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Synthesis by an Established Lymphocyte Cell Line From a Rheumatoid Synovium.

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330
SYNTHESIS BY AN ESTABLISHED
LYMPHOCYTE CELL LINE FROM A
RHEUMATOID SYNOVIUM
B R U C E C. G I L L I L A N D , DENYS K . FORD, and M A R T M A N N I K
Lymphocytes derived from the synovium of a patient with rheumatoid arthritis were shown to synthesize
predominantly IgG as measured by an equilibrium binding
assay. Chromatographic separation of the radiolabeled
lymphocyte culture supernatant revealed rheumatoid factor activity associated with IgG and IgM. In addition,
immunofluorescence studies on fixed lymphocytes demonstrated that the majority of cells stained positive for both
IgC and rheumatoid factor.
From the Division of Rheumatology in the Department of
Medicine, the Department of Laboratory Medicine, University of
Washington, Seattle, Washington, and the Division of Rheumatology,
Department of Medicine, University of British Columbia, British
Columbia, Canada.
Presented in part at the Sixth Pan-American Congress on
Rheumatic Disease, Toronto, Ontario, June, 1974.
This work was supported by Research Grant AM 12849 from
the National Institute of Arthritis, Metabolism and Digestive Diseases, in part by a Clinical Research Center grant from The Arthritis
Foundation, and by a Canadian Medical Research Council grant MA4827.
Bruce C. Gilliland, M.D.: Professor of Medicine/Lab Medicine, Division of Rheumatology, Department of Medicine, University
of Washington and Providence Medical Center, Seattle, Washington;
Denys K. Ford, M.D.: Professor of Medicine and Head, Division
of Rheumatology, Department of Medicine, University of British
Columbia; Mart Mannik, M.D.: Professor of Medicine and Head,
Division of Rheumatology, Department of Medicine, University of
Washington.
Address reprint requests to Bruce C . Gilliland, M.D., Providence Medical Center, 500 17th Avenue, Seattle, Washington 98122.
Submitted for publication November 5 , 1976; accepted in
revised form November 2, 1977.
Arthritis and Rheumatism, Vol. 21, No. 3 (April 1978)
A lymphocyte cell line, established from culture
of synovium from a patient with seropositive rheumatoid arthritis, was previously shown to secrete immunoglobulins, primarily IgG a n d IgM ( I ) . Since the lymphocytes that served as the origin of the established line had
accumulated in the synovium during the course of disease, they might therefore synthesize immunoglobulins
that participate in the generation of inflammation by a
process analogous t o t h e lymphocytes in antigen induced synovitis in experimental models (2,3). Because
rheumatoid factors are found in the synovial fluid of
patients with rheumatoid arthritis and appear t o play a
role in the pathogenesis of rheumatoid arthritis (4,5),
studies were performed to determine if these cultured
cells were synthesizing and secreting immunoglobulins
possessing rheumatoid factor activity.
MATERIALS AND METHODS
Lymphocyte Cell Culture. The lymphocyte culture was
derived from the synovium of a patient with seropositive rheumatoid arthritis at the time of synovectomy in June 1971 ( I ) .
These cells were maintained in continuous culture using
Eagle’s minimum essential medium, supplemented with 15%
fetal bovine serum, until November 1971 when a portion was
frozen in liquid nitrogen and stored. For the present studies
these cells were returned to continuous culture in February
1972. The cell line handled in this fashion has been given the
designation F,. Studies performed in 1973 by Dr. Yashar
Hirshaut of the Sloan-Kettering Institute for Cancer Research,
New York, revealed Epstein-Barr virus in the cultured cell line
(6).
SYNTHESIS BY LYMPHOCYTE CELL LINE
Twenty million lymphocytes were cultured for 20
hours at 37°C in a modified Eagle’s medium supplemented
with 20 pCi of “C-labeled amino acids contining arginine 312
mCi/m mole, leucine 312 mCi/m mole, lysine 310 mCi/m
mole, valine 258 mCi/m mole (SchwardMann, Orangeburg,
New York). These amino acids were absent in the modified
Eagle’s medium. Cells were removed by centrifugation and the
sterile supernatant was stored at 4°C. The supernatant was
exhaustively dialyzed against borate buffer (0.2 M sodium
borate, 0.15 M NaCI, p H 8.0) until counts of the cell culture
supernatant were not detectable in the dialysis bath.
lnsolubilization of Antisera and IgG. Goat antisera
specific for human IgG, IgM, and IgA, and kappa and lambda
light.?chains were obtained from a commercial source (Hyland
Laboratories, Costa Mesa, California). Globulins from each
antisera were precipitated in 33% ammonium sulfate, and the
resulting precipitates were dialyzed against borate buffer. T o
assure monospecificity of the antibody, any contaminating
antibodies in the redissolved globulins were removed with
agarose-antigen immunoadsorbent columns (7).
The source of normal human IgG was Cohn Fraction
I1 (Schwarz/Mann) which was further purified by ion exchange chromatography on DEAE cellulose. The material was
eluted with 0.01 M phosphate buffer, p H 8.0. Rabbit and goat
IgG were obtained from Miles Laboratories, Kankakee, Illinois.
The globulin fractions containing specific antibodies
and normal goat, rabbit, and human IgG were insolubilized by
drop addition of 2.5% glutaraldehyde according to the methods of Avrameas and Ternynck (8). The insolubilized material
was homogenized and washed three times with phosphate
buffered saline (0.01 M PO,, 0.15 M NaCI, p H 7.0), three
times with 0.1 M glycine-HCI buffer (pH 3.0). three times with
borate buffer, and then resuspended in borate buffer. Protein
concentration of each suspension was determined by Fohn
method after solubilization of 0.5 ml of the suspension by 1 .O
ml of 1 M NaOH for 2 hours at 56°C (9).
Equilibrium Binding Assay. Increasing quantities of
both the insolubilized antiserum and the normal IgG were
added to an aliquot of radiolabeled culture supernatant to
quantify immunoglobulins or rheumatoid factor activity. Total reaction volumes for each set of determinations were made
identical by addition of 0.15 M NaCI. Tubes were incubated
for I hour at 37°C with frequent mixing, allowed to stand
overnight at 4”C, and then centrifuged for 1 hour at 1500g.
The top half of the reaction volume was precisely removed and
placed into a counting vial labeled S. The lower half, which
included the insolubilized material, was quantitatively transferred t o another counting vial labeled R. The percent of
binding was calculated from the radioactive counts as follows:
Percent binding
=
R-S X
R+S
100
The quantity of insolubilized antisera of IgG added to each
aliquot of supernatant was increased until a plateau of percent
of binding was achieved, indicating that all available immunoglobulin or rheumatoid factor was bound. The maximum percent of binding was used to express the amount of immunoglobulin or rheumatoid factor activity in the supernatant. For
all immunoglobulin and light chain determinations, maximum
percent of binding was reached with the addition of 0.05 mg or
less of insolubilized antibody preparations to 0.8 ml of culture
33 1
supernatant. At least two separate assays were performed for
each immunoglobulin or rheumatoid factor measurement. Radioactivity was measured on a Beckman Liquid Scintillation
Counter using Aquasol (New England Nuclear, Boston, Massachusetts) as the scintillant.
Chromatographic Separation of Radiolabeled Culture
Supernatant. Aliquots of culture supernatant were placed on a
calibrated Sephadex G-200 column and equilibrated with the
borate buffer (0.2 M sodium borate, 0.15 M NaCI, pH 8.0).
Other aliquots were first dialyzed against a sodium buffer (0.1
M , pH 3.5), then placed on a Sephadex G-200 column and
equilibrated with the same sodium acetate buffer. Radioactivity in fractions was used to follow the pattern of elution.
Fractions obtained were then dialyzed against the borate buffer.
Immunofluorescence Microscopy. Fluorescein-conjugated goat antihuman IgG and antihuman IgM, sheep antihuman kappa and antihuman lambda light chains (Meloy
Laboratories, Springfield, Virginia), goat antihuman kappa
and antihuman lambda light chains (Cappel, Downingtown,
Pennsylvania), and rabbit antihuman IgG (prepared in our
laboratory) were further purified. The antisera were considered
monospecific when each reacted by immunofluorescence only
with the specific antigen coupled to Sepharose 4B beads and
not with other immunoglobulins or light chains (LO).
Human IgG was conjugated with fluorescein isothiocyanate followed by chromatography with DEAE A-50,
the elution buffer being 0.01 M PO,, 0.1 M NaCI, pH 7.2.
Monomeric preparations of fluorescein-labeled IgG were obtained by chromatography on Sephadex G-200 columns equilibrated with borate buffer. Aggregated fluorescein-labeled IgG
was prepared by heating the labeled monomeric IgG at 63°C
for 20 minutes. Preparations were heat aggregated just before
immunofluorescent staining.
Lymphocytes of the same cell line (F2)were removed
from culture medium by centrifugation and washed three
times in phosphate buffered saline containing 1% bovine serum
albumin. Smears were prepared with lymphocytes that were
harvested on the third day after fresh culture medium was
added. The cells were allowed to air dry and then were frozen
and stored at -87°C. Prior to fluorescent staining the cells
were fixed in absolute ethanol for 15 minutes and rinsed with
phosphate buffered saline.
Fixed lymphocyte smears were stained with the above
antisera and IgG preparations for 2 hours a t room temperature in a moist chamber. The slides were thoroughly washed
in phosphate buffered saline for 20 minutes and mounted,
using a buffer consisting of 9 parts glycerol and 1 part phosphate buffered saline, pH 9.5. The slides were read with a Zeiss
microscope, Osram HBO-200 lamp, Ug 1 exciting filter, GG 4
barrier filter. A total of 400 cells were counted for each stained
preparation.
RESULTS
Measurement of Immunoglobulins in Radiolabeled
Culture Supernatant. T h e q u a n t i t y of IgG, IgA, IgM,
and k a p p a and l a m b d a light chains in t h e s u p e r n a t a n t is
expressed as the percent of t o t a l c o u n t s of t h e cell cult u r e s u p e r n a t a n t b o u n d to the respective insolubilized
antiserum ( T a b l e 1). Results represent t h e mean of a t
GILLILAND ET AL
332
least two separate equilibrium binding assay experiments. The percent of counts bound to insolubilized
antihuman IgG and anti-kappa light chains was almost
identical, thus indicating that the newly synthesized immunoglobulin was mainly IgG kappa. A minimal
amount of binding was observed with anti-IgM and
anti-IgA, whereas no binding occurred with anti-lambda
light chain. Prior incubation of insolubilized antihuman
IgG with unlabeled human IgG blocked radioactivity
bound to this insolubilized antiserum.
The percent of the total counts of the cell culture
supernatant contributed by entrapment and by nonspecific binding to the insolubilized antisera was determined in conjunction with each equilibrium binding
assay. Identical quantities of insolubilized goat IgG
were incubated with 0.8 ml of radiolabeled supernatant,
and the percent of counts bound was determined as
previously described (see Table I ) . Binding of counts
was not observed; therefore no correction for entrapment and nonspecific binding was required.
Measurement of Rheumatoid Factor Activity in
the Radiolabeled Culture Supernatant. The amount of
rheumatoid factor activity in the supernatant, expressed
as the percent of counts bound to insolubilized human,
rabbit, or goat IgG, is shown in Table 2. Maximum
binding of counts by rabbit IgG or human IgG required
the addition of 1 mg of each IgG t o 1 ml of supernatant.
Insolubilized goat IgG failed to bind any counts with the
addition of 1 mg to I ml of culture supernatant. The
absence of binding of counts by goat IgG served to rule
out the possibility that the binding observed with rabbit
and human IgG was due to entrapment or nonspecific
absorption. The percent of total counts of the cell culture supernatants, however, bound to insolubilized rabbit IgG was greater than the percent of bound counts
identified as immunoglobulins. The explanation for the
greater binding with rabbit IgG is not known.
Measurement of Immunoglobulins and Rheumatoid Factor in the Pools from Sephadex G-200 Chromatography, Equilibrated with Borate Buffer, pH 8.0, or
with Acetate Buffer, pH 3.5. The chromatographic elution pattern using borate buffer, pH 8.0 (Figure 1 ) was
similar to that with acetate buffer, pH 3.5. The column
fractions were pooled as depicted in Figure I , and the
percent of counts in each pool bound to insolubilized
antibodies to human immunoglobulins or to insolubilized IgG was measured by equilibrium binding
assay. Pool I corresponded to the exclusion volume of
this calibrated Sephadex G-200 column, thus containing
proteins with a molecular weight greater than 200,000,
whereas Pool 111 corresponded to the elution peak of
monomeric IgG.
Table I . Fz Culture Supernatant Radioactivity Bound to Insolubilized
Antibodies to Human Immunoglobulins
Type of Antiserum
or Immunoglobulin
Goat anti IgG
Goat anti IgM
Goat anti IgA
Goat anti kappa
Goat anti lambda
Goat IgG
Percent of Total Counts
(Mean f 1 SD)
16.6 f 3.2
1.8 f 1.3
0.7 f 0.9
17.4 f 1.7
O*
0
* Only one determination.
The quantity of immunoglobulins or rheumatoid
factor measured in the various pools is shown in Table
3 . Rheumatoid factor activity was determined using only
insolubilized rabbit IgG because of its greater reactivity
observed in binding experiments with unfractionated
culture supernatant. When the column was equilibrated
with borate buffer, pH 8.0, IgG was present mainly in
Pool I and 111, and IgM was present only in Pool I .
Rheumatoid factor was identified in both Pools I and 111.
When the column was equilibrated with sodium acetate
buffer, pH 3.5, most of the IgG were found in Pool 111
with some in Pool 11. This observation suggested that
IgG was dissociated from complexes of IgG-IgG or
Ig M-lgG under acid conditions. Rheumatoid factor
activity again was found primarily in Pools I and 111.
It should be noted that the results are expressed as percent of counts in each pool and therefore cannot be
directly compared to the percent of binding observed in
the whole culture supernatant (Tables I and 2).
Measurement of Immunoglobulins and Rheumatoid Factor in the Radiolabeled Supernatant from Lymphocytes Cultured from the Blood of a Normal Person.
The supernatant from the established cell line 1788,
derived from the blood of a healthy individual, was
processed exactly in the same manner as that derived
from the culture under study. (Lymphocytes from this
cell line were kindly sent to us by Dr. George Moore,
Denver General Hospital, Denver, Colorado.) ImmunoTable 2. Rheumatoid Factor Activity in F2 Culture Supernatants,
Determined by Radioactivity Bound to Insolubilized IgG
Type of Immunoglobulin
Percent ofTotal Counts
(Mean f I SD)
Human IgG
Rabbit IgG
Goat IgG
13.6 f 2.4
27.0 f 3.3
0
SYNTHESIS BY LYMPHOCYTE CELL LINE
Sephodex G-200 Separation of Culture
Supernotont at pH 8.0
I ,
I I ,
m , m
800 -
600-
400-
200-
30
40
50
60
Froction Number
70
80
Figure 1. Sephadex G-200 chromatographic elution pattern using borate
bufler, p H 8.0.The column fractions were combined into four pools as
depicted.
globulins and rheumatoid factor were measured as described previously. Insolubilized anti-IgM bound 1.7%
of the total counts in the supernatant; insolubilized antiIgG and insolubilized anti-IgA bound no counts. Rabbit, human, and goat IgG failed to bind counts, a finding that indicated the absence of detectable rheumatoid
factor. Only one assay for each of the above measurements was performed.
Demonstration of Immunoglobulins and Rheumatoid Factor in the Cytoplasm of Cultured Lymphocytes by
lmmunofluorescence Microscopy. Fixed lymphocyte
smears were stained with fluorescein-labeled antisera for
human IgG, IgA, IgM, kappa and lambda light chains,
333
human monomeric, or heat aggregated IgG. Results are
expressed as the percent of cells showing cytoplasmic
fluorescent staining (Table 4). In the cells that were
stained, fluorescence was observed throughout the cytoplasm but not in the nucleus (Figure 2 ) . The majority of
cells stained for the presence of IgG or kappa light
chains, indicating that most of the cells of the F, culture
line contained IgG kappa. Only 3% of the cells stained
for IgM. IgA or lambda light chains were not detectable
in the cytoplasm of these cells by fluorescence microscopy. Fluorescein-labeled aggregated human IgG
stained 74%of the cells, whereas monomeric human IgG
stained only 20%. Incubation of the fixed lymphocyte
smears with the respective nonfluorescein-labeled antisera resulted in blocking the positive reactions with fluorescein-labeled antisera. Similarly, blocking the positive
reaction with fluorescein-labeled human aggregated IgG
was observed with nonfluorescein-labeled human or
rabbit aggregated IgG, but blocking was not observed
with aggregated goat IgG.
T o further ascertain the specificity of immunofluorescent staining for immunoglobulins and rheumatoid factor shown in this cell line, three B cell lines and
one T cell line were kindly sent to us by Drs. Fu and
Kunkel of the Rockefeller University, New York. These
cell lines were fixed and stained in an identical manner.
Our interpretation of the immunofluorescent staining of
these fixed lymphocyte smears was made without prior
information on the cell types or their products, and this
analysis was found to be in agreement with the observations of Drs. Fu and Kunkel. The cytoplasm of the T
cell line stained with neither antisera to immunoglobulins and light chains nor monomeric and aggregated
Table 3. Radioactivity of Pools from G-200 Sephadex Chromatography Bound to Insolubilized Antibodies
to Human Immunoglobulins or to Insolubilized IgG
Type of Antiserum or Immunoglobulin
Pool I
Equilibration with borate buffer, pH 8.0
Goat anti IgG
11.9f 1.5*
Goat anti IgM
9.0 f 1.6
Goat anti IgA
0.9 f 0.9
Rabbit IgG
17.4 f 3.5
Goat IgG
0
Equilibration with sodium acetate bufler. pH 3.5
1.9 f 1.0
Goat anti IgG
9.5 f 0.6
Goat anti IgM
0
Goat anti IgA
13.0 f 3.0
Rabbit IgG
0
Goat IgG
Pool 11
Pool 111
Pool IV
1.0% 1.0
0.8 f 0.8
2.2 2.2
0.8 f 0.2
0
4.9 f 2.3
0
0
11.7 f 3.2
0
0
0
0.8 f 0.8
0
4.5 f 0.9
7.3 f 1.4
0
3.2 f 1.0
8.5
0
0
10.9 f 2 . 3
0
0
0
0
0
0
0
*
0
* Percent of total counts in pool bound to insolubilized anti-immunoglobulin or IgG (Mean f 1 SD).
GILLILAND ET AL
3 34
human IgG. More important was the lack of cytoplasmic
staining of all three B cell lines with fluorescein-labeled
monomeric or aggregated human IgG; this indicated
that the immunoglobulins present in the cytoplasm of
these cell lines did not have rheumatoid factor activity
and that the positive results with our cell line were not
due to nonspecific staining by the reagents used.
DISCUSSION
A previous study on lymphocytes derived from
the synovium of this patient with rheumatoid arthritis
demonstrated immunoglobulin synthesis of both IgG
and IgM ( I ) . The present study, performed on lymphocytes of the same origin but later in their culture history,
again showed synthesis of IgG and IgM. However, IgG
now appears to be the major immunoglobulin produced
by both equilibrium binding assay and immunofluorescence microscopy. The observations on this culture cell line were further extended in the current study
to show rheumatoid factor activity of the synthesized
immunoglobulins. Sephadex G-200 chromatographic
separation of the culture supernatant revealed that rheumatoid factor activity was associated with both IgG and
IgM. Rheumatoid factor activity was found in an elution pool that contained only IgG, further indicating
that this established cell line synthesized IgG rheumatoid factor. Since radioactive counts in these pools represent only a small fraction of the total counts in the
Table 4. Cytoplasmic Staining of F2 Cells by Immunopuorescence
Type of Antiserum or Immunoglobulin
Anti.IgG
Anti IgM
Anti IgA
Anti kappa
Anti lambda
Monomeric human IgG
Aggregated human IgG
* Four-hundred cells
% Positive.
65
3
0
71
0
20
14
counted.
supernatant, the quantitative data cannot be directly
compared to those from the whole culture supernatant.
Synthesis of immunoglobulins by rheumatoid synovium has been clearly shown. Sliwinski and Zvaifler
( 1 1) used a radioisotope dilution method to measure in
vivo synthesis of IgG by rheumatoid synovium, and they
found that the synovium of a single rheumatoid knee
joint could synthesize from 5 mg to 95 mg of IgG per
day. In vitro synthesis of immunoglobulins has also
been studied using “C amino acid incorporation with
explants of synovium, lymph nodes, and spleen culture
(12). Immunoglobulin synthesis by rheumatoid synovium appeared to be quantitatively similar to that of
normal human spleen and lymph node. In this same
study, 79% of the newly synthesized immunoglobulins
by rheumatoid synovium were IgG, 11% were IgA, and
Figure 2. Immunojluorescence microscopic identification of immunoglobulins in Fz cell line. On the left the lymphocytes show
cytoplasmic staining with fluorescein-labeled goat antihuman IgG. On the right, the lymphocyte was stained with jluoresceinlabeled goat antihuman kappa. A lymphocyte not staining with this antiserum is barely visible in lefi upper corner.
SYNTHESIS BY LYMPHOCYTE CELL LINE
10% were IgM. Less than 10%of the IgM had rheumatoid factor activity as measured by coprecipitation of
radiolabeled IgM with carrier rheumatoid factor and
heat aggregated IgG. I n a subsequent study, rheumatoid
factor activity of “C-IgG produced in culture by rheumatoid synovial explants was measured by absorption
of radioactivity by human IgG coupled to Sepharose
(13) or by insoluble human and rabbit IgG cross linked
by bisdiazobenzidine. The amount of IgG-rheumatoid
factor was found to represent 0-4.9% of the newly synthesized IgG. Saykaly et al. (14) observed that explant
cultures of rheumatoid synovia produced mainly IgG;
however, they were unable to demonstrate rheumatoid
factor activity. Our findings indicate that most of the
IgG produced in this continuous lymphocyte culture
had rheumatoid factor activity. The use of an equilibrium binding assay, which measures anti-IgG of low
binding affinity, may explain our detection of more IgGrheumatoid factor than observed by others.
The synovia of patients with rheumatoid arthritis
contain both B and T cells ( 1 5-1 7), both of which play a
role in immune mediated tissue injury ( 1 8). Rheumatoid
factor activity has been detected in plasma cells of rheumatoid synovium by staining with fluorescein-labeled
aggregated IgG (19). Rheumatoid factor activity was
mainly associated with plasma cells staining for IgM.
The first evidence for IgG-rheumatoid factor in plasma
cells of rheumatoid synovium was provided by the studies of Munthe and Natvig (15). These investigators
found a predominance of IgG plasma cells in the synovial membranes of both seropositive and seronegative
rheumatoid arthritis patients, which corresponds with
the predominance of IgG production observed on culture of synovial tissue (12-14). In these studies, many of
the rheumatoid synovial membranes showed few plasma
cells staining for rheumatoid factor with fluoresceinlabeled aggregated IgG even though the tissue contained
abundant IgG plasma cells. After pepsin treatment of
synovial tissue from either seropositive or seronegative
patients, the number of plasma cells staining for rheumatoid factor greatly increased. This indicated that
many IgG plasma cells have rheumatoid factor activity
that was blocked due to intracellular complexing of
IgG-rheumatoid factors (20), presumably by the selfassociation of IgG-rheumatoid factors (21-22). In our
cell line the percent of cells with IgG and with rheumatoid factor activity by immunofluorescence was approximately the same and thus did not require pepsin digestion for demonstration of rheumatoid factor.
Aggregated human IgG stained more cells than the
335
monomeric preparations of the same fluoresceinated
protein.
All of these observations indicate that B cells in
synovia of patients with rheumatoid arthritis are synthesizing IgG rheumatoid factor. Since IgG rheumatoid
factor can also serve as antigen in the immune complexes formed by the self-association of two IgG rheumatoid factors, it is conceivable that IgG rheumatoid
factor produced by plasma cells in synovium would act
as the immunogen for continued production of these
antibodies. If the synthesis of IgG rheumatoid factor is
self-perpetuating, an exogenous immunogen may not be
required to maintain the antibody-mediated inflammation in the synovitis of rheumatoid arthritis. However,
experimental proof of such self-perpetuation has not yet
been provided. The factors or mechanisms possible for
the initiation of synthesis of IgG rheumatoid factors
also need to be explored.
ACKNOWLEDGMENTS
The authors wish to acknowledge the expert technical
assistance of Ms Pamela Croll and Mrs. Lois Lussier.
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