A novel MyD-1 (SIRP-1α) signaling pathway that inhibits LPS-induced TNFα production by monocytes by Rosemary E. Smith, Vanshree Patel, Sandra D. Seatter, Maureen R. Deehan, Marion H. Brown, Gareth P. Brooke, Helen S. Goodridge, Christopher J. Howard, Kevin P. Rigley, William Harnett, and Margaret M. Harnett Blood Volume 102(7):2532-2540 October 1, 2003 ©2003 by American Society of Hematology

Ligation of MyD-1 specifically inhibits LPS-stimulated production of TNFα but not other cytokines. Ligation of MyD-1 specifically inhibits LPS-stimulated production of TNFα but not other cytokines. Human PBMCs were cultured with LPS (100 ng/mL) in the presence or absence of murine anti-human MyD-1 or the isotype control antibody for 24 hours, after which supernatants were analyzed by ELISA for cytokine production. The results shown are the mean production from triplicate cultures of (A) TNFα, (B) IL-6, (C) IL-1β, (D) IL-10, (E) IL-12 p70, (F) IL-15, (G) IFNγ, (H) IL-2, and (I) IL-8 secretion (means ± 1 SD; *P = .002). Stimulations are as follows: lane 1, medium; lane 2, anti-MyD-1 (10 μg/mL); lane 3, IgG isotype control (10 μg/mL); lane 4, LPS; lane 5, LPS plus anti-MyD-1; lane 6, LPS plus IgG. These data are representative of up to 14 experiments. Rosemary E. Smith et al. Blood 2003;102:2532-2540 ©2003 by American Society of Hematology

The effect of ligation of MyD-1 on the inhibition of TNFα induced by other pathogen products. The effect of ligation of MyD-1 on the inhibition of TNFα induced by other pathogen products. Human PBMCs were cultured for 24 hours with either 100 ng/mL LPS, 1 μg/mL Zymosan A, or 1000 U/mL PPD in the presence or absence of murine anti-human MyD-1 or the isotype control antibody, after which supernatants were analyzed by ELISA for TNFα. The results shown are the mean production of TNFα (means ± 1 SD, *P < .01, **P < .005 versus IgG1 control) for (A) LPS, (B) Zymosan A, or (C) PPD cultures. These data are representative of 3 experiments. Rosemary E. Smith et al. Blood 2003;102:2532-2540 ©2003 by American Society of Hematology

Ligation of MyD-1 induces tyrosine phosphorylation and recruitment of SHP-2. Ligation of MyD-1 induces tyrosine phosphorylation and recruitment of SHP-2. Cells of the human monocyte cell line U937, which expresses surface MyD-1 (results not shown), were incubated in the presence and absence of murine anti-human MyD-1 antibody (ILA24; 10 μg/mL) for up to 60 minutes at 37°C and immune complexes prepared. The induction of tyrosine phosphorylation after crosslinking MyD-1 was assessed by Western blot analysis of anti-MyD-1 immune complexes using the anti-phosphotyrosine mAb, 4G10 (A). Similarly, anti-MyD-1 immune complexes were assessed for MyD-1 (B), SHP-1 (C), or SHP-2 (D) expression as indicated. Rosemary E. Smith et al. Blood 2003;102:2532-2540 ©2003 by American Society of Hematology

Stimulation of human U937 monocytes with anti-MyD-1 stimulates PI 3-kinase. Stimulation of human U937 monocytes with anti-MyD-1 stimulates PI 3-kinase. (A) Uninduced [32P]-labeled U937:Δp85 cells were stimulated in the presence (▪) and absence (□) of anti-MyD-1 (10 μg/mL) for the indicated times at 37°C. Pretreatment (30 minutes) with wortmannin (1 nM) had little effect on basal PIP3 levels (▵) but suppressed anti-MyD-1-stimulated PIP3 production (▴). Similar anti-MyD-1 stimulations were carried out on cells induced to overexpress the dominant-negative form of p85. IPTG-induced U937:Δp85 cells were stimulated in the presence (•) and absence (○) of anti-MyD-1 (10 μg/mL) for the indicated times at 37°C. Lipids were extracted and [32P]PIP3 production was identified by TLC and quantified by liquid scintillation counting. Data are presented as means ± SD, n = 3, and are representative of 5 independent experiments demonstrating coupling of MyD-1 to PI 3-kinase. (B) Anti-SHP-2 immune complexes from U937 cells were assessed for p85 expression, tyrosine phosphorylation, and SHP-2 expression. Rosemary E. Smith et al. Blood 2003;102:2532-2540 ©2003 by American Society of Hematology

MyD-1 is coupled to consequent activation of PI 3-kinase, PtdCho-PLD, and sphingosine kinase. MyD-1 is coupled to consequent activation of PI 3-kinase, PtdCho-PLD, and sphingosine kinase. (A) U937 cells labeled with [3H]inositol were stimulated with isotype control (IgG1), LPS (100 ng/mL), anti-MyD-1 (10 μg/mL), or LPS plus anti-MyD-1 for 1 hour in the presence of 10 mM LiCl before measuring PIP2-PLC activation by determining total [3H]inositol phosphate generation. (B) U937 cells labeled with [3H]palmitate were stimulated with isotype control (IgG1), LPS (100 ng/mL), anti-MyD-1 (10 μg/mL), or LPS plus anti-MyD-1 for 1 hour before measuring PtdCho-PLD activation by determining [3H]PtdBut generation. (C) Uninduced [3H]palmitate-labeled U937:Δp85 cells or [3H]palmitate-labeled U937:Δp85 cells induced to overexpress the dominant-negative form of p85 were stimulated with IgG1 or anti-MyD-1 (10 μg/mL) for 1 hour at 37°C. Some samples were pretreated (30 minutes) with wortmannin (5 nM or 50 nM) as indicated. (D) [32P]-labeled U937 cells were stimulated in the presence (♦) or absence (□) of anti-MyD-1 (10 μg/mL) for the indicated times before determining levels of sphingosine-1-phosphate. (E) Uninduced [3H]serine-labeled U937:Δp85 cells or [3H]serine-labeled U937:Δp85 cells induced to overexpress the dominant-negative form of p85 were stimulated in the presence or absence of anti-MyD-1 (10 μg/mL) for 1 hour at 37°C. Some samples were pretreated (30 minutes) with wortmannin (5 nM or 50 nM) as indicated and sphingosine-1-phosphate levels determined. (F) Uninduced [3H]serine-labeled U937: Δp85 cells or [3H]serine-labeled U937:Δp85 cells induced to overexpress the dominant-negative form of p85 were stimulated in the presence or absence of anti-MyD-1 (10 μg/mL) for 1 hour at 37°C. Some samples were pretreated (30 minutes) with 0.1% butan-1-ol as indicated and sphingosine-1-phosphate levels determined. Data are expressed as means ± SD of triplicate determinations from 3 to 6 independent experiments. Rosemary E. Smith et al. Blood 2003;102:2532-2540 ©2003 by American Society of Hematology

MyD-1 coupling to PI 3-kinase and sphingosine kinase is maturation dependent. MyD-1 coupling to PI 3-kinase and sphingosine kinase is maturation dependent. U937 cells (A-B) or U937 cells induced to differentiate toward a macrophage phenotype by culture with dibutyrl cAMP (1 mM) for 48 hours (C-D) were stimulated in the presence (filled symbols) or absence (open symbols) of anti-MyD-1 (10 μg/mL) for the indicated times and PIP3 (A,C) or sphingosine-1-phosphate (B,D) measured as described in “Materials and methods.” Rosemary E. Smith et al. Blood 2003;102:2532-2540 ©2003 by American Society of Hematology

Inhibition of TNFα by MyD-1 is regulated by a wortmannin-sensitive signaling cascade, but IL-6 secretion is not. Inhibition of TNFα by MyD-1 is regulated by a wortmannin-sensitive signaling cascade, but IL-6 secretion is not. Human PBMCs were cultured in the presence or absence of 100 ng/mL LPS, 10 μg/mL ILA24 mouse anti-human MyD-1, and 1 nM wortmannin for 24 hours, after which supernatants were analyzed for TNFα or IL-6 production. The results shown are the mean production of (A) TNFα or (B) IL-6 secretion in the presence of 1 nM wortmannin. The data are generated from triplicate samples and are shown as the mean concentration ± 1 SD. In the absence of 1 nM wortmannin, the levels of TNFα produced by anti-MyD-1 plus LPS compared with LPS are significantly reduced, *P < .001, but in the presence of 1 nM wortmannin there is no significant difference between any groups. These data are representative of 4 experiments. Rosemary E. Smith et al. Blood 2003;102:2532-2540 ©2003 by American Society of Hematology

The measured decrease in TNFα production is not due to alterations in mRNA production but to retention of TNFα within monocytes. The measured decrease in TNFα production is not due to alterations in mRNA production but to retention of TNFα within monocytes. (A) Cytokine mRNA levels were assessed by TaqMan RT-PCR. The results are expressed as the relative change in mRNA compared with a calibrator. (B,C) Intracellular cytokine staining by FACS revealed that TNFα, but not IL-6, was being retained within monocytes after culture in the presence of LPS and anti-MyD-1. The results are expressed as the percentage TNFα-positive CD14+ cells, and these data are representative of 3 experiments. (D) Cell lysates were prepared from human PBMCs stimulated with either LPS (100 ng/mL) plus anti-MyD-1 (10 μg/mL) or LPS plus IgG1 (10 μg/mL) for 24 hours at 37°C. The cell lysates were then subjected to Western blot analysis of TACE expression as described in “Materials and methods.” These lysates were prepared from a single donor and were representative of those from 2 other independent donors. Rosemary E. Smith et al. Blood 2003;102:2532-2540 ©2003 by American Society of Hematology

The anti-MyD-1 mAb ILA24 does not block binding of a MyD-1Ig fusion protein to CD47 expressed on lymphocytes. The anti-MyD-1 mAb ILA24 does not block binding of a MyD-1Ig fusion protein to CD47 expressed on lymphocytes. Bovine PBMCs were incubated with MyD-1Ig (5 μg/mL) that had been preincubated with either the anti-MyD-1 mAb ILA24 (open histogram, A), the anti-MyD-1 mAb CC149 (open histogram, B), or the relevant isotype-matched control mAbs (filled histograms). In addition, PBMCs were preincubated with the anti-CD47 mAb MEM-122 (open histogram, C) prior to addition of the MyD-1 fusion protein. All antibodies were used at a concentration of 10 μg/mL. MyD-1 binding was detected by labeling with FITC-conjugated goat anti-human IgG and FACS analysis of lymphocytes gated on the basis of forward and side scatter. Rosemary E. Smith et al. Blood 2003;102:2532-2540 ©2003 by American Society of Hematology

MyD-1 signaling in monocytes. MyD-1 signaling in monocytes. Ligation of MyD-1 results in the tyrosine phosphorylation of MyD-1 and recruitment of SHP-2. Complex formation allows SHP-2 to act as an adaptor for p85 and results in the activation of PI 3-kinase. Generation of PIP3 leads to the activation of PtdCho-PLD, production of PtdOH, and consequent activation of sphingosine kinase. Rosemary E. Smith et al. Blood 2003;102:2532-2540 ©2003 by American Society of Hematology