Rheumatology Section and 1BHF Cardiovascular Medicine Unit, The Eric Bywaters Centre, Imperial College London, Hammersmith Hospital, London and 2Brighton Sussex Medical School, Brighton, UK.
Correspondence to: A. L. Hepburn, Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. E-mail: a.hepburn{at}imperial.ac.uk
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Abstract |
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Methods. Venous blood was obtained from 30 patients with SLE, 25 with RA and 25 healthy controls. Monocyte phenotype was determined by flow cytometric analysis of whole blood samples, with selective gating using forward and side scatter signals. Surface expression of Fc receptors RI (CD64), RII (CD32) and RIII (CD16), complement receptors CR1 (CD35) and CR3 (CD11b/CD18), and adhesion molecules ICAM-1 (CD54) and CD11a (LFA-1) was determined. The effects of disease activity and corticosteroid therapy on the expression of these molecules were also examined.
Results. The expression of FcRII was reduced on monocytes from patients with SLE compared with healthy controls and patients with RA (P = 0.002). This did not correlate with disease activity using conventional indices [SLEDAI (SLE disease activity index), C3/C4 levels and anti-double-stranded DNA antibody titres], and was independent of prednisolone therapy. There was no significant difference in Fc
RI or RIII expression on SLE monocytes compared with healthy controls. In contrast, the expression of Fc
RIII was increased on RA monocytes (P = 0.01), this being highest in patients with active disease. The proportion of Fc
RIII-positive monocytes was also increased in RA, and prednisolone therapy was associated with a lower proportion of Fc
RIII-positive cells. An increase in CR3 expression was seen on RA monocytes (P = 0.002), whilst CR1 was increased on monocytes from patients with active SLE or active RA. ICAM-1 expression was reduced on monocytes from patients with SLE (P = 0.002), although high-dose prednisolone therapy was associated with the lowest level of surface ICAM-1 on monocytes.
Conclusions. Peripheral blood monocytes from patients with SLE or RA display significantly altered phenotypes compared with those from healthy controls. The observed reduction in SLE of FcRII may represent a mechanism by which monocytes are protected from IC-mediated activation. Prednisolone therapy and disease activity had little effect on phagocytic receptor expression. The observed changes may reflect the different cytokine profiles seen in SLE and RA.
KEY WORDS: Monocyte, Fc receptor, Complement receptor, Rheumatoid arthritis, SLE.
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Introduction |
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Peripheral blood monocytes from patients with SLE have been shown to be abnormal in phenotype and function. They have a reduced functional capacity, with a decreased number of HLA-DR+ monocytes [3] and impaired phagocytosis of IgG-sensitized erythrocytes [4]. MHC class II molecules act by selecting and presenting immunogenic peptides to CD4+ T cells, as well as playing a role in the selection of the T-cell receptor repertoire in the thymus. As T cells recognize short amino acid sequences derived from self or foreign antigens in the peptide-binding groove, abnormalities in HLA-mediated peptide expression to T cells may be an important mechanism in the pathogenesis of a range of autoimmune diseases. It is well known that aberrant HLA class I molecule expression may occur in the context of autoimmunity, for example in Sjögrens syndrome and dermatomyositis.
Expression of CD14 is also decreased [5] in SLE and increased expression of intracellular adhesion molecule type 1 (ICAM-1) has been reported [6], although an adhesion defect has been observed when monocytes from patients with SLE are differentiated into macrophages in vitro [7]. ICAM-1 is the endothelial ligand for lymphocyte function-associated antigen-1 (CD11a/CD18), which is an important mediator of potentially proinflammatory leucocyteendothelial interactions. Expression of phagocytic receptors, including the ß2 integrin CR3, a ligand for C3b, has also been investigated in a number of studies, although the results have been variable. Either the up- or down-regulation of these receptors in autoimmune disease states could affect the severity and duration of inflammation.
There are three classes of Fc receptor for IgG on circulating monocytes and fixed tissue macrophages. Fc
RI (CD64) is a high-affinity receptor that preferentially binds monomeric IgG, whilst Fc
RII (CD32) is a low-affinity receptor for complexed or polymeric IgG. Fc
RIII (CD16) is a medium-affinity receptor for complexed IgG, but is present on only about 10% of circulating monocytes [8]. The expression of these receptors differs between monocytes and macrophages, with an increase in Fc
RII and RIII during maturation and a reduction in Fc
RI [9]. There are two subclasses of both Fc
RII and RIII on mononuclear phagocytes. Fc
RIIa and RIIIa are stimulatory receptors, whilst Fc
RIIb, only recently identified on human monocytes [10], has an inhibitory function. There are very limited data on the relative expression of Fc
receptors on peripheral blood monocytes in SLE. One study has shown reduced expression of Fc
RII on the cells, this correlating inversely with circulating IC levels [11]. An increase in Fc
RI expression was also seen. The expression of these receptors on circulating monocytes may be relevant to macrophage phenotype and function in vivo. Correlation has been found between the expression of Fc
RII and RIII and the clearance in vivo of autologous IgG-sensitized erythrocytes in patients with SLE [12].
IgG-containing IC may also play a role in the pathogenesis of rheumatoid arthritis (RA). IC containing rheumatoid factors are abundant in serum and in synovial fluid from patients with RA [13, 14]. As in SLE, other circulating autoantibodies may also form IC.
CD14+CD16+ monocytes represent a specific subpopulation of these cells in the circulation and have differing phagocytic and antigen-presenting capacities [15]. Three recent studies have noted changes in the proportion of CD14+CD16+ monocytes in patients with RA [16, 17, 18]. Further, the expression of both FcRI and RII appears to be increased on monocytes in RA [19, 20], this being normalized by treatment with corticosteroids [20]. Fc
RII and RIII expression is also increased in rheumatoid synovium, this correlating with the number of macrophages present, and on monocyte-derived macrophages from patients with the disease [18]. Higher TNF-
production was seen when RA macrophages were stimulated with a model IC, heat-aggregated IgG, compared with normal control macrophages. Some studies, however, have not observed a difference in Fc
receptor expression between RA monocytes and those from normal controls [21].
The aim of the present study was to investigate the surface expression of Fc and complement receptors (CR3 and also CR1, a C3b/iC3b receptor important in the binding and carriage of IC on cells), as well as LFA-1 (CD11a), ICAM-1, CD14 and MHC class I and II molecules, on peripheral blood monocytes from patients with SLE or RA compared with healthy controls. We have also investigated the relationship between their expression and clinical disease activity in each disease and whether concurrent glucocorticoid therapy influences Fc
receptor expression in these patient groups.
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Patients and methods |
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Twenty-five patients with seropositive RA were also studied, each fulfilling the revised 1987 American College of Rheumatology criteria for the classification of RA [24]. Their age range was 2676 yr (median 60). The female-to-male ratio was 16:9. Patients were classified as having active or inactive disease on the basis of the DAS28 [25]: 15 patients had active disease with a DAS28 4.0 whilst 10 had quiescent disease (DAS28 <4.0). The median erythrocyte sedimentation rate (ESR) in patients with active disease was 34 mm/h (range 1882) compared with 11 mm/h (range 727) in those with quiescent disease. Prednisolone was being taken by 6/15 patients with active disease (median dose 15 mg daily, range 7.530) and 3/10 patients with quiescent disease (median dose 5 mg daily, range 2.510). Other disease-modifying anti-rheumatic drugs being taken by the RA group were as follows: azathioprine (2/25), hydroxychloroquine (2/25), methotrexate (6/25), sulphasalazine (8/25), leflunomide (2/25) and parenteral gold (1/25).
The patients were sequentially selected from a number of general rheumatology out-patient clinics and specialist SLE clinics run jointly with the nephrology service, though not all patients had renal involvement. Included patients simply had to meet the respective ACR criteria (the RA patients were seropositive only). There were no specific exclusion criteria.
Twenty-five healthy controls were included from laboratory staff. None had a history of chronic inflammatory or autoimmune disease. The median age was 34 (range 2586) and the female-to-male ratio was 1.5:1.
Immunofluorescence assay
Venous blood was collected from each subject in EDTA. For detection of surface antigens, 10 µl of primary mouse anti-human monoclonal antibody at 110 µg/ml was incubated with 90 µl whole blood for 20 min on ice. The following primary antibodies were used to detect Fc receptors: 10.1 (anti-Fc
RI; Dako, Glostrup, Denmark), K6B2 (anti-Fc
RII; Dako), IV.3 (anti-Fc
RII; American Type Tissue Collection, Manassas, VA, USA) and 3G8 (anti-Fc
RIII; Immunotech, Marseille, France). KB61 reacts equally against Fc
RIIa and RIIb [26], IV.3 preferentially stains Fc
RIIa [27] and 3G8 stains both Fc
RIIIa and RIIIb, although the latter is only expressed on neutrophils. In addition, the following antigens were also detected: CD11a/CD18 (LFA-1, Ab 38; a kind gift from Dr N. Hogg, Cancer Research UK, London), CD11b/CD18 [complement receptor type 3 (CR3); Ab 44; a gift from Dr N. Hogg], CD14 (CRIS-6; Biosource, Camarillo, CA, USA), CD35 (CR1; To5; Dako), CD54 (ICAM-1; 6.5B5), MHC class I (HLA-A, B, C; BB7.5; American Type Culture Collection) and MHC class II (HLA-DR, DQ, DP; L243; American Type Culture Collection). Mouse isotype-specific negative controls were obtained from Dako. After incubation, cells were washed twice in phosphate-buffered saline then incubated with 75 µl fluorescein isothiocyanate (FITC)-conjugated F(ab')2 rabbit anti-mouse immunoglobulins for 20 min on ice. After two further washes, erythrocytes were lysed using Immunolyse solution (Beckman Coulter, High Wycombe, UK). The remaining cells were washed twice and fixed in 1% paraformaldehyde.
Flow cytometric analysis was performed using an Epics XL flow cytometer (Beckman Coulter). Monocytes were analysed selectively by gating forward and side scatter profiles, using the CD14-positive population, and 5000 cells were analysed per sample. Mean fluorescence intensity (MFI) on a logarithmic scale was measured for each sample, together with the percentage of monocytes staining positive. Relative fluorescence intensity (RFI) was obtained by dividing the MFI of the sample with that for the isotype-matched negative control sample.
Statistical analysis
Data are expressed as mean ± SEM. The statistical significance of the difference between two disease groups and normal controls was determined using the MannWhitney U test while disease subgroups were compared using one-way ANOVA with the Bonferroni post-test for multiple comparisons. Correlation coefficients were calculated using Spearmans rank correlation. These were performed using Prism software, version 3.0 (GraphPad Software, San Diego, CA, USA). Results were considered statistically significant if P < 0.05.
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Results |
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There was no difference in monocyte expression of FcRI between patients with active SLE and those with inactive disease or normal controls (RFI 2.81 ± 0.43 vs 2.35 ± 0.59 and 2.19 ± 0.17 respectively). Likewise, although reduced in patients with SLE compared with normal controls, there was no statistically significant difference in expression of Fc
RII on monocytes between patients with active or inactive disease (2.23 ± 0.25 vs 1.67 ± 0.20 vs 1.54 ± 0.07 respectively) (Fig. 2A). There was a lower percentage of Fc
RIII-positive monocytes in patients with active disease compared with normal controls (7.23 ± 1.21 vs 11.14 ± 0.89%; P = 0.04). No direct correlation was found between individual markers of disease activity in the SLE patients and expression of Fc
receptors on monocytes.
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In patients with SLE, the reduction in FcRII was independent of prednisolone use or dosage. Likewise, prednisolone therapy had no effect on the expression of Fc
RI or RIII on monocytes in patients with SLE. However, prednisolone therapy was associated with a lower percentage of Fc
RIII-positive monocytes in RA compared with untreated patients (13.92 ± 2.50 vs 18.45 ± 2.62%). The numbers of patients with SLE or RA were too small in this study to assess the effect of individual immunosuppressive drugs on Fc
receptor expression on peripheral blood monocytes.
Complement and adhesion molecule expression on monocytes from RA and SLE patients
The expression of CR3 (CD11b/CD18) was increased on monocytes from patients with RA compared with normal controls (RFI 8.26 ± 0.79 vs 5.24 ± 0.52; P = 0.002) (Fig. 3). This increase was independent of disease activity and prednisolone therapy. An increase in CR3 expression was also seen on SLE monocytes, but this was not statistically significant. However, this increase was higher in patients with active disease compared with normal controls (7.49 ± 1.07 vs 5.24 ± 0.52). The expression of CR1 (CD35) was increased on monocytes from patients with active RA compared with normal controls (5.94 ± 0.70 vs 3.42 ± 0.32; P = 0.001), but not on monocytes from patients with inactive disease. This was not affected by prednisolone therapy in either group. The effect of disease activity on the expression of complement and Fc receptors in RA and SLE is summarized in Table 2.
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Discussion |
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In contrast to our data, an increase in expression of both FcRI and RII has also been reported previously on peripheral blood monocytes from patients with RA [19, 20]. This was seen in both patients with active or quiescent disease [19], and expression normalized following corticosteroid therapy [20]. These studies used mAb IV.3, which preferentially stains the stimulatory receptor Fc
RIIa rather than the inhibitory Fc
RIIb receptor [27]. Another recent study using mAb IV.3 has observed an increase in Fc
RIIa on peripheral blood monocytes from patients with RA, although only in patients with a high ESR [28]. In the present study mAb KB61 was used (which stains both Fc
RIIa and RIIb [26]), as well as mAb IV.3. We did not observe an increase in Fc
RIIa on monocytes in RA and there was no correlation with disease activity. The observed differences in Fc
RII staining on monocytes from RA patients in the present study compared with earlier studies may therefore be explained in part by the mAb used as well as variable, albeit unknown, differences in stimulatory-to-inhibitory receptor ratios on monocytes in the diseases. It appears likely that Fc
RIIb expression is also lowered on monocytes from patients with SLE or active RA.
In the present study an increase in the proportion of CD14+CD16+ monocytes was found in RA, this being higher in patients with active disease, together with increased surface expression of CD16. Prednisolone therapy was associated with a lower proportion of CD14+CD16+ monocytes in RA patients. This increase in CD14+CD16+ monocytes in the circulation suggests activation of the monocyte/macrophage system in RA, with the potential for subsequent differentiation of the cells into macrophages in the synovium capable of producing potent proinflammatory cytokines. Such monocyte/macrophage activation in RA is supported by recent observations showing an increase in soluble FcRIIIa derived from macrophages in plasma [29]. This integral membrane protein may be released by a metalloproteinase on macrophage activation in vitro [30]. CD14+CD16+ monocytes in RA also show increased surface expression of molecules relevant to cell migration, such as ICAM-1 and chemokine receptors CCR1 and CCR5 [17], which may promote selective recruitment of these cells to the synovium. CD16 on synovial macrophages is likely to have a proinflammatory role following ligation by small IgG rheumatoid factor IC [31].
No change in the proportion of CD14+CD16+ monocytes has been found in previous studies in patients with SLE [16], although a small, non-significant decrease in FcRIII expression on peripheral blood monocytes has been described by other observers [11, 12]. However, correlation has been found previously between Fc
RIII expression on monocytes and the prolonged clearance half-times of anti-D IgG-sensitized erythrocytes from the circulation of patients with SLE [12]. One hypothesis for these observations, together with reduced monocyte Fc
RII expression, is saturation of these receptors by IC leading to their reduced function and detection. This may be a factor, although the differential effect of pro and anti-inflammatory cytokines on their expression is likely to be of more importance in vivo [10].
Reduced expression of CR1 on erythrocytes from patients with SLE is well described [32, 33], and this has also been observed in a small number of studies on RA [34, 35]. Expression of CR1 on leucocytes in these diseases has not been studied extensively. Here, CR1 has several functions, including factor I cofactor activity, decay-accelerating activity for both the classical and alternative pathway C3 and C5 convertases, and adherence of C3b/C4b opsonized particles during phagocytosis. We found an increase in the expression of CR1 on peripheral blood monocytes from patients with active RA. An increase in CR1 expression on monocytes in RA has been observed previously [20, 36], this increase having been reversed by corticosteroid treatment [20]. An increase in CR1 on circulating monocytes is consistent with cell priming prior to recruitment to the synovium. CR1 also cooperates with CR3 and Fc receptors in the phagocytosis of particulate and soluble IC by monocytes. In the present study we found an increase in the expression of CR3 on monocytes from patients with RA compared with normal controls. This is consistent with previous studies [20, 36, 37, 38] and again suggests activation of monocytes within the circulation. However, only a small increase in CR3 was observed on SLE monocytes in our study, this being greatest in patients with active disease. An increase in monocyte CR3 expression has been noted previously in SLE patients, and may be relevant to the defective clearance of apoptotic cells as well as IC by monocytes and macrophages [37]. Expression of CR4 (CD11c/CD18) on monocytes is low compared with that of CR3 [39]. No change in its expression has been found on SLE or RA monocytes [37].
In the present study, we have also investigated the expression of two other adhesion molecules on monocytes from patients with SLE or RA. ICAM-1 (CD54) is a member of the Ig superfamily and is a ligand of LFA-1 (CD11a). The expression of ICAM-1 varies depending on the activation state of cells and may be increased by proinflammatory cytokines such as interleukin (IL)-1 and TNF- [40, 41]. In one previous study, the expression of ICAM-1 was found to be increased on monocytes from patients with active SLE [6]. The percentage of ICAM-1-positive cells decreased with prednisolone treatment. In the present study, we observed a reduction in ICAM-1 expression on monocytes from patients with SLE, this being greater in patients with active disease. However, this may have been, at least in part, a corticosteroid effect since high-dose prednisolone therapy was associated with the lowest level of ICAM-1 staining. Shedding of membrane-bound ICAM-1 may be a mechanism of down-regulation of ICAM-1 expression, and consistent with this hypothesis is the observed increase in soluble ICAM-1 in the plasma of patients with SLE [42]. There was no difference in the expression of its ligand LFA-1 on monocytes between patients with SLE or RA and normal controls. Again, prednisolone therapy was associated with lower expression of LFA-1. An increase in LFA-1 has been observed previously on RA monocytes, this being reversed by corticosteroid therapy [20]. One mechanism of action of corticosteroids in these diseases may therefore be down-regulation of adhesion molecule expression on monocytes.
Corticosteroids are also known to reduce surface expression of the lipopolysaccharide receptor CD14 on monocytes [43], and this may in part explain the increased risk of infection seen in patients receiving these drugs. Soluble CD14 is increased in SLE [5, 42] and its expression on monocytes is lowered [44]. The increase in soluble CD14 occurs secondary to shedding of the membrane-bound receptor. We also observed a decrease in CD14 expression on SLE monocytes, and this is likely to have been caused by both of these mechanisms. We have observed that corticosteroids have an effect on several aspects of monocyte phenotype. However, prednisolone therapy did not appear to influence complement or Fc receptor expression to a significant degree. This is consistent with some studies [37] but not others [20]. Corticosteroids may also cause selective depletion of CD14+CD16+ monocytes [45], and we too observed a prednisolone effect on this subpopulation in RA patients.
The differences in monocyte phenotype we have observed between SLE and RA may be related to the differing cytokine profiles seen in these diseases. For example, TNF- has a central proinflammatory role in RA, while it is probably protective in SLE [46]. Interferon-
(IFN-
) may have a role in the promotion of polyclonal B-cell activation in SLE [47]. The influence of cytokines on both complement and Fc
receptor expression has been studied previously [10, 48]. IFN-
decreases the expression of the inhibitory Fc
RIIb2 on monocytes, whilst increasing the stimulatory receptor Fc
RIIa. IL-4 has the opposite effect [10]. CR3 and CR4 expression is up-regulated on monocytes by IL-4 and TNF-
[48]. These cytokines may explain the increase in MHC class II expression that we observed on RA monocytes. We did not, however, see a reduction in MHC class II on SLE monocytes, previously observed by others [3, 44].
The balance of Th1 and Th2, or perhaps more importantly pro- and anti-inflammatory cytokines, is likely to influence the degree of inflammation and clinical phenotype in chronic inflammatory diseases such as SLE and RA. One of the many contributing mechanisms for this may be the modification of monocyte phenotype by cytokines, activation in the circulation by IC, enhanced adhesion and recruitment to sites of tissue inflammation. These effects differ between SLE and RA and may be modified by therapy including treatment with corticosteroids.
The authors have declared no conflicts of interest.
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Acknowledgments |
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References |
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