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. 2015 Oct 2;4(10):e42.
doi: 10.1038/cti.2015.18. eCollection 2015 Oct.

Immunological evidence and regulatory potential for cell-penetrating antibodies in intravenous immunoglobulin

Aggeliki D Sali  1 , Ioannis Karakasiliotis  2 , Maria Evangelidou  3 , Stratis Avrameas  1 , Peggy Lymberi  1
Affiliations

Immunological evidence and regulatory potential for cell-penetrating antibodies in intravenous immunoglobulin

Aggeliki D Sali et al. Clin Transl Immunology. .

Abstract

Anti-DNA cell-penetrating autoantibodies have been extensively studied in autoimmune but not in normal sera. We investigated herein the presence and properties of cell-penetrating antibodies (CPAbs) in intravenous immunoglobulin (IVIg), a blood product of pooled normal human IgG. IVIg cell penetration was observed into various cell lines, as well as cells from several organs of mice injected intravenously with IVIg therapeutic dose. In all cell types examined in vitro and in vivo, intracellular IgG localized in the cytoplasm, in contrast to the nuclear accumulation of disease-related CPAbs. IVIg was found to rapidly enter cells via an energy-independent mode. The CPAb-fraction was isolated and found to be polyreactive to nuclear and cytoplasmic components; although it corresponded to ~2% of IVIg, it accounted for its inhibitory effect on splenocyte activation. Investigation of IVIg cell penetration capacity provides insight into its mechanisms of action and may account for some of its beneficial effects in numerous diseases.

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Figures

Figure 1
Figure 1
In vitro cell penetration of IVIg. Cells were incubated with an IVIg preparation (Sandoglobulin; 1.6 mg ml−1), IVIg-F(ab′)2 fragments (1.6 mg ml−1) or culture medium (control). Confocal sections of IVIg staining (green) after incubation with: (a) HeLa, Vero, N2a, Human fibroblasts, HepG2 or NIH-3T3 cells for 2 h at 37 °C. An anti-human IgG (H- and L-chain-specific)-Alexa488 conjugate was used for IVIg detection (green). (b) Means±s.d. of IgG concentrations measured by ELISA in cytoplasmic and nuclear extracts of HeLa cells (white bars) and human fibroblasts (gray bars) obtained after their trypsinization and lysis with hypotonic buffers. Results are obtained from three independent experiments. (c) Confocal sections of NIH-3T3 cells after incubation with IVIg for 2 h at 37 °C and 4 °C (pretreated for 30 min at 4 °C) with the use of anti-human IgG (H- and L-chain-specific)-Alexa488 conjugate as detection antibody or (d) with IVIg and F(ab')2 detected by anti-human IgG F(ab')2 antibody conjugated to fluorescein isothiocyanate (green). In all cell-imaging assays, TO-PRO-3 iodide (blue) was used for nucleus labeling, and images captured with a x63 HCX PLApo objective lens. Results are representative of at least three independent experiments.
Figure 2
Figure 2
Antibody reactivity and in vitro cell penetration capability of IVIg affinity-purified fractions. Affinity-purified specific antibody fractions were isolated from IVIg (Intraglobin F) on histone (▪), heparin (•) or DNA (○) immunoadsorbents (IADs) and examined in comparison to the whole IVIg (Δ) or respective IVIg-effluents (◊). (a) Reactivity of antibodies (150–1.2 μg ml−1) against histone, heparin and DNA by ELISA, using anti-human IgG-horseradish peroxidase conjugate as secondary antibody. (b) Confocal sections of NIH-3T3 cells after incubation for 2 h at 37 °C with the three affinity-purified antibody fractions (0.2 mg ml−1), whole IVIg (1.6 mg ml−1), effluent (1.6 mg ml−1) or culture medium (control). This effluent results from the exhaustive and successive passages of IVIg through all three IADs. IVIg staining (green) revealed with anti-human IgG (H- and L-chain-specific)-Alexa488 and TO-PRO-3 iodide nucleus labeling (blue) observed using a x63 HCX PLApo objective lens. All results are representative of three independent experiments.
Figure 3
Figure 3
Effect of IVIg cell penetration on CD25-induced upregulation in CD4+, CD8+ and double negative splenocytes. Splenocytes from BALB/c mice were incubated for 90 min with 4 mg ml−1 of whole IVIg (Intraglobin F), 4 mg ml−1 of IVIg-effluent or culture medium (control) and then stimulated for 5 h with PMA (1 ng ml−1)/ionomycin (1 μg ml−1) at 37 °C. (a) Fluorescent intensity of CD25 expression on stimulated splenocytes incubated with samples in the presence of PMA/ionomycin, and mean fluorescent intensity (MFI) of CD25 staining by FACS analysis (n=7). Results obtained from at least three independent experiments. (b) Percentages (%) of CD25-expressing cells in CD4+, or CD8+, and CD4−CD8− populations. Results are shown as mean values of 4 (control) and 3 (effluent and IVIg) samples and are representative of three independent experiments. (c) Maximum projection of confocal sections of splenocytes. IVIg (green) and CD25 (red) observed using a x63 HCX PLApo objective lens; scale bar corresponds to 20 μm. Results are representative of three independent experiments.
Figure 4
Figure 4
In vivo cell penetration of IVIg 3 h post administration. BALB/c mice received a single intravenous injection of 2 g kg−1 of IVIg (Intraglobin F; n=3), or IVIg-effluent (n=3), or physiological saline (n=3; control). Three hours later, various organs were snap-frozen and subjected to cryostat sectioning. Maximum projection of confocal sections from organs processed with cryostat (liver, kidneys, lungs, brain, ileum and heart). IVIg staining (green) and TO-PRO-3 iodide nucleus labeling (blue) observed using a x20 HC PlanApo and x63 HCX PLApo objective lenses; scale bar corresponds to either 80 or 20 μm. Results are representative of three independent experiments.
Figure 5
Figure 5
In vivo cell penetration of IVIg 6 days post administration. BALB/c mice received a single intravenous injection of 2 g kg−1 of IVIg (Intraglobin F; n=3) or of 2 g kg−1 of IVIg-effluent (n=3). Six days later, the lung, ileum and liver were snap-frozen and subjected to cryostat sectioning. Maximum projection of confocal sections from organs were processed with cryostat. IVIg staining (green) and TO-PRO-3 iodide nucleus labeling (blue) were observed using a x20 HC PlanApo objective lens; scale bar corresponds to 80 μm. Results are representative of three independent experiments.
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