Intrahepatic immune cells (IHIC) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. In the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of IHIC. These cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and DNase. The mechanical disruption yielded viable IHIC in considerably greater numbers than those obtained following enzymatic digestion. The IHIC isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which B, T, natural killer (NK), NK T cells, granulocytes and macrophages were the major populations (constituting 37·5%, 16·5%, 12·1%, 7·9%, 7·9% and 7·5% of the total number of cells recovered respectively). The IHIC obtained following enzymatic digestion contained markedly lower numbers of NK T cells (1·8%). The B, T and NK T cells among IHIC isolated employing mechanical disruption were found to be immunocompetent, i.e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin A and α-galactosylceramide respectively) and produced immunoglobulin M and interferon-γ. Thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent IHIC for various types of investigation.

Polymorphisms of the P2X7 receptor have been associated with increased risk of fractures in postmenopausal women. Although both osteoblasts and osteoclasts express P2X7 receptors, their function in osteogenesis remains controversial. Here, we investigated the role of the P2X7 receptor on osteogenic differentiation and mineralization of bone marrow mesenchymal stem cell (BMSC) cultures from postmenopausal women (age 71 ± 3 yr, n =18). We focused on the mechanisms related to intracellular [Ca ²⁺ ] _i oscillations and plasma membrane‐dynamics. ATP, and the P2X7 agonist BzATP (100 μM), increased [Ca ²⁺ ] _i in parallel to the formation of membrane pores permeable to TO‐PRO‐3 dye uptake. ATP and BzATP elicited reversible membrane blebs (zeiosis) in 38 ± 1 and 70 ± 1% of the cells, respectively. P2X7‐induced zeiosis was Ca ²⁺ independent, but involved phospholipase C, protein kinase C, and Rho‐kinase activation. BzATP (100 μM) progressively increased the expression of Runx‐2 and Osterix transcription factors by 452 and 226% (at d 21), respectively, alkaline phosphatase activity by 88% (at d 28), and mineralization by 329% (at d 43) of BMSC cultures in a Rho‐kinase‐dependent manner. In summary, reversible plasma membrane zeiosis involving cytoskeleton rearrangements due to activation of the P2X7‐Rho‐kinase axis promotes osteogenic differentiation and mineralization of BMSCs, thus providing new therapeutic targets for postmenopausal bone loss.—Noronha‐Matos, J. B., Coimbra, J., Sá‐e‐Sousa, A., Rocha, R., Marinhas, J., Freitas, R., Guerra‐Gomes, S., Ferreirinha, F., Costa, M. A., Correia‐de‐Sá, P., P2X7‐induced zeiosis promotes osteogenic differentiation and mineralization of postmenopausal bone marrow‐derived mesenchymal stem cells FASEB J. 28, 5208–5222 (2014). www.fasebj.org