MFG-E8 inhibits neutrophil migration through αvß3-integrin-dependent MAP kinase activation.

We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFG­E8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFG­E8 attenuates neutrophil migration. Recombinant human MFG­E8 (rhMFG­E8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL­60, was treated with rhMFG­E8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin­8 (IL­8) as the chemoattractant. Surface CXCR2 and intracellular G protein­coupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogen­activated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFG­E8 resulted in a significant inhibition of dHL­60 cell migration in a dose­dependent manner. There was a 46% decrease in CXCR2 expression in the rhMFG­E8­treated dHL­60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL­60 cells, treatment with rhMFG­E8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 10­30 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL­60 cell migration which was significantly inhibited treatment with rhMFG­E8. Furthermore, blocking the MFG­E8 receptors, αvß3/αvß5­integrins, by anti­αv­integrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFG­E8­induced inhibition of dHL­60 cell migration. Finally, treatment of the dHL­60 cells with SB203580 and PD98059 neutralized the rhMFG­E8­induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFG­E8 through which it inhibits neutrophil migration through αvß3-integrin-dependent MAP kinase activation.

A novel activating, germline JAK2 mutation, JAK2R564Q, causes familial essential thrombocytosis.

Along with the most common mutation, JAK2V617F, several other acquired JAK2 mutations have now been shown to contribute to the pathogenesis of myeloproliferative neoplasms (MPNs). However, here we describe for the first time a germline mutation that leads to familial thrombocytosis that involves a residue other than Val617. The novel mutation JAK2R564Q, identified in a family with autosomal dominant essential thrombocythemia, increased cell growth resulting from suppression of apoptosis in Ba/F3-MPL cells. Although JAK2R564Q and JAK2V617F have similar levels of increased kinase activity, the growth-promoting effects of JAK2R564Q are much milder than those of JAK2V617F because of at least 2 counterregulatory mechanisms. Whereas JAK2V617F can escape regulation by the suppressor of cytokine signaling 3 and p27/Kip1, JAK2R564Q-expressing cells cannot. Moreover, JAK2R564Q-expressing cells are much more sensitive to the JAK inhibitor, ruxolitinib, than JAK2V617F-expressers, suggesting that lower doses of this drug may be effective in treating patients with MPNs associated with alternative JAK2 mutations, allowing many undesirable adverse effects to be avoided. This work provides a greater understanding of the cellular effects of a non-JAK2V617F, MPN-associated JAK2 mutation; provides insights into new treatment strategies for such patients; and describes the first case of familial thrombosis caused by a JAK2 residue other than Val617.