Introduction
Retroviral infection is associated with various pathological conditions, including several cancers and immunological and neurological disorders [
1]. Human T cell leukaemia virus type I (HTLV-I) is the causative virus of acute T-cell leukaemia (ATL) [
2]. HTLV-I is estimated to infect more than 10 million people worldwide, and is endemic in several areas including southwestern Japan, especially in Kyusyu Island. Although most seropositive individuals are asymptomatic carriers, a proportion of these individuals develop ATL in adolescence. In addition, HTLV-I has also been shown to be involved in several immunological and inflammatory disorders, such as HTLV-I-associated myelopathy/tropical spastic paraparesis, bronchopneumonopathy, Sjögren syndrome and uveitis [
3,
4].
HTLV-I-associated arthropathy (HAAP) is also recognized as chronic arthritis caused by HTLV-I infection. In 1989, Nishioka and colleagues [
5] reported 11 HTLV-I carriers with chronic oligoarthritis associated with ATL-like lymphocyte infiltration. The onset of HAAP often starts acutely in a relatively large joint such as the knee, wrist or shoulder, and the associated symptoms closely resemble those of rheumatoid arthritis (RA). The histopathological changes in HAAP include marked proliferation of synoviocytes in the synovial lining layer, gross infiltration of lymphocytes, and migration of atypical lymphocytes with nuclear indentation into the synovial fluid (SF) and/or synovial tissue.
Numerous studies have demonstrated that HTLV-I can alter the oncogenic and immunogenic properties of synovial cells and lymphocytes [
6,
7]. In addition, mice overexpressing Tax, the protein encoded by the HTLV-I pX region, develop RA-like chronic and systemic synovitis [
8]. Furthermore, epidemiological studies have revealed a significant association of HTLV-I infection and RA in endemic areas in Japan [
9,
10], although studies in the USA, Europe and South Africa failed to link HTLV-I infection and RA [
11‐
14]. These pieces of evidence link HTLV-I infection to synovial proliferation; however, several clinical issues remain unresolved. There is still no established criterion for the diagnosis of HAAP because of the lack of specific symptoms and/or laboratory markers. Moreover, it is also not clear whether HAAP could exhibit other phenotypes, such as monoarthritis instead of polyarthritis.
Osteoarthritis (OA) is a degenerative disorder caused by mechanical overload and/or a consequence of imbalanced biological events between cartilage degradation and synthesis [
15]. In primary OA, age, obesity and malalignment are known as predisposing factors, but the association of virus infection has not yet been studied. In the present study we investigated a potential role of HTLV-I infection in the pathophysiology of primary OA. For this, we compared the concentrations of several inflammatory cytokines in SF taken from HTLV-I carriers and non-carrier patients who had been diagnosed with primary OA of one or both knee joints. We also studied the histopathological features of synovia of eight HTLV-I carrier patients, and determined the expression of Tax protein by immunohistochemistry.
Materials and methods
Patients and samples
Outpatients fulfilling the criteria of the American College of Rheumatology for the diagnosis of knee OA [
16] and corresponding to OA grade II or higher by the radiographic criteria of Kellgren and Lawrence [
17] were recruited to this cross-sectional study. Patients who fulfilled even one of the American College of Rheumatology criteria for RA during the later 4-year observation period were excluded. Patients with secondary arthritis, such as gout, pseudogout, purulent or traumatic arthritis or seronegative arthritis, were also excluded.
Peripheral blood and SF samples were obtained simultaneously from 22 HTLV-I carrier outpatients at the initial examination and were subjected to appropriate pretreatment as described previously [
18]. As patient control, SF and serum were also obtained from 58 HTLV-I-negative OA patients. Comparison of HTLV-I carrier and non-carrier OA patients showed no obvious differences in age, sex, affected side and disease duration (Table
1). At the initial examination, 20 HTLV-I carriers and 55 non-carrier patients felt pain in the medial femorotibial joints, a common type in Japanese primary OA, whereas the remaining patients also complained of pain in the patellofemoral joint. Joint swelling, repeated hydrops and limited range of motion were also commonly observed in the enrolled patients, without obvious differences between HTLV-I carriers and non-carriers. The major radiographic findings in the 80 patients were osteophyte formation with or without narrowing of the joint space and sclerotic change of subchondral bone, and there was no significant difference in these radiographic features between HTLV-I carriers and non-carriers as defined by the Kellgren/Lawrence scoring method (Table
1). At the time of enrolment in the present study, patients were taking a variety of medications, including non-steroidal anti-inflammatory drugs, external splints and intra-articular injections of prednisone and/or hyaluronic acid.
Table 1
Clinical and radiographic background of human T lymphotropic virus type I carriers and non-carriers
Number of patients | 8 | 14 | 58 |
Age (years)a | 68.8 (58–78) | 72.0 (56–87) | 68.6 (51–88) |
Sex (M/F) | 3/5 | 4/10 | 16/42 |
Disease duration (years)a | 7.8 (4–15) | 6.6 (4–10) | 6.3 (3–13) |
Unilateral/bilateral | 2/6 | 5/9 | 17/41 |
K/L scaleb (II/III/IV) | 7/5/2 | 10/10/3 | 49/42/8 |
Intra-articular injection was discontinued for at least 2 weeks before sample collection. The erythrocyte sedimentation rate, serum C-reactive protein and serum calcium concentrations were confirmed to be within the normal range in all patients. The study protocol was approved by the Human Ethics Review Committee of Nagasaki University School of Medicine, and a signed consent form was obtained from each subject.
ELISA and Western blotting of HTLV-I
Anti-HTLV-I antibody in sera and SF was screened by enzyme-linked immunosorbent assay (ELISA; Eitest-ATL kit; Eisai Inc., Tokyo, Japan) in accordance with the instructions provided by the manufacturer. This ELISA system is designed to detect IgG antibody. On the basis of this test, 22 patients with immunoreactivity in both serum and SF samples were defined as HTLV-I carriers.
To determine the epitope recognized by the antibody and to characterize the specificity of IgG and IgM antibodies, SF was subjected to Western blot analysis with the use of epitope-transferred membrane (Eitest-ATL WB kit; Eisai Inc.). Two envelope proteins and three core proteins derived from HTLV-I were fixed onto nitrocellulose membrane, and specific binding of IgG or IgM was distinguished by specific secondary antibody. The result of the Western blot was defined as positive when each antibody reacted with at least two antigens. In this Western blot analysis, IgG antibody was present in all 22 examined SF, whereas IgM class antibody, which is considered to be elevated in the acute phase of active viral replication [
19], was detected in the SF of 8 carriers (Table
1). There was no difference in age, sex or disease duration between carrier patients with or without IgM antibody.
Measurements of C-terminal parathyroid hormone, sIL-2R, IL-6, chondrocalcin and deoxypyridinoline
The C-terminal region (amino acids 109–141) of parathyroid hormone-related peptide (C-PTHrP) was measured with a radioimmunoassay kit (Daiichi Radioisotopes Laboratory, Chiba, Japan) as described previously [
18]. Soluble interleukin (IL)-2 receptor (sIL-2R) (Endogen Inc., Woburn, MA), IL-6 (Endogen Inc.), chondrocalcin (Teijin Co., Tokyo, Japan) and deoxypyridinoline (DPD) (PYRLINKS-D; Quidel Corporation, Santa Clara, CA) were determined by ELISA with the protocol recommended by each manufacturer.
Tissue samples and immunohistochemistry
The synovial tissues were obtained at the time of joint replacement surgery (
n = 4), synovectomy (
n = 2) or high tibial osteotomy (
n = 2) from HTLV-I carriers, and subjected to routine histopathological and immunohistochemical examinations as described previously [
20]. In brief, deparaffinized serial sections were preincubated with 3% H
2O
2 to remove endogenous peroxidase activity, and then incubated overnight at 4°C with monoclonal anti-HTLV-I Tax antigen (Lt-4, a gift from Dr Tanaka) antibody [
21]. The protein expression was detected by the ImmunoMax/catalyzed signal amplification method with 3,3-diaminobenzidine tetrahydrochloride as a substrate [
22]. The preparation incubated without the primary antibody served as the control. In accordance with our previous methods [
20], the degree of proliferation of synovial lining cells was assessed in at least five points in an area of more than 1.5 cm × 1.5 cm as follows: -, 1 or 2 cells thick; +/-, 3 or 4 cells thick; ++, 5–9 cells thick; +++, more than 10 cells thick. The overall degree of inflammatory reaction was also semi-quantified as follows: -, no infiltration; +/-, minimal and partial infiltration; +, moderately diffuse or aggregated infiltration; ++, large number of aggregates, many demonstrating germinal centres. These histological findings were evaluated independently by two authors (TT and MN).
Statistical analysis
Data are expressed as means ± SD. Mann–Whitney test and χ2 test with Yates's correction were used to compare data from two or three groups. Correlation coefficients were determined by Pearson linear regression analysis. P < 0.05 was considered significant.
Discussion
In the present study we compared the concentrations of several inflammatory cytokines in SF between HTLV-I carrier patients and HTLV-I-negative OA patients. By investigating OA patients only and excluding those with RA, we were able to demonstrate the involvement of HTLV-I infection in arthritis. Our results showed that C-PTHrP, sIL-2R and IL-6 were significantly higher in SF of HTLV-I carriers than in that of HTLV-I-negative OA patients. Furthermore, the concentrations of increased markers were higher in HTLV-I carriers positive for IgM antibody than those negative for the antibody. These results indicate that the joint inflammation is more severe in HTLV-I carriers than in HTLV-I-negative OA patients. However, we were unable to identify any differences in radiographic findings between the two groups. It is possible that the pathological changes in our carrier patients are under the limit of detection by the Kellgren scaling system, which addresses only the radiographic dimensions of arthritis. Alternatively, HTLV-I-associated arthritis might be only slowly progressive after onset, and the changes in disease status over a few years are often small and difficult to quantify.
With regard to the mechanism of HTLV-I infection-induced changes in immunogenic properties, previous studies reported that PTHrP, IL-2 receptor α subunit and IL-6 are cellular target genes of the Tax protein [
27‐
29]. In particular, direct binding of Tax to the nuclear factor-κB sequence on the IL-6 promoter is important for HTLV-I-induced IL-6 secretion in cultured synoviocytes [
30]. In our study we used immunohistochemistry to examine the expression of the Tax protein and showed the expression of Tax in synoviocytes in all samples examined. Although Tax expression was not correlated with cytokine concentration in SF, probably owing to the time discrepancy between SF aspiration and tissue preparation, Tax expression in synoviocytes might be responsible, at least in part, for the increased concentrations of PTHrP, sIL-2R and IL-6 in SF of carrier patients.
Of the cytokines examined in our study, C-PTHrP behaved as a unique marker for HTLV-I carrier patients. PTHrP was first identified as a causative peptide of humoral hypercalcaemia of malignancy in ATL patients [
31]. However, it is currently recognized that PTHrP is produced by many tissues and is involved in a variety of biological functions by binding to PTH/PTHrP receptor [
32]. In RA, PTHrP is expressed in the proliferated synovium and such expression is correlated with the inflammatory activity [
18,
33]. PTHrP seems to act as a crucial mediator for inflammatory arthritis [
34]. We previously demonstrated that PTHrP was also expressed in articular chondrocytes [
35], and treatment of cultured chondrocytes with PTHrP inhibited chondrocyte differentiation [
36]. Although the functional role of PTHrP in our carrier patients remains obscure, together with positive correlation of C-PTHrP with DPD, which is a marker for bone destruction, it is likely that PTHrP is also important in the degenerative process of the subchondral bone. Moreover, C-PTHrP in 13 carriers was elevated above the upper concentration in OA patients, indicating that C-PTHrP could be a potential marker of HTLV-I-associated arthritis, allowing it to be distinguished from primary OA. It should be noted here that the concentrations of C-PTHrP in our carrier patients were much lower than those in RA patients [
18], and were not correlated with erythrocyte sedimentation rate or C-reactive protein, suggesting that the mechanism(s) involved in the activation of PTHrP in HTLV-I associated monoarthritis differ from that of RA.
The histopathological features of HAAP are thought to be indistinguishable from RA [
5]. Despite the small number of patients in the present study, the synovia obtained from HTLV-I-infected patients showed relatively low inflammatory reaction and synovial proliferation, which could have been diagnosed as primary OA rather than RA. That synovia taken by synovectomy tended to accompany intense synovitis compared with others might be a result of the relatively short interval from onset to tissue preparation (average 0.9 versus 2.2 years; Table
2). Together with the relatively low expression of inflammatory cytokines compared with RA and no apparent differences in radiographic findings between HTLV-I carriers and non-carriers, we consider that the principal pathological features in the HTLV-I-infected synovia are equivalent to those of OA. Further studies with larger samples of synovial tissues are necessary to validate our hypothesis.
Taken together, our results suggest that HTLV-I infection can alter the pathophysiology of OA by increasing the expression of inflammatory cytokines, but this modification does not necessarily overcome the clinical outcome of simple OA except for IgM-positive patients, who could suffer from more severe symptoms as a result of the activated inflammation. Moreover, it is possible that the altered inflammatory activity is partial and transient during the natural course of OA and does not continue to the final stage. In contrast to the development of systemic arthritis in Tax transgenic mice [
8], we consider that HTLV-I infection alone is not sufficient to cause the development of monoarthritis independently of OA. Further studies including the incidence of HTLV-I-modulated primary OA in seropositive patients, in addition to longitudinal studies, are required to enhance our understanding of the association between HTLV-I infection and arthritis.
Conclusions
The present study indicates that HTLV-I infection could modify the inflammatory activity of ordinary primary OA, whereby direct activation by Tax protein might account for the increased concentrations of PTHrP, sIL-2R, IL-6 and DPD in SF. Our results also suggest that C-PTHrP could be a potential marker to distinguish HTLV-I-associated OA from simple primary OA in carrier patients.