Anti-inflammatory IgG production requires functional P1 promoter in β-galactoside α2,6-sialyltransferase 1 (ST6Gal-1) gene

Abstract

The anti-inflammatory properties associated with intravenous immunoglobulin therapy require the sialic acid modification of the N-glycan of the Fc domain of IgG. Sialylation of the Fc fragment is mediated by β-galactoside α2,6-sialyltransferase 1 (ST6Gal-1), acting on the Gal(β4)GlcNAc terminal structure of the biantennary N-glycans on the Fc domain. However, little is known regarding the in vivo regulation of Fc sialylation and its role in the progression of inflammatory processes. Here, we report that decreased Fc sialylation of circulatory IgG accompanies the acute phase response elicited by turpentine exposure or upon acute exposure to either nontypeable Haemophilus influenzae or ovalbumin. However, Fc sialylation was increased 3-fold from the base line upon transition to chronic inflammation by repeated exposure to challenge. The P1 promoter of the ST6Gal-1 gene is critical for Fc sialylation, but P1 does not drive ST6Gal-1 expression in B cells. The Siat1ΔP1 mouse, with a dysfunctional P1 promoter, was unable to produce sialylated Fc in the systemic circulation, despite the presence of Gal(β4)GlcNAc termini on the Fc glycans. The major contribution of P1 action is to synthesize ST6Gal-1 enzymes that are deposited into the systemic circulation. The data strongly indicate that this pool of extracellular ST6Gal-1 in the blood impacts the sialylation of IgG Fc and that defective Fc sialylation is likely a major contributing mechanism for the proinflammatory tendencies previously noted in Siat1ΔP1 animals.

FIGURE 1.

Circulatory Fc sialylation in NTHi-mediated pulmonary inflammation. C57BL/6 mice (WT) were subjected to biweekly intra tracked instillations of NTHi for 16 consecutive weeks as detailed under “Experimental Procedures,” and serum (n = 5) was harvested weekly and pooled (where D1 represents a post-challenge blood draw on the first day of treatment). Fc fragments of IgG were isolated from each of the weekly pools, subjected to SDS-PAGE, and probed with SNA (green), anti-Fc antibody (blue), and anti-Fab antibody (red) as displayed in the inset. The SNA reactivity of the Fc fragments is expressed as a ratio of the relative SNA to anti-Fc signals, where untreated denotes the ratio found in the serum IgG of animals without treatment.

FIGURE 2.

Circulatory Fc sialylation in OVA model of allergic pulmonary inflammation. C57BL/6 (WT) and Siat1ΔP1 mice were sensitized to OVA and subjected to acute and chronic protocols of pulmonary inflammation as described under “Experimental Procedures.” Sera from five animals representing each of the conditions were pooled, and Fc fragments of IgG were isolated from pooled sera from animals sensitized but not challenged by OVA (Primed), challenged with the acute protocol (Acute), and challenged with the chronic protocol (Chron). A, sera were subjected to SDS-PAGE analysis, probed with SNA or ECL (green), anti-Fc antibody (blue), and anti-Fab antibody (red). Resol, resolution. B, quantitation of the data from A expressed as SNA/anti-Fc or ECL/anti-Fc ratios. Ratio values <30 were regarded as random noise.

FIGURE 3.

Acute phase response is accompanied by reduced circulatory Sia-Fc. Fc fragments were isolated from the pooled sera of C57BL/6 (WT), Siat1ΔP1 (ΔP1), and Siat1-null (KO) mice at base line (Rest) or 72 h after elicitation of the acute phase response by subcutaneous injection of turpentine (T72). The samples were separated by SDS-PAGE and probed with SNA (Cy5), anti-Fc antibody (Cy3), and anti-Fab antibody (Cy2). In each lane, serum pools from five animals were used.

FIGURE 4.

Siat1ΔP1 Fc region is undersialylated. The serum pools of three C57BL/6 (WT), three Siat1ΔP1 (ΔP1), and three Siat1-null (KO) mice at rest were probed as described in the legends to Figs. 1–3, and the results are expressed as SNA/anti-Fc ratios. A, signal ratio of intact IgG; B, signal ratio of an isolated Fc fragment.

FIGURE 5.

PSL and ECL reactivity of Fc fragments. Fc fragments from serum pools of five C57BL/6 (WT), Siat1ΔP1 (ΔP1), and Siat1-null (KO) mice were subjected to SDS-PAGE and blotted for reactivity against PSL (A) and ECL (B), recognizing the α2,6-sialyl type 2 lactosamine and unsialylated type 2 lactosamine structures, respectively, as diagrammed.

FIGURE 6.

Structural composition of Fc glycans. Shown are the results from linear ion trap-MS analysis for the glycan structural composition (expressed as percent of total glycans) present on the Fc fragments in untreated WT and Siat1ΔP1 pooled sera. MALDI-TOF-MS data from which this information was derived are shown in supplemental Fig. 1.