To screen the cytokine production profiles resulting from the treatment of internalizing anti-DNA antibody, chimeric 3D8 IgG was purified from HEK293F cells. 3D8 IgG was treated to five immune cell types (THP-1 human monocytic leukemia cells, Jurkat human T lymphoblastoid cells, DC2.4 mouse dendritic cells, RAW 264.7 mouse macrophages, and BV2 mouse microglia cells) (Fig. 1A) and five non-immune cell types (A549 human lung epithelial cells, HEK293T human embryonic kidney cells, U87MG human glioblastoma cells, HeLa human cervix carcinoma epithelial cells, and MEF mouse embryonic fibroblasts) (Fig. 1B). The production of cytokines was quantified by enzyme-linked immunosorbant assay (ELISA) to detect pro-inflammatory cytokines. The screening identified the marked secretion of the IL-8 and TNF-α inflammatory cytokines from THP-1 cells.
A
Fig. 1. Production of pro-inflammatory cytokines is induced by 3D8 IgG in various cells. Measurement of the production of cytokines related to inflammation following treatment with 10 µM 3D8 IgG for 24 or 48 h in (A) immune cells (THP-1, Jurkat, DC2.4, RAW 264.7, and BV2) and (B) non-immune cells (A549, HEK293T, U87MG, HeLa, and MEF).
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B. Internalizing anti-DNA antibody, 3D8 IgG promotes the production of pro-inflammatory cytokines, IL-8 and TNF-α in THP-1 cells.
To investigate the time- and dose-dependent production of IL-8 and TNF-α in THP-1 cells, the 3D8 IgG incubation times of up to 6 h were used (Fig. 2A) and the dose of 3D8 IgG was increased by up to 10 µM (Fig. 2B). Time- and dose-dependent secretion of both cytokines was evident. The optimum treatment conditions of the antibody were set at 5 µM and 6 h. We next investigated the cytokine production in primary human monocytes. CD14+ monocyte cells isolated from the peripheral blood mononuclear cells (PBMCs) of a healthy donor were treated with 5 µM 3D8 IgG for 6 h (Fig. 2C). IL-8 and TNF-α were also secreted by 3D8 IgG, indicating that treatment of 3D8 IgG triggers the production of these pro-inflammatory cytokines in THP-1 and primary monocytes.
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Fig. 2. 3D8 IgG internalizing anti-DNA antibody promotes production of IL-8 and TNF-α pro-inflammatory cytokines in THP-1 cells. (A, B) THP-1 cells were treated with 5 µM 3D8 IgG for the indicated times at 37°C (A), or were treated with various concentrations of 3D8 IgG for 6 h at 37°C (B). (C) Cytokines secreted from in human primary CD14+ monocytes after treatment with 5 µM 3D8 IgG were analyzed by ELISA. Data are expressed as the mean ± standard error of three independent experiments. All P-values were calculated using a two-tailed Student’s t-test (ns: not significant; *P<0.05; **P<0.01; ***P<0.001 versus negative control).
C. Fc region of internalized anti-DNA antibody mediates secretion of IL-8 and TNF-α from THP-1 cells.
To determine the portion of the internalized 3D8 IgG that is responsible for the production of cytokines in THP-1 cells, we purified the different variants of the 3D8 antibody including 3D8 scFv, which comprised of VH and VL, and 3D8 scFv-Fc fusion protein (Fig. 3A). As expected, 3D8 IgG was detected at 50 kDa and 25 kDa, whereas 3D8 scFv was detected at 27 kDa on reducing SDS-PAGE; 3D8 scFv-Fc was detected at 52 kDa (Fig. 3B). When THP-1 cells were treated with 3D8 IgG, 3D8 scFv, 3D8 scFv-Fc, and polyclonal human IgG incapable of internalizing, the secretion of IL-8 and TNF-α was observed in cells treated with 3D8 IgG and 3D8 scFv-Fc, but not in 3D8 scFv and polyclonal human IgG (Fig. 3C). The expressions of the mRNAs were also correlated (Fig. 3D). 3D8 scFv, which did not induce cytokine production, is a protein that is depleted in the constant region, indicating that the immune response is due to the constant region rather than the variable region.
Moreover, the 3D8 scFv fusion protein that lacked the CH1 domain of the constant region induced cytokine production, demonstrating that the Fc region and the CH2 -CH3 domain in the constant region are involved in the induction of inflammatory responses. To investigate whether the production of cytokines is also induced by other subtypes, 3D8 IgE was treated to THP-1 cells, and the result was observed for IL-8 and TNF production (Fig. 3E). The collective results demonstrated that production of IL-8 and TNF-α is induced by the Fc region of 3D8 anti-DNA antibody.
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Fig. 3. Fc region of internalized anti-DNA antibody mediates secretion of IL-8 and TNF-α from THP-1. (A) Schematic representation of the 3D8 IgG, 3D8 scFv-Fc and 3D8 scFv. (B) SDS-PAGE analysis of purified 3D8 variants. (C) Cells were treated with 5 µM 3D8 variants and polyclonal human IgG for 6 h at 37°C. The level of IL-8 and TNF-α in culture supernatants were quantitatively measured by ELISA.
(D) Quantitative RT-PCR for the mRNA level of IL-8 and TNF-α from cells treated with 5 µM 3D8 antibody for 6 h at 37°C. (**P<0.01; ***P<0.001 versus treatment with 3D8 scFv)(E) Measurement of cytokines production by treatment of 5 µM 3D8 IgG and IgE using ELISA. Data are expressed as the mean ± standard error of three independent experiments. All P-values were calculated using a two-tailed Student’s t-test.
D. 3D8 IgG variants are internalized into the THP-1 and CD14+ monocytes.
Confocal microscopy examination of the internalization of the antibodies revealed that all formats of the internalizing 3D8 antibody localized in the cytosol of THP-1 cells. In contrast, polyclonal human IgG used as a negative control localized on the cell surface, likely due to capture by Fcγ receptors (FcγR) expressed on THP-1 cells (Fig. 4A). Confocal microscopy also showed that internalizing 3D8 IgG is localized at an area considered as the cytosol of CD14+ monocytes (Fig. 4B), indicating the production of IL-8 and TNF-α by the internalization of free 3D8 IgG in THP-1 cells and primary CD14+ monocyte cells.
Fig. 4. 3D8 IgG variants are internalized into THP-1 and CD14+ monocytes. (A, B) Confocal microscopy for intracellular localization of 3D8 variants and polyclonal human IgG. THP-1 Cells (A) and CD14+ monocytes (B) were incubated with 5 µM 3D8 proteins and human IgG (hIg) 6 h at 37°C. After fixation and permeabilization, cells were incubated with dylight550 goat α-human IgG Fc for detecting 3D8 IgG, 3D8 scFv-Fc and human IgG, with α-3D8 IgG and TRITC-α-rabbit IgG to detect 3D8 scFv. The bar denotes 5 µm.
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E. Production of pro-inflammatory cytokines by internalized IgG is not through signaling pathways via intracellular TLR9.
Pathogenesis due to anti-DNA antibodies involves the formation of DNA-IC released from dead cells and their ensuing phagocytosis by plasmacytoid dendritic cells (pDCs) through Fcγ receptors. The complex is recognized by intracellular TLR9, leading to the production of type I interferon. To investigate whether the immune response by 3D8 IgG is related or unrelated to the recognition of TLR9, we first compared the endocytosis mechanism of DNA-IC and 3D8 IgG using pharmacological inhibitors to interfere with the individual endocytic pathways (Fig.
5A). Chlorpromazine (CPZ) was used to inhibit clathrin-dependent endocytosis, methyl-β-cyclodextrin (MβCD) to inhibit caveolae/lipid raft endocytosis, and cytochalasin D (CytoD) to inhibit phagocytosis. THP-1 cells were pre-treated with these inhibitors for 30 min at 37°C, prior to the incubation with 5 µM 3D8 IgG. CPZ inhibited the production of IL-8 and TNF-α, but MβCD and CytoD did not, suggesting that 3D8 IgG is internalized via clathrin-dependent endocytosis. We next examined whether the production of cytokines was stimulated by TLR9 or Fcγ receptors signaling, which recognize DNA-ICs. In THP-1 cells pre-incubated of TLR9 inhibitor (Fig. 5B) or human Fc blocker (Fig. 5D), the levels of IL-8 and TNF-α were not affected by TLR9 inhibitor but were increased by Fc blocker. And, we confirmed that binding of 3D8 IgG to FcγR was decreased by Fc blocker by flow cytometry (Fig. 5C). The increase of cytokines reflected the increased amount of antibody that entered the cells as the 3D8 IgG partially captured by the FcγR became free. When cells were treated with DNase I and genomic DNA extracted from THP-1 cells prior to incubation with 3D8 IgG, no significant changes in the production levels of IL-8 and TNF-α were detected (Fig. 5E). These results indicated that the production of IL-8 and TNF-α by internalizing IgG is induced in a different from the TLR9-mediated pathway.
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mean fluorescence intensity (MFI)
Fig. 5. Production of inflammatory cytokines by internalized IgG is not through signaling pathways via intracellular TLR9. (A-B, D-E) Prior to treatment with 3D8 IgG for 6 h, THP-1 cells were treated with endocytosis inhibitors chlorpromazine (CPZ), methyl-β-cyclodextrin (MβCD), and cytochalasin D (CytoD) (A), TLR9 inhibitor (B), Fc blocker (D), or DNase1 and genomic DNA (0-10 µg/ml) extracted from THP-1 cells (E). Production of IL-8 and TNF-α in culture supernatants was measured by ELISA. (C) Inhibition of 3D8 IgG binding to FcγR by Fc blocker was observed by flow cytometry. 3D8 IgG was treated with THP-1 at 4℃ for 1 hour, and 3D8 IgG bound to the surface was detected using α-kappa antibody. All P-values were calculated using a two-tailed Student’s t test. Statistical significance is indicated on the graphs (ns: not significant; *P<0.05; **P<0.01;
***P<0.001).
F. 3D8 scFv binds to HSPG and CSPG on the cell membrane.
Phagocytosis of DNA-ICs is mediated by FcγR in pDCs. 3D8 IgG is internalized via the clathrin-mediated pathway but endocytic receptors is unknown.
Therefore, we next investigated the endocytic receptors of 3D8 IgG. CPPs can penetrate the cell membrane in various living cells. The numerous positively-charged amino acids that comprise CPPs interact with GAGs, such as HS and CS, which are negatively charged when they are internalized into cells. Assuming that 3D8 IgG, which contains many amino acids that are positively-charged in CDRs of the variable region similar to CPPs, is internalized via the negatively-charged surface receptors, we first confirmed the expression level of the negatively-charged proteoglycans, HSPGs and CSPGs, in HeLa cells (Fig. 6A). Confocal microscopy revealed the binding of 3D8 scFv, which possesses internalizing ability, to HSPGs and CSPGs on the cell membranes. There was no binding of the negative control, HW6 scFv specific for human TRAIL receptor 2 did not bind. 3D8 scFv was incubated at 4 °C for 1 h in HeLa cells to inhibit endocytosis and the HS or CS chains (Fig. 6B). 3D8 scFv co-localized with HS and CS chain on the cell surface. To confirm the interaction between 3D8 scFv and GAGs, 10 mIU/ml of heparinase III and 100 mIU/ml of chondroitinase ABC were used to pre-treat HeLa cells to reduce the HS and CS chains on the cell surface (Fig. 6C). Treatment of heparinase III, chondroitinase ABC, and a mixture of both enzymes reduced the binding of 3D8 scFv by 60%, 15%, and 78%, respectively (Fig. 6D). Confocal microscopy revealed the decreased binding of 3D8 scFv to the cell membrane after treatment with GAG-degrading enzymes. The findings were corroborated by flow cytometry (Fig. 6E).
The collective results indicated that 3D8 scFv binds to both HS and CS chains on the cell membrane.
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Fig. 6. 3D8 scFv binds to HSPG and CSPG on the cell membrane. (A) Examination of the endogenous expression levels of HSPGs and CSPGs in HeLa cells by flow cytometry. (B) Detection of binding between 3D8 scFv and HSPG/CSPG by confocal microscopy. 3D8 scFv and HW6 scFv (10 µM) were incubated with HeLa cells for 1 h at 4 °C, followed by detection using a combination of rabbit α-3D8 scFv antibody and mouse α-HS IgM antibody, or rabbit α-3D8 scFv antibody and mouse α-CS IgM antibody. The mixture of TRITC-conjugated α-rabbit IgG and Alexa Fluor 488-conjugated α-mouse IgM was used as secondary antibody.
(C-E) Detection of HS and CS chains (C) and 3D8 scFv on the cell surface (D) using heparinase III (10 mIU/ml) and chondroitinase ABC (100 mIU/ml) by flow cytometry (C, D) and confocal microscopy (E). Nuclei were stained with Hoechst 33342 (blue). The bar denotes 10 µm.
G. Both transmembrane and glycosylphosphatidylinositol (GPI)-anchored forms of HSPGs function as endocytic receptors for 3D8 scFv.
Transmembrane and GPI-anchored HSPGs are expressed on the cell surface.
We assessed whether 3D8 IgG can be internalized into the cells via HSPG, regardless of the structure type, using the SDC2 transmembrane receptor and GPC3 GPI-anchored receptor. HeLa cells were transfected with plasmids encoding GFP-SDC2 and HA-GPC3, to express SDC2 and GPC3 on the cell surface, and with plasmids encoding SDC2ΔTM-GFP and GPC3-nfGPI-GFP, to enable intracellular expression (Fig 7A). After transfection, cells were treated with 3D8 scFv for 1 h at 4 °C to inhibit endocytosis, or for 6 h at 37°C to permit internalization (Fig. 7B-E). When endocytosis was inhibited at 4°C, 3D8 scFv mostly co-localized with SDC2 and GPC3 on the cell surface (Fig. 7B, D). However, 3D8 scFv did not co-localize with the SDC2ΔTM and GPC3-nfGPI. When 3D8 scFv was incubated for 6 h at 37 °C, it co-localized with SDC2 and GPC3 expressed the on the cell membrane, whereas 3D8 scFv did not co-localize with intracellularly expressed SDC2ΔTM and GPC3-nfGPI (Fig. 7C, E). Since 3D8 scFv is internalized into cells with receptors expressed on the cell surface, intracellularly expressed SDC2 and GPC3 were evident as red and green fluorescence.
The endogenous expression levels of SDC2 and GPC3 in HeLa and HEK293T cells were examined before confirming whether HSPG and 3D8 scFv physically interacted. Endogenous SDC2 was highly expressed in both HeLa and HEK293T cells, but endogenous GPC3 was not expressed in both cells (Fig. 7F, H).
SDC2-GFP and HA-GPC3 overexpressing HeLa and HEK293T cells were incubated with 3D8 scFv for 6 h and then subjected to immunoprecipitation with GFP and α-GPC3 antibodies, respectively (Fig. G, I). 3D8 scFv, SDC2 and α-GPC3 that were pulled-down in the lysates were detected by western blot, indicating that 3D8 scFv interacts with transmembrane SDC2 and GPI-anchored GPC3.
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Fig. 7. Both transmembrane and GPI-anchored forms of HSPGs participate as endocytic receptors for 3D8 scFv. (A) Schematic diagrams of SD2 and GPC3 expression vectors for cell surface or intracellular expression. Abbreviations are: SP, signal peptide; Δ, deletion; TM, transmembrane domain; CD, cytoplasmic domain;
GPI, GPI anchor; GFP, green fluorescent protein; HA, HA tag. (B-E) Confocal microscopy detection of the co-localization between 3D8 scFv, SDC2, and GPC3.
HeLa cells were transfected with SDC2-GFP (B) and HA-GPC3 (D) vectors expressed on the cell surface, and the SDC2ΔTM-GFP (C) and GPC3-nfGPI-GFP (E) vectors expressed intracellularly (nf indicates non-functional). After transfection, 3D8 scFv was used to treat HeLa cells for 1 h at 4 °C or 6 h at 37 °C, and SDC2, GPC3, and 3D8 scFv were detected using the appropriate antibodies. Nuclei were stained with Hoechst 33342 (blue). The bar denotes 10 µm. (F, H) Flow cytometry examination of the cell surface expression of endogenous SDC2 (F) and GPC3 (H) in HeLa and HEK293T cells. (G, I) SDC2-GFP (G) and HA-GPC3 (I) overexpressing HeLa and HEK293T cells were incubated with 5 µM 3D8 scFv for 6 h at 37 °C. Cell lysates were immunoprecipitated using α-GFP antibody and the immunoprecipitates were detected by western blot using SDC2, GPC3, and α-His antibodies. Asterisks denote endogenous SDC2.
H. Both transmembrane and GPI-anchored forms of CSPGs participate as endocytic receptors for 3D8 scFv.
We investigated whether the distinct transmembrane and GPI-anchored CSPGs play a role as endocytosis receptors for 3D8 scFv. First, we transfected cells with plasmids encoding CD44-GFP and HA-BCAN, expressed on cell surface, and CD44ΔTM-GFP and BCANΔGPI-GFP expressed intracellularly. After transfection, the cells were treated with 3D8 scFv for 1 h at 4 °C to inhibit endocytosis or for 6 h at 37 °C. When endocytosis was inhibited, 3D8 scFv mostly co-localized with CD44 and BCAN on the cell surface. However, 3D8 scFv did not co-localize with either CD44ΔTM or BCANΔGPI. When cells were incubated with 3D8 scFv for 6 h at 37 °C, 3D8 scFv co-localized with SDC2 and GPC3 expressed on the cell membrane, but not with intracellular expressed CD44ΔTM and BCANΔGPI, respectively. Since 3D8 IgG is internalized into cells with surface-expressed receptors, intracellularly expressed CD44 and BCAN were observed as red and green fluorescence, respectively.
The endogenous expression levels of CD44 and BCAN in HeLa and HEK293T cells were examined before confirming whether CSPG and 3D8 scFv physically interacted. Endogenous CD44 was expressed only on HeLa cells, however, endogenous BCAN was not expressed in both cells (Fig. 8F, H). CD44-GFP and HA-BCAN overexpressed HeLa and HEK293T cells were incubated with 3D8 scFv for 6 h, and then lysates were immunoprecipitated with α-CD44 and α-HA antibodies, respectively (Fig. 8G, I). In pull-downed lysates, 3D8 scFv, CD44 and BCAN were detected by western blot, indicating that 3D8 scFv interacts with transmembrane CD44 and GPI-anchored BCAN.
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Fig. 8. Both transmembrane and GPI-anchored forms of CSPGs participate as endocytic receptors for 3D8 scFv. (A) Schematic diagrams of CD44 and BCAN expression vectors for cell surface or intracellular expression. Abbreviations are: SP, signal peptide; Δ, deletion; TM, transmembrane domain; CD, cytoplasmic domain;
GPI, GPI anchor; GFP, green fluorescent protein; HA, HA tag. (B-E) Confocal microscopy detection of the co-localization of 3D8 scFv, CD44, and BCAN. HeLa cells were transfected with CD44-GFP (B) and HA-BCAN (D) vectors expressed on the cell surface, and CD44ΔTM-GFP (C) and BCAN-ΔGPI-GFP (E) vectors expressed intracellularly. After transfection, 3D8 scFv was used to treat HeLa cells for 1 h at 4 °C or 6 h at 37 °C, and CD44, BCAN, and 3D8 scFv were detected using the appropriate antibodies. Nuclei were stained with Hoechst 33342 (blue). The bar denotes 10 µm. (F, H) Flow cytometry examination of cell surface expression of endogenous CD44 (F) and BCAN (H) in HeLa and HEK293T cells. (G, I) CD44-GFP (G) and HA-BCAN (I) overexpressing HeLa and HEK293T cells were incubated with 5 µM 3D8 scFv for 6 h at 37 °C. Cell lysates were immunoprecipitated by α-CD44 and α-HA antibodies, and then detected by western blot using α-CD44 and α-HA antibodies. Asterisks denote endogenous CD44.
I. HS and CS chains, but not core proteins, are responsible for endocytosis of 3D8 scFv.
We assessed the interaction between 3D8 scFv and the HSPG/CSPG core proteins of the receptor for endocytosis. GAG-degrading enzymes were used to investigate whether HS and CS chains, but not the core proteins, interacted with 3D8 scFv to cause endocytosis (Fig. 9). SDC2, GPC3, CD44, and BCAN overexpressing cells were pre-treated with heparinase III and chondroitinase ABC, respectively, for 2 h at 37°C. 3D8 scFv was used to treat GAG-degraded HeLa cells for 6 h at 37°C, followed by immunoprecipitation with α-GFP, α-GPC3, α-CD44, and α-HA antibodies. Uptake of 3D8 scFv into GAG chain-degraded HeLa cells was significantly reduced compared to enzyme-untreated HeLa cells. The results confirmed the involvement of HS and CS chains in the endocytosis of 3D8 scFv.
Fig. 9. HS and CS chains, but not core proteins, are responsible for endocytosis of 3D8 scFv. HSPG (SDC2 and GPC3) and CSPG (CD44 and BCAN) overexpressing HeLa cells were incubated with 500 mIU/ml heparinase III or 500 mIU/ml chondroitinase ABC for 2 h at 37 °C, prior to treatment with 3D8 scFv for 6 h at 37 °C. Lysates were immunoprecipitated with GFP, GPC3, CD44, or α-HA antibodies, followed by western blot.
J. Production of IL-8 and TNF-α by internalizing IgG is not mediated by TRIM21 signaling pathway.
The TRIM21 intracellular Fc receptor is important in the ADIN process of pathogen recognition involving cytosolic antibody. As an autoantigen, TRIM21 is highly expressed in SLE patients (Ben-Chetrit et al., 1988). We hypothesized that anti-DNA antibodies with high titer in SLE are associated with TRIM21. To investigate whether the cytokine signaling by freely internalized 3D8 IgG is mediated by TRIM21, we purified the 3D8 IgG N434D mutant, which cannot interact with TRIM21 due to a single amino acid change in the CH3 domain. The internalization of the TRIM21 binding mutant was confirmed by confocal microscopy (Fig. 10A). Physical interaction of IgGs (wild-type and N434D) and TRIM21 was confirmed by immunoprecipitation (Fig. 10B). The lysates of THP-1 cells treated with IgGs were precipitated with protein A/G agarose beads. TRIM21 was detected only in the pull-downed lysates from the 3D8 IgG wild type, indicating the interaction between 3D8 IgG wild-type and endogenous TRIM21. When THP-1 cells were treated with 5 µM of IgG proteins for 6 h, the secretion of IL-8 and TNF-α were increased by approximately 40% in the cells treated with 3D8 IgG N434D mutant compared to the cells treated with 3D8 IgG wild-type (Fig. 10C). In addition, IL-8 and TNF-α production by the 3D8 IgG wild-type was increased by 20% in TRIM21-knockdown THP-1 cells (Fig. 10D, E). The results indicate that TRIM21 is not responsible for the production of cytokines by 3D8 IgG, but seems to play a role as a negative regulator, and also suggests the possibility of an unknown
The TRIM21 intracellular Fc receptor is important in the ADIN process of pathogen recognition involving cytosolic antibody. As an autoantigen, TRIM21 is highly expressed in SLE patients (Ben-Chetrit et al., 1988). We hypothesized that anti-DNA antibodies with high titer in SLE are associated with TRIM21. To investigate whether the cytokine signaling by freely internalized 3D8 IgG is mediated by TRIM21, we purified the 3D8 IgG N434D mutant, which cannot interact with TRIM21 due to a single amino acid change in the CH3 domain. The internalization of the TRIM21 binding mutant was confirmed by confocal microscopy (Fig. 10A). Physical interaction of IgGs (wild-type and N434D) and TRIM21 was confirmed by immunoprecipitation (Fig. 10B). The lysates of THP-1 cells treated with IgGs were precipitated with protein A/G agarose beads. TRIM21 was detected only in the pull-downed lysates from the 3D8 IgG wild type, indicating the interaction between 3D8 IgG wild-type and endogenous TRIM21. When THP-1 cells were treated with 5 µM of IgG proteins for 6 h, the secretion of IL-8 and TNF-α were increased by approximately 40% in the cells treated with 3D8 IgG N434D mutant compared to the cells treated with 3D8 IgG wild-type (Fig. 10C). In addition, IL-8 and TNF-α production by the 3D8 IgG wild-type was increased by 20% in TRIM21-knockdown THP-1 cells (Fig. 10D, E). The results indicate that TRIM21 is not responsible for the production of cytokines by 3D8 IgG, but seems to play a role as a negative regulator, and also suggests the possibility of an unknown