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Antibody-Mediated Phagocytosis of the Amyloid -Peptide in Microglia Is Differentially Modulated by C1q¹

Scott D. Webster^*, Manuel D. Galvan^*, Erick Ferran^*, William Garzon-Rodriguez, Charles G. Glabe^* and Andrea J. Tenner^2,*

^* Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697; and Center for Pharmaceutical Biotechnology, University of Colorado Health Sciences Center, University of Colorado, Denver, CO 80262

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Microglial ingestion of the amyloid -peptide (A) has been viewed as a therapeutic target in Alzheimer’s disease, in that approaches that enhance clearance of A relative to its production are predicted to result in decreased senile plaque formation, a proposed contributor to neuropathology. In vitro, scavenger receptors mediate ingestion of fibrillar A (fA) by microglia. However, the finding that cerebral amyloid deposition in a transgenic mouse model of Alzheimer’s disease was diminished by inoculation with synthetic A has suggested a possible therapeutic role for anti-A Ab-mediated phagocytosis. Microglia also express C1qR_P, a receptor for complement protein C1q, ligation of which in vitro enhances phagocytosis of immune complexes formed with IgG levels below that required for optimal FcR-mediated phagocytosis. The data presented here demonstrate FcR-dependent ingestion of A-anti-A complexes (IgG-fA) by microglia that is a function of the amount of Ab used to form immune complexes. In addition, C1q incorporated into IgG-fA enhanced microglial uptake of these complexes when they contained suboptimal levels of anti-A Ab. Mannose binding lectin and lung surfactant protein A, other ligands of C1qR_P, also enhanced ingestion of suboptimally opsonized IgG-fA, whereas control proteins did not. Our data suggest that C1qR_P-mediated events may promote efficient ingestion of A at low Ab titers, and this may be beneficial in paradigms that seek to clear amyloid via FcR-mediated mechanisms by minimizing the potential for destructive Ab-induced complement-mediated processes.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Glial cells, particularly microglia, have been implicated in the progression of Alzheimer’s disease (AD)³ based on their ability to release inflammatory substances (1). However, microglia also are professional phagocytes and as such can provide beneficial functions by clearing targeted pathogens or cellular debris, and, potentially, in AD the pathogenic peptide amyloid (A). Hypotheses proposing therapeutic augmentation of phagocytosis of the A as a means of counteracting deposition and subsequent toxicity have been supported by a recent study by Schenk et al. (2) showing that cerebral A deposition in a transgenic mouse model that overexpresses the gene for a mutant form of the human amyloid precursor protein (APP) was considerably diminished by prior "immunization" of the animals with synthetic A peptide. This correlated with the presence of anti-A antibodies and microglia-like cells that were immunopositive for A and has led to the speculation that FcR-mediated phagocytosis of A-anti-A immune complexes by microglia contributed to the diminished deposition through clearance of soluble and/or already deposited peptide opsonized with Ab.

In the absence of Ab, uptake of A in microglia has been shown to proceed via the family of scavenger receptors (SR) (or a different, uncharacterized pathway, depending on the aggregation state of the A) (3, 4). Among the many proteins colocalized to senile plaques in the AD brain are those of the classical complement pathway, including C1q (reviewed in Ref. 1), and we have previously shown that C1q inhibits microglial uptake of A in a fashion that suggests that C1q blocks access of microglia to A (5) and thus may contribute to the accumulation of the peptide in plaques.

We recently demonstrated that microglia express the C1q receptor that enhances phagocytosis (C1qR_P) and, upon interaction with C1q, exhibit enhanced phagocytosis of particles opsonized with low levels of Ab (6). Thus, C1q may have opposing effects on ingestion of A via the SR- and FcR-mediated pathways, inhibiting naked A uptake and enhancing of A immune complex uptake. Maxfield and colleagues (7) recently reported the ability of mouse microglia to engage in SR-independent ingestion of complexes of fibrillar A (fA) and the 4G8 monoclonal anti-A Ab, which they termed IgG-fA (7) (this convention will be retained in the current study). They demonstrated that similar levels of ingestion (1.5-fold increase over fA alone) were achieved in the presence and absence of C1q for immune complexes containing saturating concentrations of 4G8.⁴ In this study, we demonstrate that C1q bound to the immune complexes enhances uptake of A for complexes containing less than saturating Ab concentrations as it has been shown to do for other phagocytic targets (6, 8, 9). In addition, we show that this enhancement of phagocytosis is mediated through C1qR_P, since similar enhancement can be seen when cells interact with C1q and other known ligands of C1qR_P, mannose binding lectin (MBL) and lung surfactant protein A (SPA), and the effect is blocked with an anti-C1qR_P Ab.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials

C1q was purified from human serum as previously described (Ref. 10 , modified as described in Ref. 11). Rabbit polyclonal anti-human A Ab purified by octanoic acid precipitation was a gift from Neil Cooper (The Scripps Research Institute, La Jolla, CA) and the mouse monoclonal anti-human A Ab was obtained from Senetek PLC (Maryland Heights, MO). Rabbit anti-C1qR_P was generated against an 11-aa sequence from the carboxyl terminus of C1qR_P as described previously (6). Fucoidan, polyinosinic acid, BSA, ferritin, and cytochalasin D were obtained from Sigma (St. Louis, MO). Human serum albumin (HSA) was obtained from the American Red Cross (Washington, DC). Recombinant rat MBL was provided by Kurt Drickamer of the University of Oxford (Oxford, U.K.). Rat SPA was provided by Dennis Voelker of the National Jewish Hospital (Denver, CO). Human A_1-42 peptides (unlabeled or conjugated with fluorescein at residue 4) were synthesized as previously described (12, 13). All solutions were prepared using water from a Millipore MilliQ Plus ultrafiltration system equipped with a LPS filter (Millipore, Bedford, MA).

Cell culture

Neonatal rat microglial cultures were prepared as previously described (5, 6). The BV-2 mouse microglial cell line (obtained from Caleb Finch, University of Southern California) was maintained in DMEM/F-12 medium (Life Technologies, Grand Island, NY) supplemented with 10% FCS (HyClone Laboratories, Logan, UT).

Formation of fA and IgG-fA

To assess phagocytosis of peptide, aggregated A_1-42 was generated by preparing a solution of 530 µM A_1-42 in 10 mM HEPES (pH 7.4) under sterile conditions and stirring overnight at room temperature. This preparation consisted of 30 µM fluorescein-conjugated A_1-42 plus 500 µM unconjugated A_1-42 and is hereafter referred to as fluorescent A_1-42. To assess the physical state of the peptides solubilized and incubated in this fashion, nonfluorescent and fluorescent A_1-42 were assessed for -sheet structure by circular dichroism and were found to exhibit profiles identical to those of fA preparations described in previous studies from this laboratory (data not shown; Ref. 14) and are therefore referred to as fA. Fluorescent fA_1-42 was diluted to 50 µM in PBS, combined with anti-A Ab or PBS, incubated for 30 min at 37°C, washed twice by centrifugation for 5 min at 14,000 x g, and resuspended to the original volume of 530 µM fluorescent fA_1-42. In this manuscript, the IgG-fA thus formed are identified according to the concentration of anti-A Ab used to prepare them, e.g., IgG-fA prepared using 10 µg/ml anti-A Ab are referred to as IgG10-fA. To control for the possibility of IgG aggregates in the anti-A Ab, in some experiments the Ab was centrifuged at 14,000 x g for 5 min to remove aggregated Ig and the supernatant was used to generate immune complexes. Results from parallel wells using centrifuged and uncentrifuged anti-A Ab were not different (data not shown). The effects of C1q on ingestion were investigated under three different paradigms: 1) C1q was immobilized to culture surfaces as previously described (6); 2) fA or IgG-fA were combined with C1q (or PBS), incubated for 30 min at 37°C, washed twice by centrifugation at 14,000 x g, and resuspended to the original volume; 3) C1q was added directly to cultures concurrently with fA or IgG-fA. In select experiments, surfaces were also coated with MBL, SPA, BSA, HSA, and ferritin.

Flow cytometric assessment of phagocytosis

Microglia were changed to serum-free medium (DMEM for primary microglia, DMEM/F-12 for BV-2) supplemented with 20 mM HEPES (pH 7.3), 1% BSA, and 10 µg/ml penicillin/streptomycin before addition of reagents. In some experiments as noted in the text, cytochalasin D was added to cultures 30 min before addition of peptide to disrupt actin filaments. SR ligands were added to cultures 25 min before addition of peptide to saturate microglial SR. To assess the inhibitory effect of anti-C1qR_P, cells were transiently permeabilized in the presence or absence of anti-C1qR_P or control Ab as previously described (6) before addition of immune complexes. In all experiments, fA-containing solutions were added to cultures based on the final concentration of fA only; i.e., the indicated concentrations (in µM) correspond to fA and do not reflect contributions of additional protein in the form of Ab and/or C1q. Following exposure to peptide for 30 min, microglia were washed twice with HBSS to remove unassociated fA and treated with 250 µg/ml trypsin/EDTA (Life Technologies) for 20 min at 4°C to eliminate surface-bound fA and to detach the cells from the culture surface, then washed twice by centrifugation at 540 x g for 5 min at 4°C. Following trypsinization and washing, microglia were fixed in solution by exposure to 3.7% formaldehyde for 20 min, washed twice in HBSS + 0.1% BSA + 0.01% NaN₃, resuspended, and cell-associated fluorescence determined using FACSCalibur (Becton Dickinson, Bedford, MA). In the figures, error bars represent SD for triplicate (see Fig. 4) or duplicate (remaining figures) data points. Results are representative of three or more similar experiments.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Visualization of uptake of IgG-fA in fucoidan-treated cells. IgG-fA were prepared with varying concentrations of Ab and administered to primary rat (A) or BV-2 (B) microglia at 2 µM with or without preincubation with 100 µg/ml fucoidan. In the presence of fucoidan, ingestion of IgG-fA was a function of the amount of anti-A applied and was markedly more effective than uptake of fA alone.

Assessment of fA and IgG-fA by protein assay, fluorometry, and Western blot

Protein concentrations of fluorescent and nonfluorescent fA solutions were assessed using the microBCA (Pierce, Rockford, IL). Sedimentable fA and IgG-fA were diluted in PBS and examined for fluorescence using a Fluorolog model 1681 spectrofluorometer (Spex Industries, Edison, NJ). fA and IgG-fA with and without C1q were assessed by SDS-PAGE/Western blot for A, Ab, and C1q.

Microscopic analysis of uptake

Following exposure to peptide and trypsinization as described above, microglia were washed, resuspended in serum-free culture medium, and allowed to adhere to coverslips for 20 min at 37°C. Following fixation with 3.7% formaldehyde for 20 min, coverslips were mounted using Vectashield (Vector Laboratories, Ingold, CA) and examined by fluorescence microscopy.

Erythrocyte phagocytosis assay

Experiments were performed according to previously published techniques (6). Briefly, solutions of 16 µg/ml C1q, MBL, SPA, BSA, HSA, or ferritin were used to coat chambered glass culture surfaces (Nalge Nunc International, Naperville, IL). Microglia were adhered to the coated surfaces and exposed to sheep erythrocytes (Colorado Serum, Denver, CO) opsonized with a suboptimal concentration of anti-sheep erythrocyte Ab (Colorado Serum). Phagocytosis of these erythrocyte/anti-erythrocyte Ab complexes (EA_IgG) is represented as the total number of EA_IgG ingested per 100 cells.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Flow cytometric characterization of microglial A phagocytosis

In a previous study, we demonstrated quantitative assessment of microglial uptake of A containing a ¹⁴C label and morphological characterization of uptake by the use of fluorescent A (5). We have extended these experiments to examine the impact of anti-A Ab on microglial uptake by the use of flow cytometric analysis following ingestion of the fluorescent peptide. Experiments have been performed with both primary rat microglia (Fig. 1A) and the BV-2 mouse microglial cell line (Fig. 1B). The background fluorescence of each cell type is indicated by the plots for cells in the absence of fA_1-42 (no A) at 37°C (dashed line) or 4°C + azide (dotted line). Both primary and BV-2 microglia exposed to 10 µM fA_1-42 at 37°C (thick solid lines) exhibited fluorescence that was markedly higher than the autofluorescence of cells in the absence of fA, whereas microglia exposed to peptide under conditions that prevent phagocytosis, i.e., 4°C + azide (thin solid lines), did not. The mean fluorescence values for primary microglia were generally greater than those for BV-2 cells at any given concentration of fA, suggesting a greater capacity for internalization in the primary cultures. Ingestion of fA in our experiments exhibited characteristics consistent with phagocytosis, i.e., it was saturable (Fig. 2A) and inhibitable by cytochalasin D (Fig. 2B).

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Flow cytometric analysis of microglial uptake of fA1-42. Primary rat microglia (A) and the BV-2 mouse microglial cell line (B) were exposed to 10 µM fluorescent fA1-42, washed, and trypsinized to remove extracellular and surface-bound peptide and assessed by flow cytometry. Histograms correspond to counts of 10,000 cells gated to exclude debris. The mean fluorescence of primary rat microglia exposed to 10 µM peptide in this experiment was 1823 U compared with 13–17 for the two control cell populations and 82 for cells exposed to 10 µM peptide at 4°C in the presence of azide. Similarly, BV-2 cells exhibited a mean fluorescence value of 668 following ingestion of 10 µM peptide compared with 4–5 for control cells and 5.2 for cells exposed to 10 µM peptide at 4°C in the presence of azide. Comparison of background-subtracted mean fluorescence values shows that the portion of the fluorescence detected for cells at 37°C that is due to trypsin-resistant association of A with the cell surface is <4% for primary rat microglia and <1% for BV-2 cells, validating that this method accurately assesses the amount of peptide ingested by these cells.

View larger version (14K): [in this window] [in a new window]   FIGURE 2. Ingestion of fA is saturable and inhibitable by cytochalasin D. A, Mean fluorescence of rat microglia increases linearly with increasing concentrations of applied fA up to 10 µM (dotted line; r2 = 0.9991). The amount of ingested fA per mole of applied fA decreases at higher concentrations however, suggesting a saturable process. B, Incubation of rat microglia with 0.0–5.0 µM cytochalasin D for 30 min before addition of 10 µM fA resulted in a marked inhibition of ingestion, indicating a requirement for actin polymerization, characteristic of a phagocytic mechanism of internalization. Error bars represent SD values in this and all subsequent figures.

Assessment of fA sedimentability, Ab effects on the aggregation state of fA, and incorporation of anti-A Ab and C1q

IgG-fA were prepared as described in the methods employing centrifugation washes as part of the procedure to separate free, unbound Ab from the IgG-fA. First, to determine whether a significant portion of our aggregated peptide alone was solubilized under this wash schedule (and thus not recoverable by centrifugation), we examined fA and its constituent sedimentable and soluble fractions using the microBCA technique. As shown in Fig. 3A, only a small proportion of aggregated peptide remained in solution following centrifugation. We consistently observed that >90% of fA was sedimentable, showing that this method can be used to reliably and reproducibly prepare aggregated/fA.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Characterization of aggregated fA and IgG-fA. A, Thirty micromolar solutions of nonfluorescent and fluorescent fA1-42 were centrifuged and the sedimented material was resuspended to the original volume and assessed for protein concentration in parallel with the supernatant (soluble) and a companion uncentrifuged solution (total). For both fA preparations, the detected concentrations of the sedimentable fractions remained at 30 µM, whereas the soluble fractions showed 10-fold less protein. B, Two micromolar fA and IgG100-fA showed quantitatively similar emissions spectra, demonstrating that this anti-A Ab does not dissociate the A fibrils into soluble forms. Inset, quantitative analysis of fluorescent fA1-42 emissions at 515 nm (excitation, 495 nm) shows a linear response over a 0–10 µM range.

Ab-mediated ingestion of fA is not inhibited by SR ligands

Previous studies have demonstrated that fA is internalized in large part via the family of SR (3, 4), and we observed 80–90% inhibition of uptake of fA by the use of the SR ligands fucoidan and polyinosinic acid (data not shown), consistent with these reports. Uptake of IgG-fA by microglia showed no appreciable change with increasing concentrations of anti-A Ab when the SR was not blocked (Fig. 4, ). However, influences of Ab were obvious when SR-mediated uptake of fA was inhibited by preincubation of microglia with 100 µg/ml fucoidan (•). Ingestion was low in the absence of Ab, with greater uptake observed with increasing concentrations of anti-A used to form immune complexes. For primary microglia (Fig. 4A), formation of IgG-fA with as little as 10 µg/ml anti-A resulted in SR-independent ingestion. Increases in the Ab concentration resulted in increased uptake of fA to a maximum level similar to that seen in the absence of fucoidan. BV-2 microglia behaved similarly, although somewhat higher levels of Ab were required to trigger SR-independent uptake (Fig. 4B). These results demonstrate that fA in the form of an immune complex is taken up through an SR-independent mechanism, likely via FcR-mediated phagocytosis.

C1q enhances uptake of immune complexes but not of fA alone

When immobilized to culture surfaces, C1q has been shown to enhance FcR-mediated phagocytosis of targets that are suboptimally opsonized with IgG (6, 8, 9). Therefore, to test the potential for C1q-mediated increases in phagocytosis, IgG-fA were prepared using suboptimal levels of Ab, i.e., concentrations which resulted in less than the maximum possible ingestion. Based on the results in Fig. 4, IgG-fA were prepared using 10 µg/ml anti-A Ab (IgG10-fA) for use with primary microglial cultures (Fig. 5A) and 25 µg/ml Ab (IgG25-fA) for use with BV-2 cells (Fig. 5B). In addition, IgG-fA were prepared with 100 µg/ml Ab (IgG100-fA) to assess maximal levels of FcR-mediated phagocytosis in both cell types. In the absence of C1q, fucoidan-treated primary rat microglia (Fig. 5A) ingested IgG10-fA () at somewhat higher levels than fA alone (), while taking up IgG100-fA () at maximal levels. Uptake of IgG10-fA by microglia adherent to immobilized C1q was enhanced in a dose-dependent fashion up to a level similar to that of IgG100-fA. This represents a 3-fold enhancement over that seen in the absence of C1q. SR-independent uptake of fA alone and uptake of IgG100-fA were not affected by any concentration of C1q. BV-2 microglia again behaved similarly to the primary microglia (Fig. 5B). In these experiments, IgG25-fA uptake occurred at a moderately higher level than fA alone and was enhanced by interaction with C1q-coated surfaces, showing a 3- to 4-fold enhancement at the highest C1q concentration used. As with primary rat microglia, uptake of fA and IgG100-fA in the presence of fucoidan was not affected by C1q in BV-2 cells.

View larger version (33K): [in this window] [in a new window]   FIGURE 5. Immobilized C1q enhances uptake of IgG-fA. fA, IgG-fA prepared with suboptimal anti-A concentrations (IgG10-fA in A; IgG25-fA in B), or IgG-fA prepared with high anti-A concentration (IgG100-fA) were added at 2 µM to microglia which were adhered to surfaces coated with 0–32 µg/ml C1q and preincubated with 100 µg/ml fucoidan. For both primary (A) and BV-2 (B) microglia, interaction with surfaces coated with C1q led to greater internalization of IgG10-fA/IgG25-fA than interaction with uncoated surfaces. In contrast, uptake of fA remained low and uptake of IgG100-fA remained high, regardless of the presence or absence of C1q on the substrate. Data from multiple experiments were normalized to the mean fluorescence for fA alone and pooled. Analysis of variance of these aggregate data showed significant effects due to the form of the peptide (p < 0.001), concentration of C1q (p 0.008), and the interaction of fA form and C1q concentration (p 0.005) for both primary microglia and BV-2 cells.

View larger version (35K): [in this window] [in a new window]   FIGURE 6. C1q enhances uptake of IgG-fA when bound to the complexes. These experiments differ from Fig. 5 in that fA and IgG-fA were incubated with 0–75 µg/ml C1q and washed before addition at 2 µM to microglia preincubated with 100 µg/ml fucoidan. Both primary (A) and BV-2 (B) microglia responded to C1q with enhanced uptake of suboptimally opsonized fA (IgG10-fA in A; IgG25-fA in B). Uptake of fA alone and IgG100-fA remained constant with changing C1q concentrations. Data from multiple experiments were normalized to the mean fluorescence for fA alone and pooled. Analysis of variance of these aggregate data showed significant effects due to the form of the peptide (p < 0.001), concentration of C1q (p 0.05), and the interaction of the fA form and C1q concentration (p 0.01) for both primary microglia and BV-2 cells.

Fluorescence microscopy was used to verify the flow cytometry observations. In these experiments, microglia were allowed to adhere to coverslips following exposure to fluorescent fA and trypsinization. Fucoidan-treated primary microglia that were not subjected to fA showed no detectable fluorescence (Fig. 7A). Exposure to fluorescent fA resulted in minimal fluorescence, in keeping with the ability of fucoidan to block SR function (Fig. 7B). IgG10-fA (Fig. 7C) resulted in cell-associated fluorescence somewhat higher than that observed for fA alone. In contrast, IgG100-fA (Fig. 7D) and IgG10-fA opsonized with 75 µg/ml C1q (Fig. 7E) resulted in markedly increased cell-associated fluorescence, similar to that seen for microglia exposed to fA in the absence of fucoidan (Fig. 7F). Results were similar for BV-2 microglia (data not shown) and reflect the data derived from the flow cytometry assays presented in Figs. 5 and 6.

View larger version (63K): [in this window] [in a new window]   FIGURE 7. Fluorescence microscopy of primary rat microglia following uptake of fA and IgG-fA. Microglia were preincubated with (A–E) or without (F) 100 µg/ml fucoidan and subjected to medium only (A) or 10 µM solutions of fA (B and F), IgG10-fA (C), IgG100-fA (D), or IgG10-fA opsonized with 75 µg/ml C1q (E). Note that similar high levels of fluorescence are observed for fucoidan-treated cells following exposure to fA opsonized with a high Ab concentration (IgG100-fA, D) or following exposure to fA opsonized with a low Ab concentration but also with C1q (IgG10-fA, E).

Other C1qR_P ligands, but not control proteins, also enhance uptake of IgG10-fA

C1q is a member of a family of proteins termed defense collagens, which influence leukocyte function by enhancing phagocytosis and modulating production of cytokines (reviewed Ref. 17). In addition to C1q, the defense collagens MBL and SPA are known to enhance FcR-mediated phagocytosis in a C1qR_P-dependent manner (18, 19). To provide further support for the participation of C1qR_P in C1q-enhanced uptake of IgG-fA, microglia were adhered to surfaces coated with MBL, SPA, C1q, HSA, BSA, or ferritin and treated with 100 µg/ml fucoidan before application of fA or IgG10-fA. As shown in Fig. 8A, ingestion of IgG10-fA () by primary rat microglia adhered to surfaces coated with the control proteins HSA, BSA, or ferritin was similar to that observed for microglia adhered to uncoated surfaces (e.g., see in Figs. 5 and 6). In contrast, interaction of microglia with immobilized C1qR_P ligands, C1q, MBL, or SPA led to a >2-fold enhancement of SR-independent uptake of IgG10-fA. Ingestion of fA alone () was not influenced by defense collagen interaction. A similar pattern of response was observed for ingestion of suboptimally opsonized EA_IgG; i.e., C1q, MBL, and SPA resulted in markedly higher levels of phagocytosis than did HSA, BSA, and ferritin (Fig. 8B), demonstrating that C1qR_P-mediated enhancement of Ab-mediated uptake is independent of the Ag (in this case A). Finally, the enhancement of phagocytosis of IgG10-fA opsonized with 75 µg/ml C1q was inhibited by pretreatment of both microglia (Fig. 8C) and BV-2 cells (data not shown) with an anti-C1qR_P but not with control Ab. This selective inhibition demonstrates that the enhancement of ingestion of immune complexes containing IgG and fA is mediated by C1qR_P.

View larger version (27K): [in this window] [in a new window]   FIGURE 8. The C1qRP ligands MBL and SPA also enhance uptake of IgG-fA. A, Two micromolar fA or IgG10-fA was added to rat microglia which were adhered to surfaces coated with 16 µg/ml of the indicated proteins and preincubated with 100 µg/ml fucoidan. Interaction with surfaces coated with C1q, MBL, and SPA led to greater internalization of IgG10-fA than interaction with surfaces coated with HSA, BSA, and ferritin. Uptake of fA was not influenced by any of the coating proteins. B, Ingestion of suboptimally opsonized EAIgG was markedly enhanced following interaction of rat microglia with surfaces coated with C1q, MBL, and SPA compared with surfaces coated with HSA, BSA, and ferritin. C, Microglia transiently permeabilized in the presence of Ab from preimmune serum (PI) or Ab raised against the C-terminal, intracellular domain of C1qRP (anti-C11) were adhered to surfaces coated with 16 µg/ml C1q or HSA, followed by application of fA or IgG-fA. Nonspecific Ab did not noticeably interfere with C1q-mediated enhancement of uptake of suboptimally opsonized IgG10-fA, whereas anti-C11 efficiently blocked the ability of C1q to enhance uptake of these targets.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The results reported here show that C1q substantially enhanced uptake of the less opsonized IgG10-fA/IgG25-fA but did not affect IgG100-fA ingestion, suggesting that at the highest concentration of anti-A used here to form IgG-fA (i.e., IgG100-fA), maximal phagocytosis was achieved. These observations are consistent with the ability of C1qR_P to specifically enhance uptake of suboptimally opsonized phagocytic targets (6, 8, 9). In the previous study of Maxfield and colleagues (7), complexes composed of synthetic fA and the monoclonal anti-A Ab 4G8 were taken up by mouse microglia at the same level in the absence of C1q as they were when C1q was bound to the complexes; i.e., no enhancement by C1q was observed. Our results are consistent with those data. In the Maxfield study (7), C1q was bound to immune complexes that contained a saturating concentration of 4G8 and therefore induced maximal FcR-mediated uptake without need for additional stimulus. It is also of note that the Maxfield study reported a 1.5-fold increase in uptake for IgG-fA in the absence of SR ligands. Similar experiments with 4G8 (an IgG2b) in our hands confirmed this observation (data not shown), whereas the polyclonal Ab used in the current study showed enhancement of uptake only in the presence of fucoidan (Fig. 4). A polyclonal Ab would contain Ab molecules with varying affinities for A as well as multiple Ab isotypes. Given that FcR subtypes differ both in their affinities for the various Ab isotypes and in their abilities to initiate phagocytosis (20), it is reasonable to predict differences in efficacy among both monoclonal and polyclonal Abs. Bard et al. (21) found that passive immunization of PDAPP mice with a polyclonal anti-A_1-42 Ab led to a greater reduction in amyloid burden than did the monoclonal anti-A Ab 10D5. Conversely, our polyclonal Ab was less efficient than the mAb 4G8 in engaging phagocytosis in culture. To the extent that immunization is likely to generate multiple clones of anti-A Ab and even multiple Ab isotypes (22), polyclonal anti-A Ab might be a better model of immunization in vivo.

It has been shown that the primary mode of microglial ingestion of A in culture systems is via SR (4), particularly for aggregated A (3). However, it is not established that clearance of A is mediated by a single SR in vivo, as Huang et al. (23) showed that knockout of the type A SR in human APP-transgenic mice did not heighten A deposition compared with APP transgenics that contained type A SR. This suggests that either other SR (e.g., type B or C) are capable of mediating this uptake or different receptors are involved in the in vivo situation. Thus, approaches that target only one SR-mediated mechanism of clearance of A as a means of counteracting A deposition in AD may meet with limited, if any, success. One recent study which has received substantial attention has been that of Schenk et al. (2), in which APP-transgenic mice were immunized with A peptide. The authors detected an increase in A-reactive Ab in the sera of immunized mice and reported decreased A deposits in their brains consistent with an increased clearance of both newly generated and already deposited A. One hypothesis for these observations is the occurrence of FcR-mediated uptake of the peptide. The in vitro results presented here demonstrate that microglia can internalize fA that is opsonized with Ab and that this uptake is independent of SR.

However, generation of Ab to self proteins in humans is rarely of benefit. The immune complexes that result can lead to inflammatory immune complex diseases such as nephritis and vasculitis, and autoantibodies have been shown to contribute to the pathology of neurodegenerative diseases such as multiple sclerosis, Rasmussen’s encephalitis, and Guillian-Barré disease (24, 25, 26). Ligation of FcR is frequently associated with induction of free radical species and proinflammatory cytokines (27, 28, 29). Thus, the potential inflammatory and oxidative stress generated by FcR ligation must be considered before immunizing humans with the A peptide to induce anti-A Ab as a therapeutic for AD. Interaction of myeloid cells with C1q enhances FcR-mediated phagocytosis in monocytes, macrophages, and microglia (6, 8, 30), but does not induce proinflammatory cytokine synthesis (N. Jasinskiene, R. Rochford, S. Ruiz, and A. J. Tenner, unpublished observations) or free radical generation (S. D. Webster, unpublished observations). Therefore, although it remains to be seen which FcR-mediated proinflammatory responses in the brain may be modulated by C1q, mechanisms that target C1qR_P function with the aim of enhancing FcR-mediated phagocytosis of IgG-fA may be of significant interest in paradigms which seek to counteract A deposition by the use of peptide immunization (i.e., less Ab and thus less FcR ligation may be sufficient to obtain equal clearance of the peptide). Furthermore, because specific titers of Ab in cerebrospinal fluid are orders of magnitude smaller than in serum (reviewed in Ref. 31), the ability of C1qR_P to enhance FcR-mediated uptake of IgG-fA containing low levels of anti-A Ab may be of particular relevance to immunization paradigms.

In addition to generation of free radical species and proinflammatory cytokines by interaction with FcR, immune complexes formed from Ab to self proteins can result in complement activation (reviewed in Ref. 32). It is notable that mice often exhibit inflammatory responses that are substantially different from those of humans. Mice in general show diminished complement activities such as C5 convertase activity (33) and a markedly altered acute phase response (34, 35). In addition, when compared with outbred colonies, inbred strains of laboratory mice show additional deficiencies in hemolytic complement activity (36, 37) which could have profound effects on the toxicity resulting from fA-mediated complement activation (in either the presence or absence of Ab). It is possible that many of the deleterious effects associated with AD complement activation that may not be apparent during the course of transgenic mouse experiments may be due to these differences. Similarly, detrimental complement-mediated consequences of anti-A Ab in humans may not be predictable by murine models, particularly in patients with significant existing amyloid pathology. It may be that recruitment of C1qR_P function will permit efficient clearance of A at Ab titers that are low enough to avoid significant A/anti-A-mediated complement activation in the microenvironment of the senile plaque.

The dramatic inhibition of A uptake by C1q observed previously has been hypothesized to derive from structural similarities between C1q and the type A SR and consequent steric hindrance of microglial access to A by C1q (5). In the current study, ingestion of fA and all IgG-fA by both primary and BV-2 microglia was diminished when much higher concentrations of C1q were added directly to the cells concurrently with the fA or IgG-fA (data not shown), suggesting that excess soluble C1q can also mediate interference of FcR-immune complex interaction. However, it remains to be determined whether such influences by soluble C1q are relevant in vivo, since the inhibitory concentrations of C1q were 300-fold greater than the known concentration of C1q in cerebrospinal fluid (38). At this time the concentration of free C1q in the interstitial fluid of the brain parenchyma has not been characterized.

Previous work has shown that the interaction of C1q with A can result in acceleration of A aggregation in the absence of C1r, C1s, and other complement proteins (39) and can lead to activation of complement when C1q is complexed with the serine proteases C1r and C1s (as the macromolecule, C1) in the presence of a functional complement system (40, 41). However, it has also long been known that C1q could be synthesized in the absence of the synthesis of other complement components (42), although C1q synthesis in the brain is a relatively newly appreciated event. Neuronal synthesis of C1q both in vitro and in vivo has been demonstrated as a result of diverse insults (43, 44, 45). Most recently, C1q was identified as one of the prominent differentially expressed genes during aging and as one likely to be responding to oxidative stress (46). These observations suggest that the synthesis of C1q may be a response to injury and that C1q and C1qR_P may play a protective role in the rapid clearance of either pathogenic agents or apoptotic cell debris or both. If damage is chronic or excessive, and other complement components become available locally (47, 48, 49), C1q in C1 would then mediate the activation of the complement system. Although this scenario remains to be proven, approaches that target C1qR_P function without the involvement of C1q or C1q-like molecules would be independent of any detrimental consequences of C1q-dependent enhancement of A aggregation or complement activation.

In summary, both primary microglia and an immortalized microglial cell line were found to exhibit SR-independent uptake of immune complexes composed of fA and anti-A Ab, and this mode of uptake was enhanced by the presence of C1q when immune complexes contained lower amounts of Ab. Animal studies have suggested that generation of anti-A Ab via peptide inoculation may constitute a viable approach to counteracting A deposition in AD. Our data suggest that mechanisms by which C1q enhances microglial clearance of A-containing immune complexes may represent a critical adjunct to inoculation therapies in humans. However, before generalizing applicability of transgenic data to the human system, further investigation of the effects of autoantibodies, and in particular complement activation by these Abs, is warranted.

	Acknowledgments

 
We acknowledge the excellent technical assistance of Christy Sweet and Karntipa Pisalyaput and thank Drs. Neil Cooper, Kurt Drickamer, and Dennis Voelker for their gifts of anti-A Ab, mannose binding lectin, and lung surfactant protein A, respectively.

	Footnotes

 
¹ This work was supported by American Federation for Aging Research Grant A-25906 (to S.D.W.) and National Institutes of Health Grants AG-17289 (to S.D.W.), AG-00538 (to A.J.T.), and P50 AG-16573-01.

² Address correspondence and reprint requests to Dr. Andrea J. Tenner, 3205 Biological Sciences II, Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697. E-mail address: atenner{at}uci.edu

³ Abbreviations used in this paper: AD, Alzheimer’s disease; A, amyloid -peptide; APP, amyloid precursor protein; C1qR_P, C1q receptor for enhanced phagocytosis; EA_IgG, erythrocyte/anti-erythrocyte Ab complexes; fA, fibrillar A; HSA, human serum albumin; MBL, mannose binding lectin; SPA, surfactant protein A; SR, scavenger receptor.

⁴ The complexes termed C1q-fA by Brazil et al. (7 ) in fact were composed of fA, 4G8 anti-A Ab, and C1q.

Received for publication December 11, 2000. Accepted for publication April 2, 2001.

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