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(Anti-)TNF alpha matters in rheumatoid arthritis
Wijbrandts, C.A.
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Wijbrandts, C. A. (2009). (Anti-)TNF alpha matters in rheumatoid arthritis
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Download date: 19 Jun 2017
Chapter 10
Synovial lymphoid neogenesis does not
define a specific clinical rheumatoid
arthritis phenotype.
Rogier M. Thurlings1, Carla A. Wijbrandts1, Reina
E. Mebius2, Tineke Cantaert1, Huib Dinant1,3,
Tineke C. T. M. van der Pouw-Kraan2, Dominique
Baeten1, Paul P. Tak1
1 Division of Clinical Immunology and
Rheumatology, Academic Medical Center/ University
of Amsterdam, The Netherlands, 2 Department of
Molecular Biology, VU Medical Center, Amsterdam,
The Netherlands. 3 Jan van Breemen Institute,
Amsterdam, the Netherlands.
Arthritis Rheum. 2008 Jun;58(6):1582-9.
Abstract
1.
2.
Objective.
3.
To determine the relationship between lymphoid neogenesis in the synovium of patients with 4.
rheumatoid arthritis (RA) and characteristics of inflammation and disease severity.
5.
6.
Methods.
7.
103 biological-naive patients with active RA (Disease Activity Score in 28 joints ≥ 3.2) underwent 8.
an arthroscopic synovial biopsy. Sections were stained and analyzed by digital image analysis. 9.
Lymphocyte aggregates were counted and graded for size (1-3). Synovial lymphoid neogenesis 10.
was defined as the presence of grade 2 or 3 aggregates and subdivided based on the presence 11.
of follicular dendritic cells (FDC).
12.
13.
Results.
14.
Lymphoid neogenesis was present in 32% of the RA synovial tissues, whereas an additional 15.
25% contained only grade 1 aggregates. FDC were present in 28% of samples with lymphoid 16.
neogenesis, corresponding to 8% of the total RA cohort. Histologically, synovia with lymphoid 17.
Chapter 10
neogenesis showed increased infiltration by T and B lymphocytes, plasma cells, and macro- 18.
140
phages, and increased expression of TNFα and LTb compared with samples without lymphoid 19.
neogenesis. Patients with lymphoid neogenesis also had higher C-reactive protein (CRP) levels, 20.
erythrocyte sedimentation rate (ESR) and elevated leukocyte and thrombocyte counts, but no 21.
increase in clinical signs and symptoms. Of importance, there was no relationship between 22.
the presence of lymphoid neogenesis and IgM rheumatoid factor or anti-citrullinated protein 23.
antibodies. The presence of lymphocyte aggregates with FDC did not define a specific clinical 24.
phenotype compared to lymphocyte aggregates without FDC.
25.
26.
Conclusion.
27.
Synovial lymphoid neogenesis is associated with more severe synovial and systemic inflamma- 28.
tion, but this is not confined to a specific clinical subset of RA.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
Synovial lymphoid neogenesis does not define clinical RA subtype
1. The extent and pattern of lymphocyte infiltration in the inflamed synovial tissue is widely
2. variable among different rheumatoid arthritis (RA) patients. In some tissues a diffuse or scarce
3. infiltration of T cells is present, while in others B and T cells are organized in perivascular aggre4. gates with surrounding fields of plasma cells [1-3]. Sometimes large aggregates are present
5. containing clusters of follicular dendritic cells (FDC), exhibiting features normally present in
6. germinal centers of lymphoid tissue [1, 3, 4]. This suggests lymphoid neogenesis occurs in
7. rheumatoid synovial tissue.
8.
9. It has been suggested that synovial tissues with diffuse infiltration and tissues with lymphoid
10. neogenesis represent different pathophysiological subtypes of RA [5]. A further subdivision has
11. been suggested into highly organized ‘germinal center-like’ lymphoid neogenesis and a less orga12. nized form of lymphoid neogenesis in which T and B cells form aggregates, but exhibit no other
13. ‘germinal center-like’ features [5]. In contrast, other studies have shown that lymphoid neogen14. esis in rheumatoid synovial tissue is a more continuous spectrum, with lymphocyte aggregates
15. exhibiting different stages of lymphoid neogenesis present in the same patient [3]. Indications
16. of a stable presence of synovial lymphoid neogenesis and a relationship with a distinct clinical
17. phenotype have been suggested in some studies. First, it was shown that lymphoid neogenesis
18. can already be found at the onset of clinical arthritis [2]. In line with this, it has been suggested to
19. be a ‘fixed’ feature of synovial inflammation within different joints and patients [5]. Furthermore,
20. synovial tissues containing lymphocyte aggregates are associated with increased expression of
21. cytokines and adhesion molecules locally in the joint as well as in peripheral blood [6-8]. Finally,
22. histologic subtypes of lymphocyte infiltration appeared to be correlated with the presence of
23. rheumatoid factor, rheumatoid nodules, and joint erosiveness in small RA cohorts [1, 6, 9].
24.
25. Since previous studies were small and mainly based on synovial tissue retrieved during joint
26. surgery from patients with end-stage, destructive disease, it is still unclear whether lymphoid
27. neogenesis defines a specific subset of RA and whether this is related to the presence of cir28. culating autoantibodies. Therefore, we analyzed synovial tissue samples from a large cohort
29. of patients with active RA for the presence of lymphoid neogenesis in relationship to clinical
30. features and autoantibody status.
31.
32.
33. Methods
34.
35. Patients.
36. Synovial tissue was obtained from 103 RA patients fulfilling the American Rheumatism Associa37. tion 1987 revised criteria [10]. All patients used methotrexate (5-30 mg/week), were naive to
38. biologicals, and had active disease defined as a disease activity score in 28 joints (DAS28) ≥ 3.2 at
39. the time of biopsy [11]. Oral corticosteroids (≤10 mg/day) and non-steroidal anti-inflammatory
141
drug (NSAIDs) were also allowed. Disease characteristics were assessed, including disease 1.
duration, number of prior disease modifying antirheumatic drugs (DMARDs), presence of IgM 2.
rheumatoid factor (IgM-RF) and anti-citrullinated protein antibodies (ACPA) as measured by 3.
the anti-CCP2 ELISA (Immunoscan RA, Mark 2, Euro Diagnostica No. RA-96RT, Arnhem, the 4.
Netherlands), and radiographic damage as evaluated by the Sharp van de Heijde score [12]. 5.
Disease activity was assessed by the DAS28, the tender and swollen joint count in 28 joints, 6.
the patients’ assessment of global disease activity (VAS gda), the erythrocyte sedimentation 7.
rate (ESR), C-reactive protein levels (CRP), hemoglobin levels, and thrombocyte and leukocyte 8.
counts. The study was conducted in compliance with the Helsinki Declaration, and the Medi- 9.
cal Ethics Committee of the Academic Medical Center, University of Amsterdam approved the 10.
protocol. All patients gave written informed consent.
11.
12.
Arthroscopy and synovial biopsy.
13.
A mini-arthroscopy was performed under local anesthesia in all patients to obtain synovial tissue 14.
samples from an actively inflamed knee, ankle or wrist joint [13]. Biopsies were taken with a 2-mm 15.
grasping forceps (Storz, Tuttlingen, Germany) from 6 or more sites within the joint to minimize 16.
sampling error. Previous work has shown that for T cell infiltration and expression of activation 17.
Chapter 10
antigens in RA synovium, a variance of less than 10% can be reached when at least six biopsy 18.
142
specimens are examined [14], suggesting that representative data can be obtained when a limited 19.
number of biopsy samples from different areas within one joint are investigated. Consistent with 20.
these data, it has been demonstrated that using about six tissue samples allows for the detection 21.
of twofold differences in gene expression by quantitative polymerase chain reaction (PCR [15]). 22.
Thus, as T cells form an important cell population within the lymphocyte aggregates, we extrapo- 23.
lated these findings to the present study and decided to examine at least 6 biopsy samples per 24.
patient. The synovial biopsy samples were snap frozen en bloc in Tissue Tek OCT (Miles, Elkhart, IN) 25.
immediately after collection. Sections of 5 mm were cut and mounted on Star Frost adhesive glass 26.
slides (Knittelgläser, Braunschweig, Germany). Sealed slides were stored at -80°C.
27.
28.
Immunohistochemical analysis.
29.
Synovial tissue sections were stained using the following monoclonal antibodies: anti-CD55 30.
(67:Serotec, Oxford, UK) to detect fibroblast-like synoviocytes (FLS), anti-CD68 (EBM11: DAKO, 31.
Glostrup, Denmark) to detect macrophages, anti-CD3 (SK7, Becton Dickinson, San Jose, CA) for 32.
T cells, anti-CD22 (CLB-B-ly/1,6B11, the Netherlands) for B cells, and anti-CD38 (HB7, Becton 33.
Dickinson, San Jose, CA) for plasma cells. For detection of follicular dendritic cells (FDC) anti- 34.
CD21 long isoform (anti-CD21L; a kind gift from dr. Y.J. Liu) was used [16].
35.
36.
The following markers were used for detection of cytokines: anti-human TNFa (52B83; Mono- 37.
san, Uden, Belgium) and anti-LTβ (c0404; Santa Cruz Biotechnology Inc, Santa Cruz, CA). Stain- 38.
ing of cellular markers was performed using a 3-step immunoperoxidase method as described 39.
Synovial lymphoid neogenesis does not define clinical RA subtype
1. previously [17]. For staining of cytokines biotinylated tyramine was used as amplification, as
2. described previously [18]. As negative control irrelevant immunoglobulins were applied to the
3. sections instead of the primary antibody, or the primary antibody was omitted.
4.
5. Assessment of lymphocyte aggregates.
6. The presence of lymphocyte aggregates was assessed on anti-CD3 stained sections. Aggre7. gates were counted and graded by size according to the method described by Manzo et al. [3]
8. with a slight modification: the aggregate size was assessed by counting the number of cells in
9. the radius starting from an imaginary center of the aggregate. Aggregate size was classified
10. into grade 1 (1-5 cells radius), grade 2 (5-10 cells radius) or grade 3 (more than 10 cells radius).
11. Tissue sections with no lymphocyte aggregates at all were graded as 0.
12.
13. The presence of FDC, as stained by CD21L, was assessed in lymphocyte aggregates that were
14. graded for size on 3 different tissue levels; T-B cell segregation was assessed at 2 different tissue
15. levels at least 50 μm apart on sequential sections stained with CD3 and CD22. Thus, multiple
16. sections representing different levels of a tissue block consisting of at least 6 biopsy samples
17. were examined to minimize sampling error.
18.
19. Digital image analysis.
20. All sections were analyzed at random by trained analysts who were blinded for clinical char21. acteristics. The analysis was done by a computer-assisted image analysis algorithm as previ22. ously described in detail [19]. Images were acquired and analyzed using a Syndia algorithm
23. on a Qwin-based analysis system (Leica, Cambridge, UK). Positive staining of cellular markers
24. was expressed as positive cells/ mm2 (counts/ mm2), and positive staining of cytokines was
25. expressed as integrated optical density/ mm2 (IOD/ mm2). CD68+ macrophages, anti-LTβ and
26. TNFa expression were analyzed separately in the intimal lining layer and the synovial sublining.
27.
28. Statistical analysis.
29. Independent t-tests or Mann Whitney U tests were used to compare synovial (CD68, CD3, CD22
30. and CD38 positive cells), serological (ESR, CRP, leukocyte and thrombocyte counts), clinical
31. inflammatory parameters (DAS28), and Sharp van de Heijde scores. The Chi-square test was
32. employed to evaluate specific features of ACPA and IgM-RF positive disease. The same test was
33. performed to compare these parameters between FDC positive and FDC negative aggregate
34. synovitis. SPSS 12.0.2 for Windows (SPSS, Chicago, IL) was used.
35.
36.
37.
38.
39.
143
Results
1.
2.
Patient characteristics.
3.
103 patients were analyzed. Demographic and clinical features are shown in Table 1. All 4.
patients used methotrexate. Oral low-dose corticosteroids were used by 25% of the patients. 5.
On average, patients failed treatment with 2.1 DMARDs prior to inclusion in the study. Data on 6.
rheumatoid nodules was available for 58 patients, of which 22 patients (38%) had nodular RA.
7.
8.
Frequencies of lymphocyte aggregates and FDC.
9.
A mixture of lymphocyte aggregates of different sizes was present in the synovial tissue of 58 10.
patients: in 16 patients (16%) aggregates up to grade 3 were present. In another 16 patients 11.
(16%) aggregates up to grade 2 were found and 26 patients (25%) had only small peri-vascular 12.
infiltrates (grade 1 aggregates). In the remaining 45 patients (44%) no lymphocyte aggregates 13.
were detectable (Figure 1).
14.
15.
Of the 103 synovial samples, 95 were analyzable for FDC staining. Eight of 16 samples with 16.
grade 3 lymphocyte aggregates showed CD21L positive staining (50% of tissues containing 17.
Chapter 10
grade 3 aggregates and 8% of all tissues). FDC containing aggregates were found next to 18.
aggregates without FDC (Figure 2). Separate clusters of T cells and B cells were found in 7 out of 19.
16 biopsies with grade 3 aggregates and did not occur in grade 1 or 2 aggregates.
20.
21.
144
22.
Table 1. Baseline patient characteristics.
23.
N=103
Demographics
Age, mean (SD) years
Female, no (%)
55 ± 13
73 (71)
130 ± 116
79 (77)
76 (74)
76 (74)
5.9 ± 1.1
33 (18-45)
12 (5-29)
Drug treatments
Previous DMARDs, median (IQR)
Methotrexate, median (IQR) mg/wk
Receiving corticosteroids, no (%)
Receiving NSAIDs, no (%)
25.
26.
27.
Disease status
Duration, mean (SD) months
Erosive disease, no (%)
Rheumatoid factor positive, no (%)
ACPA positive, no (%)
DAS28, mean (SD)
ESR, median (IQR) mm/hour
CRP, median (IQR) mg/dL
24.
2.1 ± 15
18.1 ± 8.5
26 (25)
53 (52)
*Data are presented as mean ± SD, median (interquartile range) or percentages, as appropriate. * IgM-RF
= IgM rheumatoid factor; IQR = interquartile range; ACPA = anti–cyclic citrullinated peptide; DAS28
= Disease Activity Score 28-joint assessment; ESR = erythrocyte sedimentation rate; CRP = C-reactive
protein; DMARDs = disease-modifying antirheumatic drugs.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
Synovial lymphoid neogenesis does not define clinical RA subtype
1.
2.
3.
4.
5.
6.
7.
8. Figure 1. Different patterns of lymphocyte infiltration can be detected in the synovial tissue. In a
9. proportion of patients a mixed infiltration of aggregates of T and B cells is present (A and B), together
with a high number of infiltrating macrophages (C). Both TNFα (D) and LTb (E) are abundantly expressed.
10. In other patients a diffuse or scarce infiltrate of CD3+ T cells is found (F), and little or no B cells (G), while
11. macrophages are the dominant infiltrating cell population (H). TNFα and LTb are expressed at low levels in
12. these patients (I,J). (Original magnification × 20.)
13.
See color figure page 220.
14.
15. The presence of lymphoid neogenesis is associated with synovial inflammatory cell infiltration.
16. To study the relationship between synovial lymphoid neogenesis and other inflammatory fea17. tures we divided the tissue into 2 groups: those with grade 2 or 3 aggregates (n = 32) and those
18. with no aggregates or only grade 1 aggregates (n = 71). Synovial lymphoid neogenesis was
19. defined as the presence of grade 2 or 3 aggregates, since aggregates of these sizes frequently
20. exhibit characteristics of lymphoid neogenesis, such as T-B cell separation and CXCL13 and
21. CCL21 expession [3, 20]; the remaining tissue was defined as diffuse synovitis. For both analy22. ses, there were no significant differences in potentially confounding factors such as gender,
23. age, disease duration, number of failed DMARDs, as well as use and dosage of prednisone or
24. methotrexate between these 2 groups.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
Figure 2. In 8 % of the synovial tissue samples, clusters of follicular denritic cells, expressing the CD21
long isoform (A), could be detected in lymphocyte aggregates enriched with CD22 positive B cells (B).
(Original magnification × 20, inset x 40.)
See color figures page 220
145
In tissue with lymphoid neogenesis the number of CD3+ T cells as well as CD22+ B cells and 1.
CD38+ plasma cells was higher than in diffuse synovitis (P < 0.001 for all; Table 2). Furthermore, 2.
lymphoid neogenesis concurred with higher numbers of CD68+ macrophages in both the 3.
intimal lining layer (P = 0.002) and synovial sublining (P < 0.001). Also, the pro-inflammatory 4.
cytokine TNFα in the sublining was expressed at higher levels in tissue containing lymphoid 5.
neogenesis (P = 0.018). In line with previous data, LTb, a cytokine involved in lymphoid neo- 6.
genesis, was more abundantly expressed in the synovial sublining of these tissues (P < 0.001).
7.
8.
Lymphoid neogenesis is associated with biomarkers of systemic inflammation, but not with clinical characteristics
of disease severity.
9.
10.
Subsequently, we investigated the relationship between lymphoid neogenesis, systemic 11.
inflammatory features, and disease severity (Table 2). Compared to patients with diffuse syno- 12.
vitis, patients with lymphoid neogenesis had higher ESR (P = 0.031) and serum CRP levels (P 13.
= 0.034), as well as leukocyte (P = 0.002) and thrombocyte counts (P = 0.001). In contrast, no 14.
difference was found for disease activity as measured by the DAS28, the swollen and tender 15.
joint count, or the global disease activity (VAS).
16.
17.
Chapter 10
Bone and cartilage damage was assessed by radiology in 76 patients. Patients with lymphoid 18.
146
neogenesis tended to have less erosions and a lower overall Sharp van de Heijde score (P = 19.
0.085 and P = 0.100, respectively). However, when analyzing only the ACPA positive patients, 20.
this trend towards inverse correlation between lymphoid neogenesis and joint destruction was 21.
no longer found (P = 0.178 and P = 0.332, respectively). There was also no relationship between 22.
lymphoid neogenesis and the presence of rheumatoid nodules (Table 2).
23.
24.
Finally, lymphoid neogenesis was not associated with IgM-RF positivity. Surprisingly, patients 25.
with lymphoid neogenesis were significantly less often ACPA positive (18 of 31 patients with 26.
lymphoid neogenesis versus 58 of 72 in patients with diffuse synovitis; P = 0.018).
27.
28.
Since the relatively high number of tissues containing only grade 2 aggregates in the subset 29.
of tissues categorized in the group of lymphoid neogenesis might theoretically contribute 30.
to underestimate the potential differences between the diffuse group and the subset of RA 31.
patients with more advanced features of secondary lymphoid organs, we performed a sub- 32.
analysis comparing disease severity parameters and autoantibody status between patients 33.
with diffuse synovitis (n = 71) versus those with grade 3 aggregates (n = 16), Similar results were 34.
found compared to our previous analysis described above. In tissues with grade 3 aggregates 35.
significantly more T cells, B cells, plasma cells and macrophages were found, while expression of 36.
LTb and TNFα was significantly increased. There was no significant difference in disease activity, 37.
autoantibody status, or joint destruction between these 2 groups (data not shown).
38.
39.
Synovial lymphoid neogenesis does not define clinical RA subtype
1.
Table 2. Associations of clinical, serological and synovial parameters with lymphoid neogenesis.
2.
3.
5.
6.
Serological parameters
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
lymphoid neogenesis
N= 32
P value
5.9 ± 1.1
66 (9-161)
18 (41)
6.1 ± 1.1
30 (1-77)
4 (28)
0.330
0.1001
0.308
30 (15-41)
11 (4-27)
7.8 (7.2-8.4)
7.2 (5.9-8.6)
292 (240-364)
54 (75)
58 (81)
36 (23-70)
16 (10-44)
7.8 (6.6-8.5)
9.3 (6.8-11.3)
350 (302-403)
22 (71)
18 (58)
0.031
0.034
0.307
0.002
0.001
0.422
0.018
46734 (28456-68777)
63948 (31986-97594)
30 (11-71)
46099 (36778-96977)
99811 (52118-134603)
151 (51-611)
0.134
0.018
<0.001
5345 (402-934)
72 (37-149)
0 (0-15)
30 (0-144)
261 (177-414)
296 (166-635)
569 (303-1066)
381 (220-678)
88 (35-164)
631 (253-1089)
431 (300-573)
755 (407-1081)
0.933
<0.001
<0.001
<0.001
0.002
<0.001
Clinical parameters
DAS28
Sharp vd Heijde
Nodules, no (%)
4.
Diffuse synovitis
N= 71
ESR (mm/hour)
CRP (mg/dL)
Haemoglobin (mmol/L)
Leucocytes (10E9/L)
Thrombocytes (10E9/L)
IgM-RF, no (%)
ACPA, no (%)
Cytokines (IOD/mm2 )
TNFa lining
TNFa sublining
LTb sublining (counts/mm2)
Cellular markers (Counts/mm2 )
CD55
CD3
CD22
CD38
CD68 lining
CD68 sublining
20. *Data are presented as mean ± SD or median (interquartile range) whichever appropriate. The t-value
21. is derived from the independent samples t-test. The Z-value is derived from the Mann Whitney U test.
22.
23.
24.
25.
P values < 0.05 (two-sided) are significant and shown in Italic. DAS28 = Disease Activity Score 28-joint
assessment; IgM-RF = IgM rheumatoid factor; ACPA = anti–cyclic citrullinated peptide antibodies; ESR
= erythrocyte sedimentation rate; CRP = C-reactive protein. 1 When analyzing only the ACPA positive
patients, the trend towards inverse correlation between lymphoid neogenesis and the Sharp van de
Heijde score is no longer present (P = 0.332).
26.
27. FDC positive lymphoid neogenesis is not related to increased inflammation.
28. Although previous work has shown the sensitivity of immunohistochemistry to detect CD21L
29. positive cells compared to PCR [4, 21] we performed PCR to detect CD21L mRNA in a subset
30. of 12 patients. In 2/12 samples CD21L mRNA was detected. In both of these samples we also
31. detected CD21L protein (data not shown). One sample tested positive for CD21L protein, but
32. not for CD21L mRNA (it should be noted that immunohistochemistry allows the detection of
33. scarce, isolated cells). Thus, these results suggest that we do not appear to underestimate the
34. presence of FDCs in our study.
35.
36. In a previous study it was suggested that a subdivision exists between highly organized, FDC
37. containing, ‘germinal center-like’ lymphoid neogenesis and a less organized form of lymphoid
38. neogenesis in which T and B cells form aggregates, but no FDC or other ‘germinal center-like’
39. features are present [5]. Therefore, we performed a sub-analysis comparing patients with FDC
147
positive (n = 8) versus negative (n = 21) lymphoid neogenesis. We only found a significantly 1.
higher number of CD22+ B cells and CD38+ plasma cells to be present in FDC positive compared 2.
to FDC negative lymphoid neogenesis (median 157 B cells/mm2, IQR 90-347 and median 66, 3.
IQR 31-160, respectively, P = 0.047; median 858 plasma cells/mm2, IQR 637-1370 and median 4.
593, IQR 210-902, respectively, P = 0.041). However, all other tissue and clinical parameters did 5.
not differ statistically between these forms of lymphoid neogenesis.
6.
7.
8.
Discussion
9.
10.
The current study was performed to investigate whether lymphoid neogenesis in RA synovium 11.
is related to specific clinical or immunological features. Previous data suggesting that RA 12.
characterized by lymphocyte aggregates or ectopic germinal centers represents a specific 13.
subset were obtained in small cohorts of patients with end-stage, destructive disease [1, 6, 14.
9]. In contrast, the current study was performed in a large and well characterized cohort of RA 15.
patients with active disease despite methotrexate treatment.
16.
17.
Chapter 10
In comparison with previous data, we found lymphoid neogenesis in a relatively low number 18.
148
of patients (32% vs respectively 44-90%) [2-4]. We also found lower frequency of FDC and T-B 19.
cell separation. The differences might be related to differences in antirheumatic treatments 20.
or selection of end-stage, destructive RA in previous studies. In addition, differences may be 21.
explained in part by the specific definitions of lymphocyte aggregates adopted in previous 22.
studies. We performed a detailed analysis of lymphocyte aggregates as originally proposed by 23.
C. Pitzalis [3], and made a subdivision based on aggregate size.
24.
25.
We found that lymphoid neogenesis in rheumatoid synovial tissue coincided with inflamma- 26.
tory features such as increased numbers of infiltrating macrophages and expression of TNFα 27.
at the site of inflammation and increased leukocyte and thrombocyte counts, ESR and CRP 28.
titers in peripheral blood. These findings do not explain whether the presence of lymphoid 29.
neogenesis in rheumatoid synovial tissue is a cause or a consequence of inflammation in RA. 30.
It has been suggested that synovial lymphoid neogenesis contributes to ectopic maturation 31.
of B cell responses, since clonal expansion, somatic hypermutation, and diversification of 32.
B cells have been described in RA synovial tissue [22]. Otherwise, to date no direct proof of 33.
functionality of lymphoid neogenesis has been shown. Since lymphoid neogenesis also occurs 34.
in non-autoantibody associated forms of arthritis such as psoriatic arthritis and osteoarthritis 35.
[18, 23], lymphoid neogenesis could arise as a result of non-specific inflammation.
36.
37.
We found no indication that synovial tissue containing FDC positive aggregates represents 38.
a ‘germinal-center like’ subset of lymphoid neogenesis, but we could confirm previous 39.
Synovial lymphoid neogenesis does not define clinical RA subtype
1. observations showing that lymphocyte aggregates are present in a heterogeneous mix of
2. different numbers and sizes in synovial tissue [3]. FDC positive aggregates were detected next
3. to aggregates without FDC or other germinal center-like features such as T-B cell separation.
4. Additionally, we observed no increase in macrophage infiltration or cytokine expression in
5. synovial tissue containing FDC positive aggregates. These data suggest that the presence of
6. FDC in the synovium may be a phenomenon secondary to inflammation rather than a primary
7. phenomenon.
8.
9. Lymphoid neogenesis was related to biomarkers of inflammation, but in contrast not to
10. increased clinical signs and symptoms. This suggests that the extent to which synovial tissue
11. lymphoid neogenesis relates to systemic inflammation is not such that this translates into the
12. clinical expression of the disease. This could be, because the link between lymphoid neogenesis
13. and local synovial inflammation is not pivotal or because synovial inflammation and concur14. rent lymphoid neogenesis might vary between joints, although previous work indicated that
15. there is little variation in synovial inflammation between different joints of the same patient
16. [13]. Otherwise, clinical measures of disease activity may also be influenced by factors other
17. than synovial inflammation including joint destruction. In addition, there was no indication
18. that lymphoid neogenesis is related to more erosive or nodular disease, as has been suggested
19. previously in a small patient cohort [9]. This also indicates that either the link between lym20. phoid neogenesis and synovial inflammation is not dominant or that the presence of lymphoid
21. neogenesis and concurrent synovial inflammation may vary between joints and over time.
22.
23. Previous work has indicated that the rheumatoid synovium is a potent autoantibody-producing
24. organ; the antibodies may form immune complexes in the joint leading to complement fixation
25. and macrophage activation [24]. Synovial plasma cells may synthesize and secrete rheumatoid
26. factors, ACPA, and other autoantibodies [25, 26]. In keeping with these findings, the levels of
27. rheumatoid factors and ACPA are higher in synovial fluid than in peripheral blood [27, 28]. We
28. demonstrate here that the presence of circulating autoantibodies is not related to lymphoid
29. neogenesis. We cannot completely exclude the possibility that lymphocyte aggregates were
30. not detected in some autoantibody positive, lymphocyte aggregate negative RA patients, but
31. we did minimize sampling error by analysis of 2 levels of a tissue block representing at least 6
32. synovial tissue samples. In fact, circulating ACPA were detected even less frequently in patients
33. with lymphoid neogenesis. Thus, our data do not support a critical role for lymphoid neogen34. esis in the synovium for the production of rheumatoid factor and/or ACPA.
35.
36. In conclusion, the data presented here show that lymphoid neogenesis in the synovium does
37. not define a clinically or immunologically defined subtype of RA. The presence of lymphocyte
38. aggregates and germinal center-like structures may be a secondary phenomenon due to
39. chronic inflammation.
149
Acknowledgements.
1.
The authors wish to thank Desiree Pots for assisting with the immunohistochemical stainings, 2.
Marjolein Vinkenoog for expert digital image analysis, and Natasha Cassin and Margot Colom- 3.
bijn for performing clinical assessments.
4.
5.
Chapter 10
6.
150
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