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. Author manuscript; available in PMC: 2016 Dec 1.
Published in final edited form as: J Immunol. 2015 Oct 23;195(11):5309–5317. doi: 10.4049/jimmunol.1401195

IgM-Dependent Phagocytosis in Microglia is Mediated by Complement Receptor 3, not Fcα/μ Receptor

Jonathan R Weinstein *,†, Yi Quan *,†, Josiah F Hanson *, Lucrezia Colonna §, Michael Iorga *, Shin-ichiro Honda ‡, Kazuko Shibuya ‡, Akira Shibuya ‡, Keith B Elkon §, Thomas Möller *
PMCID: PMC4655136  NIHMSID: NIHMS728082  PMID: 26500348

Abstract

Microglia play an important role in receptor-mediated phagocytosis in the CNS. In brain abscess and other CNS infections, invading bacteria undergo opsonization with immunoglobulins (Ig) or complement. Microglia recognize these opsonized pathogens by Fc or complement receptors triggering phagocytosis. Here we investigated the role of Fcα/μ receptor (Fcα/μR), the less studied receptor for IgM and IgA, in microglial phagocytosis. We showed that primary microglia, as well as N9 microglial cells, express Fcα/μR. We also showed that anti-Staphylococcus aureus (SA) IgM markedly increased the rate of microglial SA phagocytosis. To unequivocally test the role of Fcα/μR in IgM-mediated phagocytosis we performed experiments in microglia from Fcα/μR-/- mice. Surprisingly, we found that IgM-dependent phagocytosis of SA was similar in microglia derived from wild-type (WT) or Fcα/μR-/- mice. We hypothesized that IgM-dependent activation of complement receptors might contribute to the IgM-mediated increase in phagocytosis. To test this we used immunologic and genetic inactivation of complement receptor 3 (CR3) components (CD11b and CD18) as well as complement factor-3 (C3). IgM-, but not IgG-, mediated phagocytosis of SA was reduced in WT microglia and macrophages following pre-incubation with an anti-CD11b blocking antibody. IgM-dependent phagocytosis of SA was also reduced in microglia derived from CD18-/- and C3-/- mice. Taken together, our findings implicate CR3 and C3, but not Fcα/μR, in IgM-mediated phagocytosis of SA by microglia.

Keywords: microglia, Fc Receptors, Fcα/μ receptor, phagocytosis, IgM, complement receptor 3

Introduction

Phagocytosis is a multi-step and receptor-mediated process. It is initiated by particle recognition and can be separated experimentally into two steps: (a) particle attachment to the cell surface and (b) particle ingestion by cells (1). Phagocytosis is mediated by a wide variety of cell surface receptors that bind directly or indirectly, through opsonins, to particles (2). Fc receptors, specifically the Fcγ receptor subtypes that recognize IgG, are well studied for their role in phagocytosis (3, 4). However, other immunoglobulins (Ig) including IgM (5, 6) and IgA (7) are also capable of opsonizing pathogens and playing a role in phagocytosis. A recently discovered phagocytosis related Fc receptor known as Fcα/μR (CD351), recognizes IgM and IgA but not IgG (8-10). It is a type 1 transmembrane protein with an extracellular Ig-like domain. Fcα/μR is expressed on B cells and macrophages, where it has been shown to mediate uptake of IgM-antigen immune complexes (8, 9). In addition to opsonization by antibodies, phagocytic objects (microorganisms or cells) can be opsonized by complement and recognized by complement receptors including CR1, CR3, CR4 and C1qR(P) (2, 11).

Microglia, the resident tissue macrophages of the CNS, are active sensors and versatile effector cells in the normal and pathologic brain (12). Microglia shift activity states depending on the surrounding microenvironment. Under normal conditions they are characterized by a small cell body with fine, ramified processes and low expression of surface antigens. In response to brain injury, ischemia and inflammatory stimuli, microglia rapidly transform into an activated phenotype associated with morphological changes, proliferation, migration to the site of injury, elaboration of both neurotoxic and neurotrophic factors as well as increased phagocytosis (12-14). Invading pathogens require opsonization by Ig and complement fixation for efficient recognition and phagocytosis by Fc and complement receptors in microglia (2, 15). Microglial Fc and complement receptors have been implicated in the pathophysiology of bacterial brain abscesses (16). Fc and complement receptors also represent potential molecular targets for pharmacologic therapy in Multiple Sclerosis (3), Alzheimer's (17-19) and Parkinson's disease (20).

While IgG-mediated phagocytosis (via Fcγ receptors) in the CNS is well described (3), there is little characterization to date of IgM-mediated phagocytosis in brain. Here we investigate IgM-induced phagocytosis of the bacterial pathogen Staphylococcus aureus (SA) in microglia and characterize the role of Fcα/μR in this process.

Materials and Methods

Solutions and Reagents

Fluorescein-labeled SA was obtained from Invitrogen Corporation. Anti-SA monoclonal antibodies (IgM clone 11-248.2 and IgG3 clone 11-232.3) were purchased from QED Bioscience, San Diego, CA. Anti-CD11b monoclonal antibody as well as isotype controls for both anti-SA and -CD11b antibodies were obtained from BD Biosciences, San Jose, CA. Recombinant mouse granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-γ (IFN γ) and interleukin-4 (IL-4) were purchased from R&D Systems, Minneapolis, MN. All solutions were freshly prepared from frozen stock solutions or lyophilized preparations. All materials were handled in a sterile manner using endotoxin-free microfuge tubes (Eppendorf/Fisher Scientific, Santa Clara, CA), polypropylene tubes (Becton Dickinson Labware, Franklin Lakes, NJ), polystyrene culture vessels (Becton Dickinson Labware), serological pipettes (Costar/Corning, Corning, NY), precision pipette tips (Rainin Instruments, LCC, Oakland, CA), water (Associates of Cape Cod), and phosphate buffered saline (PBS) (Gibco/Invitrogen, Carlsbad, CA).

Animals and cell culture

The mouse microglial cell line N9 was a gift of Dr. M. Righi, International School for Advanced Studies, Trieste, Italy, and was cultured in accordance with the original publication (21). In brief, cells were cultured in high glucose Dulbecco's Modified Eagle's Medium (DMEM), (Gibco/Invitrogen, Carlsbad, CA), supplemented with 10% heat inactivated fetal bovine serum (FBS), (Hyclone, Logan, UT) and penicillin/streptomycin (P/S), (50 I.U./50 μg/mL), (Mediatech/Corning, Manassas, VA). Cells were passaged weekly with 0.05% trypsin (Gibco/Invitrogen) and serum starved in macrophage serum-free medium (MSFM), (Gibco/Invitrogen) for at least 24 h before each experiment as detailed below.

The Fcα/μR-/- mice used for these studies were as previously reported (10). The CD18-/- (22) and C3-/- (23) mice were from Jackson laboratory, Bar Harbor, ME. All mice were on same genetic background (C57BL/6). Primary microglia (pMG) were prepared from the cortex of newborn (p4) wild-type (WT), Fcα/μR-/-, CD18-/- or C3-/- mice as previously described (24, 25). In brief, cortical tissue was carefully freed from blood vessels and meninges, digested with 50 ng/mL DNase, triturated, and washed. Cortical cells were cultured in DMEM/10% FBS with P/S plus 2 ng/ml GM-CSF (R&D Systems) for 11–50 d (media change every 3–4 d). Microglia were separated from underlying astrocytic monolayer by gentle agitation, spun down (100g for 10 min). Cell pellet was resuspended in DMEM/10% FBS with P/S plus 2 ng/ml GM-CSF and plated on BD Primaria™ culture dishes and plates (Falcon, St. Laurent, Quebec, Canada). Non-adherent cells were removed 30-60 min after plating by changing the medium and adherent microglia were incubated for 24 h in culture medium before being serum-starved in MSFM plus 0.2 ng/ml GM-CSF for 24 h.

Phagocytosis assays

Ig induced phagocytosis assays were done as previously described (26). In brief, after reconstitution according to manufacturer's instructions, fluorescein labeled SA (Molecular probes/Life Technologies, Grand Island, NY) were incubated overnight at 4°C with equimolar concentrations of different anti-SA or control antibody isotypes (20 μg specific anti-SA IgG/mg bacteria; 20 μg isotype IgG/mg bacteria; 120 μg specific anti-SA IgM/mg bacteria; 120 μg isotype IgM/mg bacteria). Before use, antibody-opsonized SA was washed with PBS once and then spun down and resuspended in MSFM. The various antibody/SA reaction mixes were then incubated with serum starved mouse pMG or N9 cells. After 30 min, extracellular fluorescence was quenched with 0.1% trypan blue (Gibco). Then pMG were dislodged from wells with PBS containing 0.25% trypsin plus 2 mM EDTA (Life Technologies) (5 min at 37°C) and intracellular fluorescence was determined and analyzed using a FACScan2 flow cytometer (BD Biosciences, San Jose, CA) and FlowJo™ software (Tree Star Inc., Ashland, OR). For analysis, we set a threshold for basal (non-antibody mediated) phagocytosis by processing cells using the isotype control antibody reaction mixes and setting the lower border of a marker gate to encompass only the 2.5% of total cellular events that had the highest median fluorescent intensity (MFI) values. This same gate was then applied to results from reaction mixes containing specific anti-SA IgG or IgM antibodies. This allowed us to determine percentage of cells that had undergone specific antibody-mediated endocytosis (‘true positives’). Data is presented as the ratio of true positives in a specific antibody group to that of the matching isotype control antibody group. We will refer to this ratio as ‘fold increase of phagocytosis’. In a few instances (Fig. 6B and Fig. 8), in order to facilitate a mechanistic comparison of the effects of anti-CD11b antibody on anti-SA IgM- and IgG-mediated phagocytosis of SA, we normalized the fold increase of phagocytosis for each Ig class to that induced by the corresponding anti-SA IgM or IgG alone and presented the data as percent of control. N9 cells were processed for phagocytosis assay in a similar manner to pMG except no trypsin was required for dislodgement. For CD11b blocking experiments, pMG were preincubated with 1 μg/mL anti-CD11b IgG or isotype control IgG for 1 h and then reaction mixes composed of fluorescein labeled SA combined with either: (i) anti-SA IgM, (ii) isotype control IgM, (iii) anti-SA IgG or (iv) isotype control IgG were added to the cells to assess phagocytosis.

Fig. 6. Effect of anti-CD11b blocking antibody on anti-SA IgM- or anti-SA IgG-mediated phagocytosis in wild-type microglia.

Fig. 6

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A. Representative flow cytometry histogram plots showing fluorescent intensity signal for populations of WT pMG that were pre-incubated with either anti-CD11b blocking antibody or isotype control antibody and then incubated with FITC-SA opsonized either by anti-SA IgM (black lines) or isotype control IgM (shaded histograms). Marker gates indicate percentage of anti-SA IgM-induced pMG with FITC fluorescent intensity signal greater than background (see Methods for detail). B. Bar graph quantifying effects of anti-CD11b blocking antibody or isotype control antibody on anti-SA IgM- or IgG-mediated increase in SA phagocytosis in pMG (see Methods for detail). For clarity of comparison, data is normalized to values with corresponding anti-SA IgM or IgG antibody alone, respectively, and presented as percent fold increase. n.s. indicates not significantly different compared with anti-SA IgM alone group. ***p<0.001 compared to anti-SA IgM antibody alone. ###p<0.001 compared to anti-SA IgG antibody alone. Total N = 6; two wells per experimental group from three separate experiments.

Figure 8. Quantification of IgM-dependent phagocytosis of Staphylococcus aureus (SA) in wild-type vs. C3-/- microglia.

Figure 8

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Bar graph quantifying anti-SA IgM- (A) or IgG- (B) dependent fold increase in SA phagocytosis (relative to isotype control IgM) (see Methods for detail) in primary mouse microglia (pMG) derived from either WT or C3-/- mice. For clarity of comparison, data is presented as percent control of corresponding anti-SA IgM or IgG antibody in WT cells. *P<0.05 vs. WT group in A (unpaired t-test). n.s. indicates no significant difference between WT and C3-/- in B. Total N = 6 – 15 samples per group from at least three separate experiments.

We also used microscopy to confirm phagocytosis in serum starved mouse pMG. After 30 min incubation with each of the four SA/antibody reaction mixes, extracellular fluorescence was quenched with trypan blue. Primary microglia were fixed, counterstained with DAPI and then imaged and photographed under both fluorescent and differential interference contrast (DIC) illumination using a Zeiss Axiovert 200M microscope.

Quantification of cell surface expression of Fcα/μ receptor

To quantify Fcα/μR cell surface protein expression, pMG or N9 cells were cultured on poly-ornithine (Sigma-Aldrich, St. Louis, MO) coated plates in MSFM and cells were dislodged without trypsin using 2 mM EDTA/PBS. Cells were fixed for 10 min at room temperature in a phosphate buffer containing 2% paraformaldehyde/0.6% methanol (Fisher Scientific, Kent, WA), 2.5 mM sodium periodate (Fisher) and 15 mM L-lysine (Sigma-Aldrich) and then blocked with 1 mg/ml ice-cold mouse sera (BD Biosciences) plus 50 μg/ml unlabeled streptavidin (BD Biosciences) in PBS. Following a wash step, primary antibody incubations were carried out with either 50 μg/ml rat anti-Fcα/μR monoclonal antibody (clone TX-7) (27) or isotype control (BD Biosciences). For detection we used biotinylated-mouse anti-rat IgG 2b (BD Biosciences) followed by 2 μg/ml PE-Cy7-streptavidin (BD Biosciences). Staining was quantified on a FACSAria™ cytometer/sorter (BD Biosciences) and analyzed with FlowJo™ software (Tree Star). For our primary cultures, we first selected confirmed pMG by flow cytometry based on their expression of CD11b (96% of total cell population).

RNA Isolation, Reverse Transcription and Quantitative Real-Time PCR

To detect the mRNA expression of Fcα/μR in mouse microglia, RNA isolation (including on-column DNase digestion), reverse transcription and quantitative Real-Time polymerase chain reaction (qRT-PCR) were done as previously described (28). In brief, qRT-PCR was performed using the 7500 Real Time PCR System (Applied Biosystems). SYBR GreenER qPCR SuperMix Universal Kit (Invitrogen) was used to amplify Fcα/μR and endogenous control HPRT (26). The following components were combined per 40 μl reaction volume: more than 1 μg cDNA, 20 μl SYBR GreenER qPCR SuperMix, 50 nM ROX, 500 nM mouse Fcα/μR forward primer CTG CTT CTA ATT GCT GCT CTG and reverse primer GCT TAT CTG GTA GGA AAT GTG TC or 200 nM HPRT primers. Cycling conditions were as follows: (i) 50°C for 2 min; (ii) 95°C for 10 min; (iii) 40 cycles, with each cycle consisting of 95°C for 15 s and 60°C for 1 min. Fluorescent data was acquired at the 60°C step. Melting curve analysis was carried out to verify single species PCR products. All the experiments had a ‘no template’ negative control and only intron-spanning primers were used. Data was analyzed using Sequence Detection Software v1.3 (Applied Biosystems) as previously described (28). For qRT-PCR quantifying expression of CD11b, CD18 and C3 mRNA transcripts, singleplex amplification was performed on a StepOnePlus Real-time PCR System (Applied Biosystems). Cycling conditions consisted of: (i) 95°C for 10 min, (ii) 50 cycles of 95°C for 10 s, 60°C for 30 s. Primer and probe sequences are shown in Supplementary Table 1. Relative gene expression was calculated using the ΔΔCt method in which samples were normalized to the Ct geometric mean of the housekeeping genes (HPRT, EIF4a2 and ATP5b). Numerical data is given as the mean ± S.E.M of the normalized mean values from each independent experiment. All experiments were carried out in triplicate.

Statistical analysis

Statistical evaluation was carried out using PRISM software (GraphPad, San Diego, CA). Differences between two groups were analyzed by the Student's t-test. Multiple comparisons were made using one-way ANOVA with Bonferroni post-test. P<0.05 was considered to be significant. Data are given as mean ± S.E.M.

Results

Surface expression of the Fcα/μ receptor in microglia

Uptake of IgM- and IgA-, but not IgG-antigen immune complexes in macrophages is mediated by Fcα/μR (8). We assessed expression of Fcα/μR by flow cytometry and found robust cell surface expression in both pMG (Fig. 1A) and N9 cells (Fig. 1B). Expression of Fcα/μR in both pMG and N9 cells was confirmed by qRT-PCR (Fig. 4A).

Figure 1. Cell surface expression of Fcα/μR protein in microglia.

Figure 1

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Flow cytometry histogram plots showing fluorescent intensity signal for populations of CD11b+ primary microglia (pMG) (A) or N9 cells (B) that are either unstained (black shaded histograms) or stained with anti-Fcα/μR (black line) or isotype control (shaded histogram) antibodies. Detection was with biotinylated-mouse anti-rat IgG followed by PE-Cy7-streptavidin as described in Methods. Marker gates in both A and B indicate percentage of anti-Fcα/μR antibody stained-microglia that had fluorescent intensity readings greater than isotype control background (i.e. signal greater than 97.5% of microglia stained with isotype control antibody). Legend below figure shows median fluorescent intensity (FI) values for both pMG and N9 cells either unstained or stained with anti-Fcα/μR or isotype control (ISO) antibodies. Results shown are representative data from three experiments.

Figure 4. Cytokine-induced regulation of Fcα/μR mRNA and IgM-mediated phagocytosis in microglia.

Figure 4

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A. Results of qRT-PCR experiment on RNA extracted from mouse primary microglia (pMG) or N9 cells that were either unstimulated (control) or stimulated with 10 ng/ml IL-4 or 10 U/ml IFNγ for 24 hours. Findings are presented as fold increase in steady-state levels of Fcα/μR mRNA in the stimulated groups relative to unstimulated control. **P<0.01 vs. corresponding unstimulated controls. B. Bar graph quantifying IL-4 or IFNγ-induced changes in anti-SA IgM- or isotype control IgM-induced fold changes in SA phagocytosis in pMG (see Methods for detail). **P<0.01 vs. isotype control. ###P<0.001 vs. anti-SA IgM signal in unstimulated control group. Total N = 6 (two wells per experimental group from three separate experiments).

IgM-induced phagocytosis in microglia

To test the function of Fcα/μR in mouse pMG, we assessed the phagocytosis of IgM-opsonized SA and found that IgM opsonization increased phagocytosis about 18-fold over isotype IgM control (Fig. 2A,C). In N9 cells, anti-SA IgM induced a 12-fold increase in phagocytosis relative to isotype control IgM (Fig. 2D). Differing affinities/avidities of IgM and IgG for the same antigen in our experimental set up preclude direct comparisons of functional effects induced by different Ig classes. Nevertheless, we considered characterization of both IgM- and IgG-mediated phagocytosis of SA important in this setting to allow us to examine Ig class-specific mechanisms involved. Thus, we used a SA specific IgG to quantify IgG-mediated phagocytosis. The rate of IgG-opsonized SA increased relative to that of isotype IgG control (Fig. 2B,C) by a factor of about 3-fold (Fig. 2C). In N9 cells, IgG-induced phagocytosis increased 7-fold relative to the corresponding isotype control IgG group (Fig. 2D). In order to visualize internalization of SA we performed fluorescent microscopy. Mouse pMG exposed to reaction mixes containing either specific anti-SA IgM or IgG qualitatively accumulated appreciably greater amounts of SA in their cytoplasms compared with pMG exposed to isotype control antibody reaction mixes (Fig. 3). Absence of non-cellular associated SA fluorescence confirmed the effectiveness of trypan blue quenching (Fig. 3). These imaging findings are consistent with and complement the quantitative flow cytometry results from Fig. 2.

Figure 2. Quantification of immunoglobulin-dependent phagocytosis of Staphylococcus aureus (SA) in microglia.

Figure 2

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Flow cytometric histogram plots showing: A. tonic/baseline (shaded black), isotype control IgM (shaded gray)- and anti-SA IgM (black line)-dependent phagocytosis in primary mouse microglia (pMG) or B. tonic/baseline (shaded black), isotype control IgG (shaded gray)- and anti-SA IgG (black line)-dependent phagocytosis in pMG. Marker gates in both A and B indicate percentage of anti-SA antibody-dependent pMG with FITC fluorescent intensity signal greater than background (see Methods for detail). C. Bar graph quantifying anti-SA IgM or IgG-dependent fold increase in SA phagocytosis (relative to isotype control IgM or IgG, respectively) in pMG (see Methods for detail). D. Bar graph quantifying anti-SA IgM or IgG-dependent fold increase in SA phagocytosis (relative to isotype control IgM or IgG, respectively) in mouse microglia cell line N9. *P<0.05 vs. isotype control immunoglobulin group, **P<0.01 vs. isotype control immunoglobulin group. Total N = 6 (two wells per experimental group from three separate experiments).

Figure 3. Fluorescent microscopy images demonstrating immunoglobulin-dependent phagocytosis of Staphylococcus aureus (SA) in microglia.

Figure 3

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A. Phagocytosis of isotype control (iso) IgM opsonized FITC-SA in primary mouse microglia (pMG). B. Phagocytosis of anti-SA IgM opsonized FITC-SA in pMG. C. Phagocytosis of iso IgG opsonized FITC-SA in pMG. D. Phagocytosis of anti-SA IgG opsonized FITC-SA in pMG. Phagocytosis assay with SA opsonized by indicated antibody was carried out as described in Methods. Fluorescent and differential interference contrast (DIC) images were acquired to show immunoglobulin-induced phagocytosis. Results shown are representative images from three experiments. For each experiment, there were three replicates in each treatment group and three different fields of view were imaged in each replicate.

Cytokine-induced regulation of Fcα/μR mRNA and IgM-induced phagocytosis in microglia

We previously demonstrated that T-cell derived cytokines including IL-4 and IFNγ differentially regulated expression of Fcγ receptor sub-types in microglia (26). These same cytokines modulated IgG-mediated phagocytosis in microglia in a manner that was consistent with: (i) their effects on Fcγ receptor sub-type expression and (ii) the known functions of the specific Fcγ receptor sub-types that were regulated (26). We sought to determine here if a similar pattern would emerge for cytokine-induced regulation of Fcα/μR gene expression and IgM-mediated phagocytosis of SA in microglia. Treatment with IL-4 for 24 hours did not significantly alter expression of Fcα/μR steady-state mRNA levels in either pMG or N9 cells. However treatment with IFNγ did induce a marked decrease in Fcα/μR expression in both pMG and N9 cells (Fig. 4A). In contrast to the absence of an effect of IL-4 on Fcα/μR expression, treatment with IL-4 for 24 hours significantly reduced IgM-mediated phagocytosis of SA in pMG. Treatment with IFNγ despite its dramatic lowering of Fcα/μR mRNA expression, had no effect on IgM-mediated phagocytosis (Fig. 4B). Thus, for both IL-4 and IFNγcytokine-induced regulation of Fcα/μR occurred in a manner that did not readily explain the effects of these same cytokines on IgM-mediated phagocytosis of SA in microglia.

Effect of genetic deletion of Fcα/μR on IgM-induced phagocytosis in microglia

To unequivocally test for the role of Fcα/μR in IgM-mediated phagocytosis of SA in microglia, we cultured pMG derived from either WT or Fcα/μR-/- mice and characterized IgM-induced phagocytosis (Fig. 5). Surprisingly, we found no differences in IgM-induced phagocytosis of SA in WT vs. Fcα/μR-/- microglia (Fig. 5). Thus, absence of Fcα/μR had no effect on IgM-induced phagocytosis of SA in mouse microglia.

Figure 5. IgM-induced phagocytosis in wild-type vs Fcα/μR-/- derived microglia.

Figure 5

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A. Flow cytometry histogram plots showing fluorescent intensity signal for populations of wild-type (WT) and Fcα/μR-/- mouse primary microglia (pMG) that were incubated with FITC-SA opsonized either by anti-SA IgM (black lines) or isotype control IgM (shaded histograms). Marker gates indicate percentage of anti-SA IgM-induced pMG with FITC fluorescent intensity signal greater than background (see Methods for detail). B. Bar graph quantifying anti-SA IgM-induced fold increase in SA phagocytosis (relative to isotype control IgM) in WT or Fcα/μR-/- pMG (see Methods for detail). n.s. indicates no significant difference between WT and Fcα/μR -/-. Total N = 6 (two wells per experimental group from three separate experiments).

Role for complement receptor 3 in IgM-mediated phagocytosis in microglia

Besides its reported role in Fcα/μR-mediated phagocytosis, IgM is also a potent activator of complement (29). To reconcile our observations with current knowledge of microglial phagocytosis, we hypothesized that IgM opsonization of SA leads to IgM-mediated complement activation and subsequent phagocytosis via a complement receptor mediated mechanism, specifically via CR3. Complement receptor 3 (CR3) is a heterodimeric protein composed of CD11b and CD18 (30). CR3 plays a well-documented role in the phagocytic function of microglia in CNS diseases (30). To test our hypothesis, we first examined the effect of anti-mouse CD11b antibody on IgM-induced phagocytosis of SA. Pre-incubation of pMG with anti-CD11b antibody inhibited IgM-induced phagocytosis of SA by ∼70% whereas pre-incubation with an isotype control antibody resulted in no change in IgM-induced phagocytosis (Fig. 6A,B). In contrast, pre-incubation with anti-CD11b blocking antibody did not inhibit IgG-mediated phagocytosis of SA in microglia (Fig. 6B). To confirm that CR3 plays a role in IgM-mediated phagocytosis of SA, we determined if deletion of the gene encoding CD18 altered IgM-mediated phagocytosis in pMG. Genetic deletion of CD18 attenuated IgM-mediated phagocytosis of SA in pMG by ∼60% relative to WT (Fig. 7).

Figure 7. IgM-induced phagocytosis in wild-type vs. CD18-/- microglia.

Figure 7

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A. Representative flow cytometry histogram plots showing fluorescent intensity signal for populations of WT or CD18-/- pMG that were incubated with FITC-SA opsonized either by anti-SA IgM (black lines) or isotype control IgM (shaded histograms). Marker gates indicate percentage of anti-SA IgM-induced pMG with FITC fluorescent intensity signal greater than background (see Methods for detail). B. Bar graph quantifying anti-SA IgM-induced fold change in SA phagocytosis (relative to isotype control IgM) in WT and CD18-/- pMG (see Methods for details). **P<0.01 in WT vs. CD18-/-. Total N = 6 (two wells per experimental group from three separate experiments).

Role for complement factor 3 in IgM-mediated phagocytosis in microglia

Complement factor C3 is a central component of the complement system and promotes phagocytosis of pathogens by binding to cellular complement receptors including CR3 (31). Microglia have been previously reported to express and secrete C3 (32). In order to determine if C3 plays a role in IgM-mediated phagocytosis of SA in microglia we compared rates of phagocytosis in microglia derived from WT vs. C3-/- mice. We found a significant (∼30%) reduction in the rate of IgM-mediated phagocytosis in C3-/- microglia (Fig. 8A). In contrast, we found no difference in the rate of IgG-mediated phagocytosis of SA in WT vs. C3-/- microglia (Fig. 8B). It is important to note that the microglia used for these studies had been cultured exclusively in heat-inactivated serum prior to serum starvation thus reducing the potential exposure to any exogenous source of complement. It is also important to emphasize that the microglia were thoroughly washed and serum-starved prior to the phagocytosis assays, thus it is unlikely that any potential residual C3 in the heat-inactivated serum would be present in sufficient quantities to affect the results. Thus, these data strongly suggest that microglial production of C3 is required for optimal IgM-, but not IgG-, mediated phagocytosis of SA in microglia.

Cytokine-induced regulation of CD11b, CD18 and C3 mRNAs in microglia

Given that the rate of IgM-mediated phagocytosis of SA in microglia could be altered by T-cell derived cytokines such as IL-4 (Fig. 4B), as well as the fact that optimal IgM-mediated phagocytosis of SA was dependent on both CR3 (Figs. 6, 7) and C3 (Fig. 8), we sought to determine if T-cell derived cytokines could alter expression of CR3 components CD11b and CD18 as well as C3. To determine this, we carried out qRT-PCR on RNA extracted from WT pMG that had been treated for 24 hours with either IL-4 or IFNγ and quantified expression of steady state levels of mRNA for CD11b, CD18 or C3. IL-4 significantly reduced expression of CD11b by 62% (Fig. 9A) but had no effect on CD18 or C3 mRNA species (Fig. 9B-C). It is possible that this IL-4 induced reduction in CD11b expression contributed to the IL-4 induced functional down-regulation of IgM-mediated phagocytosis of SA (Fig. 4B). In contrast, IFNγ markedly increased the mRNA levels of the C3 ligand by >20-fold (Fig. 9C) but simultaneously decreased the mRNA levels for CR3 receptor components CD11b and CD18 by 63 and 92%, respectively (Fig. 9A-B). It is possible that these striking, but directionally divergent, IFNγ-induced effects on ligand and receptor expression essentially ‘canceled each other out’ and resulted in no net change in the IgM-mediated phagocytic function of the cells (Fig. 4B).

Figure 9. Expression and regulation of CD11b, CD18 and C3 mRNAs in microglia.

Figure 9

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qRT-PCR on RNA extracted from mouse primary microglia (pMG) that were either unstimulated (control) or stimulated with 10 ng/ml IL-4 or 10 U/ml IFNγ for 24 hours. Findings are presented as fold increase in steady-state levels of CD11b (A), CD18 (B) and C3 (C) mRNA species in the stimulated groups relative to unstimulated controls. *P<0.05 vs. corresponding unstimulated controls. Total N = 3 – 5 samples per stimuli from three separate experiments.

Discussion

The key findings in this study include the following: (i) microglia express the IgM receptor Fcα/μR at their cell surface, (ii) genetic deletion of Fcα/μR does not alter IgM-mediated phagocytosis of SA in microglia, (iii) disruption of another phagocytic receptor, CR3, in microglia (via either antibody blockade or genetic deletion) significantly attenuates IgM-, but not IgG-, mediated phagocytosis of SA and (iv) genetic deletion of complement factor-3 (C3) significantly reduces IgM-, but not IgG-, mediated phagocytosis of SA. Taken together, these findings suggest that biologically relevant IgM-mediated phagocytosis in brain can be carried out by microglia and that this process is dependent on CR3 and, at least in part, on C3 as well, but not on Fcα/μR.

This is the first demonstration of Fcα/μR expression in microglia. Our cell surface expression data (Fig. 1) along with the qRT-PCR data shown in Fig. 4A demonstrate that microglia express this less investigated receptor. We have previously shown that T-cell derived cytokines such as IFNγ and IL-4 can differentially regulate Fcγ receptor sub-type expression in microglia (26). These same cytokines regulated IgG-mediated phagocytosis in a manner that could be predicted based on both the direction of regulation (up or down) and the signal transduction properties of the Fcγ receptors that they regulated (26). Unlike the Fcγ receptors, Fcα/μR has no canonical cytoplasmic activating or inhibitory signaling domain (33). However, the Fcα/μR is critical for IgM-mediated phagocytosis in selected peripheral immune cell populations (8, 34). Therefore, we hypothesized that the rate of IgM-mediated phagocytosis would vary in a manner directly proportional to Fcα/μR expression. However, we found that IFNγ markedly reduced Fcα/μR expression (Fig. 4A), but had no effect on IgM-induced phagocytosis of SA (Fig. 4B). Furthermore, we found that IL-4 induced a significant reduction in IgM-mediated phagocytosis of SA (Fig. 4B), but had no effect on Fcα/μR mRNA expression (Fig. 4A). While we could not exclude the possibility that these cytokines might have effects on other components of the cellular phagocytic machinery, these findings made us question the role of Fcα/μR in IgM-induced phagocytosis in microglia.

We went on to demonstrate here that IgM-induced phagocytosis was intact in microglia derived from mice with genetic deletion of Fcα/μR (Fig. 5). This finding confirmed that Fcα/μR did not play a major role in IgM-induced phagocytosis of SA in microglia. Our findings contrast with those of a previous study demonstrating that: (i) transfection of Fcα/μR expression vector into pro-B-cell line BaF3 induced endocytosis of IgM-coated fluorescent beads and (ii) incubation with anti-SA IgM, but not anti-SA IgG, resulted in phagocytosis of FITC-SA in B220+ splenic lymphocytes (8). This functional discrepancy could be due to cell-type specific variation between microglia and splenocytes (35). One key difference may be the level of expression of Fcα/μR, as it is much lower in brain than in spleen (8) and also is lower in our cultured microglia than in acutely isolated splenocytes (data not shown).

Microglia express and secrete complement factor C3 (18) and the latter has been implicated in microglial phagocytosis (18, 36). Microglia also express CR3, the cell surface receptor for C3 (36). Thus it seemed plausible that complement pathway activation and complement receptors could play a role in the IgM-mediated phagocytosis of SA we described in Figs. 2 & 3. Our finding that CR3 mediated a significant portion of the IgM-induced response (Figs. 6 & 7) is consistent with previous reports implicating CR3 in IgM-mediated phagocytosis of the yeast Cryptococcus neoformans (37) and the parasitic protozoa Trypanosoma congolense (38) in macrophages as well as our own data on bone marrow derived macrophages (Supplementary Fig. 1). In Fig. 6B, we show that pre-incubation with anti-CD11b blocking antibody inhibits IgM-, but not IgG-, mediated phagocytosis of SA in microglia. This Ig class-specific differential effect of the anti-CD11b blocking antibody points to CR3-dependent elements in the IgM mediated mechanism that may not be in effect for IgG-mediated phagocytosis (or at least not IgG3-mediated phagocytosis). The latter may be related to class/sub-class differences in the efficiency of complement fixation (29, 39). Another caveat to consider is that complement receptor and Fc-receptor coligation can produce cooperative effects (2, 40, 41), so the concentration of target-specific IgG does have an impact on C3b/iC3b mediated phagocytosis (2, 40, 41). Thus it is possible that at lower concentrations of anti-SA IgG we might have seen a phagocytosis-inhibiting effect of the anti-CD11b blocking antibody. Nevertheless, the inhibitory effect of the anti-CD11b blocking antibody on IgM-mediated phagocytosis is clear (Fig. 6) and is consistent with our findings in the CD18-/- microglia (Fig. 7).

We found that microglial production of C3 is required for optimal IgM-, but not IgG-, mediated phagocytosis of SA in microglia (Fig. 8). These findings are consistent with our hypothesis that complement (and in particular C3) is a key link between IgM-opsonized SA and CR3-mediated phagocytosis in microglia. As noted above, microglia can produce and release C3. We confirmed robust baseline C3 mRNA expression in mouse pMG by qRT-PCR (Fig. 9). C3 release is up-regulated by cytokines such as IL-1β, IL-6 and TNFα (42) and SA induces these cytokines in microglia through activation of Toll-like receptor-2 (TLR2) (43). Consistent with this, we found here that another pro-inflammatory cytokine, IFNγ, can markedly increase C3 expression (Fig. 9). We considered an alternative explanation that serum-derived residual active complement factors (including C3), possibly cell- or extracellular matrix-bound (44), might represent an artifactual source of C3 for our pMG in tissue culture. However, we purposefully used only heat-inactivated serum to grow the microglia and then thoroughly washed and serum starved the microglia prior to phagocytosis assay making this possibility less likely. Thus, our data point to microglia themselves as the primary source of C3 in these studies.

Our data indicate that the effect of genetic deletion of C3 on IgM-mediated phagocytosis of SA was only partial (Fig. 8) and considerably less robust than the effects induced by inhibiting CR3 (Figs. 6 & 7). Approximately 70% of the phagocytic activity persists in microglia from C3-/- mice. One possible explanation is that CR3 is a multifunctional and promiscuous receptor that binds to many different ligands in addition to the C3 fragment iC3b, including fibrinogen, ICAM-1 and heparin (45-48). The latter has been shown to facilitate the ligation of IgM in immune complexes to at least one other complement receptor (49). Interestingly, CR3-mediated myelin phagocytosis has both complement-dependent and -independent components (30). Even more relevant to the current dataset, CR3 mediates IgM-dependent, yet complement-independent, phagocytosis of intracellular pathogens via IgM-induced alteration of the microbial capsule facilitating direct CR3 interaction (37). Thus, there is precedent for our finding that a component of the CR3-mediated, IgM-dependent phagocytosis of SA seen here is independent of C3 itself.

In clinical scenarios such as bacterial meningitis and brain abscess, pyogenic bacteria such as SA induce direct damage to CNS parenchyma and subsequent tissue necrosis. Microglia play a central role in the pathology of brain abscess by responding to bacteria with robust and persistent production of pro-inflammatory mediators that augment injury and expand the area of parenchymal involvement (50, 51). Microglial phagocytosis of bacteria may be an important mechanism for reducing and suppressing the persistent inflammatory response that occurs in CNS infection (52, 53). Phagocytosis reduces similar pro-inflammatory responses in peripheral macrophages and also activates anti-inflammatory pathways (54). Our data suggest that future studies examining the effects of complement and microglial CR3 on brain abscess and CNS infection pathophysiology may be warranted.

Beyond CNS infections, Ig-mediated microglial phagocytosis and complement function are thought to play a key role in the pathophysiology of many other neurological diseases including Alzheimer's disease (AD) (55) and Multiple Sclerosis (MS) (56). Given the noted differences in how phagocytic cells respond to different targets (1, 2), we need to be cautious in extrapolating from our data on Ig-mediated phagocytosis of SA to other neurological diseases. Nevertheless, our identification of C3 and CR3 as modulators of IgM-mediated phagocytosis in microglia adds to a growing body of literature implicating IgM, complement and CR3 in a variety of CNS pathologies.

Supplementary Material

1

Acknowledgments

We thank Dr. Shahani Noor for her assistance with analysis of flow cytometry data and Dr. Ashley McDonough for review of the manuscript.

Funding sources: American Heart Association Grant-In-Aid 0750078Z (TM), NIH/NIAMS grant F32AR065837 (LC), NIH/NINDS grant NS065008 (JRW)

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