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.

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