Differential effects of interleukin-15 (IL-15) and IL-2 on human neutrophils: modulation of phagocytosis, cytoskeleton rearrangement, gene expression, and apoptosis by IL-15.

Human neutrophils have been shown recently to express both the beta and the gamma chains of the interleukin-2 receptor (IL-2R). IL-15, a cytokine that has recently been cloned and characterized, was found to share many of the biological functions of IL-2 and is known to mediate signals through IL-2R beta and IL-2R gamma. In recent studies, we observed that IL-2 exerts few effects on various neutrophil functions, but information on IL-15-neutrophil interactions is lacking. In this study, we observed that IL-15, in contrast to IL-2, induces important morphological cell shape changes that are typical of activated neutrophils. Furthermore, phagocytosis of opsonized sheep red blood cells was significantly increased by IL-15 but not by IL-2. However, similar to IL-2, IL-15 did not modulate the oxidative burst response. Furthermore, we observed that de novo RNA synthesis is increased in neutrophils by IL-15 along with de novo protein synthesis, whereas no significant effect of IL-2 was noted. Among the different proteins that were found to be upregulated by IL-15, one was identified by microsequencing as the cytoskeletal protein actin. Finally, we found that IL-15 delays apoptosis of neutrophils more efficiently than IL-2 when evaluated by both microscopic observations and flow cytometry procedures. Furthermore, this phenomenon was dose-dependent (10 to 500 ng/mL), and, at 500 ng/mL, IL-15 delayed apoptosis as strongly as granulocyte-macrophage colony-stimulating factor. This study is the first to show that IL-15 is a significant neutrophil agonist. Moreover, in view of the differential effects of IL-15 and IL-2 on this cell type, our results support the existence of a specific IL-15R component(s) on human neutrophils.

Analysis of several endoglin mutants reveals no endogenous mature or secreted protein capable of interfering with normal endoglin function.

Hereditary hemorrhagic telangiectasia type 1 (HHT1) is associated with mutations in the ENDOGLIN gene which normally codes for a polypeptide of 653 amino acids expressed at the cell surface as a dimeric glycoprotein. To maximize the detection of potential mutant proteins, we analyzed by pulse-chase experiments the expression of large truncation mutants in endothelial cells from newborns with HHT1. A mutant truncated at residue 490 (Delta490) and the Delta517 mutant, previously suggested to act as dominant negative, were undetectable. Proteins Delta471 and Delta571 were barely detectable as transient monomers of 62 and 72 kDa. A de novo 13 bp deletion in exon 11 encoded a monomeric protein of 70 kDa (Delta557), present at low levels in activated monocytes. Six novel missense mutants and DeltaS411 were expressed only as the 80 kDa intracellular precursor of surface endoglin, suggesting impaired processing. All nine novel mutations reported failed to be expressed other than intracellularly. Several constructs of endoglin were expressed in COS-1 cells; only the full-length protein was processed to the cell surface. Recombinant Delta586, corresponding to the complete extracellular domain, was secreted as monomeric and dimeric glycosylated species. Our studies show that all HHT1 mutants analyzed, although expressed to various degrees in COS-1 cells, are either undetectable, present at low levels as transient intracellular forms, or expressed as partially glycosylated precursors in endogenous cells. These mutants do not form heterodimers with normal endoglin and do not interfere with its normal trafficking to the cell surface, further supporting the haploinsufficiency model.

Endoglin expression is reduced in normal vessels but still detectable in arteriovenous malformations of patients with hereditary hemorrhagic telangiectasia type 1.

Endoglin is predominantly expressed on endothelium and is mutated in hereditary hemorrhagic telangiectasia (HHT) type 1 (HHT1). We report the analysis of endoglin in tissues of a newborn (family 2), who died of a cerebral arteriovenous malformation (CAVM), and in a lung specimen surgically resected from a 78-year-old patient (family 5), with a pulmonary AVM (PAVM). The clinically affected father of the newborn revealed a novel mutation that was absent in his parents and was identified as a duplication of exons 3 to 8, by quantitative multiplex polymerase chain reaction. The corresponding mutant protein (116-kd monomer) and the missense mutant protein (80-kd monomer) present in family 5 were detected only as transient intracellular species and were unreactive by Western blot analysis and immunostaining. Normal endoglin (90-kd monomer) was reduced by 50% on peripheral blood-activated monocytes of the HHT1 patients. When analyzed by immunostaining and densitometry, presumed normal blood vessels of the newborn lung and brain and vessels adjacent to the adult PAVM showed a 50% reduction in the endoglin/PECAM-1 ratio. A similar ratio was observed in the CAVM and PAVM, suggesting that all blood vessels of HHT1 patients express reduced endoglin in situ and that AVMs are not attributed to a focal loss of endoglin.