Immunohistologic analysis of zygapophyseal joints in patients with ankylosing spondylitis

Ankylosing spondylitis (AS) is a relatively common disease in Caucasian populations, with a reported prevalence ranging from 0.2% to 0.8%. Chronic inflammation in AS primarily affects the axial skeleton, but larger peripheral joints may also be involved. Fibrosis and secondary ossification can result in complete ankylosis of the involved joints, leading to severe disability (1). In such patients fusion of the zygapophyseal joints, in addition to ankylosis of the intervertebral space, is a predominant finding. In 1958 Wilkinson and Bywaters suggested that inflammation in the zygapophyseal joints might be the lesion primarily responsible for restriction of lumbar mobility and ankylosis of the spine (2). Nevertheless, the role of the zygapophyseal joints in AS has been the subject of controversy. Some authors have proposed that involvement of these joints in AS is of little importance (3). However, a more recent study showed that impaired spinal mobility highly correlates with radiographic changes, especially in the zygapophyseal joints of the lumbar spine (4). This is further supported by the observation that the presence of bridging syndesmophytes without concomitant ankylosis of the zygapophyseal joints was an uncommon finding in advanced AS, while the presence of zygapophyseal joint ankylosis of the lumbar spine was seen in 21% of AS patients without bridging syndesmophytes (5). These observations suggest that the zygapophyseal joints might be primarily involved in the process of spinal inflammation (4).

In the present study we obtained the superior and inferior articular processes of the vertebrae from zygapophyseal joints of AS patients who had undergone polysegmental correction of rigid hyperkyphosis (6). We performed immunohistochemical analysis of these samples and compared the results with immunohistochemical findings in autopsy specimens from non-AS controls.

PATIENTS AND METHODS

RESULTS

Macroscopic, radiographic, and microscopic features of zygapophyseal joints.

In the 2 AS patients and 1 non-AS control whose zygapophyseal joints were visualized by CT, correlations between the CT results and macroscopic and microscopic features were investigated. Macroscopically, the 2 AS zygapophyseal joints exhibited narrowed joint space (Figures 1A1 and B1). CT of the joints from both patients revealed additional features: in the zygapophyseal joint of 1 patient, both articulating parts were closely attached to each other, indicating joint space narrowing (Figure 1A2). The bone end-plate was partially present. The zygapophyseal joint of the other AS patient was ankylosed, with bony bridges, and the bone end-plate was no longer detectable (Figure 1B2). However, although the zygapophyseal joints of both AS patients showed advanced joint destruction, histopathologic study revealed mononuclear cell infiltration in both, indicating persistent inflammation (Figures 1A3 and B3).

In contrast, macroscopic examination of zygapophyseal joints from the non-AS controls revealed a regular joint space (Figure 1C1). Regular joint space was also seen on CT, as was an intact bone end-plate (Figure 1C2). Histopathologic features correlated with the findings seen on CT: regular healthy cartilage with slight degenerative changes and an intact bone end-plate. The subchondral bone marrow displayed regular hematopoiesis (Figure 1C3).

Immunohistologic features.

Lymphocytes.

We first investigated whether immunohistochemical analysis of zygapophyseal joints would reveal signs of persistent inflammation in the ankylosed lumbar spine. When all AS patients were analyzed as a group and compared with non-AS controls, the AS group was found to have significantly higher levels of interstitial CD4+ T cells (mean ± SD 11.56 ± 3.57 versus 7.0 ± 1.15 per high-power field [hpf]; P = 0.002) and CD8+ T cells (8.31 ± 3.41 versus 5.33 ± 1.88 per hpf; P = 0.049) in the bone marrow of zygapophyseal joints (Figure 2). Levels of CD20+ B cells did not differ significantly between the AS patients as a whole and the non-AS controls (8.25 ± 6.2 versus 5.2 ± 3.3 per hpf; P > 0.05).

We assessed for intertrabecular CD3+ lymphocyte aggregates, defined as clusters of ≥50 CD3+ T cells, in the bone marrow of zygapophyseal joints (8) (Figure 2A). Zygapophyseal joints of 6 of the 16 AS patients, but none of the controls, had ≥2 such aggregates (2 aggregates in 2 patients, 3 aggregates in 3 patients, 4 aggregates in 1 patient). In the zygapophyseal joints of 3 non-AS controls, a single aggregate was observed.

We classified zygapophyseal joints with ≥2 lymphocytic aggregates per 10 hpf as having persistent inflammation and conducted further analyses to compare findings in joints with and those without persistent inflammation. In AS patients with persistent inflammation, interstitial CD4+ T cells (mean ± SD 14.83 ± 2.63 per hpf) and CD8+ T cells (10.0 ± 3.22 per hpf) (Figures 2B and C) were significantly more frequent compared with levels in AS patients without active inflammation (CD4+ T cells 9.8 ± 2.29 per hpf, CD8+ T cells 7.3 ± 3.26 per hpf; P = 0.004 and P = 0.04, respectively) and non-AS controls (CD4+ T cells 7.0 ± 1.15 per hpf, CD8+ T cells 5.3 ± 1.15 per hpf; P = 0.001 and P = 0.005, respectively). In AS patients without persistent inflammation, only the level of CD4+ T cells was significantly increased compared with non-AS controls (P = 0.003).

Of even greater interest was the finding that, when AS patients with persistent inflammation were analyzed separately from AS patients without active inflammation, significant differences in the level of B cells could be observed, indicating that B cells might be of importance during persistent inflammation. Two or more intertrabecular CD20+ B cell aggregates were present in the zygapophyseal joint of 1 AS patient and in none of the non-AS controls. In the AS patient the aggregates were located exactly within the T cell aggregates (Figures 2A and D). In the other 5 AS patients with ≥2 T cell aggregates, microfocal infiltrates with clusters of 10–12 CD20+ B cells were present within the T cell aggregates. Interstitial CD20+ B cells (Figure 2E) were found in significantly higher numbers in AS patients with persistent inflammation (mean ± SD 13.5 ± 6.95 per hpf) compared with those without active inflammation (6.1 ± 3.87; P = 0.03) and non-AS controls (5.2 ± 3.34; P = 0.03) (Figure 2F).

The presence or absence of T cell aggregates did not correlate with age or treatment. CD3+ T cell aggregates were found in all 3 female patients.

Macrophages.

No significant differences were observed when the levels of bone marrow macrophages in the zygapophyseal joints of patients in the AS group as a whole and the non-AS controls were compared. Furthermore, there were no significant differences when AS patient zygapophyseal joints with and those without active inflammation were separately compared with the controls (Figure 2F).

Microvessel density.

When the overall group of AS patients was analyzed in comparison with non-AS controls, significantly higher numbers of microvessels in the bone marrow of zygapophyseal joints were present in AS patients (mean ± SD 5.87 ± 2.24 versus 1.8 ± 0.79 per hpf; P < 0.001). The level of microvessels was significantly increased compared with controls in both subgroups of AS patients (5.16 ± 1.47 per hpf in patients with persistent inflammation, 6.6 ± 2.17 per hpf in patients without inflammation; both P = 0.001 versus controls) (Figures 3A and B). Microvessel density did not differ significantly between the AS patients with and those without persistent inflammation (Figure 3B).

Osteoclasts.

The number of CD68+ osteoclasts at the trabecular bone (Figure 3C) did not differ significantly when either the overall group of AS patients (mean ± SD 3.25 ± 1.06 per hpf) or the subgroup of AS patients with persistent inflammation (3.83 ± 1.16 per hpf) was compared with controls (2.6 ± 1.26 per hpf) (Figure 3D). In the zygapophyseal joints of 3 AS patients from whom consecutive slices were available, the staining of CD68+ osteoclasts was confirmed by TRAP staining (results not shown).

DISCUSSION

Strikingly, in 6 of 16 AS patients who had complete ankylosis in major areas of the spine, we detected persistent inflammation with formation of lymphoid aggregates in the bone marrow of zygapophyseal joints, indicating that these joints are frequently involved in AS and that there is ongoing inflammation even at advanced stages of disease in a substantial proportion of patients with AS. In order to better differentiate between patients with persistent inflammation and those with less or no inflammation, the presence of ≥2 T cell aggregates per 10 hpf was used to classify patients as having persistent inflammation. We found significantly higher numbers of CD4+ T cells in AS patients compared with non-AS controls, even among AS patients in whom lymphoid aggregates were absent. The number of CD8+ T cells was significantly increased in the group of AS patients overall and those who had lymphoid aggregates compared with controls, but the increase among AS patients without lymphoid aggregates was not significant compared with controls. The presence of benign lymphoid aggregates in the bone marrow rises with age. They are found in 8% of healthy individuals, but the frequency rises to 36% in persons >70 years of age (9). Thus, the frequency of lymphoid aggregates in our non-AS control group, with a mean age of 72.3 years, could be expected to be higher than that which would have been observed in controls had the control group been age-matched to the AS patients.

Another noteworthy result of our study is the significantly increased number of CD20+ B cells in AS patients with persistent inflammation in the spine. Compared with AS patients without inflammation and non-AS controls, this was the most important difference we observed. There are no previous reports of immunohistologic staining for B cells in the spine of AS patients, and for a long time B cells were not considered in the discussion of AS pathogenesis, probably because, in contrast to rheumatoid arthritis or systemic lupus erythematosus, autoantibodies did not appear to play a major role in AS.

Significantly increased numbers of B cells in the synovial membrane of 2 AS patients with peripheral joint involvement have been reported (10). In studies of anti–tumor necrosis factor α (anti-TNFα) therapy in patients with peripheral spondylarthritis, synovitis was down-regulated, with profound reduction of T cells but persistence of B cells (11). The resistance of B cells to this kind of treatment might explain why relapses occur soon after TNFα therapy has been discontinued, again suggesting a possible role of B cells in the immunopathology of the disease.

In addition to acting as precursors of antibody-secreting plasma cells, B cells can act as highly efficient antigen-presenting cells (APCs), facilitating the activation of T cells (12, 13). This notion of B cells serving as APCs could be implied by our observation that T cell aggregates were always associated with an increased number of B cells. This possibility of a potential role of B cells in the pathogenesis of AS will require more investigation in the future.

In all AS patients the density of microvessels was up-regulated compared with that in controls, independent of persistent inflammation. A high density of microvessels was found not only close to lymphocytic infiltrates, but also at sites of new bone formation, with continuous rims of osteoblasts along the trabecular bone. This indicates that angiogenesis also plays a critical role during new bone formation and ankylosis. We have recently reported and discussed similar findings in an analysis of hip joints from AS patients (8), indicating that neoangiogenesis is essential both for inflammation and for new bone formation in AS.

The number of CD68+ bone marrow macrophages did not differ between AS patients and non-AS controls, which is consistent with findings in our investigation of femoral heads (8) and of previous studies of the enthesis of peripheral joints in AS patients (14). This might indicate that in patients with longstanding AS, bone marrow macrophages do not play a major role in inflammation or repair.

Similar to the findings with regard to bone marrow macrophages, we did not observe a difference between the number of osteoclasts in AS patients with persistent inflammation and that in patients with less active AS or controls. A later stage of inflammation in patients with advanced ankylosis and kyphosis might be one possible explanation for this.

In summary, we have demonstrated that zygapophyseal joints are directly involved in inflammatory processes in AS. It is striking that even in patients with longstanding, severe disease, inflammatory activity can be present in the spine with total ankylosis. The demonstration of a role of T cells, B cells, and neoangiogenesis during persistent inflammation provides evidence that multiple mechanisms are involved in the pathogenesis of AS and could be targeted with separate therapeutic approaches.