Altered granulopoietic profile and exaggerated acute neutrophilic inflammation in mice with targeted deficiency in the sialyltransferase ST6Gal I

Abstract

Elevation of serum sialic acid and the ST6Gal-1 sialyltransferase is part of the hepatic system inflammatory response, but the contribution of ST6Gal-1 has remained unclear. Hepatic ST6Gal-1 elevation is mediated by P1, 1 of 6 promoters regulating the ST6Gal1 gene. We report that the P1-ablated mouse, Siat1DeltaP1, and a globally ST6Gal-1-deficient mouse had significantly increased peritoneal leukocytosis after intraperitoneal challenge with thioglycollate. Exaggerated peritonitis was accompanied by only a modest increase in neutrophil viability, and transferred bone marrow-derived neutrophils from Siat1DeltaP1 mice migrated to the peritonea of recipients with normal efficiency after thioglycollate challenge. Siat1DeltaP1 mice exhibited 3-fold greater neutrophilia by thioglycollate, greater pools of epinephrine-releasable marginated neutrophils, greater sensitivity to G-CSF, elevated bone marrow CFU-G and proliferative-stage myeloid cells, and a more robust recovery from cyclophosphamide-induced myelosuppression. Bone marrow leukocytes from Siat1DeltaP1 are indistinguishable from those of wild-type mice in alpha2,6-sialylation, as revealed by the Sambucus nigra lectin, and in the expression of total ST6Gal-1 mRNA. Together, our study demonstrated a role for ST6Gal-1, possibly from extramedullary sources (eg, produced in liver) in regulating inflammation, circulating neutrophil homeostasis, and replenishing granulocyte numbers.

Figure 1.

Greater leukocyte accumulation in the peritoneum of Siat1ΔP1 and Siat1-null mice after intraperitoneal thioglycollate elicitation. Peritoneal lavage was recovered from wild-type C57BL/6 (WT), Siat1ΔP1 (ΔP1), and Siat1-null (null) animals in the absence of (0 hour; A), 5 hours after (B), and 1 day after (C) elicitation with 1 mL 4% wt/vol intraperitoneal thioglycollate. Viable cells were counted by hemocytometer after staining for trypan-blue exclusion. Age- and sex-matched mice were used. (A) n = 8WT, n = 8 ΔP1, and n = 4 null mice. (B) n = 8WT, n = 8 ΔP1 mice. (C) n = 8 WT, n = 8 ΔP1, and n = 4 null mice. *Mutant animal data points where statistically significant differences with corresponding WT have been reached. Statistical significance was noted as follows: P = .002, WT and ΔP1 (B); P = .002, WT and ΔP1 (C); and P = .001, WT and null (C). Error bars represent 1 standard deviation from the mean of each group.

Figure 2.

Exaggerated peritonitis in siat1ΔP1 mice is thioglycollate dosage dependent. Peritonitis was elicited in C57BL/6 wild-type (○) and Siat1ΔP1 (▵) mice with 1 mL of 0%, 1%, 2%, 4%, or 8% wt/vol intraperitoneal thioglycollate, as shown, and peritoneal cells were harvested 24 hours later and counted. N is the number of animals used in each determination. *Mutant animal data points at which statistically significant differences with corresponding WT have been reached. Statistical significance for the differences between wild-type and Siat1ΔP1 for 4% and 8% is as shown. Error bars indicate 1 standard deviation from the mean of each group.

Figure 3.

PKH26- and CFSE-tagged donor cells migrate to the peritonea of thioglycollate-elicited recipients. Total bone marrow cells from a Siat1ΔP1 mouse were separately labeled with either PKH26-red or CFSE. The tagged donor cells were recombined, and pooled cells were injected by tail vein into a recipient mouse, also Siat1ΔP1. Concomitant with the transfer of the tagged donor cells, peritonitis was elicited by intraperitoneal injection of 1 mL of 4% (wt/vol) thioglycollate. Peritoneal cells were recovered 6 hours later, and the presence of tagged donor cells was profiled by flow cytometry. (A) Recipient animal that did not receive tagged donor cells. (B) Recipient animal receiving 10⁷ pooled donor cells. Gates (boxed regions, B) were used to determine the PKH/CFSE ratios of tagged cells, as summarized in Table 2. Typically, up to 0.4% of the labeled donor cells are recovered in the peritonea of the thioglycollate-elicited recipients.

Figure 4.

Greater neutrophilia in Siat1ΔP1 and Siat1-null animals. Peripheral blood was collected from mice in the absence of treatment (resting, A) or after the following administrations: TG 4h, 4 hours after intraperitoneal 4% (wt/vol) thioglycollate (B); EPI 30m, 30 minutes after intravenous 0.25 mg/kg epinephrine (C); GCSF 30m, 30 minutes after intravenous G-CSF administration (D-E). Collected blood was analyzed by flow cytometry after the lysis of red blood cells, and the granulocyte population (7/4⁺/1A8⁺) was calculated by taking the percentage of the appropriate PE-7/4 and FITC-1A8 events against total events for each flow cytometric acquisition. *Mutant animal data points at which statistically significant differences with corresponding WT have been reached. (A) n = 21, WT; n = 21, Siat1ΔP1; n = 4, Siat1-null. (B) n = 4, WT; n = 4 Siat1ΔP1; P = .003. (C) n = 4, WT; n = 4, Siat1ΔP1; P < .001. (D) n = 8, WT; n = 6 Siat1ΔP1; P < .001. (E) n = 4, WT; n = 4 Siat1-null; P = .03. Error bars indicate 1 standard deviation from the mean of each group.

Figure 5.

Flow cytometric analysis of siat1ΔP1 and WT bone marrow cells. Bone marrow cells were labeled with FITC-1A8 (anti-Ly6G), PE-7/4 (anti–polymorphonuclear cell, 40-kDa antigen), and biotinylated SNA, as described in “Flow cytometric profiling of inflammatory cells.” Unfractionated bone marrow cells (Total), and differentially flow-sorted populations R1, R2, and R3 were analyzed for SNA binding. SNA binding profiles of wild-type (dashed lines) and Siat1ΔP1 (solid lines) cells are compared.

Figure 6.

Real-time PCR analysis of selected genes in bone marrow of wild-type and Siat1ΔP1 mice. Expression of selected genes (A) and different mRNA forms of ST6Gal1 (B) were measured by real-time PCR relative to RPL32, a ribosomal protein mRNA, as reference standard. The procedure involved in analysis of 1 μg total RNA in duplicate. Sensitivity limit is denoted by the dotted line. Liver RNA is shown for comparison. Error bars represent 1 standard deviation from the mean of each group.