Enhanced Pro-apoptotic Effects of Fe(II)-Modified IVIG on Human Neutrophils

Abstract

Mild modification of intravenous immunoglobulin (IVIG) has been reported to result in enhanced polyspecificity and leveraged therapeutic effects in animal models of inflammation. Here, we observed that IVIG modification by ferrous ions, heme or low pH exposure, shifted the repertoires of specificities in different directions. Ferrous ions exposed Fe(II)-IVIG, but not heme or low pH exposed IVIG, showed increased pro-apoptotic effects on neutrophil granulocytes that relied on a FAS-dependent mechanism. These effects were also observed in human neutrophils primed by inflammatory mediators or rheumatoid arthritis joint fluid in vitro, or patient neutrophils ex vivo from acute Crohn's disease. These observations indicate that IVIG-mediated effects on cells can be enhanced by IVIG modification, yet specific modification conditions may be required to target specific molecular pathways and eventually to enhance the therapeutic potential.

Keywords: FAS; cell death; inflammation; modified IVIG; neutrophil.

Figure 1

Immunoglobulin modification by ferrous ions, heme or low pH results in a diverse immunoprofile with altered capacity for regulating neutrophil survival. (A) Immunoprofiles of ferrous ion-exposed Fe(II)-IVIG, heme- or low pH-exposed IVIG preparations, analyzed by glycan array technology. Hierarchical clustering analysis based on reactivity levels expressed as relative fluorescence units (RFU) and as indicated in the color key. (B) Binding reactivity to factor VIII and IX of IVIG before and after exposure to heme and/or ferrous ions as analyzed by ELISA. (C–E) Neutrophil death upon treatment with 20 mg/ml native or modified IVIG (20-h cultures), analyzed by flow cytometry. (C) Death-promoting effects of modified IVIG preparations on neutrophils in presence or absence of GM-CSF or LPS. (D,E) Concentration effect curve (D) and time course (E) for cell death induction by ferrous ion-exposed Fe(II)- or native IVIG in unprimed or primed (GM-CSF, LPS) neutrophils analyzed by flow cytometric ethidium bromide exclusion assay. (D) Specific death in 20-h cultures calculated in comparison to untreated controls as outlined in the Materials and Methods section. (E) Neutrophil death upon treatment with 20 mg/ml IVIG. Two-way ANOVA, followed by Dunnett's posttest (B) or Tukey's posttest for comparisons among groups (C). Data are representative of three (E), four (B), or at least five (C,D) experiments; mean ± SD (B) or SEM (C–E). *P < 0.05, **P < 0.01, ****P < 0.0001. Specific death was calculated in comparison to untreated controls as outlined in the Materials and Methods section.

Figure 2

Modification of IVIG by ferrous ion-exposure enhances pro-apoptotic effects of IVIG in both unprimed and primed (GM-CSF, LPS) neutrophils. Flow cytometric analysis of neutrophils upon treatment with Fe(II)- or native IVIG. (A) Representative Annexin V-FITC/PI staining of 15 h-cultures of unprimed or primed (GM-CSF, LPS) neutrophils. (B) IVIG induced neutrophil death upon pretreatment with the pan-caspase inhibitors Q-VD-OPh (QVD) or Z-VAD-fmk (ZVAD) in 20 h-cultures of unprimed and primed (GM-CSF, LPS) cells. (C) Mitochondrial potential assessed by tetramethylrhodamine ethyl ester (TMRE) staining in 5 h cultures. Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) was used as a positive control. Two-way ANOVA, followed by Tukey's posttest for comparisons among groups (B), or paired t test (C). Data are representative of at least 2 (B), three (A), or four (C) independent experiments (mean ± SEM in B,C). *P < 0.05, n.s., non-significant.

Figure 3

Increased cytotoxicity of ferrous ion-exposed IVIG involves FAS signaling in neutrophils. (A) Concentration-dependent death response of autologous neutrophils to IVIG (native or ferrous ion-exposed) following preincubation with polymorphonuclear neutrophils (PMNs) (IVIG^PMN) or medium (IVIG^medium). (B) Neutrophil death upon preincubation of native or Fe(II)-IVIG with recombinant TNF-R1-, Siglec-9- or FAS-Fc proteins in presence of absence of GM-CSF or LPS. (C) Surface expression of FAS ligand (FASL) on unprimed or primed (GM-CSF or LPS) neutrophils upon culture for 15 h in medium, native or Fe(II)-IVIG. (D) Immunoblotting indicating native and Fe(II)-IVIG reactivity to immobilized recombinant FAS-Fc protein. (E) Blocking effect of IVIG preincubation at two different concentrations on neutrophil death induced by an α-FAS monoclonal antibody (mAb, clone CH11). The dashed line represents the mean level of neutrophil death specifically induced by α-FAS mAb treatment. Students t test (B), paired t test (D), one-way ANOVA, followed by Tukey's posttest for comparisons among groups (C), or Dunnett's posttest with anti-FAS as control (E). Data are representative of three (A), four (D) five (C), or six (B,E) independent experiments (mean ± SEM). *P < 0.05, **P < 0.01, n.s., non-significant. Specific death was calculated in comparison to untreated controls as outlined in the Materials and Methods section.

Figure 4

Fe(II)-modification of IVIG enhances death of neutrophils under inflammatory conditions. (A) Human neutrophils freshly isolated from the peripheral blood of patients with active Crohn's disease (n = 3), psoriasis (n = 5), or healthy individuals (n = 5) were assessed for neutrophil death (24-h cultures; flow cytometric ethidium bromide-exclusion assay) induced by native or Fe(II)-IVIG with or without prior priming with GM-CSF or LPS. (B) Cell death of healthy neutrophils cultured in conditioned medium with 25 or 50% cell-free rheumatoid arthritis joint fluid, in presence or absence of native or Fe(II)-IVIG, analyzed as in (A). Students t test (A), one-way ANOVA, followed by Tukey's posttest for comparisons among groups (B). Data are representative of at least three (A) or four (B) independent experiments (mean ± SEM). *P < 0.05, **P < 0.01.