Agata Nowicka

Evaluation of the phenotype pattern of macrophages isolated from malignant and non-malignant pleural effusions.

Macrophages are among the infiltrate components of most malignant tumors. Tumor-associated macrophages (TAMs) may secrete a variety of humoral factors, which promote or inhibit tumor growth. In general, depending on their activation pathway, macrophages exhibit two different patterns of phenotype, M1 or M2. It is assumed that TAMs comprise pattern M2. In the malignant pleural effusion, macrophages are a frequent component of cytological evaluation. In this microenvironment, TAMs could be involved in the development of immunity. The phenotype of macrophages represented in malignant and non-malignant pleural effusions is unknown. In this study, macrophages were isolated from 38 pleural effusions (15 malignant and 23 non-malignant) and the expression of a variety of immune mediators and their receptors was assessed to determine the type of activation (M1 vs. M2). The expression of mRNA was analyzed for IL-1β, IL-4, IL-6, IL-10, IL-11, IL-18, TNFα, TGFβ1, IL1R1, IL1RAP, TLR2, TLR4, VLA4, CD62L, MMP2, MMP9, VEGFA, PDGFA, and PDGFB. In immunohistochemical evaluation, the expressions of CD68, mesothelin, MAC387, IL-1β, IL-6, IL-10, IL-12, TNFα, and CD105 were assessed. The cytoplasmic expression of IFNγ, TNFα, IL-6, and IL-10 and the surface expression of CD11a, CD14, CD15, CD16, CD23, CD25, CD45, CD54, CD62L, CD69, VLA2, VLA3, VLA4, VLA6, TLR2, TLR4, and CCR7 were tested using flow cytometry. In supernatants from macrophages cultures, TNFα, IL-1β, IL-6, IL-8, IL-10, IL-12, MCP1, and VEGF were investigated by cytometric beads array method (CBA flex sets) and TGFβ1 by ELISA. Our results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other and suggest that macrophages isolated from non-malignant effusions show a pattern comparable to M1 while those isolated from malignant effusions express similarity to M2 phenotype, but they have not shown a classical M2 pattern.

Molecules of Damage-Associated Patterns in Bronchoalveolar Lavage Fluid and Serum in Chronic Obstructive Pulmonary Disease

Chronic exposure to detrimental environmental factors may induce immunogenic cell death of structural airway cells in chronic obstructive pulmonary disease (COPD). Damage-associated molecular patterns (DAMPs) is a family of heterogeneous molecules released from injured or dead cells, which activate innate and adaptive immune responses on binding to the pattern recognition receptors on cells. This study seeks to define the content of DAMPs in the bronchoalveolar lavage fluid (BALF) and serum of COPD patients, and the possible association of these molecules with clinical disease features. Thirty COPD in advanced disease stages were enrolled into the study. Pulmonary function tests, arterial blood gas content, 6-minute walk test, and BODE index were assessed. The content of DAMPs was estimated using the commercial sandwich-ELISA kits. We found differential alterations in the content of various DAMP molecules. In the main, BALF DAMPs positively associated with age, forced expiratory volume in one second (FEV1), and residual volume (RV); and inversely with PaO2, residual volume/total lung capacity (RV/TLC) ratio, and the disease severity staging. In serum, DAMPS positively associated with the intensity of smoking and inversely with age, PaO2, and TLC. In conclusion, DAMPs are present in both BALF and serum of COPD patients, which points to enhanced both local in the lung environment as well as systemic pro-inflammatory vein in this disease. These molecules appear involved with the lung damage and clinical variables featuring COPD. However, since the involvement of various DAMPs in COPD is variable, the exact role they play is by far unsettled and is open to further exploration.

Evaluation of Neutrophils Immunophenotype in the Microenvironment of Malignant Pleural Effusions

Abstract The lung cancer is often associated with the development of pleural effusion. Neutrophils are the most numerous population of immune system cells which are an essential component of tumor leukocyte infiltration. These cells are engaged in the development and maintenance of the inflammation. It is indicated that neutrophils support the development of cancer. The aim of the study was the evaluation of neutrophils, regarding their presence and activity in pleural effusions. This was achieved by assessing of molecular structures, which are used by neutrophils in chemotaxis and phagocytosis. 60 pleural effusions and 34 peripheral blood samples received from patients and 15 peripheral blood samples from the control group were analyzed. Expression of CD11a, CD11b, CD11c, CD18 and CD62L molecules with use flow cytometry was evaluated. The concentration of the neutrophil elastase in pleural effusions were measured with use ELISA test. The number of neutrophils in the peripheral blood of patients with pleural effusion was lower than that observed in the control group. Neutrophils present in pleural effusions were characterized by an increased ability to chemotaxis and secrete significant amounts of neutrophil elastase. Neutrophils recruited into the pleura during the formation of the effusion are an essential element of the developing inflammatory reaction in this environment. The presence of neutrophils in pleural effusion may promote its further formation and support the development of cancer.

Concentrations of SP-A and HSP70 are associated with polarization of macrophages in pleural effusions of non-small cell lung cancer

Damage-associated molecular pattern (DAMP) molecules can initiate an immune response through Toll-like receptors (TLRs). DAMPs are released from cells as a response to the extracellular danger and can be by-products of tissue damage. In cancer microenvironment necrotic cells release debris which has potency to become DAMPs. Non-small cell lung cancer (NSCLC) is often accompanied by pleural effusion (PE), which contains a variety of DAMPs. Surfactant protein A (SP-A) and heat shock protein 70 (Hsp70) are important DAMPs in the respiratory tract. The aim of this study was to determine a correlation between SP-A or Hsp70 and development of PE in the course of NSCLC. Moreover, we aimed to determine relationships between DAMPs and certain humoral factors associated with formation and persistence of PE as well as pleural-residing macrophages. In 34 PE samples, we estimated concentration of SP-A, Hsp70, IL-6, IL-18, G-CSF, M-CSF, SCF, SDF1α, VEGF as well as the fraction of macrophages and their pattern of polarization. We have found correlations between the concentration of the SP-A and Hsp70 and the percentage of PE-derived macrophages, also between concentrations of SP-A and Hsp70, and cytokines which participate in inflammation and processes involved in remodeling of extracellular matrix (ECM). Our data indicate an important role of SP-A during the development of PE associated with NSCLC. We suggest that measurement of concentration level of SP-A can be helpful in the course of diagnosis of malignant PE associated with NSCLC.

Pleural Macrophages can Promote or Inhibit Apoptosis of Malignant Cells via Humoral Mediators Depending on Intracellular Signaling Pathways

Abstract Macrophages in malignant pleural effusions (MPEs) demonstrate a promalignant phenotype. They release mediators, which are a source of inflammation within the pleura. We established in vitro model proving that pleural macrophages isolated from effusions affect cancer cells in their pro- or anti-apoptotic activity via humoral mediators. Additionally, we measured concentrations of selected transcription factors in cancer cells. Pleural macrophages can affect the apoptosis of cancer cells via intercellular mediators which trigger different signal transductors in cancer cells. The observed effect is connected to the composition of exudate which may vary depending on its origin, either malignant or nonmalignant.

Body Composition, Anthropometric Indices and Hydration Status of Obstructive Sleep Apnea Patients: Can Cachexia Coexist with Obesity?

The aim of this study was to elucidate body composition, anthropometric indices, and hydration status in obstructive sleep apnea (OSA) patients, taking into account different disease stages, gender, and the possibility of the presence of cachexia. There were 98 OSA patients and 23 control subjects enrolled into the study. All study participants underwent polysomnography examination. Body mass index (BMI), fat mass index (FMI), fat free mass, muscle mass, body cell mass, total body water, and extracellular and intracellular water were evaluated. The neck, abdominal, and waist circumference was measured. We found that overweight and obesity were present in 96% of patients. Cachexia was present in one OSA individual with comorbidities. Apnea-hypopnea index correlated with the neck and waist circumference, and with BMI in OSA patients. All muscle indices and water contents above outlined were significantly higher in severe OSA compared with control subjects. BMI, FMI, neck circumference, and extracellular water were greater in a subset of severe OSA compared with a moderate OSA stage. The female OSA patients had a higher FMI than that present in males at a comparable BMI. We conclude that the most body composition indices differed significantly between severe OSA patients and control subjects. A higher FMI in females at a comparable BMI could be due to a discordance between BMI and FMI. Cachexia occurs rarely in OSA and seems to coexist with comorbidities.