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The rheumatoid rosette
The Rheumatoid Rosette A Diagnostic Test Unifying Seropositive and Seronegative Rheumatoid Arthritis
JEAN-FRAN(~OIS BACH, M.D.~ FRANCOIS DELRIEU, M.D. FLORIAN DELBARRE, M.D. Paris, France
From the Clinique de Rhumatologie, HSpital Cochin, Fg Saint Jacques, Paris, France. Requests for reprints should be addressed to Dr. Jean-Fran(;ois Bach. Manuscript received October 1, 1969. Present address: Clinique N~phrologique, H6pital Necker, 151 rue de Sevres, Paris 15, France.
Volume 49, August 1970
Rheumatoid rosettes were obtained in vitro by mixing purified lymphocytes of patients with rheumatoid arthritis and human ORh-erythrocytes coated with rabbit immunoglobulin; 497 rheumatoid rosette tests were performed in 399 subjects. More than 6 rheumatoid rosettes/I,000 lymphocytes were found in 70 per cent of 138 patients with rheumatoid arthritis and in 5 per cent of 158 control subjects, excluding gouty patients. No direct correlation was observed between the number of rheumatoid rosettes and the serum titer of rheumatoid factor, and the proportion of positive test results was not significantly higher in seropositive (76 per cent) than in seronegative cases (64 per cent). A positive rheumatoid rosette test was significantly more frequent in patients with rheumatoid arthritis of recent onset than in those with long-standing rheumatoid arthritis and in patients with pain than in those without pain. A similar correlation was not found with the serum levels of rheumatoid factor. Biologic and clinical data suggest that rheumatoid rosette-forming cells are principally observed at the onset or during the active phase of rheumatoid arthritis whereas high levels of serum rheumatoid factor are more typical of the chronic phase of the disease. The rheumatoid rosette test is proposed as a new test, unifying seropositive and seronegative rheumatoid arthritis. When this test is combined with serum agglutination reactions, rheumatoid factor is demonstrated in more than 90 per cent of the patients with rheumatoid arthritis. A negative rheumatoid rosette test in a patient not treated with corticosteroids would suggest that the disease has become inactive. Rheumatoid rosette-forming cells are probably rheumatoid factor-producing cells or antigen sensitive cells involved in immunoglobulin recognition. A high proportion of patients with arthritis associated with more than four of the criteria of the American Rheumatism Association for rheumatoid arthritis is found not to give positive rheumatoid serologic reactions. This percentage has been variously estimated (23 per cent by Coste et al. [Z], 40 per cent by Morgan et al. [2]). It would be higher still if less restrictive criteria for the definition of rheumatoid arthritis were adopted. Several hypotheses can be put forward to explain this seronegativity including (1) absence of secretion of the rheumatoid factor, (2) insufficient secretion to raise the serum level over that observed in normal subjects, and (3) fixation on targets (immunoglobulins or cells) thus making its detection in the serum impossible. If either of the two latter hypotheses were correct, the rheumatoid factor would be found only at its source, i.e., at the cellular level. The rosette test has been introduced for that purpose. In a preliminary study in 1968 by Bach and Delbarre [3], rosette-forming cells were demonstrated in twenty-three patients with rheumatoid arthritis when their blood lymphocytes were mixed with human erythrocytes coated with rabbit immunoglobulin. More than 3 rosette.forming cells/I,000 lymphocytes ( R / l , 0 0 0 Ly) were found both in seropositive and seronegative patients with rheumatoid arthritis and always less than 3 in control subjects. This study has now been extended to 138 patients with rheumatoid arthritis and 261 control subjects including patients with various rheumatologic diseases.
213
THE RHEUMATOID ROSETTE - - BACH ET AL.
(~
parinized blood 25 ml Human 0 Rhred cells at 2 % ~ ' ~
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Hypotonic
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at 37 = C 15 -45 min
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Sensitized erythrocytes (1%)
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1 Microscopic reading 9 0 9 ~ L Rheumatoid Rosette Fig. 1.
MATERIAL AND METHODS The Rosette Test. This test is performed by mixing purified human lymphocytes with human ORh-erythrocytes coated with rabbit immunoglobulin. The rosettes thus obtained are designated rheumatoid rosettes (Fig. 1). Purification of lymphocytes: Lymphocytes are isolated by sedimentation and filtration through a nylon colLmn. Twenty milliliters of heparinized blood are collected in a tube and maintained at 37~ The red cells are allowed to sediment spontaneously for twenty to forty-five minutes. The supernatant is collected and passed through a nylon column [4]. The cells are washed three times in Hanks' medium (200 g, five minutes). Before the last washing the contaminating erythrocytes are eliminated by hypotonic shock produced as follows: the sediment of the second washing is quickly mixed with 4 ml of distilled water, the cell suspension is agitated for twenty seconds, and finally 30 ml of Hanks' medium are added. Perfect isotonicity is regained after the third washing. The lymphocyte suspension is adjusted to 15 x 10 ~ lymphocytes/ml. In normal subjects, very pure preparations are obtained with more than 95 per cent lymphocytes. The purification is not as good in arthritic patients. Sensitized erythrocytes: ORh-human erythrocytes are sensitized by incubation with a subagglutinating concentration of inactivated rabbit antihuman erythrocyte serum. No rosettes are formed when nonsensitized erythrocytes are used. It is not possible to utilize the first subagglutinating concen-
214
The rheumatoid rosette test.
tration for sensitization, as recommended for the WaalerRose reaction, because at this concentration the erythrocytes will agglutinate spontaneously after centrifugation, especially when mixed with white cells. For this reason the erythrocytes must be sensitized with a lower concentration of hemagglutinin. However, when the low concentration is decreased further, sensitization will eventually be insufficient to allow rosette formation. Thus, only a rather narrow range of hemagglutinin concentrations (i.e., 1 to 3 twofold dilutions) can be used. For each series of tests, a normal control subject with a negative test and a paLient with rheumatoid arthritis and an already known positive test are included. Three different suspensions of sensitized erythrocytes are prepared using hemagglutinin concentrations 2, 4 and 8 times lower than those indicated for the Waaler-Rose reaction. The results obtained with the intermediate concentrations are first considered and accepted as valid if the control subject has a negative reaction and the patient with rheumatoid arthritis a positive reaction. If the patient with rheumatoid arthritis has a negative result, a reading is obtained with the more sensitized erythrocytes. If the control subject has a positive result, the reading is made with the less sensitized erythrocytes. A fresh suspension of sensitized erythrocytes is prepared every day. Reading: A modification of Biozzi's method [5] has been used [6]; the following reagents are mixed in a hemolysis tube: 0.5 ml of the lymphocyte suspension (i.e., 4 X 106 lymphocytes), 0.2 ml of the 1 per cent suspension of sensi-
The American Journal of Medicine
THE RHEUMATOID ROSETTE- - BACH ET AL.
tized erythrocytes (i.e., 20 X 10~ erythrocytes), 0.3 ml of Hanks' medium. This mixture is centrifuged for five minutes (200 g) at room temperature, and then resuspended by gentle agitation ten minutes (vertical rotation at 10 rpm). The microscopic reading is performed in a 1 /~1 Malassez chamber. Lymphocytes and rosettes are counted separately. The rosette is defined as the agglutination of more than 4 erythrocytes around a lymphocyte (Fig. 2). In some instances the lymphocyte is difficult to see but its presence has been demonstrated in such cases after fluorescein labelling [6]. For each test two chambers are read. The result is expressed as the number of rosettes/I,000 lymphocytes. Reproducibility." The rheumatoid rosette test was repeated in thirty-eight patients with rheumatoid arthritis two to six times with intervals of less than fifteen days (98 tests). Three categories were defined, based on the number of rosettes/I,000 lymphocytes, namely, less than 6, between 6 and 10, and more than 10. The reproducibility was fairly good, since no change of category was observed in 50 of the 60 repeat tests. Sixty-four tests were repeated at intervals of more than fifteen days in twenty-six patients. A difference of more than 4 R / l , 0 0 0 Ly was observed i'n eight of the thirty-eight repeat tests performed. Other Technics. The Waaler-Rose reaction was performed with human ORh-erythrocytes coated with subagglutinating amounts of rabbit antihuman erythrocyte antibodies (Serpasteur, rue du Dr. Roux, Paris 15, France). The latex reaction was carried out according to Singer and Plotz r7]. Results of these tests are considered positive when the titer exceeds 1/16 for the Waaler-Rose reaction and 1/180 for the latex reaction. Synovial biopsy specimens were interpreted by C. Guiraudon using the criteria of the American Rheumatism Association. Clinical Material. The assay for rheumatoid rosettes was performed in 399 subjects. On the basis of the clinical manifestations these subjects could be classified as follows: Rheumatoid arthritis (138 cases): Rheumatoid arthritis was defined by the criteria of the American Rheumatism Association. Some of the patients were seen after admission to the hospital, but most of them were outpatients. A number of clinically quiescent cases, often still seropositive, were also examined. The proportion of seronegative cases (49 per cent) was deliberately made unusually large for the purpose of better comparison of seropositive and seronegative rheumatoid arthritis. Within the two categories, however, the patients were chosen randomly. Gout (sixty-one cases): Gout was defined by the finding of a serum uric acid level of more than 7 mg/100 ml in men, and 6 mg/100 ml in women. Diseases of connective tissue (twenty-five cases): Systemic lupus erythematosus (seventeen cases), scleroderma (seven cases), polymyositis (one case). Other rheumatic diseases (eighty-one cases): Psoriatic arthritis (four cases), ankylosing spondylitis (five cases), other forms of inflammatory rheumatism (six cases), lumbalgias (seventeen cases), arthrosis (twenty-six cases), others (twenty-three cases). Diseases in which rheumatoid factor is sometimes found independently of any joint involvement [8] (seventeen cases): Cirrhosis of the liver (six cases), patients with renal transplants [9] (seven cases), subacute endocarditis (one case), chronic suppuration (two cases), lymphosarcoma (one case). Controls (seventy-seven cases): Normal subjects (forty-one), aged fifteen to sixty years; patients with miscellaneous non-
Volume 49, August 1970
Fig. 2. Rheumatoid rosette observed in the blood of a patient with seropositive rheumatoid arthritis. rheumatologic diseases (thirty-six cases): renal failure, systemic hypertension, adrenal and thyroid insufficiency, hyperthyroidism, hemochromatosis, duodenal peptic ulcer, alcoholism, breast cancer, Steinert's disease, Raynaud's syndrome, malaria, hypogonadism, erythema multiforme. In all cases a clinical examination was performed, with particular attention to joint involvement. Biologic tests were also performed including, in most cases, the measurement of erythrocyte sedimentation rate and the Waaler-Rose reaction. RESULTS
Control Subjects. In all normal subjects and in patients with nonrheumatologic diseases the number of rosettes has always been less than or equal to 6 / 1 , 0 0 0 Ly, with one exception. This patient ( 9 / 1 , 0 0 0 Ly) and three of the five patients with 6 / 1 , 0 0 0 Ly had hyperthyroidism, a disease supposed by some to be of immunologic origin. Wher~ the group of patients with hyperthyroidism was excluded, all those with more than 3 R / l , 0 0 0 Ly were found to be subjects over forty years of age (twenty-three TABLE I
Practical Interpretation of the Rosette Test
Rosette Number and Interpretation < 4 R/1,000 Ly --Negative 4 - 6 R / l , 0 0 0 Ly --Doubtful 7 - 1 0 R / l , 0 0 0 Ly = Positive > 10 R / l , 0 0 0 Ly --Strongly positive
Rheumatoid
RA 138 Cases
Normal and Rheumatic Controls (Gout Excepted) 158 Cases
20%
84%
10%
11%
28%
2%
42%
3%
215
THE
RHEUMATOID
ROSETTE
--
BACH
ET
AL.
R ]I00~ Ly 40
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30
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. . . . . . P01ya~hritis Ser0pesitive (68Cases)
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Polyarthritis Ser0negative (70 Cases)
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~'ff~'~J'~i{ Nonrheumatotogic Diseases (77 Cases)
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of the seventy-seven subjects of this control group were over forty). Three normal subjects less than twenty years old had 3 R/1,O00 Ly. Further on, we shall consider as positive the results with more than 6 rosettes/i,000 lymphocytes. Patients with Rheumatoid Arthritis. More than 6 R/ 1,000 Ly were found in ninety-seven of the 138 patients with rheumatoid arthritis (i.e., 70 per cent, Table I and Fig. 3). Correlation with the serum level of rheumatoid factor: No direct correlation has been found between the results of the Waaler-Rose reaction or the FII latex test and the number of rosette-forming cells (Fig. 4), The percentage of patients with a positive rosette test is higher among seropositive patients (76 per cent) than among serenegative patients (64 per cent), but the difference is not significant (P > 0.10) (Table II). The proportion of patients with a large number of rosettes (i.e., more than 15/1,000 Ly) is higher in seropositive patients (26 per cent) than in seronegative patients (15 per cent), but here again no significant difference is found between the two groups (P > 0.10). However, a critical difference was observed when patients with high and low titers of rheumatoid factor were compared. The number of resettes/1,000 Ly was significantly lower in patients with high titer of rheumatoid factor (over 1/128) for the Waaler-Rose reaction than in those with low titers. Table III shows a significant negative correlation between rosette number and rheumatoid factor titers. Correlation with clinical data:, The correlation between clinical data and the rheumatoid rosette test has been compared with that observed for the Waaler-Rose reaction. It can be stated first that the degree of positivity for rheumatoid rosette or Serum tests was not correlated with any of the criteria to be considered.
216
=eee ,9 Z:II|;
Fig. 3. N u m b e r of rheumatoid rosettes Gout (61 Cases)
in 138 patients with rheumatoid arthritis and 219 control subjects.
Duration of the disease: As shown in Figure 5, the percentage of patients with a positive rosette test is significantly higher in those with recent onset (P < 0.01). Conversely, the percentage of seropositive patients is higher in those with long-standing disease (P < 0.05), Number of joints involved in the disease: No correlation has been found between this criterion and rosette or Waaler-Rose test positivity. Destructive lesions and rheumatoid arthritis carpitis (x.ray examination): Destructive lesions, including signs of carpitis, were found significantly more often in serepositive than in seronegative patients, but not more often in patients with a positive rosette test than in those with a negative rosette test (Fig. 6 and 7). Pain: The pain was classified as nil, moderate or severe. A significant correlation was observed between the intensity of pain at the time of the test and the percentage of positive reactions to the rheumatoid rosette test. Such a correlation was not found for the Waaler-Rose reaction (Fig. 8). It is noteworthy that among the sixteen seropositive patients with negative rosette tests, nine had no pain and considered themselves cured; of the seven others, five had pain but received corticosteroids (the proportion of patients who received prednisone treatment is significantly higher [P < 0.01] in this small category than in the whole group of 138 patients with rheumatoid arthritis since only twenty-nine of them received corticosteroids). Correlation with biological data: Erythr0cyte sedimentation rate: When the erythrocyte sedimentation rate was over 60 mm after one hour, the percentage of positive rosette tests was significantly higher (91 per cent) than when the erythrocyte sedimentation rate was under 60 mm (76 per cent) (P < 0.01). A similar correlation was also observed between seropositive (77 per cent) and seronegative (52 per cent) patients.
The
American
Journal
of Medicine
THE RHEUMATOID ROSETTE- - BACH ET AL.
WIO00 Ly 40
30
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20
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9
9
9
9 9149149
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9 69
9149
9
9
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Fig. 4.
Correlation between the number of rheumatoid rosettes and the results of Waaler-Rose reaction.
TABLE II
< 1/32
1/32
1/128 1 / 2 5 6 1/512 V1024
Results of the Rheumatoid Rosette Test in 138 Patients with Rheumatoid Arthritis (RA) and Seventy-Seven Control Subjects Subjects
No. of Cases
Normal subjects and patients with miscellaneous nonrheumatic diseases Seropositive RA Seronegative RA
TABLE III
_< 6 R/l,000 Ly
77 68 70
Negative Correlation Between Rosette Number and Serum Titer of Rheumatoid Factor in Seropositive Patients (P < 0.05)
Rosette Number _< 20 R / l , 0 0 0 Ly > 20 R / l , 0 0 0 Ly
6-10 R/I,000 Ly
98.7% 24% 36%
TABLE IV
Waaler-Rose Reaction
TABLE V
V64
> 10 R/l,000 Ly
1.3% 27% 29%
0% 49% 35%
Correlation Between Number of Rosettes and Gamma Globulin Percentage (P < 0.01)
< 1/128
> 1/128
Gamma Globulin Level
15 75
1 29
_< 25% > 25%
Number of Rosettes _< 22/1,000 Ly
> 22/1,000 Ly
70 8
4 7
Results of the Rheumatoid Rosette Test in Rheumatic Diseases Other than Rheumatoid Arthritis Disease
Inflammatory arthritis Psoriatic rheumatism Ankylosing spondylitis Others
No. of Cases
< 6 R/l,000 Ly
4 5 6
4 (100%) 5 (100%) 6 (100%)
6-10 R/l,000 Ly 0 0 0
> 10 R/I,000 Ly 0 0 0
Gout Monoarticular Polyarticular
10 51
8 (80%) 27 (53%)
1 (10%) 9 (18%)
1 (10%) 15 (29%)
Other rheumaticdiseases
66
60 (90%)
2 (3%)
4 (7%)
Volume 49, August 1970
217
IHP_ I~HI-UMAIFOID ROSETTE - - BACH ET AL.
RHEUMATOID ROSETTETEST
WAALER- ROSE REACTION
100%
50%
0%
P
YEARS < 2
- 10
(23 cases)
(42 cases)
< 2
') 10 (40 cases)
P< O.O1 YEARS
2-i0
(23 cases)
> 1o
(42 cases)
(40 cases)
Fig. 5. Percentage of positive results in the rheumatoid rosette test and the Waaler.Rose reaction in relation to duration o f the arthritis (P < 0 . 0 1 ) .
WAALER- ROSE REACTION
RHEUMATOID ROSETTETEST
100%
II
50 %
P ( 0.01
P>0.05
0% No Destruction (49 cases)
No
Destruction (57 cases)
Destruction (49 cases)
WAAIFR- ROSE REACTION
RHEUMATOID ROSETI'E TEST
100%
I
P 3'0.05
0% No Carpitis ()4 cases)
100%.
Destruction (57 cases)
Fig. 6. Percentage of positive results in the rheumatoid rosette test and the Waaler.Rose reaction in relation to the existence and intensity of destructive joint lesions.
I
P< 0. 01
No Carpitis (34 cases)
Carpitis (33 cases)
RHEUMATOID ROSETTETEST
Carpitis (33 cases)
Percentage of positive results in the rheumatoid rosette test and the Waaler.Rose reaction in relation to the existence and intensity of carpitis. Fig. 7.
WAALER- ROSE REACTION
50%
0%
P(0.~
No Pain (31 cases)
218
Moderat, Pain
Severe Pain
(61 cases)
(11 cases)
No Pain (31 cases)
i
Moderate Pain (61 cases)
P >0. I0 Severe Pain (1.1 cases)
Fig. 8. Percentase of positive results in the rheumatoid rosette test and the Waaler-Rose reaction in relation to the existence and intensity of pain.
The American Journal of Medicine
THE RHEUMATOID ROSETTE - -
TABLE VI
BACH ET AL.
Results of the Rheumatoid Rosette Tests in Diseases Occasionally Associated with Significant Serum Titers of Rheumatoid Factor Disease
No. of Cases
< 6 R/l,000 Ly
6-10 R/l,000 Ly
> 10 R/l,000 Ly
Diseases of connective tissue Scleroderma Systemic lupus erythematosus Polymyositis Liver cirrhosis Subacute endocarditis Renal transplantation Chronic infection Lymphosarcoma
7 17 1 6 1 7 2 1
4 14 1 2 1 5 0 1
2 1 0 3 0 1 0 0
1 2 0 1 0 1 2 0
Serum gamma globulin level: No correlation was found between the serum gamma globulin level and the number of rheumatoid rosettes or serum titer of rheumatoid factor. However, as shown in Table IV, seven of fifteen patients with more than 25 per cent gamma globulin in the serum had more than 22 R / l , 0 0 0 Ly, a proportion significantly higher than that found in patients with less than 25 per cent gamma globulin in the serum. Correlation with treatment: Twenty-nine patients were treated with corticosteroids. The proportion of positive rheumatoid rosette tests in this group was not significantly lower than in the group of patients not treated with corticosteroids. Four of five patients treated with more than 10 mg of prednisone daily had a positive rosette test. The incidence of benefit of corticosteroid treatment on rheumatoid rosettes has already been men. tioned for the category of seropositive patients with a negative rosette test and pain. No correlation has been found with other treatments, including gold preparations, antimalarial therapy and salicylate. The number of patients treated with immunosuppressive drugs who could be examined was insuffi. cient to allow a definite conclusion about the influence of these drugs on the rheumatoid rosette test. Gout: A high percentage of positive results (43 per cent) was observed in gouty patients. These positive results were found significantly more often (P < 0.01) in those with polyarticular (47 per cent) than monoarticular (20 per cent) involvement. Other diseases: The results obtained in rheumatologic diseases other than rheumatoid arthritis and gout are presented in Table V. They demonstrate the relative specificity of the rheumatoid rosette test. The positive results have always been observed in patients more than forty years old. The results obtained in diseases occasionally associated with rheumatoid factor in the serum are depicted in Table VI. COMMENTS Agglutination reactions, of which the most commonly used are the Waaler-Rose and the latex test, are very useful in the diagnosis of inflammatory polyarthrit;s. However, they are inconsistently positive even in clinically typical cases. It thus seemed worthwhile to investigate the detection of rheumatoid factor in patients with
Volume 49, A u g u s t 1970
rheumatoid arthritis by means of a new and fundamentally different technic: the rosette test. This test makes possible the detection of antibodies at the cellular level. It.may be positive even when the antibodies are not present at a high level in the serum, and we have found (Bach, unpublished data) no correlation in man between the serum titer of natural antisheep red blood cell antibodies and the number of blood lymphocytes forming rosettes with sheep red blood cells. The rosette test has been used with lymphocytes from rheumatoid arthritis afflicted patients and human erythrocytes coated with rabbit gamma globulin. Rheumatoid rosettes were found in 75 per cent of cases of rheumatoid arthritis as defined by the criteria of the American Rheumatism Association. The percentage of lymphocytes forming rosettes ranged from 0.6 to 4.4 per cent; this rate of 4.4 per cent is higher than that observed in the mouse or the guinea pig after intravenous stimulation by sheep red blood cells [5]. This difference may be partly due to the fact that in these experimental studies rosettes are counted in the spleen or the lymph nodes and not in the peripheral blood. There was no direct correlation between the number of rheumatoid rosettes and the titer in the Waaler-Rose reaction. Rheumatoid rosettes were found in 76 per cent of cases of seropositive rheumatoid arthritis and in 64 per cent of cases of seronegative rheumatoid arthritis. With the exception of gout, the search for rheumatoid rosettes was negative in 98 per cent of the control cases. The specificity of the rheumatoid rosette test is comparable to that of the Waaler-Rose reaction. The rheumatoid rosette test can thus be proposed as a new method for the diagnosis of rheumatoid arthritis. The significance of the rheumatoid rosettes has been investigated [9]. It has been shown that the rheumatoid rosette is specifically inhibited by preincubation of the lymphocytes with antihuman immunoglobulin serum, and by normal human or rabbit serum. Rosette formation can also be inhibited by noncytotoxic concentrations of azathioprine, as already described for antisheep red blood cell rosettes [10]. Morphologic studies have shown that they are mostly lymphocytes with a majority of small lymphocytes. Similar properties have been reported for the rosette-forming cells observed in the mouse after stimulation by human IgG i l l ] and these rosette-forming cells
219
THE
RHEUMATOID
ROSETTE --
BACH
ET AL.
red blood cells [5]. In one strain of mice, a good correlation is observed during the course of immunization between the number of rosette-forming cells and the titer of antisheep red blood cells antibodies (a delay of one to two days must be taken into account however, because the cellular response occurs before the appearance of serum antibodies). In rheumatoid arthritis, on the other hand, no direct correlation is found between the number of rheumatoid rosettes and the serum level of rheumatoid factor. This can be interpreted in several ways: (1) The serum level of rheumatoid factor depends mainly on the secretory activity of a few cells producing large amounts of antibody. The increase of the number of rheumatoid rosettes, essentially due to small lymphocytes, would represent the immediate response to a new antigenic stimulation, preceding the eventual increase of serum antibody level; in some cases, the stimulation might be strong enough to induce a cellular response but not an increase of the serum antibody titer. (2) The antibody-producing cells are not always present in the peripheral blood. Thus Biozzi et al. [13] have shown rosette-forming cells to be present in mouse blood after intravenous immunization, but only transitorily. In some circumstances, rheumatoid rosette-forming cells might be localized to lymphoid organs, e.g., spleen or lymph nodes. (3) There is a delay of several days between the appearance of rheumatoid rosette-forming cells in blood and the increase in titer of serum rheumatoid factor, and again between their disappearance and the decrease in serum rheumatoid factor titer, exactly as observed in experimental systems. The second period of delay could be explained by the slow catabolism of rheumatoid factor. (4) All rheumatoid factor-forming cells are not antibody forming cells but partly antigen-sensitive ceils as suggested. The significantly smaller average number of rheumatold rosettes in patients with a Waaler-Rose reaction titer over 1/256, as compared to patients with a titer under or equal to 1/256, may be interpreted as a repressive effect related to the high serum antibody level, similar to that demonstrated by Brown and Epstein [11] in the mouse when human rheumatoid factor, injected into mice
have been considered to be the producers of antibodies directed against human IgG [11]. All these data suggest that rheumatoid rosettes are formed by rheumatoid factorproducing cells whether the rheumatoid factor passes into the serum or remains on the lymphocyte's surface as it is supposed to do in antigen-sensitive cells. In fact it has been demonstrated in the mouse that rosette-forming cells are antigen-sensitive cells [12,13]. However, this interpretation might not be valid for the rheumatoid rosettes found in small number in some normal subjects (as in normal mice at a comparable level [11]) and in high number in some gouty patients, because the immunologic and morphologic studies here reported have not yet been performed in these situations. If they are indeed cells secreting rheumatoid factor, this secretion would probably be secondary to some unknown immunologic reaction. If they are not rheumatoid factor producing cells, one of the following functions could be ascribed to them: (1) Formation of natural antirabbit IgG antibody. A positive WaalerRose reaction is defined by a titer over 1/16, and the agglutination of sensitized erythrocytes is observed indeed at a low titer with most control serum samples. (2) Ability to agglutinate Ig-cOated red blood cells, nonspecifically, as described by Lo Buglio et al. r14]. The formation of these nonspecific rosettes has been described mainly with blood monocytes and could be inhibited by human serum. However, it could not be inhibited by antihuman immunoglobulin serum, in contrast to rheumatoid rosettes formed with lymphocytes in patients with rheumatoid arthritis. Four categories of patients can be distinguished when considering both cellular (rosette test) and serum(Waaler-Rose reaction and F II latex test) tests for detection of rheumatoid factor.
Seropositivity, rosette positivity: This category is the most common (fifty-two cases, 37 per cent), the more so if an unselected population of patients with rheumatoid arthritis is considered. This situation is in keeping with the experimental model, i.e., rosette formation obtained in the mouse after injection of human IgG [11] or sheep
IOOY.
50?00.
o~ RR Test
220
WR Reac;tion
RR Test
WR Reaction
RR Test
I
WR Reaction
'RHEUMATOID SYNOVITIS"
"A SPECIFIC SYNOVITIS"
DIVERSE
(48 cases)
(12 cases)
(24 cases)
Fig. 9. Percentage of positive results in the rheumatoid rosette test and the Waaler-Rose reaction in relation to synovial pathology.
The American Journal of M e d i c i n e
THE RHEUMATOID ROSETTE- - BACH ET AL.
immunized with human IgG, depressed the number of rheumatoid factor cells formed with IgG-coated sheep erythrocytes. Seropositivity, rosette negativity. This group of only six. teen cases (11 per cent) includes patients without pain (eight cases) or treated with corticosteroids (six cases). In patients without pain, the disease can be considered inactive, at least temporarily. The positivity of the WaalerRose reaction could be due to the secretion of rheuma. told factor by a small number of cells producing large amounts or to the central localization of rheumatoid factorproducing cells. In practical terms, the finding of a negative rosette test in seropositive patients with rheumatoid arthritis not treated by corticosteroids could thus be taken as evidence for a favorable course. Since a comparatively large number of patients treated with corticosteroids are found in this group, corticosteroids may act upon rheumatoid rosette-forming cells, although no difference has been found in the proportion of patients with a positive rheumatoid rosette reaction when the group of corticosteroid-treated patients considered in its entirety was compared to the group of patients not treated with corticosteroids. Seronegativity, rosette positivity: Sixty-four per cent of seronegative patients have a positive rosette test (fortyfive cases). Seronegativity has been defined as a Waaler-Rose test titer under 1/32; hence, most cases are not truly "negative." The various reasons which may explain the absence of a direct correlation between the number of rheumatoid rosettes and the serum rheumatoid factor level have already been discussed and can be applied to the particular problem of the so-called seronegative patient with a positive rosette test. In clinical studies [1,2,16] only slight differences were found between the group of seropositive and the group of sero. negative cases of rheumatoid arthritis when the severity of the course and the frequency of carpitis and destruc. tive lesions were assessed. Hess and Ziff [17] showed that it was possible to fix gamma globulin complexes on leukocytes of both seropositive and seronegative patients. The fact that the proportion of patients with a positive rosette test is not significantly different in seronegative and seropositive rheumatoid patients, can be given as a further strong argument for a unified concept of the
disease. If rheumatoid rosette.forming cells are considered to be rheumatoid factor-producing cells, seronegativity could then be ascribed to several mechanisms, of which the most likely would be a low secretion or peripheral fixation of rheumatoid factor, and the least likely a complete absence of antibody release into the serum. In the case of a low secretion rate, it would be easy to explain why the rheumatoid factor titer may not rise above the level observed in normal subjects. Seronegativity, rosette negativity: This situation has been observed in twenty.five cases (18 per cent). With the simultaneous use of the Waaler-Rose reaction and the rheumatoid rosette test, rheumatoid factor can thus be demonstrated in all of an unselected population of rheumatoid arthritis patients in more than 90 per cent of cases. In the residual 10 per cent are included patients who recovered completely and also probably some diagnostic errors. It is noteworthy that among the fortyeight patients whose synovial biopsy specimen was typical of rheumatoid synovitis, 88 per cent were rosette. positive, whereas only 65 per cent were seropositive. (Fig. 9). The clinical signs were analyzed in 138 cases of rheumatoid arthritis and compared to the results of the rheumatoid rosette test and Waaler-Rose reaction. The rosette test was significantly more often positive in patients with recent onset of rheumatoid arthritis than in those with long-standing disease and in patients with pain than in patients without pain. These two correlations were not observed for the Waaler-Rose reaction. Conversely, the Waaler-Rose reaction was significantly more frequently positive in patients with destructive lesions or carpitis. This would be expected since destructive lesions and carpitis occur generally after several years. Conversely, the rheumatoid rosette test is no more frequently positive in old or destructive cases. It is thus suggested that rheumatoid rosettes may be taken as a salient feature of early or active cellular phases of rheumatoid disease, whereas rheumatoid factor will be found in the serum at significantly high levels only in chronic cases and even when the disease has become completely inactive. The significant correlation found between positive rheumatoid rosette tests and high erythrocyte sedimentation rates adds support to this hypothesis.
REFERENCES
1. Coste F, Delbarre F, Amor B, Bontoux D, Broulhet H, Guiraudon C, Menkes C, Saporta L: Etude comparEe de cent cas de polyarthrites (rhumato~des ?) sEronegatives et de cent cas de polyarthrTtes rhumatoides sero-positives. Sem Hop Paris 44: 777, 1968. 2. Morgan ES, Atwater EC: A comparison of patients with sero-positive and sero-negative rheumatoid arthritis. Med Clin NAmer 52: 533, 1968. 3. Bach JF, Delbarre F: Nouvelle methode de detection du facteur rhumatoTde au niveau cellulaire. C R Acad Sci [D] (Paris) 267: 134, 1968.
Volume 49, August 1970
4. Debray-Sachs M, Dormont J: Isolement des lymphocytes b partir du sang circulant chez I'homme normal et urEmique. Rev Franc Etud Clin Biol 13: 414, 1968. 5. Biozzi G, Stiffel C, Mouton D, Liacopoulos-Briot M, Decreusefond C, Bouthillier Y: Etude du phEnomene de I'ammunocytoadhdrence au cours de I'immunisation. Ann Inst Pasteur (Paris) 110: 7, 1966. 6. Bach JF, Dormont J, Dardenne M, Balner H: In vitro rosette inhibition by anti-human ALS. Correlation with skin graft prolongation in primates. Transplantation 8: 269, 1969.
221
THE RHEUMATOID ROSETTE - - BACH ET AL.
7. Singer JM, Plotz CM: The latex-fixation test. I. Application to the serologic diagnosis of rheumatoid arthritis. Amer J Med 21: 888, 1956. 8. Grieble HG, Bach GL, Szanto M, Anderson TO: The serologic diagnosis of rheumatoid arthritis. J Chron Dis 21: 667, 1969. 9. Bach JF, Delrieu F, Delbarre F: Etude immunologique du phL~nomEne de la rosette rhumatoTde. In preparation. 10. Bach JF, Dardenne M, Fournier C: In vitro evaluation of immunosuppressive drugs. Nature 222: 698, 1969. 11. Brown PJC, Epstein WV: Influence of human rheumatoid factor on numbers of antibody-producing cells. Arthritis Rheum 12: 1, 1969. 12. Bach JF, Dardenne M: Signification du ph~nomene de rosette spontan~e. C R Acad Sci [D] (Paris) 264: 751, 1969.
222
13. Bach JF, Dardenne M, Muller JY: In vivo specific antigen recognition by rosette-forming cells. Nature (in press). ]4. Lo Buglio AF, Cotran RS, Jandl JH: Red cells coated with immunoglobulin G: binding and sphering by mononuclear cells in man. Science 158: 1582, 1968. 15. Biozzi G, Stiffel G, Mouton D, Bouthillier Y, Decreusefond C: A kinetic study of antibody producing cells in the spleen of mice immunized intravenously with sheep erythrocytes. Immunology 14: 7, 1968. 16. Bland JH, Brown EW: Sero-negative and sero-positive rheumatoid arthritis. Clinical, radiological and biochemical differences. Ann Intern Med 60: 88, 1964. 17. Hess E, Ziff M: Reaction of rheumatoid leukocytes with fluorescent aggregated gammaglobulin. J Clin Invest 39" 996, 1960.
The American Journal of Medicine
JEAN-FRAN(~OIS BACH, M.D.~ FRANCOIS DELRIEU, M.D. FLORIAN DELBARRE, M.D. Paris, France
From the Clinique de Rhumatologie, HSpital Cochin, Fg Saint Jacques, Paris, France. Requests for reprints should be addressed to Dr. Jean-Fran(;ois Bach. Manuscript received October 1, 1969. Present address: Clinique N~phrologique, H6pital Necker, 151 rue de Sevres, Paris 15, France.
Volume 49, August 1970
Rheumatoid rosettes were obtained in vitro by mixing purified lymphocytes of patients with rheumatoid arthritis and human ORh-erythrocytes coated with rabbit immunoglobulin; 497 rheumatoid rosette tests were performed in 399 subjects. More than 6 rheumatoid rosettes/I,000 lymphocytes were found in 70 per cent of 138 patients with rheumatoid arthritis and in 5 per cent of 158 control subjects, excluding gouty patients. No direct correlation was observed between the number of rheumatoid rosettes and the serum titer of rheumatoid factor, and the proportion of positive test results was not significantly higher in seropositive (76 per cent) than in seronegative cases (64 per cent). A positive rheumatoid rosette test was significantly more frequent in patients with rheumatoid arthritis of recent onset than in those with long-standing rheumatoid arthritis and in patients with pain than in those without pain. A similar correlation was not found with the serum levels of rheumatoid factor. Biologic and clinical data suggest that rheumatoid rosette-forming cells are principally observed at the onset or during the active phase of rheumatoid arthritis whereas high levels of serum rheumatoid factor are more typical of the chronic phase of the disease. The rheumatoid rosette test is proposed as a new test, unifying seropositive and seronegative rheumatoid arthritis. When this test is combined with serum agglutination reactions, rheumatoid factor is demonstrated in more than 90 per cent of the patients with rheumatoid arthritis. A negative rheumatoid rosette test in a patient not treated with corticosteroids would suggest that the disease has become inactive. Rheumatoid rosette-forming cells are probably rheumatoid factor-producing cells or antigen sensitive cells involved in immunoglobulin recognition. A high proportion of patients with arthritis associated with more than four of the criteria of the American Rheumatism Association for rheumatoid arthritis is found not to give positive rheumatoid serologic reactions. This percentage has been variously estimated (23 per cent by Coste et al. [Z], 40 per cent by Morgan et al. [2]). It would be higher still if less restrictive criteria for the definition of rheumatoid arthritis were adopted. Several hypotheses can be put forward to explain this seronegativity including (1) absence of secretion of the rheumatoid factor, (2) insufficient secretion to raise the serum level over that observed in normal subjects, and (3) fixation on targets (immunoglobulins or cells) thus making its detection in the serum impossible. If either of the two latter hypotheses were correct, the rheumatoid factor would be found only at its source, i.e., at the cellular level. The rosette test has been introduced for that purpose. In a preliminary study in 1968 by Bach and Delbarre [3], rosette-forming cells were demonstrated in twenty-three patients with rheumatoid arthritis when their blood lymphocytes were mixed with human erythrocytes coated with rabbit immunoglobulin. More than 3 rosette.forming cells/I,000 lymphocytes ( R / l , 0 0 0 Ly) were found both in seropositive and seronegative patients with rheumatoid arthritis and always less than 3 in control subjects. This study has now been extended to 138 patients with rheumatoid arthritis and 261 control subjects including patients with various rheumatologic diseases.
213
THE RHEUMATOID ROSETTE - - BACH ET AL.
(~
parinized blood 25 ml Human 0 Rhred cells at 2 % ~ ' ~
U
Nylon
JL
Rabbit anti-human hoemagglutinin (sub-agglutinating concentration)
Hypotonic
V
at 37 = C 15 -45 min
Lymphocytes enriched leukocytes Suspension
Sensitized erythrocytes (1%)
U
1
Centrifugation
12~176 Agitation
1 Microscopic reading 9 0 9 ~ L Rheumatoid Rosette Fig. 1.
MATERIAL AND METHODS The Rosette Test. This test is performed by mixing purified human lymphocytes with human ORh-erythrocytes coated with rabbit immunoglobulin. The rosettes thus obtained are designated rheumatoid rosettes (Fig. 1). Purification of lymphocytes: Lymphocytes are isolated by sedimentation and filtration through a nylon colLmn. Twenty milliliters of heparinized blood are collected in a tube and maintained at 37~ The red cells are allowed to sediment spontaneously for twenty to forty-five minutes. The supernatant is collected and passed through a nylon column [4]. The cells are washed three times in Hanks' medium (200 g, five minutes). Before the last washing the contaminating erythrocytes are eliminated by hypotonic shock produced as follows: the sediment of the second washing is quickly mixed with 4 ml of distilled water, the cell suspension is agitated for twenty seconds, and finally 30 ml of Hanks' medium are added. Perfect isotonicity is regained after the third washing. The lymphocyte suspension is adjusted to 15 x 10 ~ lymphocytes/ml. In normal subjects, very pure preparations are obtained with more than 95 per cent lymphocytes. The purification is not as good in arthritic patients. Sensitized erythrocytes: ORh-human erythrocytes are sensitized by incubation with a subagglutinating concentration of inactivated rabbit antihuman erythrocyte serum. No rosettes are formed when nonsensitized erythrocytes are used. It is not possible to utilize the first subagglutinating concen-
214
The rheumatoid rosette test.
tration for sensitization, as recommended for the WaalerRose reaction, because at this concentration the erythrocytes will agglutinate spontaneously after centrifugation, especially when mixed with white cells. For this reason the erythrocytes must be sensitized with a lower concentration of hemagglutinin. However, when the low concentration is decreased further, sensitization will eventually be insufficient to allow rosette formation. Thus, only a rather narrow range of hemagglutinin concentrations (i.e., 1 to 3 twofold dilutions) can be used. For each series of tests, a normal control subject with a negative test and a paLient with rheumatoid arthritis and an already known positive test are included. Three different suspensions of sensitized erythrocytes are prepared using hemagglutinin concentrations 2, 4 and 8 times lower than those indicated for the Waaler-Rose reaction. The results obtained with the intermediate concentrations are first considered and accepted as valid if the control subject has a negative reaction and the patient with rheumatoid arthritis a positive reaction. If the patient with rheumatoid arthritis has a negative result, a reading is obtained with the more sensitized erythrocytes. If the control subject has a positive result, the reading is made with the less sensitized erythrocytes. A fresh suspension of sensitized erythrocytes is prepared every day. Reading: A modification of Biozzi's method [5] has been used [6]; the following reagents are mixed in a hemolysis tube: 0.5 ml of the lymphocyte suspension (i.e., 4 X 106 lymphocytes), 0.2 ml of the 1 per cent suspension of sensi-
The American Journal of Medicine
THE RHEUMATOID ROSETTE- - BACH ET AL.
tized erythrocytes (i.e., 20 X 10~ erythrocytes), 0.3 ml of Hanks' medium. This mixture is centrifuged for five minutes (200 g) at room temperature, and then resuspended by gentle agitation ten minutes (vertical rotation at 10 rpm). The microscopic reading is performed in a 1 /~1 Malassez chamber. Lymphocytes and rosettes are counted separately. The rosette is defined as the agglutination of more than 4 erythrocytes around a lymphocyte (Fig. 2). In some instances the lymphocyte is difficult to see but its presence has been demonstrated in such cases after fluorescein labelling [6]. For each test two chambers are read. The result is expressed as the number of rosettes/I,000 lymphocytes. Reproducibility." The rheumatoid rosette test was repeated in thirty-eight patients with rheumatoid arthritis two to six times with intervals of less than fifteen days (98 tests). Three categories were defined, based on the number of rosettes/I,000 lymphocytes, namely, less than 6, between 6 and 10, and more than 10. The reproducibility was fairly good, since no change of category was observed in 50 of the 60 repeat tests. Sixty-four tests were repeated at intervals of more than fifteen days in twenty-six patients. A difference of more than 4 R / l , 0 0 0 Ly was observed i'n eight of the thirty-eight repeat tests performed. Other Technics. The Waaler-Rose reaction was performed with human ORh-erythrocytes coated with subagglutinating amounts of rabbit antihuman erythrocyte antibodies (Serpasteur, rue du Dr. Roux, Paris 15, France). The latex reaction was carried out according to Singer and Plotz r7]. Results of these tests are considered positive when the titer exceeds 1/16 for the Waaler-Rose reaction and 1/180 for the latex reaction. Synovial biopsy specimens were interpreted by C. Guiraudon using the criteria of the American Rheumatism Association. Clinical Material. The assay for rheumatoid rosettes was performed in 399 subjects. On the basis of the clinical manifestations these subjects could be classified as follows: Rheumatoid arthritis (138 cases): Rheumatoid arthritis was defined by the criteria of the American Rheumatism Association. Some of the patients were seen after admission to the hospital, but most of them were outpatients. A number of clinically quiescent cases, often still seropositive, were also examined. The proportion of seronegative cases (49 per cent) was deliberately made unusually large for the purpose of better comparison of seropositive and seronegative rheumatoid arthritis. Within the two categories, however, the patients were chosen randomly. Gout (sixty-one cases): Gout was defined by the finding of a serum uric acid level of more than 7 mg/100 ml in men, and 6 mg/100 ml in women. Diseases of connective tissue (twenty-five cases): Systemic lupus erythematosus (seventeen cases), scleroderma (seven cases), polymyositis (one case). Other rheumatic diseases (eighty-one cases): Psoriatic arthritis (four cases), ankylosing spondylitis (five cases), other forms of inflammatory rheumatism (six cases), lumbalgias (seventeen cases), arthrosis (twenty-six cases), others (twenty-three cases). Diseases in which rheumatoid factor is sometimes found independently of any joint involvement [8] (seventeen cases): Cirrhosis of the liver (six cases), patients with renal transplants [9] (seven cases), subacute endocarditis (one case), chronic suppuration (two cases), lymphosarcoma (one case). Controls (seventy-seven cases): Normal subjects (forty-one), aged fifteen to sixty years; patients with miscellaneous non-
Volume 49, August 1970
Fig. 2. Rheumatoid rosette observed in the blood of a patient with seropositive rheumatoid arthritis. rheumatologic diseases (thirty-six cases): renal failure, systemic hypertension, adrenal and thyroid insufficiency, hyperthyroidism, hemochromatosis, duodenal peptic ulcer, alcoholism, breast cancer, Steinert's disease, Raynaud's syndrome, malaria, hypogonadism, erythema multiforme. In all cases a clinical examination was performed, with particular attention to joint involvement. Biologic tests were also performed including, in most cases, the measurement of erythrocyte sedimentation rate and the Waaler-Rose reaction. RESULTS
Control Subjects. In all normal subjects and in patients with nonrheumatologic diseases the number of rosettes has always been less than or equal to 6 / 1 , 0 0 0 Ly, with one exception. This patient ( 9 / 1 , 0 0 0 Ly) and three of the five patients with 6 / 1 , 0 0 0 Ly had hyperthyroidism, a disease supposed by some to be of immunologic origin. Wher~ the group of patients with hyperthyroidism was excluded, all those with more than 3 R / l , 0 0 0 Ly were found to be subjects over forty years of age (twenty-three TABLE I
Practical Interpretation of the Rosette Test
Rosette Number and Interpretation < 4 R/1,000 Ly --Negative 4 - 6 R / l , 0 0 0 Ly --Doubtful 7 - 1 0 R / l , 0 0 0 Ly = Positive > 10 R / l , 0 0 0 Ly --Strongly positive
Rheumatoid
RA 138 Cases
Normal and Rheumatic Controls (Gout Excepted) 158 Cases
20%
84%
10%
11%
28%
2%
42%
3%
215
THE
RHEUMATOID
ROSETTE
--
BACH
ET
AL.
R ]I00~ Ly 40
e
30
II
~ ~
,,
9o
,.
20
10
o 9149149 9 9149 ,. 9149149
tt~ 9u 9=~
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QIQI
9
, 9149149149 6
9
,~ 9
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O
. . . . . . P01ya~hritis Ser0pesitive (68Cases)
I9 ..,...,.
Polyarthritis Ser0negative (70 Cases)
OOIOele*I
~'ff~'~J'~i{ Nonrheumatotogic Diseases (77 Cases)
oe* .....
eeellee
eeQe
eoleleoo
.............. Rheumatol0gic Diseases (8] Cases)
of the seventy-seven subjects of this control group were over forty). Three normal subjects less than twenty years old had 3 R/1,O00 Ly. Further on, we shall consider as positive the results with more than 6 rosettes/i,000 lymphocytes. Patients with Rheumatoid Arthritis. More than 6 R/ 1,000 Ly were found in ninety-seven of the 138 patients with rheumatoid arthritis (i.e., 70 per cent, Table I and Fig. 3). Correlation with the serum level of rheumatoid factor: No direct correlation has been found between the results of the Waaler-Rose reaction or the FII latex test and the number of rosette-forming cells (Fig. 4), The percentage of patients with a positive rosette test is higher among seropositive patients (76 per cent) than among serenegative patients (64 per cent), but the difference is not significant (P > 0.10) (Table II). The proportion of patients with a large number of rosettes (i.e., more than 15/1,000 Ly) is higher in seropositive patients (26 per cent) than in seronegative patients (15 per cent), but here again no significant difference is found between the two groups (P > 0.10). However, a critical difference was observed when patients with high and low titers of rheumatoid factor were compared. The number of resettes/1,000 Ly was significantly lower in patients with high titer of rheumatoid factor (over 1/128) for the Waaler-Rose reaction than in those with low titers. Table III shows a significant negative correlation between rosette number and rheumatoid factor titers. Correlation with clinical data:, The correlation between clinical data and the rheumatoid rosette test has been compared with that observed for the Waaler-Rose reaction. It can be stated first that the degree of positivity for rheumatoid rosette or Serum tests was not correlated with any of the criteria to be considered.
216
=eee ,9 Z:II|;
Fig. 3. N u m b e r of rheumatoid rosettes Gout (61 Cases)
in 138 patients with rheumatoid arthritis and 219 control subjects.
Duration of the disease: As shown in Figure 5, the percentage of patients with a positive rosette test is significantly higher in those with recent onset (P < 0.01). Conversely, the percentage of seropositive patients is higher in those with long-standing disease (P < 0.05), Number of joints involved in the disease: No correlation has been found between this criterion and rosette or Waaler-Rose test positivity. Destructive lesions and rheumatoid arthritis carpitis (x.ray examination): Destructive lesions, including signs of carpitis, were found significantly more often in serepositive than in seronegative patients, but not more often in patients with a positive rosette test than in those with a negative rosette test (Fig. 6 and 7). Pain: The pain was classified as nil, moderate or severe. A significant correlation was observed between the intensity of pain at the time of the test and the percentage of positive reactions to the rheumatoid rosette test. Such a correlation was not found for the Waaler-Rose reaction (Fig. 8). It is noteworthy that among the sixteen seropositive patients with negative rosette tests, nine had no pain and considered themselves cured; of the seven others, five had pain but received corticosteroids (the proportion of patients who received prednisone treatment is significantly higher [P < 0.01] in this small category than in the whole group of 138 patients with rheumatoid arthritis since only twenty-nine of them received corticosteroids). Correlation with biological data: Erythr0cyte sedimentation rate: When the erythrocyte sedimentation rate was over 60 mm after one hour, the percentage of positive rosette tests was significantly higher (91 per cent) than when the erythrocyte sedimentation rate was under 60 mm (76 per cent) (P < 0.01). A similar correlation was also observed between seropositive (77 per cent) and seronegative (52 per cent) patients.
The
American
Journal
of Medicine
THE RHEUMATOID ROSETTE- - BACH ET AL.
WIO00 Ly 40
30
e
20
e
B9
9
9
9
9 9149149
;0
9 69
9149
9
9
9149
9
e,
9
9 e 9149149
9
6 W A A L E R - R O S E
Fig. 4.
Correlation between the number of rheumatoid rosettes and the results of Waaler-Rose reaction.
TABLE II
< 1/32
1/32
1/128 1 / 2 5 6 1/512 V1024
Results of the Rheumatoid Rosette Test in 138 Patients with Rheumatoid Arthritis (RA) and Seventy-Seven Control Subjects Subjects
No. of Cases
Normal subjects and patients with miscellaneous nonrheumatic diseases Seropositive RA Seronegative RA
TABLE III
_< 6 R/l,000 Ly
77 68 70
Negative Correlation Between Rosette Number and Serum Titer of Rheumatoid Factor in Seropositive Patients (P < 0.05)
Rosette Number _< 20 R / l , 0 0 0 Ly > 20 R / l , 0 0 0 Ly
6-10 R/I,000 Ly
98.7% 24% 36%
TABLE IV
Waaler-Rose Reaction
TABLE V
V64
> 10 R/l,000 Ly
1.3% 27% 29%
0% 49% 35%
Correlation Between Number of Rosettes and Gamma Globulin Percentage (P < 0.01)
< 1/128
> 1/128
Gamma Globulin Level
15 75
1 29
_< 25% > 25%
Number of Rosettes _< 22/1,000 Ly
> 22/1,000 Ly
70 8
4 7
Results of the Rheumatoid Rosette Test in Rheumatic Diseases Other than Rheumatoid Arthritis Disease
Inflammatory arthritis Psoriatic rheumatism Ankylosing spondylitis Others
No. of Cases
< 6 R/l,000 Ly
4 5 6
4 (100%) 5 (100%) 6 (100%)
6-10 R/l,000 Ly 0 0 0
> 10 R/I,000 Ly 0 0 0
Gout Monoarticular Polyarticular
10 51
8 (80%) 27 (53%)
1 (10%) 9 (18%)
1 (10%) 15 (29%)
Other rheumaticdiseases
66
60 (90%)
2 (3%)
4 (7%)
Volume 49, August 1970
217
IHP_ I~HI-UMAIFOID ROSETTE - - BACH ET AL.
RHEUMATOID ROSETTETEST
WAALER- ROSE REACTION
100%
50%
0%
P
YEARS < 2
- 10
(23 cases)
(42 cases)
< 2
') 10 (40 cases)
P< O.O1 YEARS
2-i0
(23 cases)
> 1o
(42 cases)
(40 cases)
Fig. 5. Percentage of positive results in the rheumatoid rosette test and the Waaler.Rose reaction in relation to duration o f the arthritis (P < 0 . 0 1 ) .
WAALER- ROSE REACTION
RHEUMATOID ROSETTETEST
100%
II
50 %
P ( 0.01
P>0.05
0% No Destruction (49 cases)
No
Destruction (57 cases)
Destruction (49 cases)
WAAIFR- ROSE REACTION
RHEUMATOID ROSETI'E TEST
100%
I
P 3'0.05
0% No Carpitis ()4 cases)
100%.
Destruction (57 cases)
Fig. 6. Percentage of positive results in the rheumatoid rosette test and the Waaler.Rose reaction in relation to the existence and intensity of destructive joint lesions.
I
P< 0. 01
No Carpitis (34 cases)
Carpitis (33 cases)
RHEUMATOID ROSETTETEST
Carpitis (33 cases)
Percentage of positive results in the rheumatoid rosette test and the Waaler.Rose reaction in relation to the existence and intensity of carpitis. Fig. 7.
WAALER- ROSE REACTION
50%
0%
P(0.~
No Pain (31 cases)
218
Moderat, Pain
Severe Pain
(61 cases)
(11 cases)
No Pain (31 cases)
i
Moderate Pain (61 cases)
P >0. I0 Severe Pain (1.1 cases)
Fig. 8. Percentase of positive results in the rheumatoid rosette test and the Waaler-Rose reaction in relation to the existence and intensity of pain.
The American Journal of Medicine
THE RHEUMATOID ROSETTE - -
TABLE VI
BACH ET AL.
Results of the Rheumatoid Rosette Tests in Diseases Occasionally Associated with Significant Serum Titers of Rheumatoid Factor Disease
No. of Cases
< 6 R/l,000 Ly
6-10 R/l,000 Ly
> 10 R/l,000 Ly
Diseases of connective tissue Scleroderma Systemic lupus erythematosus Polymyositis Liver cirrhosis Subacute endocarditis Renal transplantation Chronic infection Lymphosarcoma
7 17 1 6 1 7 2 1
4 14 1 2 1 5 0 1
2 1 0 3 0 1 0 0
1 2 0 1 0 1 2 0
Serum gamma globulin level: No correlation was found between the serum gamma globulin level and the number of rheumatoid rosettes or serum titer of rheumatoid factor. However, as shown in Table IV, seven of fifteen patients with more than 25 per cent gamma globulin in the serum had more than 22 R / l , 0 0 0 Ly, a proportion significantly higher than that found in patients with less than 25 per cent gamma globulin in the serum. Correlation with treatment: Twenty-nine patients were treated with corticosteroids. The proportion of positive rheumatoid rosette tests in this group was not significantly lower than in the group of patients not treated with corticosteroids. Four of five patients treated with more than 10 mg of prednisone daily had a positive rosette test. The incidence of benefit of corticosteroid treatment on rheumatoid rosettes has already been men. tioned for the category of seropositive patients with a negative rosette test and pain. No correlation has been found with other treatments, including gold preparations, antimalarial therapy and salicylate. The number of patients treated with immunosuppressive drugs who could be examined was insuffi. cient to allow a definite conclusion about the influence of these drugs on the rheumatoid rosette test. Gout: A high percentage of positive results (43 per cent) was observed in gouty patients. These positive results were found significantly more often (P < 0.01) in those with polyarticular (47 per cent) than monoarticular (20 per cent) involvement. Other diseases: The results obtained in rheumatologic diseases other than rheumatoid arthritis and gout are presented in Table V. They demonstrate the relative specificity of the rheumatoid rosette test. The positive results have always been observed in patients more than forty years old. The results obtained in diseases occasionally associated with rheumatoid factor in the serum are depicted in Table VI. COMMENTS Agglutination reactions, of which the most commonly used are the Waaler-Rose and the latex test, are very useful in the diagnosis of inflammatory polyarthrit;s. However, they are inconsistently positive even in clinically typical cases. It thus seemed worthwhile to investigate the detection of rheumatoid factor in patients with
Volume 49, A u g u s t 1970
rheumatoid arthritis by means of a new and fundamentally different technic: the rosette test. This test makes possible the detection of antibodies at the cellular level. It.may be positive even when the antibodies are not present at a high level in the serum, and we have found (Bach, unpublished data) no correlation in man between the serum titer of natural antisheep red blood cell antibodies and the number of blood lymphocytes forming rosettes with sheep red blood cells. The rosette test has been used with lymphocytes from rheumatoid arthritis afflicted patients and human erythrocytes coated with rabbit gamma globulin. Rheumatoid rosettes were found in 75 per cent of cases of rheumatoid arthritis as defined by the criteria of the American Rheumatism Association. The percentage of lymphocytes forming rosettes ranged from 0.6 to 4.4 per cent; this rate of 4.4 per cent is higher than that observed in the mouse or the guinea pig after intravenous stimulation by sheep red blood cells [5]. This difference may be partly due to the fact that in these experimental studies rosettes are counted in the spleen or the lymph nodes and not in the peripheral blood. There was no direct correlation between the number of rheumatoid rosettes and the titer in the Waaler-Rose reaction. Rheumatoid rosettes were found in 76 per cent of cases of seropositive rheumatoid arthritis and in 64 per cent of cases of seronegative rheumatoid arthritis. With the exception of gout, the search for rheumatoid rosettes was negative in 98 per cent of the control cases. The specificity of the rheumatoid rosette test is comparable to that of the Waaler-Rose reaction. The rheumatoid rosette test can thus be proposed as a new method for the diagnosis of rheumatoid arthritis. The significance of the rheumatoid rosettes has been investigated [9]. It has been shown that the rheumatoid rosette is specifically inhibited by preincubation of the lymphocytes with antihuman immunoglobulin serum, and by normal human or rabbit serum. Rosette formation can also be inhibited by noncytotoxic concentrations of azathioprine, as already described for antisheep red blood cell rosettes [10]. Morphologic studies have shown that they are mostly lymphocytes with a majority of small lymphocytes. Similar properties have been reported for the rosette-forming cells observed in the mouse after stimulation by human IgG i l l ] and these rosette-forming cells
219
THE
RHEUMATOID
ROSETTE --
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red blood cells [5]. In one strain of mice, a good correlation is observed during the course of immunization between the number of rosette-forming cells and the titer of antisheep red blood cells antibodies (a delay of one to two days must be taken into account however, because the cellular response occurs before the appearance of serum antibodies). In rheumatoid arthritis, on the other hand, no direct correlation is found between the number of rheumatoid rosettes and the serum level of rheumatoid factor. This can be interpreted in several ways: (1) The serum level of rheumatoid factor depends mainly on the secretory activity of a few cells producing large amounts of antibody. The increase of the number of rheumatoid rosettes, essentially due to small lymphocytes, would represent the immediate response to a new antigenic stimulation, preceding the eventual increase of serum antibody level; in some cases, the stimulation might be strong enough to induce a cellular response but not an increase of the serum antibody titer. (2) The antibody-producing cells are not always present in the peripheral blood. Thus Biozzi et al. [13] have shown rosette-forming cells to be present in mouse blood after intravenous immunization, but only transitorily. In some circumstances, rheumatoid rosette-forming cells might be localized to lymphoid organs, e.g., spleen or lymph nodes. (3) There is a delay of several days between the appearance of rheumatoid rosette-forming cells in blood and the increase in titer of serum rheumatoid factor, and again between their disappearance and the decrease in serum rheumatoid factor titer, exactly as observed in experimental systems. The second period of delay could be explained by the slow catabolism of rheumatoid factor. (4) All rheumatoid factor-forming cells are not antibody forming cells but partly antigen-sensitive ceils as suggested. The significantly smaller average number of rheumatold rosettes in patients with a Waaler-Rose reaction titer over 1/256, as compared to patients with a titer under or equal to 1/256, may be interpreted as a repressive effect related to the high serum antibody level, similar to that demonstrated by Brown and Epstein [11] in the mouse when human rheumatoid factor, injected into mice
have been considered to be the producers of antibodies directed against human IgG [11]. All these data suggest that rheumatoid rosettes are formed by rheumatoid factorproducing cells whether the rheumatoid factor passes into the serum or remains on the lymphocyte's surface as it is supposed to do in antigen-sensitive cells. In fact it has been demonstrated in the mouse that rosette-forming cells are antigen-sensitive cells [12,13]. However, this interpretation might not be valid for the rheumatoid rosettes found in small number in some normal subjects (as in normal mice at a comparable level [11]) and in high number in some gouty patients, because the immunologic and morphologic studies here reported have not yet been performed in these situations. If they are indeed cells secreting rheumatoid factor, this secretion would probably be secondary to some unknown immunologic reaction. If they are not rheumatoid factor producing cells, one of the following functions could be ascribed to them: (1) Formation of natural antirabbit IgG antibody. A positive WaalerRose reaction is defined by a titer over 1/16, and the agglutination of sensitized erythrocytes is observed indeed at a low titer with most control serum samples. (2) Ability to agglutinate Ig-cOated red blood cells, nonspecifically, as described by Lo Buglio et al. r14]. The formation of these nonspecific rosettes has been described mainly with blood monocytes and could be inhibited by human serum. However, it could not be inhibited by antihuman immunoglobulin serum, in contrast to rheumatoid rosettes formed with lymphocytes in patients with rheumatoid arthritis. Four categories of patients can be distinguished when considering both cellular (rosette test) and serum(Waaler-Rose reaction and F II latex test) tests for detection of rheumatoid factor.
Seropositivity, rosette positivity: This category is the most common (fifty-two cases, 37 per cent), the more so if an unselected population of patients with rheumatoid arthritis is considered. This situation is in keeping with the experimental model, i.e., rosette formation obtained in the mouse after injection of human IgG [11] or sheep
IOOY.
50?00.
o~ RR Test
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WR Reac;tion
RR Test
WR Reaction
RR Test
I
WR Reaction
'RHEUMATOID SYNOVITIS"
"A SPECIFIC SYNOVITIS"
DIVERSE
(48 cases)
(12 cases)
(24 cases)
Fig. 9. Percentage of positive results in the rheumatoid rosette test and the Waaler-Rose reaction in relation to synovial pathology.
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THE RHEUMATOID ROSETTE- - BACH ET AL.
immunized with human IgG, depressed the number of rheumatoid factor cells formed with IgG-coated sheep erythrocytes. Seropositivity, rosette negativity. This group of only six. teen cases (11 per cent) includes patients without pain (eight cases) or treated with corticosteroids (six cases). In patients without pain, the disease can be considered inactive, at least temporarily. The positivity of the WaalerRose reaction could be due to the secretion of rheuma. told factor by a small number of cells producing large amounts or to the central localization of rheumatoid factorproducing cells. In practical terms, the finding of a negative rosette test in seropositive patients with rheumatoid arthritis not treated by corticosteroids could thus be taken as evidence for a favorable course. Since a comparatively large number of patients treated with corticosteroids are found in this group, corticosteroids may act upon rheumatoid rosette-forming cells, although no difference has been found in the proportion of patients with a positive rheumatoid rosette reaction when the group of corticosteroid-treated patients considered in its entirety was compared to the group of patients not treated with corticosteroids. Seronegativity, rosette positivity: Sixty-four per cent of seronegative patients have a positive rosette test (fortyfive cases). Seronegativity has been defined as a Waaler-Rose test titer under 1/32; hence, most cases are not truly "negative." The various reasons which may explain the absence of a direct correlation between the number of rheumatoid rosettes and the serum rheumatoid factor level have already been discussed and can be applied to the particular problem of the so-called seronegative patient with a positive rosette test. In clinical studies [1,2,16] only slight differences were found between the group of seropositive and the group of sero. negative cases of rheumatoid arthritis when the severity of the course and the frequency of carpitis and destruc. tive lesions were assessed. Hess and Ziff [17] showed that it was possible to fix gamma globulin complexes on leukocytes of both seropositive and seronegative patients. The fact that the proportion of patients with a positive rosette test is not significantly different in seronegative and seropositive rheumatoid patients, can be given as a further strong argument for a unified concept of the
disease. If rheumatoid rosette.forming cells are considered to be rheumatoid factor-producing cells, seronegativity could then be ascribed to several mechanisms, of which the most likely would be a low secretion or peripheral fixation of rheumatoid factor, and the least likely a complete absence of antibody release into the serum. In the case of a low secretion rate, it would be easy to explain why the rheumatoid factor titer may not rise above the level observed in normal subjects. Seronegativity, rosette negativity: This situation has been observed in twenty.five cases (18 per cent). With the simultaneous use of the Waaler-Rose reaction and the rheumatoid rosette test, rheumatoid factor can thus be demonstrated in all of an unselected population of rheumatoid arthritis patients in more than 90 per cent of cases. In the residual 10 per cent are included patients who recovered completely and also probably some diagnostic errors. It is noteworthy that among the fortyeight patients whose synovial biopsy specimen was typical of rheumatoid synovitis, 88 per cent were rosette. positive, whereas only 65 per cent were seropositive. (Fig. 9). The clinical signs were analyzed in 138 cases of rheumatoid arthritis and compared to the results of the rheumatoid rosette test and Waaler-Rose reaction. The rosette test was significantly more often positive in patients with recent onset of rheumatoid arthritis than in those with long-standing disease and in patients with pain than in patients without pain. These two correlations were not observed for the Waaler-Rose reaction. Conversely, the Waaler-Rose reaction was significantly more frequently positive in patients with destructive lesions or carpitis. This would be expected since destructive lesions and carpitis occur generally after several years. Conversely, the rheumatoid rosette test is no more frequently positive in old or destructive cases. It is thus suggested that rheumatoid rosettes may be taken as a salient feature of early or active cellular phases of rheumatoid disease, whereas rheumatoid factor will be found in the serum at significantly high levels only in chronic cases and even when the disease has become completely inactive. The significant correlation found between positive rheumatoid rosette tests and high erythrocyte sedimentation rates adds support to this hypothesis.
REFERENCES
1. Coste F, Delbarre F, Amor B, Bontoux D, Broulhet H, Guiraudon C, Menkes C, Saporta L: Etude comparEe de cent cas de polyarthrites (rhumato~des ?) sEronegatives et de cent cas de polyarthrTtes rhumatoides sero-positives. Sem Hop Paris 44: 777, 1968. 2. Morgan ES, Atwater EC: A comparison of patients with sero-positive and sero-negative rheumatoid arthritis. Med Clin NAmer 52: 533, 1968. 3. Bach JF, Delbarre F: Nouvelle methode de detection du facteur rhumatoTde au niveau cellulaire. C R Acad Sci [D] (Paris) 267: 134, 1968.
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4. Debray-Sachs M, Dormont J: Isolement des lymphocytes b partir du sang circulant chez I'homme normal et urEmique. Rev Franc Etud Clin Biol 13: 414, 1968. 5. Biozzi G, Stiffel C, Mouton D, Liacopoulos-Briot M, Decreusefond C, Bouthillier Y: Etude du phEnomene de I'ammunocytoadhdrence au cours de I'immunisation. Ann Inst Pasteur (Paris) 110: 7, 1966. 6. Bach JF, Dormont J, Dardenne M, Balner H: In vitro rosette inhibition by anti-human ALS. Correlation with skin graft prolongation in primates. Transplantation 8: 269, 1969.
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7. Singer JM, Plotz CM: The latex-fixation test. I. Application to the serologic diagnosis of rheumatoid arthritis. Amer J Med 21: 888, 1956. 8. Grieble HG, Bach GL, Szanto M, Anderson TO: The serologic diagnosis of rheumatoid arthritis. J Chron Dis 21: 667, 1969. 9. Bach JF, Delrieu F, Delbarre F: Etude immunologique du phL~nomEne de la rosette rhumatoTde. In preparation. 10. Bach JF, Dardenne M, Fournier C: In vitro evaluation of immunosuppressive drugs. Nature 222: 698, 1969. 11. Brown PJC, Epstein WV: Influence of human rheumatoid factor on numbers of antibody-producing cells. Arthritis Rheum 12: 1, 1969. 12. Bach JF, Dardenne M: Signification du ph~nomene de rosette spontan~e. C R Acad Sci [D] (Paris) 264: 751, 1969.
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13. Bach JF, Dardenne M, Muller JY: In vivo specific antigen recognition by rosette-forming cells. Nature (in press). ]4. Lo Buglio AF, Cotran RS, Jandl JH: Red cells coated with immunoglobulin G: binding and sphering by mononuclear cells in man. Science 158: 1582, 1968. 15. Biozzi G, Stiffel G, Mouton D, Bouthillier Y, Decreusefond C: A kinetic study of antibody producing cells in the spleen of mice immunized intravenously with sheep erythrocytes. Immunology 14: 7, 1968. 16. Bland JH, Brown EW: Sero-negative and sero-positive rheumatoid arthritis. Clinical, radiological and biochemical differences. Ann Intern Med 60: 88, 1964. 17. Hess E, Ziff M: Reaction of rheumatoid leukocytes with fluorescent aggregated gammaglobulin. J Clin Invest 39" 996, 1960.
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