Perturbations of Humoral and Cellular Immunity in a Patient with Pigmented Villonodular Synovitis

T. DOUGLAS KINSELLA, M.D., F.R.C.P.(C)’ FRANK VASEY, M.D. M. A. ASHWORTH, M.D., F.R.C.S.(C) Kingston, Ontario, Canada

Several parameters of the immune system have been studied in a patient with pigmented villonodular synovitis. Numerous immunoglobulin-synthesizing cells were found by immunofluorescent technics in both synovium and synovial fluid. Phytohemagglutinininduced in vitro blastogenesis of peripheral blood T lymphocytes was absent whereas B-lymphocyte blastogenesis was preserved. These results indicate a necessity for evaluating immune responsiveness in other patients with pigmented villonodular synovitis. Pigmented villonodular synovitis, an uncommon disease of synovial membranes, has been the subject of several histologic studies designed to elucidate the pathogenesis of this condition [l-3]. Although no specific conclusions have derived from such studies, suggestions have been advanced that pigmented villonodular synovitis might represent a response to trauma [4], to low grade neoplastic disease [5] or, more recently, to viral-like inclusion bodies in synovial endothelial cells [6]. Curiously, however, the significance of the frequent and sometimes striking infiltration of synovium in pigmented villonodular synovitis by lymphocytes and plasma cells has elicited only passing comment [ 1,2]. Our results in the present case suggest that additional studies should be undertaken to ascertain. if aberrations of immune responsiveness occur in this condition. CASE REPORT

From the Rheumatic Diseases Unit, Departments of Medicine and Orthopedic Surgery, Queen’s University, Kingston, Ontario, Canada. This study was supported by Grant CARS-6142-70 from the Canadian Arthritis and Rheumatism Society. Requests for reprints should be addressed to Dr. T. Douglas Kinsella. Manuscript accepted April 5, 1974. Present address: McGill University Rheumatic Diseases Unit, Royal Victoria Hospital, 11 Medical, 687 Pine Avenue West, Montreal 112, Quebec. l

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A 12 year old girl (J.D.) presented with a 3 year history of recurrent pain and stiffness in the right knee following a fall from a bicycle. Anterior synovectomies had been performed at other hospitals, 2 months and 15 months after the episode of trauma, at which time histologic confirmation of pigmented villonodular synovitis was obtained. Eight months after the second synovectomy, the child was referred to the Rheumatic Diseases Unit at Queen’s University because of recurrence of pain, stiffness and swelling of the right knee. Except for the right knee, which was warm, swollen and tender and lacked 10 degrees of extension, no abnormalities were elicited in the history or on physical examination. Routine laboratory examinations were within normal limits.

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Aspiration and biopsy of the right knee with a ParkerPearson needle yielded 20 cc of hemorrhagic fluid and a synovial histologic reaction compatible with pigmented villonodular synovitis (Figure 1). Total white blood cell count of the fluid was 5,900/mm3 with 5 per cent polymorphonuclear leukocytes, 68 per cent small lymphocytes, 7 per cent plasma cells and 20 per cent miscellaneous mononuclear cells (type A and C synovial lining cells [7] and macrophages).. Paired serum and synovial fluid specimens were negative for rheumatoid factor by a slide latex test (Gibco) and by a sensitized sheep cell test standardized with reagents donated by the World Health Organization ]8] _ Quantitative immunoglobulin and complement values were determined by commercial immunoplates (Behring Diagnostics): serum values were immunoglobulin G (IgG) 1,025 mg/ 100 ml, immunoglobulin A (IgA) 175 mg/ 100 ml, immunoglobulin M (IgM) 73 mg/lOO ml and C3 150 mg/ 100 ml; synovial fluid values were IgG 950 mg/iOO ml, IgA 156 mg/lOO ml, IgM 73 mg/lOO ml and C3 139 mg/lOO ml. Anterior synovectomy of the right knee, followed by posterior synovectomy 10 days later, was carried out. Eight weeks later 8 mCi of radioactive gold was instilled into the knee. Reevaluation 5 months later disclosed no clinical evidence of recurrence, and arthroscopy and synovial biopsy similarly showed no evidence of recurrence. At the latest evaluation, 14 months after synovectomy, there was clinical evidence of recurrent pigmented villonodular synovitis.

Synovial biopsy specimen of right knee shows thickening of lining layer and extensive infiltration of subsynovium by mononuclear cells. Hematoxylin and eosin stain; original magnification X 80, reduced by 5 per cent.

Figure 1.

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Figure 2. Synovial fluid smear in which the three largest cells display nuclear and cytoplasmic characteristics of plasma cells. Giemsa stain; original magnification X 800 (oil), reduced by 20 per cent.

SPECIAL STUDIES lmmunofluoresceni Studies. Synovium obtained at the third synovectomy was digested with trypsin as described previously [7], with the exceptions that Hank’s balanced salt solution served as the incubation medium and the tissue was more extensively minced so as to release subsynovial cells. The resultant cell suspension contained a preponderance of typical mature plasma cells (Figure 2) and numerous small lymphocytes, with an admixture of synovial lining cells, multinucleated giant cells, macrophages and polymorphonuclear leukocytes. lmmunofluorescent staining was performed on cytocentrifuge smears (Shandon Scientific) derived from the trypsin-digested synovium, as well as on synovial fluid cells, prepared as described previously [ 71. In brief, fluorescein isothiocyanate (FITC)conjugated rabbit antiserums specific for heavy chains of human IgG, IgA, IgM and for C3 were applied to the smears in a direct fluorescent antibody reaction; appropriate controls were employed to ensure specificity of the staining reactions [ 71. Lymphocyte Transformation Studies. The in ,vitro blastogenie response of the patient’s peripheral blood lymphocytes to phytohemagglutinin (PHA), autologous synovial fluid and an allogeneic synovial fluid from a patient with rheumatoid arthritis were evaluated as described previously [9]. In brief, 1 X lo6 small lymphocytes were established in each of three replicate cultures to which were added PHA on day 3, and autologous and allogeneic synovial fluids on day 0, the latter diluted in gamma globulin free fetal calf serum to final dilutions of 1:10, 1:20 and 1:40. Two microcuries of tritiated thymidine (3HTdr, Amersham-Searle, specific activity 5.0 Cilmmol) were added for the final 18 hours of incubation, and cultures were terminated after 5$& days. These studies were conducted 2 weeks after synovectomy when the patient was receiving

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Lymphocyte transformation results are summarized in Table I. The patient’s peripheral blood lymphocytes failed to respond to PHA in the presence of either autologous plasma or fetal calf serum. In the presence of autologous synovial fluid a weak, but not significant, blastogenic response occurred at a dilution of 1:40, 127 f 16 cpm versus 59 f 10 cpm in unstimulated control cultures. In the cultures containing allogeneic rheumatoid arthritis synovial fluid a significant response occurred at a dilution of l:lO, 260 f 21 cpm versus 59 f 10 cpm in control cultures. COMMENTS

edanti-IgG demonstrating toplasmic staining. Similar encountered employing magnification X 800 (oil cent.

Fear stained with FITC-conjugattwo plasma cells with diffuse-cybut less prominent results were anti-IgA and anti-IgM. Original immersion), reduced by 20 per

no medication. Results are expressed in counts per minute @pm); in this laboratory a significant blastogenic response in stimulated cultures represents counts per minutes three times greater than those encountered in unstimulated cultures. In other studies employing 12 normal controls a mean “stimulation index” (mean counts per minute of stimulated cultures divided by mean counts per minute of control cultures) of 18.6 f 4.7 has been obtained in this laboratory in response to PHA.

Although experimental data derived from a single case study must be interpreted with caution, the results of this study provide two striking observations which might have relevance to other subjects with pigmented villonodular synovitis. Specifically, it has been found (1) that synovial fluid and synovium of the patient contained large numbers of immunoglobulincontaining cells and (2) that the in vitro blastogenic response of the patient’s blood lymphocytes to PHA was not normal.

The immunofluorescent studies in our case indicated that the immunoglobulin-containing cells in synovial fluid and synovium

were immunoglobulin-synthesiz-

ing plasma cells. This interpretation is supported by a number of observations, including elevated synovial fluid immunoglobulin levels which approximated those in serum [lo], light microscopic studies which revealed numerous typical plasma cells in both synovial fluid and synovium, a diffuse pattern of the immu-

RESULTS lmmunofluorescent staining of synovial fluid smears revealed numerous mononucleated cells, which resembled plasma cells, with diffuse cytoplasmic staining which was predominant for IgG; IgA and IgM were present in lesser amounts but there was no staining for C3 (Figure 3). No positive staining was encountered in synovial fluid polymorphonuclear leukocytes, synovial lining cells or macrophages. Smears derived from the trypsin-digest of synovium, when subjected to immunofluorescent staining, contained numerous mononuclear cells which predominantly stained diffusely for IgG; although IgA and IgM were present in lesser amounts there was no staining for C3. These cells appeared to be typical mature plasma cells and were present in large numbers, often in clusters (Figure 4). Positive staining was occasionally encountered in some cells with the morphologic characteristics of small lymphocytes (Figure 4) but negative stainin{: reactions occurred with all other cell types recovered in the suspension.

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ization of minced synovium, stained with FITC-conjugated anti-IgG. Numerous plasma cells and occasional small Iymphocytes show diffuse cytoplasmic staining. Similar results were encountered employing anti-IgM and anti-IgA. Original magnification X 160, reduced by 30 per cent.

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I

TABLE

Lymphocyte

Transformation

Responses to PHA and Synovial Fluids Counts per Minute in Presence of Synovial

Fluid Allogeneic

Autologous* Medium

No Stimulant

Autologous plasma FCS

140 z!z 23 59 i 10

NOTE:

PHA = phytohemagglutinin;

* Synovialfluidsdiluted

PHA

169 + 31 52 f 3

1:lO

. 65 f

RA = rheumatoid

l:lO, 1:ZOand 1:40in

1:20

1:40

... 3

...

57 dz 4 arthritis;

1:lO

127 k

1:20

... 16

260 xk 21

FCS = gamma globulin-free

RA 1:40

... 143 i

. 10

92 i

13

fetal calf serum.

FCS.

nofluorescent stain within the cytoplasm of these cells, and the selective presence of positive staining for immunoglobulins whereas staining for C3 was negative. Thus, although these findings strongly support the presence of an intra-articular humoral immune response in this patient with pigmented villonodular synovitis, the data do not define the type of the immune response nor whether any specificity existed between this response and pigmented villonodular synovitis per se. Indeed, the absence of some of the usual markers of humoral immune inflammation, such as, depressed C3 levels [ 111, a polymorphonuclear pleocytosis and immunofluorescent intracellular immune complexes [7], suggests that the patient’s effector phase of humoral inflammation might not have been active at the time of this study or, alternatively, that the entire observation was a reflection of “chronic nonspecific inflammation,” perhaps provoked by previous surgical procedures. Clearly, the immunofluorescent results alone do not provide definitive answers to the foregoing possibilities, but the immunofluorescent studies and the lymphocyte transformation studies, when taken in concert, suggest that the demonstrated intra-articular immunoglobulin synthesis was not simply a nonspecific response. The in vitro lymphocyte transformation studies demonstrated that the patient’s peripheral blood lymphocytes, whether incubated in fresh autologous plasma or fetal calf serum, were unable to undergo blastogenic transformation in response to PHA, a powerful T-lymphocyte mitogen [ 121. Indeed, these studies ruled out the possibility of an impaired blastogenie response due to a circulating inhibitor in the patient’s plasma and clearly indicated an abnormality of blastogenesis of T lymphocytes per se. In addition, the patient’s lymphocytes failed to respond to autologous synovial fluid but did undergo blastogenesis in the presence of allogeneic rheumatoid arthritis synovial fluid. Since other studies conducted in this laboratory indicate that immune complexes present in

rheumatoid arthritis synovial fluids exert a blastogenic effect on B lymphocytes in vitro [ 131, it can be assumed that the patient under discussion retained a B-lymphocyte blastogenic response in the absence of a T-cell response to PHA. Parenthetically, it might also be inferred that the negative results employing cultures of autologous pigmented villonodular synovitis synovial fluid indicate an absence of immune complexes in the latter. Loss of PHA responsiveness in vitro, without concomitant loss of cell-mediated immunity in vivo, has been observed in various circumstances, including acute surgical trauma [ 141, neoplasia [ 151 and viral infections [ 161, although the mechanism(s) underlying these observations are not fully understood. In the context of the present case, it can be concluded that there was an absence of PHA-mediated T-lymphocyte blastogenesis but preservation of both synovial fluid-induced B-lymphocyte blastogenesis in vitro and intra-articular immunoglobulin synthesis in vivo, the latter demonstrated by immunofluorescent studies. Thus, these studies of a case of pigmented villonodular synovitis reveal perturbations of the patient’s immune system similar to those described in patients with neoplasia and viral infections, both of which have been considered as possible etiologic factors in pigmented villonodular synovitis [ 5,6]. Since the clinical course and histologic findings in our case are typical of those described in other cases of pigmented villonodular synovitis, it may be that similar perturbations of the immune system are present in other patients with pigmented villonodular synovitis. In view of the relatively uncommon occurrence of this disease, we have reported this case in some detail in the hope that other cases of pigmented villonodular synovitis will be evaluated by similar technics by other investigators. ACKNOWLEDGMENT We gratefully acknowledge the technical of Liliane LeBrun and Susan Sheldon.

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Jaffe HL, Lichtenstein L, Sutro CJ: Pigmented villonodular synovitis, bursitis and tenosynovitis. Arch Pathol 31: 731, 1941. Byers PD, Cotton R, Deacon OW, Lowy M, Newman PH, Sissons HA, Thomson AD: The diagnosis and treatment of pigmented villonodular synovitis. J Bone Joint Surg 50B: 290, 1968. Wyllie JC: The stromal cell reaction of pigmented villonodutar. synovitis; an electron microscopic study. Arthritis Rheum 12: 205, 1969. Fisk GR: Hyperplasia and metaplasia in synovial membrane. Ann R Coll Surg Engl 11: 157, 1952. Stewart MJ: Benign giant-cell synovioma and its relation to xanthoma. J Bone Joint Surg 308: 522, 1948. Molnar Z, Stern WH, Stoltzner GH: Cytoplasmic tubular structures in pigmented villonodular synovitis. Arthritis Rheum 14: 784, 1971. Kinsella TD, Baum J, Ziff M: Studies of isolated synovial lining cells of rheumatoid and non-rheumatoid synovial membranes. Arthritis Rheum 13: 734, 1970. Anderson SG, Bentzon MW, Houba V: International refer-

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ence preparation of rheumatoid arthritis serum. Bull WHO 42: 311, 1970. Kinsella TD: Induction of autologous lymphocyte transformation by synovial fluids from patients with rheumatoid arthritis. Clin Exp lmmunol 14: 187, 1973. Kushner, I, Somerville JA: Permeability of human synovial membrane to plasma proteins. Arthritis Rheum 14: 560, 1971. Ruddy S, Gigli I, Austen KF: The complement system of man. N Engl J Med 287: 642, 1972. Greaves MF, Bauminger S, Janossy G: Lymphocyte activation. Ill. Clin Exp lmmunol 10: 537, 1972. Kinsella TD: Manuscript in preparation. Powell JN, Radford SG: Immunosuppressive effect of surgery. Lancet 2: 1091, 1971. Edwards AJ, Rowland GF, Lee MR: Reduction of lymphocyte transformation by a factor produced by gastrointestinal cancer. Lancet 1: 687, 1973. Olson GB, Dent PB, Rawls WE: Abnormalities of in vitro lymphocyte responses during rubella virus infections. J Exp Med 128: 47, 1968.

Perturbations of humoral and cellular immunity in a patient with pigmented villonodular synovitis.

Several parameters of the immune system have been studied in a patient with pigmented villonodular synovitis. Numerous immunoglobulin-synthesizing cel...
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