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Neutrophils are important effector cells of acute inflammation because of their potential capacity to synthesize various proinflammatory mediators, and inhibition of their production is expected to result in anti-inflammatory effects. In this study, we investigate the effects of the anti-inflammatory cytokines, interleukin-10 (IL-10) and IL-4, on prostanoid synthesis in human neutrophils. Neutrophils isolated from healthy donors constitutively produced a small amount of prostaglandin E2 (PGE2 ) without any stimulations, whereas they produced a large amount of PGE2 after lipopolysaccharide (LPS) stimulation. IL-10 and IL-4 selectively inhibited their LPS-induced PGE2 production. Inhibition by both cytokines occurred at an early stage of LPS stimulation. Anti–IL-10 treatment of LPS-stimulated neutrophils resulted in enhanced PGE2 production. LPS-induced PGE2 and thromboxane B2 (TXB2 ) production in aspirin-treated neutrophils was significantly inhibited by IL-10, IL-4, and NS-398. Moreover, IL-10 and IL-4 inhibited LPS-induced cyclooxygenase (COX) activity in neutrophils. Western blot and immunocytochemical analysis showed that COX-2 protein was clearly induced in LPS-stimulated neutrophils and that its induction was inhibited by both IL-10 and IL-4. Moreover, both of these cytokines inhibited COX-2 mRNA expression in LPS-stimulated neutrophils. These results raise the possibility that these two cytokines may both offer potent clinical utility as anti-inflammatory agents in the future.

We have previously demonstrated that interleukin-4 (IL-4) induces tyrosine phosphorylation of a protein closely related or identical to the c-fes proto-oncogene product (FES) and association of this protein with the IL-4 receptor alpha chain (IL-4R alpha). IL-4 is known to induce association of phosphatidylinositol-3 (PI3) kinase with the IL-4R alpha. Since FES contains the consensus motifs for PI3 kinase binding, we tested the possibility that FES may associate with PI3 kinase upon IL-4 stimulation. We demonstrate herein that IL-4 stimulation induced rapid association of FES or a related protein with PI3 kinase in mouse T-cell lines. We also show an association of human FES (hFES) with the src homology 2 (SH2) domain of PI3 kinase in a COS7 cell expression system. The in vitro PI3 kinase assay using COS7 cells suggested that hFES partly contributes to the association between the hIL-4R alpha and PI3 kinase. We have further identified the important region in the cytoplasmic domain of the hIL-4R alpha for association of tyrosine-phosphorylated hFES with the hIL-4R alpha and SH2 domain of PI3 kinase using a COS7 cell expression system. These results suggest that FES or a related protein/PI3 kinase pathway may play a role in the pleiotropic effects of IL-4.

We have investigated the ability of glycoprotein (GP) Ibα, a megakaryocytic gene product, to sequester the signal transduction protein 14-3-3ξ and to influence megakaryocytopoiesis. Using a Gp1ba–/– mouse colony, we compared the rescued phenotypes produced by a wild-type human GP Ibα allele or a similar allele containing a 6-residue cytoplasmic tail truncation that abrogates binding to 14-3-3ξ. The observed phenotypes illustrate an involvement for GP Ibα in thrombopoietin-mediated events of megakaryocyte proliferation, polyploidization, and the expression of apoptotic markers in maturing megakaryocytes. We developed a hypothesis for the involvement of a GP Ibα/14-3-3ξ/PI-3 kinase complex in regulating thrombopoietin-mediated responses. An observed increase in thrombopoietin-mediated Akt phosphorylation in the truncated variant supported the hypothesis and led to the development of a model in which the GP Ibα cytoplasmic tail sequestered signaling proteins during megakaryocytopoiesis and, as such, became a critical regulator in the temporal sequence of events that led to normal megakaryocyte maturation.

A W127X mutation in platelet glycoprotein (GP)IX is the most common genetic defect in Japanese Bernard-Soulier Syndrome (BSS). Patients having this mutation are often misdiagnosed as having immune thrombocytopenia (ITP) because of residual expression of GPIbα on the platelet surface. The purpose of the present investigation was to determine whether the residual amount of GPIbα present on the surface of GPIXW127X BSS platelets might be sufficient to support adhesive functions of the GPIb complex. Flow cytometric analysis of platelets from our GPIXW127X BSS patient revealed 7–11% of the normal level of GPIbα (3,700 receptors/platelet), although the expression of GPIbα estimated by western blot analysis was only 1% of normal. Expression levels of other receptors such as GPIIb, GPVI, and α2 integrin were 3–4 times higher per platelet than that of normal controls–probably due to the increased size of the BSS platelets. To characterize the ability of GPIXW127X BSS platelets to bind VWF, we evaluated their agglutination in the presence of various concentrations of ristocetin, and found that ristocetin-induced agglutination was absent at 1.2 and 1.5 mg of ristocetin/ml, but present at 2.0 mg/ml. We then evaluated the ability of GPIXW127X BSS platelets to adhere under conditions of flow to immobilized VWF, type 1 collagen, and type III collagen. Whereas platelets derived from a Type I BSS patient having a TGTG deletion within the GPIbα gene in which GPIbα is completely absent (Thrombosis and Haemost 77:1055,1997) were unable to adhere to immobilized VWF under conditions of high shear, GPIXW127X BSS platelets bound well, demonstrating that the residual amount of GPIbα present GPIXW127X BSS platelets is sufficient to mediate adhesion to VWF. Finally, recent studies (J Thromb Haemost 5:797,2007) have shown that platelet adhesion to Type III collagen can be mediated by either interaction of GPIbα with VWF, or by interaction of the integrin α2β1 with collagen under high shear flow. In support of this notion, adhesion of GPIbαTGTG del BSS platelets to immobilized Type III collagen could be completely inhibited by addition of the anti-α2β1 blocking mAb, Gi9, while adhesion of GPIXW127X BSS platelets to Type III collagen was only marginally affected. Taken together, we conclude that platelets having a W127X mutation in GPIX express residual amounts of functional GPIbα, and both GPIb-VWF and integrin α2β1-collagen interactions are physiologically important under conditions of high shear stress. Residual expression of GPIbα in GPIXW127X platelets, therefore, likely accounts for the relatively mild bleeding phenotype in this variant form of BSS.

The hereditary bleeding disorder Bernard-Soulier syndrome (BSS) is caused by an absent or dysfunctional GPIb/IX complex. Lopez et al. have reported that three subunits of the complex (GPIbα, GPIbβ and GPIX) are all necessary for efficient translocation to the cell surface. In contrast, Meyer et al. have demonstrated that GPIbα alone can incorporate into the cell surface. We have established a cell culture system with inducible levels of GPIbβ and GPIX. Using these cell lines, we characterize an intrinsic ability of GPIbα to form dimers and the relevance of specific GPIbα sequences for dimer formation and the assembly of a GPIb/IX complex. A stable CHO cell line was established that expresses GPIbβ and GPIX under the control of Tet-responsive elements. This cell line was then transfected with WT GPIbα and truncated GPIbα mutants. Expression was monitored by Western blot analysis and flow cytometry. We observed that expression of GPIbα, by itself, targets GPIbα to the cell surface as a dimer albeit at reduced levels. The GPIbα binding domain for filamin (residues 545–605) is essential for forming a dimer but not for assembly of a GPIb/IX complex. We also observed GPIbα dimer formation in a transgenic mouse that expresses human GPIbα and a mouse chimeric GPIb-IX complex suggesting dimer formation can occur in the platelet. To investigate the role of filamin in the dimerization process, we established a stable cell line deficient in filamin by targeting filamin expression with RNAi. We observed GPIbα dimers in the absence of filamin expression. These results illustrate the importance of expressing all three subunits of the GPIb/IX complex, but also document as intrinsic potential within GPIbα for surface expression via intermolecular homodimerization.

Glycoprotein VI (GPVI) is an essential platelet receptor for collagens that is exclusively expressed in the megakaryocytic lineage. Transcription of the human gene GP6 is driven largely by GATA-1, Sp1 and Fli-1. However, the mere presence of these is not sufficient to initiate transcription during megakaryocyte differentiation, and other mechanisms are suggested to be involved in the regulation of megakaryocyte-specific expression of GPVI. In this study, we show that GPVI expression during megakaryocytic differentiation is dependent on CpG demethylation that can be initiated by thrombopoietin (TPO). Sodium bisulfite genomic sequencing established that a CpG-rich island within the GP6 promoter region is fully methylated at 10 CpG sites in GPVI non-expressive cell lines, such as UT-7/EPO and C8161, but completely unmethylated in GPVI expressive cell lines, including UT-7/TPO and CHRF288-11. To further confirm the relationship between CpG demethylation and expression of GPVI in primary cells, we treated human cord blood cells with TPO. The GP6 promoter is highly methylated in cord blood mononuclear cells (progenitors) but not in CD41+enriched cells obtained after TPO differentiation. Furthermore, when UT-7/EPO-Mpl cells, which stably express human c-mpl, were treated with TPO, demethylation of the GP6 promoter was induced. In every case, demethylation of the GP6 promoter correlated with an increase in mRNA level. Thus, megakaryocyte-specific expression of the GP6 gene is regulated, in part, by CpG demethylation which can be directly initiated by TPO. Our results establish, for the first time, a role for TPO in dynamic changes in CpG methylation status that are involved in the epigenetic regulation of megakaryocyte-specific gene expression.