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, 24 (9), 4075-82

Targeted Disruption of SPI3/Serpinb6 Does Not Result in Developmental or Growth Defects, Leukocyte Dysfunction, or Susceptibility to Stroke

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Targeted Disruption of SPI3/Serpinb6 Does Not Result in Developmental or Growth Defects, Leukocyte Dysfunction, or Susceptibility to Stroke

Katrina L Scarff et al. Mol Cell Biol.

Abstract

Protease inhibitor 6 (PI-6/SERPINB6) is a widely expressed nucleocytoplasmic serpin. It inhibits granulocyte cathepsin G and neuronal neuropsin, and it is thought to protect cells from death caused by ectopic release or internalization of protease during stress such as infection or cerebral ischemia. To probe the biological functions of PI-6, we generated mice lacking its ortholog (SPI3/Serpinb6). SPI3-deficient mice developed normally and were fertile, and no abnormal pathology or increased sensitivity to cerebral ischemia was observed. There were no perturbations in leukocyte development or numbers, and recruitment of leukocytes to the peritoneal cavity was normal. SPI3-deficient mice were equally susceptible as wild-type mice to systemic Candida albicans infection, although there was a slight decrease in the ability of neutrophils from SPI3-deficient mice to kill C. albicans in vitro. Increased levels of a related inhibitor Serpinb1 (monocyte/neutrophil elastase inhibitor) in the tissues of targeted mice suggests that compensation by other serpins reduces the impact of SPI3 deficiency in these animals and may explain the lack of a more obvious phenotype.

Figures

FIG. 1.
FIG. 1.
Targeted disruption of the SPI3 gene. (A) Exon 2 of the SPI3 gene was replaced with EGFP fused in frame with the SPI3 initiation codon. The pgk-loxPneo selection cassette was removed by crossing SPI3 heterozygotes with Cre-deleter mice. St, StuI; S, SmaI; X, XbaI; B, BamHI; Nh, NheI; N, NotI; WT, wild type; KO, knockout. (B) XbaI-cleaved DNA from wild-type cells (+/+) and recombinant cells (+/−) was hybridized to the 3′ probe, which distinguishes the wild-type allele (9.5 kb) from the targeted allele (7.5 kb). (C) To confirm correct integration of the 5′ arm, DNA from wild-type (+/+) and heterozygous (+/−) cells was subjected to PCR using primers 461 and 467, which amplify a 4-kb product from the wild-type allele, and primers 462 and 467, which amplify a 4-kb product from the targeted allele. (D) Wild-type (+/+), heterozygous (+/−), and knockout mice were typed by PCR of tail DNA with primers 152, 688, and 462 simultaneously.
FIG. 2.
FIG. 2.
Distribution of SPI3 mRNA and protein. (A) RT-PCR analysis of SPI3 mRNA in normal C57BL/6 mouse tissues. cDNA synthesized in the presence (+) or absence (−) of RT was amplified with SPI3-specific primers 259 and 370. LI, large intestine; LN, lymph node; SI, small intestine; R8, mouse cytotoxic T-cell line; ES, embryonic stem cells. (B) cDNA synthesized in the presence (+) or absence (−) of RT from wild-type (+/+) and SPI3-deficient (−/−) mouse brains was amplified with SPI3-specific primers 259 and 370 (top panel) or GAPDH-specific primers (bottom panel). (C) Immunoblots of marrow, spleen, and brain protein from wild-type (+/+), heterozygous (+/−), and SPI3-deficient (−/−) mice were probed with either a rabbit anti-SPI3 serum (top panel) or a rat anti-GFP antibody (bottom panel).
FIG. 3.
FIG. 3.
Expression of GFP in immune cells. Leukocytes from the spleen, thymus, blood, or bone marrow were stained with the indicated antibodies and analyzed by flow cytometry. The appropriate cell populations were gated and analyzed for GFP expression.
FIG. 4.
FIG. 4.
Recruitment of cells to the peritoneal cavity. Wild-type (+/+) and SPI3-deficient (−/−) mice were injected intraperitoneally with either (A) thioglycolate (TG) (n = 11 to 20 for each genotype) or (B) C. albicans culture filtrate (YF) (n = 7 for each genotype) and were killed 4 or 72 h later, and the contents of the peritoneal cavity were harvested and counted. Peritoneal cells from noninjected mice (no thioglycolate) were also evaluated (n = 9 to 16 for each genotype). Results represent the mean ± one standard deviation. (C) Peritoneal cells were stained with antibodies to Mac-1 and Gr-1 and analyzed by flow cytometry to determine the cell types elicited.
FIG. 5.
FIG. 5.
Response to C. albicans in vitro and in vivo. (A) Wild-type (open bar) and SPI3-deficient (grey bar) mice were injected intraperitoneally with culture filtrate from C. albicans. Neutrophils were isolated from the peritoneal cavity 4 h later and tested for the ability to kill either the blastoconidial or hyphal form of C. albicans. Results represent the mean (± one standard error of the mean) (n = 6 for each genotype) proportion of C. albicans surviving after being incubated with peritoneal cells compared to that of C. albicans incubated in the absence of cells. (B) Wild-type (open symbols) and SPI3-deficient (closed symbols) mice were injected intravenously with 2.5 × 106 CFU of C. albicans and killed 3 weeks later, and the yeast load in the liver, lungs, spleen, and kidneys was determined. Yeast load is expressed as CFU/mg of tissue, each symbol represents data from one mouse, and the bar represents the mean for each group (n = 6 for each genotype).
FIG. 6.
FIG. 6.
Up-regulation of EIA in SPI3-deficient mice. Immunoblots of protein from tissues of wild type (+/+), heterozygous (+/−), and SPI3-deficient (−/−) mice were probed with a rabbit anti-elastase inhibitor serum (EIA) followed by either a goat anti-human actin antibody or a mouse anti-cytochrome c antibody.

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