Kazimierz Węglarczyk

Tumour-derived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes

This study was designed to determine the characteristics of tumour cell-derived microvesicles (TMV) and their interactions with human monocytes. TMV were shed spontaneously by three different human cancer cell lines but their release was significantly increased upon activation of the cells with phorbol 12-myristate 13-acetate (PMA). TMV showed the presence of several surface determinants of tumour cells, e.g. HLA class I, CD29, CD44v7/8, CD51, chemokine receptors (CCR6, CX3CR1), extracellular matrix metalloproteinase inducer (EMMPRIN), epithelial cell adhesion molecule (EpCAM), but their level of expression differed from that on cells they originated from. TMV also carried mRNA for growth factors: vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), interleukin-8 (IL-8) and surface determinants (CD44H). TMV were localized at the monocytes surface following their short exposure to TMV, while at later times intracellularly. TMV transferred CCR6 and CD44v7/8 to monocytes, exerted antiapoptotic effect on monocytes and activated AKT kinase (Protein Kinase B). Thus, TMV interact with monocytes, alter their immunophenotype and biological activity. This implicates the novel mechanism by which tumour infiltrating macrophages may be affected by tumour cells not only by a direct cell to cell contact, soluble factors but also by TMV.

Expansion and differentiation of CD14+CD16− and CD14++CD16+ human monocyte subsets from cord blood CD34+ hematopoietic progenitors

To determine whether monocytes can be generated from CD34+ hematopoietic progenitors in large numbers, cord blood CD34+ cells were first expanded for 3–10 days in X‐VIVO 10 medium supplemented with FCS, stem cell factor (SCF), thrombopoietin (TPO), and Flt‐3 Ligand (Flt‐3L), and then differentiated in IMDM medium supplemented with FCS, SCF, Flt‐3L, IL‐3 and M‐CSF for 7–14 days. These two step cultures resulted in up to a 600‐fold mean increase of total CD14+ cells. Using this approach, two subpopulations of monocytes were obtained: CD14+CD16− and CD14++CD16+ occurring at 2:1 ratio. 1.25(OH)2 Vitamin D3 added to the differentiation medium altered this ratio by decreasing proportion of CD14++CD16+ monocytes. In comparison to CD14+CD16−, the CD14++CD16+ cells showed different morphology and an enhanced expression of CD11b, CD33, CD40, CD64, CD86, CD163, HLA‐DR, and CCR5. Both subpopulations secreted TNF and IL‐12p40 but little or no IL‐10. CD14++CD16+ monocytes released significantly more IL‐12p40, were better stimulators of MLR but showed less S. aureus phagocytosis. These subpopulations are clearly different from those present in the blood and may be novel monocyte subsets that represent different stages in monocyte differentiation with distinct biological function.

Caspase-8 Activation Precedes Alterations of Mitochondrial Membrane Potential during Monocyte Apoptosis Induced by Phagocytosis and Killing of Staphylococcus aureus

ABSTRACT Human peripheral blood monocytes become apoptotic following phagocytosis and killing of Staphylococcus aureus. Although this type of monocyte apoptosis is known to be initiated by Fas-Fas ligand (FasL) interactions, the downstream signaling pathway has not been determined. In this work the involvement of mitochondria and the kinetics of caspase-8 and caspase-3 activation after phagocytosis of S. aureus were studied. Caspase-8 activity was measured in cell lysates by using the fluorogenic substrate Ac-IETD-AFC. Active caspase-3 levels and mitochondrial membrane potential (Δψm) were measured in whole cells by flow cytometry using monoclonal antibodies reacting with activated caspase-3 and chloromethyl-X-rosamine, respectively. The results show that caspase-8 was activated shortly after phagocytosis of bacteria. Caspase-8 activation was followed by progressive disruption of Δψm, which is associated with the production of reactive oxygen intermediates. The irreversible caspase-8 inhibitor zIETD-FMK prevented the disruption of Δψm and the release of cytochrome c from S. aureus-exposed monocytes. Caspase-3 activation occurred following disruption of Δψm. These results strongly suggest that apoptosis of monocytes that have phagocytosed and killed S. aureus is driven by the Fas-FasL-initiated pathway, which is typical for type II cells.

Modulation of monocyte–tumour cell interactions by Mycobacterium vaccae

Immunotherapy with Mycobacterium vaccae as an adjuvant to chemotherapy has recently been applied to treatment of patients with cancer. One of the mechanisms of antitumour activity of Mycobacterium bovis bacillus Calmette-Guérin (BCG), the prototype immunomodulator, is associated with activation of monocytes/macrophages. These studies were undertaken to determine how M. vaccae affects monocyte–tumour cell interactions and, in particular, whether it can prevent or reverse deactivation of monocytes that occurrs following their contact with tumour cells during coculture in vitro. Deactivation is characterised by the impaired ability of monocytes to produce tumour necrosis factor α (TNF-α), interleukin 12 (IL-12), and enhanced IL-10 secretion following their restimulation with tumour cells. To see whether deactivation of monocytes can be either prevented or reversed, three different strains of M. vaccae—B 3805, MB 3683, and SN 920—and BCG were used to stimulate monocytes before or after exposure to tumour cells. Pretreatment of monocytes with M. vaccae MB 3683, SN 920 and BCG before coculture resulted in increased TNF-α and decreased IL-10 production. All strains of M. vaccae and BCG used for treatment of deactivated monocytes enhanced depressed TNF-α secretion. Strain SN 920 and BCG increased IL-12 release but only BCG treatment inhibited an enhanced IL-10 production by deactivated monocytes. Thus, although some strains of M. vaccae may either prevent or reverse tumour-induced monocyte deactivation, none of them appears to be more effective than BCG.

Hyaluronan carried by tumor-derived microvesicles induces IL-10 production in classical (CD14(++)CD16(-)) monocytes via PI3K/Akt/mTOR-dependent signalling pathway.

Tumor-derived microvesicles (TMV) can mimic effects of tumor cells leading to an increased anti-inflammatory cytokine production, such as interleukin 10 (IL-10), by tumor-infiltrating monocytes and macrophages. Yet, the mechanism of IL-10 induction by TMV in monocytes remains unclear. The co-incubation of TMV derived from the human pancreas carcinoma cell line (HPC-4) with human monocytes resulted in a nearly 30-fold increase in IL-10 protein production. This effect operates at the level of transcription since monocytes transduced with an adenovirus containing IL-10-promoter luciferase reporter gene showed a 5-fold induction of luciferase activity after treatment with TMV. Since tumor cells can express hyaluronan (HA), which participates in tumor invasion and metastases, we have tested its effect on IL-10 expression. We showed that HA at the concentration of 100μg/ml induces IL-10 protein expression and the IL-10 promoter activation in monocytes. Moreover, hyaluronidase treatment of TMV reduced IL-10 protein production by 50% and promoter activity by 40%. Inhibitors of the PI3K/Akt/mTOR pathway reduced both, TMV-induced IL-10 promoter activity and protein production, and the same was observed in monocytes when stimulated by HPC-4 cells or HA. Inhibition of PI3K activity down-regulated phosphorylation of the Akt and (to a lesser extent) mTOR proteins in monocytes following TMV or HA stimulation. When comparing monocyte subsets, TMV induced IL-10 protein and mRNA synthesis only in classical CD14++CD16- but not in CD16-positive monocytes. Our data show that TMV induce IL-10 synthesis in human classical monocytes via HA, which, in turn, activates the PI3K/Akt/mTOR pathway.

Biological activity of dendritic cells generated from cord blood CD34+ hematopoietic progenitors in IL-7- and IL-13-conditioned cultures

IntroductionDendritic cells (DCs) are required for initiation of the immune response and may therefore be used for the production of cancer vaccines. As mature DCs (mDCs) are the most potent antigen-presenting cells, there is increasing interest in generating them ex vivo. The present study was designed to obtain mDCs from CD34+ hematopoietic progenitors by culturing them in different media.Materials and MethodsCord blood CD34+ hematopoietic progenitors were expanded for 7 days in FST medium ontaining fms-related tyrosine kinase 3 ligand (Flt3-L), stem cell factor (SCF), and thrombopoietin (TPO). Then the cells were divided into three parts and cultured for 21 days in different media: FST medium or FST enriched in interleukin (IL)-3 (FST3 medium) or supplemented with IL-7 and IL-13 (FST713 medium). At the end of culture part of the cells was harvested, counted, and analyzed while the other part was matured with proinflammatory cytokines for 2 days. The cellsȉ phenotypes, ability to induce proliferation of allogeneic lymphocytes in the mixed lymphocyte reaction (allo-MLR), chemotaxis, phagocytosis, and O2- production were determined.ResultsThe average fold increase of DCs at the end of culture in FST medium was 127, in FST3 1043, and in FST713 71. In comparison with the other media, FST713 medium supported the generation of mDCs that were characterized by higher expression of CD83, costimulatory molecules, and HLA-DR, enhanced ability to induce allo-MLR and migration to macrophage inflammatory protein (MIP) 3β poor phagocytosis, and O2- production.ConclusionsThis study indicates that FST713 medium allows the generation of limited numbers of more mature DCs, while FST3 medium leads to the production of immature DCs in high numbers.

Elevated level of some chemokines in plasma of gastric cancer patients

Introduction Gastric cancer is one of the most common cancer-related causes of death. This is mainly due to the lack of good noninvasive method/biomarkers suitable for early-tumour diagnosis and planning of further therapy modalities. Chemokines play an important role in cancer progression and metastasis formation. In gastric cancer patients, clinical relevance of CXCL12 and CCL5 level has been postulated. Aim of the study Efforts were undertaken to examine whether expanded chemokine range may be relevant for evaluation of preoperative staging of gastric cancer patients. Material and methods Plasma from 66 gastric cancer patients and 11 healthy controls was obtained, and CCL2, CCL3, CCL4, CCL5, CXCL8, CXCL9, and CXCL10 levels were determined by flow cytometry FlexSet system. Results In gastric cancer patients’ plasma an increased level of CCL2, CCL4, CCL5, CXCL8, CXCL9, and CXCL10 was observed. In the case of CCL2, CXCL9, and CXCL10, the chemokine levels correlated with advanced (III and IV in TNM classification) disease stage. In the case of CCL4, CCL5, and CXCL8, elevated levels were observed in all cancer patients in comparison to healthy donors. Conclusions The accuracy of preoperative diagnosis in gastric cancer may include the monitoring of a wide range of chemokines in patients’ plasma. Increased levels of chemokines may warn that the disease is more advanced than conventional diagnostic procedures suggest.

Induction of monocyte antitumor response by human cancer cells transduced with TNF-GFP fusion gene: possible implications for immunotherapy of cancer.

This study was undertaken to determine how human pancreatic cancer (HPC-4) cells transduced with the TNF-GFP fusion gene (TLG) alter the antitumor response of human monocytes in vitro and whether they could act as an antitumor vaccine. In our model, HPC-4 cells were transduced with retroviral vector harboring TLG gene and designated as HPC-4(TLG). The TLG protein expression was confirmed by Western blot and flow cytometry analysis. Monocytes were co-cultured with transduced and control HPC-4 cells. The secretion of TNF, IL-10 and IL-12 was measured by ELISA. The cytotoxicity of monocytes against HPC-4 cells was determined by MTT test. The results show that the HPC-4(TLG) cells expressed membrane-bound, intracellular and secretory TLG protein. When cultured with HPC-4(TLG) cells, monocytes released a higher amount of TNF, but IL-10 and IL-12 secretion was inhibited. The pre-exposure of monocytes to HPC-4(TLG), but not to HPC-4, cells did not decrease TNF nor increase IL-10 production, thus not leading to monocyte deactivation. Also, the antitumor cytotoxicity of monocytes stimulated with HPC-4(TLG) was not downregulated, which occurred when non-transduced HPC-4 cells were used. In conclusion, compared to parental HPC-4 cells, TLG gene transduced HPC-4 cells induced stronger antitumor response of monocytes in vitro and prevented deactivation of monocytes.

Characterization of human gastric adenocarcinoma cell lines established from peritoneal ascites

The three cell lines, designated as gastric cancer (GC)1401, GC1415 and GC1436 were derived from peritoneal effusions from patients with gastric adenocarcinoma. Cell lines were established in tissue culture and in immunodeficient, non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. All cell lines were cultured in Dulbeccos modified Eagles medium supplemented with 5% fetal bovine serum. These cell lines were grown as an adherent monolayer with doubling time ranging between 25 h (GC1436 cell line) and 30–34 h (GC1401 and GC1415, respectively). All cells showed morphological features of epithelial-like cells, forming sheets of polygonal cells. Chromosomal analysis showed that the modal numbers ranged from 52 (GC1401), 51–56 (GC1415) and 106 (GC1436). High heterogeneity, resulting from several structural and numerical chromosomal abnormalities were evident in all cell lines. The surface marker expression suggested a tumor origin of the cells, and indicated the intestinal phenotype of a GC (CD10+, MUC1). All three cell lines were tumorigenic but not metastatic, in vivo, in NOD/SCID mice. The lack of metastatic potential was suggested by the lack of aldehyde dehydrogenase 1A1 activity. In conclusion, these newly established GC cell lines widen the feasibility of the functional studies on biology of GC as well as drug testing for potential therapeutic purposes.