Heparinized venous blood, synovial fluid, and synovial tissue were obtained from patients who fulfilled the American College of Rheumatology criteria for the diagnosis of rheumatoid arthritis [27]. Synovial tissue removed at the time of either joint replacement or therapeutic synovectomy was digested with collagenase, DNAse, and hyaluronidase to obtain single-cell suspensions. Mononuclear cells (MNCs) were isolated from cell suspensions from blood, synovial fluid, and synovial tissue by density gradient centrifugation (Ficoll-Paque; Pharmacia LKB Biotechnology, Piscataway, NJ, USA).

Genomic DNA was isolated from MNCs using the Puregene DNA Isolation Kit (Gentra Systems, Mineapolis, MN, USA) and total RNA was isolated using Ultraspec RNA (Biotech Laboratories, Houston, TX, USA). Both of these reagents were used according to the manufacturer's instructions. One microgram of RNA was reverse transcribed to complementary (c)DNA using 200U Moloney murine leukemia virua (M-MLV) reverse transcriptase (GIBCO BRL Life Technologies, Grand Island, NY, USA), 1U RNAse inhibitor (5 Prime 3 Prime, Boulder, CO, USA) and 20 pmol oligo dT primer in a total volume of 20 μl. These ingredients were incubated at 42°C for 1 h, heated to 65°C for 10 min to stop the reactions, and then diluted to a final volume of 100 μl.

The original immunoglobulin V_H gene fingerprinting assay [23] was modified into two stages, starting with either genomic DNA or cDNA as templates (Fig. 1). The sequences of the primers used in these reactions were published previously [28].

DNA sequences were determined by reamplifying the original genomic DNA using the appropriate family-specific V_H leader and J_H consensus primers under the following PCR conditions: denaturation at 94°C for 45s; annealing at 62°C for 30s; and extension at 72°C for 45s. After 35 cycles, extension was continued at 72°C for an additional 10min. PCR products were then cloned into TA vector (Invitrogen, San Diego, CA, USA), processed using Wizard minipreps (Promega), and sequenced using M13 forward and reverse primers, a DNA Sequencing Kit (Perkin Elmer) and an automated sequenator (Applied Biosystems, Foster City, CA, USA).

During normal B cell development, the processes of gene segment recombination and coding end processing yield nucleotide HCDR3 lengths that are characteristic and virtually invariant for an individual B-cell clone. Therefore, these lengths can be used as signatures to identify members of a B cell clone. The immunoglobulin V_H gene fingerprinting approach [23] takes advantage of the wide range of HCDR3 lengths that can occur in human B cells (approximately 5-35 amino acids) to provide an estimate of clonal diversity in polyclonal populations. When polyclonal B lymphocytes from adults are analyzed using this assay, they display a Gaussian HCDR3 length distribution around a mean of approximately 15 amino acids. The presence of an individual dominant length that differs from this Gaussian distribution can be used as an indication of a specific B-cell clonal expansion.

Figure 1 illustrates schematically the two-stage immunoglobulin V_H gene fingerprinting approach that we utilized. Note that when cDNA prepared from normal peripheral blood B cells is used as a template for these V_H family-specific and C_H-specific assays, ladders of HCDR3 lengths that differ by three nucleotides are identified. These individual HCDR3 lengths are signatures of the various individual B-cell clones contained within the polyclonal population. The intensities of the bands in virtually all of the ladders illustrated in Figure 1 are relatively uniformly distributed around the mean. This indicates that there are no dominant HCDR3 lengths that skew the Gaussian distribution, and therefore that there are no significant clonal expansions among the B cells that express most of these V_H–C_H combinations. Similar results are obtained using the genomic DNA-based assay, although these results cannot be interpreted in a C_H-specific manner (data not shown).

In the V_H6-IgG combination (Fig. 1), however, a non-Gaussian distribution is noted, even in this normal individual. This could be a reflection of the numbers of V_H genes present in the V_H family being analyzed (it is more likely to see a non-Gaussian distribution in families with small numbers of individual genes) or of the state of activation of a specific clone (because activated B cells contain much higher levels of V gene messenger RNA than resting B cells). We believe that in the instance illustrated in Figure 1 the latter possibility is more likely, because the small V_H2 and V_H5 families (only two gene members per family) do not exhibit the same degree of oligoclonality as that observed with the only somewhat smaller V_H6 family (one gene member).

Because B-cell activation and differentiation result in dramatic increases in immunoglobulin V gene messenger RNA, these fingerprinting assays cannot readily distinguish between clonal expansion and activation when cDNA is used as a starting template. Because DNA levels are not appreciably altered by cellular activation, however, the use of genomic DNA as well as cDNA from the same sample of B cells helps to distinguish these two processes.

Thus, in the setting of specific B-cell clonal expansion without concomitant cellular activation, the DNA-based fingerprinting assay will indicate a dominant HCDR3 length, whereas the cDNA-based assay may not (data not shown). Conversely, in the setting of specific B-cell clonal activation without concomitant clonal expansion, the cDNA-based assay will indicate a dominant HCDR3 length, whereas the DNA-based assay may not. Finally, in the setting of specific B-cell clonal activation with concomitant clonal expansion, both the cDNA- and the DNA-based assays will indicate a dominant HCDR3 length.

These distinctions were very reproducible in the following studies. There were no situations in which evidence for cellular activation (either selective or accompanied by clonal expansion) was present in one set of analyses and not in a subsequent set using the same starting materials.

We analyzed the peripheral blood, synovial fluid, and synovial tissue B cells of rheumatoid arthritis patients (n = 20, 10, and 5, respectively) using the genomic DNA- and cDNA-based fingerprinting assays to develop an understanding of the diversity of the B cells in these compartments. Figures 2 and 3 are illustrations of representative patients for whom concomitant blood and synovial fluid or blood and synovial tissue samples were available. In order to simplify the Figures, only the results for two large V_H families (V_H1 and V_H3) and two small V_H families (V_H5 and V_H6) are provided, although assays for each V_H family and each major CH family (μ,γ, and α) were performed and revealed similar findings.

The genomic DNA-based assays in both patients indicated that clonal expansions are common in the blood of rheumatoid arthritis patients. This type of result was obtained with all individuals tested. It was most convincingly demonstrated by the results in Figure 3 obtained from B cells expressing genes of the V_H1 and V_H3 families. Because these V_H families contain the largest numbers of V_H genes, they would be more likely to display a polyclonal pattern.

An even more striking level of B-cell clonal dominance and expansion was seen when the genomic DNA-based assay was used to analyze B cells from the synovial fluid or synovial tissue (Figs 2 and 3). In these analyses, virtually all V_H families demonstrated extensive B-cell oligoclonality. It should be pointed out that when an individual HCDR3 length comprises more than 50% of the radioactive counts of a V_H–C_H ladder, clonality, based on DNA sequencing, is very likely; when an individual length comprises more than 70% of the radioactivity, clonality is virtually assured (data not shown).

Collectively, these data indicate that the B-cell repertoire of rheumatoid arthritis patients is skewed away from the typical, apparently random representation of normal individuals. The reason for this discrepancy is not clear, although one possibility is that restricted antigenic exposure alters the composition of the repertoire in favor of B cells reactive with the putative antigen(s). If this is so, the progressive narrowing of the repertoire from the blood to the synovial tissue is consistent with the ideas that the antigenic exposures are originating at these sites and that the synovial compartment is supporting clonal amplification. Because these are true clonal expansions (ie increased numbers of B cells per specific clone), it is likely that the antigenic exposures are chronic and therefore are increasing the numbers of memory B cells reactive with these determinants.

In order to confirm that these clonally expanded B cells were receiving ongoing antigenic stimulation and not limited solely to the memory compartment, we employed the cDNA-based assay to distinguish clonal expansions of activated B cells from resting (memory) cells. As illustrated in Figure 2, activated B-cell clones (identified by the letter 'A' in Fig. 2) expressing each of the immunoglobulin heavy-chain isotypes were easily identified in all the V_H families studied. In some instances, these activated clones were also expanded numerically (as defined by the genomic DNA-based assays, and identified by the letter 'E' in Fig. 2). In other cases, these activated clones did not appear to be numerically expanded. Similar examples can be found in Figures 3 and 4, but they are not identified by letters in order to simplify the Figures.

Thus, it appears that many discrete B-cell clones exist in the synovial compartment of rheumatoid arthritis patients, and that these are increased in number, consistent with a response to a restricted antigenic challenge(s). Furthermore, these B-cell clones appear to be of both the resting (memory) and the activated types, suggesting that these antigenic challenges are chronic and ongoing. Thus, these data support and extend previous findings [18,19,20,21,22] by indicating that specific B-cell clonal amplifications can occur both in previously stimulated B cells and in currently activated B cells. The presence of activated B cells that are not increased in number is consistent either with recent in situ synovium-specific stimulation of these B-cell clones, or with the influx of activated B cells that were stimulated by antigens outside of and not necessarily relevant to the synovial compartment.

Because the preceding data indicated that the B-cell repertoire of rheumatoid arthritis patients contains expanded clones of B cells that can be resting or activated, we investigated whether the same clones could be identified in both the blood and synovial compartments. Figures 2 and 3 illustrate data that suggest that there are expanded clones that are blood restricted, joint restricted, or are common to both compartments. Examples of blood-restricted clones are highlighted on Figures 2 and 3 with the ▶ symbol, those that are joint-restricted with the ▷ symbol, and those that are common to the two compartments with the ◆ symbol. These findings suggest that there may be a degree of cellular trafficking between the blood and the synovial tissues.

In order to address this issue, we studied the B cells of the blood and two synovial sites (right and left hip) that were obtained from the same patient within 3 h of each other (Fig. 4). In this patient, the DNA-based assay provided examples of clonal expansions that were present in only one joint (eg the V_H3–J_H and V_H5–J_H combinations in Figure 4), and the companion cDNA-based assays indicated that in some instances these expansions were either activated or resting. In only rare instances, however, did the data suggest that a similar clone was present in two different synovial tissues. DNA sequence analyses confirmed the rarity of this event (data not shown). Thus it appears that in most instances the clonal amplifications occur in situ and are not the result of trafficking from one anatomic site to another. If so, this would suggest that the antigenic challenges driving these clonal expansions may not be common to all synovial tissues, but may be generated independently at each site, possibly by ongoing tissue breakdown.

If the clonal expansions identified in the joints of rheumatoid arthritis patients are due to an ongoing response to antigen, then one would predict that at least some of the clones would persist over time. To test this, we studied the synovial fluid B cells from the same joints of three patients on two occasions spanning several months. Most of the clonal expansions detected on the initial samples were not present in the subsequent samples. In a few instances, however, B-cell clonal persistence was found.

Figure 5 illustrates the best example of this phenomenon in a patient who was studied over a 4-month interval. The DNA-based assay using V_H4 family-specific primers indicated the presence of two similar clones on days 0 and 120, whereas the other V_H4-expressing clones detected at the first analysis were no longer present at the time of the second analysis. DNA sequence analyses of one of these two clones confirmed their identity, because each displayed the same rearranged V_HDJ_H gene with identical V_H mutations and identical HCDR3 sequences (data not shown). Therefore, certain clones can persist locally over time, suggesting that a common and persistent antigenic stimulation was operable in the joint of this rheumatoid arthritis patient.

The lack of persistence of the other B-cell clones suggests two possibilities. First, the initial set of B-cell clones might have been replaced by others that recognized and responded to different antigenic epitopes on the same original immunogenic protein. This type of clonal evolution to the recognition of different epitopes on the same immunogenic moiety is common in experimental situations in which repetitive immunizations with a defined antigen are delivered [29,30,31]. The other possibility is that the B cells that disappeared over time were not reactive with tissue antigens. These could have been stimulated by irrelevant antigens in the periphery and therefore, after entering the synovial compartment, could not be restimulated and hence could not enter the memory pool and take up residence in the synovial tissue.