Decay-Accelerating Factor Ameliorates Systemic Autoimmune Disease in MRL/lpr Mice via Both Complement-Dependent and -Independent Mechanisms

【摘要】  Decay-accelerating factor (DAF) is a glycosylphosphatidylinositol-anchored membrane protein that restricts complement activation on autologous cells. Previous studies have established a significant protective activity of DAF in the MRL/lpr murine model of human systemic lupus erythematosus. To dissect the mechanism of protection by DAF in this disease model, we evaluated the effect of C3 gene ablation on disease development in MRL/lpr-Daf-1C/C mice. We found no significant difference in lymphadenopathy, splenomegaly, or anti-chromatin autoantibody titer between complement-sufficient and complement-deficient MRL/lpr-Daf-1C/C mice. On the other hand, complement deficiency strikingly reduced the incidence and severity of dermatitis in MRL/lpr-Daf-1C/C mice. To assess the contribution of DAF expression on lymphocytes versus local tissues in suppressing dermatitis, we generated BM chimeric mice between MRL/lpr-Daf-1C/C and MRL/lpr-Daf-1+/+ mice. Compared with MRL/lpr-Daf-1C/C MRL/lpr-Daf-1C/C controls, MRL/lpr-Daf-1C/C MRL/lpr-Daf-1+/+ chimeras developed significantly attenuated dermatitis, suggesting that the protective effect of DAF in suppressing dermatitis is primarily attributable to its local expression. We conclude that DAF works as a complement regulator in the skin to protect MRL/lpr mice from skin inflammation, whereas its inhibitory role in the induction phase of MRL/lpr autoimmunity is complement-independent. Together, these results reveal multiple mechanisms of action for DAF in ameliorating systemic autoimmunity.
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Decay-accelerating factor (DAF, CD55), a membrane-anchored complement inhibitor, is widely expressed on cells both within and outside the vascular space such as blood cells, endothelial cells, and many kinds of epithelial and stroma cells.1,2 In humans, deficiency of DAF and CD59, another glycosylphosphatidylinositol (GPI)-anchored membrane inhibitor of complement, on affected blood cells of paroxysmal nocturnal hemoglobinuria patients was responsible for the increased sensitivity of their erythrocytes and platelets to autologous complement injury.3-6 Although anecdotal evidence has also linked germline DAF gene mutation to an intestinal inflammatory disorder,7-9 the role of DAF in human autoimmune and inflammatory tissue injury is primarily unknown.
Using mice deficient in Daf-1, the murine homologue of human DAF, a number of recent studies have addressed the role of DAF in mouse models of inflammatory tissue injury and immune response.10-15 DAF has been shown to be protective in complement-mediated renal ischemia reperfusion injury16 and nephrotoxic serum nephritis,14,15 as well as dextran sulfate-induced colitis.17 In a previous study, we demonstrated a significant protective activity of DAF in the MRL/lpr murine model of human systemic lupus erythematosus.18 MRL/lpr mice have the faslpr mutation, which inhibits the expression of Fas, a cell surface apoptosis receptor in the tumor necrosis factor receptor gene family.19 In addition, the MRL background is autoimmune.19 MRL/lpr mice spontaneously develop an autoimmune syndrome characterized by elevated levels of immunoglobulin (Ig), multiple autoantibodies, nephritis, and dermatitis, in association with massive lymphoproliferation.19-21 We found that compared with DAF-sufficient MRL/lpr littermate controls, female MRL/lpr-Daf-1C/C mice developed increased splenomegaly and lymphadenopathy, elevated autoantibody levels, and aggravated dermatitis with high incidence.18
Apart from Daf-1, another membrane inhibitor, complement receptor 1-related gene/protein y (Crry), also exists in the mouse.22-24 Crry overlaps with Daf-1 both in its complement-regulating activity and tissue distribution.23,24 Given this redundancy, it is not understood why Crry did not compensate for the lack of Daf-1 in the setting of systemic autoimmunity in MRL/lpr mice. Indeed, there is reason to wonder whether Daf-1 acted as a complement regulator or through other mechanisms in ameliorating autoimmunity in MRL/lpr mice. In addition to functioning as a complement regulator, DAF has also been identified as a ligand for CD97, an activation-associated lymphocyte antigen with seven transmembrane domains characteristic of type II G protein-coupled receptors.25,26 Furthermore, as a GPI-anchored protein, DAF has been shown to function as a costimulatory molecule on lymphocytes.27 Several studies have demonstrated enhanced T-cell activation by antibody cross-linking of DAF.27-29 Cross-linking of DAF also caused its clustering with nonreceptor tyrosine kinases in specialized membrane domains.29 A recent study further established that DAF acted as a co-stimulatory molecule on human CD4+ T cells via its interaction with CD97. Its activity in this regard is complement-independent and associated with increased production of the inhibitory cytokine interleukin (IL)-10.30
In the present study, we have examined the mechanism of action of DAF in MRL/lpr mice. Specifically, we have attempted to address the following questions: is exacerbated autoimmune disease in MRL/lpr-Daf-1C/C mice mediated by complement? Is the inhibitory effect of DAF on dermatitis development conferred at the local or systemic level? Does the Daf-1 gene mutation require both the MRL background and the faslpr mutation to produce the observed disease exacerbation phenotype? These questions were addressed by studying MRL/lpr-Daf-1C/C/C3C/C and MRL/MpJ-Daf-1C/C mice produced by genetic breeding and chimeric mice produced by bone marrow (BM) transfer between MRL/lpr-Daf-1+/+ and MRL/lpr-Daf-1C/C mice. Our studies revealed both complement-dependent and -independent activities of DAF in ameliorating systemic autoimmune disease in MRL/lpr mice.

【关键词】  decay-accelerating ameliorates systemic autoimmune complement-dependent -independent mechanisms

Materials and Methods

Mice

The generation of MRL/lpr-Daf-1C/C mice18 and MRL/lpr-C3C/C mice31 have been described previously. To generate MRL/lpr mice deficient in both Daf-1 and C3, MRL/lpr-Daf-1C/C mice were crossed with MRL/lpr-C3C/C mice to produce F1 generation mice. F1 mice (MRL/lpr-Daf-1+/C-C3+/C) were then backcrossed with MRL/lpr-Daf-1C/C mice to generate MRL/lpr-Daf-1C/C-C3+/Cmice as breeders. The latter (MRL/lpr-Daf-1C/C-C3+/C) were intercrossed to produce MRL/lpr-Daf-1C/CC3+/+ and MRL/lpr-Daf-1C/CC3C/C mice as littermates.

To determine whether Daf-1 deficiency in MRL/MpJ mice (without the faslpr mutation) also leads to exacerbated autoimmunity, we produced MRL/MpJ-Daf-1C/C mice as follows. First, MRL/lpr-Daf-1C/C mice were crossed with MRL/MpJ mice (Jackson Laboratory, Bar Harbor, ME) to generate MRL/MpJ-+/lpr-Daf-1+/C mice. These mice were then backcrossed with MRL/MpJ mice to produce MRL/MpJ-Daf-1+/C mice as breeders. Next, MRL/MpJ-Daf-1+/C mice were intercrossed to generate MRL/MpJ-Daf-1+/+ and MRL/MpJ-Daf-1C/C mice as littermates.

Throughout the breeding steps, mutated Daf-1, C3, and lpr genes were screened by polymerase chain reaction (PCR) of tail DNA and/or fluorescence-activated cell sorting (FACS) analysis of erythrocyte DAF expression. Genotyping of the lpr allele was performed by using the following primers: 5'-AGCACACTTGCTTCTGTTTA-3', 5'-AGCTAGGGTCAGTGAGTA-3', and 5'-CTGTCCGTTGCTCCGATGT-3'.32 The wild-type (WT) allele produced a 240-bp fragment, and Fas-deficient allele, a 445-bp fragment. The C3 allele was typed with the following primers: 5-GATCCCCAGAGCTAATG-3', 5'-TCGTCCTGCAGTTCATTCAG-3', and 5'-AGGGACCAGCCCAGGTTCAG-3'.33 The WT allele produced a 373-bp fragment, and the mutant C3 allele, a 503-bp fragment. To genotype the Daf-1 alleles, mouse erythrocytes, obtained by tail vein bleeding, were stained with a phycoerythrin (PE)-conjugated hamster anti-mouse DAF monoclonal antibody (mAb) (Pharmingen, San Diego, CA). Cells were analyzed by FACScan (Becton Dickinson, San Jose, CA).

Mice were monitored for autoimmune disease de-velopment until they were 5 months old (for MRL/lpr-Daf-1C/CC3+/+ and MRL/lpr-Daf-1C/CC3C/C mice) or 6 months old (for MRL/MpJ and MRL/MpJ-Daf-1C/C mice), at which time they were sacrificed for pathological evaluation. All mice were kept in a specific pathogen-free barrier facility. Experiments with mice were conducted by following established guidelines for animal care, and all protocols were approved by the appropriate institutional committees.

Assessment of Dermatitis

Mice were inspected monthly for the development of dermatitis, and the age at which open skin lesions developed was recorded. The dimension of the skin lesion was measured using a ruler. At the time of sacrifice, skin samples from the dorsal neck area (lesional and peri-lesional when present) were fixed in 10% buffered formalin and processed for paraffin embedding and sectioning, followed by hematoxylin and eosin (H&E) staining and histological evaluation.

Assessment of Lymphoproliferation

At the time of sacrifice, spleen and lymph nodes were weighed, meshed, and cleared of erythrocytes by ACK lysis buffer (0.15 mol/L NH4Cl, 1 mmol/L KHCO3, and 0.1 mmol/L Na2EDTA). After the spleen and lymph node cells were counted by a cell counter (Beckman Coulter, Fullerton, CA), the cell suspension (1.5 x 106 cells) was incubated with the following antibodies: Thy1.2-PE, CD4-PE, CD8-PE, B220-FITC, CD69-FITC, CD44-PE, CD19-FITC, CD21-FITC, CD80-PE, CD23-PE, and CD24-PE (Pharmingen). Flow cytometry analysis was performed by using FACScan (Becton Dickinson Immunocytometry Systems).

Enzyme-Linked Immunosorbent Assay (ELISA) for Autoantibodies and Their Isotypes

Serum was collected by tail vein bleeding at monthly intervals starting at 3 months until sacrifice. Samples were frozen at C20??C until use, when they were diluted to predetermined concentrations for ELISA. Quantification was achieved with either a standard curve (total IgM, IgG, IgG2a) or a positive serum sample from 5-month-old MRL/lpr mice as an internal standard (Ag-specific autoantibodies) for interplate comparison. Sandwich ELISA was used for determining the titers of antibodies (Abs). For total serum IgM, IgG, and IgG2a, plates were coated with the appropriate capture Ab. Diluted sera, as well as serially diluted IgM, IgG, and IgG2a standards, were added to the plates and allowed to bind overnight. The plate was incubated with biotin-conjugated anti-mouse IgM, IgG, and IgG2a Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 hours at 4??C. Avidin-alkaline phosphatase (Sigma, St. Louis, MO) was added, followed by alkaline phosphatase yellow liquid substrate (Sigma) for color development. OD405 was measured on a microplate reader (Molecular Devices, Sunnyvale, CA).

Assessment of Nephritis

Urine was collected in metabolic cages at monthly intervals starting at 3 months until sacrifice. Urinary albumin concentration was measured by a mouse albumin ELISA kit (Bethyl Laboratories, Montgomery, TX) and normalized to urinary creatinine measured by a colorimetric microplate assay kit (Oxford Biomedical Research, Oxford, MI). At the time of sacrifice, one kidney was fixed in 10% buffered formalin and processed for paraffin embedding and sectioning, followed by H&E and periodic acid-Schiff staining and histological evaluation. A blinded observer (K.Y.) evaluated and scored independently the severity of glomerular injury and inflammatory infiltrates (periglomerular, perivascular, and medulla, scored separately) by light microscopy. Glomerular injury was graded as follows: 0 = normal, 1 = glomerular changes (hypercellularity, crescents, or sclerotic glomeruli in 0 to 25% of total glomeruli), 2 = 25 to 50% glomeruli, 3 = 50 to 75% glomeruli, and 4 = >90% glomeruli. Infiltrative disease was graded as follows: 0 = no infiltrates visible, 1 = few infiltrates in some fields, 2 = moderate infiltrates easily found in most fields, 3 = moderate infiltrates in all fields, and 4 = severe infiltrates with loss of normal surrounding histology.

BM Transfer

BM cells were flushed out from the femurs and tibias of MRL/lpr/Daf-1C/C mice in cell culture medium (RPMI 1640, 15 mmol/L HEPES). After meshing and clearing of erythrocytes by treating with ACK buffer, the isolated BM cells were processed for depletion of mature T and B cells. To do this, cells were first incubated with anti-CD4, -CD8, -CD19, and -CD90 antibody-conjugated magnetic microbeads (Miltenyi Biotec, Auburn, CA) and then processed on a fully automated Clinimacs device (Miltenyi) equipped with TS separation columns using the program DepleteS by following the manufacturer??s instructions. Recipient mice, MRL/lpr-Daf-1+/+, and MRL/lpr-Daf-1C/C mice, were lethally irradiated with two 525-rad doses spaced 3 hours apart (totally 1050 rads). T/B-depleted BM cells were injected into recipient mice through the tail vein (1 x 107 cells per mouse).

Repopulation of the immune system was monitored by flow cytometric analysis of the blood cells using PE-conjugated hamster anti-mouse DAF. In the chimeric mice, 90% of the T cells and more than 95% of the B cells and myeloid cells were derived from donor BM. Mice were monitored for 5 months after BM transfer. Similar procedures as described above were used to assess autoimmune disease phenotype in the chimera mice.

Statistical Analysis

Data were analyzed by Student??s t-test or Mann-Whitney test as specified in the text.

Results

In previous studies, we generated MRL/lpr-C3C/C and MRL/lpr-Daf-1C/C mice by backcrossing.18,31 There was no apparent disease modification by C3 deficiency except increased glomerular IgG deposition.31 In contrast, Daf-1 deficiency significantly exacerbated autoimmune disease in MRL/lpr mice, particularly in females.18 Compared with Daf-1-sufficient littermate controls, MRL/lpr-Daf-1C/C mice had increased lymphadenopathy and splenomegaly and elevated anti-chromatin autoantibody levels and, most strikingly, developed severe dermatitis with high incidence. To determine whether this phenotype was caused by the lack of Daf-1 as a complement regulator, we deleted the C3 gene from the MRL/lpr-Daf-1C/C mouse by crossing it with the MRL/lpr-C3C/C mouse.

By genotyping, we identified a total of 56 MRL/lpr-Daf-1C/CC3+/+ and 31 MRL/lpr-Daf-1C/CC3C/C female mice. At monthly intervals, they were bled and their skin lesions evaluated. Figure 1, ACD , shows that C3 deficiency strikingly ameliorated dermatitis development in MRL/lpr-Daf-1C/C mice. The percentage of mice with visible open skin lesions in the MRL/lpr-Daf-1C/C cohort was 25, 79, and 98% at 3, 4, and 5 months, respectively (Figure 1E) . The corresponding percentages, at 10, 29, and 49%, were significantly lower in the MRL/lpr-Daf-1C/CC3C/C group (Figure 1E) . Furthermore, in those MRL/lpr-Daf-1C/CC3C/C mice that did develop dermatitis, the lesions were smaller and milder (Figure 1, A, C, and F) . For example, the average open lesion size in MRL/lpr-Daf-1C/C and MRL/lpr-Daf-1C/CC3C/C mice at 5 months of age were 3.44 ?? 0.33 cm2 (n = 55) and 1.46 ?? 0.45 cm2 (n = 14), respectively. On the other hand, we detected no substantive difference in the histology of fully developed lesions between the two strains of mice (Figure 1, B and D) . In each strain, the lesions were characterized by acanthosis, infiltration of inflammatory cells, fibrosis, and loss of epidermal integrity (Figure 1, B and D) .

Figure 1. C3 deficiency ameliorates dermatitis in MRL/lpr-Daf-1C/C mice. A: Representative appearance of severe dermatitis in 5-month-old MRL/lpr-Daf-1C/CC3+/+ mice. B: Representative appearance of 5-month-old MRL/lpr-Daf-1C/CC3C/C mice. Most of them had no or very mild skin disease. Only a few mice developed severe skin lesions. C and D: There was no qualitative difference in lesion histology between MRL/lpr-Daf-1C/CC3+/+ mice (B) and the most severe cases of MRL/lpr-Daf-1C/CC3C/C mice (D). Both are characterized by skin thickening, mostly attributable to acanthosis of the epidermis, and the presence of scattered inflammatory cells as well as some dermal fibrosis. E: Percentage of mice with visible open skin lesions at 3, 4, and 5 months for MRL/lpr-Daf-1C/CC3+/+ and MRL/lpr-Daf-1C/CC3C/C mice. F: Scatter plot of open lesion size in dermatitis-positive MRL/lpr-Daf-1C/CC3+/+ and MRL/lpr-Daf-1C/CC3C/C mice. KO, MRL/lpr-Daf-1C/CC3+/+ mice; d-KO, MRL/lpr-Daf-1C/CC3C/C mice. P value refers to Student??s t-test. Histology shown was by H&E staining. Original magnifications, x40 (B, D).

In contrast to the clear amelioration of dermatitis, C3 deficiency did not significantly affect the degree of lymphadenopathy and splenomegaly caused by DAF deficiency. The spleen and lymph node weights (g) for a cohort of MRL/lpr-Daf-1+/+ mice (n = 11) maintained in our colony were 0.182 ?? 0.009 and 1.171 ?? 0.069, respectively. In comparison, spleen and lymph node weights (g) for the MRL/lpr-Daf-1C/C and MRL/lpr-Daf-1C/CC3C/C mice were 0.264 ?? 0.001 versus 0.234 ?? 0.014 and 1.965 ?? 0.079 versus 2.178 ?? 0.147, respectively (P < 0.05 between DAF-sufficient and DAF-deficient groups; no significant difference between C3-sufficient and C3-deficient groups, Student??s t-test). Likewise, C3 deficiency did not significantly influence the levels of serum anti-chromatin and anti-ds-DNA autoantibody titers in MRL/lpr mice (Figure 2, A and B) . There was also no significant difference in the number and composition of cell subpopulations (CD4+, CD8+, B200+, and so forth) and activation markers (CD69, CD44, CD80, CD23, CD24) (data not shown). Unexpectedly, C3 deficiency increased serum total IgG, IgG2a, and IgM levels, although the elevation in total serum IgG did not reach statistically significance (Figure 2, CCE) . The development of nephritis in the two groups of mice showed no significant differences when assessed by morphological scoring of glomerular inflammation and injury (5.6 ?? 0.4 versus 6.5 ?? 0.7 for MRL/lpr-Daf-1C/C and MRL/lpr-Daf-1C/CC3C/C mice). On the other hand, urinary albumin excretion (µg/mg creatinine) in MRL/lpr-Daf-1C/CC3C/C mice was significantly higher than in MRL/lpr-Daf-1C/C mice (1097 ?? 999.5 versus 4438 ?? 2889, P = 0.0225, Mann-Whitney test). These results suggested a dissociation in mechanisms between exacerbated local inflammation in the skin and systemic autoimmunity in MRL/lpr-Daf-1C/C mice.

Figure 2. Effect of C3 deficiency on serum antibody levels. C3 deficiency had no impact on serum anti-chromatin (A) and anti-dsDNA (B) autoantibody titers. On the other hand, C3 deficiency increased serum total IgG (C), IgG2a (D), and IgM (E) levels, although only the elevations in IgG2a and IgM were statistically significant. KO, MRL/lpr-Daf-1C/CC3+/+; d-KO, MRL/lpr-Daf-1C/CC3C/C. Data shown in BCE are for 5 months. P values refer to Student??s t-test.

DAF is highly expressed on lymphocytes and other BM-derived blood cells, as well as on virtually all peripheral tissues including the skin.1,34,35 Although the C3 ablation experiment implied that DAF acted as a complement regulator in suppressing dermatitis development in MRL/lpr mice, it did not reveal where DAF exerted its role in such a capacity. To address this question, we generated BM chimera mice between MRL/lpr-Daf-1C/C and MRL/lpr mice. Mature T- and B-lymphocyte-depleted BM cells from MRL/lpr-Daf-1C/C mice were harvested and adoptively transferred via tail vein injection into either MRL/lpr-Daf-1C/C (group I, control group) or MRL/lpr mice (group II, experimental group). In the latter group of mice, all tissues except lymphocytes and other BM-derived cells expressed DAF. Figure 3A shows that 2 months after BM transfer, the donor mouse BM cells successfully reconstituted the hematopoietic system of the recipient mice, and greater than 95% of the circulating erythrocytes in the recipient mice had the donor mouse genotype. We found that both groups of chimera mice were susceptible to dermatitis development, with the incidence of visible skin lesions in group II mice (MRL/lpr-Daf-1C/C MRL/lpr chimera) being slightly reduced compared with group I mice (MRL/lpr-Daf-1C/C MRL/lpr-Daf-1C/C) (Figure 3D) . However, the lesion size was strikingly smaller in group II mice than in group I mice (Figure 3, B, C, and E) . We observed a trend of increased spleen weight, elevated serum anti-chromatin autoantibody titer, and urinary albumin excretion in group I mice (Figure 4, A, C, and D) . However, none of the differences reached statistical significance.

Figure 3. DAF ameliorates dermatitis mainly by acting locally in peripheral tissues. A: FACS analysis of erythrocyte DAF expression in chimeric mice 2 months after BM transfer. KO, MRL/lpr-Daf-1C/C; WT, MRL/lpr-Daf-1+/+. The result demonstrates that donor mouse BMs have successfully reconstituted the hematopoietic system of the recipient mice. The WT KO chimera was used as a control for the efficiency of BM reconstitution. B and C: Representative pictures at 5 months after BM transfer showing that MRL/lpr-Daf-1C/C MRL/lpr-Daf-1+/+ chimeras (C) developed significantly attenuated dermatitis compared with MRL/lpr-Daf-1C/C MRL/lpr-Daf-1C/C chimeras (B). D and E: Although the incidence of dermatitis (D) was only slightly lower in MRL/lpr-Daf-1C/C MRL/lpr-Daf-1+/+ chimeras (group II, n = 6) than in MRL/lpr-Daf-1C/C MRL/lpr-Daf-1C/C chimeras (group I, n = 7), the dermatitis was significantly less severe in group II mice as measured by the size of open skin lesions (E). P value refers to Student??s t-test.

Figure 4. Effect of peripheral DAF expression on lymphoproliferation, anti-chromatin autoantibody production, and proteinuria in MRL/lpr mice. Spleen (A) and lymph node (B) weights, anti-chromatin autoantibody titer (C), and proteinuria (D) were not significantly different between MRL/lpr-Daf-1C/C MRL/lpr-Daf-1C/C chimeras (group I, n = 7) and MRL/lpr-Daf-1C/C MRL/lpr-Daf-1+/+ chimeras (group II, n = 6). There seemed to be a trend of increased spleen weight, higher anti-chromatin titer, and increased proteinuria in group I mice, but the difference did not reach statistical significance (Student??s t-test). Data in A, B, and D are 5 months after BM transfer.

Because no obvious autoimmune phenotype was seen with Daf-1C/C mice on other genetic backgrounds at a comparable age,36 the MRL background and/or the faslpr mutation must have acted as critical modifiers to reveal the proautoimmune consequences of Daf-1 gene deficiency. To determine whether daf-1 gene deficiency in combination with the MRL background alone, without the faslpr mutation, was sufficient to recapitulate the exacerbated autoimmune phenotype of MRL/lpr-Daf-1C/C mice, we produced and studied 21 female MRL/MpJ-Daf-1+/+ and 23 female MRL/MpJ-Daf-1C/C mice as littermates. Figure 5, A and B , shows that on the MRL/MpJ background, Daf-1 deficiency also increased the incidence and severity of dermatitis. However, only 30% of MRL/MpJ-Daf-1C/C mice developed open skin lesions as opposed to nearly 100% in MRL/lpr-Daf-1C/C mice (Figure 1) . Both the lymph node and spleen weights were also higher in MRL/MpJ-Daf-1C/C mice, although the difference in spleen weight was not statistically significant (Figure 5, C and D) . Thus, the faslpr mutation was not necessary for exposing the proautoimmune consequence of Daf-1 gene deficiency, but it clearly amplified the autoimmune phenotypes of MRL/MpJ-Daf-1-deficient mice.

Figure 5. Effect of Daf-1 gene deficiency on dermatitis development and lymphoproliferation in MRL/MpJ mice. A: Dermatitis incidence was higher in MRL/MpJ-Daf-1C/C mice (KO, n = 23, 5 of 23, 7 of 23, 7 of 23, and 7 of 23 for 3, 4, 5, and 6 months, respectively) than in MRL/MpJ mice (WT, n = 21, 0 of 21, 0 of 21, 3 of 21, and 3 of 21 for 3, 4, 5, and 6 months, respectively). B: In those mice that developed skin disease, the average size of open skin lesions in MRL/MpJ-Daf-1C/C mice was bigger than in MRL/MpJ mice at 5 and 6 months, although the differences did not reach statistical significance. C and D: MRL/MpJ-Daf-1C/C mice (KO, n = 23) also had increased spleen (C) and lymph node (D) weights than MRL/MpJ mice (WT, n = 21) (P = 0.1656 for spleen, P = 0.0012 for lymph node; Mann-Whitney test).

Discussion

We previously demonstrated a significant anti-autoimmune activity of DAF in MRL/lpr mice.18 Although an apparent explanation for the observed effect of DAF in MRL/lpr mice was its complement regulatory activity, DAF has additional physiological functions that may be relevant.26-30 In the present study, we have combined the C3 deletion with Daf-1 deletion in MRL/lpr mice to evaluate the mechanisms of action of DAF in MRL/lpr mice. By intercrossing MRL/lpr-Daf-1C/C/C3+/C mice, we obtained 56 female MRL/lpr-Daf-1C/C/C3+/+ and 31 MRL/lpr-Daf-1C/C/C3C/C mice. The apparent deviation from Mendelian inheritance was notable but not understood. We observed no significant mortality in the MRL/lpr-Daf-1C/C/C3C/C group by the experimental end point (5 months). However, the possibility cannot be excluded that MRL/lpr-Daf-1C/C/C3C/C mice had a higher mortality postnatally (ie, before genotyping at 4 weeks old), even though these mice were kept in a specific pathogen-free facility and a similar phenotype has not been observed in C57/BL6 C3C/C mice.33 It should also be noted that mild renal disease and lack of significant dermatitis and mortality at 5 months of age were characteristics of the MRL/lpr mice in our colony. These features contrasted with those observed in other MRL/lpr mouse colonies37,38 and presumably reflected genetic drifting in our MRL/lpr mouse line after prolonged in-house breeding.

Our results suggested that the aggravated dermatitis in MRL/lpr-Daf-1C/C mice was mainly caused by the lack of DAF as a complement inhibitor because it was ameliorated in DAF/C3 double-deficient mice. Nevertheless, the development of skin injury in some MRL/lpr-Daf-1C/C-C3C/C mice was still a notable observation, as it contrasted with MRL/lpr mice, which rarely develop comparable dermatitis at that age. This observation implied the existence of a secondary, complement-independent effect of DAF in ameliorating skin inflammation. Such an effect may be exerted at the systemic level on T/B cells, in which the influence of DAF on lymphoproliferation was shown to be independent of complement (see discussion below).

We previously showed that DAF deficiency in MRL/lpr mice exacerbated lymphadenopathy and splenomegaly and elevated serum anti-chromatin titers.18 To determine the contribution of complement to these phenotypes, we examined the spleen and lymph nodes and measured anti-chromatin titers in MRL/lpr-Daf-1C/C and MRL/lpr-Daf-1C/C-C3C/C mice. Because complement plays a role in B-cell function39 and is important for immune complex processing,40 we also measured several other antibodies to determine the potential effect of C3 deletion in the context of DAF deficiency. We found that C3 deficiency had no impact on the degree of lymphadenopathy and splenomegaly or on anti-chromatin and anti-ds-DNA autoantibody titers of MRL/lpr-Daf-1C/C mice. This implied that DAF worked to suppress T/B cell autoimmunity in MRL/lpr mice via a pathway that is independent of complement. This result contrasted with findings of recent studies of C57BL/6-Daf-1C/C mice, wherein increased T-cell recall responses were observed in Daf-1C/C mice and shown to be primarily complement-dependent in models of active immunization, using ovalbumin or myelin oligodendrocyte glycoprotein peptide as model antigens.11 Thus, DAF could inhibit adaptive immunity via both complement-dependent and -independent mechanisms depending on the context. The complement-independent mechanism by which DAF inhibits lymphoproliferation and autoantibody production in MRL/lpr mice remains unknown. Antibody cross-linking of DAF during anti-CD3-inducd human CD4+ T-cell activation increased IL-10 production.30 In the active immunization model, C57BL/6-Daf-1C/C mouse T cells secreted significantly less IL-10 in response to antigen restimulation, and this defect in IL-10 production was not rescued by C3 deficiency.11 Whether diminished production of IL-10, an inhibitory cytokine, was involved in the complement-independent exacerbation of lymphoproliferation and autoantibody production in MRL/lpr-Daf-1C/C mice remains to be determined. Another possibility that cannot be discounted at this time is the influence of 129J mouse strain-derived genes. The Daf-1 gene is localized to the distal region of chromosome 1, a region where several lupus susceptibility genes are known to reside.41-43

C3 deficiency also did not influence kidney disease development in MRL/lpr-Daf-1C/C mice as assessed by pathology scores. On the other hand, MRL/lpr-Daf-1C/C-C3C/C mice developed more severe proteinuria than MRL/lpr-Daf-1C/C mice. These results were not unexpected because neither Daf-1 gene deficiency nor C3 deficiency was previously shown to affect kidney pathology in MRL/lpr mice, and C3 deficiency likewise worsened proteinuria in these animals, possibly reflecting a role of C3 in immune complex solubilization in the kidney.31 Surprisingly, we found that C3 deficiency significantly increased serum total IgM and IgG2a levels in MRL/lpr-Daf-1C/C mice. It is possible that this was a reflection of the failure by the C3-deficient MRL/lpr-Daf-1C/C mice to clear persistent infections, although the animals were housed in a specific pathogen-free facility. On the other hand, elevated IgG2a may have reflected impaired immune complex handling in the MRL/lpr-Daf-1C/C-C3C/C mice. Whatever the underlying mechanism may be, it seems to be related to the Daf-1C/C background because elevated serum IgM or IgG2a was not observed in either MRL/lpr-C3C/C31 or C57BL/6-C3C/C mice.44

BM transplantation experiments showed that MRL/lpr-Daf-1C/C MRL/lpr-Daf-1+/+ chimeras developed significantly attenuated dermatitis compared with MRL/lpr-Daf-1C/C MRL/lpr-Daf-1C/C chimeras. Our interpretation of this result is that DAF mainly acted as a complement inhibitor in peripheral tissues (most likely in the skin) to suppress skin inflammation. This conclusion was consistent with our previous demonstration of C3 deposition, often used as a surrogate to track tissue complement activation, along the dermal-epidermal junction in prelesional MRL/lpr-Daf-1C/C mouse skins.18 Nevertheless, the fact that MRL/lpr-Daf-1C/C MRL/lpr-Daf-1+/+ chimeras, unlike MRL/lpr mice, did develop dermatitis, implied that exacerbated systemic autoimmunity at the T/B lymphocyte level also contributed to dermatitis development in MRL/lpr-Daf-1C/C mice. It is notable that although not statistically significant, there seemed to be a trend for increased spleen weight and serum anti-chromatin autoantibody titer and elevated proteinuria in MRL/lpr-Daf-1C/C MRL/lpr-Daf-1C/C chimeras compared with MRL/lpr-Daf-1C/C MRL/lpr-Daf-1+/+ chimeras, suggesting that peripheral DAF expression may also impact these parameters.

Breeding experiment with MRL/MpJ mice showed that faslpr mutation was not necessary to expose the autoimmune consequence of Daf-1 gene deficiency (Figure 5) . This finding suggests that DAF may be a significant disease-modifying gene in other animal and human autoimmune disease settings where the faslpr mutation does not occur. Nevertheless, the faslpr mutation clearly had an amplifying effect on the phenotype of Daf-1C/C mice, because only 30% of female MRL/MpJ-Daf-1C/C mice developed dermatitis as compared with close to 100% observed in MRL/lpr-Daf-1C/C mice (Figure 1) .

In summary, we have revealed multiple mechanisms of action for DAF in ameliorating systemic autoimmunity. We conclude that DAF works as a complement regulator locally to protect MRL/lpr mice from skin inflammation, whereas its inhibitory role in the induction phase of MRL/lpr autoimmunity is complement-independent. The proautoimmune consequence of DAF dysfunction does not require a faslpr mutation and may manifest in broad settings of systemic autoimmunity in animals and human patients. This further characterizes the complex role of complement and its regulators in the development of both systemic and organ-specific autoimmunity.

Acknowledgements

We thank Dr. Victoria Werth (University of Pennsylvania) for skin pathology examination and useful discussions.

【参考文献】
  Medof ME, Walter EI, Rutgers JL, Knowles DM, Nussenzweig V: Identification of the complement decay-accelerating factor (DAF) on epithelium and glandular cells and in body fluids. J Exp Med 1987, 165:848-864

Miwa T, Sun X, Ohta R, Okada N, Harris CL, Morgan BP, Song WC: Characterization of glycosylphosphatidylinositol-anchored decay accelerating factor (GPI-DAF) and transmembrane DAF gene expression in wild-type and GPI-DAF gene knockout mice using polyclonal and monoclonal antibodies with dual or single specificity. Immunology 2001, 104:207-214

Medof ME, Kinoshita T, Silber R, Nussenzweig V: Amelioration of lytic abnormalities of paroxysmal nocturnal hemoglobinuria with decay-accelerating factor. Proc Natl Acad Sci USA 1985, 82:2980-2984

Nicholson-Weller A, March JP, Rosenfeld SI, Austen KF: Affected erythrocytes of patients with paroxysmal nocturnal hemoglobinuria are deficient in the complement regulatory protein, decay accelerating factor. Proc Natl Acad Sci USA 1983, 80:5066-5070

Pangburn MK, Schreiber RD, Muller-Eberhard HJ: Deficiency of an erythrocyte membrane protein with complement regulatory activity in paroxysmal nocturnal hemoglobinuria. Proc Natl Acad Sci USA 1983, 80:5430-5434

Rosse WF, Parker CJ: Paroxysmal nocturnal haemoglobinuria. Clin Haematol 1985, 14:105-125

Telen MJ, Hall SE, Green AM, Moulds JJ, Rosse WF: Identification of human erythrocyte blood group antigens on decay-accelerating factor (DAF) and an erythrocyte phenotype negative for DAF. J Exp Med 1988, 167:1993-1998

Lublin DM, Mallinson G, Poole J, Reid ME, Thompson ES, Ferdman BR, Telen MJ, Anstee DJ, Tanner MJ: Molecular basis of reduced or absent expression of decay-accelerating factor in Cromer blood group phenotypes. Blood 1994, 84:1276-1282

Wang L, Uchikawa M, Tsuneyama H, Tokunaga K, Tadokoro K, Juji T: Molecular cloning and characterization of decay-accelerating factor deficiency in Cromer blood group Inab phenotype. Blood 1998, 91:680-684

Kim DD, Song WC: Membrane complement regulatory proteins. Clin Immunol 2006, 118:127-136

Liu J, Miwa T, Hilliard B, Chen Y, Lambris JD, Wells AD, Song WC: The complement inhibitory protein DAF (CD55) suppresses T cell immunity in vivo. J Exp Med 2005, 201:567-577

Heeger PS, Lalli PN, Lin F, Valujskikh A, Liu J, Muqim N, Xu Y, Medof ME: Decay-accelerating factor modulates induction of T cell immunity. J Exp Med 2005, 201:1523-1530

Lin F, Kaminski HJ, Conti-Fine BM, Wang W, Richmonds C, Medof ME: Markedly enhanced susceptibility to experimental autoimmune myasthenia gravis in the absence of decay-accelerating factor protection. J Clin Invest 2002, 110:1269-1274

Sogabe H, Nangaku M, Ishibashi Y, Wada T, Fujita T, Sun X, Miwa T, Madaio MP, Song WC: Increased susceptibility of decay-accelerating factor deficient mice to anti-glomerular basement membrane glomerulonephritis. J Immunol 2001, 167:2791-2797

Lin F, Emancipator SN, Salant DJ, Medof ME: Decay-accelerating factor confers protection against complement-mediated podocyte injury in acute nephrotoxic nephritis. Lab Invest 2002, 82:563-569

Yamada K, Miwa T, Liu J, Nangaku M, Song WC: Critical protection from renal ischemia reperfusion injury by CD55 and CD59. J Immunol 2004, 172:3869-3875

Lin F, Spencer D, Hatala DA, Levine AD, Medof ME: Decay-accelerating factor deficiency increases susceptibility to dextran sulfate sodium-induced colitis: role for complement in inflammatory bowel disease. J Immunol 2004, 172:3836-3841

Miwa T, Maldonado MA, Zhou L, Sun X, Luo HY, Cai D, Werth VP, Madaio MP, Eisenberg RA, Song WC: Deletion of decay-accelerating factor (CD55) exacerbates autoimmune disease development in MRL/lpr mice. Am J Pathol 2002, 161:1077-1086

Cohen PL, Eisenberg RA: Lpr and gld: single gene models of systemic autoimmunity and lymphoproliferative disease. Annu Rev Immunol 1991, 9:243-269

Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA, Nagata S: Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 1992, 356:314-317

Theofilopoulos AN, Dixon FJ: Murine models of systemic lupus erythematosus. Adv Immunol 1985, 37:269-390

Molina H, Wong W, Kinoshita T, Brenner C, Foley S, Holers VM: Distinct receptor and regulatory properties of recombinant mouse complement receptor 1 (CR1) and Crry, the two genetic homologues of human CR1. J Exp Med 1992, 175:121-129

Li B, Sallee C, Dehoff M, Foley S, Molina H, Holers VM: Mouse Crry/p65. Characterization of monoclonal antibodies and the tissue distribution of a functional homologue of human MCP and DAF. J Immunol 1993, 151:4295-4305

Holers VM, Kinoshita T, Molina H: The evolution of mouse and human complement C3-binding proteins: divergence of form but conservation of function. Immunol Today 1992, 13:231-236

Gray JX, Haino M, Roth MJ, Maguire JE, Jensen PN, Yarme A, Stetler-Stevenson MA, Siebenlist U, Kelly K: CD97 is a processed, seven-transmembrane, heterodimeric receptor associated with inflammation. J Immunol 1996, 157:5438-5447

Hamann J, Vogel B, van Schijndel GM, van Lier RA: The seven-span transmembrane receptor CD97 has a cellular ligand (CD55, DAF). J Exp Med 1996, 184:1185-1189

Tosello AC, Mary F, Amiot M, Bernard A, Mary D: Activation of T cells via CD55: recruitment of early components of the CD3-TCR pathway is required for IL-2 secretion. J Inflamm 1998, 48:13-27

Stefanov? I, Horejsi V, Ansotegui IJ, Knapp W, Stockinger H: GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science 1991, 254:1016-1019

Shenoy-Scaria AM, Kwong J, Fujita T, Olszowy MW, Shaw AS, Lublin DM: Signal transduction through decay-accelerating factor. Interaction of glycosyl-phosphatidylinositol anchor and protein tyrosine kinases p56lck and p59fyn 1. J Immunol 1992, 149:3535-3541

Capasso M, Durrant LG, Stacey M, Gordon S, Ramage J, Spendlove I: Costimulation via CD55 on human CD4+ T cells mediated by CD97. J Immunol 2006, 177:1070-1077

Sekine H, Reilly CM, Molano ID, Garnier G, Circolo A, Ruiz P, Holers VM, Boackle SA, Gilkeson GS: Complement component C3 is not required for full expression of immune complex glomerulonephritis in MRL/lpr mice. J Immunol 2001, 166:6444-6451

Maldonado MA, Eisenberg RA, Roper E, Cohen PL, Kotzin BL: Greatly reduced lymphoproliferation in lpr mice lacking major histocompatibility complex class I. J Exp Med 1995, 181:641-648

Circolo A, Garnier G, Fukuda W, Wang X, Hidvegi T, Szalai AJ, Briles DE, Volanakis JE, Wetsel RA, Colten HR: Genetic disruption of the murine complement C3 promoter region generates deficient mice with extrahepatic expression of C3 mRNA. Immunopharmacology 1999, 42:135-149

Lublin DM, Atkinson JP: Decay-accelerating factor: biochemistry, molecular biology, and function. Annu Rev Immunol 1989, 7:35-58

Werth VP, Ivanov IE, Nussenzweig V: Decay-accelerating factor in human skin is associated with elastic fibers. J Invest Dermatol 1988, 91:511-516

Sun X, Funk CD, Deng C, Sahu A, Lambris JD, Song WC: Role of decay-accelerating factor in regulating complement activation on the erythrocyte surface as revealed by gene targeting. Proc Natl Acad Sci USA 1999, 96:628-633

Reilly CM, Mishra N, Miller JM, Joshi D, Ruiz P, Richon VM, Marks PA, Gilkeson GS: Modulation of renal disease in MRL/lpr mice by suberoylanilide hydroxamic acid. J Immunol 2004, 173:4171-4178

Bao L, Haas M, Boackle SA, Kraus DM, Cunningham PN, Park P, Alexander JJ, Anderson RK, Culhane K, Holers VM, Quigg RJ: Transgenic expression of a soluble complement inhibitor protects against renal disease and promotes survival in MRL/lpr mice. J Immunol 2002, 168:3601-3607

Carroll MC: The complement system in regulation of adaptive immunity. Nat Immunol 2004, 5:981-986

Walport MJ: Complement. Second of two parts. N Engl J Med 2001, 344:1140-1144

Wakui M, Morel L, Butfiloski EJ, Kim C, Sobel ES: Genetic dissection of systemic lupus erythematosus pathogenesis: partial functional complementation between Sle1 and Sle3/5 demonstrates requirement for intracellular coexpression for full phenotypic expression of lupus. J Immunol 2005, 175:1337-1345

Chen Y, Perry D, Boackle SA, Sobel ES, Molina H, Croker BP, Morel L: Several genes contribute to the production of autoreactive B and T cells in the murine lupus susceptibility locus Sle1c. J Immunol 2005, 175:1080-1089

Xu Z, Duan B, Croker BP, Wakeland EK, Morel L: Genetic dissection of the murine lupus susceptibility locus Sle2: contributions to increased peritoneal B-1a cells and lupus nephritis map to different loci. J Immunol 2005, 175:936-943

Wessels MR, Butko P, Ma M, Warren HB, Lage AL, Carroll MC: Studies of group B streptococcal infection in mice deficient in complement component C3 or C4 demonstrate an essential role for complement in both innate and acquired immunity. Proc Natl Acad Sci USA 1995, 92:11490-11494

作者单位：From the Institute for Translational Medicine and Therapeutics and Department of Pharmacology,* and the Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; and the Department of Medicine, Medical University of South Carolina, Charleston, South Carolina