Grzegorz Skibinski

Immunosuppression by human seminal plasma-extracellular organelles (prostasomes) modulate activity of phagocytic cells

PROBLEM: Prostasomes are trilamellar to multilamellar vesicles produced by the acinar cells of the human prostate and are present in appreciable amounts in normal human semen. The aim of this work was to study the effect of prostasomes on human polymorphonuclear cell and monocyte function.

The role of hepatocyte growth factor and its receptor c‐met in interactions between lymphocytes and stromal cells in secondary human lymphoid organs

Secondary lymphoid tissue consists of two major populations of cells: lymphoid cells and stromal cells. It is generally accepted that these two cell populations influence each other however, factors mediating these processes are poorly understood. In this paper we characterize one of the possible means of communication between stroma and lymphocytes namely through hepatocyte growth factor/c‐met receptor interactions. Hepatocyte growth factor (HGF) is a pleiotropic factor that is mainly produced by mesenchymal cells and acts on cells of epithelial origin which express the HGF receptor c‐met. Here we demonstrate that biologically active HGF is constitutively produced by fibroblast‐like stromal cells from human lymphoid tissues. HGF secretion from stromal cells was increased by direct contact with activated T cells. This increase was abrogated when activated T cells were separated physically from stromal cells. Using neutralizing antibody or cytokine inhibitors we provide evidence that enhancement of HGF production was due to additive effects of T‐cell membrane‐associated interleukin‐1 (IL‐1) and CD40 ligand. Finally, we also show that B lymphocytes activated with CD40L/anti‐µ or phorbol 12‐myristate 13‐acetate (PMA) express c‐met receptor. Co‐culture of activated B cells with stromal cells from spleen leads to enhanced production of immunoglobulins. This can be partially inhibited by introduction of anti‐HGF neutralizing antibodies to the culture system. Substitution of stromal cells with recombinant HGF did not produce enhancement of immunoglobulin secretion. On the other hand stimulation of c‐met receptor with HGF leads to enhanced integrin‐mediated adhesion of activated B cells to vascular cell adhesion molecule (VCAM‐1) and fibronectin. On the basis of the above experiments we conclude that HGF production by fibroblast‐like stromal cells can be modulated by activated T cells, thus providing signals for the regulation of adhesion of c‐met expressing B cells to extracellular matrix proteins. In this way HGF may indirectly influence immunoglobulin secretion by B cells.

ENHANCEMENT OF TERMINAL B LYMPHOCYTE DIFFERENTIATION IN VITRO BY FIBROBLAST-LIKE STROMAL CELLS FROM HUMAN SPLEEN

Stromal elements are major components of lymphoid tissues contributing to both tissue architecture and function. In this study we report on the phenotype and function of fibroblast‐like stromal cells obtained from human spleen. These cells express high levels of CD44 and ICAM‐1 and moderate levels of VLA‐4, VCAM, CD40 and CD21. They fail to express endothelial, epithelial, lymphocyte and monocyte/macrophage markers. We show that these cells interact with B cell blasts induced in vitro by anti‐CD40 and anti‐μ  stimulation. As a result of these interactions both IL‐6 and IgG secretion into culture medium is increased. The enhanced secretion of IgG is partly inhibited by abolishing B cell blast‐stromal cell contact or by anti‐IL‐6, anti‐VCAM or anti‐CD49d antibodies. Our studies also suggest that the ability of stromal cells to promote B cell survival is most likely the underlying mechanism of the enhanced immunoglobulin secretion. Comparison of stromal cells from different lymphoid and non‐lymphoid organs revealed that bone marrow‐ and spleen‐derived stromal cells are the most effective in promoting B cell blast differentiation.

Relative immunosuppressive activity of human seminal prostaglandins

Human seminal plasma contains uniquely high concentrations of prostaglandins of the E series which are believed to contribute to its immunosuppressive effects in vivo. In order to obtain further insight into their activity we have compared the immunosuppressive properties in vitro of PGE1, PGE2 and 19-OH PGE using three immunological systems known to be modulated by prostaglandins, namely, mitogen induced lymphocyte proliferation, IL-2 and transferrin receptor expression and NK-cell mediated cytotoxicity. These studies revealed that PGE1 and PGE2 exerted a greater immunosuppressive effect than 19-OH PGE, but considerably higher levels of 19-OH PGE in semen might contribute the majority of immunosuppressive activity in vivo. Our studies also show that the lower stability of 19-OH PGE in culture media may be responsible for its lower immunosuppressive effect observed in vitro.

Immunomodulatory effects of extracellular secretory vesicles isolated from bovine semen

We demonstrate that extracellular secretory vesicles isolated from bovine seminal plasma have immunomodulatory properties. They inhibit mitogen induced proliferation of bovine and human peripheral blood lymphocytes in a dose dependent fashion. They also inhibit phagocytosis of latex particles by bovine neutrophils. Phagocytosis of opsonised Staphylococcus aureus however was not affected. Furthermore phorbol ester and chemotactic peptide induced superoxide production was decreased especially when a suboptimal dose of stimulants was used. We suggest that extracellular secretory vesicles may preserve sperm survival in the female reproductive tract.

The effects of human seminal plasma and PGE2 on mitogen induced proliferation and cytokine production of human splenic lymphocytes and peripheral blood mononuclear cells

The effects of human seminal plasma (HSP) and prostaglandin E2 (PGE2) on the proliferative responses of human splenic lymphocytes and peripheral blood mononuclear cells (PBMCs) to phytohaemagglutinin (PHA), anti-CD3 and anti-CD3 plus anti-CD28 mAb have been studied. Th1 and Th2 cytokines were also measured in the supernatants of selected cultures. Both HSP and PGE2 reproducibly inhibit the proliferative response to PHA and anti-CD3 mAb in a dose dependent manner. These effects were observed with both fresh and frozen human PBMCs and splenic lymphocytes. HSP and PGE2 however were less effective in inhibiting the co-stimulatory response induced by anti-CD3 plus anti-CD28 mAb. In addition, the HSP and PGE2 treatment used inhibited the production of the Th1 cytokines IL-2 and IFNg but had a differential modulatory effect on Th2 cytokine production, namely enhancing the production of IL-6 whilst simultaneously impairing the synthesis of IL-4 and IL-10.

Organ culture of human lymphoid tissue I. Characteristics of the system

The major aim of three-dimensional tissue culture is to preserve the natural architecture of the tissue and thereby allow the cells to retain their original functions during in vitro cultivation. Here we describe a method for the rapid preparation of three-dimensional tissue explants from human lymphoid organs. The precision-cut tissue slices are of uniform size and thickness and can be cryopreserved and stored in liquid nitrogen without substantial loss of viability or functionality of the cells. Upon in vitro culture, cells within the explants survived as well as their counterparts cultured in single cell suspension. However, spontaneous immunoglobulin (Ig) production in explants started more promptly and often reached considerably higher levels than that in suspension cultures run in parallel. Lymphocytes within the slices could be activated by polyclonal stimuli such as PHA, as shown by the upregulation of the activation markers CD23 and CD25 on B and T cells, respectively. However, approximately five-fold higher concentrations of mitogen than those used for suspension cultures were needed. Taken together, the system presented here constitutes a potent tool for the investigation of the complex interactions leading to activation and differentiation of B and T cells in lymphoid organs.

Tonsil Stromal-Cell Lines Expressing FDC-Like Properties: Isolation, Characterization, and Interaction with B Lymphocytes

The microenvironment of secondary lymphoid organs consists of two major populations of cells, the lymphoid cells and a population of stromal cells that contribute to both tissue architecture and function. Interactions of both populations are essential for the development and control of humoral immune responses. In this study, stromal-cell preparations were obtained by a multistage process. This involved culturing 300-400-μm slices of human tonsil for 6-8 days at 25°C, trypsin digestion of the residual explant, followed by CD45-positive-cell depletion using magnetic beads, and a final period of culture for 4 days to remove remaining nonadherent cells. Phenotypifig with a panel of monoclonal antibodies revealed that the cells express HLA-DR, CD54 (ICAM-1), CD44, but no CD45 nor a range of other markers for epithelial and endothelial cells. Immunoassays of supernatants from stromal cells revealed that IL-6 was produced constitutively, and its production was increased by treatment with TNF-α and IFN-γ. In contrast IL-1, IL-2, IL-4, IL-7, IL-8, IL-10, IL-12, TNF-α, and IFNγ were not produced. Functional tests showed that these cells express follicular dendritic cell-like properties. Coculturing of tonsilar B cells with stromal cells resulted in enhanced proliferation and also led to increased production of immunoglobulins and IL-6, suggesting crucial signaling between these populations.