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Fc gamma receptor IIb participates in maternal IgG trafficking of human placental endothelial cells.

Ishikawa T, Takizawa T, Iwaki J, Mishima T, Ui-Tei K, Takeshita T, Matsubara S, Takizawa T - Int. J. Mol. Med. (2015)

Bottom Line: The internalization and transcytosis of IgG was significantly higher in the pFCGR2B2-EGFP-transfected cells than in the mock-transfected cells, and the majority of the internalized IgG was co-localized with the FCGR2B2-EGFP signals.Furthermore, we isolated FCGR2B2 compartments from the human placenta and found that the Rab family of proteins [RAS-related protein Rab family (RABs)] were associated with FCGR2B2 compartments.The downregulation of RAB3D by small interfering RNA (siRNA) resulted in a marked reduction in the FCGR2B2-EGFP signals at the cell periphery.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine and Anatomy, Nippon Medical School, Tokyo 113-8602, Japan.

ABSTRACT
The human placental transfer of maternal IgG is crucial for fetal and newborn immunity. Low-affinity immunoglobulin gamma Fc region receptor IIb2 (FCGR2B2 or FcγRIIb2) is exclusively expressed in an IgG-containing, vesicle-like organelle (the FCGR2B2 compartment) in human placental endothelial cells; thus, we hypothesized that the FCGR2B2 compartment functions as an IgG transporter. In this study, to examine this hypothesis, we performed in vitro bio-imaging analysis of IgG trafficking by FCGR2B2 compartments using human umbilical vein endothelial cells transfected with a plasmid vector containing enhanced GFP-tagged FCGR2B2 (pFCGR2B2-EGFP). FCGR2B2-EGFP signals were detected as intracellular vesicular structures similar to FCGR2B2 compartments in vivo. The internalization and transcytosis of IgG was significantly higher in the pFCGR2B2-EGFP-transfected cells than in the mock-transfected cells, and the majority of the internalized IgG was co-localized with the FCGR2B2-EGFP signals. Furthermore, we isolated FCGR2B2 compartments from the human placenta and found that the Rab family of proteins [RAS-related protein Rab family (RABs)] were associated with FCGR2B2 compartments. Among the RABs, RAB3D was expressed predominantly in placental endothelial cells. The downregulation of RAB3D by small interfering RNA (siRNA) resulted in a marked reduction in the FCGR2B2-EGFP signals at the cell periphery. Taken together, these findings suggest that FCGR2B2 compartments participate in the transcytosis of maternal IgG across the human placental endothelium and that RAB3D plays a role in regulating the intracellular dynamics of FCGR2B2 compartments.

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Validation of antibodies and characterization of pFCGR2B2-EGFP-transfected human umbilical vein endothelial cells (HUVECs). (A) Validation of antibodies. The anti-FCGR2A, anti-FCGR2B and anti-FCGRT antibodies used in this study were validated by western blot analysis. Lysates of Cos-7 cells transfected with a plasmid designed to overexpress EGFP or EGFP-tagged FCGRT, FCGR1A, FCGR2A, FCGR2B1 or FCGR2B2 were used for this validation. The upper band in each lane corresponds to an EGFP-conjugated Fc receptor and the lower band corresponds to an Fc receptor without EGFP. (B and C) Representative immunofluorescence images of CD31 and CD144 in HUVECs plated on cell culture inserts. (B) CD31 and (C) CD144 are localized along the lateral membrane of adjacent cells. (D and E) Comparison of the subcellular localization and IgG binding property of FCGR2B2-EGFP with those of FCGR2B2. (D) Representative immunofluorescence image of a pFCGR2B2-transfected HUVEC. FCGR2B2 signals are detected as intracellular vesicular and tubular structures; some signals are also present on the cell surface. Note that a pFCGR2B2-EGFP-transfected cell (Fig. 2A) shows similar localization of FCGR2B2 in the pFCGR2B2-transfected cell. (E) The affinities of human IgG for FCGR2B2-EGFP and FCGR2B2 detected by enzyme-linked immunosorbent assay (ELISA). FCGR2B2-EGFP (white circles) shows an almost identical affinity for FCGR2B2 (black circles). By contrast, EGFP from mock-transfected pEGFP-N1 cells do not exhibit selectivity for IgG (black triangles). Three independent experiments were performed; error bars represent the means ± SD. FCGR2B2, low-affinity immunoglobulin gamma Fc region receptor IIb2.
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f1-ijmm-35-05-1273: Validation of antibodies and characterization of pFCGR2B2-EGFP-transfected human umbilical vein endothelial cells (HUVECs). (A) Validation of antibodies. The anti-FCGR2A, anti-FCGR2B and anti-FCGRT antibodies used in this study were validated by western blot analysis. Lysates of Cos-7 cells transfected with a plasmid designed to overexpress EGFP or EGFP-tagged FCGRT, FCGR1A, FCGR2A, FCGR2B1 or FCGR2B2 were used for this validation. The upper band in each lane corresponds to an EGFP-conjugated Fc receptor and the lower band corresponds to an Fc receptor without EGFP. (B and C) Representative immunofluorescence images of CD31 and CD144 in HUVECs plated on cell culture inserts. (B) CD31 and (C) CD144 are localized along the lateral membrane of adjacent cells. (D and E) Comparison of the subcellular localization and IgG binding property of FCGR2B2-EGFP with those of FCGR2B2. (D) Representative immunofluorescence image of a pFCGR2B2-transfected HUVEC. FCGR2B2 signals are detected as intracellular vesicular and tubular structures; some signals are also present on the cell surface. Note that a pFCGR2B2-EGFP-transfected cell (Fig. 2A) shows similar localization of FCGR2B2 in the pFCGR2B2-transfected cell. (E) The affinities of human IgG for FCGR2B2-EGFP and FCGR2B2 detected by enzyme-linked immunosorbent assay (ELISA). FCGR2B2-EGFP (white circles) shows an almost identical affinity for FCGR2B2 (black circles). By contrast, EGFP from mock-transfected pEGFP-N1 cells do not exhibit selectivity for IgG (black triangles). Three independent experiments were performed; error bars represent the means ± SD. FCGR2B2, low-affinity immunoglobulin gamma Fc region receptor IIb2.

Mentions: Rabbit anti-human FCGR2B antibody directed against the C-terminal cytoplasmic tail of human FCGR2B (MHPDALEEPDDQNRI) and rabbit anti-human FCGRT antibody (DADLKDVNVIPATA) directed against the C-terminal cytoplasmic tail of human FCGRT were generated (Peptide Institute, Osaka, Japan). Rabbit anti-human low-affinity immunoglobulin FCGR2A antibody directed against the C-terminal cytoplasmic tail of human FCGR2A (YLTLPPNDHVNSNN) was also generated and used to test the specificity of the anti-FCGR2B antibody. Cos-7 cells (Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer Tohoku University, Miyagi, Japan) were transfected with a plasmid designed to overexpress EGFP or EGFP-tagged FCGRT, FCGR1A, FCGR2A, FCGR2B1 or FCGR2B2; the specificity of these affinity-purified antibodies was validated by western blot analysis (Fig. 1A). Rabbit anti-von Willebrand factor antibody (catalog no. F3520; Sigma-Aldrich), mouse anti-caveolin-1 antibody (catalog no. C-37120-150; BD Transduction Laboratories, Franklin Lakes, NJ, USA), mouse anti-CD31 antibody (catalog no. BBA7; R&D Systems, Minneapolis, MN, USA), mouse anti-CD144 (clone 16B1; eBioscience, San Diego, CA, USA; CD144 also known as VE-cadherin and CDH5), rabbit anti-human RAB1 antibody (catalog no. FL-205), rabbit anti-human RAB3 antibody (catalog no. FL-220) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-human RAB3D (catalog no. 12320-1-AP; ProteinTech Group, Chicago, IL, USA), mouse anti-human RAB11 antibody (catalog no. 610656; BD Transduction Laboratories), mouse anti-human RAB33B (catalog ID H00083452-M01; Abnova, Taipei, Taiwan), mouse anti-human Ras-related protein Ral-A antibody (catalog no. 610221; BD Transduction Laboratories), Alexa Fluor-labeled secondary antibodies (Invitrogen), and HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were obtained from commercial sources.


Fc gamma receptor IIb participates in maternal IgG trafficking of human placental endothelial cells.

Ishikawa T, Takizawa T, Iwaki J, Mishima T, Ui-Tei K, Takeshita T, Matsubara S, Takizawa T - Int. J. Mol. Med. (2015)

Validation of antibodies and characterization of pFCGR2B2-EGFP-transfected human umbilical vein endothelial cells (HUVECs). (A) Validation of antibodies. The anti-FCGR2A, anti-FCGR2B and anti-FCGRT antibodies used in this study were validated by western blot analysis. Lysates of Cos-7 cells transfected with a plasmid designed to overexpress EGFP or EGFP-tagged FCGRT, FCGR1A, FCGR2A, FCGR2B1 or FCGR2B2 were used for this validation. The upper band in each lane corresponds to an EGFP-conjugated Fc receptor and the lower band corresponds to an Fc receptor without EGFP. (B and C) Representative immunofluorescence images of CD31 and CD144 in HUVECs plated on cell culture inserts. (B) CD31 and (C) CD144 are localized along the lateral membrane of adjacent cells. (D and E) Comparison of the subcellular localization and IgG binding property of FCGR2B2-EGFP with those of FCGR2B2. (D) Representative immunofluorescence image of a pFCGR2B2-transfected HUVEC. FCGR2B2 signals are detected as intracellular vesicular and tubular structures; some signals are also present on the cell surface. Note that a pFCGR2B2-EGFP-transfected cell (Fig. 2A) shows similar localization of FCGR2B2 in the pFCGR2B2-transfected cell. (E) The affinities of human IgG for FCGR2B2-EGFP and FCGR2B2 detected by enzyme-linked immunosorbent assay (ELISA). FCGR2B2-EGFP (white circles) shows an almost identical affinity for FCGR2B2 (black circles). By contrast, EGFP from mock-transfected pEGFP-N1 cells do not exhibit selectivity for IgG (black triangles). Three independent experiments were performed; error bars represent the means ± SD. FCGR2B2, low-affinity immunoglobulin gamma Fc region receptor IIb2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380207&req=5

f1-ijmm-35-05-1273: Validation of antibodies and characterization of pFCGR2B2-EGFP-transfected human umbilical vein endothelial cells (HUVECs). (A) Validation of antibodies. The anti-FCGR2A, anti-FCGR2B and anti-FCGRT antibodies used in this study were validated by western blot analysis. Lysates of Cos-7 cells transfected with a plasmid designed to overexpress EGFP or EGFP-tagged FCGRT, FCGR1A, FCGR2A, FCGR2B1 or FCGR2B2 were used for this validation. The upper band in each lane corresponds to an EGFP-conjugated Fc receptor and the lower band corresponds to an Fc receptor without EGFP. (B and C) Representative immunofluorescence images of CD31 and CD144 in HUVECs plated on cell culture inserts. (B) CD31 and (C) CD144 are localized along the lateral membrane of adjacent cells. (D and E) Comparison of the subcellular localization and IgG binding property of FCGR2B2-EGFP with those of FCGR2B2. (D) Representative immunofluorescence image of a pFCGR2B2-transfected HUVEC. FCGR2B2 signals are detected as intracellular vesicular and tubular structures; some signals are also present on the cell surface. Note that a pFCGR2B2-EGFP-transfected cell (Fig. 2A) shows similar localization of FCGR2B2 in the pFCGR2B2-transfected cell. (E) The affinities of human IgG for FCGR2B2-EGFP and FCGR2B2 detected by enzyme-linked immunosorbent assay (ELISA). FCGR2B2-EGFP (white circles) shows an almost identical affinity for FCGR2B2 (black circles). By contrast, EGFP from mock-transfected pEGFP-N1 cells do not exhibit selectivity for IgG (black triangles). Three independent experiments were performed; error bars represent the means ± SD. FCGR2B2, low-affinity immunoglobulin gamma Fc region receptor IIb2.
Mentions: Rabbit anti-human FCGR2B antibody directed against the C-terminal cytoplasmic tail of human FCGR2B (MHPDALEEPDDQNRI) and rabbit anti-human FCGRT antibody (DADLKDVNVIPATA) directed against the C-terminal cytoplasmic tail of human FCGRT were generated (Peptide Institute, Osaka, Japan). Rabbit anti-human low-affinity immunoglobulin FCGR2A antibody directed against the C-terminal cytoplasmic tail of human FCGR2A (YLTLPPNDHVNSNN) was also generated and used to test the specificity of the anti-FCGR2B antibody. Cos-7 cells (Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer Tohoku University, Miyagi, Japan) were transfected with a plasmid designed to overexpress EGFP or EGFP-tagged FCGRT, FCGR1A, FCGR2A, FCGR2B1 or FCGR2B2; the specificity of these affinity-purified antibodies was validated by western blot analysis (Fig. 1A). Rabbit anti-von Willebrand factor antibody (catalog no. F3520; Sigma-Aldrich), mouse anti-caveolin-1 antibody (catalog no. C-37120-150; BD Transduction Laboratories, Franklin Lakes, NJ, USA), mouse anti-CD31 antibody (catalog no. BBA7; R&D Systems, Minneapolis, MN, USA), mouse anti-CD144 (clone 16B1; eBioscience, San Diego, CA, USA; CD144 also known as VE-cadherin and CDH5), rabbit anti-human RAB1 antibody (catalog no. FL-205), rabbit anti-human RAB3 antibody (catalog no. FL-220) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-human RAB3D (catalog no. 12320-1-AP; ProteinTech Group, Chicago, IL, USA), mouse anti-human RAB11 antibody (catalog no. 610656; BD Transduction Laboratories), mouse anti-human RAB33B (catalog ID H00083452-M01; Abnova, Taipei, Taiwan), mouse anti-human Ras-related protein Ral-A antibody (catalog no. 610221; BD Transduction Laboratories), Alexa Fluor-labeled secondary antibodies (Invitrogen), and HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were obtained from commercial sources.

Bottom Line: The internalization and transcytosis of IgG was significantly higher in the pFCGR2B2-EGFP-transfected cells than in the mock-transfected cells, and the majority of the internalized IgG was co-localized with the FCGR2B2-EGFP signals.Furthermore, we isolated FCGR2B2 compartments from the human placenta and found that the Rab family of proteins [RAS-related protein Rab family (RABs)] were associated with FCGR2B2 compartments.The downregulation of RAB3D by small interfering RNA (siRNA) resulted in a marked reduction in the FCGR2B2-EGFP signals at the cell periphery.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine and Anatomy, Nippon Medical School, Tokyo 113-8602, Japan.

ABSTRACT
The human placental transfer of maternal IgG is crucial for fetal and newborn immunity. Low-affinity immunoglobulin gamma Fc region receptor IIb2 (FCGR2B2 or FcγRIIb2) is exclusively expressed in an IgG-containing, vesicle-like organelle (the FCGR2B2 compartment) in human placental endothelial cells; thus, we hypothesized that the FCGR2B2 compartment functions as an IgG transporter. In this study, to examine this hypothesis, we performed in vitro bio-imaging analysis of IgG trafficking by FCGR2B2 compartments using human umbilical vein endothelial cells transfected with a plasmid vector containing enhanced GFP-tagged FCGR2B2 (pFCGR2B2-EGFP). FCGR2B2-EGFP signals were detected as intracellular vesicular structures similar to FCGR2B2 compartments in vivo. The internalization and transcytosis of IgG was significantly higher in the pFCGR2B2-EGFP-transfected cells than in the mock-transfected cells, and the majority of the internalized IgG was co-localized with the FCGR2B2-EGFP signals. Furthermore, we isolated FCGR2B2 compartments from the human placenta and found that the Rab family of proteins [RAS-related protein Rab family (RABs)] were associated with FCGR2B2 compartments. Among the RABs, RAB3D was expressed predominantly in placental endothelial cells. The downregulation of RAB3D by small interfering RNA (siRNA) resulted in a marked reduction in the FCGR2B2-EGFP signals at the cell periphery. Taken together, these findings suggest that FCGR2B2 compartments participate in the transcytosis of maternal IgG across the human placental endothelium and that RAB3D plays a role in regulating the intracellular dynamics of FCGR2B2 compartments.

Show MeSH
Related in: MedlinePlus