Carla Olsnes

MAPKs ERK and p38, but not JNK Phosphorylation, Modulate IL-6 and TNF-α Secretion Following OK-432 In Vitro Stimulation of Purified Human Monocytes

Interaction between the immune system and cancer allows for the use of biological response modifiers, e.g. OK‐432, in cancer therapy. OK‐432, penicillin‐killed Streptococcus pyogenes, is used in treating carcinomas, but also lymphangiomas. We have studied the role of monocytes (MOs) in the immune response to OK‐432 by examining IL‐6 and tumour necrosis factor (TNF)‐α secretion after in vitro MO stimulation with OK‐432, to some extent in comparison with lipoteichoic acid (LTA) and lipopolysaccharide (LPS). LTA stimulation of whole blood gave IL‐6 but not TNF‐α secretion, as previously shown with OK‐432 stimulation, whereas both cytokines were secreted following LPS stimulation. Addition of the MAPK kinase (MAPKK) MEK inhibitor U0126 inhibited IL‐6/TNF‐α secretion in a dose‐dependent manner. Flow cytometry and to some extent Western blot (Wb) analyses showed that MAPK ERK, located downstream of MEK1/2, is predominantly phosphorylated at isolation from peripheral blood. Addition of the p38 MAP kinase inhibitor SB202190 decreased MO IL‐6/TNF‐α production upon OK‐432 stimulation in a dose‐dependent manner. Addition of the MAPK JNK inhibitor SP600125 did not systematically change the MO IL‐6/TNF‐α OK‐432 response. Flow cytometry showed that when stimulating the MOs before isolation from blood, LPS yielded ERK phosphorylation and LPS/LTA p38 phosphorylation, whereas OK‐432 had no effects on phosphorylation levels. In conclusion, we have shown that OK‐432 resembles TLR2 more than TLR4 stimulation of MOs and depends on MAPKK MEK and MAPK p38, but not on JNK phosphorylation. The MEK and p38 MO OK‐432 stimulation dependence is possibly related to the differentiation of cells of the MO lineage.

Co-culture of Head and Neck Squamous Cell Carcinoma Spheroids with Autologous Monocytes Predicts Prognosis

Co‐culture of monocytes with autologous fragment (F) spheroids originating from malignant (M) tumour or benign (B) control mucosa of head and neck squamous cell carcinoma (HNSCC) yields interleukin (IL)‐6 and monocyte chemo‐attractant protein (MCP)‐1 secretion. This study investigates the association between this cytokine co‐culture response and prognosis. Analysis of IL‐6 and MCP‐1 content of supernatants from monocytes in vitro co‐culture with autologous MF‐ or BF‐spheroids was investigated in a cohort of HNSCC patients (nu2003=u200365) diagnosed between 1998 and 2005, all of whom were treated with curative intent by primary surgery. The IL‐6 response was expressed as a fraction of the lipopolysaccharid response of the same batch of monocytes. Recurrence, survival and causes of death were then established following the second part of 2005. MCP‐1 levels did not predict prognosis. We found that increased levels of IL‐6 from autologous monocytes in co‐culture with MF‐spheroids predicted recurrence with a hazard ratio (HR) of 1.5 [confidence interval (CI): 1.01–2.60; Pu2003=u20030.05] and co‐culture with BF‐spheroids and monocytes predicted recurrence (HRu2003=u20034.17; CI: 1.54–11.29; Pu2003=u20030.005). The same results where obtained in addition with TNM stage of the patients. Simultaneous analysis of BF‐ and MF‐spheroid co‐culture IL‐6 responses as well as adjustment for age and TNM stage of the patients allowed prediction of total survival (HRu2003=u20033.1; CI: 1.11–8.56; Pu2003=u20030.03) based on BF co‐culture levels. IL‐6 secreted upon in vitro co‐culture with monocytes and BF‐spheroids predicts recurrence and prognosis, whereas co‐culture with monocytes and MF‐spheroids predicts recurrence.

Human Autologous Monocytes and Monocyte‐Derived Macrophages in Co‐Culture with Carcinoma F‐Spheroids Secrete IL‐6 by aNon‐CD14‐Dependent Pathway

The secretion of interleukin (IL)‐1β, IL‐6 and tumour necrosis factor (TNF)‐α were compared when freshly isolated autologous monocytes or monocytederived macrophages (MDMs) were co‐cultured in vitro with autologous fragment (F)‐spheroids established from a series of head and neck squamous cell carcinoma (HNSCC) patients. F‐spheroids were generated from the malignant tumour (M‐spheroids) or from benign mucosa (B‐spheroids) from which the tumour originated control. If monocytes maturated towards MDMs before co‐culture, the IL‐6 secretion declined dependent on the extent of the MDM maturation by both M‐ and B‐spheroid stimulation. When MDMs maturated in continuous co‐culture, a steady‐state secretion of IL‐6 continued for several days but diminished when the culture medium was changed every 24u2003h. No co‐culture‐induced IL‐1β or TNF‐α was determined. Both the cytokine secretion and the mRNA gene expression revealed a different monocyte/MDM activation when co‐culture and lipopolysaccharide (LPS)‐stimulation were compared. Addition of anti‐CD14 (10u2003µg/ml) decreased monocyte LPS‐stimulated, but increased monocyte co‐culture stimulated IL‐6 secretion. In conclusion, M‐ and B‐spheroids similarly stimulated monocytes and to a lesser extent MDMs. MDMs that maturated with F‐spheroids present, retained responsiveness at the monocyte level. Co‐culture‐induced monocyte stimulation, as measured by IL‐6 secretion, was not dependent on activation via the CD14 molecule.

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.

Mechanisms for monocyte activation in co-culture with autologous tumor spheroids

Biopsies from carcinoma tissue and benign control mucosa from head and neck squamous cell carcinoma patients were used to establish fragment (F)-spheroids in vitro. We have previously shown that autologous monocytes co-cultured with F-spheroids in vitro secrete interleukin (IL)-6 upon 24h in co-culture. Presently, the aim was to study the mechanisms of this monocyte secretion. Paraformaldehyde (0.1% for 2min) or actinomycin-D (1 microg/ml for 24h) pre-treatment of the F-spheroids abolished the monocyte IL-6 co-culture response. Addition of glucose (100mM) or mannose (100mM), and to some extent galactose (100mM), but not fructose (100mM) to the co-cultures, partly inhibited the monocyte IL-6 co-culture response, but such addition did not inhibit the in vitro monocyte lipopolysaccharide (LPS)-generated IL-6 secretion. When mannose was added to the co-cultures, monocyte IL-6 mRNA expression was eradicated in malignant co-cultures and reduced to a low level in benign co-cultures. Addition of mouse anti-human beta(1)-integrin (anti-CD29) antibody (2 microg/ml) diminished the IL-6 co-culture response but not the monocyte LPS-generated IL-6 response. In conclusion, the monocyte IL-6 co-culture response is dependent on live spheroids and to some extent on direct contact with the F-spheroids, possibly via lectin-like receptor(s), the mannose receptor and beta(1)-integrin.

Monocyte and monocyte-derived macrophage secretion of MCP-1 in co-culture with autologous malignant and benign control fragment spheroids

Abstractu2002This study was performed in order to determine how monocytes and macrophages in co-culture with autologous head and neck squamous cell carcinoma (HNSCC) tumor tissue regulate the secretion of monocyte chemotactic protein-1 (MCP-1). The levels of MCP-1 were measured when autologous monocytes or monocyte-derived macrophages (MDMs) were co-cultured in vitro with autologous fragment (F)-spheroids established from HNSCC tumors or benign mucosa serving as control. MCP-1 secretion from co-culture stimulated monocytes and MDMs was increased compared to spontaneous MCP-1 secretion. With prolonged co-culture, MDMs showed a steady-state MCP-1 secretion above background levels for up to 96u2009h, even with change of co-culture media every 24u2009h. Addition of an anti-MCP-1 antibody to the medium decreased co-culture-induced monocyte IL-6 secretion. Addition of lipopolysaccharide (LPS) (1u2009μg/ml) reduced MCP-1 secretion compared to spontaneous secretion in monocyte cultures. F-spheroids also secrete MCP-1, but at insignificant levels compared to the MCP-1 secretion from monocytes and MDMs. MCP-1 secretion from monocytes/MDMs is regulated differently when co-culture stimulation is compared to LPS-stimulation. Monocytes and MDMs expressed MCP-1 mRNA at a high level in all tested conditions: stimulated in co-culture, not stimulated or stimulated with LPS, indicating post-transcriptional regulation of MCP-1 secretion. The secretion of MCP-1 from tumor-derived F-spheroids, and the maintenance of co-culture MCP-1 secretion from MDMs in vitro, suggests that tumor-associated macrophages are a source of MCP-1 in HNSCC tumors.

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

BackgroundOK-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-1α/β 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.ResultsOK-432 stimulated MNPs to secrete MCP-1 and MIP-1α/β in healthy individuals and in head and neck squamous cell carcinoma (HNSCC) patients, except for OK-432 stimulation of WB giving a minimal MIP-1α/β 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-1α production being increased upon Syk inhibitor addition and an increased MCP-1 response upon addition of both inhibitors. Adhesion may possibly involve β1 and/or β3 integrins, not β2, whereas β1–3 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-1β and MCP-1 production may occur upon binding to CD36.ConclusionAdherent human MOs produce MCP-1 and MIP-1α/β upon stimulation with OK-432. CD36 modulates MIP-1β 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-1α/β, in part explaining why OK-432 is suited as a biological response modifying drug.

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.

Conclusion 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). Objective 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. Material and methods 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)-α were determined. Results 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-α. 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.

TNF‐α is Secreted by Monocytes in Transit to become Macrophages, but not by Peripheral Blood Monocytes, following OK‐432 (Lyophilized S. pyogenes) Stimulation

OK‐432, penicillin‐killed Streptococcus pyogenes, is used in treating lymphangiomas and carcinomas. We have studied proinflammatory interleukin (IL) secretion following OK‐432 stimulation of total blood, peripheral blood mononuclear cell (PBMC) and purified monocytes in vitro. OK‐432 stimulation of purified monocytes gave IL‐1β, IL‐1RA, IL‐6, IL‐12p40 and tumour necrosis factor (TNF)‐α response. OK‐432 stimulation of cells within blood did, however, not yield TNF‐α secretion. When PBMC or monocytes were cultured in low‐attachment wells a decreased IL secretion was observed compared to adherent cells. Inhibition of Syk kinase with piceatannol, only at high, non‐specific doses, but not PI3 kinase inhibition with LY294002 or Wortmannin, decreased monocyte IL response to OK‐432. This shows that β1–3‐integrin receptor function is not necessary for monocyte OK‐432‐stimulated TNF‐α secretion. Direct blockage of the β2‐integrin (CD18) receptor by anti‐CD18 antibody was also unable to prevent the stimulating effects of OK‐432 in human monocytes. On the other hand, Syk phosphorylation is elevated upon adherence of monocytes and this is further increased by OK‐432 stimulation, as shown by Western blot. The Fc‐receptor was also ruled out as a main receptor of the OK‐432 monocyte response. In conclusion, TNF‐α secretion is only found in monocytes removed from blood. This TNF‐α secretion is not mediated through the β1–3‐integrin receptors. OK‐432 may act as a target‐seeking substance whereby only monocytes adhered, e.g. to a tumour cell, become cytotoxic in part explaining why OK‐432 is well suited as a cancer treatment drug.

In vitro cholesteatoma growth and secretion of cytokines

Abstract 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.