Macrophage and Tumor Cell Cross-Talk Is Fundamental for Lung Tumor Progression: We Need to Talk.

Regardless of the promising results of certain immune checkpoint blockers, current immunotherapeutics have met a bottleneck concerning response rate, toxicity, and resistance in lung cancer patients. Accumulating evidence forecasts that the crosstalk between tumor and immune cells takes center stage in cancer development by modulating tumor malignancy, immune cell infiltration, and immune evasion in the tumor microenvironment (TME). Cytokines and chemokines secreted by this crosstalk play a major role in cancer development, progression, and therapeutic management. An increased infiltration of Tumor-associated macrophages (TAMs) was observed in most of the human cancers, including lung cancer. In this review, we emphasize the role of cytokines and chemokines in TAM-tumor cell crosstalk in the lung TME. Given the role of cytokines and chemokines in immunomodulation, we propose that TAM-derived cytokines and chemokines govern the cancer-promoting immune responses in the TME and offer a new immunotherapeutic option for lung cancer treatment.

Epithelial cell plasticity defines heterogeneity in lung cancer.

Lung cancer is the leading cause of cancer death for both men and women and accounts for almost 18.4% of all deaths due to cancer worldwide, with the global incidence increasing by approximately 0.5% per year. Lung cancer is regarded as a devastating type of cancer owing to its high prevalence, reduction in the health-related quality of life, frequently delayed diagnosis, low response rate, high toxicity, and resistance to available therapeutic options. The highly heterogeneous nature of this cancer with a proximal-to-distal distribution throughout the respiratory tract dramatically affects its diagnostic and therapeutic management. The diverse composition and plasticity of lung epithelial cells across the respiratory tract are regarded as significant factors underlying lung cancer heterogeneity. Therefore, definitions of the cells of origin for different types of lung cancer are urgently needed to understand lung cancer biology and to achieve early diagnosis and develop cell-targeted therapies. In the present review, we will discuss the current understanding of the cellular and molecular alterations in distinct lung epithelial cells that result in each type of lung cancer.

N-3 vs. n-6 fatty acids differentially influence calcium signalling and adhesion of inflammatory activated monocytes: impact of lipid rafts.

BACKGROUND: Anti-inflammatory n-3 fatty acids (FA) like docosahexaenoic acid (DHA) opposed to the pro-inflammatory n-6 FA arachidonic acid (AA) might modulate lipid rafts within the cell membrane by differential incorporation. In inflammation, monocyte adhesion to endothelial cells is a crucial step mediated by intracellular calcium changes. We investigated whether lipid rafts mediate FA-induced modulation of adhesion and intracellular calcium. METHODS: In isolated human monocytes and monocytic U937 cells we measured adhesion to human umbilical vein endothelial cells (HUVEC) using a parallel flow chamber and a static assay, adhesion molecules by FACScan, and intracellular calcium by fluorescence. Monocyte lipid rafts were isolated by ultracentrifugation and submitted to gas chromatography for FA analysis. RESULTS: Pre-incubation with AA or DHA resulted in a predominant incorporation of the respective FA into raft compared to non-raft fraction. DHA as compared to AA significantly reduced monocyte adhesion and calcium release after stimulation with TNF-α while expression of adhesion molecules remained unchanged. Pre-treatment with a calcium chelator abolished the effect of FA on calcium and adhesion. Disruption of lipid rafts prevented FA-induced modulations. CONCLUSION: Incorporation of FA into lipid rafts seem to be crucial for modulation of adhesion under inflammatory conditions.

cAMP phosphodiesterase inhibitors increases nitric oxide production by modulating dimethylarginine dimethylaminohydrolases.

BACKGROUND: Pulmonary arterial hypertension is characterized by a progressive increase in pulmonary vascular resistance caused by endothelial dysfunction, inward vascular remodeling, and severe loss of precapillary pulmonary vessel cross-sectional area. Asymmetrical dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, and its metabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) play important roles in endothelial dysfunction. We investigated whether combined phosphodiesterase (PDE) 3 and 4 inhibition ameliorates endothelial function by regulating the ADMA-DDAH axis. METHODS AND RESULTS: We investigated the effects of the PDE3/4 inhibitor tolafentrine in vitro on endothelial cell survival, proliferation, and apoptosis. Effects of tolafentrine on the endothelial nitric oxide synthase/nitric oxide pathway, DDAH expression, DDAH promoter activity, and cytokine release from endothelial cells and their subsequent influence on DDAH expression were investigated. In monocrotaline-induced pulmonary arterial hypertension in rats, the effects of inhaled tolafentrine on DDAH expression and activity were investigated. Real-time-polymerase chain reaction, immunocytochemistry, and PDE activity assays suggested high expression of PDE3 and PDE4 isoforms in endothelial cells. Treatment of endothelial cells with PDE3/4 inhibitor significantly decreased ADMA-induced apoptosis via a cAMP/PKA-dependent pathway by induction of DDAH2. Chronic nebulization of PDE3/4 inhibitor significantly attenuated monocrotaline-induced hemodynamic, gas exchange abnormalities, vascular remodeling, and right heart hypertrophy. Interestingly, PDE3/4 inhibitor treatment reduced ADMA and elevated nitric oxide/cGMP levels. Mechanistically, this could be attributed to direct modulatory effects of cAMP on the promoter region of DDAH2, which was consequently found to be increased in expression and activity. Furthermore, PDE3/4 inhibitor suppressed apoptosis in endothelial cells and increased vascularization in the lung. CONCLUSION: Combined inhibition of PDE3 and 4 regresses development of pulmonary hypertension and promotes endothelial regeneration by modulating the ADMA-DDAH axis.

Induction of lymphocyte apoptosis in a murine model of acute lung injury--modulation by lipid emulsions.

Acute lung injury (ALI) and sepsis are the major causes of mortality in intensive care units. Lymphocytes apoptosis is a hallmark feature of late detrimental sepsis. Parenteral nutrition in critically ill patients is based on lipid emulsions, but the impact of ALI and lipid emulsions on lymphocytes has not been defined. The effects of intravenously infused conventional soybean oil (SO)-based and new olive oil (OO)-based emulsions on splenic and blood lymphocytes were investigated in a murine model of endotoxin-induced ALI. After LPS challenge and infusion of lipid emulsions, apoptosis of lymphocytes and lung injury were assessed by flow cytometry, Western blot, and histology. Induction of ALI led to a time-dependent decline in splenic and circulating lymphocyte numbers and an increase in apoptosis, with engagement of the extrinsic apoptotic pathway. Both SO- and OO-based emulsions promoted the apoptosis of splenic lymphocytes before induction of ALI. The OO-based emulsions exhibited lower proapoptotic activity than did SO-based emulsions, an observation paralleled by the induction of survival factors. Induction of ALI increased the mortality of mice receiving SO-based emulsions compared with OO-based emulsions and normal saline. Splenic lymphocyte apoptosis is apparent in murine ALI, which may be linked to detrimental outcome. Infusion of lipid emulsions per se provoked splenic lymphocyte apoptosis. Infusion of SO-based emulsions further augmented the apoptosis of splenic and circulating lymphocytes in ALI and led to increased mortality in mice. These findings may be of relevance for patients experiencing ALI that require parenteral nutrition.

Acute lung injury is reduced in fat-1 mice endogenously synthesizing n-3 fatty acids.

RATIONALE: Acute lung injury (ALI) remains an important cause of mortality in intensive care units. Inflammation is controlled by cytokines and eicosanoids derived from the n-6 fatty acid (FA) arachidonic acid (AA). The n-3 FA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and mediators derived from EPA and DHA possess reduced inflammatory potency. OBJECTIVES: To determine whether the ability of fat-1 mice to endogenously convert n-6 to n-3 FA, and thus generate an increased ratio of n-3 to n-6 FA, impacts experimental ALI. METHODS: We investigated ALI induced by intratracheal instillation of endotoxin in fat-1 and wild-type (WT) mice, assessing leukocyte numbers, protein concentration, and prostaglandin and cytokine levels in bronchoalveolar lavage fluid, as well as free FA in plasma, and lung ventilator compliance. Body temperature and motor activity of mice--markers of sickness behavior--were also recorded. MEASUREMENTS AND MAIN RESULTS: In ALI, fat-1 mice exhibited significantly reduced leukocyte invasion, protein leakage, and macrophage inflammatory protein-2 and thromboxane B(2) levels in lavage fluid compared with WT mice. Free AA levels were increased in the plasma of WT mice in response to endotoxin, whereas EPA and DHA were increased in the fat-1 group. Ventilator compliance was significantly improved in fat-1 mice. Body temperature and motor activity were decreased in ALI. fat-1 Mice recovered body temperature and motor activity faster. CONCLUSIONS: fat-1 Mice exhibited reduced features of ALI and sickness behavior. Increasing the availability of n-3 FA may thus be beneficial in critically ill patients with ALI.

Immunomodulation by n-3- versus n-6-rich lipid emulsions in murine acute lung injury--role of platelet-activating factor receptor.

OBJECTIVE: Cytokines, platelet-activating factor (PAF), and eicosanoids control local and systemic inflammation. Conventional soybean oil-based lipid emulsions used for parenteral nutrition may aggravate the leukocyte inflammatory response or adhesion to the vessel wall. Fish oil-based lipid emulsions, in contrast, may exert an anti-inflammatory effect. DESIGN: We investigated the impact of lipid emulsions on leukocyte invasion, protein leakage, and cytokines in two murine models of acute inflammation. SETTING: Research laboratory of a university hospital. SUBJECTS: Wild-type mice and PAF-receptor knockout mice. INTERVENTIONS: Mice received an infusion of normal saline, fish oil- or soybean oil-based lipid emulsions before lipopolysaccharide challenge. MEASUREMENTS AND MAIN RESULTS: Preinfusion with soybean oil resulted in increased leukocyte invasion, myeloperoxidase activity, and protein leakage and exaggerated release of tumor necrosis factor (TNF)-alpha as well as macrophage inflammatory protein (MIP)-2 into the alveolar space after intratracheal lipopolysaccharide challenge. In contrast, preinfusion with fish oil reduced leukocyte invasion, myeloperoxidase activity, protein leakage, and TNF-alpha as well as MIP-2 generation. Corresponding profiles were found in plasma following intraperitoneal lipopolysaccharide application: Soybean oil increased but fish oil decreased the TNF-alpha and MIP-2 formation. When PAF-receptor-deficient mice were challenged with lipopolysaccharide, leukocyte invasion, lung tissue myeloperoxidase, cytokine generation, and alveolar protein leakage corresponded to those observed in wild-type animals. Fish oil and soybean oil lost their diverging effects on leukocyte transmigration, myeloperoxidase activity, leakage response, and cytokine generation in these knockout mice. Similarly, the differential impact of both lipid emulsions on these lipopolysaccharide-provoked changes was suppressed after pretreating animals with a PAF-receptor antagonist. CONCLUSIONS: Fish oil- vs. soybean oil-based lipid infusions exert anti- vs. proinflammatory effects in murine models of acute inflammation. The PAF/PAF-receptor-linked signaling appears to be a prerequisite for this differential profile.

Activation of Ca2+ -activated potassium channels is involved in lysophosphatidylcholine-induced monocyte adhesion to endothelial cells.

OBJECTIVE: Ca(2+)-activated K(+)-channels (BK(Ca)) play an important role in lysophosphatidylcholine (LPC)-induced endothelial dysfunction. Aim of our study was to investigate whether LPC-induced activation of BK(Ca) is also involved in monocyte adhesion to endothelial cells (EC). METHODS AND RESULTS: Measurement of membrane potential (MP) was performed using the fluorescence dye DiBAC. Adhesion of the monocytotic cell line U937 to EC was analysed by (3)[H]-thymidine-adhesion-assay. Expression of ICAM-1 and VCAM-1 were analyzed by FACS. LPC induced a hyperpolarization of EC in a dose-dependent manner with the maximum seen with 2 microM. This was prevented by the BK(Ca)-inhibitor iberiotoxin (IBX, 100nM). Adhesion of U937 cells to EC was increased after stimulation of EC with LPC. This effect was time-dependent with the maximum seen after 4h. LPC-induced adhesion was significantly reduced when EC were co-incubated with IBX, or NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI, 5 microM) and also blocked by addition of 2-aminoethoxydiphenylborate (2-APB, 100 microM) or the calcium-chelator BAPTA (10 microM). Stimulation of U937 cells with LPC did not result in an increased adhesion to unstimulated EC. CONCLUSION: Activation of the endothelial BK(Ca) plays an important role in monocyte adhesion to endothelial cells.

Lipopolysaccharide-induced proliferation and adhesion of U937 cells to endothelial cells involves barium chloride sensitive hyperpolarization.

The adhesion of monocytes to the endothelium and their proliferation in the subendothelial space play an important role in atherosclerosis. Since the proliferation and migration of cells are influenced by the activity of ion channels, the aim of this study was to examine whether barium chloride (Ba(2+))-sensitive potassium channels (K(iCa)) are involved in lipopolysaccharide (LPS)-induced proliferation of monocytic U937 cells, and in the adhesion of these cells to endothelial cells. The adhesion of LPS-stimulated U937 cells to endothelial cells reached a maximum at a concentration of 5 microg/ml. This effect of LPS was completely abolished in the presence of Ba(2+) (100 micromol/l). In addition, LPS-induced proliferation was significantly reduced by Ba(2+) (control, 100%; LPS 5 microg/ml, 175%; LPS + Ba(2+) 100 micromol/l, 136%; n = 12, P < 0.05). To examine whether K(iCa) are activated by LPS, changes of U937 membrane potential were determined. LPS (5 microg/ml) caused a hyperpolarization of U937 cells indicating a flux of K(+) ions out of the cells. This effect was completely blocked by Ba(2+) (100 micromol/l). In conclusion, we demonstrate that LPS activates K(iCa) in U937 cells, which is responsible for LPS-induced adhesion of these cells to endothelial cells, and to the proliferation of U937 cells.

Statins inhibit hypoxia-induced endothelial proliferation by preventing calcium-induced ROS formation.

Pathological hypoxia plays an important role in many diseases, such as atherosclerosis, cancer, and rheumatoid arthritis. The aim of the present study was to examine the effects of different statins on hypoxia-induced endothelial cell signalling. Human umbilical cord vein endothelial cells (HUVEC) were treated with NaCN (CN, 2.5 mmol/l) to simulate a transient hypoxia. The CN-induced increase of endothelial cell numbers was significantly (n = 10, p < 0.01) reduced by the Ca(2+) chelator BAPTA (10 micromol/l), or the reactive oxygen species (ROS) scavenger N-acetylcysteine (ACC, 1 mmol/l), or the NAD(P)H-oxidase inhibitor diphenyleneiodonium (DPI, 5 micromol/l). In detail, cell numbers were (in percentage of control): 163.24 (CN), 90.06 (CN+ACC), 92.06 (CN+DPI). Intracellular-Ca(2+) and -ROS, analysed by fluorescence imaging, were significantly increased by CN. Interestingly, the CN-induced increase of ROS was in part Ca(2+)-dependent, whereas the Ca(2+) increase was not ROS-dependent. Simvastatin (5 micromol/l), fluvastatin (2.5 micromol/l), and cerivastatin (0.1 micromol/l) all reduced CN-induced proliferation, ROS generation and Ca(2+) increase. Cell viability was not reduced by the statins and the antiproliferative effect was completely reversed by mevalonate (500 micromol/l). In conclusion our study demonstrates that statins block hypoxia-associated endothelial proliferation by preventing the increase of Ca(2+) and ROS.