Platelet Supernatant Suppresses LPS-Induced Nitric Oxide Production from Macrophages Accompanied by Inhibition of NF-κB Signaling and Increased Arginase-1 Expression

<div><p>We previously reported that mouse bone marrow-derived macrophages (BMDMs) that had been co-cultured with platelets exhibited lower susceptibility to bacterial lipopolysaccharide (LPS) and produced lower levels of nitric oxide (NO) and inflammatory cytokines including TNF-α and IL-6. The suppression of macrophage responses was mediated, at least in part, by platelet supernatant. In the present study, we assessed phenotypic changes of BMDMs induced by incubation with the supernatant from thrombin-activated platelets (PLT-sup) and found that BMDMs cultured with PLT-sup (PLT-BMDMs) expressed a lower level of inducible NO synthase (iNOS) and a higher level of arginase-1, both of which are involved in the L-arginine metabolism, upon stimulation with LPS or zymosan. We also examined possible modulation of the NF-κB signaling pathway and observed suppression of IκBα phosphorylation and a decrease of NF-κB p65 expression in LPS-stimulated PLT-BMDMs. These results suggest that PLT-sup suppresses inflammatory responses of BMDMs via negative regulation of NF-κB signaling leading to lowered expression of iNOS and enhanced L-arginine catabolism by arginase-1.</p></div

Attenuation of LPS-induced NO production from BMDMs by PLT-sup.

<p>BMDMs (2.5 × 10⁶ cells) were cultured for 24 h with PLT-sup (PLT-BMDMs) or 0.5 U/mL thrombin alone (control BMDMs) in a 3.5 cm dish, and stimulated with complete medium containing LPS (50 ng/mL) for 0–24 h. The production levels of NO₂^- in the culture supernatant were determined by Griess reaction using a calibration curve for NaNO₂. Experiments were performed in quintuplicate and repeated four times. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01 vs. control BMDMs. Representative results of the four experiments are shown.</p

Expression of iNOS and arginase-1 in BMDMs after LPS stimulation.

<p>(A) PLT-BMDMs and control BMDMs (2.5 × 10⁶ cells) were stimulated with LPS (50 ng/mL) for 24 h, and the gene expressions of <i>Nos2</i> and <i>Arg1</i> were analyzed by RT-qPCR with the relative standard curve method using <i>Gapdh</i> as an internal control. The gene expression is represented as the value relative to gene expression in the original BMDMs. Experiments were performed in quintuplicate and repeated four times. The data are presented as the mean ± SEM. ***p < 0.005 vs. control BMDMs. Representative results of the four experiments are shown. (B) PLT-BMDMs and control BMDMs (2.5 × 10⁶ cells) were stimulated with LPS (50 ng/mL) for 0–24 h. Cells were then lysed in 1 × SDS sample buffer, and the cell lysates were subjected to western blotting analysis with antibodies against iNOS, arginase-1 or GAPDH. The relative intensity of each iNOS or arginase-1 band after normalization to the levels for GAPDH is shown in the lower panel. Experiments were repeated four times, and representative results are shown. (C) PLT-BMDMs and control BMDMs (2.5 × 10⁶ cells) were stimulated with zymosan (25 or 100 μg/mL) for 12 h, and then cell lysates were subjected to western blotting analysis with antibodies against iNOS, arginase-1 or GAPDH. <i>C</i>, control BMDMs; <i>P</i>, PLT-BMDMs. The relative intensity of each iNOS or arginase-1 band after normalization to the levels for GAPDH is shown in the lower panel. Experiments were repeated four times, and representative results are shown.</p

Induction of arginase-1 expression by PLT-sup in BMDMs.

<p>(A) The gene expressions of <i>Tnf</i>, <i>Il6</i>, <i>Il1b</i>, <i>Nos2</i>, <i>Arg1</i>, <i>Fizz1</i>, <i>Ym1</i>, and <i>Mrc1</i> in PLT-BMDMs and control BMDMs were analyzed by RT-qPCR with the relative standard curve method using <i>Gapdh</i> as an internal control. The gene expression in PLT-BMDMs was represented as the value relative to gene expression in control BMDMs. Experiments were performed in triplicate and repeated three times. Data are presented as the mean ± SEM, and representative results of the three experiments are shown. (B) PLT-BMDMs and control BMDMs were lysed in 1 × SDS sample buffer, and the resultant lysates were subjected to western blotting analysis with antibodies against arginase-1 or GAPDH. The relative intensity of each arginase-1 band after normalization to the corresponding levels of GAPDH is shown above each blot. Experiments were repeated five times, and representative results are shown. (C) PLT-BMDMs and control BMDMs (4 × 10⁵ cells) were lysed with 0.1% Triton X-100 (100 μL) containing a protease inhibitor cocktail for 10 min at room temperature, and the lysate was assayed for arginase activity as described in the Materials and Methods. Experiments were performed in quintuplicate and repeated five times. Data are presented as the mean ± SEM. **p < 0.01 vs. control BMDMs. Representative results of the five experiments are shown.</p

Cell surface expression of CD14 and the TLR4/MD-2 complex in PLT-BMDMs.

<p>PLT-BMDMs and control BMDMs were incubated with anti-mouse CD16/32 at 4°C for 10 min, and then incubated with biotin anti-mouse CD14 antibody or rat anti-mouse TLR4/MD-2 complex antibody at 4°C for 30 min. After incubation with streptavidin-PE or anti-rat IgG-Alexa Fluor 647 antibody at 4°C for 30 min, cells were analyzed by FACSVerse (BD Biosciences). The data are shown as histograms representing the number of cells (Y-axis) against the log of fluorescence intensity (X-axis). The black lines represent cells with primary antibody, and the gray-shaded areas represent cells without primary antibody. Experiments were repeated three times, and representative results are shown.</p

Oligodeoxynucleotide primers used in qPCR experiments.

<p>Oligodeoxynucleotide primers used in qPCR experiments.</p

Western blotting analyses of NF-κB and MAPK signaling pathways in BMDMs after LPS stimulation.

<p>(A) PLT-BMDMs and control BMDMs (2.5 × 10⁶ cells) were stimulated with LPS (50 ng/mL) for 0–120 min. Cells were then lysed in 1 × SDS sample buffer, and the cell lysates were subjected to western blotting analysis with antibodies against phospho-IκBα, total IκBα, phospho-NF-κB p65, total NF-κB p65, phospho-p38 MAPK, total p38 MAPK, phospho-JNK, total JNK, phospho-ERK1/2, total ERK1/2 or GAPDH. The relative intensity of each band after normalization to the corresponding level of GAPDH is shown in the right panel. Experiments were repeated three times, and representative results are shown. (B) PLT-BMDMs and control BMDMs (2.5 × 10⁶ cells) that had been stimulated with LPS (50 ng/mL) for 0–120 min were separated into cytoplasmic and nuclear fractions. Each fraction was subjected to western blotting analysis with antibodies against NF-κB p65. GAPDH and histone H3 were used as controls for the cytoplasmic and nuclear fractions, respectively. The relative intensity of each band after normalization to the level of GAPDH or histone H3 is shown in the lower panel. Experiments were repeated three times, and representative results are shown.</p

Staphylococcal Superantigen-Like Protein 5 Inhibits Matrix Metalloproteinase 9 from Human Neutrophils▿

Staphylococcal superantigen-like proteins (SSLs) constitute a family of exoproteins exhibiting structural similarities to superantigens and enterotoxins but no superantigenic activity. In this article, we present evidence that SSL5 specifically binds to matrix metalloproteinase 9 (MMP-9) and inhibits its enzymatic activity. When human neutrophil cell lysate was applied to recombinant His-tagged SSL5 conjugated to Sepharose, the bound fraction gave a major band of approximately 100 kDa in SDS-polyacrylamide gel electrophoresis. This protein was identified as the proform of MMP-9 (proMMP-9) by peptide mass fingerprinting analysis. The recombinant SSL5-Sepharose also bound to proMMP-9 secreted by interleukin 8 (IL-8)-stimulated neutrophils and HT1080 fibrosarcoma cells. Surface plasmon resonance analysis revealed that recombinant SSL5 bound to proMMP-9 with rather high affinity (dissociation constant [KD] = 1.9 nM). Recombinant SSL5 was found to effectively inhibit MMP-9-catalyzed hydrolysis of gelatin and a synthetic fluorogenic peptide in a noncompetitive manner (Ki = 0.097 nM), as assessed by zymography and the fluorescence quenching method. Finally, the transmigration of neutrophils across Matrigel basement membranes in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) was suppressed by the presence of recombinant SSL5. We discuss possible roles that SSL5 may play in immune evasion of staphylococci by inhibiting MMP and interfering with leukocyte trafficking

Coronin-1 is phosphorylated at Thr-412 by protein kinase Cα in human phagocytic cells

Coronin-1, a hematopoietic cell-specific actin-binding protein, is thought to be involved in the phagocytic process through its interaction with actin filaments. The dissociation of coronin-1 from phagosomes after its transient accumulation on the phagosome surface is associated with lysosomal fusion. We previously reported that 1) coronin-1 is phosphorylated by protein kinase C (PKC), 2) coronin-1 has two phosphorylation sites, Ser-2 and Thr-412, and 3) Thr-412 of coronin-1 is phosphorylated during phagocytosis. In this study, we examined which PKC isoform is responsible for the phosphorylation of coronin-1 at Thr-412 by using isotype-specific PKC inhibitors and small interfering RNAs (siRNAs). Thr-412 phosphorylation of coronin-1 was suppressed by Gö6976, an inhibitor of PKCα and PKCβI. This phosphorylation was attenuated by siRNA for PKCα, but not by siRNA for PKCβ. Furthermore, Thr-412 of coronin-1 was phosphorylated by recombinant PKCα in vitro, but not by recombinant PKCβ. We next examined the effects of Gö6976 on the intracellular distribution of coronin-1 in HL60 cells during phagocytosis. The confocal fluorescence microscopic observation showed that coronin-1 was not dissociated from phagosomes in Gö6976-treated cells. These results indicate that phosphorylation of coronin-1 at Thr-412 by PKCα regulates intracellular distribution during phagocytosis