The role of complement activation in the brains of MRL/lpr lupus mice was determined using the potent C3 convertase inhibitor, CR1-related y (Crry), administered both as an overexpressing Crry transgene and as Crry-Ig. Prominent deposition of complement proteins C3 and C9 in brains of MRL/lpr mice was indicative of complement activation and was significantly reduced by Crry. Apoptosis was determined in brain using different independent measures of apoptosis, including TUNEL staining, DNA laddering, and caspase-3 activity, all of which were markedly increased in lupus mice and could be blocked by inhibiting complement with Crry. Complement activation releases inflammatory mediators that can induce apoptosis. The mRNA for potentially proinflammatory proteins such as TNFR1, inducible NO synthase, and ICAM-1 were up-regulated in brains of lupus mice. Crry prevented the increased expression of these inflammatory molecules, indicating that the changes were complement dependent. Furthermore, microarray analysis revealed complement-dependent up-regulation of glutamate receptor (AMPA-GluR) expression in lupus brains, which was also validated for AMPA-GluR1 mRNA and protein. Our results clearly demonstrate that apoptosis is a prominent feature in lupus brains. Complement activation products either directly and/or indirectly through TNFR1, ICAM-1, inducible NO synthase, and AMPA-GluR, all of which were altered in MRL/lpr mouse brains, have the potential to induce such apoptosis. These findings present the exciting possibility that complement inhibition is a therapeutic option for lupus cerebritis.

Human systemic lupus erythematosus (SLE)3 is a multiorgan inflammatory autoimmune disease characterized by the loss of tolerance to self Ags and the production of autoantibodies. The CNS is a target organ for this disease, with potentially debilitating neuropsychiatric consequences. Several different pathogenic mechanisms appear to be involved in CNS lupus. These include B cell-produced autoantibodies that form immune complexes and are deposited in the CNS, cytokine-induced brain inflammation, microthrombosis, vasculopathy, and aberrant MHC class II Ag expression with T cell-mediated disease (1, 2). These processes are not mutually exclusive, and the actual mechanisms of injury remain poorly defined.

The complement system is a powerful arm of innate immunity that protects tissues against invading pathogens. However, complement has also been implicated as an etiologic factor for the inflammation and degeneration that occur in autoimmune diseases of the CNS (3). Activation of the complement cascade leads to cleavage of C3 and C5 with generation of anaphylatoxins C3a and C5a, both of which attract neutrophils and monocytes to the site of activation (4). In addition to anaphylatoxin receptors on inflammatory cells, there is increasing evidence of a role for these receptors on fixed tissue cells. C3b binds to immune complexes, whereas C5b initiates the assembly of the C5b-9 membrane attack complex (MAC) that can result in cellular death or activation after membrane insertion (5, 6). To prevent injury of host tissue, regulatory proteins strictly control the spontaneous and immune complex-induced activation of the complement system (7). The rodent complement protein, CR1-related y (Crry), is a potent inhibitor of the pivotal C3 convertase of the complement cascade (8). Similarly to human CR1, Crry demonstrates decay-accelerating activity in both the classical and alternative pathways of complement as well as cofactor activity for factor I-mediated cleavage of C3b (9).

Support that complement can contribute to CNS pathology in human SLE includes the increased concentration of the central complement proteins, C3 and C4, in the cerebrospinal fluid (CSF) of SLE patients (10, 11). In addition, systemic complement activation, as reflected by increased serum levels of the anaphylatoxins, C3a and C5a, has been shown to correlate with CNS disease (12). Once complement activation occurs, it can lead to increased migration of cells toward the site of inflammation, alter the release of cytokines, and cause cell necrosis or apoptosis (3, 13, 14). Apoptosis has an important role in the development, maintenance of constant tissue mass, and expression of disease processes. Although apoptosis was observed in the brains of lupus mice by TUNEL staining, the underlying mechanism remains to be defined (15).

Complement proteins have the potential to activate intracellular signaling pathways such as the TNF cascade, which can lead to activation of downstream mediators, thereby amplifying its inflammation. Activation of C3 via the alternative pathway can induce TNF release (16), which is increased in the circulation of SLE patients (17, 18). The two main receptors for TNF are TNFR1 and TNFR2. The main biological responses, such as gene induction, cytokine increase, and cell death, are mediated by TNFR1 (19). One of the best-studied effects of TNF in different brain cells is up-regulated expression of ICAM-1 (20), which is an important mediator in leukocyte transmigration through the endothelium and their accumulation at the site of injury and can also promote apoptosis (21, 22, 23). TNF also induces increased expression of inducible NO synthase (iNOS) through ERK and NF-κB in glial cells, which, in turn, produces the signaling molecule, NO (24). Increased iNOS activity can ultimately lead to apoptosis (25). Furthermore, both the complement cascade and TNF can modulate the calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-glutamate (AMPA-Glu) R1, leading to excitotoxicity (26, 27, 28). An influx of Ca2+ through AMPA-GluR1 can also cause an increase in intracellular cGMP, which can activate iNOS and result in potential neurotoxicity (29, 30).

This study was designed to determine the cross-talk that occurs in CNS lupus among the proteins of the complement cascade; the proinflammatory mediators, TNF, iNOS, and ICAM-1; and AMPA-GluR in promoting apoptosis. For this, we used the MRL/MpJ-Tnfrsf6lpr (MRL/lpr) strain, an extensively studied lupus mouse model, which differs from the congenic MRL/MpJ (MRL+/+) strain by the nearly complete absence of the proapoptotic membrane Fas protein due to a retroviral insertion in the Tnfrsf6 gene (31, 32). Two strategies were used to inhibit complement activity using Crry in MRL/lpr mice: transgenic overexpression of soluble (s) Crry (33) and administration of Crry-Ig, a chimeric molecule incorporating mouse IgG1 (34). We measured relevant proapoptotic mediators, TNFR1, ICAM, iNOS, and AMPA-GluR1, and apoptosis itself in lupus brains as well as determined how the complement system influenced these manifestations. These results reveal a critical role for the complement system in lupus cerebritis.

To study the effect of Crry on the CNS in lupus mice, MRL/lpr mice were divided into two groups. Group 1 contained Crry transgenic mice originally produced on a CD-1 background and backcrossed into MRL/lpr mice (The Jackson Laboratory) until the ninth generation, thereby retaining ≤0.4% CD-1 genes (35). The presence of the Crry transgene was determined by PCR and was confirmed by serum ELISA, as described previously, showing the presence of sCrry, which is not present in wild-type mice (36). Littermate mice that lacked the Crry transgene were used as controls. In group 2, wild-type MRL/lpr mice were given Crry-Ig 3 mg/kg i.p. every other day from 12–24 wk of age, after which they were killed. Because Crry-Ig contains mouse IgG1, control groups were MRL/lpr mice injected with either saline or irrelevant mouse IgG1 at the same dose and schedule as Crry-Ig (37). In previous measurements of alternative pathway activity in these MRL/lpr mice, Crry-Ig inhibited complement activity by ∼60%, whereas sCrry in Crry transgenic mice inhibited systemic complement activity by ∼30% (35, 37). Soluble Crry is also present, as determined by Western blotting, in CSF, but not in wild-type controls, indicative of CNS expression of the Crry transgene (38). To determine whether Crry-Ig also had access to CSF, separate studies were performed in which three MRL/lpr mice were given a single 3-mg dose of Crry-Ig i.p., followed in 24 h by CSF harvest for Crry Western blotting (38). All animal work in this study was approved by the University of Chicago Animal Care and Use Committee.

Brains were isolated from animals killed at 24 wk of age. Cerebellum and brainstem were discarded. One section of the brain was snap-frozen in OCT compound (Miles) for immunofluorescence (IF) microscopy and TUNEL staining, a second section was frozen for protein isolation, a third section was processed for genomic DNA isolation, and the remainder was processed for RNA.

Mouse C3 was detected on 4-μm cryostat sections using FITC-conjugated Abs to mouse C3 (Cappel Laboratories), whereas C9 and AMPA-GluR1 were detected by indirect IF microscopy. Anti-rat C9 (obtained from Dr. B. P. Morgan, University of Wales, Cardiff, U.K.), which is cross-reactive with mouse C9, and anti-mouse AMPA-GluR1 (Chemicon International; catalog no. AB1504) Abs were used at 1/300 dilutions. Because both Abs were raised in rabbits, they were detected using FITC-conjugated anti-rabbit IgG, which lacks reactivity with mouse IgG. The sections were photographed maintaining the exposure time constant (Axiocam version 3.1; Zeiss). The sections from mice in each group were scored by an observer masked to origin from 0 to 4, with 0 being no difference from control background staining and 4 being the most intensely stained.

TUNEL staining was performed with the TdT-FragEL DNA fragmentation detection kit (Oncogene) according to the manufacturer’s instructions. In brief, 8-μm cryostat sections were washed in Tween 20/TBS and fixed in 4% formaldehyde for 30 min. This was followed by proteinase K treatment for 10 min and incubation in 3% H2O2/methanol for 7 min. Specimens were incubated with biotin-TdT at 37°C for 60 min, blocked in dilute BSA for 20 min, and incubated in streptavidin-HRP for 30 min, followed by detection with diaminobenzidine reagent for 15 min. Sections were counterstained with methyl green, and a blinded observer counted the number of positively stained nuclei per high-power field and recorded the average of 10 fields for each sample. For dual localization studies by IF, a similar protocol was followed, using the TACS 2 TdT-Fluor reagent kit (Trevigen). In this instance, biotin-TdT in apoptotic nuclei was identified with streptavidin-FITC, and neurons were stained with rabbit anti-mouse neurofilament Ab (Chemicon International), followed by rhodamine-conjugated anti-rabbit IgG (Sigma-Aldrich).

DNA was purified from frozen brain tissue using the DNeasy kit (Qiagen) according to the manufacturer’s instructions. The extent of DNA laddering was amplified and detected by LM-PCR (BD Clontech). DNA isolated from each animal was ligated with the supplied primer targets for 18 h at 16°C. The ligated DNA was then used as the substrate for PCR, using supplied primers and Advantage DNA polymerase (BD Clontech) for 23 cycles of 94°C for 1 min and 72°C for 3 min. The reaction product for each animal was electrophoresed through a 1.2% agarose gel, and ethidium bromide-stained bands were detected with UV light illumination.

Brains were homogenized in cell lysis buffer (25 mM HEPES (pH 7.4) buffer containing 2 mM DTT, 5 mM EDTA, and 10 mM digitonin) (39). Lysates were then incubated on ice for 15 min and centrifuged at 10,000 × g for 10 min at 4°C, and protein concentrations were determined using the bicinchoninic acid assay. Soluble supernatants were either used immediately or were stored at −80°C. Aliquots of protein (50 μg) were incubated at 37°C with assay buffer (50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 1 mM EDTA, and 10% glycerol) and 200 mM Ac-DEVD-pNA (BIOMOL). Hydrolysis of the DEVD-AFC substrate was followed for 15 min by fluorometry of the released AFC (excitation, 400 nm; emission, 505 nm), and activity was calculated from the slope. Addition of the caspase-3 inhibitor, Ac-DEVD-CHO (0.1 mM; BIOMOL), to the reaction mixture was used to confirm the specificity of the assay.

RNA was isolated from brains of MRL+/+ mice, and control and Crry-Ig-treated MRL/lpr mice using TRIzol reagent (Invitrogen Life Technologies). The integrity of all RNA was assured by analysis on a 2100 Bioanalyzer (Agilent Technologies). Five micrograms of RNA was used as the template for the first-strand cDNA synthesis in a reaction primed with oligo(dT) containing a T7 RNA polymerase promoter sequence. The second cDNA strand was synthesized using Escherichia coli DNA polymerase I and then used as a template to make biotinylated RNA probe by in vitro transcription. The RNA probes were hybridized to MG-U74Av2 arrays (Affymetrix) according to the manufacturer’s instructions. The hybridized arrays were scanned using a Genearray scanner (Agilent Technologies).

Data mining was performed using both Affymetrix and GeneSpring software (Silicon Genetics). For Affymetrix software, global scaling was performed to a target intensity of 500. Single-array analysis was performed to ascertain that the arrays were of good and comparable quality. The parameters included expression of GAPDH and its 3′/5′ signal ratio and the ratio of the expression of the spiked bacterial genes bioB, bioC, bioD, and cre as well as the scaling factors. Comparative analysis was performed using the statistical algorithms of Microarray Suite (MAS; version 5.0; Affymetrix). Standard GeneSpring analysis involved importing Affymetrix signal data, which were then normalized per chip and then per gene such that the median value of each gene was 1.0. Restrictions were performed to eliminate genes scored as absent and to select for genes that showed at least a 1.3-fold increase in saline-treated MRL/lpr brains compared with those from MRL+/+ control mice.

Real-time qRT-PCRs for iNOS, ICAM-1, AMPA-GluR1, and TNFR1 were performed on RNA isolated from brain. To remove all traces of genomic DNA, samples were treated with RQ1 DNase (Promega) at 37°C for 30 min, followed by addition of 1 μl of 20 mM EGTA (pH 8.0). Subsequently, samples were incubated at 65°C for 10 min to inactivate the DNase. cDNA was generated from RNA using random hexamers as primers with the SuperScript first-strand synthesis kit (Invitrogen Life Technologies) according to the manufacturer’s instructions. Real-time qPCR was performed using a Smart Cycler (Cepheid). The iNOS and GAPDH probes were labeled at the 5′ end with the reporter dye molecule FAM (emission maximum, 518 nm) and at the 3′ end with the quencher dye molecule TAMRA (emission maximum, 582 nm). Each reaction was conducted in a total volume of 25 μl with 1 μl of TaqMan Master Mix (Applied Biosystems), 3 μl of sample or standard cDNA, primers at 200 nM each, and probe at 100 nM. PCR was conducted with a hot start at 95°C (5 min), followed by 45 cycles of 95°C for 15 s and 60°C for 30 s. For each sample, the number of cycles required to generate a given threshold signal was recorded. Using a standard curve generated from serial dilutions of splenic cDNA, the ratio of iNOS expression relative to GAPDH expression was calculated for each experimental and control animal. Measurements of ICAM-1, AMPA-GluR1, and TNFR1 mRNA expression relative to that of GAPDH were performed in an analogous fashion, but using the QuantiTect SYBR Green RT-PCR kit (Qiagen) on an ABI 7700 sequence detector (Applied Biosystems). Primers were synthesized by Integrated DNA Technologies, and probes by Synthegen. For each set of primers, pilot studies confirmed the presence of a single PCR product. The sequences of the primers/probes were as follows: GAPDH forward, 5′-GCAAATTCAACGGCACAGT-3′; GAPDH reverse, 5′-AGATGGTGATGGGCTTCCC-3′; GAPDH probe, 5′-AGGCCGAGAATGGGAAGCTTGTCATC-3′; iNOS forward, 5′-CAGCTGGGCTGTACAAACCTT-3′; iNOS reverse, 5′-TGAATGTGATGTTTGCTTCGG-3′; iNOS probe, 5′-CGGGCAGCCTGTGAGACCTTTGA-3′; ICAM-1 forward, 5′-CGCAAGTCCAATTCACACTGA-3′; ICAM-1 reverse, 5′-CAGAGCGGCAGAGCAAAAG-3′; AMPA-GluR1 forward, 5′-TCCTGAAGAACTCCTTAGTG-3′; AMPA-GluR1 reverse, 5′-ATCATGTCCTCATACACAGC-3′; TNFR1 forward, 5′-GACCGGGAGAAGAGGGATAG-3′; and TNFR1 reverse, 5′-GTTCCTTTGTGGCACTTGGT-3′.

Data were analyzed with Minitab and Microsoft Excel software. All data are expressed as the mean ± SEM. When two groups were compared, the two-sample t test was used. Otherwise, one-way ANOVA, followed by Fisher’s pairwise comparisons, were used. A value of p < 0.05 was considered significant.

In these studies we examined the effect of complement inhibition in MRL/lpr mice given exogenous Crry-Ig or in which sCrry was produced endogenously by a constitutively active transgene. As previously noted, the MRL/lpr mice given Crry-Ig had a greater extent of systemic alternative pathway inhibition (∼60%) than those with constitutive overexpression of sCrry (∼30%) (35, 37). In contrast, although sCrry was present in the CSF of Crry transgenic mice (38), we were unable to find Crry-Ig in the CSF after administration of Crry-Ig to MRL/lpr mice (data not shown).

There was evidence for complement activation in MRL/lpr mouse brains, as shown by IF staining for C3 (Fig. 1). As expected in the setting of complement inhibition with Crry, there was less C3 deposition in the brains of Crry transgenic mice compared with transgene-negative controls and in mice treated with Crry-Ig compared with the saline- and normal mouse IgG1-treated controls.

FIGURE 1.

Complement inhibition with Crry decreases C3 deposition in brains of MRL/lpr mice. Representative sections of the choroid plexus (a and b) and the thalamic region (c and d) from MRL/lpr mice (a and c), MRL/lpr mice treated with Crry-Ig (b), and those with the Crry transgene (d) were stained by IF for C3. Widespread C3 deposits were significantly increased in MRL/lpr mice (a and c) and were reduced by Crry (b and d).

FIGURE 1.

Complement inhibition with Crry decreases C3 deposition in brains of MRL/lpr mice. Representative sections of the choroid plexus (a and b) and the thalamic region (c and d) from MRL/lpr mice (a and c), MRL/lpr mice treated with Crry-Ig (b), and those with the Crry transgene (d) were stained by IF for C3. Widespread C3 deposits were significantly increased in MRL/lpr mice (a and c) and were reduced by Crry (b and d).

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Productive complement activation can result in formation of the C5b-9 MAC in cell membranes with injurious effects. The deposition of C9 as a marker for MAC (6) was significantly increased in MRL/lpr brains and was reduced in expression in Crry transgenic mice and those animals treated with Crry-Ig (Fig. 2). Semiquantitative IF scores for C3 and C9 in all animals are shown in Fig. 3 documenting the efficacy of Crry to reduce complement activation in brains of lupus mice.

FIGURE 2.

Complement inhibition with Crry decreases C9 deposition in brains of MRL/lpr mice. Representative sections of the choroid plexus (a and b) and the thalamic region (c and d) from MRL/lpr mice, MRL/lpr mice treated with Crry-Ig (b), and mice with the Crry transgene (d) were stained by IF for C9. Widespread C9 deposits were significantly increased in MRL/lpr mice (a and c) and were reduced by Crry (b and d).

FIGURE 2.

Complement inhibition with Crry decreases C9 deposition in brains of MRL/lpr mice. Representative sections of the choroid plexus (a and b) and the thalamic region (c and d) from MRL/lpr mice, MRL/lpr mice treated with Crry-Ig (b), and mice with the Crry transgene (d) were stained by IF for C9. Widespread C9 deposits were significantly increased in MRL/lpr mice (a and c) and were reduced by Crry (b and d).

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FIGURE 3.

Semiquantitative scores for complement deposition in brains of Crry-Ig-treated MRL/lpr lupus mice. The sections stained for complement proteins C3 and C9 were scored in a blinded fashion from 0 to 4, and the results are represented graphically. ∗, p < 0.005 compared with the other two groups.

FIGURE 3.

Semiquantitative scores for complement deposition in brains of Crry-Ig-treated MRL/lpr lupus mice. The sections stained for complement proteins C3 and C9 were scored in a blinded fashion from 0 to 4, and the results are represented graphically. ∗, p < 0.005 compared with the other two groups.

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There is indirect evidence for increased apoptosis occurring in the neurons of MRL/lpr mice compared with their MRL+/+ counterparts (15). The relevance of the complement system to apoptosis in brains of lupus mice was studied using the TUNEL staining technique. There was a substantial number of apoptotic cells in brain sections in control MRL/lpr mice, which was significantly reduced in littermate Crry transgenic animals (Fig. 4, a and b). To investigate whether this apoptosis was occurring in neurons, a double-labeling technique was used, which showed that anti-neurofilament-stained neurons (Fig. 4,c) contained TdT-labeled apoptotic nuclei (Fig. 4,d), confirming neuronal apoptosis in these lupus mice. To substantiate this histological evidence for apoptosis, we also assessed apoptosis by the extent of DNA laddering. As shown by this method in Fig. 4 f, apoptosis increased from 12 to 24 wk in brains of MRL/lpr mice, which was significantly greater than that in their congenic MRL+/+ controls. Furthermore, complement inhibition by either overexpression of sCrry in Crry transgenic mice or administration of exogenous Crry-Ig reduced apoptosis in brains of MRL/lpr mice. Thus, apoptosis in brains of lupus mice occurs in a complement-dependent fashion.

FIGURE 4.

Apoptosis occurs in MRL/lpr brains in a complement-dependent fashion. As shown by TUNEL staining, complement inhibition with sCrry reduced apoptosis in brains of MRL/lpr mice (a) compared with littermate MRL/lpr controls without the Crry transgene (b). A representative thalamic section from an MRL/lpr mouse was stained for neurons with anti-neurofilament Ab (c) and for TUNEL-stained apoptotic nuclei (d, arrows), showing the presence of apoptotic neurons. As shown by LM-PCR, apoptosis increased with age in MRL/lpr brains and was reduced by complement inhibition with Crry (e). Shown are representative samples from untreated 12- and 24-wk-old MRL/lpr mice and 24-wk-old MRL+/+ controls (left panel). The complement dependence of apoptosis is shown by the reduction in DNA laddering in 24-wk-old MRL/lpr animals overexpressing sCrry compared with littermate MRL/lpr controls (middle panel) or those treated with Crry-Ig compared with control MRL/lpr mice (right panel).

FIGURE 4.

Apoptosis occurs in MRL/lpr brains in a complement-dependent fashion. As shown by TUNEL staining, complement inhibition with sCrry reduced apoptosis in brains of MRL/lpr mice (a) compared with littermate MRL/lpr controls without the Crry transgene (b). A representative thalamic section from an MRL/lpr mouse was stained for neurons with anti-neurofilament Ab (c) and for TUNEL-stained apoptotic nuclei (d, arrows), showing the presence of apoptotic neurons. As shown by LM-PCR, apoptosis increased with age in MRL/lpr brains and was reduced by complement inhibition with Crry (e). Shown are representative samples from untreated 12- and 24-wk-old MRL/lpr mice and 24-wk-old MRL+/+ controls (left panel). The complement dependence of apoptosis is shown by the reduction in DNA laddering in 24-wk-old MRL/lpr animals overexpressing sCrry compared with littermate MRL/lpr controls (middle panel) or those treated with Crry-Ig compared with control MRL/lpr mice (right panel).

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Key effectors of apoptosis are the proteolytic enzymes of the caspase family. Caspase-3, a critical mediator of apoptotic pathways in mammalian cells (40), was increased in MRL/lpr mouse brains, as assessed by hydrolysis of the DEVD-AFC substrate (Fig. 5). This increase in caspase-3 activity was significantly reduced in MRL/lpr mice that were complement inhibited by both overexpression of sCrry and systemic administration of Crry-Ig.

FIGURE 5.

Caspase-3 activity is increased in brains of MRL/lpr mice in a complement-dependent fashion. The activity of caspase-3 was significantly increased in brains from MRL/lpr mice, as assessed by hydrolysis of DEVD-AFC substrate. Complement inhibition with Crry-Ig or sCrry in Crry transgenic mice maintained caspase-3 activity comparable to that in the congenic MRL+/+ controls. Shown are data from individual mice, with the means depicted as horizontal lines. ∗, p < 0.001 compared with MRL/+ and matched MRL/lpr mice exposed to Crry.

FIGURE 5.

Caspase-3 activity is increased in brains of MRL/lpr mice in a complement-dependent fashion. The activity of caspase-3 was significantly increased in brains from MRL/lpr mice, as assessed by hydrolysis of DEVD-AFC substrate. Complement inhibition with Crry-Ig or sCrry in Crry transgenic mice maintained caspase-3 activity comparable to that in the congenic MRL+/+ controls. Shown are data from individual mice, with the means depicted as horizontal lines. ∗, p < 0.001 compared with MRL/+ and matched MRL/lpr mice exposed to Crry.

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In this study we examined the expression of TNFR1 and how this was affected by complement inhibition in brains of lupus mice. TNFR1 mRNA expression was significantly increased in lupus mice and was prevented by complement inhibition with sCrry and Crry-Ig (Fig. 6). Complement inhibition with sCrry in Crry transgenic mice and with Crry-Ig given as a recombinant protein had similar effects, with Crry-Ig consistently greater than sCrry (i.e., normalizing expression toward MRL+/+ animals).

FIGURE 6.

Expression of TNFR1 is increased in brains of MRL/lpr mice in a complement-dependent fashion. As shown by qRT-PCR, TNFR1 mRNA expression was increased in brains of MRL/lpr mice. Complement inhibition with Crry-Ig or sCrry in Crry transgenic mice maintained TNFR1 expression at a level comparable to that in the congenic MRL+/+ controls. Data from individual mice are shown, with mean values depicted as horizontal lines. ∗, p < 0.001 compared with MRL/+ and matched MRL/lpr mice exposed to Crry.

FIGURE 6.

Expression of TNFR1 is increased in brains of MRL/lpr mice in a complement-dependent fashion. As shown by qRT-PCR, TNFR1 mRNA expression was increased in brains of MRL/lpr mice. Complement inhibition with Crry-Ig or sCrry in Crry transgenic mice maintained TNFR1 expression at a level comparable to that in the congenic MRL+/+ controls. Data from individual mice are shown, with mean values depicted as horizontal lines. ∗, p < 0.001 compared with MRL/+ and matched MRL/lpr mice exposed to Crry.

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TNF signaling through its TNFR1 has been shown to modulate the expression of iNOS and ICAM-1, which, in turn, can mediate inflammation and tissue damage (41). The expression of both iNOS and ICAM-1 was increased in MRL/lpr mouse brains compared with that in MRL+/+ controls. This increased expression of iNOS and ICAM-1 was prevented by complement inhibition with Crry-Ig (Figs. 7 and 8). Thus, the increased expression of potentially proinflammatory TNFR1, iNOS, and ICAM-1 occurs in a complement-dependent fashion in the brains of MRL/lpr mice.

FIGURE 7.

Expression of iNOS is increased in brains of MRL/lpr mice in a complement-dependent fashion. As shown by qRT-PCR, iNOS mRNA expression was increased in brains from MRL/lpr mice treated with normal mouse IgG1. Complement inhibition with Crry maintained iNOS expression at a level comparable to that in congenic MRL+/+ controls. Data from individual mice are shown, with mean values depicted as horizontal lines. ∗, p < 0.001 vs other two groups.

FIGURE 7.

Expression of iNOS is increased in brains of MRL/lpr mice in a complement-dependent fashion. As shown by qRT-PCR, iNOS mRNA expression was increased in brains from MRL/lpr mice treated with normal mouse IgG1. Complement inhibition with Crry maintained iNOS expression at a level comparable to that in congenic MRL+/+ controls. Data from individual mice are shown, with mean values depicted as horizontal lines. ∗, p < 0.001 vs other two groups.

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FIGURE 8.

Complement regulates the expression of ICAM-1 in brains of MRL/lpr mice. As shown by qRT-PCR, ICAM-1 mRNA expression was increased in brains from MRL/lpr mice treated with normal mouse IgG1. Complement inhibition with Crry maintained ICAM-1 expression at a level comparable to that in the congenic MRL+/+ controls. Data from individual mice are shown, with mean values depicted as horizontal lines. ∗, p < 0.001 vs other two groups.

FIGURE 8.

Complement regulates the expression of ICAM-1 in brains of MRL/lpr mice. As shown by qRT-PCR, ICAM-1 mRNA expression was increased in brains from MRL/lpr mice treated with normal mouse IgG1. Complement inhibition with Crry maintained ICAM-1 expression at a level comparable to that in the congenic MRL+/+ controls. Data from individual mice are shown, with mean values depicted as horizontal lines. ∗, p < 0.001 vs other two groups.

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We used an exploratory approach to determine genes that might be relevant to lupus cerebritis and that were affected by complement inhibition. As determined by microarray analysis, AMPA-GluR1, -GluR2, and -GluRδ expression was increased in the brains of lupus mice and was reduced toward control levels by complement inhibition with Crry-Ig (Fig. 9). Because these were exploratory studies performed on a subset of animals from this study, in subsequent experiments we validated the data for AMPA-GluR1 in all animals of the study with qRT-PCR (Fig. 10). Furthermore, increased IF staining for AMPA-GluR1 in brains of MRL/lpr mice and prevention of this increase by complement inhibition with Crry-Ig (Fig. 11) indicate that the observed increase in AMPA-GluR1 mRNA occurred in a complement-dependent fashion and was translated into altered protein expression. The full dataset from these microarray studies can be freely accessed at 〈http://fgf.bsd.uchicago.edu/JJ/〉.

FIGURE 9.

Microarray analysis shows that mRNA for AMPA-GluR1, -2, and -δ were up-regulated in brains of MRL/lpr mice in a complement-dependent fashion. Shown is the expression of GluRs from MRL+/+ mice or MRL/lpr mice treated with either saline or Crry-Ig from 12 to 24 wk of age. Data from Affymetrix MG-U74Av2 arrays were analyzed by GeneSpring software. The results are shown as the average for each probe set for AMPA-GluR1 and AMPA-GluRδ and as the mean ± SEM of three probes sets for AMPA-GluR2.

FIGURE 9.

Microarray analysis shows that mRNA for AMPA-GluR1, -2, and -δ were up-regulated in brains of MRL/lpr mice in a complement-dependent fashion. Shown is the expression of GluRs from MRL+/+ mice or MRL/lpr mice treated with either saline or Crry-Ig from 12 to 24 wk of age. Data from Affymetrix MG-U74Av2 arrays were analyzed by GeneSpring software. The results are shown as the average for each probe set for AMPA-GluR1 and AMPA-GluRδ and as the mean ± SEM of three probes sets for AMPA-GluR2.

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FIGURE 10.

The expression of AMPA-GluR1 is increased in brains of MRL/lpr mice in a complement-dependent fashion. The qRT-PCR was used to validate the results of microarray analyses for AMPA-GluR1. mRNA expression of AMPA-GluR1 was increased in brains of MRL/lpr mice treated with normal mouse IgG1 and was reduced by complement inhibition with Crry-Ig to levels approximating those in the MRL+/+ controls. ∗, p < 0.005 vs the other two groups.

FIGURE 10.

The expression of AMPA-GluR1 is increased in brains of MRL/lpr mice in a complement-dependent fashion. The qRT-PCR was used to validate the results of microarray analyses for AMPA-GluR1. mRNA expression of AMPA-GluR1 was increased in brains of MRL/lpr mice treated with normal mouse IgG1 and was reduced by complement inhibition with Crry-Ig to levels approximating those in the MRL+/+ controls. ∗, p < 0.005 vs the other two groups.

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FIGURE 11.

Complement-dependent increases in AMPA-GluR1 protein occur in MRL/lpr mice. Representative sections from the cortex (a) and thalamus (b) of control MRL/lpr mouse brains are shown. Reduced AMPA-GluR1 expression was observed in cortical (c) and thalamic (d) sections from MRL/lpr mice treated with Crry-Ig. Limited expression of AMPA-GluR1 was observed in brain sections from MRL+/+ mice (e). Sections were scored in a blinded fashion, and the results are given in f. ∗, p < 0.005 vs the other two groups.

FIGURE 11.

Complement-dependent increases in AMPA-GluR1 protein occur in MRL/lpr mice. Representative sections from the cortex (a) and thalamus (b) of control MRL/lpr mouse brains are shown. Reduced AMPA-GluR1 expression was observed in cortical (c) and thalamic (d) sections from MRL/lpr mice treated with Crry-Ig. Limited expression of AMPA-GluR1 was observed in brain sections from MRL+/+ mice (e). Sections were scored in a blinded fashion, and the results are given in f. ∗, p < 0.005 vs the other two groups.

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Neurological involvement is one of the debilitating complications that occurs in SLE patients. Approximately 50–70% of lupus patients develop neuropsychiatric manifestations, such as headaches, seizures, cognitive disorders, cerebrovascular disease, and psychosis at some point in their illness (2). Magnetic resonance and other imaging techniques reveal lesions and increased diffusivity that may be due to loss of tissue integrity in the brains of SLE patients (42). However, the exact pathogenic mechanisms causing the aberrations leading to lupus cerebritis remain unknown.

Abnormal neurological functioning similar to that seen in SLE patients is detectable in MRL/lpr lupus mice by 8–10 wk of age and is severe by 18 wk of age (43). Deterioration in behavioral performance and atrophy of pyramidal neurons in the parietal cortex and the hippocampal CA1 area were observed with the onset of the disease in MRL/lpr mice (44). CNS inflammation, which was considered to be due to immune complex deposition and complement activation in the choroid plexus and was accompanied by perivascular leakage of IgG, was observed as early as 8 wk of age in MRL/lpr mice (45). Recently, we have demonstrated that this phenomenon occurs in a complement-dependent fashion in diseased MRL/lpr mice (46). Several additional lines of evidence indicate that complement activation plays a very important role in the pathology of SLE. Although the brain is an immunologically privileged organ, it has the ability to synthesize complement components (47). Furthermore, when the blood-brain barrier is compromised, as can occur in the CNS in lupus, access of plasma complement proteins to brain parenchyma may occur.

In the present study we demonstrate for the first time that apoptosis and associated pathological events that occur in brains of MRL/lpr mice are complement dependent, because they can be prevented by administration of the potent complement inhibitor, Crry, either overexpressed as sCrry or administered as Crry-Ig. By immunostaining we observed increased deposits of C3, the central protein of the complement system, and C9, a component of the MAC, supporting the idea that complement activation occurs in MRL/lpr brains. Notably, as reported in this study, Crry-Ig was twice as potent as sCrry in reducing systemic alternative pathway complement activity. One explanation for this is that Crry transgenic animals have an increase in alternative pathway activity, presumably as a compensatory response to the presence of sCrry throughout their lives (48). In contrast, sCrry was present in the CSF of transgenic animals, reflecting its ubiquitous production and small size (38), although we could not identify Crry-Ig in CSF under conditions comparable to those used in this study. The latter suggests that the blood-CSF barrier is sufficiently intact in lupus cerebritis, at least to restrict passage of a molecule the size of Crry-Ig (as well as IgG). Given that native Crry is a ubiquitously expressed, type I membrane protein, including in the CNS (49), it was not possible for us to conclusively determine levels or activities of sCrry or Crry-Ig in the brain parenchyma in these studies. Overall, the fact that Crry-Ig was as effective as sCrry, and in some cases more effective (for instance, in reducing TNFR1 mRNA), it seems likely that systemic complement inhibition can limit pathophysiological manifestations of lupus cerebritis. Because both Crry-Ig and IgG autoantibodies are present in the systemic circulation, to the extent that the BBB is disrupted in SLE (50), it does seem likely that they would both track together into brain parenchyma, where Crry-Ig could restrict complement activation. Our finding that partial systemic complement inhibition with Crry-Ig can reduce pathophysiological manifestations of CNS lupus has direct clinical relevance given the availability of human complement inhibitors with profiles comparable to that of Crry-Ig.

One significant effect of complement activation can be apoptosis, which can occur directly through cellular events stimulated by C5b-9 (13, 14), C5a (51), and Bb (52), culminating in activation of caspase-3, the effector protein that performs the downstream function of apoptosis (40). That apoptosis was increased in brains of MRL/lpr mice was observed by TUNEL staining and was substantiated by LM-PCR. In this study we have extended the findings by Sakic et al. (15) of TUNEL staining in nonlymphoid cells in MRL/lpr brains by showing colocalization with neurons, thereby confirming their conclusions that neurons were dying by apoptosis, which can account for the neuronal loss that occurs in these lupus mice. Given that MRL/lpr mice are Fas deficient, the mechanism of apoptosis cannot be through the Fas/Fas ligand pathway, but, rather, could be through the TNFR1 or other pathways. Caspase-3 was up-regulated in MRL/lpr mice, indicating that apoptosis in the brain is likely to occur in a caspase-dependent fashion.

Different components of the complement cascade, including C2, C3, C5a, and C7, are crucial for increased release of the proinflammatory molecule, TNF, in meningitis and endotoxemia (53, 54). Circulating levels of TNF were increased and correlated with disease in SLE patients (18). In our study the expression of TNFR1, which is involved in biological responses such as gene induction, cytokine increase, and cell death, was increased in brains of MRL/lpr mice. TNFR1 can regulate different inflammatory proteins, such as ICAM-1, iNOS, and AMPA-GluR1, all of which can lead to tissue damage (20, 24, 26).

The iNOS mRNA expression was higher than normal in brains of MRL/lpr mice, similar to the enhanced iNOS expression and NO production demonstrated in endothelial cells of patients with SLE (55). Increased NO production could lead to the release of cytokines and oxygen-derived free radicals. Both TNF and iNOS mediate up-regulation of the adhesion molecule, ICAM-1 (56). We found increased mRNA expression of ICAM-1 in MRL/lpr brains. This increase was similar to the increased expression of ICAM-1 observed in the choroid plexus and endothelial cells of MRL/lpr mice (57). ICAM-1 is a cytokine-induced, membrane-bound protein that facilitates the recruitment of cells into tissues undergoing inflammatory responses. Therefore, this increase in ICAM-1 expression could lead to increased leukocyte entry into the CNS and the ensuing inflammation (58). It is also possible that complement-dependent ICAM-1-up-regulation contributed to inflammatory cell accumulation and focal vascular thromboses, which can lead to ischemia and apoptosis (50).

TNF, iNOS, and components of the complement cascade modulate AMPA-GluR1 in oligodendrocytes (26). GluR signaling is necessary for the normal functioning of the CNS. In our study we identified increased expression of AMPA-GluR1, -GluR2, and -GluRδ by exploratory microarray analyses. These observations were validated at both mRNA and protein levels for AMPA-GluR1. Such up-regulation of AMPA-GluR may render lupus brains susceptible to GluR-mediated excitotoxicity (59).

In a recent study we demonstrated complement-mediated alterations in aquaporin 4 expression and deposition of IgG in MRL/lpr brains, suggesting that edema and derangement of the blood-brain barrier were part of the pathogenesis of lupus cerebritis (46). The increase in aquaporin 4 expression has the potential to cause apoptosis (60). Activated complement proteins, aquaporin 4, and inflammatory mediators, TNF, iNOS, and ICAM-1, when induced can ultimately cause apoptosis. Overall, based upon our current findings, it appears that activation of the complement system is required for up-regulated expression of these other mediators. The possibility that the complement system directly and/or indirectly contributes to apoptosis via these mediators is the subject of ongoing studies.

Our results clearly demonstrate that apoptosis is a prominent feature in the brains of lupus mice, and this occurs by a Fas-independent mechanism. More than one mechanism may mediate cell death in lupus brains. Our studies demonstrate for the first time that the neuropathology observed in lupus brains is complement dependent because complement inhibition with Crry ameliorated disease, at least as measured by apoptosis. That treatment with recombinant Crry-Ig reduced disease in lupus mice supports the idea that complement inhibition by available human recombinant proteins with activity profiles similar to that of Crry may be a potential therapeutic option for lupus cerebritis.

The authors have no financial conflict of interest.

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 National Institutes of Health Grant R01DK55357. L.B. was supported by National Institutes of Health Training Grant T32DK07510.

3

Abbreviations used in this paper: SLE, systemic lupus erythematosus; AMPA-Glu, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-glutamate; Crry, CR1-related y; CSF, cerebrospinal fluid; IF, immunofluorescence; iNOS, inducible NO synthase; LM, ligase mediated; MAC, membrane attack complex; MRL+/+, MRL/MpJ-Tnfrsf6+/+; MRL/lpr, MRL/MpJ-Tnfrsf6lpr/lpr; qRT-PCR, quantitative RT-PCR; s, soluble.

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