IVIG regulates the survival of human but not mouse neutrophils

Abstract

Intravenous immunoglobulin (IVIG) are purified IgG preparations made from the pooled plasma from thousands of healthy donors and are being tested in preclinical mouse models. Inherent challenges, however, are the pluripotency of IVIG and its xenogeneicity in animals. IVIG can alter the viability of human neutrophils via agonistic antibodies to Fas and Siglec-9. In this study, we compared the effects of IVIG on human and mouse neutrophils using different death assays. Different commercial IVIG preparations similarly induced cytokine-dependent death in human neutrophils, whereas they had no effects on the survival of either peripheral blood or bone marrow neutrophils from C57BL/6 or BALB/c mice. F(ab')₂ but not Fc fragments of IVIG induced death of human neutrophils, whereas neither of these IVIG fragments, nor agonistic monoclonal antibodies to human Fas or Siglec-9 affected the viability of mouse neutrophils. Pooled mouse IgG, which exhibited a different immunoprofile compared to IVIG, also had no effect on mouse cells. Together, these observations demonstrate that effects of IVIG on neutrophil survival are not adequately reflected in current mouse models, despite the key role of these cells in human inflammatory and autoimmune diseases.

Figure 1

IVIG induces cell death in human but not in mouse polymorphonuclear neutrophils (PMNs) ex vivo. Death of human (PMN^H), C57BL/6 (PMN^BL/6) or BALB/c (PMN^BALB/c) mouse neutrophils in the presence or absence of priming with species matched GM-CSF, as assessed by flow cytometric ethidium bromide exclusion assay. (A) Different commercial IVIG/SCIG preparations similarly induce death of human peripheral blood neutrophils, an effect that is enhanced in GM-CSF primed cells. No death is induced in mouse PB neutrophils. (B) Comparison of the concentration-effect curves of IVIG in PB PMN^H or PMN^BL/6 with or without GM-CSF priming. (C) In mouse neutrophils from both strains, no IVIG-mediated death is observed, neither in cells derived from peripheral blood (PB) nor bone marrow (BM). (D) In in vivo primed neutrophils isolated from spleen or peripheral blood (PB) no IVIG-induced cell death was observed. Results of 24-h cultures are shown. Bars show mean ± SEM. Data are representative of at least 3 (D), 4 (C), 5 (B), or 6 (A) independent experiments. *p < 0.05, **p < 0.01, Student t test. Specific death was calculated in comparison to untreated controls as outlined in the Materials and Methods section.

Figure 2

Confirmation of selective survival regulatory capacity of IVIG in human but not mouse neutrophils by different death assays. Features of cell death assessed in GM-CSF primed or unprimed human (PMN^H) or C57BL/6 mouse (PMN^BL/6) neutrophils following treatment with IVIG by flow cytometry (A–C) or light microscopy (D). (A) Annexin V-FITC/PI staining assay. Quantitative analysis is indicated as the percentage representative of each quadrant. (B) DNA-fragmentation assay. Quantitative analysis of gated subdiploid DNA is indicated. (C) Assessment of mitochondrial potential (Δψm). The mitochondrial uncoupler FCCP was used as a positive control. Results of 5- (C) or 15-hour cultures (A,B,D) are shown. (D) Morphological characterization. Cells were stained with Giemsa–May–Grünwald (Diff-Quik) (original magnification X1000). Continuous arrows indicate condensed nuclei, dashed arrows vacuoles. The lower panel represents the statistical analysis of the normal, apoptotic or vacuolated phenotypes. Data are representative for at least 3 independent experiments. Bars show mean ± SEM. *p < 0.05, **p < 0.01, one way-ANOVA followed by Dunnet’s post hoc test. Δψm = mitochondrial potential.

Figure 3

Human neutrophil susceptibility to death stimuli is specific, Fc-independent, and differs from responses of mouse neutrophils. Death of human (PMN^H) or mouse C57BL/6 (PMN^BL/6) polymorphonuclear neutrophils as assessed by flow cytometric ethidium bromide exclusion assay. Results of 24-h cultures of quiescent or GM-CSF primed cells are shown following treatment with (A) 20 mg/ml of whole IgG (Privigen^#B) or equimolar concentrations of Fc-, or F(ab’)₂ (Gammavenin)-fragments of pooled IgG, or with (B) stimuli known to trigger human neutrophil death including IVIG (Prigiven^#A), Fas-ligand, anti-human Fas (clone CH11) mAb or anti-Siglec-9 mAb. Bars show mean ± SEM. Data are representative of at least 3 (B), or 4 (A) independent experiments. *p < 0.05, **p < 0.01, Student t test. Specific death was calculated in comparison to untreated controls as outlined in the Materials and Methods section.

Figure 4

Mouse polyclonal IgG does not induce cell death in mouse neutrophils. Flow cytometric characterization of C57BL/6 mouse neutrophil cell death of unprimed or primed cells by mouse polyclonal IgG (IgG^Mice). Representative example of Annexin V-FITC/PI staining assay (A) and statistical summaries (B) of Annexin⁻/PI⁻-, Annexin⁺/PI⁻- and Annexin⁺/PI⁺-gated cell populations are indicated for analysis after 15 h incubation with polyclonal mouse IgG. (C) Death of mouse C57BL/6 mouse (PMN^BL/6) or BALB/c mouse (PMN^BALB/c) neutrophils after IgG stimulation as assessed by ethidium bromide exclusion assay. (D) Binding profiles of IgG^Mice or IVIG to 609 glycan antigens, as assessed by glycan array technology. RFU = relative fluorescence units. Bars show mean ± SEM (n = 3). *p < 0.05. One way-ANOVA followed by Dunnets posttest. Specific death was calculated in comparison to untreated controls as outlined in the Materials and Methods section.