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 alpha, 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.
Some ligands of pattern recognition receptors (PRR) are present on tumour cells. The role of PRR in signalling for cytokine and reactive oxygen intermediates (ROI) production by monocytes and ...monocyte-derived macrophages (MDM) stimulated with tumour cells was studied.
Monocytes/MDM were pretreated with PRR ligands or anti-PRR monoclonal antibodies (mAbs) and stimulated with tumour cells. Cytokine secretion was measured by enzyme-linked immunoassay (ELISA) and ROI production by luminol-dependent chemiluminescence (CL).
The ligands of scavenger receptor A (SR-A): (fucoidan, polyguanylic acid (polyG) and modified low density lipoproteins (LDL)) and B (SR-B) (native and modified LDL, phosphatidylserine (PdS)) and of mannose receptor (MR) (mannan), induced tumour necrosis factor alpha (TNF) and ROI (except LDL) release by monocytes. Production of TNF and interleukin-10 (IL-10) by MDM was stimulated by SR-A ligands and mannan. Tumour cell-induced TNF and IL-10 production by monocytes, but not MDM, was diminished by fucoidan and polyG, while ROI release was reduced by MR and SR-A ligands. Supplementation of tumour cells with modified LDL and PdS enhanced their stimulatory capacity. TNF and ROI release by tumour cells-stimulated monocytes was inhibited by anti-CD36 and anti-MR (clone PAM-1) mAbs.
SR and MR may be involved to different extents in the induction of cytokines and ROI production by monocytes, but not MDM, stimulated with tumour cells.
Introduction. In the past decade significant advances in ex vivo expansion of haematopoietic stem cells were made. Protocols of differentiating CD34+ cells into cells from hematopoietic linages, ...especially dendritic cells, were described. However, little is known about in vitro differentiation of CD34+ cells to monocytes. In peripheral blood there are two main populations of monocytes CD14++CD16− and CD14+CD16+. They represent about 10 percent of total blood monocytes. Recently we have showed that expansion and differentiation of cord blood CD34+ cells to monocytes/macrophages leads to generation of CD14+CD16− and CD14++CD16+ monocyte subpopulations (Stec et al, J Leukoc Biol. 2007).
Aim of study. To compare two monocyte subpopulation: CD14+CD16− and CD14++CD16+ obtained from our expansion and differentiation protocol of CD34+ cord blood cells.
Materials and methods. After 3–10 days expansion in X-VIVO10 medium with FBS, SCF, IL-3, FLT-3L, TPO and then 7–10 days differentiation in IMDM medium with FBS, M-CSF, FLT-3L, IL-3, SCF, CD14+CD16− and CD14++CD16+ subpopulations were isolated by FACS sorting (FACSVantage). Release of IL-6, IL8, IL-12, IP-10, MIP1 α, MIP-1 β, RANTES, VEGF and FGF after LPS/INFγ or cancer cells stimulation were analysed using CBA (Cytometric Bead Array) method. Cytotoxic activity against cancer cells were assessed by MTT test and migration capacity was assesed with uncoated porous filters (8 μm) in a 24-well Boyden chamber. The adhesion capacity of subpopulations on human umbilical vein endothelial cells (HUVEC) were assessed with 5(6)-CFDA, SE (Molecular Probes) application and fluorescent microscope. NOD-SCID mice were used to evaluate the influence of subpopulations on angiogenesis and tumour growth.
Results. CD14++CD16+ monocytes released more IL-8, IL-12, IP-10, MIP1-α, MIP1-β, and RANTES than CD14+CD16− monocytes after LPS/INFγ or cancer cells stimulation. After 24 hours culture in medium without cytokines and than stimulation with LPS/INFγ or cancer cells, CD14++CD16+ subpopulation released more RANTES, TNF, MIP1-α, IL10 than CD14+CD16−cells. CD14+CD16− subpopulation exhibited greater chemotaxis to SDF-1 and MIP1-α than CD14++CD16+ (12,2%±5,5% versus 2,2%±1%). At the same time CD14++CD16+ monocytes showed significantly higher cytotoxic activity against tumour cells in vitro than CD14+CD16− (34,04±17,45% versus 15,56±13,66%). CD14++16+ population showed increased adhesion to HUVEC than CD14+16− population (79±20 cells versus 30±13 cells). In in vivo tests in NODSCID mice both subpopulations together inhibited angiogenesis, but CD14+CD16− subpopulation inhibited stronger tumour growth than CD14++CD16+ subpopulation.
Conclusions. Using our expansion protocol it was possible to obtain two subpopulations of monocytes: CD14+CD16− and CD14++CD16+ which showed the differences in the release of cytokines/chemokines following stimulation with LPS/INFγ or tumour cells, chemotactic and antiangiogenic activity and cytotoxicity and are clearly distinct from known main subpopulations of blood monocytes CD14+CD16+ and CD14++. Additional tests, which are still in progress in our lab will provide evidence if both populations are functional monocytes.
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 alpha (TNF-alpha), 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-alpha and decreased IL-10 production. All strains of M. vaccae and BCG used for treatment of deactivated monocytes enhanced depressed TNF-alpha 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.
The in vitro model of tumour infiltrating macrophages (TIM)-tumour interactions in which monocytes and monocyte-derived macrophages (MDM) are cultured with cancer cells was used to assess ...immunophenotypic changes of interacting cells. Following short cocultures, monocytes, MDM and tumour cells were sorted out by FACS and the expression of several determinants was evaluated. Monocytes showed the induction of CD44v6 and v7/8, and up-regulation of CD16 (Fc gamma RIII), CD54 (ICAM-1), CD68 (macrophage maturation marker) and CD86 (costimulatory molecule B7.2). The increased expression of CD11a (LFA-1) and CD58 (LFA-3) was noted on some cancer cells. Up-regulation of TNFRII and HLA-DR was observed on both types of cells. MDM shared similar changes. Contact of monocytes, but not of MDM, with tumour cells led to Fas-FasL-dependent apoptosis of both types of cells. This study suggests that the immunophenotype of monocytes/macrophages and cancer cells may be modified during their bidirectional interactions in the absence of other microenvironmental elements that are present in the tumour stroma.
This study examined the role of extracellular matrix compounds (EMC) in the alteration of tumour necrosis factor-alpha (TNF alpha) and interleukin-10 (IL-10) production by human monocytes stimulated ...with cancer cells.
Monocytes were cultured with cancer cells in the absence or presence of EMC and cytokine release was measured by ELISA. In some experiments monocytes preincubated with monoclonal antibodies (mAbs) against CD29 and CD44 were used.
Fibronectin, collagen type I and type IV induced production of cytokines by monocytes and mAbs inhibited this effect. The release of TNF alpha monocytes stimulated with cancer cells was inhibited by fibronectin, collagen type I and IV and IL-10 by fibronectin and collagen type IV. Other EMC were ineffective. Both mAbs partly reversed this inhibitory effect.
These findings suggest that some EMC induced cytokine release by monocytes but inhibit monocyte-cancer cell interactions and this effect is presumably due to competition for the same receptors.
Tumour necrosis factor alpha (TNF) mRNA is detected in the macrophage infiltrate surrounding the tumour, but the cellular/molecular interactions leading to TNF gene expression in macrophages are ...unknown. The in vitro system in which human blood monocytes are stimulated with human cancer cells for TNF release was used to study such interactions. Monoclonal antibodies (MAbs) against various adhesion molecules (LFA-1, LFA-3, ICAM-1, VNR, VLA beta I chain) were unable to block TNF production in co-culture of monocytes with a human pancreatic carcinoma (HPC) cell line. However, anti-CD44 and anti-HLA-DR MAbs effectively blocked TNF release and TNF-mRNA induction in monocytes. Pre-incubation of monocytes with anti-HLA-DR and tumour cells with anti-CD44 MAbs had a similar effect. It was concluded that CD44 molecules are involved in tumour-monocyte interactions and that HLA-DR determinants of monocytes are engaged in signal transduction for TNF gene activation. These findings may suggest that certain surface determinants of tumour cells act as ligands for MHC class-II molecules and induce TNF production in monocytes.
Synthesis and localization of inducible nitric oxide synthase mRNA (iNOS-mRNA) and iNOS protein in the cultures of human monocytes (Mphi) 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 Mphi, but not in cancer cells. Mphi cultured alone did not show detectable iNOS-mRNA expression and iNOS protein. Mphi sorted out from tumor cells after 8 h of co-culture expressed iNOS protein and iNOS-mRNA, which were not detected in Mphi without previous contact with cancer cells. Prevention of NO synthesis by (L-N5-1-iminoethyl)-ornithine (L-NIO) partly inhibited Mphi 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 Mphi. This suggests that Mphi cytotoxic activity, at least in some cases, may be NO dependent. These observations provide further evidence that Mphi 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 Mphi, its role in the anti-tumor response of the host is probably limited.
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