Bożenna Mytar

Tumor cell-induced deactivation of human monocytes.

Although blood monocytes exhibit significant cytotoxic activity against tumor cells, the function of tumor infiltrating macrophages (TIM) is depressed in cancer patients. This study addresses the question of how the antitumor response of human monocytes, assessed by production of cytokines (tumor necrosis factor α, TNF; IL‐10; IL‐12p40) and cytotoxicity, is altered by exposure to cancer cells. Tumor cell−pre‐exposed monocytes restimulated with tumor cells showed significantly decreased production of TNF, IL‐12, increased IL‐10 (mRNA and release) and inhibition of IL‐1 receptor‐associated kinase‐1 (IRAK‐1) expression. This down‐regulation of cytokine production was selective, as the response of pre‐exposed monocytes to lipopolysaccharide (LPS) was unaffected. Treatment of tumor cell−pre‐exposed monocytes with hyaluronidase (HAase) improved their depressed production of TNF, while HAase‐treated cancer cells did not cause monocyte dysfunction. The response of hyaluronan (HA)−pre‐exposed monocytes to stimulation with tumor cells was also inhibited. Cytotoxic activity of monocytes pretreated with cancer cells was also decreased. This study shows that tumor cells selectively deactivate monocytes and suggests that tumor cell‐derived HA by blocking CD44 on monocytes inhibits their antitumor response. These observations may provide some explanation for the depressed function of TIM in human malignancy.

Cross-talk between human monocytes and cancer cells during reactive oxygen intermediates generation: The essential role of hyaluronan

Human monocytes exhibit considerable cytocidal activity against tumor (but not normal cells) associated, at least partly, with the generation of reactive oxygen intermediates (ROIs). The present study examined the role of surface determinants and hyaluronan (HA) in the induction of ROI production by human monocytes stimulated with cancer cells, as measured by luminol‐enhanced chemiluminescence (CL). The inhibitory effect of monoclonal antibodies (MAbs) indicated the engagement of CD18, CD29 and CD44 adhesion molecules. Preincubation of monocytes and tumor cells, expressing CD44 determinants, with either anti‐CD44 MAb or HA inhibited CL generation. Addition of HA to monocytes decreased the expression of CD44 and induced CL response. Supernatants from the cultures of tumor cells stimulated CL response of monocytes, an effect that was abolished by treatment of the supernatants with hyaluronidase (HAase) or by preincubation of monocytes with an anti‐CD44 MAb. These results indicate that several surface molecules of monocytes, including CD44, are required to trigger the generation of ROI after their contact with tumor cells, whereas HA overexpressed on some cancer cells may allow monocytes (via CD44) to distinguish between transformed and normal cells. However, blocking of CD44 on monocytes by free HA dampens their response to tumor cells. Taken together, these observations suggest that the presence of HA in the tumor stroma may modulate effector functions of infiltrating macrophages and their interactions with cancer cells in situ.

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.

Cytokine production in transient hypogammaglobulinemia and isolated IgA deficiency

BACKGROUND Transient hypogammaglobulinemia of infancy and isolated IgA deficiency are characterized by normal numbers of circulating B lymphocytes. It is likely that no single abnormality, but rather different factors, may be relevant for the delayed onset of IgG synthesis in transient hypogammaglobulinemia or for the differentiation defect of B cells in IgA deficiency. These factors may include defective production of cytokines or an abnormal response of B cells to various mediators. Alternatively, some cytokines may act as inhibitory factors of B-cell function. METHODS The ability of peripheral blood mononuclear cells from children with proved or probable transient hypogammaglobulinemia (30 patients) and IgA deficiency (15 patients) to secrete several cytokines on stimulation with phytohemagglutinin in vitro was analyzed. RESULTS An enhanced production of tumor necrosis factor (TNF)-alpha, TNF-beta, and IL-10 was observed in transient hypogammaglobulinemia; whereas secretion of IL-1, IL-4, and IL-6 was essentially similar in the control and patient groups. Increased frequency of mononuclear cells secreting TNF-alpha was seen in the patient groups. Apart from elevated production of TNF-alpha, no other abnormalities in cytokine synthesis in selective IgA deficiency were observed. In vitro observations showed that exogenously added TNF-alpha and TNF-beta inhibited IgG and IgA secretion by pokeweed mitogen-stimulated mononuclear cells. During follow-up of 10 children, normalization of serum IgG level was associated with a decrease in previously elevated TNF-alpha and TNF-beta production, but IL-10 production remained unchanged. CONCLUSION These results suggest that TNF may be involved in the regulation of IgG and IgA production and can be associated with an arrest of IgG and IgA switch of B cells in hypogammaglobulinemia. The balance between TNF and IL-10 may be important for the normal development of IgG-secreting B cells.

Blood monocytes stimulate migration of human pancreatic carcinoma cells in vitro: The role of tumour necrosis factor – alpha

In some types of cancers, tumour-infiltrating monocytes/macrophages (TIM) may be responsible for the formation of an invasive microenvironment in a manner dependent on the secretion of soluble mediators such as tumour necrosis factor-alpha (TNF). Human pancreatic carcinoma (HPC-4) cells are able to induce TNF production by monocytes. Here, the effect of human peripheral blood monocytes, precursors of TIM, on the motility of co-cultured HPC-4 cells, was directly analysed in vitro. A phenotypic transition, i.e., the appearance of rear-front polarised HPC-4 cells paralleled by their increased motility, and increased motility of monocytes, were observed. This effect was attenuated when HPC-4 cells and monocytes were co-cultured in the presence of inhibitors of TNF production and anti-TNF monoclonal antibodies, indicating the specific role of this cytokine in determining paracrine loops between monocytes and cancer cells. Moreover, exogenous TNF induced HPC-4 cell motility concomitantly to the appearance of cellular features characteristic for epithelial-mesenchymal transition (EMT) such as rear-front polarisation, rearrangements of the actin cytoskeleton characteristic for motile cells and the induction of Snail-1 expression. Since cell movement is crucial for cancer invasion and the formation of metastases, these findings demonstrate an EMT-dependent mechanism of cancer progression which acts through the phenotypic transition of pancreatic cancer cells dependent on monocyte-derived TNF.

Induction of intracellular cytokine production in human monocytes/macrophages stimulated with ligands of pattern recognition receptors.

AbstractObjective: This study addressed the role of the pattern recognition receptors (PRR), which recognize different molecular structures present on microorganisms, apoptotic, senescent and tumor cells, in the stimulation of human monocyte and monocyte-derived macrophages (MDM) for the production of intracellular cytokines. Materials and methods: Monocytes and MDM were stimulated with different ligands of scavenger receptors (SR) and mannose receptor (MR). Production of intracellular cytokines: tumor necrosis factor alpha (TNFα), interleukin 10 and 12 (IL-10, IL-12) was determined by flow cytometry following staining with anti-cytokine monoclonal antibodies (mAbs). Results: The ligands of SR type A: fucoidan, polyguanylic acid (polyG), chemically modified low density lipoproteins (LDL), ligands of SR-B: native and chemically modified LDL, and ligand of mannose receptor (MR) – mannan induced strong expression of intracellular TNFα and weaker IL-10 in monocytes, while phosphatidylserine (PdS) was without effect. IL-12 was stimulated only by fucoidan and polyG. The induction of cytokine m-RNA generally followed the pattern and the magnitude of intracellular cytokine production. In MDM, intracellular TNFα and IL-12 expression was induced by mannan, native and modified LDL, but not other ligands. Expression of IL-10 was less pronounced and occurred following stimulation with fucoidan, polyG and modified, but not native, LDL. Conclusions: These results suggest that some PRR ligands may be involved in activation of monocytes/MDM for the production of mainly proinflammatory cytokines (TNFα, IL-12) implicating their role in the response to microbial and tumor invasion.

DEMONSTRATION OF INOS-MRNA AND INOS IN HUMAN MONOCYTES STIMULATED WITH CANCER CELLS IN VITRO

Synthesis and localization of inducible nitric oxide synthase mRNA (iNOS‐mRNA) and iNOS protein in the cultures of human monocytes (Mφ) and colon carcinoma cell line (DeTa) that resulted in nitric oxide (NO) synthesis has been studied. The iNOS‐mRNA was observed around the sixth hour of culture and peaked at the twelfth hour. The iNOS‐mRNA, as determined by the in situ hybridization and iNOS protein, as detected by staining with specific anti‐iNOS monoclonal antibodies, were observed preferentially in the cytoplasm of some Mφ, but not in cancer cells. Mφ cultured alone did not show detectable iNOS‐mRNA expression and iNOS protein. Mφ sorted out from tumor cells after 8hof co‐culture expressed iNOS protein and iNOS‐mRNA, which were not detected in Mφ without previous contact with cancer cells. Prevention of NO synthesis by (L‐N 5‐1‐iminoethyl)‐ornithine (L‐NIO) partly inhibited Mφ cytotoxic activity against DeTa (NO‐inducing cancer cell line) but not against the human pancreatic cancer (HPC‐4) cell line that does not induce NO production in Mφ. This suggests that Mφ cytotoxic activity, at least in some cases, may be NO dependent. These observations provide further evidence that Mφ can be directly stimulated by cancer cells for de novo production of NO and suggest that iNOS occurring in the tumor‐infiltrating macrophages may arise as a result of their interactions with tumor cells. However, because only some tumor cells are able to induce NO production in a small proportion of Mφ, its role in the anti‐tumor response of the host is probably limited. J. Leukoc. Biol. 65: 597–604; 1999.

Monocyte - Mediated Regulation of Antigen - Driven IFNgamma Production by T Cells. The Role of Endogenously Produced TNF

The question was asked whether tumour necrosis factor alpha (TNF) is involved in regulation of interferon gamma (IFN gamma) production by T cells. Monocytes were exposed to exogenous TNF or to TNF synthesis inhibitors (pentoxifylline, PTX and adriamycin, ADR) and then used as antigen (PPD) presenting cells for autologous T cells. The ability of T lymphocytes to release IFN gamma was assessed after 3 days of culture. Preincubation of monocytes with rTNF enhanced their ability to induce IFN gamma production while TNF synthesis inhibitors decreased it. Anti-TNF and anti-TNF-R2 monoclonal antibodies (mAbs) inhibited monocyte ability to present PPD for IFN gamma production which suggested that endogenously produced TNF by monocytes had to be released and acted on TNF-R2 on the monocyte surface. The enhancing effect of exogenous TNF was also abrogated by anti-TNF-R2 mAb. Pretreatment of monocytes with rTNF enhanced, while pretreatment with PTX decreased, PPD-induced IL-6 production. An increased production of IL-4 was found in cultures of PTX-treated, PPD-pulsed monocytes with T cells. This may indicate that in the relative absence of monocyte costimulatory signal(s), probably IL-6, Th2 cells are stimulated. These results indicate that TNF is involved in control of monocyte-mediated regulation of cytokine production by T cells.

Interactions of monocyte subpopulations generated from cord blood CD34+ hematopoietic progenitors with tumor cells: Assessment of antitumor potential

Monocytes and their subsets (CD14(++)CD16(+) and CD14(+)CD16(-)) generated from cord blood CD34(+) progenitor cells were used for determination of their capacity to interact with tumor cells in vitro and in vivo. The studies in vitro included adhesion to human umbilical vein endothelial cells, cytotoxicity, production of toxic mediators: reactive oxygen and nitrogen intermediates (ROI and RNI, respectively), and finally their effect on transplantable human tumor growth in nonobese diabetic severe combined immunodeficient mice. The CD14(++)CD16(+) subset exhibited an increased adherence to human umbilical vein endothelial cells and cytotoxicity toward tumor cells in vitro. CD14(+)CD16(-) monocytes showed a higher production of reactive oxygen and nitrogen intermediates after stimulation with tumor cells, and more pronounced inhibition of tumor growth in vivo. The results revealed significant differences in the behavior of CD14(++)CD16(+) and CD14(+)CD16(-) monocyte subsets toward tumor cells, thus providing further evidence that CD34(+) cell-derived monocytes differ in this respect from blood monocytes. The protocol for generation of monocytes with antitumor reactivity described here may be useful to obtain monocytes from CD34(+) progenitor cells of cancer patients. This might offer a basis for a novel approach for various forms of cellular immunotherapy of cancer.

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.