B-cell activation and development within chronically inflamed synovium in rheumatoid and reactive arthritis

seminars in

IMMUNOLOGY, Vol 9, 1997: pp 261–268

B-cell activation and development within chronically inflamed synovium in rheumatoid and reactive arthritis Claudia Berek and Hye-Jung Kim Table 1. RA and ReA

In autoimmune diseases, B cells often accumulate in the affected tissue. In patients with rheumatoid arthritis or reactive arthritis, germinal center-like structures may develop in the inflamed synovial tissue. B cells from these structures were isolated and their V-gene repertoire determined. The majority of synovial B cells are long-term memory cells and thus are part of the chronic inflammatory reaction. In the synovium a micro-environment is built up which allows the activation of naive and memory B cells and the diversification of their V-gene repertoire. The analysis of plasma cells suggests that these cells are long lived and hence accumulate in the synovial tissue under chronic activation.

RA

ReA

Genetic susceptibility Etiology

Class II HLA DR4

Class I HLA B27

Unknown

Affected joints

polyarthritis, symmetrical inflammation RF factor TH 1

Bacterial infection at a site distant from the joint oligoarthritis

Autoantibodies Cytokine millieu

negative TH2

affinity for the antigen may determine whether a germinal center B cell will die through apoptosis or whether it will be selected to further differentiate into a plasma or a memory B cell.4 As only those cells with the highest available affinity BCRs are able to differentiate, an affinity maturation of the immune response is ensured.7 In autoimmune diseases, B cells are often not restricted to lymphoid tissue but accumulate in the affected tissue. Lymphocytic infiltrates are observed in various parts of the body. In patients with Sj¨ogren’s syndrome large cell clusters are seen in the salivary gland tissue,8 in Hashimoto’s disease in the thyroid tissue9 and in rheumatoid (RA) or reactive arthritis (ReA) in the synovial tissue (ST).10-13 In myasthenia gravis germinal center-like structures are formed in the thymus where, under normal conditions, the maturation and selection of T cells occurs.14

Key words: germinal center / memory B cell / plasma cell / rheumatic disease / somatic mutation ©1997 Academic Press Ltd

IN A T-DEPENDENT immune response antigen-activated B cells migrate into the primary follicles of the lymphoid organs where germinal centers with their unique micro-environment develop and where clonal expansion of the antigen activated B cells takes place. Within these germinal centers a hypermutation mechanism is triggered which diversifies the B-cell repertoire.1,2 Variants of different affinity for the antigen develop and those with increased affinity are selected and expanded. In a typical germinal center, as seen for example in the tonsil, a dark and a light zone can be distinguished.3,4 B-cell proliferation takes place in the dark zone. The light zone, on the other hand, is characterized by a dense network of follicular dendritic cells (FDC).3,4 Antigen complexed with antibody is bound to the surface of the FDC via the complement or the Fc receptor.5,6 Thus, in contrast to dendritic cells FDC present unprocessed antigen to B cells. It is thought that the B-cell receptor (BCR) and the antibodies bound to the surface of the FDC are in competition for the available antigen. In this way,

Rheumatic diseases RA and ReA are both diseases in which an inflammation develops in the ST. As a result a massive infiltration of mononuclear cells into the synovium may be seen. Nevertheless, the clinical course of these diseases is quite different (Table 1). The etiology of RA is still unknown.15 However, individuals positive for HLA class II allele DR4 seem to have a higher risk to progress to a more severe clinical form of the disease.16 Furthermore, it is generally believed that a high titer of rheumatoid factor (RF), an autoantibody

From the Deutsches Rheuma ForschungsZentrum, Berlin Monbijoustr. 2, 10117 Berlin, Germany ©1997 Academic Press Ltd 1044-5323/97/040261 + 08 $25.00/0/si970076

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C. Berek and H.-J. Kim has been reported that a dark and a light zone may form.22 However, this seems to be more the exception than the rule, and in our studies we never observed it.

specific for the Fc part of self IgG, is associated with disease activity and progressive joint damage.17,18 In contrast much more is known about the etiology of ReA. In general ReA is an acute form of synovitis and only a fraction of patients develop a chronic inflammation of the joint. The disease arises as a consequence of an infection with Chlamydia trachomatis, Yersinia or Shigella in the uro-genital or the gastrointestinal tract, respectively.19 As a result of the infection a humoral immune response is induced and antibodies to bacterial antigens are seen in the blood of the patients. Autoantibodies, like the RF factor seen in RA, are not found in the sera of patients with ReA. It has been discussed that the persistance of the microbe in the body for long periods of time may be responsible for the development of a chronic inflammation. Though the mechanism by which bacterial infections induce a chronic arthritis are not fully understood and despite the failure to isolate viable bacteria from the ST there is evidence that bacterial antigens are present in the joint.19,20 Thus, an immune reaction against bacterial antigens may be responsible for the development of a chronic synovitis without the involvement of self antigen. On the other hand, a cross-reaction between bacterial and self antigen may lead to a shift from an immune to an autoimmune response.

Repertoire of synovial B cells Why do B cells accumulate in the ST? Are these resting B cells which have not yet encountered antigen? Do they reflect the repertoire seen in the peripheral blood? Are synovial B cells memory cells

Immunohistology of the ST We have analysed ST from patients with RA12 and from those with ReA.13 Interestingly, the immunohistology of the ST gave comparable results. In some patients, B cells may constitute only a small fraction of immigrating cells, while in others, CD20 + B cells together with CD4 + T cells may form large cellular clusters in which roughly 2/3 of the cells are B cells (Figure 1A). The remainder are T cells. In addition, in the majority of patients, plasma cells are seen dispersed throughout the tissue. Often, they are seen in the perivascular areas where they may form dense lymphocytic sheaths. Additionally, histological analysis shows rings of plasma cells surrounding T and B lymphocytes which appear to have formed follicular structures (Figure 1B). In some of these cell clusters FDC can be observed.12,21,22 We sometimes see only a few FDC in the center of these cellular aggregates (Figure 1) while in other cases B cells are lying in a dense network of FDC. It seems that in this non-lymphoid tissue a germinal center-like structure has evolved. It

Figure 1. A follicle-like structure in the ST from a patient with RA. A lymphocytic infiltrate is shown on two consecutive sections. CD20 + B cells (a) are surrounded by a ring of plasma cells (b). In addition, plasma cells are seen in the neighbouring tissue. B cells were stained with a CD20 specific antibody (mAb L26, Dako) and plasma cells with antibody Wue-1.12

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B-cell response in rheumatic diseases which have been activated by self antigen over a long period of time and have developed into high affinity autoreactive cells? Does the cytokine millieu attract B cells into the ST where IL-6, in particular, may support a nonspecific polyclonal B-cell activation in the joint.23 The observation that synovial B cells seem to express a more restricted antibody repertoire than those in circulation suggests that they have accumulated by virtue of their BCR specificity.24,25 So what B-cell receptor specificity leads to the accumulation of these B cells into the ST? In RA one possibility would be that these are B cells with RF specificity which can bind circulating antigen/antibody complexes and in this way internalize a variety of antigens.26 Multiple peptides presented to T cells might lead to the activation of these B cells and support their accumulation in the ST. Another possibility is that locally — in the ST — an inflammation is induced. B cells may be activated by autoantigens, foreign antigens or both

and this results in a chronic inflammatory immune reaction. In order to address the question of B-cell migration into the ST we have isolated single follicle-like structures directly from frozen tissue sections and determined their V-gene repertoire.12,13 In general the results obtained from both patients with RA and patients with ReA are very similar although there are some minor differences. Follicle-like structures isolated from patients with RA have a more oligoclonal repertoire than the cellular aggregates isolated from the one patient with ReA. Here multiple B cell clones were seen. The VH gene repertoire seen in lymphocytic infiltrates from four different patients with RA and one patient with ReA is shown in Figure 2A. A diverse repertoire of different VDJ rearrangements was found. Though only a small number of follicles were analysed it seems unlikely that there is a preferential

Figure 2. VH-gene repertoire expressed in synovial and peripheral blood B cells. The upper part (A) shows VH rearrangements isolated from the ST of four patients with RA (stippled columns) and one patient with ReA (open columns). Data are taken from Schr¨oder et al12 and Schr¨oder et al13 and unpublished data. The lower part (B) shows VH rearrangements isolated from the peripheral blood. Data are taken from Brezinschek et al.28 Germline genes are named according to the nomenclature used by Cook and Tomlinson29 (DP) or Matsuda et al38 (VH 4-4 and 3-49), VH-gene families are indicated.

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C. Berek and H.-J. Kim V-gene usage in synovial B cells. Certain VH genes — DP47, DP49, DP71 and DP73 — seem to be more frequently used than others. However, it is unlikely that this is due to selection since similar VH-gene usage preferences are also seen in the normal repertoire (Figure 2B).27,28 If anything, there seems to be a slight increase in the frequency of DP71 rearrangements. Furthermore, in both groups of synovial B cells, those derived from patients with RA and those from the patient with ReA, the majority of VD rearrangements are joined to a JH6 gene element. The predominance of JH4 rearrangements reported in the literature could not be confirmed.27

Synovial B cells are memory cells The majority of sequences isolated from synovial B cells of the patients with RA were highly mutated (Figure 3B). On average 18 substitutions per variable region were seen.12 In the follicle-like structures of the patient with ReA two different groups of B cells were seen.13 About half of the sequences had low numbers of somatic mutations. The numbers of mucleotide exchanges were in the same range as that seen in resting B cells of the peripheral blood. These B cells may correspond to new immigrants into the ST. The other half of the V regions were highly mutated. Like synovial B cells isolated from patients with RA, these sequences had in the order of 20 somatic mutations per V region.13 Some of the observed diversity may reflect gene diversity in the human gene pool, though, there seems to be relatively little gene polymorphism in the human population. When germline genes have been isolated from different individuals they have in general identical or nearly identical sequences. Allelic forms differ by 1-3 nucleotides from each other.29 Two VH4 rearrangements, one isolated from the ST of a patient with RA and one from a patient with ReA, differed by 47 and 51 nucleotides, respectively from their closest germline genes. In both cases, the pattern of mutations suggested that these V regions are derived from yet unknown V genes. In some individuals there may be additional VH4 germline genes or new allelic forms which have not been isolated.29 Figure 3. Somatic diversity in B cells isolated from a patient with RA. The somatic diversity for peripheral blood B cells (A), for synovial follicle-like structures (B) and for Vλ2 expressing plasma cells (C) is shown. Sequences are grouped according to the range of somatic mutations per V region.

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B-cell response in rheumatic diseases the non-lymphoid tissue a micro-environment is built up which permits a germinal center-like reaction to take place and which supports the diversification of the B cell repertoire. From single lymphocytic aggregates V regions were isolated with identical rearrangements but different patterns of somatic mutations. A set of DPL2 rearrangements was isolated where sequences had only one single nucleotide exchange in common.13 It seems that the entire diversification of this B-cell clone occurred in the ST. Such data suggest that a naive B cell may become activated by antigen in the ST. A T-cell dependent germinal centerlike reaction is initiated locally and leads to the diversification of the B-cell repertoire. In addition we see that long-term memory cells become reactivated in the ST and during proliferation may accumulate further somatic mutation. V regions with a VH DP52 shared 18 somatic mutations but differed by up to 12 nucleotides from each other.12 The diversification of the V-gene repertoire was seen in both the ST of patients with RA and in the patient with ReA. A germinal center reaction is an antigen-dependent T-cell driven immune response. The question arises which antigens lead to B-cell activation in the ST. In patients with RA this is a completely open question. In ReA the primary immune response is induced by bacterial antigens. One might speculate whether it is a bacterial antigen that drives the chronic inflammation or whether it is an autoimmune reaction that evolves with time. In the case of our ReA patient the disease was induced by an infection with Chlamydia trachomatis via the uro-genital tract.13 The biopsy which we analysed was taken two years after onset of the synovitis. Although it was not possible to isolate viable bacteria from the synovium there is evidence from PCR analysis that bacteria are still present.13 Furthermore, in the follicle-like structures in the ST there are FDCs on which antigen can be preserved for long periods of time.5,6 These cells may play a crucial role in mediating a chronic activation of B cells.

The frequency of somatic mutations in sequences obtained from synovial B cells indicates that the majority of these cells are memory cells (Figure 3B). In order to accumulate these high numbers of somatic mutations they must have gone through multiple rounds of somatic mutations. With a hypermutation rate in the order of 10–3/base pair per cell generation30 a nucleotide exchange will be introduced with every third division. Thus, a sequence with 20 substitutions per V region must have come from a cell which divided at least 60 times after antigenic activation. Thus, CD20 + B cells isolated from the ST are an antigen selected population of memory cells which are part of the chronic inflammatory immune reaction. For one of the patients with RA the V-gene diversity seen in synovial B cells was compared with that seen in peripheral blood B cells (Figure 3A). Like in healthy individuals, the majority of V genes here were unmutated or showed a low level of somatic mutations. If these long-term memory B cells with a highly diversified repertoire are present in the peripheral blood they are only a minor subset. To look for these cells, memory and plasma cells will have to be specifically isolated from the peripheral blood of patients with chronic rheumatic diseases.

Diversification of the B-cell repertoire in the ST Identical sequences were isolated from consecutive sections of some of the infiltrates suggesting that memory cells are clonally expanded in the ST. The finding of sequences with an identical rearrangement and pattern of somatic mutations showed that these cells proliferated without further diversification of the repertoire. This result fits with data from the analysis of autoimmune mice which suggested that under chronic activation memory cells reach a stage where no further somatic mutations accumulate.31 In general it is thought that the hypermutation mechanism becomes activated only during clonal expansion of B cells in the micro-environment of the germinal center. However, mutated V-gene sequences were isolated from lymphotoxin α knock out mice which have no germinal centers in their splenic tissue.32 These data suggest that the diversification of the B-cell repertoire may take place independent of germinal centers. Another possibility is that germinal centers develop in non-lymphoid tissue. The analysis of lymphocytic cell clusters isolated from the ST showed that in autoimmune diseases in

Restricted repertoire of synovial plasma cells In some of the patients with RA numerous plasma cells accumulate in the ST. The finding of several thousand plasma cells in a small part of the ST of the size of 1 to 2 mm2 gives an idea about the very large numbers of plasma cells found in the ST. Antigenactivated B cells may have developed into pre-plasma cells and than migrated into the ST. On the other 265

C. Berek and H.-J. Kim structure is built up in the inflamed synovium. Here, the differentiation of B cells into plasma cells may occur. The circular arrangement of plasma cells, surrounding the follicle-like structures (Figure 1) suggests that plasma cells developed from CD20 + B cells in the inner part of the lymphocytic cell clusters. The enrichment of CD4 + T-helper cells in the outer parts of the lymphocytic cell clusters10 may favour the differentiation into plasma cells. Finally, these cells may migrate into the tissue and populate the neighbouring synovium (Figure 1B). Finding several hundred B-cell clones all expressing a Vλ2 rearrangement in such a small area of the ST is difficult to understand. Even if under chronic activation of the immune system a selected population of B cells is continuously drawn into a germinal center reaction and plasma cells are generated, one would not expect to find such high numbers. It seems more likely that, with time, increasing numbers of plasma cells expressing a certain repertoire have accumulated in the ST. However, this interpretation requires that plasma cells are long lived. Plasma cells, terminally differentiated into non-dividing cells, are traditionally thought of as being short-lived. The explanation for long-term antibody titers after immunization may be persisting antigen5 leading to continuous reactivation of memory cells and their differentiation into plasma cells. On the other hand, plasma cells may be longlived.34 In in-vitro cultures, the life-time of plasma cells can be extended by co-culturing with synoviocytes.33 Similarly the bone marrow seems to support the longevity of plasma cells (Radbruch, pers. communication).

hand, there is evidence that synoviocytes support the differentiation of activated B cells.33 Thus, B-cell differentiation into plasma cells may take place in the follicle-like structures of the ST. In order to look at the L-chain repertoire of synovial plasma cells DNA was prepared from a section which contained approximately 4000 plasma cells (Berek, manuscript in preparation). As there seems to be a preference for Vλ over Vκ expressing B cells in the ST the Vλ repertoire was determined. Altogether 50 V-regions were sequenced all of which were different. Differences in the CDRIII region showed that the majority of sequences are of independent clonal origin. Of these 50 sequences only two pairs of V regions were isolated where the rearrangement and the pattern of somatic mutations suggested that they are descendents of one B-cell clone. Surprisingly, the majority of V λ sequences (45 out of 50) had a rearrangement where a V gene of the Vλ2 gene family was joined with either Jλ1 or Jλ2/3. Thus, the large clonal diversity seen in the synovial plasma cells of this patient is derived from a rather restricted repertoire. Statistical analysis suggests that there may be up to 1000 different B-cell clones in this small part of the total ST. Several hundred B-cell clones expressing a Vλ2 rearrangement have accumulated here. On the other hand the analysis of the peripheral blood showed a depletion of Vλ2 sequences. When the same set of Vλ primers was used to amplify rearranged V genes of peripheral blood B cells only Vλ1, Vλ3, Vλ4 and Vλ9 sequences were isolated. A specific amplification with a Vλ2 primer in combination with Jλ primers resulted in no detectable DNA band. Thus, it seems that in this patient the peripheral blood is depleted of B cells expressing Vλ2 rearrangements. An antigenspecific immune reaction may have drawn Vλ2 expressing B cells into the ST. The V regions of these plasma cells were mutated less than those isolated from the follicle-like structures (Figure 3C). They had on average nine somatic mutations/V-region. These plasma cells must have developed from B cells which were activated by antigen and had gone through an affinity maturation process in a germinal center. In the peripheral blood we did not find B cells expressing Vλ2 L-chains though the number of memory and plasma cells in the periphery may be extremely low and little is known about lymphocyte traffic in the later stages of the chronic inflammatory reaction of patients with RA. The finding that a germinal center response takes place in the ST (see above) shows that a lymphoid-like

‘Memory’ plasma cells CD20 + B cells from the inner part of a follicle and plasma cells from the outer part of the same structure were isolated by micro-manipulation, and the repertoire of both cell populations determined (Kim et al, manuscript in preparation). In one of the clusters derived from the ST of a patient with ReA, CD20 + B cells were scarcely mutated (Figure 4A). The frequency of somatic mutations was comparable to that seen in resting mature B cells of the peripheral blood.35 The majority of the CD20 + B cells seem to represent new immigrants from peripheral blood B cells. On the other hand, V genes isolated from the neighbouring plasma cells carried multiple somatic mutations (Figure 4B). The majority of these cells seem to be derived from long-term memory cells. On 266

B-cell response in rheumatic diseases average, 19 somatic mutations per variable region were found. Clearly in this cluster, CD20 + B cells and plasma cells represent two distinct populations. A comparison of the rearrangements seen in the CD20 + B cells and the plasma cells showed that there are no sequences with identical rearrangements.

The immunization of mice with antigens such as phenyl oxazolone or nitro phenyl has demonstrated that germinal centers develop in the lymphoid organs 3 to 4 days after induction of the immune response. They increase in size until day 10 after which they rapidly decline.36,37 The kinetics of the development of germinal center-like structures in the ST may be similar. Patients with ReA go through relapsing phases of the disease. The isolated CD20 + B cells may correspond to B cells activated during the most recent relapse. The diversity seen in the V regions (Figure 4A) suggests that these B cells are new immigrants from the peripheral blood which migrated into a preexisting follicle-like structure. Memory cells generated in a previous germinal center-like reaction may have left the structure, whereas plasma cells may survive in the tissue for long periods of time. Thus, these plasma cells have to be long-lived.

Conclusion These data suggest that it is an antigen-dependent immune reaction that leads to the accumulation of B cells into the ST. During chronic inflammation a micro-environment is built up which allows a germinal center reaction to take place. In this lymphoid-like tissue not only the diversification of the B-cell repertoire, but also the differentiation into plasma and memory cells may take place. The restricted repertoire of plasma cells suggests that it is an antigen selected population that accumulates in the ST. Longevity of plasma cells might explain their high frequency in the inflamed tissue.

Acknowledgements We would like to thank B. Mu¨ ller, R.S. Jack and L. Cowell for critical reading of the manuscript and helpful discussion. This work has been supported by the Senate for Research and Education of the city of Berlin.

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Figure 4. CD20 + B cells and plasma cells are distinct population. From a single follicle-like structure in the ST of a patient with ReA CD20 + B cells (A) and plasma cells (B) were analysed. The V-gene diversity of synovial cells is shown. VH (clear column), Vκ (lightly stippled column) and Vλ (densely stippled column) sequences are shown separately.

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