Article Figures & Data
Figures
- FIG. 1.
Targeted disruption of the S100A9 locus. (A) Recombination strategy. A schematic representation of the S100A9 wild-type gene, the targeting construct, and the mutant allele are shown. Closed boxes represent the three exons of the S100A9 gene; the neomycin resistant gene (Neo) and thymidine kinase gene (HSV-TK) are indicated by open boxes. Arrows represent the direction of transcription. The region indicated as the probe was used for the screening of targeted ES cells. Arrowheads indicate PCR primers used for detection of the mutant allele. The predicted sizes of DNA fragments in Southern blot analysis and PCR are also shown. (B) Southern blot analysis of five selected ES cell clones. Genomic DNA was digested with PvuII and hybridized with the probe shown in panel A. The observed 4.3- and 3.6-kb bands represent the wild-type and mutated alleles, respectively. (C) PCR analysis of tail DNAs from 4-week-old progeny. After PCR amplification of wild-type and targeted alleles, DNA fragments of 4.7 and 5.1 kb were detected.
- FIG. 2.
Expression of S100A8 in mice lacking a functional S100A9 gene. (A) Flow cytometric profiles after S100A9 and S100A8 immunostaining of blood neutrophils are shown. Profiles are representative of three mice for each genotype. Analysis reveals the absence of both proteins in peripheral blood cells. (B) Western blot analysis of S100A9 and S100A8 expression in wild-type and S100A9-deficient mice demonstrates the absence of S100A9 and S100A8 in peripheral S100A9-deficient leukocytes. The upper part of the S100A8 filter was stained with Coomassie brilliant blue to confirm the transfer of the proteins to the filter. P, peritoneal granulocytes; B, bone marrow; S, spleen. (C) Northern blot analysis demonstrates the absence of S100A9 mRNA in thioglycolate-elicited peritoneal PMNs from S100A9-deficient mice, whereas the S100A8 gene is transcribed in wild-type as well as S100A9-deficient cells. Signals presented are shown as gene expression relative to the average expression of the housekeeping gene actin. (D) RT-PCR analysis confirms the absence of S100A9 RNA transcripts in various organs of deficient mice (right panel) and shows the presence of S100A8 RNA transcripts in the same organs (left panel). B, brain; S, spleen; L, liver; Br, brain.
- FIG. 3.
Expression of S100A8 in S100A9-deficient bone marrow and spleen cells. As indicated, spleen and bone marrow cells were stained with the S100A8 antibody. As a control, granulocytes were stained with a commercially available gr1 antibody. S100A8 is present in bone marrow but not detectable in the peripheral tissues of the spleen.
- FIG. 4.
Analysis of the hematopoietic cell population. Bone marrow from wild-type and S100A9-deficient mice were cultured for 4 days (d) in Teflon bags and analyzed daily by immunostaining of cytospin preparations for the expression of indicated cell markers in order to follow the differentiation of myeloid cells. Bars presented are the means from five separate experiments. In all panels, white bars correspond to wild-type mice and black bars correspond to S100A9-deficient mice.
- FIG. 5.
CFU experiments using wild-type and S100A9-deficient bone marrow leukocytes. Bone marrow cells from wild-type and S100A9-deficient mice were incubated with the cytokines indicated to evaluate the myelopoietic potential of the precursor cells. As a control, bone marrow cells were incubated without any cytokines.
- FIG. 6.
Chemoattractant-induced in vitro TEM assay. TEM of bone marrow cells from wild-type (white bars) and S100A9-deficient (black bars) mice through a bEnd5 monolayer is shown. Values represent the means from four experiments. The stimulation rate was calculated by dividing the number of transmigrated cells after stimulation by the number before stimulation. Values are means ± standard errors of the means and were considered significant at a P of <0.01.
- FIG. 7.
Dynamic parameters of spontaneous and chemokine-induced migration of S100A9-deficient and wild-type peritoneal neutrophils. The data show the means from four independent experiments. In all panels, white bars correspond to wild-type neutrophils and black bars correspond to S100A9-deficient neutrophils. All parameters indicate a reduced chemokinesis of the S100A9-deficient neutrophils upon stimulation with IL-8. Values were considered significant at a P of <0.05.
- FIG. 8.
IL-8-mediated surface expression of CD11b on Gr1-gated cells. Bone marrow cells were incubated at 37°C for 5 min with or without IL-8 and in the presence (upper panel) or absence (lower panel) of Ca2+ and Mg2+. At the end of the incubation period, surface expression of CD11b on Gr1-gated cells was determined by direct-immunofluorescence flow cytometry. The data presented here show one experiment representative of five experiments.
- FIG. 9.
IL-8 failed to enhance the adhesion of S100A9-deficient neutrophils to bEnd5 cells. bEnd5 cells were incubated with TNF-α for 2 h; subsequently, S100A9−/− (B) and wild-type (A) leukocytes were allowed to adhere for 1 h with or without IL-8. After washing, the myeloperoxidase activity was determined to measure the number of neutrophils that adhered. To compare the unstimulated and IL-8-stimulated conditions, the value obtained for the unstimulated neutrophils was set to 1.0.
- FIG. 10.
F-actin content in unstimulated S100A9-deficient PMNs is higher than that in wild-type PMNs. (A) The time course of IL-8-induced changes in the cellular F-actin content in Gr1-gated cells is shown. Broken lines represent data for S100A9+/+ mice, and dotted lines represent data for S100A9−/− mice. Levels of IL-8 are either 0 ng/ml (circle) or 250 ng/ml (square). The values represent the means from five separate experiments. (B) The photographs show the F-actin distribution in unstimulated (left panels) and IL-8-stimulated (right panels) CD11b+ cells isolated from bone marrow.
- FIG. 11.
IL-8-induced emigration of neutrophils into the skin. IL-8 was injected into the ears of wild-type and S100A9-deficient mice. The number of Gr1-stained cells 4 h after IL-8 injection was visually evaluated. There was no difference observed between the number of emigrated neutrophils with respect to the wild-type and S100A9-deficient mice.
- FIG. 12.
Thioglycolate-elicited leukocyte emigration into the peritoneum. (A) The total number of leukocytes migrated into the peritoneum subsequent to intraperitoneal injection of thioglycolate was determined at various time points as indicated (106). No significant difference was observed between S100A9-deficient (black bars) and wild-type (white bars) mice. Each bar represents the mean values for at least 10 mice. (B) Cellular composition of bone marrow cells was analyzed subsequent to thioglycolate-induced peritonitis (4 h). After inflammatory stimulation of the granulopoiesis, the percentage of S100A8-positive cells in the bone marrow was similar in wild-type and S100A9-deficient mice. (C) Cellular composition (percentage of positive cells) of the peritoneal exudate was determined cytochemically with various cellular surface markers as indicated. No deviation was observed between S100A9-deficient and wild-type mice.
