Research ArticleImmunology

The phytosphingosine-CD300b interaction promotes zymosan-induced, nitric oxide–dependent neutrophil recruitment

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Science Signaling  15 Jan 2019:
Vol. 12, Issue 564, eaar5514
DOI: 10.1126/scisignal.aar5514
  • Fig. 1 CD300b deficiency reduces zymosan-induced neutrophil accumulation in the mouse model of dorsal air pouch inflammation.

    (A to D) Numbers of neutrophils recruited into the dorsal air pouches of WT or CD300b−/− [knockout (KO)] mice before or at the indicated times after treatment with zymosan (n = 5 mice) (A); 4 hours after treatment with LPS (n = 4 mice), Pam3CSK4 (n = 6 mice), or FSL-1 (n = 6 mice) (B); 4 hours after treatment with curdlan or mannan (n = 6 mice per group) (C); or 4 hours after treatment with HKSC or HKCG (n = 6 mice per group) (D). (E and F) The amounts of NO2 + NO3 (n = 6 mice per group) (E) or LTB4 (n = 5 mice per group) (F) in the dorsal air pouch exudates of WT or CD300b−/− mice before or after the indicated times of zymosan treatment. Data are means ± SD and are representative of two independent experiments. *P < 0.05 and **P < 0.01 by Student’s t test (A to E) or t test with Welch’s correction (D).

  • Fig. 2 Reduced neutrophil accumulation results from the decreased NO production in zymosan-treated air pouches of CD300b−/− mice.

    (A) Numbers of neutrophils recruited to the dorsal air pouches of WT and CD300b−/− KO mice that were pretreated with vehicle or l-NAME for 30 min and then treated with zymosan for 4 hours (n = 5 mice per group). (B and C) The amounts of NO2 and NO3 (n = 6 mice per group) (B) and LTB4 (n = 5 mice per group) (C) in the pouch exudates of the indicated mice after 30-min pretreatment with vehicle or l-NAME and then 1.5 hours after injection with zymosan (n = 5 mice per group). (D) Numbers of neutrophils recruited to the dorsal air pouches of the indicated mice 4 hours after treatment with SNP alone, zymosan alone, SNP and zymosan, or vehicle (n = 5 mice per group). (E and F) The amounts of NO2 and NO3 (E) and LTB4 (F) in the pouch exudates of the indicated mice that were pretreated with an Ab against Gr-1 (anti–Gr-1) or a control Ab and then treated with zymosan for 1.5 hours (n = 4 mice per group). Data are means ± SD and are representative of two independent experiments. *P < 0.05 and **P < 0.01 by analysis of variance (ANOVA) with Holm-Šídák multiple comparison test (A to F). ns, not significant.

  • Fig. 3 NO-producing inflammatory DCs are responsible for the CD300b-dependent recruitment of neutrophils to zymosan-treated air pouches.

    (A) The dorsal air pouches of WT and CD300b−/− mice were treated for 4 hours with zymosan, and then, frozen sections of the skin covering the pouches were treated with Abs against the indicated targets or with 4′,6-diamidino-2-phenylindole (DAPI) and then were analyzed by confocal microscopy. Scale bars, 20 μm. The images are representative of three experiments. (B to D) WT and CD300b−/− mice were treated for 4 hours with zymosan. (B) Cells in air pouch exudates were analyzed by flow cytometry with Abs against the indicated markers and were classified as F4/80Ly-6Cint (R1), F4/80+Ly-6Chigh(R2), or F4/80+Ly-6Clow (R3). (C and D) Cells of the indicated groups from WT mice were stained with Abs against the indicated markers and were analyzed by flow cytometry. (A to D) Data are representative of four independent experiments. (E) Number of F4/80+Ly-6Chigh cells in the pouches (n = 6). (F to I) WT and CD300b−/− mice were pretreated with control liposomes or clodronate liposomes before being treated for 4 hours (F and G) or 1.5 hours (H and I) with zymosan. (F) Percentages of cells in the air pouch exudates of WT mice that were stained with indicated Abs and analyzed by flow cytometry. Data are representative of four independent experiments. (G to I) Numbers of neutrophils (n = 5 mice per group) (G) and amounts of NO2 and NO3 (n = 4 mice per group) (H) and LTB4 (n = 6 mice per group) (I) in the dorsal air pouches of the indicated mice. Data are means ± SD and are representative of two independent experiments (E and G to I). **P < 0.01 by Mann-Whitney test (E) or by ANOVA with Holm-Šídák multiple comparison test (G to I).

  • Fig. 4 Phytosphingosine, a component of zymosan, is a potential ligand of CD300b.

    (A and B) 2B4-GFP or CD300b–2B4-GFP cells were incubated in the presence of zymosan, or phorbol 12-myristate 13-acetate (PMA) and ionomycin, or on plates coated with either zymosan-derived lipids (A) or the indicated lipids (B) for 24 hours. DHS, dihydrosphingosine; PHS, phytosphingosine; PS, phosphatidylserine. The cells were then analyzed by flow cytometry to detect GFP. (C) CD300b–2B4-GFP cells were incubated for 24 hours on plates coated with phytosphingosine in the presence of anti-CD300b or control Ab (each at 10 μg/ml). The cells were then analyzed by flow cytometry to detect GFP. (A to C) Data are representative of three independent experiments. (D) Plates coated with the indicated concentrations of phytosphingosine (C-18) were incubated with CD300b-Fc or control Fc (each at 10 μg/ml). The amount of either CD300b-Fc or control Fc that was bound to the wells was quantified by enzyme-linked immunosorbent assay (ELISA). Data are means ± SD of three biological replicates.

  • Fig. 5 Phytosphingosine acts as a potential ligand of CD300b both in vitro and in vivo.

    (A) Left: GM/IL-4–DCs were stained with anti-CD11c and anti–major histocompatibility complex II (MHC-II) Abs and then analyzed by flow cytometry. R1 and R2 indicate populations of cells characterized as CD11c+MHC-IIhigh and CD11c+MHC-II+ cells, respectively. Middle and right: GM/IL-4–DCs in the R1 or R2 gates were stained with an Ab against CD300b and analyzed by flow cytometry. Data are representative of three independent experiments. (B) CD11c+MHC-IIhigh cells sorted from WT or CD300b−/− GM/IL-4–DCs were treated with zymosan or LPS or were cultured on phytosphingosine-coated plates for 12 hours. The amounts TNF-α (left) and NO2 and NO3 (right) were determined by ELISA and colorimetric NO2/NO3 assay, respectively. (C) CD11c+MHC-IIhigh cells sorted from WT or CD300b−/− GM/IL-4–DCs were treated with zymosan or were cultured on phytosphingosine-coated plates for 12 hours in the presence of an Ab against CD300b (anti-CD300b) or a control Ab. The amounts of TNF-α produced by the cells were determined by ELISA. (B and C) Data are representative of three independent experiments. Data are means ± SD of three biological replicates and are representative of three independent experiments. (D) Numbers of neutrophils recruited to the dorsal air pouches of WT and CD300b−/− mice 4 hours after treatment with phytosphingosine-containing vesicles (n = 5 mice per group). Data means ± SD and are representative of three independent experiments. *P < 0.05 and **P < 0.01 by Student’s t test (B and D) or by ANOVA with Holm-Šídák multiple comparison test (C).

  • Fig. 6 CD300b deficiency ameliorates zymosan-induced arthritis by reducing NO production.

    (A to G) WT and CD300b−/− (KO) mice were intra-articularly injected with zymosan. (A) The change in knee thickness of the indicated mice was monitored over time (n = 8 mice per group). (B) Knee sections from zymosan-treated WT and KO mice were taken at the indicated times and stained with hematoxylin and eosin. Scale bars, 20 μm. Data are representative of three independent experiments. (C) The indicated histological scores were determined on days 3 (n = 7 mice per group), 7 (n = 12 mice per group), or 28 (n = 12 mice per group). Data are means ± SD and are pooled from two independent experiments. (D) Numbers of neutrophils in the knee joint cavities of zymosan-treated WT and KO mice at the indicated times (n = 4 to 6 mice per group). (E) Cells isolated from the synovial tissues of clodronate liposome– or control liposome–pretreated WT mice after zymosan treatment were stained with Abs against the indicated marker and analyzed by flow cytometry. The percentages of F4/80Ly-6Cint cells (R1), F4/80+Ly-6Chigh cells (R2), and F4/80+Ly-6Clow cells (R3) in CD11b+ cell populations were determined. Data are representative of five independent experiments. (F and G) Numbers of neutrophils isolated from the knee joint cavities of WT and KO mice that were pretreated with clodronate liposomes or control liposomes (F) or with vehicle or l-NAME (G) and then treated with zymosan (n = 5 mice per group). Data are means ± SD and are representative of two independent experiments (A, D, F, and G). *P < 0.05 and **P < 0.01 by Student’s t test (C and D) or by ANOVA with Holm-Šídák multiple comparison test (F and G).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/564/eaar5514/DC1

    Fig. S1. DAP12 deficiency does not influence zymosan-induced neutrophil recruitment in the mouse model of dorsal air pouch inflammation.

    Fig. S2. sCD300b is not involved in zymosan-induced neutrophil accumulation in the mouse model of dorsal air pouch inflammation.

    Fig. S3. Both l-NAME and 1400 W dihydrochloride reduce neutrophil recruitment and the amounts of NO, LTB4, KC, and MIP-2 in the air pouches of zymosan-treated WT mice.

    Fig. S4. An Ab against Gr-1 decreases the number of neutrophils recruited to air pouches in response to zymosan.

    Fig. S5. Pretreatment with clodronate liposomes decreases the number of inflammatory DCs, but not of macrophages, in the air pouch exudates of zymosan-treated WT mice.

    Fig. S6. Pretreatment with clodronate liposomes reduces the amounts of KC and MIP-2 in the air pouch exudates of WT mice to levels comparable to those in CD300b−/− mice.

    Fig. S7. Phytosphingosine is present in zymosan.

    Fig. S8. CD300b deficiency does not affect the phagocytosis of zymosan by GM/IL-4–DCs.

    Fig. S9. Delipidation of zymosan decreases the number of neutrophils recruited to the air pouches of zymosan-treated WT mice.

  • This PDF file includes:

    • Fig. S1. DAP12 deficiency does not influence zymosan-induced neutrophil recruitment in the mouse model of dorsal air pouch inflammation.
    • Fig. S2. sCD300b is not involved in zymosan-induced neutrophil accumulation in the mouse model of dorsal air pouch inflammation.
    • Fig. S3. Both L-NAME and 1400 W dihydrochloride reduce neutrophil recruitment and the amounts of NO, LTB4, KC, and MIP-2 in the air pouches of zymosan-treated WT mice.
    • Fig. S4. An Ab against Gr-1 decreases the number of neutrophils recruited to air pouches in response to zymosan.
    • Fig. S5. Pretreatment with clodronate liposomes decreases the number of inflammatory DCs, but not of macrophages, in the air pouch exudates of zymosan-treated WT mice.
    • Fig. S6. Pretreatment with clodronate liposomes reduces the amounts of KC and MIP-2 in the air pouch exudates of WT mice to levels comparable to those in CD300b−/− mice.
    • Fig. S7. Phytosphingosine is present in zymosan.
    • Fig. S8. CD300b deficiency does not affect the phagocytosis of zymosan by GM/IL-4–DCs.
    • Fig. S9. Delipidation of zymosan decreases the number of neutrophils recruited to the air pouches of zymosan-treated WT mice.

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