As far as we know, this is the first study on the relative frequency of immune cells and cytokines in open sacroiliac biopsies of patients with AS at different stages of the disease.
Although our samples were obtained decades ago, most specimens could still be nicely stained. However, we cannot exclude the possibility that the failure to stain for IL4 was due to the age of the material.
In contrast with sacroiliac needle biopsies, the major advantage of this study is the rather exact localisation of cells and cytokines within the SIJ. Because it is very difficult to obtain such material, the number of samples was limited. Therefore no advanced statistical analyses were performed. However, this is unique and rare material, and similar biopsies have not been performed for decades.
A remarkable observation in this study is the relatively large number of CD3+ T cells and CD68+ cells (mainly macrophages and osteoclasts) in the BM of patients with AS. While T cells were mainly seen in early cases, macrophages were seen similarly often in both disease stages. This finding, confirming previous data obtained by needle biopsy in active sacroiliitis in patients with AS4
, is consistent with a pathogenetic role for such cells in the BM in early AS.
Involvement of the BM in AS has already been suspected in earlier studies.6,10,11,12,13,14,15,16
Increased numbers of T cells, predominantly CD8+ cells, have been described in the BM adjacent to entheses at specimens obtained from hip and knees during endoprosthetic surgery.17
In contrast, a paucity of lymphocytes was seen in needle biopsies of calcaneal insertions18
where macrophages were the predominant cells in the infiltrate, but the bone component was limited in that study. In our study T cells were observed in the synovium of the patient with very early AS and in an inflammatory infiltrate in the sacroiliac ligament in a patient with advanced AS. In contrast, no T cells were seen in the cartilage. There were aggregates of CD8+ T cells. No CD4+ staining was possible for technical reasons. In an earlier study on sacroiliac needle biopsies more CD4+ than CD8+ T cells had been found.4
In synovial knee biopsies of patients with AS about equal amounts of CD4+ and CD8+ T cells have been described.19
In the present study, macrophages and chondroclasts or osteoclasts were present in the eroded articular cartilage of patient 2 with early disease and in the chondro‐osseous ankylotic area of patient 4. Recent work has emphasised the role of macrophages in SpA by showing an increased expression of the scavenger receptor CD163 in the synovium as well as in the colonic lamina propria in specimens of patients with AS.20
Our earlier study4
had already reported a large amount of TNFα messenger RNA close to cellular infiltrates in inflamed SIJs. In the present study, this finding is clearly confirmed at the protein level with better localisation. Large numbers of cells containing TNFα, clearly more than in the control cases, were seen in all three tissues of patient 1 with early AS, and also in the CT and the cartilage of the other patient with early disease (No 2). This does not exclude the possibility that the other patients also had an increase in TNFα secretion in another area that was not detected. The cellular source of this proinflammatory cytokine is not clear. It is known that mainly macrophages but also lymphocytes can produce TNFα. This observation lends further pathophysiological support to the treatment of AS with anti‐TNFα agents. At least it might partly explain the impressive efficacy of anti‐TNFα therapy in active AS which has been demonstrated both clinically 21,22,23
and by magnetic resonance imaging (MRI) studies.24
In follow up immunohistological studies after anti‐TNFα therapy a reduction of histopathological changes in the synovium of peripheral joints has been seen25,26
: decreasing lining layer thickness, less vascularity, lower numbers of macrophages, and down regulated matrix metalloproteinases. Anti‐TNF agents may influence a compromised Th1 cytokine pattern, including low TNFα secretion of patients with AS.27,28,29,30,31
The data on serum levels of TNFα in AS are similarly conflicting.32,33
Taken together, there is a clear role for TNFα in the pathogenesis of AS. Studies on this subject should include the BM as a possible early source and the potential origin of this important cytokine.
The present study demonstrates IL6 in all tissues of 4/5 patients with AS, not enough material being available to test the remaining case. The highest levels were found in the very active inflammatory and destructive changes of AS patient 2. Raised levels of IL6 have been recovered in the synovial fluid of patients with different forms of arthritis, including some cases of AS.34,35
In accordance, we found more IL6 in the CT and in the cartilage but not in the BM, in the one early case as compared with the three late cases. Increased serum levels of IL6 have been detected in patients with AS by several32,36,37
but not all investigators.33
A positive correlation between IL6 and erythrocyte sedimentation rate was described in patients with SpA.38
Taken together, there is some evidence for a role of IL6 in the pathogenesis of AS.
In the BM of AS patients 2 and 4 we did also find IL1β. Peripheral blood mononuclear cells of patients with AS were shown to produce IL1β to a greater extent than controls,39
whereas serum levels of IL1β were reportedly normal.32,33,40
Nevertheless, on the basis of recent genetic findings which point to an increased prevalence of an IL1 polymorphism in AS, a role for IL1β seems probable.41,42,43
IL10 was recovered in the BM of all our patients with AS in higher amounts than in the controls, with a maximum in AS patient 2 with early disease. IL10 plasma levels have been shown to correlate with disease activity in patients with SpA.44
This has also been interpreted as an indication of a Th2 pattern of cytokine response. However, there has been quite an argument about this, and it is not clear which type of immune response predominates at which stage of disease.
was recovered in high amounts in the connective tissues and cartilage of the three patients with more advance AS (Nos 3–5), in sharp contrast with AS patient 2 with early disease. This is not surprising in the repair phase after destruction, because repair is characterised by cartilage and bone proliferation, and because TGFβ1
is known to be one of the most important anabolic factors in articular cartilage and bone.45,46
In the newborn rat femur, TGFβ injected subperiosteally induced localised intramembranous bone formation and chondrogenesis; the dose of TGFβ determined what type of tissue would predominate, a high dose giving rise to more cartilage than bone.46
TGFβ is autoregulatory for itself and T cells, limiting their clonal expansion,47
and more so for CD4+ than for CD8+ T cells.48
Serum levels of TGFβ1
were found to be increased in oriental but not in European patients with AS, independently of TGFβ1
Patients with AS produced more TGFβ1
upon non‐specific stimulation than controls,49
while synovial levels of TGFβ1
were found to be increased in patients with SpA and rheumatoid arthritis (RA) compared with osteoarthritis.51
However, genetic studies have not found a strong role for TGFβ1
gene polymorphisms in AS.52
Chondro‐osseous ankylosis is one of the characteristic changes of AS16
—one of the possible features distinguishing AS from RA. Such a difference could be the consequence of TGFβ in the tissue, which may favour chondrogenesis over direct ossification.46
Whether synovial tissue levels of TGFβ are higher in AS than in RA is not known. Plasma,53
levels of TGFβ are raised in RA, and TGFβ mRNA is expressed in rheumatoid synovial fluid cells.56
A higher secretion of TGFβ by T cells, plasma cells, and macrophages was seen in patients with reactive arthritis than in patients with RA.57
The particular tendency towards chondrogenesis in AS has been explained by a high response to TGFβ1
in mice with progressive ankylosis.58
On the whole, the task of studying mediators of new bone formation in AS continues and there is some evidence that TGFβ1
may have a role in that very characteristic feature of this disease.
In summary, the present work confirms the important role played by the BM in AS. It shows a different tissular pattern of cytokines in patients with early disease and in more advanced cases, with more TNFα and IL6 in early cases, and more TGFβ1 in late cases.