Lupus-prone BXSB mice develop monocytosis characterized by selective accumulation of the Gr-1– monocyte subset. The aim of this study was to explore the possible role of activating IgG Fc receptors (FcγR) in the development of monocytosis and to characterize the functional phenotype of the Gr-1– subset that accumulates in lupus-prone mice bearing the NZB-type defective Fcgr2b allele for the inhibitory FcγRIIB.
The development of monocytosis was analyzed in BXSB and anti-IgG2a rheumatoid factor–transgenic C57BL/6 mice deficient in activating FcγR. Moreover, we assessed the expression levels of activating FcγR and inhibitory FcγRIIB on Gr-1+ and Gr-1– monocyte subsets in C57BL/6 mice bearing the C57BL/6-type or the NZB-type Fcgr2b allele.
We observed monocytosis with expansion of the Gr-1– subset in anti-IgG2a–transgenic C57BL/6 mice expressing IgG2a, but not in those lacking IgG2a. Moreover, monocytosis barely developed in BXSB and anti-IgG2a–transgenic C57BL/6 mice deficient in activating FcγR. The Gr-1– subset that accumulated in lupus-prone mice displayed a unique hyperactive phenotype. It expressed very low levels of inhibitory FcγRIIB, due to the presence of the NZB-type Fcgr2b allele, but high levels of activating FcγRIV. This was in contrast to high levels of FcγRIIB expression and no FcγRIV expression on the Gr-1+ subset.
Our results demonstrated a critical role of activating FcγR in the development of monocytosis and in the expansion of a Gr-1–FcγRIIBlowFcγRIV+ hyperactive monocyte subset in lupus-prone mice. Our findings further highlight the importance of the NZB-type Fcgr2b susceptibility allele in murine lupus, the presence of which induces increased production of hyperactive monocytes as well as dysregulated activation of autoreactive B cells.
The BXSB strain of mice spontaneously develops an autoimmune syndrome with features of systemic lupus erythematosus (SLE) that affects males much earlier than females (1). The accelerated development of SLE in male BXSB mice results from the genetic abnormality Yaa (Y-linked autoimmune acceleration), which is present on the Y chromosome in the BXSB mouse (2). Recently, the Yaa mutation was shown to be a consequence of a translocation from the telomeric end of the X chromosome onto the Y chromosome (3–5). Based on the presence of the gene encoding Toll-like receptor 7 (TLR-7) in this translocated segment of the X chromosome, Tlr7 gene duplication has been proposed as the etiologic basis for Yaa-mediated enhancement of disease.
One of the cellular abnormalities linked to the Yaa mutation is monocytosis (6), which is strongly associated with autoantibody production and the subsequent development of lupus nephritis (7–9). At 8 months of age, monocytes reach a frequency of ∼50% of peripheral blood mononuclear cells (PBMCs) in male BXSB mice with the Yaa mutation. Circulating monocytes are divided into 2 phenotypically and functionally distinct subsets in mice (10, 11). The first subset, which is classified as “inflammatory” monocytes and characterized by a Gr-1+CX3CR1lowCCR2+CD62L+ phenotype, is preferentially recruited to inflamed tissue. The second Gr-1–CX3CR1highCCR2–CD62L– subset is classified as “resident” monocytes and considered to be a source of tissue-resident macrophages and dendritic cells. Significantly, monocytosis in lupus-prone mice was characterized by a selective expansion of the Gr-1– “resident” subset (12). However, the molecular basis for the development of monocytosis with expansion of the Gr-1– monocyte subset has not yet been defined.
The analysis of Yaa plus non-Yaa mixed bone marrow chimeras demonstrated no selective production of monocytes of Yaa origin over those of non-Yaa origin, thus indicating that the development of monocytosis is not due to an intrinsic abnormality in the growth potential of monocyte lineage cells from Yaa mice (12). Therefore, we hypothesized that Yaa-mediated monocytosis might result from an excessive production of a monocyte-specific growth factor(s) by macrophages due to hyperresponsiveness of their IgG Fc receptors (FcγR) to immune complexes (ICs).
It has been well established that among the 3 different types of activating FcγR expressed on murine immune effector cells, low-affinity FcγRIII and FcγRIV play a major role in the pathogenesis of IC-mediated vascular and glomerular injuries (13). However, FcγR-mediated inflammatory responses are down-regulated through coengagement of the low-affinity inhibitory FcγRIIB. Thus, competitive engagement of these 2 types of FcγR and their relative expression on immune effector cells could be critical for the development of IC-mediated inflammatory lesions in SLE. Notably, lupus-prone NZB, BXSB, and MRL strains share the NZB-type Fcgr2b allele (14, 15), and because of deletion polymorphism in its promoter region and additional polymorphism in the putative regulatory region in intron 3 (14–17), levels of FcγRIIB expression on peritoneal macrophages in these mice were shown to be down-regulated as compared with mice carrying the B6-type Fcgr2b allele (9). However, it remains to be determined whether the expression levels of FcγRIIB on 2 different monocyte subsets and polymorphonuclear cells (PMNs), the immune effector cells which initially interact with circulating ICs, are similarly modulated in mice bearing the NZB-type Fcgr2b allele.
In the present study, we explored the possible role of activating FcγR in the development of monocytosis with expansion of the Gr-1– resident monocyte subset and characterized the functional phenotype of this subset. Our results demonstrated that the development of monocytosis and the selective expansion of the Gr-1– resident monocyte subset in lupus-prone male BXSB Yaa mice were dependent on IC-mediated activation of FcγR. Moreover, this subset displayed a unique hyperactive phenotype, as indicated by a very low level of expression of inhibitory FcγRIIB but a high level of expression of activating FcγRIV, in contrast to the Gr-1+ inflammatory subset, which had high expression of FcγRIIB but no expression of FcγRIV.
MATERIALS AND METHODS
C57BL/6 (B6) mice deficient in common γ-chains of the Fc receptor (FcRγ) were generated by gene targeting in B6-derived embryonic stem cells, as described previously (18). FcRγ−/− BXSB mice lacking expression of activating FcγRI, FcγRIII, and FcγRIV were established by selective backcrossing of (BXSB × FcRγ−/− B6)F1 mice to BXSB mice, as described previously (19). The chromosome segment of FcRγ−/− B6 mice introduced into the BXSB genetic background was identified using microsatellite marker polymorphisms. (NZB × NZW)F1 and B6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). FcγRIII−/− mice, which were generated by gene targeting in 129 mouse–derived embryonic stem cells (20), were backcrossed for 5 generations on a B6 background. FcγRIIB−/− mice were recently generated using B6 mouse–derived embryonic stem cells in the laboratory of one of us (JSV).
Mice expressing the 6-19 IgG3 anti-IgG2a rheumatoid factor (RF) transgene (21) were backcrossed for 8 generations on a B6 background bearing either the Igha or Ighb allele. FcRγ−/− 6-19–transgenic mice bearing the Igha allotype were produced through intercross between corresponding B6 mice. The presence of the 6-19 transgene and the FcRγ genotype were determined by polymerase chain reaction analysis, as described previously (21, 22). The expression of the Igha and Ighb alleles was determined by enzyme-linked immunosorbent assay (ELISA), as described elsewhere (23).
Animal studies were approved by the Ethical Committee for Animal Experimentation, Faculty of Medicine, University of Geneva.
Flow cytometric analysis.
Flow cytometry was performed using 3-color or 4-color staining of peripheral blood cells and analyzed with a FACSCalibur instrument (BD Biosciences, San Jose, CA). The following antibodies were used: M1/70 anti-CD11b, anti-F4/80, anti–Gr-1, AFS98 anti-CD115 (macrophage colony-stimulating factor receptor) (24), K9.361 anti-Ly17.2 (B6-type FcγRIIB), 9E9 anti-FcγRIV (25), 2.4G2 anti-FcγRIIB/III, and RA3-6B2 anti-B220 monoclonal antibodies (mAb).
The mean ± SD percentage of CD11b+F4/80+ monocytes (distinguished from PMNs by their lower level of granularity, as reflected in a low side light–scatter pattern) among PBMCs in 8-month-old male B6 mice (n = 15) was 10.3 ± 3.0%. Mice displaying percentages of monocytes that were more than 3SD above the mean in male B6 mice (>19.3%) were considered to be positive for monocytosis.
Serum levels of gp70–anti-gp70 ICs and IgG3 anti-IgG2a RF were determined by ELISA, as described previously (8, 26). Cryoglobulins were isolated from sera as described elsewhere (26). Concentrations of IgG2a and IgG2c in cryoglobulins were determined by ELISA using polyclonal goat anti-IgG2a antibodies that were cross-reactive with IgG2c (SouthernBiotech, Birmingham, AL) by referring to standard curves established with serum pools from B6 mice bearing either the Igha or Ighb allotype.
In vitro binding of IgG2a ICs to monocytes.
IgG2a ICs were prepared in vitro by incubation of fluorescein isothiocyanate (FITC)–labeled Hy1.2 IgG2a anti-dinitrophenyl (anti-DNP) mAb (50 μg/ml) and DNP15–bovine serum albumin (DNP15-BSA; 50 μg/ml) at 37°C for 2 hours. Then, PBMCs from B6 mice were incubated with a mixture of FITC-labeled Hy1.2 mAb plus DNP15-BSA or FITC-labeled Hy1.2 mAb alone in the presence of either hamster 9E9 FcγRIV-blocking mAb (25) or polyclonal hamster IgG (Jackson ImmunoResearch Europe, Suffolk, UK) as a control. The cells were simultaneously stained with phycoerythrin-labeled anti–Gr-1 and biotinylated AFS98 anti-CD115 mAb, followed by staining with allophycocyanin. Binding of FITC-labeled Hy1.2 mAb on Gr-1+ and Gr-1– monocytes was analyzed with a FACSCalibur instrument.
Analyses for percentages of monocytes and their subsets were performed with the Mann-Whitney U test. Unpaired comparison of the mean fluorescence intensity (MFI) of 9E9 and 2.4G2 staining of monocyte subsets was analyzed by Student's t-test. Probability values >5% were considered insignificant.
Suppression of monocytosis and of the expansion of the Gr-1– subset in lupus-prone male BXSB Yaa mice deficient in FcRγ.
As described previously (6, 12), 8-month-old male BXSB Yaa mice developed monocytosis, with 31–57% of PBMCs being CD11b+F4/80+ monocytes (mean ± SD 45.3 ± 8.1%) and a predominance of the Gr-1– resident subset (Figure 1). However, when the development of monocytosis was assessed in male FcRγ-deficient BXSB Yaa mice lacking the functional expression of all 3 activating receptors (FcγRI, FcγRIII, and FcγRIV), neither monocytosis nor expansion of the Gr-1– monocyte subset was observed at 8 months of age (Figure 1). Notably, as shown previously (19), male FcRγ-deficient BXSB Yaa mice still developed high titers of IgG anti-DNA autoantibodies and nephritogenic gp70–anti-gp70 ICs at levels comparable with those in male wild-type (WT) BXSB Yaa mice (data not shown). These results indicated that the development of monocytosis and expansion of the Gr-1– subset in aged BXSB Yaa mice resulted from FcγR-dependent activation of immune effector cells by the accumulation of ICs in lupus-prone mice.
Development of monocytosis with expansion of the Gr-1– monocyte subset in lupus-prone (NZB × NZW)F1 mice.
The implication of FcγR in monocytosis occurring in BXSB Yaa mice prompted us to explore the possible development of monocytosis in (NZB × NZW)F1 mice, another lupus-prone strain. Percentages of monocytes in the peripheral blood were only slightly increased in female (NZB × NZW)F1 mice at 4 months of age (mean ± SD 10.5 ± 1.6%) (Figure 2). In contrast, at 8 months of age, 5 of the 8 mice displayed significant increases in monocytes, with a mean value of 26.4 ± 8.6% (P < 0.001). Although the extent of monocytosis was moderate as compared with that in male BXSB Yaa mice (Figure 1B), monocytosis occurring in aged female (NZB × NZW)F1 mice was characterized by a selective increase in the Gr-1– monocyte subset (4.5 ± 0.7% Gr-1+ monocytes and 21.9 ± 8.3% Gr-1– monocytes) (Figure 2), as was the case in male BXSB Yaa mice.
FcγR-dependent monocytosis with expansion of the Gr-1– monocyte subset in 6-19 IgG3 anti-IgG2a RF–transgenic mice.
The role of IgG ICs and activating FcγR in the development of monocytosis was further examined in B6 mice expressing the 6-19 IgG3 anti-IgG2a RF transgene and bearing either the Igha or Ighb allele. Since 6-19 RF is specific for IgG2a (but not IgG2c), 6-19 IgG3–IgG2a ICs were only formed in B6.Igha 6-19–transgenic mice, which express IgG2a, but not in conventional B6 mice, which bear the Ighb allele, an allele that expresses IgG2c, but not IgG2a. This finding was confirmed by the presence of IgG2a, but not IgG2c, in 6-19 cryoglobulins isolated from the sera of B6.Igha and B6 (Ighb) 6-19–transgenic mice (data not shown), since 6-19 mAb generated cryoglobulins because of the unique physicochemical property of the IgG3 subclass (21, 27).
At 3–4 months of age, all B6.Igha 6-19–transgenic mice developed monocytosis (mean ± SD 27.6 ± 5.7%), whereas none of the B6 (Ighb) 6-19–transgenic mice displayed monocytosis (10.6 ± 0.8%; P < 0.0001) (Figure 3). Again, the development of monocytosis in B6.Igha 6-19–transgenic mice was due to an accumulation of the Gr-1– monocyte subset (6.2 ± 2.1% Gr-1+ monocytes and 21.9 ± 5.6% Gr-1– monocytes) (Figure 3). Furthermore, the implication of activating FcγR in the development of monocytosis was confirmed by the absence of monocytosis in FcRγ−/− B6.Igha 6-19–transgenic mice (Figure 3). It should also be mentioned that the extent of monocytosis in B6.Igha 6-19–transgenic mice was not exacerbated by the presence of the Yaa mutation (Figure 3).
Selective expression of activating FcγRIV on Gr-1–, but not Gr-1+, monocyte subsets and higher capacity of Gr-1– than Gr-1+ monocytes for binding to IgG2a ICs in B6 mice.
It has been shown that the monocytes that repopulated the circulation after monocyte depletion by liposome treatment were exclusively of the Gr-1+ subset (11) and that these cells were the only monocytes initially labeled after in vivo treatment with bromodeoxyuridine (12). These results support the idea that Gr-1+ and Gr-1– monocytes represent 2 different stages of maturation in the bloodstream. To determine the possible functional differences between Gr-1+ and Gr-1– monocytes, we compared the expression levels of low-affinity FcγR (FcγRIIB, FcγRIII, and FcγRIV), all of which efficiently bind circulating ICs. The expression of FcγRIIB and FcγRIV on monocytes was assessed with B6-type FcγRIIB-specific K9.361 and with FcγRIV-specific 9E9 mAb, respectively, whereas the expression of FcγRIII was evaluated by the staining of FcγRIIB-deficient monocytes with 2.4G2 anti-FcγRIIB/III mAb because of the lack of FcγRIII-specific mAb.
The extent of surface staining for FcγRIIB and for FcγRIII on Gr-1+ monocytes in B6 mice was comparable and slightly higher, respectively, as compared with their staining on Gr-1– monocytes (Figure 4A). In contrast, FcγRIV was expressed only on the Gr-1– subset, and not on the Gr-1+ subset. These data suggested that Gr-1– monocytes that newly express FcγRIV could more efficiently interact with IgG2a ICs than Gr-1+ monocytes. Indeed, the binding analysis of in vitro–prepared IgG2a ICs between Hy1.2 anti-DNP mAb and DNP15-BSA revealed a higher capacity of Gr-1– monocytes than Gr-1+ monocytes to bind to IgG2a ICs (Figure 4B). This enhanced binding was no longer observed in the presence of 9E9 FcγRIV–blocking mAb, thereby confirming the implication of FcγRIV in an increased IgG IC–binding activity of Gr-1– monocytes.
Very low level of expression of inhibitory FcγRIIB on the Gr-1–FcγRIV+ monocyte subset in B6 mice bearing the NZB-type Fcgr2b allele.
We have previously shown that the expression of FcγRIIB on resident peritoneal macrophages from B6 mice bearing the NZB-type Fcgr2b allele, which is shared by lupus-prone mice (14–16), was markedly diminished as compared with that from conventional B6 mice bearing the B6-type Fcgr2b allele (9). Since our analysis showed comparable expression of the B6-type FcγRIIB allelic form on Gr-1+ and Gr-1– monocytes in B6 mice (Figure 4A), we compared the expression levels of FcγRIIB on these 2 subsets of monocytes in B6 mice with the NZB-type Fcgr2b allele. Because of the lack of a mAb that was able to specifically recognize the NZB-type allelic form of FcγRIIB, we used 2.4G2 anti-FcγRIIB/III mAb to determine the expression levels of FcγRIIB on monocytes from FcγRIII−/− B6 mice carrying the 129-derived NZB-type Fcgr2b allele, which was cotransferred with the Fcgr3 mutant gene during backcrossing of the mutated 129 interval to B6 mice (9).
Flow cytometric analysis of FcγRIII−/− monocytes revealed that the expression level of FcγRIIB on Gr-1– monocytes was much lower than that on Gr-1+ monocytes, whereas both Gr-1+ and Gr-1– subsets in FcRγ−/− B6 mice carrying the B6-type Fcgr2b allele stained equally with the 2.4G2 mAb (Figure 5A). Notably, a similar down-regulated expression of FcγRIIB was observed on PMNs bearing the NZB-type Fcgr2b allele, but not on circulating B cells (Figure 5B). These data indicated a selective down-regulation of the expression of FcγRIIB on Gr-1–FcγRIV+ monocytes and PMNs bearing the NZB-type Fcgr2b allele as compared with those bearing the B6-type Fcgr2b allele.
Selective expansion of FcγRIIBlowFcγRIV+ monocytes in aged BXSB mice bearing the NZB-type Fcgr2b allele.
To confirm that Gr-1– monocytes accumulating in aged male BXSB Yaa mice bearing the NZB-type Fcgr2b allele indeed displayed the FcγRIIBlowFcγRIV+ phenotype, the expression levels of FcγRIV and FcγRIIB were determined by flow cytometric analysis. As expected, Gr-1– monocytes from 8-month-old male BXSB Yaa mice with monocytosis highly expressed FcγRIV on their surface (mean ± SD MFI 327.2 ± 28.1; n = 3 mice) at levels comparable with those on Gr-1– monocytes from B6 mice (MFI 362.3 ± 42.5; n = 5 mice) (Figure 5C). Although the expression of FcγRIIB could not be directly determined because of the lack of antibodies specific for the NZB-type FcγRIIB allelic form, the majority of Gr-1– monocytes displayed limited staining with 2.4G2 anti-FcγRIIB/III mAb, as compared with the Gr-1+ subset. Notably, 2.4G2 staining of Gr-1– monocytes from BXSB mice (MFI 39.4 ± 13.4) was clearly weaker than that of Gr-1– monocytes from B6 mice (MFI 74.2 ± 4.6; P < 0.005), whereas Gr-1+ monocytes displayed comparable MFI of 2.4G2 staining in both strains of mice (MFI 115.9 ± 28.7 in BXSB mice and 144.7 ± 10.7 in B6 mice). Collectively, our data suggested that Gr-1–FcγRIIBlowFcγRIV+ monocytes accumulated in the peripheral blood of male BXSB Yaa mice as a result of exposure of IgG ICs during the course of the disease.
The present study was designed to define the molecular mechanisms responsible for the development of monocytosis, which is characterized by a selective expansion of the Gr-1– monocyte subset in lupus-prone BXSB Yaa mice. Analysis of BXSB Yaa and 6-19 anti-IgG2a–transgenic B6 mice deficient in activating FcγR demonstrated that the development of monocytosis was a consequence of IC-triggered activation of immune effector cells, such as monocyte/macrophages, through activating FcγR. Moreover, the Gr-1– monocyte subset accumulating in BXSB Yaa mice bearing the NZB-type Fcgr2b allele, which is common in lupus-prone mice, was revealed to display a hyperactive phenotype in response to IgG ICs because of low expression of inhibitory FcγRIIB but high expression of activating FcγRIV.
Our previous analysis of Yaa plus non-Yaa mixed bone marrow chimeras showed that there was no selective production of monocytes of Yaa origin over those of non-Yaa origin (12). This result suggests that monocytosis associated with the Yaa mutation is a result of excessive production of monocyte-specific growth factors by immune effector cells in response to ICs during the course of lupus-like autoimmune disease. Indeed, the absence of monocytosis in male BXSB Yaa mice deficient in activating FcγR, despite high production of autoantibodies (19), indicates that IC-mediated, FcγR-dependent activation of immune effector cells is crucial for the development of monocytosis. This conclusion was further supported by 2 findings. First, the expression of the 6-19 IgG3 anti-IgG2a RF transgene induced monocytosis only in B6 mice expressing IgG2a, but not in those deficient in activating FcγR or lacking IgG2a. Second, the development of monocytosis was also observed in (NZB × NZW)F1 mice, another lupus-prone strain, at 8 months of age, when high titers of autoantibodies started to accumulate.
In light of these results, it seems plausible that persistent FcγR-mediated activation of monocyte/macrophages by IgG ICs may result in the production of excessive amounts of monocyte-specific growth factors, thereby leading to the development of monocytosis. Notably, interactions of IgG ICs with FcγR on macrophages trigger the production of macrophage colony-stimulating factor and granulocyte–macrophage colony-stimulating factor by macrophages (28, 29).
It should be stressed that the extent of monocytosis in (NZB × NZW)F1 mice and in 6-19 anti-IgG2a RF–transgenic B6 mice was less severe than that observed in BXSB Yaa mice. This indicates that the Yaa mutation somehow plays a unique role in the development of monocytosis. The recently identified Tlr7 gene duplication resulting from a translocation from the telomeric end of the X chromosome (containing the Tlr7 gene) onto the Y chromosome has been proposed as the etiologic basis for Yaa-mediated enhancement of SLE (3–5). Accordingly, the development of monocytosis was strongly suppressed in B6 Yaa mice congenic for the Nba2 (NZB autoimmunity 2) locus following the introduction of the Tlr7-null mutation on the X chromosome (30). This suggests that IgG ICs containing endogenous nuclear antigens could excessively activate Yaa-bearing macrophages through interaction with FcγR and then with TLR-7, which is expressed at increased levels in endosomes of these macrophages, to secrete high levels of monocyte-specific growth factors. In addition, because of the Tlr7 gene duplication, the Yaa mutation selectively enhances the production of autoantibodies against nuclear antigens that are capable of interacting with TLR-7, thereby further promoting the activation of Yaa-bearing macrophages (3–5, 30, 31). This is consistent with the finding that the presence of the Yaa mutation failed to aggravate the extent of monocytosis in 6-19 IgG3 anti-IgG2a–transgenic mice, since IgG3–IgG2a ICs are not expected to interact with TLR-7.
Analysis of FcRγ−/− BXSB mice and 6-19 anti-IgG2a–transgenic B6 mice revealed that IC-mediated, FcγR-dependent activation was also responsible for an expansion of the Gr-1– subset, 1 of the 2 major monocyte subsets that are present in the circulation. Since recent immigrants from bone marrow appear to enter the circulation as Gr-1+ monocytes (11, 12), it has been suggested that the Gr-1+ subset consecutively becomes the Gr-1– subset while still in the bloodstream. In this regard, our demonstration that activating FcγRIV was expressed on the Gr-1– subset, but not on the Gr-1+ subset, supports the idea that the Gr-1– subset represents a more mature stage of monocytes as compared with the Gr-1+ subset, although this has not yet been formally proven. The selective accumulation of the Gr-1– subset in lupus-prone mice developing monocytosis is likely to be due to a longer half-life of the Gr-1– subset as compared with that of the Gr-1+ subset (10, 11).
It is significant that the Gr-1– monocyte subset that accumulated in lupus-prone mice carried a unique functional phenotype, since it expressed very low levels of inhibitory FcγRIIB but high levels of activating FcγRIV, in contrast to the high levels of FcγRIIB and no FcγRIV expression on the Gr-1+ monocyte subset. It has been well established that the relative balance of engagement of activating and inhibitory FcγR is critical for the development of IC-mediated tissue lesions (13). Thus, it is reasonable to assume that the Gr-1– monocyte subset accumulating in lupus-prone mice could be excessively activated in the presence of circulating IgG ICs, thereby actively participating in the development and progression of IC-mediated tissue injury in SLE.
Previous reports have shown a considerable role of infiltrating monocyte/macrophages in the progression of glomerular lesions (32) and of FcγR in glomerulonephritis, including murine lupus nephritis (18, 33, 34). Thus, monocytosis could actively participate in the development of glomerular inflammation and injury through increased secretion of proinflammatory cytokines, reactive oxygen species, and matrix-specific proteases as a result of IC-mediated, FcγR-dependent activation of infiltrating monocyte/macrophages. Moreover, down-regulated expression of FcγRIIB on PMNs bearing the NZB-type Fcgr2b allele could additionally contribute to the development of glomerular lesions in lupus-prone mice, since a considerable role of PMNs in IC-mediated inflammatory disorders has been well established (35, 36).
The findings of our study further underline the importance of the NZB-type Fcgr2b allele as a lupus susceptibility gene in murine SLE. The down-regulated expression of FcγRIIB on monocyte/macrophages, PMNs, as well as activated B cells in lupus-prone mice appears to contribute not only to increased production of autoantibodies as a result of dysregulated activation of autoreactive B cells (19, 37), but also to enhanced IC-mediated glomerular and vascular inflammation as a result of excessive activation of monocyte/macrophages and PMNs. The selective expression of FcγRIV on Gr-1– resident monocytes in mice appears to have an analogy in humans, since FcγRIIIA, the human homolog of FcγRIV (38), is expressed on the resident, but not the inflammatory, subset in humans (39). In view of the critical role of FcγR and the accumulation of a hyperactive monocyte subset in parallel with the progression of disease in lupus-prone mice, the enumeration of blood monocytes and the analysis of their subsets, especially the FcγRIIBlowFcγRIIIA+ subset, might be useful predictive markers in patients with SLE.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Izui had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Santiago-Raber, H. Amano, Hirose, Izui.
Acquisition of data. Santiago-Raber, H. Amano, E. Amano, Baudino, Otani.
Analysis and interpretation of data. Santiago-Raber, H. Amano, E. Amano, Baudino, Otani, Hirose, Izui.
Generation of FcRγ−/−BXSB mice. Lin, Hirose.
Provision of anti-FcγRIV monoclonal antibody. Nimmerjahn, Ravetch.
Provision of FcγRIIB−/−and FcγRIII−/−mice. Verbeek.
We thank Dr. T. Moll for critical reading of the manuscript, and Mr. G. Celetta, Mr. G. Brighouse, and Mr. G. Sealy for excellent technical help.