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. 1999 Sep 28;96(20):11452-7.
doi: 10.1073/pnas.96.20.11452.

Multiple, targeted deficiencies in selectins reveal a predominant role for P-selectin in leukocyte recruitment

S D Robinson  1 , P S Frenette, H Rayburn, M Cummiskey, M Ullman-Culleré, D D Wagner, R O Hynes
Affiliations

Multiple, targeted deficiencies in selectins reveal a predominant role for P-selectin in leukocyte recruitment

S D Robinson et al. Proc Natl Acad Sci U S A. .

Abstract

We extend our previous analyses of mice deficient in selectins by describing the generation and comparative phenotype of mice lacking one, two, or three selectins after sequential ablation of the murine genes encoding P-, E-, and L-selectins. All mice deficient in selectins are viable and fertile as homozygotes. However, mice missing both P- and E-selectins (PE(-/-)), and mice missing all three selectins (ELP(-/-)) develop mucocutaneous infections that eventually lead to death. Mice deficient in multiple selectins display varying degrees of leukocytosis, resulting in part from alterations in leukocyte rolling and recruitment. PE(-/-) mice, ELP(-/-) mice, and mice missing both P- and L-selectins (PL(-/-)) show drastic reductions in leukocyte rolling and in extravasation of neutrophils in thioglycollate-induced peritonitis. In a separate inflammatory model (ragweed-induced peritoneal eosinophilia), we demonstrate P-selectin to be both necessary and sufficient for the recruitment of eosinophils. The phenotype of mice missing both E- and L-selectins (EL(-/-)) is less severe than those seen in the other double knockouts. Comparisons among the double knockouts suggest that P-selectin normally cooperates with both E- and L-selectins. Our results indicate a preeminent role for P-selectin in regulating leukocyte behavior in mice. Data from the ELP(-/-) mice indicate, however, that all three selectins are important to leukocyte homeostasis and efficient neutrophil recruitment.

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Figures

Figure 1
Figure 1
Targeting strategy and L-selectin genotyping of progeny from double heterozygous crosses. (A) Overall scheme for generating mice deficient in one, two, or three selectins. P-, E-, and L-selectin loci were sequentially mutated by homologous recombination in ES cells by using replacement targeting constructs carrying drug-resistance genes for neomycin, hygromycin, and puromycin, respectively. By targeting the L-selectin locus in two different heterozygous ES cell clones with null mutations in P- and E-selectins, all possible combinations of heterozygous selectin-deficient ES cell clones were generated. Targeting of the P- and E-selectin loci has been described (2, 5). (B) The WT L-selectin locus is shown in the upper line. To construct the targeting vector, the PUROr cassette was inserted into 6.4 kb of L-selectin sequence. The exons encoding the lectin (LEC) domain and most of the epidermal growth factor (EGF) domain were deleted in this process. Resistant clones were screened with a 3′ probe that identifies the targeted 22-kb mutant and 25-kb WT bands upon EcoRI digestion. The XbaI–EcoRI fragment 3′ of the replacement vector is approximately 17 kb long; the XbaI–EcoRV probe comprises the first 1.6 kb of this segment. (C) Southern blot analyses of the L-selectin locus from EcoRI digests. Genomic DNA was extracted from tail biopsies of representative litters derived from mating heterozygous animals known to be carrying the L-selectin mutation. The first lane shows a digest of an ES cell clone, heterozygous for the L-selectin mutation, for comparison. W, WT allele; M, mutated allele. (D) PCR analysis of L-, E-, and P-selectins of progeny from an intercross of animals carrying heterozygous mutations in all three alleles. The targeted E-selectin band is 231 bp in length, the targeted L-selectin band is 200 bp in length, and the targeted P-selectin band is 479 bp in length. The null mutations segregate together during meiosis, indicating the mutations exist in cis. Null progeny from two such crosses were used to establish a homozygous triple-deficient line.
Figure 2
Figure 2
Verification of null alleles. (A) Leukocytes from WT and homozygous selectin-deficient mice were analyzed for L-selectin expression by flow cytofluorometric analysis. Whole blood was collected and leukocytes were stained for L-selectin surface expression. M1 indicates the region of MEL-14-expressing cells. The double peak in the IgG control and L-selectin null genotypes is the result of two cell populations that demonstrate different background binding to the antibodies. (B) WT and homozygous selectin-deficient animals were analysed for expression of E-selectin mRNA by Northern blot analysis. Total RNA was isolated from pooled lungs and hearts of lipopolysaccharide-stimulated animals. Samples were electrophoresed on a 1.0% agarose gel and sequentially probed for E-selectin and β-actin transcripts. Note the underloading of the L and PL lanes. (C) WT and homozygous selectin-deficient animals were analyzed for expression of P-selectin by Western blot analysis. Platelet-rich plasma was isolated from whole blood. Samples were electrophoresed on a 6% SDS-polyacrylamide gel and sequentially stained for P-selectin and vinculin proteins.
Figure 3
Figure 3
Peripheral blood counts. Whole blood was collected and a total leukocyte count was obtained on a Coulter counter. Leukocyte subpopulations were determined by differential counts on Diff-Quick-stained smears of the same samples. Ten to 20 animals were used for each genotype. ∗, P < 0.05; ∗∗, P < 0.01, in comparison to WT mice.
Figure 4
Figure 4
Leukocyte rolling in TNF-α-stimulated venules. Mice were prepared for intravital microscopy of mesenteric venules 3.5 hr after administration of TNF-α. Venular blood flow and diffractive, rolling leukocytes were videotaped for 20 min. Averages were obtained from at least five animals of each genotype. ∗, P < 0.05; ∗∗, P < 0.01, in comparison to WT mice.
Figure 5
Figure 5
Neutrophil influx after thioglycollate injection. Peritoneal lavages were performed at intervals after thioglycollate administration. Total cell numbers were determined with a hemocytometer, and the percentages of neutrophils were determined from cytospun, Diff-Quick-stained samples. Five to 31 animals of each genotype, for each time point, were used.
Figure 6
Figure 6
Eosinophil influx after ragweed sensitization and challenge. (A) Peripheral eosinophil counts. Blood was collected by using Eosinophil Unopettes for Manual Methods and eosinophils were counted by using a hemocytometer. (B) Peritoneal eosinophil counts after ragweed challenge. Ragweed-sensitized mice were challenged with an i.p. injection of ragweed allergen. Forty-eight hours later, peritoneal lavages were performed. Total cell numbers were determined with a hemocytometer and the percentages of eosinophils were determined from cytospun, Diff-Quick-stained samples. n = 12–23. ∗, P < 0.05; ∗∗, P < 0.01, in comparison to WT mice. All strains showing decreased levels relative to WT mice were statistically indistinguishable from each other.
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