Cell recovery from synovium-rich tissue during adoptively transferred arthritis
Recipients received 2 × 108 TD cells from arthritic donors by intravenous injection. Consistent with previous findings [18], mild transient inflammation was observed at day 3 after adoptive transfer and sustained paw swelling commenced from day 6, reaching a maximum at day 12 (Figure 1). Total viable cells recovered from each pair of hind paws by collagenase digestion had increased by day 3 after adoptive transfer and reached approximately 1.5 × 107 between days 9 and 14. Thus, cell yields and arthritis severity followed similar time courses.
Dual-fluorochrome analysis of CD45+cells prepared from synovium-rich tissue
The forward and side scatter of light by cells in an SRT preparation is shown in Figure 2a. Gate 'a' (the 'DC gate') is based on the light scatter of afferent lymph DCs (Materials and methods). In addition to containing DCs, this gate contains monocytes, PMN cells, fibroblasts, and endothelial cells but excludes essentially all lymphocytes (M. Moghaddami, L.G. Cleland, G. Radisic, G. Mayrhofer, unpublished data). All analyses described below were confined to cells in the 'DC gate', whereas gate 'b' contained the fluorescent beads used for measurement of cell numbers (see below).
Cells from SRT (n = 5 for each time point) were analysed first by dual-fluorochrome flow cytometry (Figure 2b–i). When cells from healthy rats were labelled with mAbs against CD45 (OX1, indirect FITC) and MHC class II (OX6, PE conjugate), the CD45+ cells within the DC gate were found to consist of MHC IIhi, MHC IIlo, and MHC II- subpopulations (Figure 2b). The MHC IIhi and MHC IIlo/int subsets comprised approximately 30% and 12%, respectively, of the total CD45+ cells in SRT from normal rats (Figure 2b). We have shown previously that the MHC IIhi cells in healthy SRT consist mainly of CD11c-CD163- 'indeterminate cells' and CD163+-activated Mϕ, whereas MHC IIlo/int cells consist mainly of CD163+ Mϕ [20]. SRT preparations from healthy rats contained very few PMN cells.
A distinct subset of CD45lo MHC II- cells was present at 14 days after adoptive transfer (Figure 2f). This population is seen clearly when CD45+ events in Figure 2f are plotted as a histogram (Figure 2j). Cytospin preparations of sorted CD45loMHC II- cells showed that greater than 80% were PMN cells (not shown). In contrast, the CD45hi cells incorporated the MHC IIhi and MHC IIlo/int subsets and, when sorted, consisted entirely of mononuclear cells (not shown). CD45hiMHC IIhi cells comprised 52% of the CD45+ cells at this time, compared with 30% in healthy rats. The sorted CD45hiMHC IIhi cells were heterogeneous in morphology (Figure 2k). Approximately 70% resembled DCs at various stages of differentiation and approximately 2% had veiled morphology (Figure 2k, upper inset). Of the remaining cells, approximately 20% resembled monocytes and 8% had the morphology of Mϕ (Figure 2k, lower inset). When stained by the indirect immunoperoxidase technique, the cells with Mϕ morphology were found to express CD163 (Figure 2m) but not CD11c (Figure 2l and inset). Conversely, the cells with monocyte-like and DC morphology were found to express CD11c (Figure 2l and inset) but not CD163 (Figure 2m). These immunohistochemical findings support the identification of Mϕ and DCs [20] and suggest that the monocyte-like cells are related to DCs.
Further investigation and enumeration of CD45+subsets in synovium-rich tissue after adoptive transfer of arthritogenic thoracic duct cells
Importantly, only CD45+ cells were found to express high levels of MHC class II molecules. Thus, by gating MHC IIhi cells, it was possible to study the expression of other molecules by this subset, using only two fluorochromes. When aliquots of cells were dual-labelled to detect MHC class II molecules plus either CD11c or CD163, 90% of the MHC IIhi cells were found to express CD11c (Figure 2g), 15% expressed CD163 (Figure 2h), and very few cells expressed neither marker. This contrasted with SRT from normal rats (Figure 2c,d), in which the proportions of MHC IIhi cells that expressed either CD11c or CD163 were 27% and 30%, respectively, whereas 43% were negative for both markers. Therefore, compared with healthy SRT, there are very few 'indeterminate' cells (expressing neither CD11c nor CD163) in the inflamed SRT.
The maturity of the MHC IIhi cells in SRT prepared from normal rats and from rats with adoptively transferred AA was assessed by examining expression of CD36 [24], using mAb UA009 [23]. As shown in Figures 2e and 2i, the molecule was expressed by most of the MHC IIhi cells. Thus, relative to DCs in pseudo-afferent lymph, where most of the cells are not stained by mAb UA009 (M. Moghaddami, L.G. Cleland, G. Radisic, G. Mayrhofer, unpublished data), the majority of the MHC IIhi cells in SRT have an immature phenotype. However, the proportion of CD36+ cells was slightly higher in SRT from healthy rats (87%) than in SRT from animals 14 days after adoptive transfer (78%). This finding suggests that the proportion of mature DCs is greater in SRT from inflamed paws.
Incorporation of CaliBRITE beads (Figure 2a) in the analysis [19] allowed the numbers of cells in each subset to be calculated per pair of hind paws (Figure 3a,b,d). The mean total of CD45+ cells in healthy SRT was 1 ± 0.18 × 106 per pair of hind paws (Figure 3a). In SRT preparations from rats 3, 6, 9, 12, and 14 days after adoptive transfer, the numbers increased slightly during the first 6 days and then rose steeply to reach 6.4 ± 2.8 × 106 at day 14 after transfer.
MHC IIhi cells outnumbered MHC IIlo/int cells at all times. The MHC IIhi cells had increased approximately 10-fold by day 14 after adoptive transfer (p < 0.01 compared with healthy, p < 0.05 compared with day 3 or day 6), and MHC IIlo/int cells were also more numerous than either in healthy rats (p < 0.01) or at days 3 to 6 after transfer (p < 0.05) (Figure 3a). Within these subsets, presumptive Mϕ were identified by expression of CD163 (Figure 3b). The numbers of MHC IIhiCD163+ cells increased progressively over the 14 days after adoptive transfer. Numbers of MHC IIlo/-CD163+ cells also increased until day 12 but then decreased to levels similar to normal SRT by day 14. Thus, except at day 14, MHC IIlo/- Mϕ predominated in both normal and inflamed SRT.
MHC II- cells also increased from approximately day 6 after adoptive transfer and, by day 14, were as numerous as the MHC IIhi cells (Figure 3a). As discussed above, most of the MHC II- cells were PMN cells. However, this population also contains presumptive monocytes and the kinetics of recruitment of monocytes and PMN cells are discussed separately below.
In addition, dual-fluorochrome analysis of CD80, CD86, and CD54 expression was performed on the MHC IIhi cells (not shown). The proportions of MHC IIhi cells that express these markers are shown in Figure 3c. The proportions of CD86+ cells remained relatively constant (70% to 80%) after adoptive transfer, whereas CD54+ cells increased slightly from 84% in healthy SRT to 96% in SRT 14 days after transfer. In contrast, there were significant increases in the proportions of CD11c+ and CD80+ cells over the same period. Importantly, the proportion of CD163+ Mϕ remained unchanged, indicating that the increased proportion of MHC II+CD80+ cells must be due to an increase in DC-like cells. This conclusion is supported by a parallel increase in the proportion of CD11c+ cells (Figure 3c). Estimates were also made of the numbers of MHC IIhi cells expressing CD11c, CD54, CD80, CD86, and CD163 (Figure 3d). The subsets expressing each of these markers increased during the period following adoptive transfer and for each, the increase was roughly in proportion to the increase in total MHC IIhi cells. Whereas there was an increase in Mϕ during adoptively transferred arthritis (Figure 3d), there was no appreciable change in the ratio of MHC IIhiCD163+ cells (Mϕ) to MHC IIhiCD163- cells (presumptive DCs) in SRT (Figure 3c).
Four-fluorochrome analysis of CD11b+ and CD11b-subsets in synovium-rich tissue
A number of subsets of mDCs have been described in rats [25–28]. A CD4+ subset is believed to be stimulatory, whereas it is suggested that a CD4- subset has tolerizing functions [25]. Furthermore, cells that resemble pDCs (MHC II+, CD4+, CD11b-, and CD11c-) have been identified [29]. CD11b, which is expressed by the myeloid lineage, distinguishes mDCs from pDCs. Care is needed in making this distinction because, under some circumstances, mDCs may express only low tointermediate levels of CD11b [30, 31].
Four-color analysis was applied to some of the SRT samples described above (n = 2 for each time point) in order to extend the phenotype of the MHC IIhi cells isolated from arthritic hind paws. MHC IIhi cells (selected as in Figure 4a) could be divided into four subpopulations: CD4+CD11b-, CD4-CD11b-, CD4+CD11b+, and CD4-CD11b+ (Figure 4b). Analysis of the CD4+CD11b+ and CD4-CD11b+ subpopulations showed that both consist mainly of CD11c+ cells (Figure 4c). In the CD4+CD11b+ subset, the proportions of CD11c- and CD163+ cells were approximately 43.5% and 33.5%, respectively (Figure 4d), suggesting that most CD11c- cells are CD163+MHC IIhi Mϕ. This was confirmed using cytospin preparations of sorted CD4+CD11b+ cells, in which most had DC-like or monocyte-like morphology (Figure 4e) but some were typical Mϕ (inset). Approximately 10% of CD4+CD11b+ cells appear to express neither CD11c nor CD163. Of the CD4-CD11b+ cells, most had either DC-like (Figure 4f) or monocyte-like (inset) morphology and most expressed CD11c (75%) and only a few (11.5%) expressed CD163 (Figure 4c,d). As in the case of the CD4+CD11b+ subset, a small proportion of MHC IIhiCD4-CD11b+ cells (approximately 14%) did not express either CD11c or CD163 and both subsets contain, therefore, a small number of 'indeterminate' cells.
The subpopulations of MHC IIhi cells in the CD11b- quadrants (Figure 4b) do not appear to be of myeloid origin because they do not express even low levels of CD11b. When sorted by fluorescence-activated cell sorting, the morphologies of the CD4+CD11b- (approximately 1% of the MHC IIhi cells in the DC gate) and CD4-CD11b- (approximately 4% of the MHC IIhi cells in the DC gate) subsets were similar. Both consist of small- to medium-sized mononuclear cells (Figure 4g,h) resembling pDCs isolated from rat spleen [29]. However, only approximately 5% to 10% express the B220 isoform of CD45 (not shown), which has been described on most rat spleen pDCs [29]. Nevertheless, some of the cells in each subset expressed CD45RC (20% and 40%, respectively), another isoform of CD45 described by the same workers on rat spleen pDCs. Furthermore, approximately half of the cells in these subsets expressed CD86 and some expressed CD80 (20% and 32%, respectively), consistent with at least some being DCs. The CD4+CD11b- and CD4-CD11b- subsets have similarities in expression of CD11c (75% and 65%), CD54 (70% and 90%), CD172a (60% and 66%), CD11a (60% and 96%), CD32 (50% and 95%), and/or CD5 (20% and 32%). The two subsets also have similarities with putative MHC II+CD4-CD11b-CD11c+ precursors of CD4+ pDCs in mice [32]. Further work is required to determine the functional characteristics of these cells, in particular their response to stimulation with Toll-like receptor 9 (TLR9) agonists [29].
Changes in numbers of myeloid-derived and CD11b-cells during adoptively transferred arthritis
As a proportion of CD45+MHC IIhi cells, the CD4+CD11b+CD163- and CD4-CD11b+CD163- subsets remained relatively constant throughout adoptively transferred disease (Figure 5b). However, the numbers of cells in both subsets increased approximately 10-fold by day 14 after transfer (Figure 5a). The numbers of MHC IIhiCD11b- cells were also estimated (not shown). The CD4+CD11b- and CD4-CD11b- subsets increased approximately 5- and 10-fold, respectively, by day 14 after transfer but remained unchanged as a proportion of MHC IIhi cells (1% and 5%, respectively).
As noted using dual-fluorochrome analysis (Figure 3b), numbers of CD163+ Mϕ increased during adoptively transferred AA. Four-fluorochrome analysis resolved Mϕ into CD4+ and CD4- subsets (Figure 5c,d). In both the MHC IIhi and MHC IIlo/- subsets, there was a consistent trend toward increased numbers of CD4- Mϕ late in the disease (days 9 to 14), as shown in Figure 5c. The significance of the sharp increase in CD4+ Mϕ at day 12, in both the MHC IIhi and MHC IIlo/- subsets, is unclear and requires further investigation in additional animals.
To compare the influx of DCs and Mϕ with other inflammatory cells, data from four-fluorochrome analysis were used to identify monocytes (MHC II-CD163lo) [20] and PMN cells (deduced phenotype MHC II-CD11b+CD4-CD163-) amongst the MHC II- cells in SRT. Numbers of PMN cells in normal SRT were small and there were no significant changes during the first 6 days after adoptive transfer (Figure 5e). By day 9, there was a small increase in the numbers of PMN cells and this accelerated rapidly over the following 5 days. These results were consistent with the numbers of PMN cells (CD45+MHC II-) observed by dual-flurochrome analysis (Figure 3a) and with histological examination of synovial tissue at day 9 after adoptive transfer. Monocytes could be divided into MHC II-CD11b+CD4+CD163lo and MHC II-CD11b+CD4-CD163lo subsets. Only small numbers of CD4+ and CD4- monocytes were observed in healthy SRT. The numbers of CD4+ monocytes did not change significantly throughout adoptively transferred disease (Figure 5e). In contrast, CD4- monocytes increased approximately 30-fold between days 9 and 12 compared with healthy rats and remained at 15-fold that of healthy levels at day 14.
Surface antigen phenotype of the CD4+CD11b+ and CD4-CD11b+subsets of putative myeloid dendritic cells
Individual markers expressed by the CD4+CD11b+ and CD4-CD11b+ subsets during the course of adoptively transferred disease are shown in Figure 6. The proportion of cells expressing CD11c increased markedly between days 9 to 14 of the disease (Figure 6a,b). This increase was more pronounced in the CD4-CD11b+ subset, in which between 75% and 90% of CD4-CD11b+ cells expressed CD11c during this period (Figure 6b) compared with 55% to 60% of the CD4+CD11b+ subset (Figure 6a). The proportions of cells expressing CD80 and CD11a also increased during this time in both populations, but the proportion expressing CD86 remained essentially unchanged. At all times, essentially all cells in both subpopulations expressed CD54 and CD32 (Figure 6a,b). In contrast to DCs in afferent lymph [25], the majority of both CD4- and CD4+ cells in SRT expressed CD172a (Figure 6). Interestingly, most of the cells in both subsets expressed CD90 (data not shown).
Location of putative dendritic cells and macrophages in synovium-rich tissue
The soft tissues of the skinned hind paws contain multiple diarthrodial joints and tendon sheaths, with their associated synovial linings and subintimal connective tissues (Figure 7a). However, they also contain tendons, loose areolar connective tissue, adipose tissue, and muscle, plus vascular and nerve tissues. Nine days after adoptive transfer, the subintimal tissues contained many mononuclear cells (Figure 7a) and granulocytes (Figure 7b). Immunohistochemical examination revealed a dense infiltration of the inflamed synovium and surrounding connective tissues with CD45+ cells (Figure 7d), consisting of both mononuclear and PMN leukocytes. Densely stained MHC II+ mononuclear cells (Figure 7e) were present in both the synovial lining and the subintimal tissues, and a large number of lightly stained CD11c+ cells (Figure 7g) were scattered in the subintima. The latter cells exhibited both monocyte and DC morphology (Figure 7h and inset). In contrast, CD163+ cells were fewer in number (Figure 7f) and most were located in the synovial lining (type A synoviocytes), with only scattered cells in the subintimal connective tissue. These observations provide qualitative support to the quantitative studies on inflammatory cells obtained from SRT by enzymatic digestion (Figure 3c). Importantly, they show that Mϕ are a numerically small subset of the total CD45+ cells in the inflamed synovial tissues during adoptively transferred AA whereas MHC II+ and CD11c+ cells are much more abundant.