During early pregnancy, uterine mucosa decidualization is accompanied by a drastic enrichment of CD56 high CD16 ؊ natural killer (NK) cells. Decidual NK (dNK) cells differ from peripheral blood NK (pbNK) cells in several ways, but their origin is still unclear. Our results demonstrate that chemokines present in the uterus can support pbNK cell migration through human endothelial and stromal decidual cells. Notably, we observed that pregnant women's pbNK cells are endowed with higher migratory ability compared with nonpregnant women's or male donors' pbNK cells. Moreover, NK cell migration through decidual stromal cells was increased when progesterone-cultured stromal cells were used as substrate, and this correlated with the ability of progesterone to up-regulate stromal cell chemokine expression. Furthermore, we demonstrate that dNK cells migrate through stromal cells using a distinct pattern of chemokines. Finally, we found that pbNK cells acquire a chemokine receptor pattern similar to that of dNK cells when they contact decidual stromal cells. Collectively these results strongly suggest that pbNK cell recruitment to the uterus contributes to the accumulation of NK cells during early pregnancy; that progesterone plays a crucial role in this event; and that pbNK cells undergo reprogramming of their chemokine receptor profile once exposed to uterine microenvironment. IntroductionNatural killer (NK) cells represent a distinct population of circulating and tissue-resident lymphocytes that play an important role in the early phases of immune responses against microbial pathogens by exhibiting cytotoxic functions and secreting a number of cytokines and chemokines. NK cells develop from a lymphoid precursor resident in the bone marrow (BM), considered the main site of NK cell generation, however, the existence of a pathway of NK cell development in the thymus has been recently suggested and evidence also indicates that final maturation of NK cell precursors can occur in the periphery. [1][2][3] During development and activation, NK cells acquire a multiple cell surface receptor system including both activating and inhibitory receptors that finely control their functional activation. 4 Some of these receptors are oligoclonally distributed and/or are expressed at different densities on circulating NK cells. Based on cell surface density of these receptors, phenotypically distinct peripheral blood NK (pbNK) cell populations have been identified and suggested to represent specialized subsets capable of performing different functions and endowed with distinct migratory properties. 5 Mature NK cells circulate mainly in the peripheral blood, but are also present in several lymphoid and nonlymphoid organs such as spleen, lymph nodes, tonsils, liver, lungs, intestine, and uterus. 1,[6][7][8] Interestingly, NK cells are the most abundant class of lymphocytes found in the mucosal tissues of maternal uterus where their number reaches 70% to 80% of the total leukocytes in the first trimester of pregnancy, then start to decline,...

Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the in vitro studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate in vitro, as a suitable model to follow the release of inflammatory and repair mediators in response to TNFα or IL-1β. Different treatment conditions (presence or absence of serum) and differentiation stimuli (increase in cell density as a function of time in culture and elevation of extracellular calcium) were considered. ELISA and Multiplex measurement technologies were used to monitor the production of cytokines and chemokines. Taken together, the results highlight that Ca2+ concentration in the medium, cell density, and presence of serum influences at different levels the release of proinflammatory mediators by HaCaT cells. Moreover, HaCaT cells maintained in low Ca2+ medium and 80% confluent are similar to normal keratinocytes in terms of cytokine production suggesting that HaCaT cells may be a useful model to investigate anti-inflammatory interventions/therapies on skin diseases.

Our data indicate that chemerin is up-regulated during decidualization and might contribute to NK cell accumulation and vascular remodeling during early pregnancy.

Uterine natural killer (uNK) cells represent the predominant lymphocytes in the uterus during early pregnancy and in the secretory phase of the menstrual cycle. They are CD56(high)CD16(-) and have low cytotoxicity, but constitutively secrete a number of cytokines, chemokines and angiogenic molecules. uNK cells differ from CD56(high) blood NK cells in several ways, including the killer cell immunoglobulin-like receptor repertoire and expression of some genes induced by hormone environment. uNK cells may arise by in-utero proliferation and differentiation of NK cell progenitors under the control of the sex steroid hormones and/or cytokines, such as interleukin-15, and/or be recruited from CD56(+) blood NK cells that would undergo tissue-specific differentiation in the uterine microenvironment. There is evidence showing that uNK cells display a different pattern of chemokine receptors and adhesion molecules, thus leading to a different migratory response. It has not yet been fully defined which uNK cell function(s) are critical for successful pregnancy. The close encirclement of spiral arteries by NK cells, together with their ability to produce angiogenic factors, suggests that they might influence mucosal vascularization. Their proximity to the extravillous trophoblast supports the idea that uNK cells could recognize these cells as fetal, and regulate their invasion during placentation.

IntroductionThe Wiskott-Aldrich syndrome protein (WASp) is a multidomain member of the WASp/suppressor of cAMP receptor/WASP family verprolin-homologous protein family whose expression is restricted to hematopoietic cells. WASp has been identified as one of the main regulators of actin cytoskeletal dynamics. 1,2 WASp was initially discovered as the product of the gene whose mutations are responsible for the Wiskott-Aldrich syndrome (WAS), a rare X-linked recessive primary immunodeficiency characterized by eczema, thrombocytopenia, and impaired cellular and humoral immunity. [3][4][5][6] This syndrome is highly heterogeneous in terms of clinical severity with some cases showing an attenuated clinical and immunologic phenotype referred to as X-linked thrombocytopenia (XLT). Most of the patients with XLT have missense mutations in exons 1 and 2 of the WASP gene, leading to decreased but detectable levels of protein expression. In contrast, WASP mutations that lead to total absence of WASp are usually associated with a more severe clinical phenotype.In resting cells, WASp is found in a closed, autoinhibited form in which the guanosine triphosphate (GTPase) binding domain is bound to the carboxyl-terminal verprolin homology, cofilin homology, acidic region. 1,2,7 After cell activation, WASp interacts with the GTP-bound form of the small Rho GTPase Cdc42 by its GTPase binding domain; this association results in a conformational change that allows WASp interaction with the Arp2/3 complex, thus initiating actin polymerization. WASp also contains a proline-rich region that is responsible for its association with several tyrosine kinases and adaptor proteins. Moreover, the N-terminal located at ENA-Vasp homology 1 domain binds to the WASp-interacting protein, known to modulate WASp functions. The ability of WASp to interact with and activate the Arp2/3 complex is regulated in a cooperative manner by the GTP-bound form of Cdc42 and the phosphatidylinositol 4,5 bisphosphate. Moreover, it has been reported that WASp functional activation is also enhanced by its tyrosine phosphorylation after ligation of immune or adhesion receptors. [8][9][10][11] Because of its ability to induce cytoskeletal remodeling, WASp has been implicated in the regulation of many cellular functions, including T-cell activation and proliferation, phagocytosis, cell migration, and chemotaxis. 1,2 Moreover, we and others have reported that patients with WAS or XLT phenotype are impaired in both natural and CD16-mediated natural killer (NK)-cell cytotoxicity as a result of disturbed cytoskeletal reorganization. 12,13 NK cells are a CD3 Ϫ CD16 ϩ CD56 ϩ lymphocyte subpopulation that plays an important role in the early phase of immune responses against viruses, parasites, and microbial pathogens by exhibiting cytotoxic functions and secreting a number of cytokines and chemokines. 14,15 NK cells are mainly found in the peripheral blood, but they are also present in several lymphoid and nonlymphoid organs such as spleen, lymph nodes, tonsils, liver, lungs, i...