CXCL1/KC prevents the induction of iNOS protein expression in hearts of mice challenged with cardiac-specific α-myosin-heavy chain-derived peptide M7Aα.

<p>Frequency of positive cells in heart sections from mice immunized with M7Aα in CFA 21 days after the initial immunization. Percentage of cells (±SD) staining for iNOS in cardiomyocytes, or CD68 and iNOS in inflammatory cells, was calculated after counting cells per visual field at magnification of x160 on sections from hearts of 3 mice per group. A minimum 100 cells in at east 10 different visual fields was evaluated per heart. One result representative of 3 independent experiments is shown. * p<0.05 when compared to the other groups. n.d., cells were not detected in sufficient numbers, i.e., there were <100 cells per visual field.</p

<i>LPS</i>^def mice are highly susceptible to induction of autoimmune inflammatory heart disease.

<p>Genetic loss of TLR4 function leads to severe autoimmune myocarditis in mice challenged with heart-specific autoantigen and complete Freund's adjuvant (CFA), a complex mixture of TLR agonists. (A) <i>LPS</i>^def. mice lacking functional TLR4 developed significantly more severe autoimmune myocarditis that wild type BALB/c control mice. Histopathological disease severity, as described in Methods, was determined 21 days after the initial immunization with heart-specific M7Aα peptide and CFA. * p<0.05. One representative result out of 5 independent experiments is shown. (B) Serum IgG autoantibodies reactive to heart specific epitope M7Aα were determined 21 days after initial immunization with heart-specific M7Aα peptide and CFA. * p<0.05. One representative result out of 5 independent experiments is shown. (C) Heart inflammatory infiltrate. CD3ε⁺ T cells expressing IL-17A were evaluated 21 days after initial immunization with heart-specific M7Aα peptide and CFA. Squares represent the percentage of IL-17A⁺ cells per CD3ε⁺ T cells as determined by immunohistochemistry in heart-sections from individual mice, lines indicate mean values. * p<0.05. One representative result out of 5 independent experiments is shown. (D) Representative photomicrograph of heart section from a <i>LPS</i>^def mouse immunized with autoantigen and CFA. Inflammatory infiltrate consisting mostly of mononuclear cells is present throughout the myocardium often surrounding necrotic cardiomyocytes (arrow). Original magnifications x10 and x200 are shown. Hearts were analyzed 21 days after initial immunization with heart-specific M7Aα peptide in CFA. Staining was with hematoxylin and eosin (H&E). (E) Histopatholgy in a heart section from a BALB/c mouse immunized with heart-specific M7Aα peptide in CFA. Inflammatory infiltrate consisting mostly of mononuclear cells is present as an inflammatory focus (arrow). Original magnifications x100 is shown. (F) <i>LPS</i>^def. mice fail to resolve autoimmune myocarditis by day 28 after the initial autoantigen challenge but not wild type BALB/c control mice. Histopathological disease severity, as described in Methods, was determined 28 days after the initial immunization with heart-specific M7Aα peptide and CFA. * p<0.05. Squares represent individual mice, lines indicate mean values. One representative result out of 3 independent experiments is shown. Student's t Test was used for statistical analysis.</p

Sphingosine Kinase 1 Mediation of Expression of the Anaphylatoxin Receptor C5L2 Dampens the Inflammatory Response to Endotoxin

The complement anaphylatoxin C5a has a pathogenetic role in endotoxin-induced lung inflammatory injury by regulating phagocytic cell migration and activation. Endotoxin and C5a activate the enzyme sphingosine kinase (Sphk) 1 to generate the signaling lipid sphingosine-1-phosphate (S1P), a critical regulator of phagocyte function. We assessed the function of Sphk1 and S1P in experimental lung inflammatory injury and determined their roles in anaphylatoxin receptor signaling and on the expression of the two C5a receptors, C5aR (CD88) and C5L2, on phagocytes. We report that Sphk1 gene deficient (Sphk12/2) mice had augmented lung inflammatory response to endotoxin compared to wild type mice. Sphk1 was required for C5a-mediated reduction in cytokine and chemokine production by macrophages. Moreover, neutrophils from Sphk12/2 mice failed to upregulate the anaphylatoxin receptor C5L2 in response to LPS. Exogenous S1P restored C5L2 cell surface expression of Sphk12/2 mouse neutrophils to wild type levels but had no effect on cell surface expression of the other anaphylatoxin receptor, CD88. These results provide the first genetic evidence of the crucial role of Sphk1 in regulating the balance between expression of CD88 and C5L2 in phagocytes. S1P-mediated up-regulation of C5L2 is a novel therapeutic target for mitigating endotoxin-induced lung inflammatory injury

Anaphylatoxin C5a-mediated reduction in cytokine and chemokine production depends on Sphk1.

<p>BMDMs from <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> mice were stimulated with LPS (500 ng/ml), with LPS concomitant with C5a (1 nM), or without C5a. Tissue culture supernatants were harvested 2 h (TNF-α) or 8 h (IL-6 and KC) after the addition of stimuli. (<b>A</b>) TNF-α; (<b>B</b>); IL-6; (<b>C</b>) KC. Error bars represent s.d. *p<0.05 by Student's t-test; n = 5 for each genotype; representative for at least three independent experiments. (<b>D</b>) Reduced C5a-induced ERK1/2 and activation. BMDMs from <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> mice were stimulated with C5a (10 nM), LPS (500 ng/ml), LPS and C5a, or saline for 5 min. Cell lysates were processed for immunoblotting with indicated antibodies. Representative of 3 independent experiments showing similar results. UD, undetected. (<b>E</b>) Model: Reduction of inflammatory cytokine production by phagocytes stimulated with C5a requires Sphk1. The model links Sphk1 activity to the cell surface expression of the anaphylatoxin receptor C5L2. LPS, C5a and inflammatory cytokines activate Sphk1 which is required to maintain S1P during inflammation. S1P regulates C5L2 cell surface expression on phagocytes. C5a, via C5L2 expressed on the cell surface, reduces neutrophil inflammation and inflammatory cytokine production by macrophages.</p

Sphingosine-1-phosphate (S1P) and anaphylatoxin C5a concentrations in plasma and lung tissue.

<p>S1P concentrations in plasma (<b>A</b>) or lung tissue lysate (<b>B</b>) or C5a concentrations in plasma (<b>C</b>) or lung tissue lysates (<b>D</b>) from <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> mice were determined before or after i.p. LPS challenge (0.5 mg/kg), using the LC-MS/MS (46) or ELISA techniques. Error bars represent s.d. *p<0.005, **p<0.001, by Student's t-test. No significant differences in (<b>B, C, D</b>). n = 10 for each genotype and time point.</p

Genetic deletion of Sphk1 amplifies lung inflammation and lethality in mice.

<p>(<b>A</b>) Lung MPO activity. <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> (n = 10 per time point of each genotype) mice were given LPS i.p. (0.5 mg/kg) and lungs were removed at the indicated times. Error bars represent s.d. *p<0.01 by Student's t-test. (<b>B, C</b>) Increased LPS-induced cytokine and chemokine production. TNF-α, IL-6, IL-1β, and KC were measured in plasma (<b>B</b>) and TNF-α, IL-6 and KC in lung tissue lysates (<b>C</b>) from <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> mice, 1 h after i.p. LPS or saline injection. Error bars represent s.d. *p<0.05 by Student's t-test; n = 5 for each genotype. (<b>D</b>) Increased LPS-induced lethality. <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> mice (n = 10/genotype, representative of three independent experiments) were given LPS i.p. (<b>E</b>) Increased LPS-induced lethality. <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> mice (n = 10/genotype, representative of three independent experiments) were given LPS i.p. Differences in mortality were assessed by log-rank test (p<0.05). UD, undetected.</p

Sphk1 regulates cell surface expression of anaphylatoxin receptor C5L2.

<p>(<b>A</b>) <i>in vivo</i> cell surface expression of anaphylatoxin receptors CD88 and C5L2 in circulating Gr1⁺ neutrophils and F4/80⁺ peritoneal macrophages from <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> mice was determined by flow cytometry. The percentage of C5L2⁺CD88⁺ cells is shown. There is a significant reduction of C5L2⁺ cells in <i>Sphk1^−/−</i> mice. (<b>B</b>) Bar graph depicts the mean fluorescence intensity (MFI ± s.d.) of C5l2 or CD88 cell surface expression of cells from five mice per genotype. (<b>C</b>) Total C5L2 expression (MFI) assessed by fixing and permeabilizing cells before staining with specific Ab to C5L2. Cell surface expression only, on non-permeabilized cells, is shown for comparison. Representative histograms of C5L2 expression by circulating neutrophils and peritoneal macrophages from <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> mice are shown. (<b>D</b>) Percentage change of anaphylatoxin receptors CD88 and C5L2 neutrophil cell surface expression after <i>in vitro</i> stimulation with LPS (1 µg/ml) for 1 h compared to non-stimulated controls. (<b>E</b>) Exogenous S1P (250 nM) restores C5L2 cell surface expression of <i>Sphk1^−/−</i> PMNs to the level of <i>Sphk1</i>^+/+ PMNs. Error bars represent s.d. *p<0.05 by Student's t-test. Representative of at least 3 independent experiments with similar results. n.s., not significant.</p