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The macrophage-lymphocyte rosette. Its increased incidence among cells from rheumatoid synovial fluid

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The Macrophage-LymphocyteRosette
Its Increased Incidence Among Cells from Rheumatoid Synovial Fluid
Bonnie Hepburn, F. C. McDuffie and R. E. Ritts, Jr.
Macrophage-lymphocyte rosettes are a prominent feature of rheumatoid
synovial fluid cell cultures. They suggest the presence of antigen in or
on synovial fluid leukocytes. The majority of lymphocytesfound in macrophage-lymphocyte rosettes have been identified as T cells.
Interaction between lymphocytes and
macrophages has for some time been
thought to play an important role in the
immune response to antigen. Macrophagelymphocyte (M-L) clusters or rosettes have
been described as precursors to blastogenesis in mixed lymphocyte cultures (1) and
in antigen stimulated lymphocyte cultures
(2). They have also been observed in autologous guinea pig cell mixtures in which
the stimulus for this phenomenon has been
unknown (3). T h e present report describes
the phenomenon of the macrophage-lymphocyte rosette in autologous cell mixtures
obtained from synovial fluid of patients
with rheumatoid arthritis. T h e majority of
lymphocytes participating in rosette formaFrom the Mayo Graduate School of Medicine and
the Departments of Microbiology and Medicine,
Mayo Clinic and Mayo Foundation, Rochester,
Minnesota 55901.
Supported in part by a grant from The Minnesota Chapter of The Arthritis Foundation.
BONNIE HEPBURN, MD: Fellow in Rheumatology,
Mayo Graduate School of Medicine; F c MCDUFFIE,
MD: Associate Professor of Medicine and Microbiology, Mayo Medical School: R E RITTS, JR, MD:
Professor and Chairman, Department of Microbiology, Mayo Medical School.
Address reprint requests to Dr R E Ritts, Mayo
Clinic, Rochester, Minnesota 55901.
Submitted for publication December 31, 1975;
accepted May 13, 1974.
1020
tion have been identified as T cells by the
sheep cell-lymphocyterosette.
MATERIALS AND METHODS
Patient Selection
T h e cells examined in this study were obtained
from the peripheral blood and synovial fluid of 5
patients with classic and 5 patients with definite
rheumatoid arthritis (4), from the blood of healthy
laboratory personnel and from the synovial fluid of
6 additional patients. Most patients were taking
aspirin until 10 hours before cells were obtained.
Three patients were taking 6 mg prednisone or less
per day.
Preparation of Cell Cultures
Peripheral blood lymphocytes were obtained by
collecting 25 ml of blood in a heparinized syringe.
The syringe was placed in an upright position until the red cells had settled. The plasma and upper
red cell layer were removed and diluted 1:2 in
Hepes 199 medium ( 5 ) . A Ficoll-Hypaque solution'
was layered under the cell suspension at a ratio of
one part Ficoll-Hypaque to three parts cell suspension. The layered suspension was spun at 4OOxg for
30 minutes. The cells at the interface were removed
and washed twice with Hepes 199 medium. The
washed cells were counted and diluted in Hepes
199 medium with 25% fetal calf serum, penicillin
100 units/ml and streptomycin 100 pg/ml. The final
'Twenty-four parts of 9% w/v Ficoll (Pharmacia)
and 10 parts 34% w/v Hypaque (sodium diatrizoate, Winthrop).
Arthritis and Rheumatism, Vol. 17, No. 6 (November-December 1974)
MACROPHAGE-LYMPHOCYTE ROSETTE
cell concentration was adjusted to 0.5 X loe lymphocytes per milliliter.
Synovial fluid was obtained by aspirating diseased
knees. Sodium heparin (without preservative) was
added to the synovial fluid immediately. The synovial fluid was diluted 1:3 with Hepes 199 medium
and preparations for cell cultures were otherwise
identical to those described above. Hyaluronidase
was not used.
Final blood cell suspensions contained from 56%93y0 lymphocytes, 50/,-320/, monocytes and 270-2670
polymorphonuclear cells and synovial cell suspensions contained 58%-870/, lymphocytes, 9y0-23YO
monocytes and 20/,-23Y0 polymorphonuclear cells.
Aliquots (0.2 ml) of cell suspension were distributed
in quadruplicate in sterile microtiter plates and incubated at 37°C. On the third day of culture 1
pCi of SH-thymidine (2Ci/mM) was added to each
well. T h e cells were harvested by a modified
MASH' apparatus. The washed trichloracetic acid
precipitate of these cells was counted in a Beckman
liquid scintillation counter. Viability of the cultured cells was assessed by the trypan blue exclusioh method.
Morphologic Examination
T o assure sufficient numbers of cells for morphologic study, the original cell suspension was also
divided into 2.5-ml aliquots. After 4 days at 37"C,
the supernate was aspirated from the cell pellet.
The pellet was then resuspended in several drops
of fetal calf serum. A smear was prepared from
this suspension and stained with Wright's stain.
The degree of macrophage-lymphocyte adherence
was then evaluated by determining the percent of
M-L rosettes in the total population of macrophages. The number of lymphocytes in each rosette
was also recorded. T h e macrophages counted were
randomly determined as in a differential cell count
except that tightly clumped macrophages whose
surfaces might not have been available for lymphocyte binding were not counted. The macrophages,
with or without lymphocytes attached to their surfaces, retained the ability to phagocytose latex particles. Unlike blasts, their cytoplasm stained lightly.
Their nuclei were eccentric and were either indented or lobulated.
'From Microbiological Associates, Inc., Bethesda,
Maryland. Modifications included replacement of
the plastic suction tubes with stainless steel needle
stock and the addition of a manually operated control for aspiration and washing.
T-cell assays (6) were performed by removing the
supernate from the 2.5-ml aliquots as above and
adding sheep red cells to the white cell pellets. The
cells were mixed and incubated overnight at 0°C.
Following a 2-hour incubation at room temperature, the cells were resuspended in fetal calf serum.
Smears were made and stained with Wright's stain.
Lymphocytes with two or more sheep cells adhering
were identified as T cells. Assays were performed at
the start of the 4-day incubation period to determine the percent of T cells present in the culture.
Assays were performed on day 4, on separate aliquots, to determine the nature of the macrophage
adherent lymphocytes.
Lymphocytes without attached sheep cells could
not be definitely identified as B or null cells. The
incubation time necessary for macrophage-lymphocyte adherence (at least 24 hours) has precluded reliable identification of the immunoglobulin markers
on the B cells. T h e number of these markers appears to decrease with the increased age of the
culture.
RESULTS
Without exception the macrophage-lymphocyte adherence was greater in rheumatoid synovial fluids than in either rheumatoid or normal peripheral blood (Table 1,
Figure 1). Adherence was low in synovial
fluid from 2 patients with acute attacks of
pseudogout (chondrocalcinosis) and 2 patients with Reiter's syndrome.
After 4 days of nonmitogen stimulated
cell culture, viability ranged between 70y0
and 99%. There was no correlation between viability and degree of rosette formation.
Synovial fluid lymphocytes of 3 rheumatoid patients demonstrated increased thymidine incorporation, exceeding twice the
mean level of incorporation in peripheral
blood cultures. At 6-7 days there was some
increase in thymidine incorporation by both
synovial fluid and blood lymphocytes of
rheumatoid as compared to normal control
lymphocytes. The small number of samples
and the wide variation in response limit
analysis of these data.
Arthritis and Rheumatism, Vol. 17, No. 6 (November-December 1974)
1027
HEPBURN E l AL
Table 1.
Patient No.
andcode*
RF
Peripheral blood
1 c
2 c
3 c
4 c
5 c
6 C
7 c
8 C
9 RA
10 RA
11 RA
12 RA
13 RA
14 RA
15 RA
-+
16 RA
-
+
+
+
+
+
Synovial fluid
9 RA
10 RA
11 RA
12 RA
13 RA
14 RA
15 RA
16 RA
17 CC
18 CC
19 R
20 R
21 R
22 P
Thymidine
Incorporation
(CPM/Culture)
Percent Macrophages
With Lymphocytes Adhering
+
++
+
+
+-
-
-_
CH,t
55
96
63
1.2
6.1
0
0
6.5
4.0
4.9
9.2
9.7
9.9
6.8
0
1
2
3
24
4th Day
6-7 Days
89
71
80
85
91
84
91
86
88
77
93
73
95
98
84
69
10
18
13
6
9
8
8
10
9
13
3
21
1
2
13
20
1
9
4
3
0
6
0
3
2
4
2
5
4
0
3
7
0
1
2
4
0
0
0
1
0
1
0
0
0
0
0
1
0
486
231
104
455
227
103
163
275
277
452
321
349
1285
220
136
390
319
131
964
836
21
49
30
55
52
35
54
29
86
88
18
89
83
62
31
29
27
20
35
35
34
24
13
9
28
9
13
26
15
11
20
14
10
18
9
13
1
3
22
2
2
8
13
6
8
8
2
10
3
5
0
0
14
0
1
2
20
5
15
3
1
2
0
29
0
0
18
0
1
2
974
365
292
10
461
1190
282
1
1
2
0
2
1
0
1
5
2
1
0
0
0
3
281
462
419
3501
810
375
798
2227
1041
1071
213
206
2804
1743
672
460
*C = control, RA = rheumatoid arthritis, CC = chondrocalcinosis, R = Reiter’s syndrome, P = psoriatic arthritis
tSynovial fluid CHra is expressed as u n i t d l 0 mg protein; < 2 u n i t d l 0 mg is below normal
Most lymphocytes adhering to macrophages were identified as T cells by their
ability to bind sheep cells (Table 2). The
number of T cells adhering to macrophages
appeared proportional to the number of T
cells present in the culture. Most M-L ro1028
settes contained only T cells (Figure 2).
Some of the M-L rosettes contained both T
cells and unmarked cells. Many of the unmarked cells however formed kl-L rosettes
that failed to bind any sheep red cells
(Figure 3). The significance of the different
Arthritis and Rheumatism, Vol. 17, No. 6 (November-December 1974)
MACROPHAGE-LYMPHOCYTEROSETTE
Fig 1. Macrophage-lymphocyte rosette containing six lymphocytes,
from a 4-day culture of synovial fluid
leukocytes. Patient 11. Magnification
2000 before enlarging.
binding patterns is not clear, but the apparent exclusion of T cells from certain
macrophages suggests that the macrophagelymphocyte binding may not be an entirely
random event.
DISCUSSION
The macrophage-lymphocyte rosette appears to be a phenomenon of cellular interaction that occurs in' a complement-free
system in the presence of antigen. T h e interaction becomes apparent after 24 hours
of cell culture when lymphocytes become
Table 2. T-cell Assay of Synovial Fluid
Lymphocytes
Patient
No.
9
11
16
23*
T Cells/100
Synovial Fluid
Lymphocytes
T Cells/100
Synovial Fluid
Lymphocytes
Bound to
Macraphages
83
90
73
63
86
84
87
80
'Patient 23 had seropositive rheumatoid arthritis
firmly attached to macrophages by means
of their foot processes. U p to 10 or more
lymphocytes may surround the central
macrophage. McFarland and Heilman (1)
described this phenomenon in mixed lymphocyte cultures, where one population of
cells acts as the antigenic stimulus to the
second population of cells. Sulitzeanu described it in cultures in which he used bovine serum albumin as the antigen to stimulate rabbit lymphocytes previously sensitized to bovine serum albumin (2). The
adherence of autologous guinea pig cells
described by Siege1 (3) appears to be an entirely different phenomenon. It occurred
between peritoneal macrophages and thymocytes shortly after the cells were mixed
together, without addition of antigen and
without a 24-hour incubation.
The M-L rosette in synovial fluid from
rheumatoid patients appears to be the same
phenomenon described in these other
models. If it also has the same significance
as the other models, it suggests the presence
of antigen in rheumatoid synovial fluid. If
antigen is present, it must be in or on the
Arthritis and Rheumatism, Vol. 17, No. 6 (November-December 1974)
1029
HEPBURN ET AL
Fig 2. Macrophage-lymphocyte rosette containing four T cells, from a
culture of synovial fluid leukocytes
incubated with sheep cells on day 4.
Patient 11. Magnification 2000 before
enlarging.
cells themselves, as the cells have been
washed and the cultures do not contain the
synovial fluid itself. It seems quite likely
that any antigen related to the pathogenesis of rheumatoid arthritis would be present in the highest concentration a t . the
major site of disease-ie,
in the joint as
opposed to peripheral blood. The increased
M-L adherence in synovial fluid cell cultures however may reflect not only the
presence of antigen but also increased numbers of cells sensitized to that antigen. Since
the cells of four nonrheumatoid joints did
not demonstrate any significant degree of
rosetting, the stimulus for this phenomenon
is not common to all synovial fluids.
It is unlikely that rheumatoid factor itself
can explain the rosetting. Either IgG or
IgM rheumatoid factor might be expected
to bind to IgG on the surface of B cells,
enhancing macrophage-B cell adherence.
However such a mechanism does not explain the high degree of macrophage-T cell
adherence found in this study. Although
1030
IgM rheumatoid factor was present in the
blood of 6 rheumatoid patients, peripheral
blood lymphocytes from these patients
showed minimal M-L adherence. In addition, the cells from two rheumatoid joints
that lacked IgM rheumatoid factor demonstrated a high degree of macrophage-lymphocyte adherence.
It is possible that immune complexes
may play a role in stimulating maaophagelymphocyte adherence. IgG complexes of
high molecular weight are found in higher
frequency in synovial fluid than in peripheral blood (7). The normal synovial fluid
complement levels in patients 11, 16 and
19, however, suggests that complexes were
not present in these three synovial fluids to
any measurable degree (8). Some complexes
may have been present in phagocytic cells
(9) however and may therefore have been
present in the cultures of the synovial fluid
cells.
Although viruses have never been
identified in rheumatoid joints, in spite of
Arthritis and Rheumatism, Vol. 17, No. 6 (November-December 1974)
MACROPHAGE-LYMPHOCYTE ROSETTE
Fig 3. Macrophage-lymphocyte rosette containing no T cells, from a culture of synovial
fluid leukocytes incubated with sheep cells on day 4. Patient 11. Magnification 1600 before
enlarging.
attempts to do so (lo), we cannot exclude
the possibility that the macrophage-lymphocyte adherence observed in cultures of these
joints is virus mediated. T h e Sendai viruses
are known to be capable of fusing macrophages and lymphocytes (11). In the fusion
process, the lymphocyte nuclei enter the
main body of macrophage cytoplasm much
as they do in emperiopolesis (1). Although
most of the adhering lymphocytes in our
cultures appear at the periphery of the
macrophage, the lymphocytes occasionally
do lie within the macrophage.
Whatever the antigenic stimulus, bind-
ing of the lymphocyte and macrophage a p
pears to initiate a series of cellular events
which result in an immune response to
that antigen. McFarland and Heilman (1)
described the role of the macrophage-adherent lymphocyte in initiating blastogenesis in culture. Adherent lymphocytes began
to transform around the third day of culture, gradually detaching themselves from
the macrophage by day 5 and 6. T h e work
of Sulitzeanu et a1 (2) supports this observation with reports of increased thymidine
uptake in cultures with increased M-L adherence.
Arthritis and Rheumatism, Vol. 17, No. 6 (November-December 1974)
1031
HEPBURN ET AL
We have been unable to correlate the increased adherence in our cultures with increased thymidine uptake (Table l), or
with actual numbers of blasts seen on morphologic examination. (At day 4, 1000 cpm
represents approximately 2-3 lymphoblasts
per 100 lymphocytes.) At this time, we have
no explanation for the increased thymidine
uptake by some of the peripheral blood
cultures where M-L rosetting was minimal.
It is apparent that most of these macrophage-adherent lymphocytes from synovial
fluid, a predominantly T-cell population,
fail to go on to blastogenesis. Recent studies
from our own laboratory and from others
(12), have shown that synovial fluid lymphocytes from rheumatoid patients do not
respond to PHA as well as do peripheral
blood lymphocytes. This decreased response
is not due to decreased numbers of T cells
(Table 2). It may however reflect a defect
in the T cells themselves, a previous immunologic commitment, or the presence of
an inhibitor substance in synovial cell cultures, which could diminish blastogenesis.
Although the M-L rosette fails to function here as a precursor to any marked degree of blastogenesis, it appears otherwise
similar to the antigen induced models previously described. The failure of these
macrophage-adherent lymphocytes to transform in the presence of an implied antigen
may indeed reflect a similar abnormality in
the rheumatoid patient. Further evaluation
of the synovial fluid derived M-L rosette
may help to elucidate cellular interaction
and antigen processing in rheumatoid arthritis.
ACKNOWLEDGMENTS
The authors wish to express their appreciation
to Mrs. D. Kimmel and Mr. D. Johnson for their
expert technical assistance and to the members of
the Division of Rheumatology for referring their
patients to us for this study.
1032
REFERENCES
1. McFarland W, Heilman DH: Lymphocyte
foot appendage: its role in lymphocyte
function and in immunological reactions.
Nature 205:887-888, 1965
2. Sulitzeanu D, Kleinman R, Benzra D, et al:
Cellular interactions and the secondary response in vitro. Nature (New Biol) 229:254255, 1971
3. Siege1 I: Autologous macrophage-thymocyte
interactions. J Allergy 46: 190-194, 1970
4. Ropes MW, Bennett GA, Cobb S, et al:
1958 Revision of diagnostic criteria for
rheumatoid arthritis. Bull Rheum Dis 9:
175-176, 1958
5. Ritts RE, Jr: Immune functions of leukocytes, Laboratory Procedures in Clinical
Microbiology. Edited by JA Washington 11.
Little, Brown and Co. I n Press
6. Hepburn B, Ritts RE: A method for preparing permanently fixed slides of human T
lymphocytes. Mayo Clin Proc (In press)
7. Winchester RJ, Kunkel HG, Agnello V:
Occurrence of gamma globulin complexes
in serum and joint fluid of rheumatoid arthritis patients: Use of monoclonal rheumatoid factors as reagents for their demonstration. J Exp Med 134:286-295s, 1971
8. Winchester RJ, Agnello V, Kunkel HG:
T h e joint-fluid gamma globulin complexes
and their relationship to intraarticular complement diminution. Ann NY Acad Sci 168:
195-203, 1969
9. Hurd ER, Kinsella D, Ziff M: Immunohistologic studies of synoviocytes and synovial
exudate cells. J Exp Med 134:296-297s, 1971
10. Person DA, Sharp JT, Rawls WE: A search
for viruses and mycoplasmas in connective
tissue diseases. Arthritis Rheum 16:677-687,
1973
11. Harris H: Cell fusion, the Dunham Lectures, Chap 1. Cambridge, Mass, Harvard
University Press, 1970, p p 1-31
12. Reynolds MD, Abdou NI: Comparative
study of the in vitro proliferative responses
of blood and synovial fluid leukocytes of
rheumatoid arthritis patients. J Clin Invest
52:1627-1631, 1973
Arthritis and Rheumatism, Vol. 17, No. 6 (November-December 1974)
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