Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway

Abstract

High-dose intravenous immunoglobulin is a widely used therapeutic preparation of highly purified immunoglobulin G (IgG) antibodies. It is administered at high doses (1-2 grams per kilogram) for the suppression of autoantibody-triggered inflammation in a variety of clinical settings. This anti-inflammatory activity of intravenous immunoglobulin is triggered by a minor population of IgG crystallizable fragments (Fcs), with glycans terminating in α2,6 sialic acids (sFc) that target myeloid regulatory cells expressing the lectin dendritic-cell-specific ICAM-3 grabbing non-integrin (DC-SIGN; also known as CD209). Here, to characterize this response in detail, we generated humanized DC-SIGN mice (hDC-SIGN), and demonstrate that the anti-inflammatory activity of intravenous immunoglobulin can be recapitulated by the transfer of bone-marrow-derived sFc-treated hDC-SIGN(+) macrophages or dendritic cells into naive recipients. Furthermore, sFc administration results in the production of IL-33, which, in turn, induces expansion of IL-4-producing basophils that promote increased expression of the inhibitory Fc receptor FcγRIIB on effector macrophages. Systemic administration of the T(H)2 cytokines IL-33 or IL-4 upregulates FcγRIIB on macrophages, and suppresses serum-induced arthritis. Consistent with these results, transfer of IL-33-treated basophils suppressed induced arthritic inflammation. This novel DC-SIGN-T(H)2 pathway initiated by an endogenous ligand, sFc, provides an intrinsic mechanism for maintaining immune homeostasis that could be manipulated to provide therapeutic benefit in autoimmune diseases.

Figure 1. Human DC-SIGN conveys sFc anti-inflammatory activity

a, A map of the human chromosome 19 BAC clone containing DC-SIGN and DC-SIGN-R genes. Cen, centromere. b, Wild-type (WT; black bars), SIGN-R1^−/− (white bars), or hDC-SIGN⁺/SIGN-R1^−/−(grey bars) mice were administered K/BxN sera and sFc. **P >0.001 determined by a Fisher least significant difference (LSD) post-hoc test. c, AsialoFc- (black bars) or sFc- (white bars) treated wild-type and hDC-SIGN⁺ bone-marrow derived macrophages (BMMΦ) were administered to K/BxN-sera-treated wild-type recipients. *P >0.05 determined by Tukey’s post-hoc test. d, PBS (black bars) or sFc-treated hDC-SIGN⁺ bone-marrow-derived macrophages (white bars) were administered to SIGN-R1^−/− and FccRIIB^−/− recipients. Means and standard deviations are plotted; **P >0.001 determined by Tukey’s post-hoc test.

Figure 2. IL-4 requirements of sFc anti-inflammatory activity

a, sFc-treated hDC-SIGN⁺bone-marrow-derived macrophages (BMMΦ; white bars) or PBS was administered to K/BxN-treated wild-type or IL-4^−/− recipient mice. **P >0.002 determined by Fisher LSD post-hoc test. b, Wild-type (black bars) and IL-4^−/− (white bars) mice were treated with K/BxN sera and IVIG. *P >0.01 determined by Mann–Whitney’s U test. c, Wild-type (black bars), IL-4Rα^−/− (white bars), or Stat6^−/− mice (grey bars) were given K/BxN sera and IVIG. *P >0.01 determined by Tukey’s post-hoc test. d, Wild-type (black bars) and FccRIIB^−/− mice (white bars) were administered cytokine immune complexes (IL-4ic, IL-3ic, IL-13ic) and K/BxN sera. Means and standard deviations are plotted; *P >0.01, **P >0.001 determined by Mann–Whitney’s U test.

Figure 3. IL-33 triggers IL-4 anti-inflammatory activity

a, Cytokine expression 1 h after IVIG administration in wild-type (black bars) or SIGN-R1^−/− mice (white bars) determined by quantitative polymerase chain reaction (qPCR). n.d., not detected. b, K/BxN-treated wild-type mice received PBS, IL-33, IL-25, or TSLP. *P >0.05 determined by Tukey’s test. c, K/BxN-treated wild-type (black bars) or IL-4Rα^−/− (white bars) mice received PBS, IL-4ic, or IL-33. **P >0.001 determined by Tukey’s test. d, hDC-SIGN⁺/SIGN-R1^−/− mice received K/BxN sera, sFc and anti-IL-33Rα. **P >0.001 determined by Fisher LSD test. e, sFc-treated hDC-SIGN⁺ bone-marrow-derived macrophages were administered to wild-type mice, K/BxN- and anti-IL-33Rα-treated wild-type mice. Means and standard deviations are plotted; *P >0.05 determined by Tukey’s test. f, Individual mean fluorescence intensities (MFI) of bone marrow monocyte (CD11b⁺ Ly6G⁻) FcγRIIB surface expression 24 h after PBS, IL-4, IL-33, or IL-25 treatment by FACS. **P >0.01 determined by Tukey’s test.

Figure 4. Anti-inflammatory activity mediated by basophils

a, hDC-SIGN⁺/SIGN-R1^−/− mice were administered K/BxN sera, sFc and anti-FcεRI or an isotype control. **P >0.001, *P >0.05 determined by Fisher LSD test. b, 4get mice were administered K/BxN sera and sFc. Circulating IL-4⁺ basophils (grey bars, DX5⁺ FcεRI⁺ GFP⁺) and clinical scores (black bars) are plotted. *P >0.05 determined by Tukey’s test. c, PBS or IL-33-treated basophils (DX5⁺ FcεRI⁺ c-Kit⁻) were administered to K/BxN-treated wild-type mice. Control mice received PBS or IVIG. d, Basophils from IL-33-treated FcγRIIB^−/− mice were administered to K/BxN-treated wild-type recipients. Clinical scores (black) and serum IL-6 levels (grey) are plotted. Means and standard deviations are plotted;*P >0.05 determined by Mann–Whitney’s U test.