin, does it mean we have got rabbit 7-globulin in us as an antigen?
DR. MILGROM:Antibody is defined as a serologic reaction evolved by immunizing stimulus.
DR. BENACERRAF:I share your view on this, Dr. Vaughan. Usually the
customary thing considered in specificity of the antibody is “what is the
immunologic stimulus?” The cross-reactivity is what you see in vitro according to what you add to your antibody, but the total specificity is what the
461
SPECIFICITY OF RHEUMATOID FACTOR FOR OTHER GG
Sheep c e l l s coated with rabbit A 9 anti-SC serum
Inhibitor
Conc.
rnglrnl
-
None
7s RGG
1.0
0.01
Agg RGG
1.0
0.01
Normal R serum
1:lO
1:LOOO
7s HGG
1.0
0.01
Agg
=
1.0
0.01
Normal H serum 1:lO
1:looo
* All
(-) a t
28
None
-
-----
+
f
D i 1 u t i o n J o r.
56 112 224 448 896
c
*
- * *
- . ,
*
f
*
-
*
-
*
+
- - - - * * + -
#
* * *
.
-
-
3500
+*
*
-
* * - I + *
* * *
- * * *
- - * + +*
*
* * +
*-I+
t
-
1792
-
-
+
- - - -
* - - * * - -
+
-
-*
-
+ * * * *
- - - *
- ** ** +* * * * -
-
1:7000
Fig. 14.-Agglutination of sensitized sheep cells by Jor.Inhibition by human and
rabbit y-globulin.
antibodies have been designed for by the immunized animal. The trouble in
this situation is that one assumes that this is some form of denatured autologous 7-globulin; since you don’t know what it is. That gives rise to this
degree of confusion.
DR. HEIMER:I have great difficulty understanding one aspect of Dr. Milgrom’s experiment which deals with the dispersion of the mixed agglutinate
from alligator-rabbit sensitized cell and the rabbit-rabbit sensitized cell on
adding human 7-globulin to it. If you add sufficient amounts of human
7-globulin to this mixed agglutinate you generally should get a dispersion
because, if you reverse the procedure and absorb out the serum first with
human 7-globulin, you would not get a reaction with any of the rabbit systems.
DR. MILGROM:You are right. However, these experiments are mutally controlled in a quantitative way. One per cent human y-globulin does not disperse agglutinates of red blood cells sensitized by rabbit antibody and the
rabbit 7-globulin will disperse these agglutinates not only at a 1 per cent
concentration but also when much more diluted. For the agglutinates of red
blood cells sensitized by human antibodies, the reverse is true. Admittedly if
one used very concentrated human 7-globulin, it would also disperse the agglutinates of red blood cells sensitized by rabbit antibodies but I don’t think
that this is the purpose of experiments of this type.
DR.VAUGHAN:
I think it would be very interesting if you would denature
some of your human 7-globulin and use it in the normal quantities to see
whether this now would disperse.
DR. MILGROM:On the basis of your data I am almost positive that it would
produce the dispersion.
462
IMMUNOLOGIC ASPECTS OF RHEUMATOID ARTHRITIS A N ) SLE
DR. WILLIAMS:
We have been interested in a little different approach to
this same problem. We have utilized polyamino-polystyrene particles diazotized to different forms of 7-globulin after the methodology developed by
Gyenes and Sehon.lo
When one takes polyamino-polystyrenecolumns the particles of which have
been diazotized to human F-I1 (the total column containing probably 120 mg.
of human F-11), and then passes 16 cc. of sheep cell-absorbed and heat-inactivated rheumatoid serum over the column, one notices that the early effluent
material, which is pretty much like serum, has retained sensitized sheep cell
activity but completely lost latex activity. Thereafter, fractions in the effluent
column retain both sheep cell and latex activity.
The material which has been adsorbed on the column in this rather complicated form of adsorption is then eluted with reagents that are known to
dissociate antigen-antibody complexes such as 6M urea or acid buffers at pH
3.5 or 4.5 and immediately dialyzed back to normality against buffered saline.
In the eluted fractions one gets some relative concentration of rheumatoid
factors. In this particular instance the material that was eluted from the
column had a very high titer in the latex test but also a relatively high titer
in the sheep cell reaction.
The interesting finding in this column work which is a rather crude preparative method in contradistinction to the exquisite method previously described
by Dr. Milgrom is that the material which is eluted from the column is both
7s and 19s y-globulin on immunoelectrophoresis. There are no other serum
proteins noted. The finding that is still unexplained is what the 7s material
constitutes.
When the same serum was run over two different types of antigen column
diazotized to polyamino-polystyrene particles-rabbit 7-globulin and human
aggregates-different patterns were obtained. When the serum was passed
over a rabbit 7-globulin column where 120 mg. of rabbit F-I1 had been
diazotized to the polyamino-polystyrene particles, there was a striking ahsence of sheep cell activity in the first five fractions, whereas latex activity in
titers of 1:10,240 was still retained. It was only until the sixth fraction that
minute amounts of sheep cell activity did appear in the effluent.
When this material was eluted from the column with 6M urea and dialized
immediately to get rid of the urea, tests in the sensitized sheep cell reaction
showed titers of 1:2O,OOOin the sheep cell reaction and very low titers ( I:%
1:80)in the latex fixation test. Again, this material was both 7s and 19s on
immunoelectrophoresis and on analytic ultracentrifugation about 60 per
cent was 19s material and 40 per cent was 7s material.
The same serum was then passed over an aggregated human 7-globulin
column and there was a striking absence of reactivity to human 7-globulin in
the effluent material up t o about the sixth fraction where sheep cell-like activity
was retained to titers of 1:2,560 and the latex activity was completely void
in the first six fractions. These first fractions of the column effluent were
similar to human serum in terms of protein concentration and patterns on
immunoelectrophoresis.
SPECIFICITY OF RHEUMATOID FACTOR FOR OTHER GG
463
This then is a demonstration of the possible application of such antigen
columns to the problem of trying to separate out the specific reactivity of
rheumatoid factors, some of which may react with only human 7-globulin or
only rabbit 7-globulin. However, the majority of rheumatoid factors isolated
by these procedures reacted with both human and rabbit 7-globulins.
DR. CHRISTIAN:
Regarding the fractions that had high sheep cell activity
and very low latex titers, were other systems utilizing human 7-globulin
comparable to the latex tests?
DR.WILLIAMS:
Yes, the latex agglutination followed most closely the anti-Rh
coat testing in the ‘effluentsor the material eluted. One thing that concerned us
was that when we diazotized an antigen-like 7-globulin to columns, how much
of the diazotized material would possibly be eluted with such things as 6M
urea. So we tested this by taking some of Dr. Harboe’s serum, kindly lent by
him, which was Gm( a+b- ). This was passed over a human aggregate column.
If any of the aggregates were eluted by the 6M urea, the effluents should be
Gm(a+b+), but instead effluents retained the specific Gm activity of the
testing system.
We are fairly sure at this time that the material which we elute from the
column is not what we diazotized to it originally.
Have you tested for 7s rheumatoid factors with density gradDR. EPSTEIN:
ients?
DR.WILLIAMS:
No,these experiments have not yet been done. We do know
that in every experiment to date the ubiquitous 7s 7-globulin appears. It does
not make any digerenee which high titer rheumatoid serum we test. We
thought perhaps in the 19s rheumatoid factor reaction with the diazotized
7-globulin that somehow 7s may be trapped as a complex and comes off at thc
same time as the eluting material is applied. The other possibility is that this
is active 7s rheumatoid factor or anti-7-globulin antibody.
DR. FUDENBERG:
May I suggest that you use a control serum other than
Dr. Harboe’s. The data in the literature indicate that Gm(b-) does not
become Gm(b+ ) upon degradation; rather, Gm( b+ ) becomes Gm( b- ).
Gm(a+) does not lose it. So, if you wanted a G m ( a - b f ) put on, then aggregated by urea and come off, you would have Gm ( a+ ) rather than Gm (a- )
and Gm(b-) rather than Gm(b+).
DR. HARBOE:
The key question here is whether some of the aggregates get
through on elution. The material used to diazotize on the column was Gm( b+ )
and what got through the column was Gm( b- ).
DR. MZLGROM:
In 1956 we published a paper on an agglutinating factor
found in 0.5 per cent of normal and pathologic human sera.ll We called it
anti-antibody because it specifically combined with the antibody portion of
the antigen-antibody complex.
I was recently able to dernonstratel2that sera of 2 to 3 per cent of apparently normal rabbits contain a factor with serologic properties that are very similar
to those of the factor previously demonstrated in human sera. The most convenient way to detect rabbit anti-antibody was the agglutination of rabbit
blood group G red blood cells that had been sensitized by incomplete anti-G
464
IMMUNOLOGIC ASPECTS OF RHEUMATOID ARTHRITIS AND SLE
Table 3.-Znhibition of Agglutinating Activity of Rabbit Sera Containing (A)
Anti-Antibody and ( B ) Serum Group RGGZZ Zsoantibody
Inhibiting
specimen
Agglutination of blood group G erythrocytea (sensitized by incomplete
anti-G serum #log?) after addition of 10 agglutinating units d A or
B mixed with the inhibiting specimen at a dilution of 1 to:
1
3
___-___---___--
++
-
++-I-
9
+++
-
27
81
+-I-+
243
+++
+++
-
729
-I-++
2187
++-I-
4%F~tionII
of pooled
rabbit serum
A
B
RabbitRGGI
serum
A
RabbitRGGII
serum
A
B
Pre-immune
sample of
serum #lo97
A
B
+++ -I-++ -H-+ +-kk -I-++ +-k-k
+++ +++ +++ +++ +++ +++ +++
+++
f4-f
+++
+f+ +f+ S-f-k
- ++-I- +f+
- +++
ff+ +++ +++ +++ fS.+ -H--k
+++
+++
Saline
A
B
+++
+++
B
-
-
-
+U
-t++ f f - k
serum of rabbit origin. The same indicator system was also agglutinated by
rabbit antisera containing serum group antibodies. This gave us the possibility
of comparing the serologic properties of anti-antibody on the one hand and
serum group isoantibodies on the other.
We had antisera against serum group antigens available which were kindly
supplied by Dr. Dray. They were anti-RGGI and anti-RGGII sera.13 Only
the anti-RGGII serum agglutinated the red blood cells sensitized by the anti-G
serum under investigation. Obviously the latter antiserum was of RGGII
serum group, which could be confirmed by double diffusion gel precipitation
procedures.
In table 3 an inhibition experiment is presented. Anti-antibody containing
serum and the anti-RGGII serum were used at concentrations corresponding
to 10 agglutinating units. Various specimens were tested for their activity
in inhibition of the agglutination of sensitized blood group G erythrocytes.
F-I1 of pooled rabbit serum did not inhibit anti-antibody but it strongly
inhibited the anti-RGGII serum. Normal rabbit Serum of RGGI serum group
did not inhibit any of them. Rabbit serum of RGGII group did not inhibit
anti-antibody but it strongly inhibited the anti-RGGII serum. We also tested
a pre-immune serum sample obtained from the same rabbit which, after
proper immunization, served as a source of anti-G serum used in this experiment. As is to be seen, this serum sample did not inhibit the anti-antibody but
it strongly inhibited the serum group isoantibodies.
In table 4 an experiment is presented in which the anti-antibody-containing
serum was mixed with anti-G serum. Both sera were used at various increasing
dilutions and the experiment was performed as a box titration. The mixture
of the two sera was left for several hours and then a suspension of blood group
G erythrocytes was added. As is to be seen, the erythrocytes were strongly agglutinated; the mixed serum behaved as if it contained complete G antibodies.
An experiment which was performed under identical conditions but in which
465
SPECIFICITY OF RHEUMATOID FACTOR FOR OTHER GG
Table I.-AggEutination of Rabbit Blood Group G Erythrocytes by a
Mixture of Anti-Antibodg and Anti-G
Dilutions of
anti-antibody
containing
serum 1 to:
Dilutions of G antiserum 1 to:
10
128
+++
+++
+++
+++
+++
+++
++
+
Saline
Zk
1
2
4
8
I6
32
64
80
40
20
+++
+++
+++
+++
+++
++
+-
+++
+++
+++
+++
++
+
saline
160
+
+
+-
+
Table 5.-Absorption of Anti-Antibody Containing Serum by Immune Agglutinates
Agglutination of rabbit blood group G erythrocyten
(sensitized by incomplete anti-G serum)
at serum XU01 dilutions of 1:
Serum P1201 absorbed with
sheep red blood cells
2
Agglutinated by rabbit
anti-sheep erythroqteserurnatadilution of 1 :
76
160
300
600
Agelutinatedbyhuman
infectious mononucleosis serum at
dilution of 1 :
10
Nonagglutinatsd
20
-
4
_____--
-
+
+
+++ ++
fff
-kff
+++ f f - t
+++ +++
8
''
++
16
-
++
+f+ +++
64
128
-
+
*
-+
$+
-t
rl:
32
-
-
--
+++ +++ f+ + *
+++ +++ ++ ++ +
anti-RGCII serum was used instead of anti-antibody gave completely negative
results.
The findings presented indicate that anti-antibody combined with a different
antigenic determinant of the G antibody than the serum group antibody did.
The latter antibody acted upon a serum group antigen RGGII which is also
present in some whole rabbit sera and in the 7-globulin fraction of pooled rabbit serum. In contrast, anti-antibody acted upon the antigenic structure that
is revealed on the antibody molecule during its reaction with the antigen and
which resulted from the transformation of this molecule.
The only way to remove anti-antibody from the serum was by absorption
with an immune complex containing rabbit antibody. In the experiment presented in table 5, anti-antibodywas absorbed by sheep red blood cells agglutinated by the corresponding rabbit antiserum. However, the same sample of
sheep red blood cells was incapable of removing anti-antibody if it was agglutinated by human infectious mononucleosis sera.
We also obtained positive absorption experiments using other agglutinates,
e.g., Proteus bacilli agglutinated by rabbit anti-Proteus serum. Similarly, immune precipitates consisting of any antigen and the corresponding antibody
of rabbit origin were capable of absorbing anti-antibody.
466
IMMUNOLOGIC ASPECTS OF RHEUMATOID ARTHRITIS AND SLE
Rabbit anti-antibody may be considered as a quite specific reagent capable
of distinguishing between rabbit immunoglobulin that suffered denaturation
in the serologic reaction and normal or immune rabbit 7-globulin as it appears
free in serum or in the solution of the chemically isolated fraction. Similarly,
as the affinity of human anti-antibody was restricted to the transformed human
7-globulin, the affinity of rabbit anti-antibodywas restricted to the transformed
rabbit immunoglobulin.
DR. HARBOE:
We have found that the identical substance (anti-antibody)
in human serum is 19S, and it reacts strongly with the B piece of the antibody
on the cells because if one coats cells with pepsin there is no longer agglutination of this kind.
REFERENCES
1. Butler, V. P., Jr., Gleich, G. J., and
Vaughan, J. H.: An immunochemical
basis for the cross-reactivity of rheumatoid factor with animal gamma
globulin. Arth. & Rheumat. 5:104,
1962.
2. LoSpalluto, J., and Ziff, M.: Chromatographic studies of the rheumatoid
factor. J. Exper. Med. 110:169, 1959.
3. Heimer, R., and Schwartz, E. R.: ISOlation of rheumatoid factors. Arth. &
Rheumat. 4:153, 1961.
4. Milgrom, F., Witebsky, E., Goldstein,
R., and Loza, U.: Studies on the
rheumatoid and related serum factors.
11. Relation of anti-human and antirabbit gamma globulin factors in
rheumatoid arthritis sera. J.A.M.A.
181:476, 1962.
5. Cohen, E., Neter, E., Mink, I., and Norcross, R. M.: Use of alligator erythrocytes for demonstrating agglutination
activating factor in rheumatoid arthritis. Am. J . Clin. Path. 30:32,
1958.
6. Milgrom, F., Witebsky, E., Abeyounis,
C . J., and Wilson, D. M.: Red blood
cells sensitized by incomplete rabbit
antibodies as a reagent for detecting
the rheumatoid factor. Acta rheumat.
scandinav. In press.
7. Kellner, A., and Hedal, E.: Experimental erythroblastosis fetalk in rabbits. I. Characterization of a pair of
nllelic blood group factors and their
specific immune isoantibodies. J. Exper. Med. 97:33, 1953.
8. Waller, M. V., and Vaughan, J. H.
Z7se of anti-Rh sera for demonstrating
agglutination activating factor in
rheumatoid arthritis. Proc. SOC. Exper. Biol. & Med. 92:198, 1956.
9. Fudenberg, H. H., and Kunkel, H. G.:
Specificity of the reaction between
rheumatoid factors and gamma globulin. J. Exper. Med. 114:257, 1961.
Prepara10. Gyenes, L., and Sehon, A .H.:
tion and evalutation of polystyrene
antigen conjugates for the isolation
of antibodies. Canad. J. Biochem. &
Physiol. 38: 1235, 1960.
11. Milgrom, F., Dubiski, S., and Womiczko, G.: Human sera with “antiantibody.” Vox Sang. 1: 172, 1956,
12. -: Rabbit sera with “anti-antibody.”
Vox Sang. 7:545, 1962.
13. Dray, S., and Young, G.: Differences in
the antigenic components of sera of
individual rabbits as shown by induced isoprecipitins. J. Immunol. 81:
142, 1958.
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