OK-432-stimulated chemokine secretion from human monocytes depends on MEK1/2, and involves p38 MAPK and NF-κB phosphorylation, in vitro.

Interaction between the immune system and cancer cells allows for the use of biological response modifiers, like OK-432, in cancer therapy. We have studied the involvement of monocytes (MOs) in the immune response to OK-432 by examining MCP-1, MIP-1α and MIP-1ß secretion, in vitro. OK-432-induced IL-6/TNF-α secretion has previously been shown to depend on mitogen-activated protein kinases (MAPKs) ERK1/2 and p38, and we therefore investigated the role of these MAPKs in OK-432-induced chemokine secretion. Here we demonstrate that pharmacological MEK1/2 kinase inhibition generally impaired chemokine secretion from MOs, whereas p38 MAPK inhibition in particular reduced MIP-1α production. Furthermore, simultaneous inhibition of MEK1/2 and Syk kinase was seen to have an additive impact on reduced MCP-1, MIP-1α and MIP-1ß secretion. Based on single cell flow cytometry analyses, OK-432, lipoteichoic acid (LTA) and lipopolysaccharide (LPS) were seen to induce p38 MAPK and NF-κB phosphorylation in MOs with different time kinetics. LTA and LPS have been shown to induce ERK1/2 phosphorylation, whereas the levels of phosphorylated ERK1/2 remained constant following OK-432 treatment at the time points tested. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, and we demonstrate increased TLR2 cell surface levels on the MO population, most profoundly following stimulation with LTA and OK-432. Together these results indicate that modulation of MEK1/2 and p38 MAPK signalling could affect the response to OK-432 treatment, having the potential to improve its therapeutic potential within cancer and lymphangioma treatment.

In vitro cholesteatoma growth and secretion of cytokines.

CONCLUSION: Our results show a significant difference between skin and cholesteatoma biology in vitro. OBJECTIVES: Cholesteatoma disease is a process of destruction characterized by uncontrolled growth of squamous epithelial cells in the middle ear or temporal bone. The pathophysiology behind the cholesteatoma development is controversial, and the mechanisms driving the cholesteatoma growth, migration and destructive properties is still unclear. We aimed to provide a method to study the effect of various compounds on cholesteatoma and skin tissue growth, as well as to further investigate the biological differences between normal skin and cholesteatoma tissue. METHODS: We have established a method to study cholesteatoma biopsy tissue in vitro. Cholesteatoma tissues from patients undergoing surgery for chronic otitis were grown in culture medium and compared to growth patterns and behaviour of normal retroauricular skin. Conditioned medium was analysed for various secreted cytokines. RESULTS: We found a radial outgrowth of keratinocyte epithelium from the circular biopsies. After 5 days of culture we found a significant growth of both cholesteatoma and skin-derived cells. Cholesteatoma samples showed higher growth rate as compared with skin control cultures from the same patient. Moreover, the cholesteatoma cells showed higher production of monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-6 as compared with normal skin.

Chemokines are secreted by monocytes following OK-432 (lyophilized Streptococcus pyogenes) stimulation.

BACKGROUND: OK-432, penicillin-killed Streptococcus pyogenes, is used in treating lymphangiomas and carcinomas. We have studied in vitro the role of mononuclear phagocytes (MNPs), including purified monocytes (MOs), in the immune response to OK-432. MIP-1alpha/beta and MCP-1 secretions were assessed in whole blood (WB), peripheral blood mononuclear cells (PBMCs) and purified MOs, after in vitro stimulation with OK-432 with or without adherence for 24 hours. RESULTS: OK-432 stimulated MNPs to secrete MCP-1 and MIP-1alpha/beta in healthy individuals and in head and neck squamous cell carcinoma (HNSCC) patients, except for OK-432 stimulation of WB giving a minimal MIP-1alpha/beta response. Upon culture on low-attachment wells, a spontaneous chemokine secretion was observed, with an unchanged secretion following OK-432 stimulation. Inhibition of Syk kinase and/or PI-3 kinase did not significantly change the chemokine response to OK-432, except for MIP-1alpha production being increased upon Syk inhibitor addition and an increased MCP-1 response upon addition of both inhibitors. Adhesion may possibly involve beta1 and/or beta3 integrins, not beta2, whereas beta(1-3) integrins may act as co-stimulatory receptors for OK-432. Based on direct blockage of CD36 or CD18 by antibodies, MCP-1 production may be mediated by CD18 while MIP-1beta and MCP-1 production may occur upon binding to CD36. CONCLUSION: Adherent human MOs produce MCP-1 and MIP-1alpha/beta upon stimulation with OK-432. CD36 modulates MIP-1beta and MCP-1 response. Thus, to some extent OK-432 acts as a substance whereby only MOs adhered to surfaces secrete MCP-1 and MIP-1alpha/beta, in part explaining why OK-432 is suited as a biological response modifying drug.

Tumour-associated macrophages secrete IL-6 and MCP-1 in head and neck squamous cell carcinoma tissue.

Conclusion. Tumour-associated macrophages (TAMs) in head and neck squamous cell carcinomas (HNSCCs) secrete interleukin 6 (IL-6) and monocyte chemotactic protein (MCP-1) that can be down-regulated by L-leucine-methylester (LLME); however, there is no qualitative difference between function of TAMs and tissue macrophages in mucosa as measured by IL-6 and MCP-1 secretion. Objectives. TAMs play an important role in the interaction with tumour cells in malignant tumours. The cells in the tumours that are the main sources of the various signal substances need to be further elucidated. The aim of this investigation was to reveal whether TAMs in HNSCCs secrete IL-6 and MCP-1. These cytokines influence tumour cell growth and macrophage influx in tumours, respectively. Materials and methods. In order to inhibit macrophage function in F-spheroids, in some experiments the tissue fragments were initially incubated with LLME, a substance that selectively inhibits function of phagocytes. IL-6 and MCP-1 secretion from untreated F-spheroids was compared to cytokine secretion from LLME-treated F-spheroids as measured by ELISA. Results. LLME did not affect the viability of F-spheroids and reduced IL-6 and MCP-1 secretion from monocyte-derived macrophages in vitro. F-spheroids from LLME-treated tissue fragments showed lower IL-6 and MCP-1 secretion compared with F-spheroids from tissue fragment untreated with LLME.

Viable head and neck tumor spheroids stimulate in vitro autologous monocyte MCP-1 secretion through soluble substances and CD14/lectin-like receptors.

Biopsies from carcinoma tissue and benign control mucosa from head and neck squamous cell carcinoma (HNSCC) patients were used to establish fragment (F)-spheroids in vitro. We have previously shown that autologous monocytes co-cultured with F-spheroids in vitro augment their secretion of monocyte chemotactic protein-1 (MCP-1). Presently, the aims of the present work were to study whether the metabolic activity, secreted products and/or specific receptor/ligand on the surface of the F-spheroids and monocytes are necessary for stimulation of the monocyte MCP-1 secretion upon F-spheroid co-culture. Actinomycin D (1 mug/ml for 24 h) pre-treatment of the F-spheroids abolished the monocyte MCP-1 co-culture response. Co-culture of monocytes and F-spheroids separated by a semi-permeable membrane showed a decreased, but still present, monocyte MCP-1 co-culture response. Conditioned medium from F-spheroids stimulated allogenous monocytes to secrete MCP-1. The addition of glucose or galactose, but not mannose, to co-cultures partially inhibited the monocyte MCP-1 co-culture response. The addition of anti-CD14 antibody diminished the MCP-1 co-culture response. In conclusion, the monocyte MCP-1 co-culture response is dependent on metabolically active spheroids, secreted stimuli, and is augmented by direct contact with F-spheroids, possibly via lectin-like receptors and the CD14 receptor.

Head and neck squamous cell carcinoma spheroid- and monocyte spheroid-stimulated IL-6 and monocyte chemotactic protein-1 secretion are related to TNM stage, inflammatory state and tumor macrophage density.

Monocyte fragment (F)-spheroid-stimulated and F-spheroid IL-6 and monocyte chemotactic protein (MCP)-1 secretion are related to inflammatory state, macrophage density and the TNM stage of patients with head and neck squamous cell carcinoma (HNSCC). Fragment (F)-spheroids from HNSCC patients in vitro secrete and stimulate autologous monocytes to secrete IL-6 and MCP-1. The aim of this investigation was to study this cytokine secretion in relation to other cytokines, spheroid composition and host factors.In series I (n=14) the densities of epithelial cells, fibroblasts and macrophages were determined in sections from F-spheroids and donor tissue. In series II (n=17) the TNM stage, donor inflammatory state, macrophage density and the secretion of F-spheroid- and monocyte F-spheroid-stimulated IL-6, MCP-1 and tumor necrosis factor (TNF)-alpha were determined. Epithelial cells were partly replaced by interstitial tissue during spheroid formation. Malignant (M) F-spheroids secreted more MCP-1 than benign (B) F-spheroids. No F-spheroid secreted measurable amounts of TNF-alpha. Monocytes secreted more IL-6 when co-cultured with MF- compared to BF-spheroids. Monocyte IL-6 MF- and MCP-1 MB-spheroid-stimulated secretion correlated with macrophage density. In addition, there was an association between MF- and BF-spheroid-stimulated monocyte cytokine secretion, as well as between BF- and MF-spheroid-stimulated MCP-1 secretion. An inverse relation was also noted between the erythrocyte sedimentation rate at monocyte harvest and the monocyte MCP-1 F-spheroid responses.