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Immunology and Microbiology  |   October 2013
Endogenous IgG Affects the Cell Biology of RPE Cells and Involves the TLR4 Pathway
Author Affiliations & Notes
  • Na Niu
    Department of Pathology, Weifang Medical University, Weifang, China
  • Jie Zhang
    Department of Human Anatomy, Weifang Medical University, Weifang, China
  • Michael A. McNutt
    Department of Pathology, School of Basic Medical Sciences, Peking University, Beijing, China
  • Correspondence: Na Niu, Department of Pathology, Weifang Medical University, Weifang, 261053, China; naniubjmu@gmail.com
Investigative Ophthalmology & Visual Science October 2013, Vol.54, 7045-7052. doi:10.1167/iovs.13-12531
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      Na Niu, Jie Zhang, Michael A. McNutt; Endogenous IgG Affects the Cell Biology of RPE Cells and Involves the TLR4 Pathway. Invest. Ophthalmol. Vis. Sci. 2013;54(10):7045-7052. doi: 10.1167/iovs.13-12531.

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Abstract

Purpose.: RPE is a key component of the blood-ocular barrier (BOB) and is equipped with immunological molecules such as toll-like receptors (TLRs) and complement receptors, which together orchestrate the innate and adaptive immunity of the eye. Immunoglobulin G (IgG) in the aqueous humor and vitreous body has traditionally been thought to be derived from serum via transcytosis across the BOB. Our previous work validated production of endogenous IgG by RPE cells locally. However, the function and role of this IgG in the intraocular immunity is poorly understood.

Methods.: After confirming IgG production in a human RPE cell line (ARPE-19) with immunofluorescence, in situ hybridization, and RT-PCR, we further investigated the function of endogenous IgG in RPE biology with MTS, flow cytometry, and cell invasion analysis after downregulation of IgG by siRNA. Involvement of the TLR4 pathway was also studied using Western blot, ELISA and confocal microscopy.

Results.: Endogenous IgG is crucial for support of proliferation, mitosis, migration, and inhibition of apoptosis of RPE. Moreover, production of endogenous IgG by RPE is regulated by the TLR4 pathway in a concentration- and duration-dependent manner, and IgG affects the activation of the TLR4 pathway in a synergistic manner. Activation of the FcγR I pathway and production of IL-10 could be induced by IgG derived from RPE.

Conclusions.: These data suggest that endogenous IgG may be a molecule that is essential for the physiological function of RPE, and suggest IgG is important for regulating intraocular immune responses under physiologic and pathologic conditions.

Introduction
RPE is a single cell layer derived from neural ectoderm residing between the neural retina and the blood-rich choroid. 1 As RPE cells are neuronal progenitor-like cells, they have the capacity for proliferation, phagocytosis, migration, and dedifferentiation when stimulated under pathologic or physiologic conditions. 24 Serving as a resident antigen-presenting cell in the retina, this epithelial layer is a rich source of proinflammatory or anti-inflammatory molecules. Moreover, as it is endowed with toll-like receptors (TLRs), complement receptors, FcγR I (CD64), and MHC class I and II molecules, 1,3 it plays a pivotal role in the immune homeostasis and integration of the innate and adaptive immune responses of the eye. Damage and atrophy of RPE cells result in several types of retinopathy such as AMD, which is the leading cause of blindness in elderly adults in developed countries. 1  
TLRs are a family of evolutionary conserved molecules that recognize microbial pathogens. They serve as a first line of defense, initiate innate immune responses, and regulate adaptive immune reactions. 5 RPE cells are equipped with several subtypes of TLRs and among these TLR4 has been well described in physiologic and pathologic processes. 68 When triggered by the ligand lipopolysaccharide (LPS), TLR4 is activated and functions in a Myd88-dependent or -independent manner. Myd88 and toll/interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-β (TRIF) are the adaptor molecules for Myd88-dependent and -independent function, respectively. The nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) pathway is the terminal pathway that brings about production of several inflammatory cytokines such as TNF-α and IL-6. 6  
The TLR4 pathway has received a great deal of attention regarding its regulation of the immunologic balance in the eye, and the chronic inflammation caused by excessive activation of this pathway is known to be involved in a number of retinal disease such as AMD and endophthalmitis. 1,6  
IgG is the most abundant and important immunoglobulin in the immune system and has traditionally been thought to be produced by mature B-lymphocytes only. As it was observed to be a molecule normally present in the aqueous humor and vitreous body of the human eye, it was presumed to be transcytosed across the blood-ocular barrier (BOB) from the serum. 913 However, Murray et al. 12 and Bloch-Michel et al. 13 proposed there is local production of IgG in the eye. They studied patients with Fuchs heterochromic cyclitis and senile cataracts and found the relative concentration ratios of IgG/albumin in the eye were increased independent of the serum IgG concentration. 12,13 Our previous study verified local synthesis of IgG by several intraocular cells including the ciliary epithelium, retinal ganglion cells, and the retinal RPE cells. 14 Here, we further investigate the function of this endogenous IgG in an RPE cell line, ARPE-19. Our results show that endogenous IgG is crucial for the proliferation, apoptosis, mitosis, and migration of RPE cells. However, here we must note that this study involves only the function of IgG in RPE, and not in other intraocular cells. The function of IgG in these other cells is not known and needs further investigation. However, most importantly, IgG it is involved with the TLR4 pathway in a synergistic manner. 
Materials and Methods
Cell Lines
The ARPE-19 cell line (ATCC, Manassas, VA) was a gift from Dr. Xiaoguang Dong of Shandong Eye Institute. It was cultured in DMEM:F12 medium (Gibco, Carlsbad, CA) with 10% fetal bovine serum containing ultralow IgG (Gibco). The study was approved by the Ethical Committee of Weifang Medical University. 
Immunofluorescence and Confocal Microscopy
To investigate the expression of IgG in RPE cells and translocation of NFκB, immunofluorescence staining was performed on ARPE-19 cells as described previously. 15 Primary rabbit anti-human IgG, κ chain (Igκ), and p-NFκB p65, mouse anti-human cellular retinaldehyde-binding protein (CRALBP, a marker for RPE) and IgG (γ chain specific, Igγ) antibodies were used and their specific features are listed in Supplementary Table S1. Goat anti-rabbit IgG-tetramethyl rhodamine isothiocyanate (1:100; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and goat anti-mouse IgG-fluorescein isothiocyanate (1:100; Jackson ImmunoResearch Laboratories, Inc.) were used as secondary antibodies. Slides were evaluated with a confocal microscope (FV1000; Olympus, Tokyo, Japan) after mounting with medium containing DAPI (Sigma-Aldrich, St. Louis, MO). 
In Situ Hybridization
To confirm IgG expression in ARPE-19 at the mRNA level, in situ hybridization (ISH) was performed as previously described. 16 The specific cRNA antisense probe directed against the human immunoglobulin G1 heavy chain was the same probe used and well characterized in our previous study of the eyes. 14  
RT-PCR and Real-Time PCR
Total RNA was extracted from ARPE-19 cells with TRIzol reagent (Invitrogen, Carlsbad, CA). Contamination of genomic DNA in RNA samples was excluded with RQ1 RNase-free DNase (Promega Corp., Madison, WI). Five micrograms of total RNA isolated was reverse transcribed with the reverse transcriptase kit (SuperScript III; Invitrogen) according to the manufacturer's protocol. 
PCR or nested PCR was performed with a commercial PCR enzyme (PrimeSTAR HS DNA Polymerase; Takara Bio, Inc., Dalian, China). Specific primers used for the constant region of IgG1(IGHG1), V-D-J sequence of the IgG variable region (VDJ), Igκ, and recombination activating gene −1 and 2 (RAG1 and RAG2) were identical to those used and described previously. 14 CD19 was amplified to exclude contamination by B-lymphocytes and β-actin was amplified as the internal reference. 14 PCR products were identified and confirmed by DNA sequencing and blasting. Raji cells, a cell line of B cell leukemia, were employed as the positive control. 
To check the levels of IgG transcripts and the effect of IgG administration on TLR4 and CD64 in ARPE-19 cells, real-time PCR was performed in triplicate with a real-time PCR system (ABI PRISM 7500; Applied Biosystems, Foster City, CA) using a SYBR detection kit (SYBR Premix Ex Taq II; Takara Bio, Inc.) according to the standard protocol. Specific primers for IgG were identical to those used for PCR. Primers for TLR4 and CD64 were used as described previously. 17,18 β-actin was amplified for normalization. Water was substituted for template as the negative control. 
Knockdown of IgG Expression
SiRNA (5′-CCAAGGACACCCUCAUDAUTT-3′, 5′-AUCAUGAGGGUGUCCUUGGTT-3′) against the constant region of endogenous IgG was designed by Shanghai GenePharma Co., Ltd. (Shanghai, China) and transfected into ARPE-19 cells with a transfection reagent (X-tremeGene SiRNA Transfection Reagent; Roche Applied Science, Mannheim, Germany) according to the manufacturer's protocol. Scrambled siRNA was used as the isotype control. PBS instead of siRNA was employed as the negative control. Transfection rate was evaluated by carboxyfluorescein (FAM)-siRNA using flow cytometry. Effectiveness of IgG knockdown was verified with Western blot and real-time PCR at both protein and mRNA levels. 
Viability of ARPE-19 used for functional experiments was tested with PI (Sigma-Aldrich) staining and analyzed with flow cytometry according to standard protocol. 
Western Blot (WB)
Cytosol proteins from ARPE-19 cells (40 μg/lane) were analyzed with 10% SDS-PAGE as described previously. 19 Specific information about the primary antibodies to IgG, TLR3, TLR4, Myd88, TRIF, p-NFκB p65, and β-actin that were used is listed in Supplementary Table S1. HRP-labeled goat anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.) were employed as secondary antibodies. 
Cell Proliferation Analysis
The MTS cell proliferation assay (CellTiter 96 AQ One Solution Cell Proliferation Assay Kit; Promega Corp.) was carried out in a 96-well plate according to the manufacturer's instructions. ARPE-19 cells (5 × 103/well) were seeded followed by knockdown of endogenous IgG and incubated for 6 days. OD490 was recorded and analyzed at 24-hour intervals. 19 All experiments were performed in triplicate. 
Apoptosis and Cell Cycle Assay
ARPE-19 cells (2 × 104/well) were seeded in a 6-well plate and IgG was knocked down as described above. Decreased mitochondrial membrane potential in ARPE-19 cells was analyzed as a maker for early apoptosis with the mitochondrial membrane potential assay kit with JC-1 (Beyotime Institute of Biotechnology, Shanghai, China) according to the manuscript's protocol. The cell cycle was analyzed with a commercial analysis kit (Cell Cycle and Apoptosis Analysis Kit; Beyotime Institute of Biotechnology) and the percentage of cells in each stage of the cell cycle was evaluated with flow cytometry (BD, Franklin Lakes, NJ) according to the manufacturer's protocol. 
Cell Invasion Assay
To investigate the effect of endogenous IgG on the capacity for invasion in ARPE-19 cells, the migration assay was performed as described previously. 15 The membrane was stained with hematoxylin and eosin. Five random fields per membrane were observed at ×400 magnification with a research microscope (BX51; Olympus Corporation of the Americas, Melville, NY) and the invading cells were counted and average numbers were calculated. 
TLR4 Triggering and ELISA
To study the involvement of endogenous IgG in the TLR4 pathway, LPS (Sigma-Aldrich) was used to stimulate TLR4 with graduated concentrations of 0.1, 1.0, and 10 μg/mL. At a concentration of 1.0 μg/mL, LPS was employed to trigger the TLR4 pathway for durations of 3, 6, 12, 24, and 48 hours, and IgG expression levels were evaluated by WB as described above. 
After seeding into a 6-well plate and transfection with IgG and scrambled siRNA, ARPE-19 cells (1 × 104/well) were stimulated with LPS at a concentration of 1.0 μg/mL for 3 hours. Protein levels of TLR4, Myd88, TRIF, and p-NFκB p65 were evaluated with WB. Translocation of NFκB into the nucleus was observed with confocal microscopy as described above. Levels of TNF-α (terminal protein of TLR4 pathway) and IFN-γ (terminal protein of TLR3 pathway) in the supernatant were evaluated with development kits (Duoset Human TNF-α and IFN-γ ELISA Development kits; R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. 
Extraction and Purification of Endogenous IgG
Endogenous IgG was extracted and purified from ARPE-19 cells with a purification kit (Montage Antibody Purification Kit; Millipore Corp., Billerica, MA) according to the manufacturer's protocol and assessed with Western blot as described above. Standard serum IgG of human (Sigma-Aldrich) was used as the reference. 
Effect of Endogenous IgG on TLR4 and CD64 Pathway
To investigate the effect of endogenous IgG on the TLR4 and CD64 pathways, purified IgG was administered into the medium of ARPE-19 for 12 hours at doses of 0.2, 2.0, 20, 200, and 2000 μg/mL. Normal human serum IgM (Sigma-Aldrich) at identical concentrations was used as an isotype control. TNF-α and IL-10 (a terminal protein of CD64 pathway) in the supernatant were evaluated with development kits (Duoset Human TNF-α and IFN-γ; ELISA Development kits; R&D Systems) according to standard protocol. The most effective concentration of neuron-derived IgG was selected and used in the following experiments. 
Endogenous IgG and the isotype control (IgM) at 20 μg/mL were administrated into the medium of ARPE-19 cells, and mRNA levels of CD64 and TLR4 were analyzed with real-time RT-PCR as described above. 
Results
Expression and Distribution of IgG Protein and mRNA in ARPE-19 Cells
Using double labeling and confocal microscopy, expression of IgG protein was found in a nonhomogeneous distribution. IgG protein mainly localized in the two polar areas of the cytoplasm in ARPE-19 cells, marked by CRALBP (Fig. 1A). Colocation of Igγ and Igκ (Fig. 1B) was observed, which also supports interpretation of this data as IgG protein functioning in this system as an intact whole molecule. 
Figure 1
 
IgG expression in the ARPE-19 cell line. (A) Coexpression of IgG (green) and CRALBP (red) in the cytoplasm of ARPE-19 cells. (B) Colocation of Igγ (red) and Igκ (green) in ARPE-19 cells. (C) Photo of ISH for localization of IgG mRNA, visualized with NBT/BCIP (purple). (D) Agarose gel electrophoresis of RT-PCR amplification products. DM, DNA marker; Neg, negative control; R, DNase-treated RNA used as a template; C, cDNA used as a template. Scale bars: 20 μm.
Figure 1
 
IgG expression in the ARPE-19 cell line. (A) Coexpression of IgG (green) and CRALBP (red) in the cytoplasm of ARPE-19 cells. (B) Colocation of Igγ (red) and Igκ (green) in ARPE-19 cells. (C) Photo of ISH for localization of IgG mRNA, visualized with NBT/BCIP (purple). (D) Agarose gel electrophoresis of RT-PCR amplification products. DM, DNA marker; Neg, negative control; R, DNase-treated RNA used as a template; C, cDNA used as a template. Scale bars: 20 μm.
At the mRNA level, IgG expression was verified with ISH and RT-PCR. With ISH, positive signals for the constant region of Igγ were distributed mainly in the perinuclear cytoplasm of ARPE-19 cells (Fig. 1C). With RT-PCR, transcripts of the variable (VDJ) and constant region (IGHG1) of Igγ and Igκ were all successfully amplified from total RNA isolated from ARPE-19 cells (Fig. 1D). Moreover, RAG1 and RAG2, which are essential enzymes for IgG rearrangement, were also found. CD19 transcripts (which are a marker of B-lymphocytes) were found only in Raji cells but not in ARPE-19 cells, which excluded the possibility of contamination by B-lymphocytes among the ARPE-19 cells. No band was detectable in the negative control. The specificity of the probes and primers that were used for these experiments had been well established in our previous studies. 14  
Endogenous IgG Augments the Capacity for Proliferation, Mitosis, and Migration in ARPE-19 Cells, and Decreases Apoptosis
The IgG siRNA used here was designed against the constant region of endogenous IgG whose sequence was described in our previous study. With FAM-siRNA, the transfection rate of siRNA in the ARPE-19 cell line was 92.1% ± 2.3% as confirmed by flow cytometry (Supplementary Fig. S1A). Viability of the tested cells was identical to the negative control (Supplementary Fig. S1B). The downregulating effect of siRNA was confirmed by WB and real-time PCR. More than 50% of the IgG protein was knocked down with IgG siRNA compared with the negative control as shown in Figure 2A. At the mRNA level, IgG in RPE cells was diminished by 69.0% ± 5.3% with IgG siRNA compared with the negative control, while scrambled siRNA had no significant effect (Fig. 2B). These results indicated that IgG siRNA was effective and specific. 
Figure 2
 
Effect of IgG knock out on behaviors of ARPE-19 cells. (A, B) Confirmation of IgG downregulation at protein and mRNA levels with WB (A) and real time RT-PCR (B). (C) Evaluation of proliferation with the MTS essay showing IgG knockout significantly inhibits growth of ARPE-19 cells. (DG) Cell cycle analysis shows that IgG downregulation leads to a lower percentage of cells in S phase (8.2% ± 1.6%) as compared with the negative control (35.8% ± 2.3%) and the scramble group (30% ± 5.2%). (H) Analysis of early apoptosis. IgG knockdown induced a decrease of mitochondrial membrane potential in ARPE-19 cells. (I) Migration essay with ARPE-19 cells in which IgG was knocked down, with control. Scr, scramble siRNA group; Si, IgG siRNA group. Scale bars: 20 μm. **Indicates this is the group of significance.
Figure 2
 
Effect of IgG knock out on behaviors of ARPE-19 cells. (A, B) Confirmation of IgG downregulation at protein and mRNA levels with WB (A) and real time RT-PCR (B). (C) Evaluation of proliferation with the MTS essay showing IgG knockout significantly inhibits growth of ARPE-19 cells. (DG) Cell cycle analysis shows that IgG downregulation leads to a lower percentage of cells in S phase (8.2% ± 1.6%) as compared with the negative control (35.8% ± 2.3%) and the scramble group (30% ± 5.2%). (H) Analysis of early apoptosis. IgG knockdown induced a decrease of mitochondrial membrane potential in ARPE-19 cells. (I) Migration essay with ARPE-19 cells in which IgG was knocked down, with control. Scr, scramble siRNA group; Si, IgG siRNA group. Scale bars: 20 μm. **Indicates this is the group of significance.
As a cell line derived primarily from human RPE, ARPE-19 cells share characteristics of RPE, such as the capacity for proliferation, apoptosis, and migration. The role played by endogenous IgG in the cellular biology of ARPE-19 cells was studied. 
With the MTS essay, the rate of proliferation in ARPE-19 cells decreased markedly with downregulation by IgG. As shown in Figure 2C, apparent differences in the proliferation rate in the control and test cells became apparent on the third day of subculture, and this difference continued to increase throughout the following 4 days. In addition, the effect of endogenous IgG on the cell cycle of RPE cells was investigated with ARPE-19 cells. As shown in Figures 2D through 2G, downregulation of IgG resulted in fewer cells in S phase (8.2% ± 1.6%) as compared with the scramble (30% ± 5.2%) and negative (35.8% ± 2.3%) groups. Using early apoptosis analysis, percentages of ARPE-19 cells with decreased mitochondrial membrane potential in the negative control, scramble, and test groups were 8.35%, 7.51%, and 51.9%, respectively (Fig. 2H), indicating that IgG knockdown induced a significant increase in early apoptosis in ARPE-19 cells. These data suggest that endogenous IgG functions in augmenting proliferation and mitotic rate, and in inhibiting apoptosis in ARPE-19 cells. 
To determine whether endogenous IgG is associated with capacity for migration of RPE cells, a cell migration assay using matrigel and a multiporous membrane was carried out with ARPE-19 cells in which endogenous IgG was downregulated. The average number of migrating cells was significantly lower in the test cells (12 ± 5.1/cm2), than in the scramble and negative control cells (29 ± 6.4, 30 ± 4.4 /cm2, both P < 0.05) as shown in Figure 2I. These results suggest that endogenous IgG supports the capacity for migration in RPE cells. All these functional data shed light on the possibility that endogenous IgG may be involved in the immune regulating function of RPE, as the cellular behaviors evaluated above are crucial events in pathologic processes such as inflammation. 
Endogenous IgG Is Involved in the TLR4 Pathway
The TLR4 pathway is crucial for innate immunity in the retina. We further investigated the relationship of endogenous IgG with function of the TLR4 pathway for which LPS is the special ligand. As shown in Figure 3A, the protein levels of IgG in ARPE-19 cells became elevated as the concentration of LPS increased to 1.0 μg/mL, but decreased at a concentration of 10 μg/mL. ARPE-19 cells were then exposed to LPS at 1.0 μg/mL, for various lengths of time including 0, 3, 6, 12, 24, and 48 hours. The amount of IgG increased sharply after 3 hours, and then decreased slowly from 6 to 48 hours (Fig. 3B). The concentration of 1.0 μg/mL with duration of 3 hours was therefore selected as the most effective condition for use of LPS for the following experiments. These data suggest that endogenous IgG was produced in a concentration- and duration-dependent manner when stimulated by LPS, indicating that endogenous IgG may be a terminal effector of the TLR4 pathway. 
Figure 3
 
Correlation of endogenous IgG and the TLR4 pathway in ARPE-19 cells. (A, B) Evaluation of LPS stimulation on IgG production with graduated concentrations (A) and duration (B). (C) Effect of IgG knockdown on key proteins in the TLR3 and TLR4 pathways. (D, E) IgG knockdown inhibits the translocation of NFκB p65 to the nucleus and production of TNF-α. Scale bars: 20 μm. **Indicates this is the group of significance.
Figure 3
 
Correlation of endogenous IgG and the TLR4 pathway in ARPE-19 cells. (A, B) Evaluation of LPS stimulation on IgG production with graduated concentrations (A) and duration (B). (C) Effect of IgG knockdown on key proteins in the TLR3 and TLR4 pathways. (D, E) IgG knockdown inhibits the translocation of NFκB p65 to the nucleus and production of TNF-α. Scale bars: 20 μm. **Indicates this is the group of significance.
In addition, the effect of endogenous IgG on the TLR4 pathway was studied. Under stimulation with LPS, protein levels of TLR4, Myd88, TRIF, and p-NFκB p65 in ARPE-19 cells in which IgG had been knocked down decreased significantly compared with the negative control and the scrambled group (Fig. 3C), but no significant change was found between the latter two groups. Considering the possibility that a siRNA of 21 nucleotides may affect the TLR3 pathway, 20 we also studied the possible involvement of TLR3 in biological effects induced by IgG siRNA. As shown in Figure 3C, with LPS stimulation, the TLR3 protein level was decreased by scrambled siRNA and IgG siRNA, but there was no significance difference when compared with the negative control. As TRIF is a downstream molecule for the TLR4 and TLR3 pathways, decrease in its protein level may mainly result from TLR4 inhibition induced by IgG siRNA. In addition to the key molecules in the TLR4 and TLR3 pathways, translocation of NFκB p65 to the nucleus was also studied. NFκB p65 was distributed mainly in the cytoplasm of ARPE-19 cells under normal culture conditions with scrambled siRNA, but no LPS stimulation (Fig. 3D, the upper line), which ruled out a nonspecific effect of scrambled siRNA. Once triggered by LPS, almost all NFκB p65 translocated into the nucleus (Fig. 3D, middle line). When IgG was knocked down followed by LPS triggering, translocation to the nucleus of NFκB p65 was diminished (Fig. 3D, lower line). Additionally, when stimulated with LPS, the expression level of TNF-α in test ARPE-19 cells (141.83 ± 11.3 pg/mL) was significantly lower than in the negative control (564.57 ± 9.1 pg/mL) and scrambled (510.27 ± 11.4 pg/mL) groups (Fig. 3E). However, no significant change in IFN-γ (one of the terminal effector of TLR3 pathway) was found in these three groups. These data suggest that reduction of the TLR3 pathway induced by nonspecific siRNA of 12 nucleotides do not alter the effects of the TLR4 pathway that are induced by IgG siRNA. IgG and the TLR4 pathway may have a mutually positive regulatory effect as the TLR4 pathway increases expression of IgG, and IgG enhances activation of the TLR4 pathway, and both Myd88-dependent and -independent effects were altered in conjunction with these mutually positive effects. 
Endogenous IgG Affects RPE Cells via the TLR4 and CD64 Pathways
TLR4 and CD64 are widely known molecules which are expressed on RPE cells, and they play key roles in immunological regulation and inflammation reactivation under physiologic and pathologic conditions. 3,21 Although TNF-α is known as a terminal effector of the TLR4 pathway and take part in inflammatory injury, accumulating evidence suggested that it can protect cells (such as neurons) from inflammatory injury at low concentrations. 22 IL-10 is an anti-inflammatory cytokine produced by RPE and is one of the final products of CD64 pathway. In the present study, in order to investigate the effect of endogenous IgG protein on RPE, we first extracted IgG from ARPE-19 cells and confirmed the purity with WB in which human serum IgG was used as a reference (Fig. 4A). It was then administrated into the medium of ARPE-19 cells. We found that IgG triggered a significant increase in TNF-α and IL-10 levels in the supernatant of ARPE-19 cells with a reverse U-shaped dose-response curve (Fig. 4B). Maximal levels of TNF-α and IL-10 were 254.1 ± 8.7 and 203.8 ± 6.4 pg/mL (12.1- and 18.2-fold of the control IgM), respectively, and were induced by IgG at 20 μg/mL, which we finally chose as the most effective concentration. When exposed to IgG at 20 μg/mL, mRNA levels of TLR4 and CD64 in ARPE-19 were elevated by 6.1 ± 0.5- and 2.5 ± 0.3-fold, respectively (Fig. 4C), as compared with the control (IgM, 20 μg/mL). These data suggest that endogenous IgG may trigger TLR4 and CD64 pathways and affect the biology of RPE cells. 
Figure 4
 
Endogenous IgG triggers TLR4 and CD64 pathways in RPE. (A) Confirmation of endogenous IgG purified from ARPE-19 with WB. (B) Release levels of TNF-α and IL-10 from ARPE-19 evaluated with ELISA, after exposure to a series of concentrations of purified IgG and the isotype control (IgM). (C) Relative levels of CD64 and TLR4 in ARPE-19 cells treated with purified IgG (20 μg/mL) and the isotype control (IgM). **Indicates this is the group of significance.
Figure 4
 
Endogenous IgG triggers TLR4 and CD64 pathways in RPE. (A) Confirmation of endogenous IgG purified from ARPE-19 with WB. (B) Release levels of TNF-α and IL-10 from ARPE-19 evaluated with ELISA, after exposure to a series of concentrations of purified IgG and the isotype control (IgM). (C) Relative levels of CD64 and TLR4 in ARPE-19 cells treated with purified IgG (20 μg/mL) and the isotype control (IgM). **Indicates this is the group of significance.
Discussion
Murray et al. 12 proposed the possibility of IgG production locally in the eye in the 1990s, 12 and we conclusively demonstrated for the first time that IgG could be locally produced by intraocular cells in humans and mice in 2011. 14 The present study is a follow-up of our previous work that furthers functional investigation of local endogenous IgG production by RPE cells. 
We confirmed IgG is expressed in the ARPE-19 cell line at both protein and mRNA levels, which is generally accepted as representative of RPE cells. It is noteworthy that IgG protein strongly localizes in the polar cytoplasm, which bears a spatial relationship to the growth pattern of RPE cells that grow in a direction parallel to the long axis of these characteristically spindle-shaped cells. Whether this intriguing spatial relationship reflects a functional relationship is undetermined, but it raises the possibility that IgG may be mechanically involved in the growth and mitotic activity of RPE. At the mRNA level, several transcripts including the heavy chain constant and variable regions, Igκ and key enzymes for rearrangement of IgG were found and the result of sequencing the VDJ region showed strong identity with the sequence that we reported previously. 14 All these data serve to verify our previous identification of intrinsic IgG production by RPE cells. 
siRNA was designed against the constant region of IgG produced by RPE. Functional experiments with ARPE-19 cells in which IgG was knocked down indicated that endogenous IgG functions to stimulate growth, mitosis, and migration of RPE cells, as well as inhibiting RPE apoptosis. These functional behaviors of RPE cells are key events under physiological and pathological conditions. Under induction by injury or inflammatory factors, RPE cells may be activated to proliferate and migrate to the site of the lesion, 24 contributing to the pathologic processes of several diseases such as autoimmune uveitis and endophthalmitis. 1 Excessive RPE apoptosis is the proximate mechanism of AMD. 23 It is possible that endogenous IgG produced by RPE is a molecule that is both of great importance in maintenance of normal function of RPE, which is also involved in immune homeostasis of the eye. 
As a specialized organ of the brain, the eye is a classic privileged immune site in which immune responses are suppressed. Because of the BOB, entry of immunologic cells and molecules was considered to be forbidden in the eye. Recently, based on accumulating evidence, it was suggested that immune privilege is an immunological niche in which a specialized, well-orchestrated collaboration between the innate and adaptive arms of the immune system is constructed. 3 RPE cells, beneath the neural retina, are the key cellular component of the BOB. TLRs, complement receptor, CD64, and MHC I and II molecules are expressed on the surface of RPE. 3 Orchestrating innate and adaptive immunity, RPE cells play a crucial role in regulating the immune homeostasis of the eye. 1 TLR pathways comprise the main component of the innate immune system. After binding with their special ligands, TLRs initialize a “toll rush” and generate effectors. Endowed with many TLRs, RPE cells are strategically situated to provide a rapid defense for the retina. 6 The various roles played by the TLR4 pathway in intraocular disease have been well documented. Oxidative stress, mitochondrial DNA damage, chronic inflammation, and angiogenesis (basic pathologic features of AMD) in the retina can be induced by activation of the TLR4 pathway. 7,8 For example, appropriate stimulation by LPS at a low dose can decrease the levels of IL-6 and IL-8 in corneal stroma cells, and prevent excessive inflammatory response to Aspergillus fumigatus . 24 In the present study, we found that expression of IgG in RPE cells can be elevated by LPS in a concentration- and duration-dependent fashion. Moreover, downregulation of IgG in RPE resulted in a decrease of TLR4, adaptor molecules, the NFκB pathway, and final generation of TNF-α when under stimulation by LPS. TNF-α is a classic effector of the TLR4 pathway, and has been confirmed to protect neurons from damage induced by inflammation at low concentrations. 22,25 The present study showed that upon exposure to graded concentrations of IgG, expression of TLR4 and production of TNF-α by RPE is elevated in a dose-depended manner that plots a reverse U-shaped curve, and the maximal level of TNF-α is under the noninjurious threshold in a slice culture of the brain. 25 These data raise the possibility that IgG may be a terminal effector of the TLR4 pathway, and it in turn has a positive regulatory effect on this pathway and may be protective of RPE under physiologic conditions. 
CD64 is a high-affinity receptor of IgG, expressed on microglia and RPE in the retina. 26 It was suggested that Fc-CD64 interaction contributes to the inflammatory retinal diseases such as AMD by producing cytokines, and this interaction may be involved in complications associated with intravitreal bevacizumab-associated clinical therapy. 21,27 However, IL-10 is one of the effectors of the CD64 pathway and functions as an inflammatory suppressor. It is produced by RPE, and can significantly suppress the activation of intraocular T-cells. 2830 In the present study, we found exogenous IgG can induce a significant increase of CD64 and an elevation of IL-10 in a dose-dependent manner. This suggests that endogenous IgG may exert a protective function in an autocrine manner. 
In conclusion, our findings demonstrate that IgG produced by RPE is essential for proliferation, apoptosis, mitosis, and migration of RPE cells and also shows a mutually positive effect with TLR4-mediated innate immune responses and is involved in activation of CD64 pathway. Endogenous IgG may play a significant role in the immune homeostasis of the eye, and is self-protective in intraocular physiologic and pathologic processes. The mechanisms of this IgG activity warrant further investigation, but this study may contribute to our understanding of intraocular immunity and translate into better therapy for retinopathy. 
Supplementary Materials
Acknowledgments
Supported by grants from the National Natural Science Foundation of China (No. 81100885 [NN]) and the Young and Middle-Aged Scientists Research Awards Foundation of Shandong Province (No. BS2011SW051 [NN]). 
Disclosure: N. Niu, None; J. Zhang, None; M.A. McNutt, None 
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Figure 1
 
IgG expression in the ARPE-19 cell line. (A) Coexpression of IgG (green) and CRALBP (red) in the cytoplasm of ARPE-19 cells. (B) Colocation of Igγ (red) and Igκ (green) in ARPE-19 cells. (C) Photo of ISH for localization of IgG mRNA, visualized with NBT/BCIP (purple). (D) Agarose gel electrophoresis of RT-PCR amplification products. DM, DNA marker; Neg, negative control; R, DNase-treated RNA used as a template; C, cDNA used as a template. Scale bars: 20 μm.
Figure 1
 
IgG expression in the ARPE-19 cell line. (A) Coexpression of IgG (green) and CRALBP (red) in the cytoplasm of ARPE-19 cells. (B) Colocation of Igγ (red) and Igκ (green) in ARPE-19 cells. (C) Photo of ISH for localization of IgG mRNA, visualized with NBT/BCIP (purple). (D) Agarose gel electrophoresis of RT-PCR amplification products. DM, DNA marker; Neg, negative control; R, DNase-treated RNA used as a template; C, cDNA used as a template. Scale bars: 20 μm.
Figure 2
 
Effect of IgG knock out on behaviors of ARPE-19 cells. (A, B) Confirmation of IgG downregulation at protein and mRNA levels with WB (A) and real time RT-PCR (B). (C) Evaluation of proliferation with the MTS essay showing IgG knockout significantly inhibits growth of ARPE-19 cells. (DG) Cell cycle analysis shows that IgG downregulation leads to a lower percentage of cells in S phase (8.2% ± 1.6%) as compared with the negative control (35.8% ± 2.3%) and the scramble group (30% ± 5.2%). (H) Analysis of early apoptosis. IgG knockdown induced a decrease of mitochondrial membrane potential in ARPE-19 cells. (I) Migration essay with ARPE-19 cells in which IgG was knocked down, with control. Scr, scramble siRNA group; Si, IgG siRNA group. Scale bars: 20 μm. **Indicates this is the group of significance.
Figure 2
 
Effect of IgG knock out on behaviors of ARPE-19 cells. (A, B) Confirmation of IgG downregulation at protein and mRNA levels with WB (A) and real time RT-PCR (B). (C) Evaluation of proliferation with the MTS essay showing IgG knockout significantly inhibits growth of ARPE-19 cells. (DG) Cell cycle analysis shows that IgG downregulation leads to a lower percentage of cells in S phase (8.2% ± 1.6%) as compared with the negative control (35.8% ± 2.3%) and the scramble group (30% ± 5.2%). (H) Analysis of early apoptosis. IgG knockdown induced a decrease of mitochondrial membrane potential in ARPE-19 cells. (I) Migration essay with ARPE-19 cells in which IgG was knocked down, with control. Scr, scramble siRNA group; Si, IgG siRNA group. Scale bars: 20 μm. **Indicates this is the group of significance.
Figure 3
 
Correlation of endogenous IgG and the TLR4 pathway in ARPE-19 cells. (A, B) Evaluation of LPS stimulation on IgG production with graduated concentrations (A) and duration (B). (C) Effect of IgG knockdown on key proteins in the TLR3 and TLR4 pathways. (D, E) IgG knockdown inhibits the translocation of NFκB p65 to the nucleus and production of TNF-α. Scale bars: 20 μm. **Indicates this is the group of significance.
Figure 3
 
Correlation of endogenous IgG and the TLR4 pathway in ARPE-19 cells. (A, B) Evaluation of LPS stimulation on IgG production with graduated concentrations (A) and duration (B). (C) Effect of IgG knockdown on key proteins in the TLR3 and TLR4 pathways. (D, E) IgG knockdown inhibits the translocation of NFκB p65 to the nucleus and production of TNF-α. Scale bars: 20 μm. **Indicates this is the group of significance.
Figure 4
 
Endogenous IgG triggers TLR4 and CD64 pathways in RPE. (A) Confirmation of endogenous IgG purified from ARPE-19 with WB. (B) Release levels of TNF-α and IL-10 from ARPE-19 evaluated with ELISA, after exposure to a series of concentrations of purified IgG and the isotype control (IgM). (C) Relative levels of CD64 and TLR4 in ARPE-19 cells treated with purified IgG (20 μg/mL) and the isotype control (IgM). **Indicates this is the group of significance.
Figure 4
 
Endogenous IgG triggers TLR4 and CD64 pathways in RPE. (A) Confirmation of endogenous IgG purified from ARPE-19 with WB. (B) Release levels of TNF-α and IL-10 from ARPE-19 evaluated with ELISA, after exposure to a series of concentrations of purified IgG and the isotype control (IgM). (C) Relative levels of CD64 and TLR4 in ARPE-19 cells treated with purified IgG (20 μg/mL) and the isotype control (IgM). **Indicates this is the group of significance.
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