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Fas Death Receptor Signaling Represses Monocyte Numbers and Macrophage Activation In Vivo¹

Nathaniel J. Brown^*, Jack Hutcheson^*, Emily Bickel^*, John C. Scatizzi^*, Lee D. Albee^*, G. Kenneth Haines, III, Joy Eslick^*, Kathleen Bradley^*, Elsa Taricone and Harris Perlman^2,*

Departments of ^* Molecular Microbiology and Immunology and Comparative Medicine, St. Louis University School of Medicine, St. Louis, MO 63104; and Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Over 1 billion monocytes are produced daily, with a small percentage differentiating into macrophages, suggesting that excess monocytes are deleted through a tightly regulated process. Although the in vivo mechanism governing monocyte/macrophage homeostasis is unknown, deletion of monocytes in culture is mediated by the Fas death pathway and is blocked by M-CSF. To determine the in vivo significance of Fas in monocyte development, mice lacking Fas (lpr/lpr) and mice deficient in Fas and M-CSF were examined. Compared with congenic control C57BL/6 (B6) mice, lpr/lpr mice displayed increased numbers of circulating monocytes. The lack of Fas in M-CSF-deficient mice resulted in an enhanced percentage, but not total numbers, of monocytes. Fas deficiency led to an increase in myeloid bone marrow progenitor potential only in M-CSF-intact mice. Although lpr/lpr and B6 mice had similar numbers of tissue macrophages, the loss of Fas in M-CSF-deficient mice was sufficient to increase the number of macrophages in a subset of tissues. Additionally, after stimulation with thioglycolate, lpr/lpr and B6 mice showed equivalent numbers of peritoneal macrophages. However, Fas-deficient peritoneal macrophages displayed a marked increase in spontaneous and LPS-induced proinflammatory molecule production. Moreover, Fas-deficient mice showed enhanced systemic inflammatory arthritis associated with up-regulation of IL-1 and CCL2 secretion, elevated numbers of inflammatory monocytes, and increased numbers of tissue macrophages. Collectively, these data suggest that Fas may be required for maintaining circulating monocytes and for suppressing macrophage activation and recruitment that are stimulus dependent.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Monocytes and macrophages are critical effector cells in the innate immune response, contributing to acute and chronic inflammation and protecting against microbial infection and tumor formation (1). Although numerous investigations examined the functionality of monocytes and macrophages during various immune responses (2), little or no information is available concerning their growth, development, and deletion in vivo under normal conditions or during inflammation. The vast majority of studies that examined the mechanism of monocyte turnover used human monocytes isolated from peripheral blood. Human monocytes cultured in the absence of serum or growth factors undergo a marked spontaneous apoptosis, with <5% of monocytes remaining by 3 days in culture (3, 4). Even in the presence of serum, 75% of monocytes die by spontaneous apoptosis within the first 3 days of culture (3). Spontaneous monocyte apoptosis in culture is inhibited by neutralizing Abs to the death receptor Fas or its cognate ligand Fas ligand (FasL)³ (3, 4, 5, 6). Although these data suggest that Fas-FasL are vital for spontaneous monocyte apoptosis in vitro, the essential in vivo role for Fas-FasL in regulating monocyte/macrophage homeostasis has yet to be shown.

M-CSF deficient (op/op) mice contain a single base pair insertion (thymidine) in the M-CSF coding region, resulting in an early stop codon 21 bp downstream of the start site (7). op/op mice display a marked reduction in the number of circulating monocytes and tissue macrophages (8, 9, 10, 11, 12, 13, 14, 15, 16, 17). These data suggest that, in vivo, monocytes require M-CSF to survive and differentiate into macrophages (18, 19, 20). Moreover, exogenous M-CSF is sufficient to inhibit the spontaneous monocyte apoptosis that occurs in culture and is mediated by Fas-FasL-signaling (19). Taken together, these data suggest that one of the functions of M-CSF may be to inhibit Fas-induced monocyte apoptosis. It is unknown, however, whether the loss of Fas is sufficient to rescue monocyte and macrophage numbers in M-CSF-deficient mice.

Rheumatoid arthritis (RA) is a systemic chronic inflammatory and destructive arthropathy of unknown etiology (21). Monocytes/macrophages contribute directly to synovial inflammation, hyperplasia of the synovial lining, and destruction of cartilage and bone, in part by being the main producers of TNF- and IL-1 (22). Inhibition of TNF- or IL-1 activity suppresses synovial inflammation and bone destruction in RA patients and is associated with reduced numbers of monocytes and macrophages (23, 24, 25). Although Fas and FasL are present on synovial macrophages (26), increased FLIP expression (26) and enhanced levels of soluble FasL in the joint (27) may suppress the Fas death signal. The fact that increased numbers of monocytes and macrophages are observed in the RA joint and that they are highly activated suggest that a dysfunctional Fas-signaling pathway may contribute to RA pathogenesis.

In this study we determined the role of Fas death receptor signaling in regulating monocyte homeostasis during normal growth and with inflammation. Fas-deficient mice (lpr/lpr, C57BL/6 background) display elevated numbers of circulating resident and inflammatory monocytes compared with congenic control C57BL/6 (B6) mice. Mice lacking both Fas and M-CSF show a marked increase in the percentage, but not the total number, of circulating monocytes compared with M-CSF-deficient mice. Moreover, lpr/lpr mice exhibit enhanced numbers of myeloid bone marrow progenitors, but Fas- and M-CSF-deficient mice have reduced numbers of myeloid bone marrow progenitors. Despite the fact that lpr/lpr mice display increased numbers of peripheral blood monocytes, no differences are observed in the number of lpr/lpr and B6 tissue macrophages. In contrast, Fas deficiency is sufficient to enhance the number of macrophages in op/op mice in a subset of tissues. Even though the number of peritoneal macrophages is the same in B6 and lpr/lpr mice after stimulation with thioglycolate, the isolated thioglycolate-treated lpr/lpr peritoneal macrophages display a marked increase in spontaneous and LPS-induced TNF-, IL-1, IL-6, and CCL2 production. Moreover, compared with B6, Fas-deficient mice exhibit worse experimental inflammatory arthritis, which is associated with elevated levels of IL-1 and CCL2 and increased numbers of macrophages. Collectively, these data support the essential role of the Fas-signaling pathway in regulating monocyte development, activation, and recruitment during normal growth and with inflammation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

B6.MRL-Tbfrsf6lpr (^lpr/lpr), C57BL/6 (congenic control for lpr/lpr mice), and B6C3Fe a/a-CSF1^op (op/+) mice were purchased from Jackson Laboratory (Bar Harbor, ME); nonobese diabetic (NOD) mice were purchased from Taconic Farms (Germantown, NY); and homozygous KRN TCR transgenic mice (C57BL/6 background) were a gift from Drs. D. Mathis and C. Benoist (Harvard Medical School, Boston, MA) and Institute de Gene-Tique et de Biologie Moleculaire et Cellulaire (Strasbourg, France). The op/+ mice (B6:C3H) were crossed with lpr/lpr (B6) mice, and the progeny were screened for the mutant M-CSF gene and the Fas gene as previously described (28, 29). Brother-sister op/+lpr/+ mice (F₁) were crossed to yield progeny (op/+, op/+/lpr/lpr, and +/+) that would become the breeder pairs for all experiments. The F₂ progeny +/+ (designated B6:C3H), op/op, and op/op/lpr/lpr were used for all experiments. Because op/op and op/op/lpr/lpr mice lack incisors, mice were fed high fat powdered chow (Research Diets, New Brunswick, NJ). All experiments with mice were approved by the animal care and use committee at St. Louis University.

Immunophenotyping

Peripheral blood was isolated by cardiac sticks from 5- to 8-wk-old mice after euthanasia. Unstimulated peritoneal and 5-day 4% thioglycolate-stimulated peritoneal cells were isolated by peritoneal lavage. Total leukocyte numbers were determined on the automated hematology analyzer ABX Pentra 60 (Diamond Diagnostics, Holliston, MA). Cells were blocked with anti-CD16/32 Ab (BD Pharmingen, San Diego, CA), then stained with fluorescent-conjugated anti-CD45, anti-CD11b, anti-Gr-1, anti-CD62L, anti-CD23, anti-CD69, anti-I-A^b, (BD Pharmingen), or anti-F4/80 (Caltag Laboratories, Burlingame, CA) Abs. After incubation with Abs, RBC were lysed, and cells were fixed with BD FACS lysing solution (BD Pharmingen) and acquired on a FACSCalibur (BD Pharmingen) using CellQuest software at the St. Louis University Core flow cytometry facility. All analysis was performed using FlowJo software (TreeStar, San Carlos, CA).

Immunohistochemistry

After euthanasia, mouse tissues were isolated, fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned. To stain for F4/80 positivity, Ags were retrieved using a target retrieval solution (DakoCytomation, Copenhagen, Denmark). After Ag retrieval, sections were blocked in hydrogen peroxide, incubated with anti-F4/80 Ab or isotype control, then incubated with secondary biotinylated anti-rat Ab (DakoCytomation). Sections were treated with streptavidin peroxidase conjugate (DakoCytomation), color was visualized with diaminobenzidine, and sections were counterstained with hematoxylin. All F4/80 staining was performed an autostainer (DakoCytomation). F4/80⁺ cells were scored by examining three fields per section at x400 by a pathologist or an observer blinded to the study. A minimum of 75 cells were counted in each field. Ankle sections were also stained with H&E or with Safranin O and methyl green. The median synovial lining thickness, inflammatory score (0–5), cartilage destruction score (0–5), bone erosion score (0–5), pannus formation (0–5), and macrophage positivity (0–5) were determined by a pathologist blinded to the study (30). Photographs were taken on a Nikon microscope equipped with the Nikon digital camera DMX1200 (Nikon, Melville, NY).

Colony formation assays

Single-cell suspensions of bone marrow cells were isolated from tibias. The marrow shaft of each tibia was flushed with IMDM supplemented with 2% FBS and 1/200 penicillin/streptomycin and filtered through a 100-µm pore size filter. For op/op and op/op/lpr/lpr mice, tibias were crushed with mortar and pestle and filtered through a 40-µm pore size filter. RBC were lysed with PharM Lyse (BD Pharmingen). Triplicate cultures of bone marrow cells were plated at a concentration of 2 x 10⁴/ml in M3434 medium (Stem Cell Technologies, Vancouver, Canada) and incubated at 37°C. GM-CFU and erythroid burst-forming units (BFU-E) were scored on days 9–10 as described by the manufacturer (Stem Cell Technologies).

Cell culture

Four percent thioglycolate (Sigma-Aldrich, St. Louis, MO)-stimulated peritoneal macrophages isolated by peritoneal lavage were plated onto 24-well tissue culture plates at 500,000 cells/well for 1 h in RPMI 1640. The adherent cells (peritoneal macrophages) were washed with PBS and cultured in 20% heat-inactivated FBS/RPMI 1640. Peritoneal macrophages were verified by F4/80-positive staining using flow cytometry, rested for 24 h, then stimulated for 24 h with LPS (Sigma-Aldrich) at a concentration of 100 ng/ml in 20% FBS/RPMI 1640. Supernatants were collected and assessed for TNF-, IL-1, IL-6, and CCL2 secretion by ELISAs.

Serum transfer-induced arthritis

The 7-wk-old progeny from KRN mice crossed with NOD mice (K/BxN) were euthanized, peripheral blood was isolated, and serum was collected and pooled. One hundred and fifty microliters of K/BxN serum was i.p. injected on each flank of 6-wk-old B6 and lpr/lpr mice. As a control B6 or lpr/lpr were treated with saline in place of serum (not shown). At each time point and before euthanasia, the degree of arthritis, as indicated by joint swelling, was quantitated by measuring two perpendicular diameters of the ankles using a caliper (Lange Caliper; Cambridge Scientific Industries, Cambridge, MA). Joint circumference was calculated using the geometric formula of ellipse circumference: (2 x (a² + b²)/2), as previously described (30). Ankle joints were removed, and one was fixed in 10% neutral buffered formalin for 24 h, then in EDTA-decalcification buffer for 2 wk, embedded in paraffin, and sectioned. Before decalcification, ankles were examined for bone destruction using microcomputed tomography at Washington University School of Medicine. The other ankle was placed in liquid nitrogen, ground into a fine powder by mortal and pestle, digested in protein lysis buffer, and homogenized on ice for 20 s (3, 31, 32).

ELISA

For detection of mouse TNF-, IL-1, IL-6, and CCL2, sandwich ELISAs were performed according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). The sensitivity was 5 pg/ml for TNF-, 15 pg/ml for IL-1, 15 pg/ml for IL-6, and 3.9 pg/ml for CCL2. ELISAs were quantitated by absorbance at 450 nm on a microplate reader (Bio-Rad, Hercules, CA). All data were normalized by cell number (peritoneal macrophages) or by protein concentration (ankle extracts).

Statistics

Results were expressed as the mean ± SE. Differences between groups were analyzed using Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Loss of Fas enhances monocyte numbers in the circulation

The Fas-FasL apoptotic pathway plays an important role in mediating the deletion of monocytes in vitro. However, the in vivo role of the Fas death pathway in regulating monocyte development is unknown. In this study we investigated the effects of the loss of Fas on circulating monocytes, myeloid progenitors, and tissue macrophages under normal growth conditions and during inflammation in M-CSF-intact and -deficient mice. To examine the effect of Fas deficiency on circulating monocytes, peripheral blood was harvested from 5- to 8-wk-old C57BL/6 (B6) mice and lpr/lpr (C57BL/6, donor strain MRL/MPJ-Tnfrsf6lpr) mice before development of the autoimmune disease phenotype. Circulating monocytes, identified as two populations, were determined as recently described by Geissmann et al. (33). Specifically, PBMC are divided into two different populations, one that will become resident macrophages (CD45⁺CD11b⁺⁺Gr-1^–CD62L^–; resident monocytes) and another that will become inflammatory macrophages (CD45⁺CD11b⁺⁺Gr-1⁺CD62L⁺⁺; inflammatory monocytes) (33). By using this strategy, the different populations of monocytes (Fig. 1, R2 and R3 regions) are readily discernable by flow cytometry (Fig. 1). As shown in Fig. 2A, compared with B6 mice, lpr/lpr mice displayed a 63% increase (p < 0.001) in percentage of resident-monocytes and a 42% increase (p < 0.02) in percentage of inflammatory monocytes in peripheral blood (Fig. 2A). The lpr/lpr mice had a 95% increase (p < 0.0001) and a 67% (p < 0.0001) increase, respectively, in the number of resident monocytes and inflammatory monocytes in peripheral blood (Fig. 2B). These data suggest that Fas deficiency results in a substantial increase in the number of circulating monocytes.

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Representative FACS scatter plots of myeloid lineage cells in mouse peripheral blood. Peripheral blood was isolated by cardiac sticks from 5- to 8-wk-old B6 and lpr/lpr mice. Cells were blocked, then stained with anti-CD45, anti-CD11b, anti-Gr-1, and anti-CD62L Abs. After incubation with Abs, RBC were lysed, and cells were fixed with BD FACS lysing solution. R1, CD45+ population; R2, resident monocytes; R3, inflammatory monocytes; R4, neutrophils; R5, eosinophils; R6, NK cells. The NK cell population was confirmed by NK1.1-positive staining.

View larger version (12K): [in this window] [in a new window]   FIGURE 2. Fas-deficient mice display an elevated percentage (A) and number (B) of circulating monocytes. Quantitative analysis of PBMC isolated from B6 (n = 20) and lpr/lpr (n = 27) mice were analyzed as described in Fig. 1. CD45+CD11b++Gr-1–CD62L– cells are resident monocytes, whereas CD45+CD11b++Gr-1+CD62L++ cells are inflammatory monocytes. Values represent the mean ± SE, which were compared by Student’s t test.

View larger version (14K): [in this window] [in a new window]   FIGURE 3. Fas deficiency enhances the percentage of resident and inflammatory monocytes (A), but not the total numbers of monocytes (B), in peripheral blood from M-CSF-null mice. Peripheral blood was isolated from 5- to 8-wk-old B6:C3H (n = 14), op/op (n = 27), and op/op/lpr/lpr (n = 23) mice and analyzed as described in Fig. 1. Values represent the mean ± SE, which were compared by Student’s t test.

The observation that lpr/lpr mice display enhanced numbers of monocytes compared with B6 mice suggests that the elevated number of PBMC may be due to an increased efflux from bone marrow. To examine whether lpr/lpr mice exhibit increased numbers of myeloid progenitors that may contribute a higher efflux of monocytes from bone marrow, myeloid progenitor assays were performed on isolated bone marrow. Although lpr/lpr mice have equivalent numbers of bone marrow cells compared with B6 mice (not shown), lpr/lpr mice showed a 33% (p < 0.0001) increase in GM-CFU (Fig. 4A) compared with B6 mice. In contrast, there were no differences in the number of BFU-E in lpr/lpr compared with B6 mice (Fig. 4A). Similar to previous studies (17), op/op compared with B6:C3H mice showed a 42% (p < 0.0001) decrease in myeloid progenitor cell potential, as measured by the number of CFU-GM colonies (Fig. 4B). Although Fas-deficient mice displayed elevated CFU-GM in M-CSF-intact mice (Fig. 4A), Fas and M-CSF-deficient mice exhibited 60% (p < 0.0001) and 27% (p < 0.07) decreases in CFU-GM compared with B6:C3H and op/op mice, respectively (Fig. 4B). These data suggest that the impact of Fas on myeloid progenitor proliferation/survival is dependent on M-CSF.

View larger version (12K): [in this window] [in a new window]   FIGURE 4. Fas deficiency only enhances myeloid progenitor potential in M-CSF intact mice. A, The lpr/lpr mutant mice show increased myeloid progenitor cell potential. Colony formation assays were performed on single-cell suspensions from bone marrow of B6 (n = 17) and lpr/lpr (n = 12) mice as described in Materials and Methods. GM-CFU and BFU-E were scored 9–10 days after plating. Values represent the mean ± SE, which were compared by Student’s t test. B, Progenitor cell potential is decreased in op/op and op/op/lpr/lpr mice. Colony formation assays were performed on single-cell suspensions from B6:C3H (n = 5), op/op (n = 7), and op/op/lpr/lpr (n = 6) bone marrow. Values represent the mean ± SE, which were compared by Student’s t test.

Fas deficiency is associated with more circulating monocytes; therefore, we determined whether an elevation in the number of monocytes translates into an increase in the number of tissue macrophages. Immunohistochemical and flow cytometric analysis using the macrophage-specific Ab, F4/80 (34, 35), revealed similar numbers of macrophages in liver, spleen, and peritoneum in lpr/lpr and B6 mice (Table I and Fig. 5). In contrast, the numbers of tissue macrophages in liver, spleen, and lung were enhanced in op/op/lpr/lpr mice compared with op/op mice (Table I). There were no appreciable differences in the percentage of peritoneal or synovial lining macrophages in op/op/lpr/lpr mice compared with op/op mice (Table I). These data suggest that in M-CSF-intact mice, additional signals independent of the Fas death signal are required to attract monocytes to tissues and for monocyte differentiation. These data also suggest that when M-CSF is absent, suppression of the Fas death signal is sufficient for monocyte differentiation into a macrophage, but only in a subset of tissues.

View larger version (79K): [in this window] [in a new window]   FIGURE 5. Loss of functional Fas protein is sufficient to restore macrophage numbers in M-CSF-deficient mice, but not in M-CSF-intact mice. Representative photomicrographs of F4/80-positive cells in liver and spleen of B6, lpr/lpr, B6:C3H, op/op, and op/op/lpr/lpr mice are shown. Tissues were isolated from 5- to 8-wk-old mice, fixed in 10% formalin, embedded in paraffin, sectioned, incubated with isotype control (not shown) or anti-F4/80 Ab (brown), and counterstained with hematoxylin (blue).

In the absence of M-CSF, Fas deficiency results in increased numbers of macrophages in a subset of tissues, whereas B6 and lpr/lpr mice display equivalent numbers of tissue macrophages. Thus, we examined whether a deficiency in Fas alters the number of macrophages in response to an acute inflammatory stimuli. As presented in Table II, stimulation with 4% thioglycolate failed to increase the number of peritoneal macrophages in lpr/lpr compared with B6 mice (Table II). However, lpr/lpr, compared with B6 peritoneal macrophages, displayed enhanced spontaneous and LPS-induced proinflammatory molecule production. Untreated lpr/lpr peritoneal macrophages produced increased spontaneous IL-1 (2.7-fold; p < 0.001), CCL2 (1.7-fold; p < 0.001), and TNF- (1.8-fold; p < 0.01) compared with B6 peritoneal macrophages (Fig. 6). Additionally, LPS induced a marked increase in IL-1 (3.9-fold; p < 0.001), CCL2 (1.7-fold; p < 0.001), IL-6 (6.0-fold; p < 0.0001), and TNF- (3.6-fold; p < 0.0001) secretion in lpr/lpr peritoneal macrophages compared with B6 mice (Fig. 6). However, although Fas deficiency resulted in enhanced activity, there were no differences in the number or intensity of (not shown) of MHC class II⁺ or CD69⁺ expression on peritoneal macrophages in B6 and lpr/lpr mice (Table II). These data suggest that Fas deficiency has no effect on recruitment of monocytes to the inflamed peritoneum, but suppresses cytokine and CCL2 production in peritoneal macrophages after activation.

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Fas-deficient peritoneal macrophages express increased spontaneous and LPS-induced proinflammatory cytokine and chemokine production. B6 (n = 6) and lpr/lpr (n = 6) mice were i.p. injected with 4% thioglycolate broth. Five days after i.p. injection, peritoneal cells were isolated by peritoneal lavage. Peritoneal cells (PCs) were plated at 500,000 cells/well in a 24-well plate in RPMI 1640 medium without serum for 1 h, which was then replaced with 20% FBS/RPMI 1640. After a 24-h rest period, peritoneal macrophages were either untreated or stimulated with LPS (100 ng/ml) for 24 h. Supernatants were collected and analyzed for IL-1, CCL2, IL-6, and TNF- production using ELISA. All data were normalized to cell number. Values represent the mean ± SE, which were compared by Student’s t test.

The lpr/lpr mice inbred on a C57BL/6 background do not develop arthritis spontaneously (36); therefore, we used an experimental model of arthritis, the serum transfer model of arthritis. The progeny of KRN TCR transgenic mice crossed with NOD mice (K/BxN) spontaneously develop arthritis. Serum isolated from K/BxN mice induces arthritis in allogeneic hosts (37, 38, 39, 40). The arthritis that occurs in these hosts is similar to RA, in that, in common with humans, mice develop symmetric joint disease with pannus formation, cartilage destruction, and bone erosion. As shown in Fig. 7, A and B, compared with B6 mice, lpr/lpr mice displayed enhanced arthritis, as demonstrated by a 3.7-fold (p < 0.05) increase in ankle circumference. The lpr/lpr mice exhibited increased inflammation, pannus formation, and bone and cartilage destruction compared with B6 mice (Fig. 7, C and D, and Fig. 8). Analysis of peripheral blood after the induction of arthritis revealed a 93% (p < 0.003) increase in the percentage of inflammatory monocytes in lpr/lpr compared with B6 mice (Table III). Although the percentage of resident monocytes in lpr/lpr compared with B6 mice at 7 days after the injection of K/BxN serum was elevated, the differential increase was 51% (p < 0.09). More than a 100-fold increase and a 3.8-fold (p < 0.03) increase in IL-1 and CCL2 production, respectively, were also observed in ankle joints from lpr/lpr compared with B6 mice (Fig. 9). In contrast, there were no differences in IL-6 and TNF- production in lpr/lpr compared with B6 mice (Fig. 9). Because lpr/lpr mice exhibited an increased percentage of inflammatory monocytes in the circulation and because IL-1 was markedly elevated, we examined the number of macrophages in the joint. The lpr/lpr mice displayed an elevated number of F4/80⁺ macrophages, associated with enhanced cellularity of the joint, compared with B6 mice (Fig. 7, C and D, and Fig. 10). Taken together, these data demonstrate that Fas functions to repress arthritis development in the serum transfer model of arthritis.

View larger version (25K): [in this window] [in a new window]   FIGURE 7. The lpr/lpr mice display enhanced arthritis. A, Photomicrograph showing increased ankle thickness in lpr/lpr mice. B, Quantitative analysis of the differential ankle circumference. Three hundred microliters of pooled serum from K/BxN mice was injected i.p. into B6 (n = 11) and lpr/lpr (n = 13) mice. As a negative control, B6 mice (n = 4) were i.p. injected with saline (not shown). Ankle joints were examined for arthritis by measuring two perpendicular diameters of both joints (anterior-posterior and medio-lateral) by calipers. The ankle circumference is defined as the difference in ankle circumference compared with the day 0 measurement. The values represent the mean ± SE of ankles per time point, which were compared by Student’s t test. *, p < 0.05 compared with B6 at the parallel time point. C and D, Histological scores of ankle sections from B6 (C) and lpr/lpr (D) mice. Ankle sections stained with H&E or with Safranin O and methyl green and for F4/80 positivity were evaluated by a pathologist blinded to the study, as described in Materials and Methods.

View larger version (88K): [in this window] [in a new window]   FIGURE 8. Increased inflammation and bone destruction in Fas-deficient mice after serum transfer. A, Enhanced histological evidence of inflammation in lpr/lpr mice. At 7 days after serum transfer, ankles were harvested (n = 6/genotype), sectioned, and stained with hematoxylin (blue) and eosin (pink) or with Safranin O (blue) and methyl green. C, cartilage; SL, synovial lining; B, bone; P, pannus. Black arrows denote sites of invading pannus. B, The lpr/lpr mice display increased bone destruction. Ankles from lpr/lpr and B6 (n = 6/genotype) mice, as described above, were fixed and examined by microcomputed tomography. Shown are representative anterior-posterior and oblique views of the ankles.

View larger version (22K): [in this window] [in a new window]   FIGURE 9. Fas-deficient mice express increased levels of IL-1 and CCL2 in ankle joints after serum transfer induced arthritis. B6 (n = 6) and lpr/lpr (n = 6) mice were isolated 7 days after serum transfer-induced arthritis. Ankle joints were isolated, snap-frozen, ground into a fine powder, lysed, and examined for IL-1, CCL2, IL-6, and TNF- production by ELISA. All data were normalized by the total protein concentration. Values represent the mean ± SE, which were compared by Student’s t test.

View larger version (71K): [in this window] [in a new window]   FIGURE 10. Increased F4/80-positive macrophages in lpr/lpr synovium. Ankles were isolated from B6 and lpr/lpr mice 7 days after injection of K/BxN serum. Ankles were fixed, decalcified, and stained with anti-F4/80 Ab (brown staining) or isotype control (not shown) and counterstained with hematoxylin. Shown are representative photomicrographs of the synovium from four different lpr/lpr and B6 mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Recently, op/op/lpr/lpr mice have been established on an MRL background (44), which is known to enhance the autoimmune disease phenotype that occurs in lpr/lpr mice. Although op/op mice on an MRL background and total macrophage numbers in multiple tissues in young mice (<2 mo) were not examined in this study, aged op/op/lpr/lpr mice display reduced splenomegaly, lymphadenopathy, glomerular nephritis, and cellular infiltrate in lung and salivary and lacrimal glands compared with op/+/lpr/lpr (MRL) mice. The decrease in autoimmune disease is attributed to reduced numbers of B cells, which are also observed in op/op/lpr/lpr mice on a B6 background compared with op/op and B6 mice (not shown). In addition, op/op/lpr/lpr (MRL) had lower numbers of macrophages in kidney and decreased numbers of leukocytes in spleen. Taken together, this study supports the idea that the function of M-CSF in Fas-deficient mice is tissue specific and potentially age and background specific. Thus, future studies of op/op/lpr/lpr (B6) mice will examine the effects of aging and background specificity on the number of macrophages in tissues, splenomegaly, lymphadenopathy, and autoantibody production.

A nonapoptotic function has also been assigned to the Fas-FasL death-signaling pathway. In culture, increased ligation of Fas by anti-Fas Ab on human PBMC and monocyte-derived macrophages results in cytokine and chemokine production and elevated NF-B DNA binding activity (45). However, the proinflammatory effect of preventing constitutive binding of Fas to FasL, which normally occurs in human PBMC and monocyte-derived macrophages (4, 5, 46), was not examined. Additionally, the impact of increasing Fas oligomerization on LPS signaling in monocytes and macrophages was not examined. In our study we show that endogenous Fas oligomerization functions to repress spontaneous and LPS-induced cytokine and CCL2 production. Although a recent report shows that Fas-deficient peritoneal cells display reduced cytokine production in response to LPS, the difference in time that the isolated peritoneal cells from thioglycolate-stimulated mice rested on plates (3 days vs 1 day), the difference in the time of cell isolation after LPS stimulation (4 vs 24 h), and the amount of LPS stimulation (10 vs 100 ng) may explain potential discrepancies in the observed data. Moreover, our data are supported by a recent study that showed that a deficiency in FADD, a critical adaptor molecule in the Fas death-signaling pathway, leads to an increase in cytokine production in response to IL-1 and LPS (47). However, in contrast to Fas-deficient peritoneal macrophages, LPS-induced similar levels of proinflammatory cytokine and CCL2 production in BH3-interacting domain death agonist-deficient and B6 peritoneal macrophages (not shown). Taken together, these data suggest that an intact Fas-signaling pathway, including FADD, but independent of BH3-interacting domain death agonist, is required to inhibit LPS-induced cytokine and CCL2 production.

Mice that carry a mutation in Fas on the C57BL/6 or DBA background do not spontaneously develop arthritis, suggesting that additional loci are required for spontaneous arthritis development (36). In contrast, mice carrying a functional mutation in Fas on the MRL background spontaneously develop autoimmune disease, including glomerulonephritis, lupus-like disease, vasculitis, and inflammatory arthritis (48, 49). However, the roles of monocytes and macrophages in these mice are unclear, because at the time these mice manifest the autoimmune disease phenotype, significant levelsof circulating autoantibodies and autoreactive T and B cells are observed (41). Using the serum transfer model, which requires the effector phase of inflammatory arthritis and bypasses the need for lymphocytes, we show that Fas-deficient mice exhibit a marked increase in all aspects of inflammatory arthritis, including joint swelling, inflammation, pannus formation, and cartilage and bone destruction. A recent investigation demonstrated that lpr/lpr mice on a DBA background display reduced collagen-induced arthritis (50, 51), a model that requires both adaptive and innate immune responses. However, as early as 10 wk of age, the level of autoantibody production is markedly elevated, and the number of double-negative peripheral T lymphocytes is significantly enhanced in lpr/lpr mice, which may confound the results of collagen-induced arthritis because in this model, 6- to 8-wk-old mice are observed over a 10-wk period (51). In this study we examine the effect of Fas deficiency on the innate immune response during inflammatory arthritis over a 1-wk period. However, Fas deficiency in the absence of M-CSF is insufficient to enhance serum transfer-induced arthritis (not shown). Both op/op and op/op/lpr/lpr mice display reduced serum transfer-induced arthritis compared with B6:C3H mice (not shown). Because op/op and op/op/lpr/lpr mice lack synovial macrophages (Table I), these data indicate that synovial macrophages and/or circulating monocytes may be required for serum transfer-induced arthritis.

In lpr/lpr mice, the milieu of the joint supports the idea that inflammatory monocytes are critical for disease development. After serum transfer in lpr/lpr compared with B6 mice, CCL2, which induces the recruitment of inflammatory monocytes (33), is elevated and corresponds to a 93% increase in the percentage of inflammatory monocytes in lpr/lpr compared with B6 mice. Under normal resting conditions, a 42% increase in the percentage of inflammatory monocytes is observed in lpr/lpr mice compared with B6 mice. Additionally, IL-1, which is required for serum transfer-induced arthritis (39, 52) and is mainly secreted by macrophages (53), is significantly increased in lpr/lpr compared with B6 mice. Moreover, there is a marked elevation in the number of macrophages in lpr/lpr compared with B6 mice after serum transfer. These data indicate that the inactivation of Fas during a systemic immune response, similar to inflammatory arthritis, may lead to aberrant recruitment of inflammatory monocytes, differentiation of macrophages, and secretion of proinflammatory molecules, resulting in pathogenesis. In RA, FLIP, a known activator of NF-B (54, 55) and suppressor of Fas-induced apoptosis (56), is highly expressed in monocytes and macrophages in the joint (26). Moreover, elevated levels of circulating soluble FasL (27), which block Fas-FasL interactions, are detected in RA synovial fluid. Taken together, the milieu of the RA joint resembles that in Fas-deficient mice after serum transfer. Thus, although Fas mutations have never been observed in RA, a defect in Fas signaling through increased FLIP expression or soluble FasL expression may be one of the contributors to RA pathogenesis.

	Acknowledgments

 
We thank Dr. Richard E. Stanley for his advice on our colony formation assays, and Dr. Tripathi Rajavashisth for M-CSF-deficient mice husbandry. We thank Drs. Michael Green and Cliff Bellone for their insightful comments on the manuscript.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the American Heart Association and the National Institutes of Health (AR02147 and AR050250).

² Address correspondence and reprint requests to Dr. Harris Perlman, Department of Molecular Microbiology and Immunology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104. E-mail address: perlmanh{at}slu.edu

³ Abbreviations used in this paper: FasL, Fas ligand; BFU-E, erythroid burst-forming unit; NOD, nonobese diabetic; RA, rheumatoid arthritis.

Received for publication April 26, 2004. Accepted for publication October 5, 2004.

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