Fibroblast-like synoviocytes support B-cell pseudoemperipolesis via a stromal cell–derived factor-1– and CD106 (VCAM-1)–dependent mechanism (original) (raw)
FLSs can function as NLCs for human B cells. We examined whether FLSs isolated from patients with RA or OA could function as NLCs for normal B cells and B-cell lines. RA-derived FLSs (RA FLSs) or OA-derived FLSs (OA FLSs) were cultured with a pro–B cell line (Nalm-6), a pre–B cell line (Reh), a B-cell line (Ramos), a B-lymphoblastoid cell line (BJAB), or normal adult blood B cells. Pseudoemperipolesis was monitored by phase contrast microscopy. We found that Nalm-6, Reh, Ramos, or blood B cells spontaneously migrated beneath RA FLSs or OA FLSs within hours of coculture (Figure 1). During this time, the lymphocytes migrated beneath or became trapped by cytoplasmatic projections of the adherent FLSs (13, 20). The B cells that migrated into the same focal plane as the FLSs had a dark appearance (Figure 1, a and b). In contrast, the lymphoblastoid B-cell line BJAB did not migrate under similar conditions and retained a light appearance (Figure 1c).
Representative phase contrast photomicrographs demonstrating pseudoemperipolesis of B-cell lines cultured for 2 hours on FLSs from RA patients. Cells that had not migrated beneath the FLSs were washed off, and the FLS layer containing the migrated B cells was photographed (200× magnification). In contrast to Nalm-6 (a) and Ramos B cells (b), BJAB B cells did not display pseudoemperipolesis (c). (A few BJAB cells were added back to the well after washing in panel c to demonstrate the bright appearance of nonmigrated cells relative to that of cells that had migrated into the same focal plane as the FLSs, as seen in panels a and b).
To measure relative B-cell migration beneath FLSs, we performed titration experiments in which 105 to 107 input B cells were added to wells containing a layer of confluent FLSs. BJAB cells did not migrate beneath RA FLSs or OA FLSs at any input cell number (data not shown). However, all other B-cell lines migrated beneath the RA FLSs (Figure 2a). No significant differences were noted between RA FLSs and OA FLSs in their ability to support pseudoemperipolesis of Nalm-6 or Ramos (Figure 2b). In either case, the numbers of migrated cells increased proportionately with the numbers of input cells provided that 5 × 106 or fewer input B cells were used. When greater numbers of input cells were added, we did not observe any further increase in the numbers of migrating cells over that seen with 5 × 106 input B cells (Figure 2). As such, 5 × 106 input cells were used for all subsequent experiments.
(a) Measurement of B-cell pseudoemperipolesis on FLSs from RA patients. Nalm-6, Reh, Ramos, or BJAB B cells were added to separate wells of confluent RA FLSs from a representative RA patient at the input numbers indicated on the horizontal axis. After 2 hours, nonmigrated cells were removed and the adherent FLS layer was detached and made into single-cell suspensions. The numbers of B cells contained within the adherent FLS layer were assessed by flow cytometry. Displayed are the mean (± SEM) numbers of events acquired in 20 seconds in the lymphocyte scatter gate at high flow from duplicate wells. (b) Comparison between RA FLSs and OA FLSs in their capacity to support pseudoemperipolesis. Nalm-6 B cells or Ramos B cells were incubated on confluent layers of FLSs from a representative patient with RA or a patient with OA. After 2 hours, the B cells that had migrated beneath the FLSs were assessed, as outlined in a. Displayed are the mean relative numbers (± range) of migrated cells, collected from duplicate wells with the numbers of input cells indicated on the horizontal axis.
High numbers of cells from the immature B-cell lines Nalm-6 (111,870 ± 2,263 cells, mean ± range from duplicate wells) or Reh (58,943 ± 2,345 cells), and lower numbers from the Ramos cell line (24,161 ± 6,484 cells), migrated beneath RA FLSs. In contrast, dermal fibroblasts were relatively ineffective in supporting pseudoemperipolesis of the various B-cell lines. Spontaneous migration of Nalm-6 beneath dermal fibroblasts was only 13 ± 2.5% or 12.6 ± 1.7% of that noted with OA FLSs or RA FLSs. Moreover, the migration of Ramos cells beneath dermal fibroblasts was 50 ± 1.8% or 42 ± 5% of that noted with OA FLSs or RA FLSs.
Coculture of purified normal B cells with RA FLSs or OA FLSs protected the B cells from apoptosis. As shown in Figure 3a, only a small proportion of normal B cells retained characteristics of viable cells (high mitochondrial membrane potential and PI exclusion) when cultured in medium alone for a period of 5 days. In contrast, the majority of B cells remain viable when cultured with RA FLSs or OA FLSs. In contrast, dermal fibroblasts were significantly less effective in supporting B-cell survival than either RA FLSs or OA FLSs at all time points (Figure 3, a and b). The effect of RA FLSs or OA FLSs on B-cell survival was most prominent during the initial 5–7 days of culture (Figure 3b).
(a) FLSs from RA and OA patients protect normal blood B lymphocytes from spontaneous apoptosis in vitro. The viability of purified B cells was determined by staining with DiOC6 and PI. Presented are contour maps of B cells from a representative donor defining the relative green (DiOC6) and red (PI) fluorescence intensities of the B cells on the horizontal and vertical axes, respectively. The vital cell population (DiOC6bright, PI-exclusion) was determined for B cells cultured in medium alone, or on a confluent layer of FLSs from a patient with RA (RA FLSs) or OA (OA FLSs), or dermal fibroblasts. The vital cells were gated as indicated by the lines. The relative proportions of vital cells are displayed above each of these gates. (b) The viability of B cells from three different donors was determined by DiOC6/PI staining at the time points indicated on the horizontal axis. Displayed is the mean (± SD) viability of B cells cultured with RA FLSs (diamonds), OA FLSs (circles), dermal fibroblasts (triangles), or medium alone (squares).
Expression of SDF-1 and CXCR4. SDF-1 is a powerful chemoattractant cytokine (chemokine) that promotes the migration and activation of B lymphocytes and other hematopoietic cells (15, 21). To determine whether SDF-1 plays a role in the B-cell pseudoemperipolesis mediated by FLSs, we examined B cells, RA FLSs, OA FLSs, and dermal fibroblasts for expression of SDF-1 and its receptor CXCR4 by RT-PCR. CXCR4 mRNA was detected in Reh, Nalm-6, Ramos, and normal B cells (Figure 4a, lanes 2–5). However, RT-PCR of BJAB cDNA generated relatively small amounts of the CXCR4-specific 1,058 bp product (Figure 4a, lane 6). This did not appear to be due to differences in the amounts of input cDNA, as indicated by control RT-PCR for the GAPDH gene (Figure 4a, lanes 8–12). Using SDF-1β–specific primers we found expression levels of SDF-1 mRNA among RA FLSs isolated from three different patients with RA (Figure 4b, lanes 1–3) or OA (Figure 4b, lanes 4–6), respectively. SDF-1 mRNA was also detected in dermal fibroblasts by RT-PCR (Figure 4b, lane 7).
(a) RT-PCR analysis for CXCR4 expressed by different B-cell lines or normal blood B cells. A CXCR4 PCR product of the expected size (1,058 bp) was generated using cDNA obtained from Reh, Nalm-6, Ramos, normal blood B cells, or BJAB cells in lanes 2–6, respectively. Similarly, a GAPDH PCR product was generated using the cDNA from Reh, Nalm-6, Ramos, normal blood B cells, or BJAB cells in lanes 8–12, respectively. Lanes 1 and 7 contain DNA fragments of known size, allowing for calibration of the migration distances, as indicated on the far left-hand side of the figure. (b) RT-PCR analysis for SDF-1 expression by FLSs from RA and OA patients, or dermal fibroblasts. SDF-1 PCR products (top row) or GAPDH PCR products (bottom row) were generated using cDNA obtained from the FLSs of three different RA patients (lanes 1–3), three different patients with OA (lanes 4–6), dermal fibroblasts (lane 7), or a DNA plasmid containing the human SDF-1β cDNA (lane 8). All tested samples displayed amplification of a PCR fragment of the expected size for SDF-1.
We examined FLSs for expression of SDF-1 protein by immunofluorescence microscopy. For this we stained RA FLSs with anti-SDF-1 antibodies by intracytoplasmic immunofluorescence staining. Figure 5 displays examples of representative FLSs from RA patients stained with anti-SDF-1 (Figure 5a) or stained with anti-SDF-1 and anti-vimentin Ab’s (Figure 5b). In both images, specific granular staining for cytoplasmatic SDF-1 can easily be seen as red fluorescence. These cells did not display nonspecific staining with either mouse IgG1 or biotinylated goat IgG (data not shown).
SDF-1 protein detection in RA FLSs by immunofluorescence microscopy. This figure depicts fluorescence micrographs of representative RA FLSs stained with anti-SDF-1 Ab’s (red fluorescence) and Hoechst 33258 (blue; a), or anti-SDF-1 Ab’s (red fluorescence), anti-vimentin mAb’s (green fluorescence), and Hoechst 33258 (blue; b).
Flow cytometry revealed high-level expression of CXCR4 on Reh, Nalm-6, Ramos, and normal B cells, but relatively low levels of this receptor on BJAB (Figure 6a). In addition, we also examined for CD49d. This molecule was expressed at high levels on all B cells examined, with the exception of a lower display of CD49d on BJAB cells (Figure 6b).
Expression of CXCR4 or CD49d (VLA-4) by different B-cell lines or normal blood B cells, as indicated. Displayed are fluorescence histograms depicting the relative red fluorescence intensity of the cells stained with anti-CXCR4 mAb’s (a, shaded histograms) or with anti-CD49d mAb’s (b, shaded histograms) compared with that of the same cells stained with a PE-conjugated isotype control mAb of irrelevant specificity (open histograms). The mean fluorescence intensity ratio of each specifically-stained cell population is displayed above the histograms.
Functional significance of CXCR4 on human B cells. To determine whether differences in the capacity of B cells or B-cell lines to migrate beneath FLSs correlated with the function of CXCR4 on the respective cells, we examined the CXCR4 function by actin polymerization and chemotaxis in response to 100 ng/ml SDF-1α. In contrast to all other cell lines tested, BJAB cells did not show the characteristic transient increase in filamentous actin (F-actin) following SDF-1α stimulation (Figure 7a). Moreover, normal blood B cells and all B-cell lines except BJAB displayed chemotaxis with 100 ng/ml SDF-1 (Figure 7b). Background migration was always less than 1% of the input cells for all cell lines examined. As such, BJAB cells apparently lack a functional CXCR4 receptor.
Responses of B-cell lines or normal blood B cells to SDF-1α. (a) Intracellular F-actin was measured using FITC-labeled phalloidin after the addition of 100 ng/ml SDF-1α at time 0. Results are displayed as percent of intracellular F-actin relative to that prior to the addition of SDF-1α, for each cell line, as indicated above the box for each graph. The lines connect the data points that are the mean ± the range of two independent experiments. (b) SDF-1α induces chemotaxis of B-cell lines or normal blood B cells, but not BJAB B cells. Blood B lymphocytes or B-cell lines, as indicated, were assayed in the bare filter chemotaxis assay for migration toward 100 ng/ml of SDF-1α. The bars represent the mean (± range) relative proportion of input B cells that had migrated in response to SDF-1. Migration to control wells containing medium alone was less than 1% in all cases.
To determine whether B-cell activation through CXCR4 was required for pseudoemperipolesis, Nalm-6 or Ramos B cells were preincubated with either pertussis toxin, anti-CXCR4 mAb (12G5), or a control mAb of irrelevant specificity prior to culture with RA FLSs from different patients. Pertussis toxin, which inhibits signaling through G-protein coupled, seven transmembrane receptors, such as CXCR4, significantly reduced pseudoemperipolesis of Nalm-6 or Ramos cells to levels that were 16 ± 9.2% or 16.8 ± 0.9%, respectively, of those noted of untreated controls (n = 4, P < 0.005; Figure 8a). Anti-CXCR4 antibody pretreatment also significantly inhibited migration of Nalm-6 or Ramos cells beneath RA FLSs (Nalm-6: 46.9 ± 21.6%, n = 4, P < 0.005; Ramos: 49.5 ± 10.1% of the untreated controls, n = 4, P < 0.005; Figure 8a). Control antibody pretreatment had no effect (92.1 ± 1.8% or 105 ± 19.4%, n = 4). Similar results were obtained using purified blood B cells. For such cells, migration beneath RA FLSs was inhibited significantly by pretreatment with pertussis toxin (14 ± 7.1%, n = 5, P < 0.05), or anti-CXCR4 mAb (45.3 ± 19.8%, n = 5, P < 0.05), but not with a control mAb (95.1 ± 10%, n = 7; Figure 8b). Migration beneath OA FLSs was inhibited to a similar degree with either pertussis toxin or anti-CXCR4 mAb (data not shown).
Inhibition of B-cell pseudoemperipolesis of RA FLSs by CXCR4 and VLA-4 antagonists. (a) Nalm-6 (open bars) or Ramos cells (filled bars) were preincubated with pertussis toxin (PT), αCXCR4 mAb’s, control mAb’s, αVLA-4 mAb’s, 100 μg/ml or 10 μg/ml of CS1 peptide, or a control peptide, as indicated on the left-hand side. Cells then were incubated on RA FLSs and allowed to migrate beneath the FLSs for 2 hours. Then, the FLS layer containing the migrated cells was harvested, and the relative numbers of migrated cells were determined by flow cytometry. The bars represent the mean (± SD) B-cell migration relative to that of untreated samples. AThe difference between the percent migration under a given condition is significantly less than that noted for same cell population for FLSs in the absence of inhibitors (e.g., P values < 0.05, Bonferroni’s t test). (b) Pseudoemperipolesis of normal blood B cells beneath RA FLSs was also inhibited by PT, αCXCR4 mAb’s, or CS1 peptide. The bars represent the mean (± SD) relative B-cell migration of B cells from five different donors. ASignificant inhibition of migration with P values < 0.05 using Bonferroni’s t test.
Pseudoemperipolesis is dependent on interaction of CD106 with VLA-4. Interactions between CD49d/CD29 (VLA-4) on B cells and its respective ligands (CD106 and the CS1 portion of fibronectin) play an important role in the adhesion between B cells and FLSs (3) or NLCs derived from RA synovium (5, 10). We examined the influence of a VLA-4 mAb or a synthetic CS1 peptide on B-cell migration beneath RA FLSs. Both anti-VLA-4 and CS1 peptide were active inhibitors of pseudoemperipolesis for Ramos and normal B cells (Figure 8, a and b), but had little or no inhibitory effect on the migration of immature Nalm-6 cells (Figure 8a). For instance, Nalm-6 migration was not inhibited by anti-VLA-4 mAb (108 ± 7% of untreated controls, n = 4), but the antibody reduced migration of Ramos cells to 38 ± 2% of respective controls (n = 4, P < 0.05; Figure 9a). Moreover, CS1 peptide inhibited Ramos cell migration to levels that were 4 ± 0.6% (100 μg/ml CS1) and 8.7 ± 1.3% (10 μg/ml CS1) of untreated controls (n = 4, P < 0.05; Figure 9a), while 100 μg/ml of CS1 was required for significant inhibition of pseudoemperipolesis with Nalm-6 (71.2 ± 2.6%, n = 4, P < 0.05; Figure 8a). One hundred micrograms per milliliter of CS1 peptide also inhibited the migration of normal B cells to 37.9 ± 12% of cultures without added peptide (mean ± SD, n = 4, P < 0.05). In contrast, the scrambled CS1 control peptide did not inhibit migration of Nalm-6 or Ramos even at the highest peptide concentration of 100 μg/ml (Nalm-6: 132.2 ± 2.3%, n = 4; Ramos: 91.8 ± 20%, mean ± SD, n = 4). Moreover, normal B-cell pseudoemperipolesis in the presence of 100 μg/ml of scrambled CS1 peptide was 111 ± 20% of control cultures without added peptide (mean ± SD, n = 4; Figure 8b).
(a–d) CD106 (VCAM-1) expression by RA FLSs (a), OA FLSs (b), dermal fibroblasts (c), or IL-4–stimulated dermal fibroblasts (d). a–d depict histogram graphs that display the relative red fluorescence intensity of cells stained with anti-CD106 mAb’s (shaded histograms) or a nonspecific isotype control antibody (open histograms). The mean fluorescence intensity ratios of cells stained for CD106 relative to that of control antibody–stained cells are displayed in the upper right-hand corner of each histogram. (e) B-cell migration beneath dermal fibroblasts (DFs) is significantly enhanced by IL-4 treatment of dermal fibroblasts. The bars represent the mean (± SD) Nalm-6 or Ramos B-cell migration beneath IL-4–treated dermal fibroblasts relative to the migration beneath untreated dermal fibroblasts, corresponding to 100%. ASignificant inhibition of migration with P values < 0.05 using Bonferroni’s t test. (f) Migration of Ramos B cells and Nalm-6 B cells beneath IL-4–treated dermal fibroblasts is significantly inhibited by anti-CD106 mAb’s. Compared with the migration beneath IL-4–treated dermal fibroblasts without antibody treatment (column 1), control antibody treatment did not significantly affect pseudoemperipolesis (PEP) of Ramos and Nalm-6 cells (column 2). In contrast, treatment with anti-CD106 mAb significantly decreased the migration of Ramos cells (column 3). ASignificant inhibition of migration compared with that of control cultures (P < 0.05, Bonferroni’s t test). The bars represent the mean (± SEM) PEP of Ramos B cells in test conditions relative to that of Ramos B cells in control conditions without antibody (n = 6).
Because CD49d appeared necessary for B-cell pseudoemperipolesis, we examined FLSs and dermal fibroblasts for expression of the ligand for this β1 integrin, namely CD106 (VCAM-1). We found that RA FLSs and OA FLSs express high levels of cell-surface CD106 (Figure 9, a and b). However, nonstimulated dermal fibroblasts did not express detectable levels of this adhesion molecule (Figure 9c). We hypothesized that this could account for the inability of dermal fibroblasts to support B-cell pseudoemperipolesis.
To test this hypothesis, we stimulated dermal fibroblasts with exogenous IL-4, a cytokine that we noted could induce fibroblasts to express CD106 (Figure 9d). We next tested whether IL-4–stimulated dermal fibroblasts could support B-cell pseudoemperipolesis. Migration of Nalm-6 or Ramos cells beneath IL-4–stimulated dermal fibroblasts was comparable to that observed for RA FLSs or OA FLSs. Whereas only 5,691 ± 1,076 (mean ± SD, n = 4; Figure 9e) Ramos cells migrated beneath untreated dermal fibroblasts, 29,000 ± 5,733 cells migrated beneath IL-4–stimulated dermal fibroblasts (P < 0.05, Student’s t test). The effect on Nalm-6 migration was more modest in that 31,312 ± 4,307 Nalm-6 cells migrated beneath untreated dermal fibroblasts, but 38,786 ± 2,255 cells migrated beneath IL-4–treated dermal fibroblasts. Nevertheless, the difference between Nalm-6 migration beneath IL-4–stimulated dermal fibroblasts versus that noted for IL-4–treated dermal fibroblasts was statistically significant (P < 0.05, Student’s t test).
We examined whether pseudoemperipolesis mediated by IL-4–treated dermal fibroblasts was dependent upon CD106. Pseudoemperipolesis of Ramos or Nalm-6 beneath IL-4–treated dermal fibroblasts was significantly reduced by anti-CD106 mAb’s to levels that were 57.9 ± 8.5% (mean ± SEM, n = 6) or 85.4 ± 3.1% (n = 6), respectively, of that observed with each cell line beneath IL-4–treated dermal fibroblasts without mAb’s (100%) (P < 0.05). In contrast, addition of control mAb’s (MOPC21) to the cultures did not significantly inhibit pseudoemperipolesis beneath IL-4–treated dermal fibroblasts (90.8 ± 5.5%, n = 6; Figure 9f). Pseudoemperipolesis of Nalm-6 cells was also significantly inhibited by anti-CD106 mAb’s (85.4 ± 3.1% of untreated controls, P < 0.05, n = 6), whereas control mAb’s also did not display a significant effect on Nalm-6 pseudoemperipolesis (106.1 ± 4, n = 6; Figure 9f).