Ceramide, a product of agonist-stimulated sphingomyelinase activation, is known to be generated during the phagocytosis of antibody-coated erythrocytes by polymorphonuclear leukocytes. Agonist-stimulated formation of ceramide-1-phosphate is now shown to occur in 32 PO 4 -labeled neutrophils. Ceramide-1-phosphate is formed by a calcium-dependent ceramide kinase, found predominately in the neutrophil plasma membrane. The neutrophil kinase is specific for ceramide because, in contrast to the bacterial diglyceride kinase, ceramide is not phosphorylated under conditions specific for diglyceride phosphorylation. Conversely, 1,2-diacylglycerol does not serve as substrate for the neutrophil ceramide kinase. Ceramide kinase activation occurs in a time-dependent fashion, reaching peak activity 10 min after formyl peptide stimulation and challenge with antibody-coated erythrocytes. The lipid kinase activity is optimal at pH 6.8. Because the formation of the phagolysosome is a critical event in phagocytosis, the effect of ceramide-1-phosphate in promoting the fusion of liposomes was determined. Both the addition of increasing concentrations of sphingomyelinase D and ceramide-1-phosphate promoted liposomal fusion. In summary, ceramide-1-phosphate is formed during phagocytosis through activation of ceramide kinase. Ceramide-1-phosphate may promote phagolysosome formation.

IntroductionMigration inhibitory factor (MIF) is a pleotropic cytokine which plays a pivotal role in inflammatory and immune-mediated diseases such as rheumatoid arthritis (RA) and atherosclerosis. MIF is secreted by T lymphocytes and macrophages on lipopolysaccharide (LPS) exposure and induces secretion of tumor necrosis factor-␣ (TNF-␣) by mouse macrophages. 1,2 In RA, MIF is highly expressed in macrophages, endothelial cells, synovial tissue (ST) fibroblasts, serum, and synovial fluids. 1,2 MIF stimulates macrophage release of proinflammatory cytokines such as TNF-␣, interleukin 1 ␤ (IL-1␤), 4 MIF up-regulates IL-1␤, matrix metalloproteinases (MMPs) MMP-1, MMP-3, MMP-9, and MMP-13 in RA ST fibroblasts. 5,6 In rodent arthritis models, administration of anti-MIF antibody ameliorates arthritis with profound inhibition of clinical and histologic features of disease. [7][8][9] Anti-MIF treatment ameliorates acute encephalomyelitis and experimental autoimmune myocarditis in mice. 10,11 These studies show a key role of MIF in the pathogenesis of immunologic and inflammatory diseases.We have shown that MIF is a potent angiogenic factor. 12 Anti-MIF inhibits tumor growth and tumor-associated angiogenesis, and MIF is a required factor for tumor-initiated endothelial cell proliferation and tumor neovascularization. 13,14 Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in soluble forms (sVCAM-1 and sICAM-1, respectively) are potent angiogenic mediators, and RA synovial fluid-induced angiogenesis is blocked by anti-VCAM-1. 15,16 MIF is found in human vascular endothelial cells, which have been considered to play a pivotal role in systemic inflammatory and immunologic diseases by producing cytokines and growth factors. 17 Adhesion of inflammatory cells to vascular endothelium is the initial step in leukocyte recruitment and is mediated by a number of cell adhesion molecules such as ICAM-1, VCAM-1, E-, P-, and L-selectin, as well as integrins. MIF up-regulates ICAM-1 on endothelial cells. 18 Rat kidney VCAM-1 and ICAM-1 expression are decreased by anti-MIF treatment, blocking the development of glomerulonephritis. 19 Similarly, anti-MIF prevents VCAM-1 up-regulation on endothelial cells and improves acute encephalomyelitis in mice. 11 Cell adhesion molecules mediate and amplify the inflammatory response by allowing the ingress of leukocytes into diseased tissues. 20-22 VCAM-1 and ICAM-1 may be used as a reliable measure of the extent of atherosclerotic progression, and focal expression of adhesion molecules is consistently found in atherosclerotic plaques in humans. [22][23][24] The most compelling data for the necessity of adhesion molecules in the development of atherosclerotic plaques came from a report indicating that mice deficient in adhesion molecules are protected against atherosclerosis when fed an atherogenic diet. 25 Those studies support the role of adhesion molecules in immune-mediated diseases. For personal use only. on May 11, 2018. by guest www.bloodjournal.org From ...

A precipitating factor in the development of atherosclerotic lesions is the inappropriate migration and proliferation of vascular smooth muscle cells (SMC) within the intima of the vessel wall. Focusing on the role of extracellular matrix proteins in SMC migration, we have demonstrated that thrombospondin (TSP) itself is a potent modulator of SMC motility and acts to potentiate platelet-derived growth factor (PDGF)-mediated SMC migration as well. Migration of SMC to TSP was dose dependent. Interestingly, maximal SMC migration to TSP exceeded that to either PDGF or basic fibroblast growth factor (bFGF). The distal COOH terminus of TSP was shown to mediate SMC migration as demonstrated by complete inhibition of the response by monoclonal antibody (mAb) C6.7. Nevertheless, proteolytic fragments of TSP were not as potent as intact TSP in mediating SMC migration. Only by combining the heparin-binding domain (HBD) with the 140 kD COOH terminal fragment was SMC migration restored to levels seen with intact TSP. Based on antibody inhibition studies, an alpha v-containing integrin receptor, but not alpha v beta 1 or alpha v beta 3, appeared to be involved in SMC migration to TSP. The coincidental expression of PDGF and TSP at sites of vascular injury and inflammation led us to evaluate the effect of suboptimal levels of TSP on SMC responsiveness to PDGF. SMC migration in response to PDGF was enhanced nearly 60% in the presence of suboptimal concentrations of TSP. This effect was specific for PDGF and dependent on the concentration of TSP with maximal potentiation obtained between 50-100 nM TSP, concentrations tenfold lower than those necessary for SMC migration to TSP itself. mAb C6.7 completely inhibited enhancement but, as with SMC migration to TSP alone, TSP proteolytic fragments did not possess the effectiveness of the intact molecule. Additional experiments assessing SMC migration to PDGF demonstrated that PDGF stimulated SMC motility indirectly by inducing TSP synthesis. These studies suggested that TSP functions as an autocrine motility factor to modulate SMC migration, which in conjunction with PDGF could serve to aggravate and accelerate development of atherosclerotic lesions at sites of vascular injury or inflammation.

Objective. Interleukin-18 (IL-18) is a proinflammatory cytokine implicated in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to examine the role of IL-18 in up-regulating secretion of the angiogenic factors stromal cell-derived factor 1␣ (SDF-1␣)/CXCL12, monocyte chemoattractant protein 1 (MCP-1)/CCL2, and vascular endothelial growth factor (VEGF) in RA synovial tissue (ST) fibroblasts, and the underlying signaling mechanisms involved.Methods. We used enzyme-linked immunosorbent assays, Western blotting, and chemical inhibitors/ antisense oligodeoxynucleotides to signaling intermediates to assess the role of IL-18.Results.