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Reactions of Aggregated Mercaptoethanol Treated Gamma Globulin with Rheumatoid Factor Precipitin and Complement Fixation Studies.

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Arthritis and Rheumatism
O&ial Journal
4 the gmerican Rheumatismassociation
VOL. XI, NO. 4
AUGUST, 1968
Reactions of Aggregated Mercaptoethanol Treated
Gamma Globulin with Rheumatoid Factor Precipitin
and Complement Fixation Studies
-
By NATHAN
J. ZVAIFLER
AND PETER
SCHUR
Reduction and alkylation of human gamma globulin prior to aggregation results
in a loss of complement-fixing ability,
and retention of the capacity to react
with rheumatoid factor. The addition
of aggregated, reduced gamma globulin
to serum containing high titers of
rheumatoid factor produces a typical
bell-shaped precipitin curve with an
equivalence point and little precipitate
in the region of antigen excess. Since
aggregated, reduced gamma globulin
fixes little complement by itself, it was
possible to determine whether its reaction with rheumatoid factor fixes com-
I
plement. Under appropriate conditions
rheumatoid factor can fix complement.
When aggregated, reduced gamma globulin was added to heat-inactivated sera
with varying titers of rheumatoid f a e
tor, no fixation of guinea pig or human
complement was observed. But preformed complexes of rheumatoid factor
and gamma globulin will fix human complement at 37 C. Intradermal injection
of 40 pg N of aggregated, r e d u d gamma globulin into control subjects or patients with rheumatoid arthritis produced no immediate or delayed skin
reactions.
of the structure of gamma
globulin have defined 2 distinct portions of the molecule: one that contains the
antibody combining site, and the other responsible for a variety of biologic properties. These properties include the ability to
fix to skin and give passive cutaneous anaphylaxis, the capacity to cross the placental
membrane, reactivity with rheumatoid factors, and complement fixation.1 The struc-
tures essential for the induction of biologic
activities also appear to be dependent
on disulfide linkages, since treatment with
sulfhydryl reducing agents followed by alkylation results in a loss of many of these
properties. Wiedermann, Miescher and
Franklin observed that when 7s human
antibodies or heat-aggregated human gamma globulin were reduced with 2-mercaptoethanol, they lost the ability to fix com-
Supported in part (N.J.Z.) b y grants from the
Public Health Service (AM45042 and AM-05140).
NATHANJ. ZVAIEZER,M.D.: Associate Professor
of Medicine, Director, Division of Rheumatic Diseases, Georgetown Unioersity Medical Center,
Washington, D . C . 20007.PETERH. SCWR, M.D.:
Associate in Medicine, Harvard Medical School,
Robert B . Brigham Hospital, Boston, Massochusetts 02120.
NVESTIGATIONS
ARTHRITISAND
~ E U M A T I S M , VOL.11,
No. 4 (AUGUST1968)
523
524
ZVAIFLER AND S C H U R
plement while retaining the capacity to
react with rheumatoid factors., This dissociation of these 2 biologic properties is of
interest, for a variety of studies have examined the interrelationships of rheumatoid
factor, antigen antibody complexes (or aggregated gamma globulin) and complement. Most investigations have shown that
rheumatoid factor inhibits complement fixation by antigen antibody comple~es.~-7
It
has also been inferred that the reaction of
rheumatoid factor with aggregated gamma
globulin is not complement fixing, but direct testing of this hypothesis has not been
possible because aggregated gamma globulin is, of itself, too anticomplementary. The
availability of a reagent, aggregated mercaptoethanol treated gamma globulin,
which does not fix complement, but does
react with rheumatoid factor, provides an
opportunity to study this question.
MATERIALSAND METHODS
Human gamma globulin-Cohn fraction I1 was
obtained from the American Red Cross. 2-mercaptoethanol (2ME) in a final concentration of 0.1
M was added to a solution containing 10 mgm./
ml. of human fraction I1 in phosphate buffered
saline (0.15 M, pH 7.3). After incubation for 3
hours at room temperature, the reduced gamma
globulin solution was dialyzed for 4 hours against
large volumes of 0.02 M iodoacetamide, and then
overnight in the cold against several changes of
buffered saline. In some experiments sulfhydryl
reduction was obtained by dialyzing the gamma
globulin preparation against an excess of 0.1 M
2-mercaptoethanol for 3 hours at room temperature. The subsequent steps were the same. Aggregation of the gamma globulin preparations was
produced by heating at 63 C for 15 to 20 minutes.
The SS, and SS, precipitates were prepared according to the method of Christian.8 Fifty ml. of
a 1 per cent solution of human gamma globulin
was reduced with 0.1 M 2-mercaptoethanol and
then heat-aggregated. Ten ml. of 2.18 M sodium
sulfate was added. The precipitate which formed
(SS,) was removed by centrifugation. An additional 10 ml. of 2.18 M sodium sulfate was added
to the supernatant and a second precipitate
formed (SS,). The two precipitates (SS, and SS,)
were combined and dissolved in 10 ml. of buffer
saline.
Precipitin reactions were performed as described by Vaughan.9 Increasing concentrations
of gamma globulin solution were introduced into
ice-cold phosphate buffered saline (0.15 M, pH
7.2) and then 0.5 ml. of a serum with a high titer
of rheumatoid factor was added. The final reaction volume was 5.0 ml. The reaction was allowed
to proceed at room temperature for 3 hours, and
then at 4 C for 7 days. The tubes were spun at
2,000 RPM for 60 minutes in PR2 International
refrigerated centrifuge. The supernatant was decanted and analyzed for rheumatoid factor. The
precipitates were washed twice with ice cold buffered saline, packed by centrifugation and analyzed for their nitrogen content by the micro
Kjeldahl method.10 Rheumatoid factor was measured by the bentonite flocculation test.11 A single
serum containing a high titer (1:4096) of rheumatoid factor was utilized for the precipitin studies.
Precipitates of rheumatoid factor and reduced
aggregated gamma globulin were made as follows:
a pool of serum containing high titers of rheumatoid factor was inactivated at 56 C for 30 minutes, centrifuged at 2,000 RPM in a refrigerated
centrifuge, and then 7.5 ml. was added to 27.5
ml. of iced buffered saline containing 0.01 M
EDTA. Subsequently 15 ml. of reduced, aggregated FII (10 mgm./ml.) was added dropwise.
These proportions were chosen to be at or near
equivalence. The mixture was kept at room temperature for one hour and then at 4 C for 72
hours. The precipitates which formed were
washed 3 times with phosphate saline and then
suspended at the desired protein concentration.
These are referred to in the text as rheumatoid
factor-reduced gamma globulin complexes or preformed precipitates.
Quantitative complement fixation was performed by the method of Barbaro and Becker.12
Hemolysis was measured spectrophotometrically.
Guinea pig complement containing approximately
100 C'H,, units per 0.1 ml. was added to iced
test tubes containing 0.25 ml. of heat-aggregated
mercaptoethanol gamma globulin (10 mgm./ml.)
and 0.25 ml. of heat-inactivated human serum
containing rheumatoid factor. Sufficient triethanol
amine buffered saline ( T B S ) containing 0.1 per
cent gelatin was added to bring the total volume
to 1.0 ml. The tubes were incubated at 37 C for
80 minutes, then 19 ml. of ice cold TBS was
added, and they were centrifuged in the cold at
2,000 RPM for 15 minutes. Residual complement
was determined in the supernatants. Controls of
REACXIONS OF
525
TREATED GAMMA GLOBULW WITH RF
*...............a
A YC
t
R.F. Serum
A ZMEYC t R.F. Serum
Total
pgN ppt10.5 ml
I
I
I
I
I
I
I
1
2
3
4
5
6
7
MJ YGlobulin N added10.5 ml
Fig. 1.-The
globulin
(a2ME
effect of increasing concentrations of aggregated human gamma
(A 6)
or
+)
aggregated mercaptoethanol-treated human gamma globulin
on precipitable N from 0.5 ml. of serum containing rheumatoid
factor.
complement alone, complement and rheumatoid
factor serum, and complement and aggregated
mercaptoethanol gamma globulin were included
with each experiment.
In experiments with preformed precipitates
0.25 ml. of the rheumatoid factor complexes was
incubated with 1.75 ml. of fresh human serum at
37 C for 2 hours or overnight at 4 C. Controls
included 0.25 ml. of reduced, aggregated FII of
an equal protein concentration or 0.25 ml. of saline handled in identical fashion, In some experiments 1.75 ml. of guinea pig serum was substituted
as a complement source. Following incubation the
mixtures were centrifuged in the cold and the
supernatants analyzed for residual total hemolytic
complement and residual activity of the second
and third components of complement. C'2hu
was measured by the method of Austenl3 and C'3
by immune adherence14 and conversion of B,C
to B,A globulin by micro-immunoelectrophoresisla against an anti human B,C/BIA globulin
antiserum prepared in rabbits by the zymosan
adsorption method of Mardiney and Muller-Eberhard.16 The per cent complement fixed (%C
fixed) was calculated as:
C'H,, units fixed
x loo
total CH,,
526
ZVAIFLW AND S C R V R
0.-
........+
A7C +
-
m... ........m
*.........
d
A
R.F. Serum
rC + A R J . Serum
AtC
+ R.F. Serum + EDTA
A 2ME% + R.F. Serum
r2NYrC + AR.F. Serum
A~METG+ R.F. Serum + EDTA
175.
150,
Tdal
pgN ppU0.5 ml
125
la,
15
50
25
I
1
I
I
3
2
I
4
I
5
I
6
1
7
Mg rClabulin N ddedn.5 ml
Fig. 2.-The effect on the amount of nitrogen precipitated from rheumatoid
serum produced by preheating rheumatoid serum at 56 C. for 30 minutes or the
addition of 0.01M EDTA to rheumatoid factor serum prior to reaction with
aggregated gamma globulin (A yC) or aggregated mercaitoethanol treated gamma globulin. (A 2ME +).
RESULTS
Precipitin reaction. The precipitin curve
obtained when increasing concentrations
of aggregated gamma globulin were added
to a serum containing a high titer of rheumatoid factor is shown by the dotted lines
in Fig. 1. There is a slow rise in precipitable nitrogen, followed by a long flat zone
which lacks a clear-cut equivalence or solu-
bilization of the precipitate in the region of
antigen excess. The curve obtained with
aggregated mercaptoethanol gamma globulin differs in several particulars. The total
nitrogen precipitated is less, but there are
well-defined zones of equivalence and antigen excess. Tests for rheumatoid factor
in the supernatants showed activity up to
the points designated by the arrows; be-
527
REACJXONS OF TREATED G A M M A GLOBULIN WlTH RF
-A
200
2ME7G
f
R.F. Serum
- - 4 A 2ME SS, - SS2
+
R.F. Serum
175
150
125
I
I
Total
I
WN ppt10.5 ml
n
t
'
'
100
15
50
25
I
1
1
2
I
3
1
4
I
5
i
;
Mg7Globulin N added10.5 mi
Fig. 3.-Comparison of the amount of N precipitated from 0.5 ml. of serum containing rheumatoid factor by aggregated mercaptoethanol treated human gamma
globulin (A 2ME yC) and by sodium sulfate fractions from aggregated mercaptoethanol treated human gamma globulin (A 2ME SS1 - SS2).
yond this there was complete neutralization of rheumatoid agglutinating activity
in both systems.
An analysis of the contribution of complement or components of complement to
the precipitable nitrogen is shown in Fig.
2. Precipitin reactions were performed as
described, except that in one experiment
the serum containing rheumatoid factor
was heated at 56 C. for 30 minutes prior
to reacting with the aggregated gamma
globulin or to the mercaptoethanol treated
gamma globulin. In another experiment
the reaction was performed in the pres-
ence of 0.01 M EDTA. Heat inactivation
and EDTA treatment results in a decrease
in the amount of nitrogen precipitated in
the reaction with aggregated gamma globulin, but there is a similar decrease with
the aggregated mercaptoethanol-treated
gamma globulin. In spite of these decreases
there is no significant change in the overall codguration of the 2 precipitin curves.
The precipitin reaction of aggregated
mercaptoethanol treated SS1 and S S 2 fractions of human gamma globulin with rheumatoid factor serum is shown in Fig. 3.
The shape of the precipitin curve obtained
528
ZVAIFLER AND S c E U R
90 -
100
80 70
o
x
L
-
60-
50 -
U
P
40-
30 20
-
10
I
resembles that with the aggregated mercaptoethanol gamma globulin, and is not
like the broad flat curve seen in the reactions with aggregated gamma globulin.
There is an abrupt rise in precipitable nitrogen, a narrow zone of equivalence and
a well-defined solubilization of antigen in
the region of antigen excess.
Complement Fixation
A comparison of the complement fixing
ability of aggregated gamma globulin and
aggregated mercaptoethanol-treated gamma globulin is shown in Fig. 4. As little
as 100 pgm. of aggregated gamma globulin
fixes 50 per cent of the available guinea
pig complement, while 10 mgm. of aggregated mercaptoethanol gamma globulin is
unable to give a simliar degree of complement fixation. The results were similar with
fresh human serum as the complement
source.
The results of 8 experiments analyzing
complement fixation by the reaction of
rheumatoid factor with aggregated reduced
gamma globulin are summarized in Fig. 5.
1
Each experiment was performed with a
sera of a different rheumatoid factor titer.
Column I, in each instance, shows the residual complement after 60 minutes of incubation with only TBS. Column I1 is the
complement left after the reaction of serum
containing rheumatoid factor and aggregated mercaptoethanol gamma globulin.
Column I11 shows the residual complement
with rheumatoid factor serum alone, and
IV the residual complement after reaction
with only aggregated mercaptoethanol
gamma globulin. The titer of rheumatoid
factor for the individual sera is recorded
beneath. There is no significant difference
in the amount of residual complement in
Column 111-rheumatoid factor serum plus
complement, when compared with Column
11-rheumatoid factor serum and aggregated mercaptoethanol gamma globulin
plus complement. Altogether 18 sera with
varying titers of rheumatoid factor were
analyzed; no evidence for complement fixation could be demonstrated. An additional
6 sera were examined comparing human
complement (approximately 50 C’HSo
REACITONS OF
529
TREATED G A M M A GLOBULIN M"H RF
64
256
4096
4096
C'H%
32
512
1024
4096
Fig. 5.-The residual guinea pig C'H,, in experiments with 8 different human
sera containing rheumatoid factor: I-Complement alone; II-cmmplement, serum
and aggregated mercaptoethanol treated gamma globulin; III-complement and
serum; IV-complement and mercaptoethanol treated gamma globulin. The number
beneath each experiment is the rheumatoid factor titer of the serum tested.
units) with guinea pig complement; no
complement fixation was noted with either.
In all of the experiments performed the
residual complement in the presence of
heat inactivated rheumatoid factor serum
(Column 111) was equal to, or greater
than, the complement remaining after incubation with only TBS gelatin. The reason
for the apparent protective effect of rheumatoid serum is analyzed in Fig. 6. I n
3 separate experiments, complement was
incubated at 37 C. with TBS-gelatin, or
in the presence of 6 normal sera or one
serum with a high titer of rheumatoid factor. The residual complement was measured at 0, 30 and 60 minutes (only 0 and
60 minutes are shown). The inactivation
of complement in TBS-gelatin was 13 per
cent of the initial value: 6 per cent in the
presence of normal serum and 7 per cent
for the serum containing rheumatoid fac-
tor. Thus, the protection noted at the end
of 60 minutes of incubation reflects the
ability of serum to prevent the deterioration of complement, and is not unique to
serum containing rheumatoid factor.
Complexes of rheumatoid factor and aggregated, reduced gamma globulin were
incubated with fresh human serum at 37 C.
for 2 hours, or overnight at 4 C., and the
residual complement determined. This was
compared with an equal concentration of
aggregated, reduced gamma globulin
alone. The results are shown in Table 1.
At 4 C. there is very little complement
fixed by either the preformed complexes
or the aggregated, reduced gamma globulin; but at 37 C. the complex fkes 43 per
cent of the available complement. The
complement fixation must be due to the
complex, and not the reduced gamma globulin in the complex, for if all the protein
530
ZVAIFLER AND SCHUR
- 0 c
0-
C +Serum
.-e
_-___..
A C
R.F. (40961Serum
loo Q
+
\
\
95
\
%
\
\-
'\
90
CH50
85
m
rs
MINUTES
effect of heat inactivated normal human serum (C' + serum), heat
inactivated human serum containing rheumatoid factor, (C' + R.F. 4096 serum)
or triethanohmine buffered saline with 0.1%gelatin (C') on the loss of guinea pig
complement activity during incubation at 37 C. for 60 minutes.
Fig. &-The
in the complex were derived from aggrea
otnrl
6 " C V U '
rJta.-J 6
nnmmn
"""""
A V U U - U
rrlnha-lin ;
t xV w
w~lA
&L
V U U l U
6 A " " U U
only fix 3 per cent of the available complement. Neither the aggregated, reduced
gamma globulin, nor the complexes fix
significant amounts of guinea pig complement whether tested at 4 C. or at 37 C.
(Table 2). The effect of a change in concentration of the complexes on the amount
of complement fixed is shown in Fig. 7.
Increasing the concentration of complex
75
" minmnrsmr
6'""'" tn
c u R R mnm n F nrntnin
6- u- r--results in increasing complement fixation.
2 mgm. of preformed complex fixes about
50 per cent of the available complement
and 8 mgm. fixes more than 90 per cent.
Analysis of the second and third components of complement was performed to see
if the rheumatoid factor-reduced gamma
globulin complexes fix complement in the
Frnm
llUlll
I
A l l A U l U
V."
1-1
531
REACTIONS OF TREA'ITD GAMMA GLOBULIN Wl'lX RF
10D-1
-
1
1000
100
10
10.000
vgms Protein
Fig. 7.-The effect of increasing concentrations of rheumatoid factor-reduced
gamma globulin complexes on ftration of human complement. Preformed complexes
were incubated with fresh human serum at 37 C. for 120 minutes.
Table l.-lncubatwn
of Aggregated, Reduced
Gamma Globulin and RF with Human Serum
Reagent
Residual C'&o % C' Fired
Human C'-S7"C for 2 hours:
RF
AME FII*
55
AME FII*
94
Saline
97
+
Human Ct-4'for 18 hours:
RF
AME FII*
107
AME FII*
95
+
Saline
104
*3.0 mgm. protein/ml.
43
3
0
8
-
@loConversion
+0
0
0
0
0
tration of aggregated, reduced gamma
globulin.
Skin Tests
The intravenous injection of aggregated
gamma globulin into experimental animals
has been shown to produce profound effects, kcluding lowering of serum complement, and the induction of vascular and
renal lesi0n.l7-~~Intradermal injection of
less than 1.0 microgram N of aggregated
human gamma globulin results in a marked
infiammatory response characterized by
usual manner. The results are shown in
Table 3. At a protein concentration of 75 Table 2.-lncubation of Aggregated, Reduced
pgm. the rheumatoid factor-reduced gamma Gamma Globulin and RF with Guinea Pig Serum
globulin complex fixed 35 per cent of the
Residual C'Hro 46 C' Fixed @lC Conversion
Reagent
total hemolytic complement and 33 per cent Guinea pig C'37"C for 2 hours:
of the available C'2, and reduced the im- RF + AME FII*
219
14
mune adherence titer by 50 per cent. When
AMEFII*
234
8
Saline
255
the protein concentration was increased to
6.1 mgm. the preformed complexes &ed 95 Guinea pi0 C'-4"C for 18 hours:
per cent of the total complement, 97 per RF + AME FII*
212
9
cent of the available C'2 and reduced the
AME FII*
203
13
Saline
234
immune adherence titer to 13 per cent of
the value obtained with a similar concen*3.0 mgm. protein/ml.
532
ZVAIFLER AND S C H U R
Table 3.-Fixation of Human Conlplement
Components (C2 and C 3 ) by Different
Concentrations of Rheumatoid FactorAggregated, Reduced Gamma Globulin
Complexes
Reagent
AMEFII
RF
- AMEFII
RF
- AME FII
AME FII
Protein
(mgm.)
0.75
0.75
6.1
6.1
C't
C't
Total
C'Hro
160
108
300
8
1600
800
1600
200
67
100
5
(34
erythema, edema and a wheal and flare
reaction which begins in several minutes
and reaches a peak intensity in 15 to 20
minutes.20~21
To assess the phlogistic properties of heat aggregated 2-mercaptoethan01 gamma globulin the following studies
were performed: human gamma globulin
(FII)was reduced and alkylated and then
passed through a 0.22 p millipore filter prior
to heat aggregation. The final protein concentration was adjusted to contain 15 to 40
micrograms N in 0.1 ml. This volume was
injected intradermally into 7 normal subjects or patients with non-rheumatic diseases. No inflammatory response was observed immediately or at 24 or 48 hours.
The same test was performed in 15 subjects with definite or classical rheumatoid
arthritis, 12 of whom had rheumatoid factor in their serum. No inflammatory response was noted in any of them.
Gamma globulin was isolated by DEAE
cellulose chromatography from the serum
of 5 patients with seropositive rheumatoid
arthritis. This was reduced and alkylated,
millipore filtered and then heat aggregated
at 63 C. for 15 minutes. The separated
gamma globulin was introduced intradermally into the subjects who supplied the
serum; this autologous mercaptoethanoltreated gamma globulin produced no immediate or delayed reactions.
DISCUSSION
The findings reported in this paper are
an extension of the studies of Wiedermann.
Miescher and Franklin who showed that
when 7s human antibodies or heat aggregated gamma globulin were reduced with
2-mercaptoethanol they lost their ability to
fix complement while retaining the capacity
to react with rheumatoid factor.
Their observations on the reaction of
mercaptoethanol treated heat aggregated
fraction I1 of human plasma with rheumatoid factor serum were made utilizing a
semiquantitative capillary tube precipitation method. As can be seen in Fig. 1,
when this reaction is analyzed by the classical precipitin reaction there are both quantitative and qualitative differences from the
results obtained with non-reduced gamma
globulin. The point of maximum precipitation on the precipitin curve with non-reduced gamma globulin shows no constant
relationship to the point of complete neutralization of agglutination, and there is not
a well-defined solubilization of the precipitates in the region of antigen
The
reaction of mercaptoethanol-treated, heataggregated gamma globulin is remarkable
because it does have the configuration of a
classical antigen-antibody reaction, with a
well-defined zone of equivalence and solubilization of the precipitate in the region of
antigen excess. Examination of the supernatants for residual rheumatoid agglutinating activity did not demonstrate this activity beyond the point of equivalence.
Several explanations for the differences
in the precipitin reaction were considered.
Since mercaptoethanol treatment interferes with complement fixation, the role of
complement was studied. Precipitin curves
performed in the presence of 0.01 M EDTA
showed a reduction of precipitate nitrogen
in the reactions with both reduced and nonreduced aggregated gamma globulin. Heating the serum containing rheumatoid factor to 56 C. prior to the addition of gamma
globulin resulted in a definite lowering of
REAClTONS OF TREATED GAMMA GLOBULIN WITH RF
precipitate nitrogen with both preparations.
The reduction of precipitable nitrogen by
both of these procedures suggests some
complement fixation in the reaction of rheumatoid factor with gamma globulin, but
cannot explain the differences in the curves
obtained with reduced and non-reduced
gamma globulin, because the overall configuration of the 2 precipitin curves remained the same.
Another possible effect of the mercaptoethanol treatment might be to make gamma
globulin less readily aggregatable and thus
provide less “reactant” gamma globulin to
combine with rheumatoid factor. This possibility was excluded by the studies in
which sodium sulfate fractions were prepared from mercaptoethanol treated gamma globulin. Christian has shown that
these fractions (SS1
SS2) contain the
majority of the reactant material capable
of combining with rheumatoid factor.* With
this more concentrated reactant gamma
globulin, the precipitin curve showed a
better defined zone of equivalence and solubilization of the precipitate in antigen
excess.
It is apparent that none of the studies
performed have elucidated the reason for
the differences between the reaction of reduced and non-reduced gamma globulin
with rheumatoid factor. It is possible that
all gamma globulin molecules are not reduced to a similar degree by mercaptoethanol treatment and that the observed
precipitin reaction is between rheumatoid
factor and a species of mercaptoethanolresistant gamma globulin. A more likeIy explanation is that the mercaptoethanoltreated gamma globulin complexes with
rheumatoid factor when aggregated, but
when non-aggregated is less able to solubilize these complexes in the zone of antigen excess.
A number of studies have analyzed the
influence of rheumatoid factor on comple-
+
533
ment fixation. Heimer and his associates
described an unusual macroglobulin in
rheumatoid serum which inhibited fixation
of guinea pig complement by rabbit antigen antibody complexes, but had no effect
on complement fixation by latex particles
coated with human FII. Isolated, purified
rheumatoid factor had the opposite effect,
that is, it inhibited complement fixation to
latex particles coated with FII, but not with
antigen antibody c o m p l e ~ e s . When
~ * ~ latex
particles coated with FII were incubated
with human or guinea pig serum prior to
mixing with dilutions of rheumatoid factor,
then a 3- to 4-tube decrease in agglutination titer was ob~erved.~
Zvaifler and Block
showed that serum containing rheumatoid
factor interfered with complement fixation
by both human and rabbit antigen-antibody
systems.6 Euglobulin preparations from
rheumatoid serum will protect a complement-sensitive enzyme system. Davis and
Bollet found that the degree of protection
paralleled the latex titer and was removed
by prior incubation of the euglobulin preparation with sensitized sheep cells.’ An interesting extension of these studies to an in
vivo situation showed that rheumatoid factor could prevent the lowering of serum
complement that accompanies the intraperitoneal injection of antigen antibody
complexes. This is presumably accomplished by trapping the complexes in the
extravascular space.23Thus, in all the studies reported, isolated rheumatoid factor,
rheumatoid euglobulin preparations, or
heat-inactivated serum containing rheumatoid factor has been shown to compete with
complement for binding sites on gamma
globulin. The ability of mercaptoethanol
reduction to impair complement fixation by
gamma globulin without inhibiting its combination with rheumatoid factor suggests
that the sites are separate but possibly
close together, and that rheumatoid factor
interferes by steric hinderance or by pro-
534
ducing conformational changes in the gamma globulin?
From all of these studies it has been inferred that the precipitation reaction between rheumatoid factor and aggregated
gamma globulin does not fix complement.
However, this has not been shown heretofore, because aggregated gamma globulin,
by itself, is so markedly anticomplementary.
The use of mercaptoethanol-treated gamma
globulin makes such an analysis possible.
The results obtained show that under appropriate conditions rheumatoid factor can
fix complement. When aggregated, reduced
gamma globulin is added to heat-inactivated human serum containing rheumatoid
factor, there is no fixation of either guinea
pig or human complement. But preformed
complexes of rheumatoid factor and reduced gamma globulin will fix human complement at 37 C. Several features of this
reaction are worthy of comment: 1) the
greater efficiency of preformed complexes;
2) the need for complement fixation to occur in the warm; and 3) a requirement for
human complement.
There is a tendency to assume that complement fixation is more efficient when
complement is present during the reaction
of antigen and antibody than when it is
added to preformed precipitate^.^^ While
this is true for rabbit antibody, it does not
hold for all antisera.12 Washed preformed
precipitates made with sheep or horse antibodies & complement better than those
aggregates that combine in the presence of
complement. In an investigation of complement hation by 19s antibodies it has
been shown that there are certain requiiements for the antigen. These include a
large molecular volume and critically
spaced repeating determinants on the antigenic molecule.25 It may be that in the
rheumatoid factor precipitates, the reduced gamma globulin assumes a more ap-
ZVAIFLER AND SCHUR
propriate form for initiating complement
hation.
The greater efficiency of complement
fixation at 4 C. is well recognized, but
there are exceptions. Stollar has shown
that rabbit and human 19s antibodies are
actually more efficient at 37 C. than at
4 C., which was never the case with 7s
antibodies.25 In studies with human complement this daerence might be exaggerated, since fixation of the first component
of human complement to antigen-antibody
complexes does not proceed well in the
cold.26 The finding that the rheumatoid
factor-reduced gamma globulin complex
fixes human complement in the warm but
not in the cold is consistent with these
observations.
The absolute requirement for human
complement, and the inability to fm guinea
pig complement, is most surprising, but not
without some precedent. In a study of the
ability of naturally occurring anti A and
anti B antibodies to fix complement, Ostgard found a sigdcantly higher frequency
of positive results with human complement
(43 of 54 samples) than with guinea pig
complement (6 of 54). The titers were also
higher using human complement?’ The
immunoglobulin class of the anti A and B
antibodies was not investigated, but the
“naturally” occurring form is usually a
macroglobulin.28
The relevance of these findings to patients with rheumatoid arthritis can only
be speculative. The rheumatoid factorreduced gamma globulin complexes are
not a very efficient complement fixing system, since approximately 2 mgm. of preformed complexes are required to fix 50
per cent of the available human complement, whereas only 100 pgm. of aggregated human gamma globulin will fix a
similar amount. One clinical situation has
been described where there are mixed cryoglobulin complexes consisting of Ig M im-
535
m<;TIONS OF TREATED GAMMA GLOBULIN WlTH RF
munoglobulins with rheumatoid factor ac-
tivity and Ig G molecules; these patients
usually have depressed serum complement
levels.29
Small amounts of aggregated gamma
globulin produce skin reactions, and treatment with urea removes this property.20.21
Mercaptoethanol treatment of rabbit 7s
antibody interferes with its ability to fix to
skin and give PCA reactions.s0 Therefore,
the finding that intradermal injection of
aggregated mercaptoethanol treated FII into normal subjects produced no reaction,
even in amounts as great as 40 micrograms
N, was not surprising. That it produced no
reactions in patients with classicd rheumatoid arthritis might be taken as supporting
evidence for the inability of rheumatoid
suMMA€uo IN
factor-gamma globulin complexes to fix
complement; for the two properties, complement fixation and phlogistic response,
are parallel phenomena. Such an explanation requires that the conditions for antigen-antibody combination are similar in
vivo as in uitro, and that rheumatoid factor
can get from the intravascular space to an
extravascular space before the reduced
gamma globulin diffuses away from the injection site.
ACKNOWLEDGMENTS
The authors wish to thank Mrs. Jennifer Robiuson and Mrs. Annelore Serenyi for excellent technical assistance. Part of these studies were
performed in the laboratories of Dr. Elmer Becker,
whose interest and encouragement is gratefully
acknowledged.
hTElUI"TERLINUA
Le reduction e alcoylation de globulina gamma human ante le aggregation resulta in
un perdita da capacitate pro le fixation de complemento sed in le retention del capacitate pro reager con factor rheumatoide. Le addition de aggregate, reducite glob&
gamma a sero que contine alte titros de factor rheumatoide resulta in le production de un
typicamente campaniliforme curva de precipitina con un puncto de equivalentia e
pauc precipitato in le region de excess0 de antigeno. Viste que aggregate, reducite
globulina gamma per se fixa pauc complemento, il esseva possibile determinar si su
reaction con factor rheumatoide resulta in le fixation de complemento. Le constatation
esseva: Sub appropriate conditiones factor rheumatoide pote fixar complemento. N d e
fixation de complemento de porco de India o de complemento human esseva observate
quando aggregate, reducite globulina gamma esseva addite a thermoinactivate seros con
varie titros de factor rheumatoide, sed preformate complexos de factor rheumatoide e
globulina gamma fixa complemento human a 37 C. Le injection intradennal de 40 pg N
de aggregate, reducite globulina gamma ad in subjectos de control0 o ad in patientes
con arthritis rheumatoide produce nulle immediate o tardive reactions cutanee.
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