A novel, somatic, transforming mutation in the extracellular domain of Epidermal Growth Factor Receptor identified in myeloproliferative neoplasm.

We describe a novel ERBB1/EGFR somatic mutation (p. C329R; c.985 T > C) identified in a patient with JAK2V617F Polycythaemia Vera (PV). This substitution affects a conserved cysteine residue in EGFR domain 2 and leads to the formation of a ligand-independent covalent receptor dimer, associated with increased transforming potential. Aberrant signalling from the EGFRC329R receptor is cell type-dependent and in the TF1.8 erythroid cell line expression of this mutant suppresses EPO-induced differentiation. Clonal analysis shows that the dominant JAK2V617F-positive clone in this PV patient harbors EGFRC329R, thus this mutation may contribute to clonal expansion. Somatic mutations affecting other ERBB and related receptor tyrosine kinases are observed in myeloproliferative neoplasms (MPN), and we show elevated EGFR levels in MPN samples, consistent with previous reports. Thus activation of this group of receptors, via multiple mechanisms, may contribute to clonal growth and survival of the JAK2V617F disease clone in MPN.

Dysregulated hematopoiesis and a progressive neurological disorder induced by expression of an activated form of the human common beta chain in transgenic mice.

Previously we described activating mutations of hbetac, the common signaling subunit of the receptors for the hematopoietic and inflammatory cytokines, GM-CSF, IL-3, and IL-5. The activated mutant, hbetacFIDelta, is able to confer growth factor-independent proliferation on the murine myeloid cell line FDC-P1, and on primary committed myeloid progenitors. We have used this activating mutation to study the effects of chronic cytokine receptor stimulation. Transgenic mice were produced carrying the hbetacFIDelta cDNA linked to the constitutive promoter derived from the phosphoglycerate kinase gene, PGK-1. Transgene expression was demonstrated in several tissues and functional activity of the mutant receptor was confirmed in hematopoietic tissues by the presence of granulocyte macrophage and macrophage colony-forming cells (CFU-GM and CFU-M) in the absence of added cytokines. All transgenic mice display a myeloproliferative disorder characterized by splenomegaly, erythrocytosis, and granulocytic and megakaryocytic hyperplasia. This disorder resembles the human disease polycythemia vera, suggesting that activating mutations in hbetac may play a role in the pathogenesis of this myeloproliferative disorder. In addition, these transgenic mice develop a sporadic, progressive neurological disease and display bilateral, symmetrical foci of necrosis in the white matter of brain stem associated with an accumulation of macrophages. Thus, chronic hbetac activation has the potential to contribute to pathological events in the central nervous system.

Chronic expression of P-selectin on endothelial cells stimulated by the T-cell cytokine, interleukin-3.

P-selectin expressed on the surface of endothelium mediates leukocyte adhesion in vitro and rolling in vivo. Several inducers of cell-surface P-selectin expression on endothelial cells (EC) have previously been identified, all of which yield transient cell-surface expression of P-selectin lasting minutes to a few hours. We now show that a T-lymphocyte product, interleukin-3 (IL-3), stimulates the long-term endothelial cells (HUVEC). IL-3 induced cell-surface P-selectin expression in two phases. An initial peak at 10 minutes was followed by a prolonged upregulation beginning 16 hours after IL-3 addition and lasting at least 4 days. The level of P-selectin expression induced by IL-3 added for 48 hours was similar to that induced by treatment of HUVEC for 10 minutes with thrombin, and the effect of adding IL-3 for 48 hours followed by thrombin for 10 minutes was additive. Induction of cell-surface P-selectin expression by IL-3 was blocked by pretreatment of EC with a blocking monoclonal antibody against the IL-3 receptor alpha-chain. Lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha) and a mutant form of IL-3 with decreased potency did not induce cell-surface P-selectin expression after 48 hours' incubation with HUVEC, suggesting that the effect was specific to IL-3. The increase in cell-surface P-selectin expression occurring after 16 hours of incubation with IL-3 was accompanied by a similar prolonged increase in the steady-state mRNA level that was not observed at 10 minutes after IL-3 addition. As T-lymphocyte infiltration is a hallmark of chronic inflammation, our observations suggest that the secretion of IL-3 by T lymphocytes may serve to maintain the inflammatory state during chronic inflammation.

P-selectin interacts with a beta 2-integrin to enhance phagocytosis.

P-selectin is an adhesion molecule for myeloid cells that seems to be essential for the development of cellular inflammatory responses. We show that adhesion of neutrophils to purified and recombinant P-selectin enhances the phagocytosis of unopsonized zymosan particles as judged by the number of cells ingesting particles (30.2 +/- 5.8 vs 14.5 +/- 4.0, p = 0.002) and the number of particles ingested per cell (percentage increase 58.3 +/- 4.4%. p = 0.0002). The enhanced phagocytosis was inhibited by Abs to CD18 or CD11b, suggesting that P-selectin alters beta 2-integrin function. The enhancement was only seen in the presence of cations allowing the integrin to assume a particular extracellular conformation. Furthermore, P-selectin, although not altering the total expression of CD18 on neutrophils, significantly increased the binding of mAb 24, which detects an activation-dependent epitope. Our results support a signaling role for P-selectin in influencing beta 2-integrin function.