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The tumor milieu can influence dendritic cell (DC) differentiation. We analyzed DC differentiation in a 3-dimensional tumor model and propose a new mechanism of DC modulation by the tumor environment. Monocytes were cultured in the presence of IL-4 and GM-CSF within multicellular tumor spheroids (MCTSs) generated from different tumor cell lines. Monocytes invaded the MCTSs and differentiated into tumor-associated dendritic cells (TADCs). The antigen expression was altered on TADCs independent of the culture conditions (immature/mature DCs, Langerhans cells) and IL-12 secretion was reduced. Supernatants of MCTSs could partially transfer the suppressive effect. Conditioned media from urothelial carcinoma cell lines contained high levels of M-CSF and IL-6, both cytokines known to modulate DC differentiation. In contrast, melanoma and prostate carcinoma MCTS cocultures produced little M-CSF and IL-6, but high levels of lactic acid. Indeed, addition of lactic acid during DC differentiation in vitro induced a phenotype comparable with TADCs generated within melanoma and prostate carcinoma MCTSs. Blocking of lactic acid production in melanoma MCTS cocultures reverted the TADC phenotype to normal. We therefore conclude that tumor-derived lactic acid is an important factor modulating the DC phenotype in the tumor environment, which may critically contribute to tumor escape mechanisms.

Terminal maturation of human blood monocytes to macrophages (MAC) in vivo is believed to be important for the morphology, antigen expression and functional activity of the resulting MAC population. This process is modulated by the specific tissue micro-environment to which blood monocytes migrate upon leaving the vasculature. Tumor-associated macrophages (TAM) are a special type of MAC, and little is known about the modulating capacity of the tumor environment on monocyte-to-MAC differentiation. By co-culturing 3-dimensional multicellular spheroids (MCS) of the urothelial-bladder-carcinoma cell lines J82 and RT4 with human monocytes/MAC we generated TAM in vitro. For comparison, monocytes/MAC were co-cultured with the non-tumorigenic urothelial cell line HCV29. The effects on monocyte differentiation were analyzed, particularly with respect to cytokine release. Monocyte maturation was modulated within the tumor spheroid dependent upon the tumor cell type. Monocytes co-cultured with MCS of the poorly differentiated J82 carcinoma spontaneously produced high amounts of IL-1beta and IL-6, but only low amounts of TNF-alpha, which could be further increased by the addition of LPS. This cytokine pattern is characteristic for monocytes and remained constant for up to 8 days in J82-MCS co-cultures. However, in RT4-MCS and HCV29-MCS co-cultures, the initial cytokine pattern changed and after 8 days corresponded well to that of MAC differentiated in vitro without tumor contact. In addition to functional parameters, we analyzed the morphology of J82-MCS-TAM and found that they displayed a monocyte-like morphology. Our data indicate that (1) tumor cells can influence monocyte-to-MAC differentiation, giving rise to TAM with monocyte-specific phenotypic properties; and (2) this capacity is dependent on the type of tumor cell.

We studied phenotype, function and differentiation of mononuclear phagocytes in 11 cancer patients treated subcutaneously with 10O microg/kg recombinant human (rhu) GM-CSF for 7 d. The rhuGM-CSF treatment induced (1) a 5.9-fold increase in the number of blood monocytes (MO), (2) a decrease of CD14bright/CD16bright cells with a diminution of the mean fluorescence intensity (MFI) of CD14, and (3) a decrease of MO cellular cytotoxicity. In patients' sera, tumour necrosis factor (TNF)-alpha, interleukin (IL)-10, IL-12, neopterin, macrophage-colony-stimulating factor (M-CSF), and IL-1 receptor antagonist (IL-1RA) increased, whereas GM-CSF and granulocyte-colony-stimulating factor (G-CSF) decreased after an initial peak. In whole blood samples the lipopolysaccharide (LPS)-stimulated release of TNF-alpha, IL-6 and IL-1RA increased initially, whereas IL-1beta, IL-10 and IL-12 decreased. During differentiation from MO to macrophages (MAC), interferon (IFN)-gamma-stimulated tumour cytotoxicity increased, but both MO and MAC were less cytotoxic upon rhuGM-CSF treatment. The differentiation-associated increase of LPS-induced TNF-alpha, IL-1RA and IL-10 secretion was reduced by the rhuGM-CSF treatment, and the expression of CD14 on MAC as well as the proportion of CD14+/CD16+, CD14+/MAX.1+ and CD14+/CD71+ cells in 7-d cultured MAC declined. We interpret these findings as (1) an increase of immature MO upon rhuGM-CSF therapy, (2) a priming effect on the LPS-induced proinflammatory cytokine repertoire of MO, and (3) an impact of rhuGM-CSF on the capacity of MO to differentiate to MAC in vitro.

The expression of many cytokines is dysregulated in individuals infected with the human immunodeficiency virus-1 (HIV-1). To determine the effects of HIV-1 infection on cytokine expression in individual cells (at the single cell level), we investigated the intracellular levels of proinflammatory cytokines (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, IL-6, and IL-8) and hematopoietic growth factors (granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF]) in monocyte-derived macrophages, mock-infected, or infected with HIV-1 by immunocytochemical staining for cytokine protein and compared this with secreted cytokine levels as determined by specific enzyme-linked immunosorbent assay (ELISA). No difference in the frequency or intensity of cell-associated immunocytochemical cytokine staining could be observed between HIV-1 and mock-infected cells even though the level of secreted proinflammatory cytokines increased and the hematopoietic growth factors decreased in HIV-1-infected cultures. Furthermore, equal expression of cytokine mRNA was observed in all cells in the culture regardless of whether the cells were productively infected with HIV-1 as determined by double-labelling immunocytochemical staining for HIV-1 p24 antigen and in situ hybridization for cytokine mRNA expression. These results indicate that HIV-1 infection results in dysregulation of intracellular cytokine mRNA expression and cytokine secretion not only in HIV-1-infected cells, but also through an indirect way(s) affecting cells not producing virus.

Monocytes (MO) and macrophages (MAC) are important producers of cytokines involved in the pathophysiology of bacterial sepsis. Most studies concentrate on the effects of bacterial lipopolysaccharides (LPS) regarding the induction of cytokine gene expression and secretion in MO/MAC. Here we report that besides LPS, the synthetic lipoprotein analogue lipopeptide N-palmitoyl-S-(2,3-bis(palmitoyl)-(2RS)-propyl)-(R)-cysteinyl-alanyl- glycine (Pam3-Cys-Ala-Gly), another component of the outer membrane of Gram-negative bacteria, as well as heat-killed Staphyloccocus aureus (S. aureus/SAC) are potent stimuli for cytokines in human MO. For all three investigated stimuli we found an individual pattern of cytokine induction: LPS was most potent in inducing interleukin-6 (IL-6) synthesis, whereas for tumour necrosis factor-alpha (TNF-alpha) secretion SAC was the best stimulus. Comparable amounts of IL-8 were induced by either LPS or Pam3-Cys-Ala-Gly, with SAC being less effective even at higher concentrations. The addition of serum led to an increase in LPS-, SAC- and Pam3-Cys-Ala-Gly-stimulated TNF-alpha secretion, indicating that the presence of serum is critical not just for LPS stimulation. Furthermore, as is known for LPS, Pam3-Cys-Ala-Gly and SAC rendered MO refractory to a second bacterial stimulus. Pam3-Cys-Ala-Gly and SAC induced tolerance for itself, but LPS could partially overcome this effect. As the CD14 molecule is discussed as a common receptor for different bacterial components, we investigated whether the TNF-alpha response of MO could be blocked by anti-CD14 antibodies. MY4, a CD14 antibody, selectively blocked the TNF-alpha secretion induced by LPS but not by Pam3-Cys-Ala-Gly or SAC. In summary, we conclude that besides LPS, lipopeptide Pam3-Cys-Ala-Gly and SAC are potent stimuli for human MO, while the mechanisms of activation seem to be partially different from LPS.

It is well known that adherence of monocytes (MO) to extracellular matrix substrates or tissue culture plastic activates these cells and induces the expression of a multitude of genes. Especially, it was described, that MO are primed by cell adhesion to produce higher amounts of some cytokines, e.g. interleukin (IL)-8 and tumor necrosis factor alpha (TNF-alpha). In order to investigate adherence-induced effects upon cytokine production, we seeded MO into tissue cultures and stimulated cells by lipopolysaccharide (LPS) simultaneously or at later time points. An increasing time-lag between cell adhesion and LPS-stimulation led to differential effects upon cytokine production: whereas TNF was upregulated (in accordance with reports by others), granulocyte colony-stimulating factor (G-CSF) was considerably down-regulated. In contrast, G-CSF production did not change, when cells were kept under non-adherent conditions in whole blood. In adherent cultures down-regulation of G-CSF could already be observed after two hours with a maximum after 24 h and was paralleled by a much lower abundance of G-CSF mRNA. Adhesion induced a significant suppression of G-CSF comparable to MO, if mature macrophages derived from MO in vitro were examined. Furthermore, two other cytokines, granulocyte-macrophage (GM)-CSF and IL-6, were also down-regulated following adhesion. In conclusion, activation of mononuclear phagocytes by adhesion can lead to "priming" for the production of some cytokines and at the same time to "silencing" for the production of others.

Functional competence as well as phenotype heterogeneity of macrophages depend on the completion of their maturation pathway. Differentiation of committed myeloid progenitor cells is induced by colony-stimulating factors (CSF), but no consistent data exist on which factor(s) induce the terminal maturation from the circulating blood monocyte to the mature macrophage. In vitro, monocyte to macrophage transformation occurs in the presence of serum and can be followed by the expression of the maturation-associated antigens gp65-MAX.1, gp68-MAX.3, and CD51. We describe that the differentiation-inducing activity in serum cannot be replaced by any of the known and available purified recombinant cytokines. In the absence of serum monocytes die in suspension cultures while surviving as non-differentiating cells when cultured adherent to plastic. In serum-free suspension cultures survival can be significantly improved by the addition of recombinant human macrophage (rhM)-CSF whereas other cytokines do not. At any stage of serum-free adherent culture, monocyte to macrophage differentiation can be induced rapidly by the addition of serum, whereas cytokines (rhM-CSF, recombinant human granulocyte macrophage [rhGM]-CSF, recombinant human granulocyte [rhG]-CSF, recombinant human interleukin [rhIL]-1, rhIL-3, rhIL-4, rhIL-6, tumor necrosis factor [TNF]-alpha, interferon [IFN]-alpha, IFN-gamma) alone or in combination are not effective. Serum-induced maturation, however, was suppressed in the presence of neutralizing anti-M-CSF antibodies. In addition to phenotype analysis, the secretory repertoire of rhM-CSF cultured monocytes was analyzed in comparison to serum cultured monocytes which further characterized them to be immature cells, i.e., low release of maturation-associated products such as alpha-2-macroglobulin, neopterin, fibronectin, and TNF-alpha, but high IL-6 secretion, an attribute of blood monocytes. We conclude that for monocyte survival in vitro the presence of endogenous M-CSF and possibly other autocrine factors elicited by cell adherence are required for the induction of macrophage maturation; however, yet undefined additional factor(s) are necessary. They are present in serum and may act in conjunction with M-CSF but are distinct from all known cytokines. Our in vitro system may be useful in the screening and discovery of these serum factor(s).

Under inflammatory conditions, the pleiotropic cytokine interleukin-10 (IL-10) is released in many tissues. It mediates anti-inflammatory effects in particular by inhibiting the release of T helper type 1 (Th1) cytokines. In contrast, we show here that NK cell cytotoxicity against autologous macrophages is elevated if both cell types are cultured with IL-10. The expression of most activatory NK receptors is increased after culture in the presence of IL-10. On the other hand, macrophages cultured in the presence of IL-10 show elevated expression of the NKG2D ligands major histocompatibility complex (MHC) class 1-like molecules (MIC) – A and – B, as well as UL-16 binding proteins (ULBP) – ULBP-1, ULBP-2 and ULBP-3. By masking the interaction of NK cells with macrophages through interruption of the NKG2D receptor with its ligands, we could reverse the IL-10-induced lysis of macrophages. Our data therefore reveal that IL-10 may exert a novel immunomodulatory role by stimulating NKG2D ligand expression on macrophages, thereby rendering them susceptible to NK cell elimination. This suggests that NK cells would delete macrophages and potentially other immature antigen-presenting cells (APC) or their precursors under inflammatory conditions as a feedback mechanism to shut off uncontrolled immune responses. [ABSTRACT FROM AUTHOR]

Until recently, the only accepted mechanism of tumour vascularization was the sprouting of endothelial cells (EC) from pre-existing vessels, while recent studies suggest the contribution of stem cell-derived endothelial progenitors as well as cells from the myeloid lineage. Here, we show a new way of endothelial differentiation that involves the specific modulation of monocytes by the tumour environment. The tumour milieu is characterized by the presence of cytokines and lactate which induce the differentiation of tumour-invading monocytes into tumour-associated dendritic cells (DC). Additional incubation of tumour-associated DC with pro-angiogenic factors, such as vascular endothelial growth factor and oncostatin M, led to transdifferentiation into endothelial-like cells. The cells showed strong expression of von Willebrand factor and VE-Cadherin, both classical EC markers, while leukocytic markers were reduced. In addition, they were able to form network-like structures on matrigel, which could be blocked by the DNA-based drug Defibrotide. This finding may be of great therapeutic relevance for tumour therapy. [ABSTRACT FROM AUTHOR]

Monocytes (MO) and macrophages (MAC) are important producers of cytokines involved in the pathophysiology of bacterial sepsis. Most studies concentrate on the effects of bacterial lipopoly-saccharides (LPS) regarding the induction of cytokine gene expression and secretion in MO/MAC. Here we report that besides LPS, the synthetic lipoprotein analogue lipopeptide N-palmitoyl-S-( 2,3-bis (palmitoyl)-(2RS)-propyl)-(R)cysteinyl-alanyl-glycine (Pam 3-Cys-Ala-Gly), another component of the outer membrane of Gram-negative bacteria, as well as heat-killed Staphyloccocus aureus (S. aureus/SAC) are potent stimuli for cytokines in human MO. For all three investigated stimuli we found an individual pattern of cytokine induction: LPS was most potent in inducing interleukin-6 (IL-6) synthesis, whereas for tumour necrosis factor-α (TNF-α) secretion SAC was the best stimulus. Comparable amounts of IL-8 were induced by either LPS or Pam3-Cys-AlaGly, with SAC being less effective even at higher concentrations. The addition of serum led to an increase in LPS-, SAC- and Pam3-Cys-Ala-Gly-stimulated TNT-α secretion, indicating that the presence of serum is critical not just for LPS stimulation. Furthermore, as is known for LPS, Pam3-Cys-Ala-Gly and SAC rendered MO refractory to a second bacterial stimulus. Pam3-Cys-Ala-Gly and SAC induced tolerance for itself, but LPS could partially overcome this effect. As the CD14 molecule is discussed as a common receptor for different bacterial components, we investigated whether the TNF-α response of MO could be blocked by anti-CD 14 antibodies. MY4, a CD14 antibody, selectively blocked the TNF-α secretion induced by LPS but not by Pam3-Cys-AIa-Gly or SAC. In summary, we conclude that besides LPS, lipopeptide Pam3-Cys- Ala-Gly and SAC are potent stimuli for human MO, while the mechanisms of activation seem to be partially different from LPS. [ABSTRACT FROM AUTHOR]