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Departments of
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Medicine and Immunology,
Laboratory Medicine and Pathology,
Health Services Research, and
Orthopedic Surgery, Mayo Clinic, Rochester, MN 55905
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsHistopathological evaluationsRT-PCR and cytokine...AntibodiesImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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and of B
lymphocyte chemoattractant (BLC; BLC/CXCL13), that were predictors for
FDC recruitment and synovial GC formation. LT- and BLC/CXCL13 were
found to be independent variables that could, in part, compensate for
each other to facilitate GC formation. Prediction models incorporating
in situ transcription of LT- and BLC/CXCL13 had high negative yet
moderate positive predictive values, suggesting that LT- and
BLC/CXCL13 are necessary but not sufficient. LT- protein was
detected on a subset of mantle zone and GC B cells, but also on T cells
in follicular structures. BLC/CXCL13 was produced by FDCs in follicular
centers, but was predominantly found in endothelial cells and synovial
fibroblasts, suggesting heterotypic signaling between cells of the
synovial membrane and infiltrating lymphocytes in regulating extranodal
lymphoid neogenesis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsHistopathological evaluationsRT-PCR and cytokine...AntibodiesImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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A close relationship between inflammation and lymphoid organogenesis
has been suggested by the finding that proinflammatory cytokines, such
as members of the TNF superfamily, have a critical role in both
processes (4, 5). Initially, it was found that mice
deficient in lymphotoxin (LT) had no lymph nodes or Peyer’s patches
and failed to form GCs in the spleen (6, 7). Despite the
absence of GCs, LT-
-/- mice still produced
high-affinity IgG1 responses, provided the mice were immunized with
high doses of Ag (8). Gene targeting has been successfully
used to implicate other molecules in the process of lymphoid
organogenesis, including LT-, type I TNFR, and the LT-R
(9, 10, 11). Mice with defects in these genes display
different structural abnormalities and functional impairment of
secondary lymphoid organs. In essence, signals transmitted by
LT-12 appear to be pivotal in the ontogeny of secondary lymphoid
organs (12, 13).
More recently, studies of lymphoid organogenesis have focused on the contribution of chemokines that provide the cues to guide cell movement inside lymphoid organs (14, 15). Much progress has been made in understanding how the two major populations of lymphocytes are directed either to B cell or T cell zones and how chemokines control the movements of such cells during the development of Ag-specific immune responses. Mice homozygous for the spontaneous mutation, paucity of lymph node T cells (plt), are characterized by major abnormalities in T cell trafficking into lymph nodes and disturbances in the organization of T cell zones (16, 17). These mice lack expression of secondary lymphoid chemoattractant (SLC/CCL21) (18), which binds to CCR7. Defects in the movement of lymphocytes and DCs through the T cell areas of spleen, lymph nodes, and Peyer’s patches are shared by plt and CCR7-deficient mice, establishing a critical role of this receptor-ligand pair in compartmental homing of T cells (19). Homeostatic trafficking of B cells into lymphoid tissue and B cell follicles appears to be critically controlled by B lymphocyte chemoattractant (BLC/CXCL13) (20, 21). The receptor for BLC/CXCL13, CXCR5, is expressed on recirculating B cells; in in vitro chemotaxis assays, BLC/CXCL13 attracts B cells. In mice with a targeted inactivation of CXCR5, the normal development of Peyer’s patches, inguinal lymph nodes, splenic follicles, and peripheral lymphocytes is disrupted, making the BLC/CXCL13-CXCR5 receptor-ligand pair critical in lymphoid tissue organization (15).
We studied a large cohort of patients with RA who presented with
different phenotypes of lymphoid microarchitectures in the synovial
lesions and examined whether cytokines and chemokines implicated in the
genesis of secondary lymphoid organs are involved in the process of
lymphoid neogenesis in RA. Analysis of the cellular elements in the
synovium demonstrated that T cells, B cells, macrophages, and DCs were
universally present in rheumatoid synovitis irrespective of the
topographical organization of the infiltrate. In contrast, follicular
DCs (FDCs) were limited to a subset of patients. Their presence
perfectly correlated with the formation of secondary follicles and GCs.
Multivariate regression analysis identified LT- and BLC/CXCL13 as
independent critical variables in distinguishing patients with and
without synovial GCs. These data suggest that seeding with FDCs or
their precursors is the critical step in follicle formation in the
synovium and occurs in some, but not all, patients. LT-12 and
BLC/CXCL13 may recruit or retain this highly specialized cell to
extranodal tissue sites, thus determining the ultimate organization and
function of tissue-invading lymphocytes in rheumatoid synovitis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsHistopathological evaluationsRT-PCR and cytokine...AntibodiesImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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Synovial tissue was obtained from 64 patients with active RA who fulfilled the American College of Rheumatology 1987 revised criteria for RA and who underwent joint surgery. All patients provided informed consent. The study was approved by the Mayo Clinic Internal Review Board.
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Histopathological evaluations |
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TopAbstractIntroductionMaterials and MethodsHistopathological evaluationsRT-PCR and cytokine...AntibodiesImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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RT-PCR and cytokine semiquantification |
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TopAbstractIntroductionMaterials and MethodsHistopathological evaluationsRT-PCR and cytokine...AntibodiesImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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-actin-specific sequences by semiquantitative PCR-ELISA and then
adjusted to contain equal numbers. Adjusted cDNA was amplified by PCR
for 30 cycles under nonsaturating conditions with cytokine-specific
primers (Table I
) in parallel with a standard containing a known number of cytokine
sequences (23, 24). Each PCR amplification cycle consisted
of denaturation at 94°C for 30 s, annealing at either 55°C
(-actin, BLC/CXCL13, LT-, MCP-1/CCL2, and DC-derived C-C
chemokine (DC-CK1/CCL18)), 58°C (LT-, and LT-R), or 60°C
(SLC/CCL21) for 1 min, and polymerization at 72°C for 1 min with
10-min denaturation at 94°C at the start of the reaction and a final
10-min extension at 72°C. Amplified products were labeled with
digoxygenin-11-dUTP (Roche Molecular Biochemicals, Indianapolis,
IN) and then semiquantified in a liquid hybridization assay with
biotinylated internal probes (Table I) using a commercially available
PCR-ELISA kit (Roche Molecular Biochemicals). Labeled PCR products were
hybridized for 2.5 h with 200 ng/ml probe at 42°C for -actin,
at 50°C for DC-CK1/CCL18 and macrophage chemoattractant protein 1
(MCP-1)/CCL2, and at 55°C for LT-, LT-, LT-R, BLC/CXCL13,
and SLC/CCL21. Hybrids were immobilized on streptavidin-coated
microtiter plates and, after washing, were detected with a
peroxidase-labeled anti-digoxigenin Ab. Plates were developed by a
color reaction using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic
acid) (diammonium salt) substrate and quantified using a kinetic
microplate reader (Molecular Devices, Sunnyvale, CA). The number of
cytokine-specific sequences was determined by interpolation with a
standard curve and was expressed as the number of cytokine sequences
per 1 x 106 -actin sequences. A ratio of
one cytokine-specific sequence per 1 x 106
-actin sequences was arbitrarily defined as 1 U.
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)
(25). Amplified products were analyzed by 2% agarose gel
electrophoresis. PCR conditions were as described for amplification of
products for PCR-ELISA. The annealing temperature for the primer set
was 55°C. |
Antibodies |
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TopAbstractIntroductionMaterials and MethodsHistopathological evaluationsRT-PCR and cytokine...AntibodiesImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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mAb, B9.C9 (AC10) and B27.B2 (26, 27) (from J. L. Browning, Biogen, Cambridge, MA), mouse
anti-human TNF- (LT-) mAb (R&D Systems), and mouse
anti-human CXCR5 (BLR-1) mAb (R&D Systems). Secondary Ab used were
peroxidase-labeled goat anti-mouse IgG (1:300; Kirkegaard & Perry
Laboratories, Gaithersburg, MD) and biotinylated rabbit anti-goat
Ig (1:300; Dako). |
Immunohistochemistry |
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TopAbstractIntroductionMaterials and MethodsHistopathological evaluationsRT-PCR and cytokine...AntibodiesImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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For double-staining with LT-12 and CD20 Abs, the
EnVision+ system (Dako) was used. Sections were
fixed as above, blocked with 5% swine serum for 15 min, and incubated
with mouse anti-human LT-12 mAb (1:200) for 30 min at room
temperature. Thoroughly washed sections were treated for 30 min with
the EnVision+ reaction system and developed with
3,3'-diaminobenzidine tetrahydrochloride. The 3,3'-diaminobenzidine
tetrahydrochloride-stained slides were washed in tap water. Nonspecific
binding was blocked for 15 min with 5% goat serum, and sections were
stained with mouse anti-human CD20 mAb (1:100; Dako) for 60 min at
room temperature. After incubation with biotinylated rabbit
anti-mouse Ig Ab (1:300; Dako), the slides were incubated with the
VectaStain avidin-biotin complex-alkaline phosphatase kit
(Vector Laboratories, Burlingame, CA) for 30 min and then developed
with a Vector Red substrate kit (Vector Laboratories) for 3 min. Slides
were counterstained with hematoxylin for 5 s and permanently
mounted in Cytoseal-60 (Stephens Scientific, Riverdale, NJ). Negative
controls were stained with secondary Ab without the primary
Ab.
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Statistical analysis |
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TopAbstractIntroductionMaterials and MethodsHistopathological evaluationsRT-PCR and cytokine...AntibodiesImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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Results |
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TopAbstractIntroductionMaterials and MethodsHistopathological evaluationsRT-PCR and cytokine...AntibodiesImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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Synovial tissues from 64 RA patients were obtained at the time of
synovectomy or joint replacement surgery and were analyzed for the
topographical organization of the inflammatory infiltrate. The
arrangements of tissue-invading cells in rheumatoid synovitis were
diverse, but three distinct patterns could be distinguished (Fig. 1). In 36 tissues (56.3%), T cells, B cells, macrophages, and DCs were
arranged as diffuse infiltrates without specific microstructures. In
tissues from the other 28 patients, B cells formed clusters, generally
combined with T cells. Such cell clusters were either organized as
secondary follicles with GC reactions (15 tissues or 23.4%) or
presented as T cell-B cell aggregates lacking GCs (13 tissues or
20.3%). These two patterns were mutually exclusive; lymphoid
aggregates with and without GCs were not found in parallel in the same
tissues. GC+ follicles were characterized by B
cell proliferation, loss of central IgD-expressing B cells, and the
presence of FDC networks. B cells in T cell-B cell aggregates without
GCs did not actively proliferate and FDC networks were not detectable
(3). Both types of tissues with clustering of B cells and
T cells contained plasma cells that tended to accumulate under the
synovial lining layer. CD83+ DCs were present in
all variants of rheumatoid synovitis.
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FDCs are an essential cellular component of B cell follicles in
secondary lymphoid tissues. To assess whether all synovial tissue
samples contained these cells, immunohistochemical stains for CD23 were
performed (Fig. 1). To avoid sampling artifacts, PCR for a constitutive
marker of FDCs, CD21L (25), was performed in parallel
(Fig. 2). Both approaches yielded the same result. FDCs were found in 15 of 64
tissues (23.4%) analyzed. In all biopsies with a positive signal for
CD21L mRNA, follicles with GC reactions were present. In all tissues
lacking GC+ follicles, CD21L was undetectable. In
particular, tissues with T cell-B cell aggregates without GCs lacked
FDCs, indicating that these structures are different from primary
follicles. This finding demonstrated that the lymphoid neogenesis in
the rheumatoid synovium fundamentally differed from secondary lymphoid
organs. The exclusive finding of GCs but no primary follicles suggested
that the lymphoid neogenesis in the synovium is strictly dependent on
an Ag recognition event, in distinction to normal lymph nodes. The
selective presence of FDCs also indicated that these cells are not a
regular component of the synovial tissue.
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To identify cytokines and chemokines contributing to the formation
of synovial GCs, LT- and LT- transcripts were semiquantified
after adjustment for the number of -actin transcripts. As shown in
Fig. 3, tissues with GC+ follicles contained
significantly more LT- and LT- mRNA than the two other tissue
types. Both cytokines were expressed at distinctly low levels in
tissues with aggregates or diffuse synovitis. In biopsies with
GC+ synovitis, the median for LT- was 182 U;
LT--specific sequences were present with a median of 441 U. In
synovial tissue samples with T cell-B cell aggregates, the median
values for LT- and LT- were 42 and 35 U, respectively. Tissues
with diffuse synovitis were essentially negative for LT--specific
sequences and had low LT- mRNA.
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12 binds to the LT-R. Functional activity of LT-,
therefore, depends on the availability of
LT-R+ cells. Expression of LT-R in the
synovial microenvironment was analyzed by PCR. All tissue extracts
contained mRNA for LT-R. There was a trend for GC tissues to have
higher numbers of LT-R transcripts, but this was not statistically
significant. Correlation of tissue chemokine profiles and lymphoid microstructures
BLC/CXCL13 and SLC/CCL21 are critical players in the formation of
secondary lymphoid tissues (28, 29, 30) and are expressed in
chronic inflammatory lesions. BLC/CXCL13 mRNA was detected in synovial
tissue extracts at varying levels depending on the lymphoid
microstructure encountered in the biopsy (Fig. 4). The highest quantities of BLC/CXCL13 mRNA were detected in
GC-positive synovium, 15- to 30-fold higher than in tissues with T
cell-B cell aggregates (p = 0.001) or diffuse
lymphocytic infiltrates (p < 0.001). There was
a trend for BLC/CXCL13 to be higher in aggregate-positive tissues than
in diffuse tissues (median of 159 vs 89 U) that did not reach
statistical significance (p = 0.262).
Essentially all samples with secondary follicles contained BLC/CXCL13
mRNA in severalfold higher quantities than the average GC-negative
cases. However, the aggregate as well as the diffuse synovitis group
included four and three samples, respectively, with BLC/CXCL13 results
above 1000 U, indicating that BLC/CXCL13 can be transcribed in the
absence of lymphoid follicles.
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To rule out the possibility that the overrepresentation of BLC/CXCL13
and SLC/CCL21 in the GC+ follicles was simply a
reflection of increased inflammatory activity, two additional
chemokines, DC-CK1/CCL18 and MCP-1/CCL2 were analyzed. Results are
given in Fig. 4. All tissue extracts contained transcripts for
DC-CK1/CCL18 and MCP-1/CCL2. Tissues with secondary follicles had the
highest values for DC-CK1/CCL18, but they were not significantly
different from those in the aggregate-positive or diffuse cases.
MCP-1/CCL2 levels were comparable in all synovitis subsets. Median
values varied between 700 U in the diffuse samples and 1050 U in the
tissues with follicles.
In summary, we found preferential expression of SLC/CCL21 and BLC/CXCL13, two chemokines implicated in regulating trafficking of T cells and B cells in lymphoid tissues, in synovial tissue specimens containing GCs.
Predicting GC+ synovitis by logistic regression modeling
Several variables distinguished tissues with and without GC
formation, including expression of LT-, LT-, BLC/CXCL13, and
SLC/CCL21. In an attempt to estimate the relative contribution of these
parameters to the process of synovial GC formation, data were analyzed
using logistic models. Because lymphoid aggregate and diffuse tissues
did not differ for any of the variables (Figs. 3 and 4),
GC+ tissues were compared with the remaining
tissues. None of the variables had a Gaussian distribution. Each
continuous variable was, therefore, transformed into a discrete
variable by recursive partitioning. Logistic regression analysis was
performed to determine whether the dichotomized variables correlated
with GC formation. In the univariate analysis, high mRNA of all seven
markers were predictive of synovial GCs (data not shown). However,
tissue concentrations of LT- and BLC/CXCL13 transcripts were by far
the most powerful predictors of GC reactions in the synovial lesion.
The probability of encountering synovial GCs was 31-fold higher if
LT- transcripts were present at units above 315. Similarly, tissue
expression of BLC/CXCL13 sequences at >1800 U increased the
probability of GC formation by a factor of 31.
To explore whether the different cytokines were dependent or
independent variables in the molecular pathways generating synovial
GCs, multivariate analysis was performed. As shown in Table II, LT- and BLC/CXCL13 were independent predictors of GC formation.
Significance for SLC/CCL21 was lost after bootstrapping was performed
for validation of the variables in the model (43). None of the other
parameters continued to be significant after correction for LT- and
BLC/CXCL13 measurements. The model that best predicted GC formation was
based on the following two assumptions, reflecting the independent
contribution of BLC/CXCL13 and LT-: 1) patients with low BLC/CXCL13
(<1800 U) and low LT- (<315 U, defined by the recursive
partitioning) have no GCs, and 2) patients with high BLC/CXCL13 and/or
LT- have GCs. The model had a sensitivity of 86.7% and a
specificity of 87.8% to correctly predict GC formation (Table III). The positive predictive value was 68.4%, i.e., only 11 of
the 17 tissues predicted were true GC formers. Six of the 17 tissues
fulfilled the cytokine criteria but did not form GCs, indicating that
the mere presence of high LT- and/or BLC/CXCL13 transcripts was not
sufficient to guarantee follicle formation and that other variables,
e.g., cytokines not included in our analysis, may play a
role. The negative predictive value of the model was excellent (95.6%,
Table III), suggesting that GC formation does not occur if both of
these mediators are below a certain threshold. The alternative model,
requiring high concentrations of either cytokine, lacked
sensitivity.
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and BLC/CXCL13 in rheumatoid
synovitis
The strong predictive value of either LT- or BLC/CXCL13 in
identifying tissues with GC reactions raised the question of which cell
types supplied these two factors in the synovial microenvironment.
Immunohistochemical staining was used to detect membrane-bound LT-
in rheumatoid synovitis. Staining of peripheral blood B cells served as
positive controls; 80–85% of all circulating
CD20+ B cells expressed LT-. In secondary
lymphoid tissues, such as tonsils, weak staining could be localized to
few cells in secondary follicles. More prominent staining results were
obtained in synovial tissue sections. Sections from biopsies with
diffuse synovitis or T cell-B cell aggregates were negative. Synovial
tissue B cell follicles with GC reactions yielded a positive signal for
LT- (Fig. 5). A subset of the CD20+ B cells in the follicular
centers stained with anti-LT- Ab. A subpopulation of B cells in
the mantle zone was also positive for surface LT- protein. No
morphological or topographical characteristics were found
distinguishing LT-- and
LT-+ B cells. LT- staining was not
restricted to CD20+ cells. Follicular structures
also included lymphoid non-B cells staining positive. These cells
expressed CD4, identifying them as CD4+ T cells.
The majority of T and B cells in the tissue, however, did not stain for
LT-. LT- positivity of CD20+ and
CD20null cells was associated with follicles and
was not encountered in interfollicular regions.
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). Frequently, the cytoplasm of follicular center B cells also stained
with the Ab, whereas mantle zone cells were consistently negative.
BLC/CXCL13 production in the synovial tissue was not restricted to
follicular centers. In patients with or without
GC+ follicles, BLC/CXCL13 protein was detected on
endothelial cells of capillaries and small arterioles. Yet another cell
population contributed to BLC/CXCL13 production in the synovial
lesions; intense staining was consistently found on synovial lining
cells. In addition, isolated synoviocytes, dispersed throughout the
tissue, expressed BLC/CXCL13 protein.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsHistopathological evaluationsRT-PCR and cytokine...AntibodiesImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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and BLC/CXCL13. Interestingly,
LT- and BLC/CXCL13 were independently regulated and could, in part,
substitute for each other. LT-12 originated from lymphoid cells,
mainly B cells, whereas BLC/CXCL13 was predominantly supplied by
ancillary cells of the synovial membrane. The ability of synovial
tissue cells to participate in BLC/CXCL13 production is obviously a
factor guiding lymphoid neogenesis to this tissue site. Recruitment of
FDCs or their precursors was identified as the ultimate determinant in
regulating lymphoid organization in the joint. Emergence of GC+ follicles in extranodal sites is considered as a critical step in the generation of autoimmune process (31). It is not unique for RA and has also been observed in other chronic inflammatory syndromes such as hepatitis C infection, Sjögren’s syndrome, Heliobacter pylori-associated gastric mucosa-associated lymphoid tissue, and Hashimoto’s thyroiditis (32, 33, 34, 35), but usually in only a subset of cases. It is not known why only some patients can generate these highly structured lymphoid organizations. Evidence for a critical role of host-response factors in determining whether or not GCs are formed in the synovium comes from prospective monitoring of multiple successive tissue lesions in RA patients. We have found that the pattern of lymphoid arrangement in the joint lesions is stable over time in individual patients and that samples harvested from multiple different joints will contain the same type of synovitis (S. Takemura, P. J. Kurtin, J. J. Guronzy, and C. M. Weyand, manuscript in preparation). An additional possible explanation is that Ags driving rheumatoid synovitis are diverse and that the particular topography of the lymphoid infiltrates is a reflection of the type of Ag encountered in the synovial membrane. Precedence for this model comes from the observation that lymphadenitis is characterized by preferential activation of submicroenvironments in lymph nodes, depending on the Ag that elicits the immune response. For example, EBV-induced lymphadenitis is known to lead to profound activation of paracortical T cell zones, whereas bacterial infections with streptococci can be expected to produce follicular lymphoid hyperplasia (36).
Results of the current study indicate that the molecules implicated in
the development of secondary lymphoid organs are also critically
involved when tertiary lymphoid structures are formed. Elegant studies
in gene-targeted mice have suggested that aberrant expression of LT-
may be sufficient to induce the generation of lymph node-like
structures in nonlymphoid tissues (5). Induction of
chemokines and adhesion molecules on endothelial cells and, thus,
regulation of cell recruitment have been suggested as an underlying
mechanism through which LT- could determine lymphoid neogenesis
(37). A recent study provided evidence for an ultimately
critical position of BLC/CXCL13 in the process of lymphoid neogenesis
(38). In transgenic mice expressing BLC/CXCL13 in the
pancreatic islets, lymph node-like structures emerged with B cell and T
cell zones, high endothelial venules, and production of SLC/CCL21.
Studies in H. pylori-induced mucosa-associated lymphoid
tissue have shown high-level expression of BLC/CXCL13 in all lymphoid
aggregates (39). From these studies, it could be concluded
that the isolated presence of LT- or BLC/CXCL13 would be sufficient
to initiate the process of extranodal lymphoid neogenesis. Human
studies have the limitation that the process of GC induction and
establishment has to be retrospectively analyzed. However, in this
study, multivariate logistic regression analysis allowed for the
identification and hierarchical modeling of markers associated with
GC+ synovial follicles.
In the univariate analysis, all seven cytokines/chemokines correlated
with synovial GC reactions, provided they were present at high
concentrations. However, the multivariate analysis demonstrated that
only two markers emerged as independent determinants, LT- and
BLC/CXCL13. All other molecules were dependent variables. In
particular, LT- that is found in the soluble LT-3 cytokine, as
well as in the LT-12 molecule, was a dependent variable,
suggesting that LT-12, and not LT-3, is important in GC
formation in the synovial tissue. Although high transcription of
BLC/CXCL13 in the tissue increased the likelihood of finding GCs, the
isolated expression of BLC/CXCL13-specific sequences was not sufficient
to predict GC formation. Several patients had high BLC/CXCL13 mRNA in
the synovium but failed to form typical follicles, indicating that the
relationship between BLC/CXCL13 expression and structuring of lymphoid
infiltrates is more complex in rheumatoid synovitis than in the murine
models. Modeling of the data suggested that BLC/CXCL13 and LT-
concentrations were critical but could substitute for each other to
some degree, raising the interesting question how the chemokine
BLC/CXCL13 and the membrane-integrated cytokine LT-12 could
compensate for each other in lymphoid neogenesis. The negative
predictive value of this model was excellent, indicating that the
formation/maintenance of synovial follicles is essentially impossible
if the concentrations of both mediators are below a critical threshold.
The positive predictive value was moderate, suggesting that high
concentrations of either mediator was not always sufficient to
guarantee the process of GC formation. In particular, there were
patients with high BLC/CXCL13 and high LT- who did not form GCs,
suggesting that additional factors are required besides aberrant
expression of LT- and BLC/CXCL13. One such factor could be the
availability of FDCs. The absence of FDCs, documented by
immunohistochemical staining as well as PCR for CD21L, in tissue
samples free of GCs was surprising and established that the synovial
membrane differs fundamentally from lymphoid organs. Not a single
example was found of a primary follicle, i.e., the presence of FDCs
without a fully developed GC reaction. FDCs participated in GC
reactions whenever they were present. Whether patients with the ability
to form synovial GCs are carriers of FDCs at that tissue site or
recruit the precursor or mature FDCs to that site needs to be explored.
The ontogeny of FDCs is not completely understood. A recent publication
has suggested that synovial fibroblasts may share functional
characteristics with FDCs, including the ability to cluster B cells on
their surface (40, 41). We have seen BLC/CXCL13 expression
in isolated synovial fibroblasts (Fig. 5), but we have not been able to
induce CD21L expression or the production of BLC/CXCL13 in synovial
fibroblast lines, two features that would be suggestive of a
relationship between synoviocytes and FDCs (data not shown). The
perfect correlation between FDC presence and GC reaction emphasizes the
critical contribution of Ag recognition events in the process of
lymphoid neogenesis in RA. The total lack of primary follicles suggests
that Ag-specific T cells and B cells precede FDCs and may actually be
necessary for the recruitment, differentiation, or survival of FDCs in
the synovial microenvironment.
Our finding that the B cell is an origin of LT- is in-line with
previous studies, providing evidence of a critical role of B cells and
LT- in the process of secondary lymphoid tissue development. LT-
is expressed on the vast majority of circulating B cells (data not
shown). However, in the tissue, only a subset of B cells in the mantle
zone and a small proportion of B cells in the GCs express LT-. T
cell-B cell aggregates, although rich in B cells, contained very few
LT- transcripts. The reason that B cells appear to lose LT-
expression as they infiltrate into the tissue and that only selected B
cells continue to produce LT- once in the tissue lesion requires
additional studies. At least two different mechanisms have been
identified to up-regulate membrane LT-12 expression on B cells.
One mechanism implicates BLC/CXCL13 in inducing B cell surface
expression of LT-12; the second mechanism is BLC/CXCL13
independent (15). In our studies, BLC/CXCL13 and LT-
did not correlate, and the multivariate modeling demonstrated their
relative independence in the induction of GCs.
Identifying B cells as a cellular origin of LT-, critically involved
in the decision process of how the lymphocytic infiltrate in RA joints
is organized, emphasizes that B cells have multiple functions in
rheumatoid synovitis extending far beyond a simple role of releasing
autoantibodies (42). However, T cells participating
in follicles also expressed LT-, raising the question whether
additional cell populations contribute to the decision process to
establish a GC reaction in this extranodal site.
BLC/CXCL13 derived from several different cellular sources. Endothelial cells lining capillaries and small arterioles were by far the most frequent cell types that stained with anti-BLC Abs. Endothelial cells are generally not considered a major source of this chemokine. Therefore, it cannot be excluded that the BLC detected was passively adsorbed. However, BLC/CXCL13-positive endothelial cells were present in synovial tissues of all types, including tissues with no GC reaction and no FDCs, suggesting that endothelial cells may indeed be producer cells. BLC/CXCL13 was consistently expressed in GCs, in-line with the interpretation that FDCs are an important source for this chemokine. Nevertheless, BLC/CXCL13 expression was not restricted to FDCs and was even found in synoviocytes. This observation emphasizes that the tissue structure hosting the immune reaction, in this case, the synovial membrane, may be directly involved in directing the organization of infiltrating lymphocytes. Heterotypic signaling between the diverse cell types within rheumatoid lesions may ultimately provide an explanation why the synovial membrane is chosen as a site for extranodal lymphoid neogenesis.
This study has several clinical implications. It is important to
realize that the rheumatoid lesion not only has the ability to
facilitate tissue destruction but can also function like a lymph node.
The complexity of lymphoid microstructures amplifies Ag-specific
responses, and much lower concentrations of Ag are required for
Ag-specific reactions to occur (31). One of the
interesting questions is whether immune responses in the synovial
membrane are restricted to Ags present in the local environment or
whether Ag trapping and enrichment could also become relevant for Ags
not primarily involved in the disease process. If that were the case,
the joint could provide the infrastructure to support immune responses
against nonarthritogenic Ags. This could be of particular relevance for
patients producing rheumatoid factor, an autoantibody binding to the Fc
portion of IgG. It is known that synovial fluid is rich in
immunocomplexes, possibly providing a wide spectrum of Ags to be
handled in the tertiary lymphoid tissue structures of the joint.
Another important lesson from the current study is that the sharing of
molecular mechanisms in the formation of secondary and tertiary
lymphoid organizations may limit the use of BLC/CXCL13 and LT- as
targets for immunosuppressive therapy in RA. Obviously, BLC/CXCL13- or
LT--directed therapy would pose the risk of also destroying lymphoid
follicles in lymph nodes, spleen, and Peyer’s patches. The interesting
finding that FDCs are not a constitutive cell population of the
synovial membrane should initiate studies into the processes of FDC
recruitment to sites of tertiary lymphoid tissue formation. Inhibition
of that process could provide an elegant approach to suppress
rheumatoid synovitis.
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Acknowledgments |
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. We also thank Tammy J. Dahl and James W.
Fulbright for assistance with manuscript preparation and graphics. |
Footnotes |
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2 S.T. and A.B. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Cornelia M. Weyand, Mayo Clinic, Guggenheim 401, 200 First Street SW, Rochester, MN 55905. E-mail address: weyand.cornelia{at}mayo.edu
4 Abbreviations used in this paper: RA, rheumatoid arthritis; GC, germinal center; BLC, B lymphocyte chemoattractant; CD21L, CD21 long isoform; DC-CK1, dendritic cell-derived C-C chemokine 1; FDC, follicular DC; LT, lymphotoxin; MCP, macrophage chemoattractant protein; plt, paucity of lymph node T cells; SLC, secondary lymphoid chemoattractant.
Received for publication January 24, 2001. Accepted for publication May 9, 2001.
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TopAbstractIntroductionMaterials and MethodsHistopathological evaluationsRT-PCR and cytokine...AntibodiesImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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