Altered eosinophil profile in mice with ST6Gal-1 deficiency: an additional role for ST6Gal-1 generated by the P1 promoter in regulating allergic inflammation

Abstract

Cumulative evidence indicates that the sialyltransferase ST6Gal-1 and the sialyl-glycans, which it constructs, are functionally pleiotropic. Expression of the ST6Gal-1 gene is mediated by six distinct promoter/regulatory regions, and we hypothesized that these promoters may be used differentially to produce ST6Gal-1 for different biologic purposes. To examine this hypothesis, we compared a mouse with a complete deficiency in ST6Gal-1 (Siat1 null) with another mouse that we have created previously with a disruption only in the P1 promoter (Siat1DeltaP1). We noted previously greater neutrophilic inflammation associated with ST6Gal-1 deficiency. Here, we report that ST6Gal-1-deficient mice also have significantly elevated eosinophilic responses. Upon i.p. thioglycollate elicitation, eosinophils accounted for over 20% of the total peritoneal inflammatory cell pool in ST6Gal-1-deficient animals, which was threefold greater than in corresponding wild-type animals. A principal feature of allergic respiratory inflammation is pulmonary eosinophilia, we evaluated the role of ST6Gal-1 in allergic lung inflammation. Using OVA and ABPA experimental models of allergic airways, we showed that ST6Gal-1 deficiency led to greater airway inflammation characterized by excessive airway eosinophilia. The severity of airway inflammation was similar between Siat1DeltaP1 and Siat1 null mice, indicating a role for P1-generated ST6Gal-1 in regulating eosinophilic inflammation. Colony-forming assays suggested greater IL-5-dependent eosinophil progenitor numbers in the marrow of ST6Gal-1-deficient animals. Moreover, allergen provocation of wild-type mice led to a significant reduction in P1-mediated ST6Gal-1 mRNA and accompanied decline in circulatory ST6Gal-1 levels. Taken together, the data implicate ST6Gal-1 as a participant in regulating not only Th1 but also Th2 responses, and ST6Gal-1 deficiency can lead to the development of more severe allergic inflammation with excessive eosinophil production.

Figure 1.

Elevated peritoneal eosinophil abundance in ST6Gal-1-deficient animals. Peritoneal lavage was recovered from Siat1ΔP1 mice 24 h after challenge with thioglycollate i.p., as stated in Materials and Methods, immobilized on glass slides by cytospin, and stained by Hema 3® (A). (B) FACS analysis of the elicited peritoneal cells using FITC-anti-CCR3 and PE-Ly6G, where the fractions labeled A and B, representing Ly6G^hi:CCR3^neg and Ly6G^lo:CCR3^hi populations, were flow-sorted and visualized by cytospin. (C) Cells from peritoneal lavage of wild-type (WT, open bars), Siat1ΔP1 (ΔP1, hatched bars), or Siat1 null (Null, shaded bars) were assessed for total cell numbers by hemocytometer and for eosinophil (Eos) numbers by FACS and by visual evaluation of stained cytospin preparations. Peritoneal lavage was harvested at baseline (0 h, without thioglycollate treatment) and at 24 h after thioglycollate elicitation. The numbers above each bar represent the number of animals used for each determination. Statistical significant differences between wild-type and mutant animals are denoted by * (P<0.01) or ** (P<0.002).

Figure 2.

H&E histologic evaluation of animals undergoing acute allergic pulmonary inflammation. Representative views of lung sections from wild-type and Siat1ΔP1 mice undergoing OVA-provoked acute allergic pulmonary inflammation are shown in A and B, respectively. (C and D, respectively) Representative lung sections from wild-type and Siat1ΔP1 mice subjected to the experimental ABPA protocol. (A–D) Micrographs using the 4× objective; insets using a ×40 objective.

Figure 3.

Elevated eosinophil presence in the BAL of ST6Gal-1-deficient mice. BALF was recovered from wild-type, Siat1ΔP1, or Siat1 null animals undergoing acute OVA-provoked allergic pulmonary inflammation. (A) Total cell content, expressed as BAL cells/animal, as determined on a coulter counter. n is the number of animals used for each respective data point. (B) BAL cell composition of wild-type (open bars) and Siat1ΔP1 (hatched bars), as determined by FACS analysis and outlined in Materials and Methods. The data shown are the mean of four to five wild-type and four to six Siat1ΔP1 animals. **, Indicates statistical significance of P < 0.003. neu, Neutrophil; mac, macrophage.

Figure 4.

Serum Ig and cytokine profiles of OVA-challenged wild-type and Siat1ΔP1 animals. Profiles of IgE, IgG1, IgG2a, and IgG3 (A) and IL-4, IL-5, IL-6, and IL-13 (B) in sera of animals undergoing OVA-provoked allergic airway were determined as outlined in Materials and Methods. End-point titer for corresponding serum IgE, IgG1, IgG2a, and IgG3 was zero in resting (rest) animals in all cases (data not shown). For cytokine assays (B), shaded areas show the lower assayable limit for each cytokine. n = 4 for all three genotypes; *, indicates statistical significance of P < 0.05.

Figure 5.

Apoptotic profiles of BAL eosinophils (A) and estimation of eosinophil precursor level cells in bone marrow at baseline (B). Cells from BAL of wild-type (open bar, n=5) and Siat1ΔP1 (hatched bars, n=5) were assayed for viability as described in Materials and Methods. In FACS analysis, granulocytes were gated based on their forward-scatter and side-scatter, and the percentage of different fractions in the gated region was measured (A). Colony-forming cell assay was performed on bone marrow cells as outlined in Materials and Methods. Colonies were counted, and total CFU in 10⁵-nucleated bone marrow cells was calculated for WT (open bar, n=7) and Siat1ΔP1 (hatched bar, n=7). **, Indicates statistical significance between WT and Siat1ΔP1, P < 0.0003 (B).

Figure 6.

ST6Gal-1 profiles of animals undergoing OVA or ABPA models of allergic pulmonary inflammation. (A) Serum sialyltransferase activity profiles: Serum was harvested from wild-type and Siat1ΔP1 mice at rest (base) or undergoing OVA or ABPA protocols of allergic airway inflammation and tested for sialyltransferase activity as described in Materials and Methods. Shown is the ³[H] incorporation into the synthetic acceptor substrate from 30 Ci/mmol CMP-³[H]NeuNAc by 10 μl serum after 2 h incubation at 37°C. The numbers immediately beneath the abscissa indicate the number of animals comprising each data bar. Statistical significance is reached (P<0.01) for wild-type undergoing OVA or ABPA when compared with baseline (*) and also for the difference between wild-type and Siat1ΔP1 upon OVA provocation. (B) Real-time RT-PCR analysis of liver ST6Gal-1 mRNA in wild-type and Siat1ΔP1 mice at rest (baseline) or undergoing the OVA protocol. Open bars represent wild-type (n=3), and hatched or shaded bars represent Siat1ΔP1 mice (n=3). Statistical significance was reached (P< 0.05) for wild-type upon OVA provocation compared with baseline.