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Macrophage migration inhibitory factor in rheumatoid pericarditis.

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969
MACROPHAGE MIGRATION
INHIBITORY FACTOR IN
RHEUMATOID PERTCARDITIS
MARIO ANDREIS and ERIC R. HURD
The pericardial effusion in a case of rheumatoid
pericarditis was studied to determine whether immune
complexes and mediators of cellular immunity, represented by migration inhibitory factor (MIF), were present. MIF-like activity was detected in the pericardial
fluid, but only traces of immune complexes were revealed
by ultracentrifugation. The MIF-like activity was partially characterized by column fractionation and sugar
inhibition tests. The role of lymphokines in the pathogenesis of this case of rheumatoid pericarditis is strongly
suggested.
Although articular manifestations are the dominant factor in rheumatoid arthritis, it is considered to be
a systemic disease which may involve almost any organ
or system (1-3). Pericardial involvement in chronic
rheumatoid arthritis, described initially by Charcot in
From the Department of Internal Medicine, University of
Texas Southwestern Medical School at Dallas, and the Dallas Veterans Administration Hospital. Dallas, Texas.
Supported by USPHS Research Grants AM 09989 and
Training Grant AM 05154. and by an Arthritis Foundation Clinical
Study Center Grant.
Mario Andreis, M.D.: Assistant Professor of Medicine, University of Texas Southwestern Medical School at Dallas, and the
Dallas Veterans Administration Hospital; Eric R. Hurd, M.D.: Associate Professor of Internal Medicine. University of Texas Southwestern Medical School at Dallas.
Address reprint requests to Mario Andreis. M.D., Department of Internal Medicine, The University of Texas Southwestern
Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235.
Submitted for publication December 20. 1976: accepted January 10. 1977.
Arthritis and Rheumatism, Vol. 20, No. 4 (May 1977)
188 I , is now a well-recognized manifestation of rheumatoid disease (4-8). The presence of pericarditis in rheumatoid patients has been reported to range from 1 1 to
50%. This variation in the percentage of incidence seems
to reflect the different methods used to assess pericardial
involvement. I t is agreed, however, that pericarditis can
be present in rheumatoid arthritis with very few symptoms, or can even be first discovered upon autopsy
(4.9- I 1 ).
Clinically, rheumatoid pericarditis seems to correlate better with more severe disease and with the presence of other extraarticular manifestations of the rheumatoid disease, such as subcutaneous nodules (9).
Usually it has little clinical consequence, but in some
cases it can lead to cardiac tamponade and constrictive
pericarditis ( 1 1 ). Pericardial fluid is characterized by
decreased levels of sugar, high levels of gammaglobulin
and lactic acid dehydrogenase, a positive latex fixation
test, and, frequently, low complement levels (9,12). Although the exact etiology of rheumatoid arthritis remains unknown, most of the evidence accumulated in
the last 20 years implicates immune mechanisms in the
pathogenesis of the disease manifestations (13).
The findings of immune complexes in the serum
and synovial fluid of patients with rheumatoid arthritis,
together with low complement values in the joint effusions, emphasizes the role of immune complexes in the
pathogenesis of rheumatoid disease (14,15). In a recently reported case of rheumatoid pericarditis, studies
of the pericardial effusion showed decreased total hemolytic complement and immune complexes similar to
970
ANDREIS AND HURD
those described in synovial fluid (16). These findings
strongly support the concept of immune complexes as
mediators of rheumatoid chronic inflammation. They
also provide evidence linking the synovial and pericardial manifestations of rheumatoid arthritis.
The role of immune complexes i n mediating
tissue injury in rheumatoid arthritis has been well
established; mediators of cellular immunity (lymphokines) have also been claimed to maintain the chronic
synovial inflammation ( I 7,18). The present report
describes the studies performed on the pericardial
effusion of a patient with rheumatoid pericarditis who
underwent surgery. It reports the demonstration and
partial characterization of a macrophage migration inhibitory factor in this fluid which is similar to the mediator that has been described in rheumatoid synovial fluid
(17).
CASE REPORT
RS is a 68-year-old white woman who was in good
health until April 1972, when she began having arthritis in her
hands. Eventually the proximal interphalangeal and metacarpal phalangeal joints of both hands, and the wrists, shoulders,
knees, and ankles became involved. Large nodules were present over both elbows. From January to April 1973 she was
given a series of gold salt injections with some subsequent
improvement. In December 1973 the diagnosis of carpal tunnel syndrome was made. Surgery was performed on the left
hand and the symptoms improved. I n March 1974 she developed thrombophlebitis in the left leg and was hospitalized and
treated with heparin and coumadin. Her therapy has consisted
primarily of aspirin and acetaminophen with intermittent therapy with prednisone.
She was doing well until July 1974. when she experienced an abrupt onset of palpitations with shortness of breath
associated with a feeling of substernal tightness. Her heart
rhythm was irregular and an EKG showed atrial fibrillation
with some slight ST elevations. A grade W V I systolic ejection
murmur was heard along the left sternal border with radiation
into the neck. A fourth heart sound was present at the apex.
Bruits were heard over the abdominal aorta and both femoral
arteries. A few rales were heard in the right lung base posteriorly. Chest X ray showed that the heart was markedly enlarged
and revealed a small right pleural effusion. An echocardiogram disclosed a large pericardial effusion. An attempt
at pericardiocentesis was unsuccessful. A pericardiectomy was
performed and 250 cms of serosanguinous fluid were removed.
After surgery she was started on 5 mg of prednisone
per day and digitalis. Her atrial fibrillation converted to a
normal sinus rhythm on the fourth postoperative day. Her
heart rhythm continued in normal sinus rhythm, her pleural
effusion disappeared, and she was discharged from the hospital on July 19, 1974. Although her cardiac status has remained
stable, her arthritis has been quite active unless she is taking
prednisone. To taper the patient off the prednisone, she was
started on 100 mg of cyclophosphamide per day, and this
regimen has been continued until the present time.
At the time of pericardiectomy, she had 4800 WBC
with a differential of 78 segmented, 3 bands, and 19 lymphocytes. Her hematocrit was 26.4%, hemoglobin 8.19 g%, and
erythrocyte sedimentation rate 66. LE prep, anti-DNA, and
anti-ENA tests were negative. Antinuclear antibodies were
positive at I :80 with a homogeneous pattern. Her latex titer in
tube dilution was positive at 1:640. The pericardial fluid
showed a cell count of 200/mms with a differential of 81%
mononuclear cells and 19% polymorphonuclear cells. The
pathologic analysis of segments of pericardium (done by Dr.
V. Q. Telford, Pathology Department, Presbyterian Hospital
of Dallas) showed acute and chronic inflammation with
marked fibrinous exudate (Figure I).
METHODS AND RESULTS
Attempts were made to demonstrate both immune complexes as well as macrophage migration inhibitory factor (MIF). To assess the presence of immune
complexes in the pericardial effusion as well as in the
serum, three methods were used. First, analytical ultracentrifugation was performed in a Spinco Model E ultracentrifuge (Beckman Instruments, Palo Alto, California). Ultracentrifugation was carried out at 56,100
rpm at 25" in 1:3 dilutions with phosphate-buffered
saline at pH 7.2. The pattern in the serum showed an
abnormal peak with a sedimentation coefficient of 2 2 s
or greater. The pericardial fluid also showed a small
peak with the same characteristics. These findings are
consistent with the presence of immune complexes probably formed by the interaction of IgM rheumatoid factor with complexes consisting of IgG combined with 7s
rheumatoid factor (19).
Second, double diffusion studies by the Ouchterlony technique, described in detail elsewhere (20),
were performed in 0.6% agarose in pH 7.2 phosphatebuffered saline. Purified rheumatoid factors, one monoclonal and three polyclonal, were used as reagents.
Neither the serum nor the pericardial effusion showed
precipitin lines with the rheumatoid factors, and thus
the absence of demonstrable immune complexes was
verified.
Third, normal polymorphonuclear cells were incubated, according to the technique described by Hurd
et al (21), with the patient serum and pericardial fluid,
and normal human serum was used as a control. After
incubation the cells were stained with fluorescein-conjugated goat monospecific antisera directed against each
of the three major human immunoglobulins and C3.
As Table 1 shows, only the patients' serum induced
phagocytosis of inclusions of IgG, IgM, and C3 which
correlates to some extent with the ultracentrifugal findings.The pericardial fluid failed to induce intracellular
inclusions.
97 1
RHEUMATOID PERICARDITIS
Fig I . Photomicrograph of the pericardium, which is infiltrated with numerous neurrophils, lymphocytes, and
a Jew plasma cells. Reactive pericardial mesothelial cells and prominent fibrous organization can also be seen
( H & E X 2501.
Pericardial fluid was studied for the presence of
migration inhibitory factor by using normal guinea pig
peritoneal macrophages as target cells in a capillary
tube migration assay (22). The sample's inhibition of
macrophage migration was less than 80% of the control's area of migration. All the migration assays were
carried out at 37" in RPMI 1640 with the addition
of antibiotics and 20% fetal calf serum (Grand lsland
Biological Company, New York). When the pericardial
fluid was assayed in a 1 :5 dilution of the medium described above, its migration area was 63% of the control
area, a result demonstrating the presence of MIF.
To further characterize the migration inhibitory
factor present in this pericardial effusion, the fluid was
fractionated on a G-100 Sephadex column and seven
fractions were obtained. The fractions were dialyzed
against distilled water and lyophilized. The lyophilized
fractions were reconstituted to the original volume of
the sample with RPMI 1640 with 20% fetal calf serum.
Fractions I and VI represent pre- and postcolumn controls. As Figure 2 shows, the inhibitory activity eluted in
the peak 1V corresponding to the chymotrypsinogen
marker which is similar to the MIF produced by human
peripheral lymphocytes stimulated in vitro with streptokinase-streptodornase (23). Because migration inhib-
itory factor seems to act by binding to a membrane
receptor in the macrophage through an L-fucose group
present in this receptor, the identification of MIF activity can also be studied by its specific inhibition by incubation with a-L-fucose. This inhibition cannot be accomplished by using the other sugars (24).
I n order to identify better the migration inhibTable I. Presence of Immune Complexes in Serum and Pericardial Fluid
Method
Analytical ultracentrifugation
Intermediate (9-16s)complexes
Large (222s) complexes
ImmunodiKusion against purified
rheumatoid factor
Phagocytosed inclusions by PMN
kc
kA
IgM
c3
Serum
PeriCard i a 1
Fluid
-
-
+
-
+
+
+
f
-
-
-
972
ANDREIS AND H U R D
Fraction
1
I
I
I1
I
I11
I
IVa
I
IVb
I I I
V
VI
EFFLUENT VOLUME (MILLILITERS)
Fig 2. Chromatographic separation of pericardial Puid on Sephadex G-100. Seven fractions were collected
and assayed,for macrophage migration inhibitory activity. Migration was significantly inhibited by Fraction
I Vb, which contained material which eluted in the region similar to chymotrypsinogen. EA, Chym, and Myo
are regions of elution of egg albumin, chymotrypsinogen. and myoglobin. Hatched bars represent the percentage oJ macrophage migration. Solid line illustrates optical density at 280 nm.
itory activity present in the pericardial fluid, the G-100
Sephadex fractions shown in Figure 2 were studied after
the addition of a final concentration of 0.1 M of either
a-L-rhamnose or a-L-fucose (Schwartz-Mann). As
Table 2 shows, the MIF activity in fraction IVb was not
modified by rhamnose but was completely abolished by
fucose-results demonstrating a definite similarity with
MIF induced in vitro (24).
DISCUSSION
The clinical characteristics of this case of rheumatoid pericarditis do not differ from the general descriptions reviewed by others (9,l l , l 2 ) . Our patient had
circulating immune complexes in her serum which were
of the heavy type (22s or more). Such complexes are not
infrequently seen in patients with rheumatoid arthritis.
Table 2. Macrophage Migration Inhibition by Rheumatoid Pericardial
Fluid Fraction: Effect of L-Fucose
Percent Migration
G-100
Fractions
I
I1
111
IVa
IVb
V
VI
N o Sugar
Added
0.1 M
L-Rhamnose
0.1 M
L-Fucose
I07
I30
73
73
25
83
I09
I10
I04
88
67
22
75
70
135
90
I15
77
I00
77
69
These complexes were presumably responsible for the
induction of inclusions when incubated with normal
polymorphonuclear cells. Analytical ultracentrifugation
of the pericardial effusion, done in the same way as for
the serum, showed only traces of these large complexes
and, accordingly, the phagocytosis of inclusions was
shown to be negative. The double immunodiffusion of
both serum and pericardial fluid against purified rheumatoid factors failed to demonstrate soluble immune
complexes.
These results can be interpreted in at least three
different ways. The amounts of complexes in the pericardial fluid may be minimal and cannot be picked up
by the inclusion test or the Ouchterlony technique. Alternatively, an excess of IgM rheumatoid factor might
interfere with the formation of a precipitin line in the
double diffusion test. Finally, it is possible that the
pericardial fluid lacked the complexes that were detected
in the patient’s serum. The available data strongly support the role of immune complexes in rheumatoid synovitis. Furthermore, the case reported by Ball ef af (16)
extends this concept to the pericardial manifestations of
rheumatoid disease. The patient reported here is clinically indistinguishable from the classic descriptions of
rheumatoid pericarditis. Nevertheless, the study of immune complexes in the pericardial fluid failed to demonstrate clear-cut evidence of these aggregates, as was
found in the case referred to above. Based on the data
presented here, an alternative explanation for these findings can be offered.
Experimental studies have demonstrated the in
R H E U M A T O I D PERICARDITIS
vitro production of M I F by synovial tissue from rabbits
with a chronic antigen-induced synovitis that resembles
rheumatoid arthritis (25). Moreover, supernatants from
immune lymphocytes cultured in the presence of the
antigen a n d containing MIF activity exhibited inflammatory properties when assayed in normal experimental animals. These inflammatory properties were
characterized in the skin by a tuberculin-type reaction
and in the synovium by a mononuclear cell infiltration
mainly composed of histiocytes and macrophages (26).
The studies performed on the pericardial fluid of our
patient clearly demonstrate t h e presence of M I F actiyity
similar to the one previously described in the synovial
fluid from rheumatoid patients ( 1 7).
The case reported by Ball et al (16) emphasizes
a n d extends the role of immune complexes as common
offenders responsible for chronic inflammation in synovium as well as pericardium in rheumatoid arthritis. The
case reported here is intended t o illustrate two main
points: a ) Soluble products from lymphocytes or
lymphokines represented by macrophage migration inhibitory factor can be isolated from rheumatoid pericardial fluid during periods of active inflammation. b )
T h e similarity of these findings with the finding in rheumatoid synovial fluid (17), plus the lack of significant
amounts of complexes in the pericardial effusion of our
patient, strongly suggest a role for lymphokines in the
chronic inflammatory process that characterizes rheumatoid synovitis and pericarditis. The relative importance of lymphokines in tissue injury, as compared with
immune complexes, cannot be precisely defined. However, it is most likely that the combination of both
factors is responsible for the lesions present in rheumatoid disease.
ACKNOWLEDGMENTS
The authors express their gratitude to Dr. Lige B.
Rushing, Jr., for allowing them to study this patient. They
would also like to thank Ms. Li-Chu Hsu for her excellent
technical assistance.
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974
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ANDREIS AND HURD
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