PLSCR1 amplifies degranulation <i>in vitro</i> in primary culture of BMMC and PCMC.

<p>Wild-type (WT) and <i>Plscr1</i>^<i>-/-</i> (KO) BMMC (A, C, D) and PCMC (B) were sensitized for 24 hours with anti-DNP IgE. After two washes, the IgE-sensitized cells were stimulated with different doses of specific antigen (DNP-HSA) for 30 minutes (A, B) or at different times to the optimal antigen dose of 10 ng/ml (C). Statistical analysis was done by a Two-way ANOVA followed by Sidak’s multiple comparisons test. Data of n independent experiments with n = 12 (A), n = 5 (B) and n = 6 (C) are presented as mean ± s.e.m. *: P <0.05; **: P <0.01; ***: P <0.001. (D) WT and KO BMMC generated in the presence of IL3 and SCF (black bars) or of IL3 alone (open bars) were stimulated for 30 min with 10 ng/ml DNP-HSA. Data of n independent experiments with n = 7 (IL3 + SCF) and n = 3 (IL3) are presented as mean ± s.e.m. Statistical analysis was done by an unpaired Student <i>t</i> test. **: p < 0.01.</p

PLSCR1 amplifies anaphylactic reaction through increased mast cell degranulation.

<p>A passive systemic anaphylaxis (PSA) was carried out on wild-type (WT) and <i>Plscr1</i>^<i>-/-</i> (KO) mice. (A) PSA was monitored by following the drop in body temperature every five minutes after antigen injection (t = 0) using a thermal probe reader. Data are presented as mean ± s.e.m. with 5 mice (3 experiments). Two-way ANOVA followed by Sidak’s multiple comparisons test was used to compare the two groups. *: P < 0.05; ****: P < 0.0001. (B) PSA was monitored by measurement of the histamine released in the serum 25 minutes after antigen injection in the two groups of mice. Histamine was assayed using an EIA kit. The values obtained for each individual mouse are shown. Data are presented as mean ± s.e.m. Statistical analysis: unpaired Student <i>t</i> test. **: P <0.01.</p

Sensitivity of <i>Plscr1</i>^<i>-/-</i> mice to histamine.

<p>Thermal probes were inserted under the dorsal skin of wild-type (WT) and <i>Plscr1</i>^<i>-/-</i> (KO) mice. The following day mice were injected i.v. with 5 μmol histamine. The drop in their body temperature was monitored using a thermal probe reader. Data are presented as ± s.e.m. with n = 6 mice per group (3 experiments). Statistical analysis was done using a two-way ANOVA. ns: non significant.</p

<i>Ex vivo</i> and <i>in vivo</i> analysis of <i>Plscr1</i>^<i>-/-</i> mast cells.

<p>(A) Histamine content of peritoneal mast cells from WT and <i>Plscr1</i>^<i>-/-</i> (KO) mice was measured by EIA and expressed as pg per mast cell. Values obtained for each individual mouse are shown. Data are presented as mean ± s.e.m.. Statistical analysis was done by an unpaired Student <i>t</i> test and showed no significant difference. (B) Number of peritoneal mast cells (PMC). The peritoneal cavity of WT and <i>Plscr1</i>^<i>-/-</i> (KO) mice was washed and the number of PMC per mouse was counted after staining with toluidine blue. Data are presented as mean ± s.e.m.. Statistical analysis done by an unpaired Student <i>t</i> test showed no significant difference. (C) The surface expression of FcεRIα chain by WT and <i>Plscr1</i>^<i>-/-</i> peritoneal mast cells was analyzed by flow cytometry. Blue line: WT, red line: <i>Plscr1</i>^<i>-/-</i>, gray line: isotype controls. Representative data of three independent experiments. (D) Number and tissue localization of <i>Plscr1</i>^<i>-/-</i> mast cells. Left panels: Representative histological analysis of the ear skin and of the stomach submucosa of wild-type (WT), <i>Plscr1</i>^<i>-/-</i> (KO) and Kit^{<i>W-sh/W-sh</i>} (W^sh) mice with toluidine blue staining. Arrowheads: mast cells, stained in purple. Selected mast cells (empty arrowheads) are shown under higher magnification in the insets. Scale bar: 100μm. Right panels: Quantitative analysis of countable mast cells in these tissues using CaloPix software. Data are presented as mean + s.e.m. of four mice. Statistical analysis done by an unpaired Student <i>t</i> test showed no significant difference.</p

Phenotypic analysis of <i>Plscr1</i>^<i>-/-</i> BMMC and PCMC.

<p>(A) Cytologic analysis. Wild-type and <i>Plscr1</i>^<i>-/-</i> BMMC (top panels) and PCMC (bottom panels) were cyto-centrifuged and stained with May-Grünwald Giemsa (left, MGG) or toluidine blue (right, TB) and observed with an optical microscope. Scale bar = 100 μm. (B) The expression of FcεRI on the surface of WT (blue line) and <i>Plscr1</i>^<i>-/-</i> (red line) BMMC and PCMC was analyzed by flow cytometry. Gray line: isotype controls. Representative data of three independent experiments. (C) Expression of major effector molecules of FcεRI signaling. Lysates of WT and <i>Plscr1</i>^<i>-/-</i> (KO) BMMC and PCMC were analyzed by immunoblotting with specific antibodies for the presence of PLSCR1, Fyn, Lyn, Syk, LAT1, PLCγ1, AKT, Erk1/2 and actin.</p

Disruption of the CD4/PLSCR1 interaction by SLPI.

<p>A) In vitro inhibition of the CD4/PLSCR1 interaction by SLPI. GST or GST-PLSCR1 (left panel, coomassie blue) was incubated with equal amounts of lysates from Jurkat CD4-positive T cells in the presence of the indicated concentrations of either GST-SLPI or GST-ARF1 (left panel) used as a control. Bound proteins were analyzed by Western blot with anti-CD4 (right panel). B) Mapping of the PLSCR1 determinants required for binding to CD4 and SLPI. L40 yeast strain expressing either the cytoplasmic domain of CD4 (CD4c) or SLPI fused to LexA in combination with each of the deleted forms of the Gal4AD-PLSCR1 hybrids indicated on the left was analyzed for histidine auxotrophy and beta-gal activity. The interactions between hybrid proteins were scored as follows: (+), cell growth on medium without histidine and development of a β-gal activity; (−), no growth on medium without histidine and no β-gal activity.</p

Mapping of the CD4 determinants required for PLSCR1 binding by co-immunoprecipitation assay.

<p>A) Schematic representation of the human CD4 mutants. The extracellular and transmembrane (TM) domains of CD4 are represented in grey and hatched, respectively. The a.a. sequences of the cytoplasmic tail of the CD4 mutants are aligned with that of the wild-type CD4 (CD4 WT). Dashes (−) indicate a.a. identities with the wild type protein and a.a. substitutions are identified. B) Co-immunoprecipitation assay. Protein extracts were prepared 48 h post transfection from 293T cells co-expressing HA-PLSCR1 together with wild type or mutated CD4 as indicated at the top. Crude extracts were then subjected to CD4 immunoprecipitation followed by Western blot analysis with anti-CD4 (upper panels) and anti-HA (lower panels).</p

Mapping of the CD4 determinants required for PLSCR1 binding by ELISA.

<p>A) Primary a.a. sequences of the CD4 long (405–433) and short (405–426) peptides used in the ELISA test. The primary sequence of the entire cytoplasmic domain of CD4 is shown at the top; the 2 Cys residues required for p56Lck binding and mutated in Ala in both peptides are indicated in red. B) ELISA interaction test. 96-well plates were coated with the CD4c long (left panel) or short (right panel) peptides at a final concentration of 0.5 µM. After washings and blocking of non-specific binding sites, GST or GST-PLSCR1 was incubated at concentrations ranging from 1 nM to 300 nM. Binding was then revealed with an anti-GST antibody and a secondary peroxidase-conjugated anti-mouse IgG. The peroxidase substrate solution was incubated for 10 min and the optical density was measured at 450 nm.</p

Interaction of PLSCR1 and PLSCR4 with the CD4 receptor.

<p>A) In vitro interaction. Lysates from Jurkat CD4-positive T cells were incubated with equal amounts of GST or GST-PLSCR1 (upper panel, coomassie blue) immobilized on GSH-sepharose beads. Bound proteins were then analyzed by immunoblotting with anti-CD4 (lower panel). B) Co-immunoprecipitation of endogenous proteins from T lymphocytes. Jurkat T cells were lyzed and CD4 was precipitated with either anti-CD4 (OKT4) or a control isotypic antibody. Precipitates were analyzed by Western blot with anti-CD4 (upper panel) or anti-PLSCR1 (lower panel). C) Co-precipitation of overexpressed CD4 and PLSCR1 proteins. 293T cells expressing HA-tagged PLSCR1 (lower panel, Cell lysate) in combination with wild-type CD4 (WT) or a mutant of CD4 deleted of its cytoplasmic domain (ΔCT) were lyzed and the CD4 forms were precipitated with anti-CD4. Precipitates were then analyzed by Western blot with anti-CD4 (upper panel) or anti-HA (middle panel). D) Co-precipitation of overexpressed CD4 and PLSCR4 proteins. 293T cells expressing GFP- PLSCR1, GFP-PLSCR3 or GFP-PLSCR4 (lower panels, Cell lysate) in combination with CD4 WT (right panels) or CD4 ΔCT (left panels) were lyzed and the CD4 forms were precipitated with anti-CD4. Precipitates were then analyzed as in (C). Of note, the differences in migration observed between PLSCR1 (318 a.a.), PLSCR3 (295 a.a.) and PLSCR4 (329 a.a.) is likely related to the respective amino acid lengths of the GFP fusion proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005006#pone.0005006-Zhou1" target="_blank">[23]</a>. E) Interactions in the two-hybrid system. L40 yeast strain expressing the LexA-CD4c hybrid in combination with the indicated Gal4AD hybrids was analyzed for histidine auxotrophy and β-gal activity. Transformants were patched on medium with histidine (upper panels) and then replica-plated on medium without histidine (middle panels) and on Whatman filter for β-gal assay (lower panels).</p

Anti-HIV-1 activity of recombinant SLPI.

<p>A) Inhibition of virus replication. Primary T lymphocytes were isolated from peripheral blood mononuclear cells and used for infection with the HIV-1JR-CSF isolate in the presence of increasing concentrations (0–50 µg/ml) of purified GST-SLPI (see inset, coomassie blue) or GST (50 µg/ml). After washing, infected cells were cultured for 6 days and the viral production was quantified by measuring the p24 production in the cell-culture medium. B) Inhibition of virus transfer from dendritic cells to T lymphocytes. Dentritic cells were derived from purified primary monocytes, and incubated with either HIV-1Bal (R5 strain, upper panel) or HIV-1NDK (×4 strain, lower panel) for 1 h at 37°C in the presence of 50 µg/ml of purified GST-SLPI or GST. After washing, IL-2 activated-lymphocytes were added in a 1/5 ratio and maintained in co-culture for 72 h. Viral production was quantified by measuring the p24 production in the cell culture medium.</p