Ectodomain shedding of interleukin-2 receptor beta and generation of an intracellular functional fragment.

Interleukin-2 (IL-2) regulates different functions of various lymphoid cell subsets. These are mediated by its binding to the IL-2 receptor (IL-2R) composed of three subunits (IL2-Ralpha, -beta, and -gamma(c)). IL-2Rbeta is responsible for the activation of several signaling pathways. Ectodomain shedding of membrane receptors is thought to be an important mechanism for down-regulation of cell surface receptor abundance but is also emerging as a mechanism that cell membrane-associated molecules require for proper action in vivo. Here, we demonstrate that IL-2Rbeta is cleaved in cell lines of different origin, including T cells, generating an intracellular 37-kDa fragment (37beta ic) that comprises the full intracellular C-terminal and transmembrane domains. Ectodomain shedding of IL-2Rbeta decreases in a mutant deleted of the juxtamembrane region, where cleavage is predicted to occur, and is inhibited by tissue inhibitor of metalloproteases-3. 37Beta ic is tyrosine-phosphorylated and associates with STAT-5, a canonic signal transducer of IL-2R. Finally, lymphoid cell transfection with a truncated form of IL-2Rbeta mimicking 37beta ic increases their proliferation. These data indicate that IL-2Rbeta is subject to ectodomain shedding generating an intracellular fragment biologically functional, because (i) it is phosphorylated, (ii) it associates with STAT5A, and (iii) it increases cell proliferation.

Prolactin in eyes of patients with retinopathy of prematurity: implications for vascular regression.

PURPOSE: Disruption of the anti-angiogenic environment of the retina leads to neovascular eye diseases, including retinopathy of prematurity (ROP). Prolactin (PRL), the hormone originally associated with milk secretion, is proteolytically processed to 16K-PRL, a fragment with potent antiangiogenic, proapoptotic effects. Whether 16K-PRL is produced in eyes of patients with ROP and promotes the regression of intraocular blood vessels associated with this disease was investigated. METHODS: PRL was quantified in the aqueous humor, subretinal fluid, and serum from patients with stage 5 ROP and in patients with non-neovascular eye disorders. Intraocular expression of PRL was evaluated by RT-PCR, in situ hybridization, and Western blot analysis. AntiPRL antibodies were injected intravitreously in neonatal rats, and apoptosis of hyaloid vessels determined by TUNEL and ELISA. RESULTS: PRL was elevated in ocular fluids and serum from ROP patients. There was no correlation between PRL in ocular fluids and its level in serum, whereas PRL in aqueous humor and subretinal fluid were significantly correlated. PRL mRNA was expressed in blood vessels and leukocytes within retrolental fibrovascular membranes of ROP patients, and these membranes contained a 16 kDa immunoreactive PRL. The 16K-PRL isoform was more concentrated in subretinal fluid than in serum and was generated from PRL by subretinal fluid proteases. Intravitreous injection of neutralizing antiPRL antibodies inhibited the apoptosis of hyaloid vessels in neonatal rats. CONCLUSIONS: 16K-PRL derived from PRL internalized from the circulation or synthesized intraocularly can stimulate apoptosis-induced vascular regression and contribute to the development and progression of ROP.

Helper T cell subpopulations from women are more susceptible to the toxic effect of sodium arsenite in vitro.

Arsenic is known to produce inhibition as well as induction of proliferative responses in animal and human cells depending on the doses. Despite the amount of information on the immunotoxic effects of arsenic exposure in different animal models, little is known in humans. Arsenic susceptibility of lymphocyte subpopulations (T helper (Th), CD4+; T cytotoxic (Tc), CD8+) and whether arsenic effects are gender related are still to be determined. This work evaluated the in vitro toxicity of sodium arsenite on human T lymphocyte subpopulations from men and women. Peripheral blood mononuclear cells (PBMC) obtained from healthy young men and women were treated with sodium arsenite (0.01, 0.1, and 1 microM). We assessed cell viability, cell proliferation, and the proportion of Th and Tc cells after 48 or 72h of arsenic exposure in resting and phytohemagglutinin M (PHA)-activated PBMC. We observed that sodium arsenite at 1 microM was more toxic for Th than for Tc cells in PBMC from women. Besides, T lymphocytes from women were more affected by the cell proliferation inhibition induced by arsenic, suggesting that women could be more susceptible to the toxic and immunotoxic effects caused by arsenic exposure.

Prolactin stimulates integrin-mediated adhesion of circulating mononuclear cells to endothelial cells.

Attachment of leukocytes to endothelial cells is an essential step for the extravasation and recruitment of cells at sites of inflammation. The pituitary hormone prolactin (PRL) is involved in the inflammatory process. Here, we show that treatment with PRL of human peripheral blood mononuclear cells (PBMC) stimulates their adhesion to human umbilical vein endothelial cells (HUVEC) activated by interleukin-1beta. Stimulation of adhesion by PRL is mediated via integrins leukocyte functional antigen-1 (LFA-1) and very late antigen-4 (VLA-4), because immunoneutralization of both integrins prevents PRL action. Also, PRL promotes the adhesion of PBMC to immobilized intercellular adhesion molecule-1 and fibronectin, ligands for LFA-1 and VLA-4, respectively. Stimulation of integrin-mediated cell adhesion by PRL may involve the activation of chemokine receptors, because PRL upregulates the expression of the G-protein-coupled chemokine receptor CXCR3 in PBMC, and pertussis toxin, a specific G-protein inhibitor, blocks PRL stimulation of PBMC adhesion to HUVEC. In addition, PRL stimulates tyrosine phosphorylation pathways leading to leukocyte adhesion. PRL triggered the tyrosine phosphorylation of Janus kinase-2, of signal transducer and activator of transcription-3 and 5, and of the focal adhesion protein paxillin. Furthermore, genistein, a tyrosine kinase inhibitor, blocked PRL-stimulated adhesion of PBMC and Jurkat T-cells to HUVEC. These results suggest that PRL promotes integrin-mediated leukocyte adhesion to endothelial cells via chemokine receptors and tyrosine phosphorylation signaling pathways.