Pleural mesothelioma and lung cancer: the role of asbestos exposure and genetic variants in selected iron metabolism and inflammation genes.

Two of the major cancerous diseases associated with asbestos exposure are malignant pleural mesothelioma (MPM) and lung cancer (LC). In addition to asbestos exposure, genetic factors have been suggested to be associated with asbestos-related carcinogenesis and lung genotoxicity. While genetic factors involved in the susceptibility to MPM were reported, to date the influence of individual genetic variations on asbestos-related lung cancer risk is still poorly understood. Since inflammation and disruption of iron (Fe) homeostasis are hallmarks of asbestos exposure affecting the pulmonary tissue, this study aimed at investigating the association between Fe-metabolism and inflammasome gene variants and susceptibility to develop LC or MPM, by comparing an asbestos-exposed population affected by LC with an "asbestos-resistant exposed population". A retrospective approach similar to our previous autopsy-based pilot study was employed in a novel cohort of autoptic samples, thus giving us the possibility to corroborate previous findings obtained on MPM by repeating the analysis in a novel cohort of autoptic samples. The protective role of HEPH coding SNP was further confirmed. In addition, the two non-coding SNPs, either in FTH1 or in TF, emerged to exert a similar protective role in a new cohort of LC exposed individuals from the same geographic area of MPM subjects. No association was found between NLRP1 and NLRP3 polymorphisms with susceptibility to develop MPM and LC. Further research into a specific MPM and LC "genetic signature" may be needed to broaden our knowledge of the genetic landscape attributed to result in MPM and LC.

A genetic variant of NLRP1 gene is associated with asbestos body burden in patients with malignant pleural mesothelioma.

The presence of asbestos bodies (ABs) in lung parenchyma is considered a histopathologic hallmark of past exposure to asbestos fibers, of which there was a population of longer fibers. The mechanisms underlying AB formation are complex, involving inflammatory responses and iron (Fe) metabolism. Thus, the responsiveness to AB formation is variable, with some individuals appearing to be poor AB formers. The aim of this study was to disclose the possible role of genetic variants of genes encoding inflammasome and iron metabolism proteins in the ability to form ABs in a population of 81 individuals from North East Italy, who died after having developed malignant pleural mesothelioma (MPM). This study included 86 genetic variants distributed in 10 genes involved in Fe metabolism and 7 genetic variants in two genes encoding for inflammasome molecules. Genotypes/haplotypes were compared according to the number of lung ABs. Data showed that the NLRP1 rs12150220 missense variant (H155L) was significantly correlated with numbers of ABs in MPM patients. Specifically, a low number of ABs was detected in individuals carrying the NLRP1 rs12150220 A/T genotype. Our findings suggest that the NLRP1 inflammasome might contribute in the development of lung ABs. It is postulated that the NLRP1 missense variant may be considered as one of the possible host genetic factors contributing to individual variability in coating efficiency, which needs to be taken when assessing occupational exposure to asbestos.

Physical interactions between mast cells and eosinophils: a novel mechanism enhancing eosinophil survival in vitro.

BACKGROUND: Mast cells (MCs) and eosinophils (Eos) are the key effector cells of the allergic reaction. Although classically associated with different stages of the response, the cells co-exist in the inflamed tissue in the late and chronic phases in high numbers and are likely to cross-talk. While some mediators of MCs are known to affect Eos biology and vice versa, paracrine and physical interplay between the two cells has not been described yet. We aimed to investigate whether intercellular MC-Eos communication could take place in the allergic response and exert functional bidirectional changes on the cells. METHODS: Tissue sections from various allergic disorders were specifically stained for both cells. Human cord blood-derived MCs and peripheral blood Eos, co-cultured under different conditions, were studied by advanced microscopy and flow cytometry. RESULTS: Several co-localized MC-Eos pairs were detected in human nasal polyps and asthmatic bronchi, as well in mouse atopic dermatitis. In vitro, MCs and Eos formed stable conjugates at high rates, with clear membrane contact. In the presence of MCs, Eos were significantly more viable under several co-culture conditions and at both IgE-activated and steroid-inhibited settings. MC regulation of Eos survival required communication through soluble mediators but was even more dependent on physical cell-cell contact. CONCLUSIONS: Our findings provide the first evidence for a complex network of paracrine and membrane interactions between MCs and Eos. The prosurvival phenotype induced by this MC-Eos interplay may be critical for sustaining chronic allergic inflammation.

Actin-dependent tumour cell adhesion after short-term exposure to the antimetastasis ruthenium complex NAMI-A.

Imidazolium trans-imidazoledimethylsulphoxidetrachlororuthenate (NAMI-A) was tested in vitro on the pro-adhesive properties, evaluated as resistance to trypsin treatment, which is a bona fide measure of adhesion strength, of KB and HeLa carcinoma cell lines and on human polymorphonuclear neutrophils (HPMN). NAMI-A increased the pro-adhesive activity of KB cells at 0.001 mM concentration, after few minutes incubation and this effect was not influenced by the vehicle used for cell challenge, neither did it depend on NAMI-A concentration or on temperature. The same effect occurred on HeLa cells at 0.01 mM NAMI-A. This effect, detected at concentrations up to 100 times lower than those necessary to block cells at the G(2)-M premitotic phase of cell cycle, or to inhibit matrix metalloproteinase release or cell invasion, was not related to ruthenium uptake by tumour cells. HeLa cells and healthy HPMN, following short exposure to 0.1 mM NAMI-A, assumed a different shape, with the extrusion of filopodia (HeLa) and of large lamellopodia (HPMN), which increased their interactions with the substrate. This effect was attributed to stabilisation, altered turnover and sensitivity to cytochalasin D of actin filaments. Provided that adhesion is associated with cell motility and invasion, these data suggest that NAMI-A may exert antimetastatic properties at concentrations lower than those observed in the lungs at the end of a conventional intraperitoneal treatment in vivo.

Isolation of a murine metastatic cell line and preliminary test of sensitivity to the anti-metastasis agent NAMI-A.

We have isolated a new cell line (metGM) obtained from the spontaneous lung metastases of the mouse MCa mammary carcinoma. MetGM is a stable cell line which, after one year from its isolation, grows in vitro in suspension, forming cell aggregates, with cells that show irregular blabbing borders, active protein synthesis and convoluted nuclei and which have the capacity of invading matrigel membranes on which they give rise to a network of branching colonies. The preliminary study of the effects of the anti-metastasis ruthenium complex NAMI-A on metGM showed no direct cytotoxicity, with a mild reduction of cell proliferation, independent of the concentration of the ruthenium complex and not evident before 24 hours from treatment. A 10% DNA fragmentation was also measured on metGM cells 24 hours after challenge for 1 hour with 10(-5)M NAMI-A, suggesting that this compound is probably capable of apoptosis in a metastasis-derived cell line. Besides these effects on a limited percent of the cell population, NAMI-A changed the shape of the metGM cells and these alterations might account for the non-cytotoxic anti-metastatic properties of this innovative ruthenium complex. Thus MetGM appears to be a novel cell line suitable for the in vitro study of compounds endowed with anti-metastatic properties and for the development of new drugs with this activity.

In vitro growth of TS/A adenocarcinoma and of the gene transfected TS/A-IL4 line on biological substrates.

The growth capacity and adaptation of TS/A and TS/A-IL4 lines on laminin, fibronectin, collagens I and IV and matrigel compared to plastics were studied by flow cytometry. On plastic plates, TS/A-IL4 grows in vitro more slowly than the TS/A line and shows a more differentiated phenotype. TS/A-IL4 cells loose the capacity to bind lymphocytes and peroxidase positive cells obtained from mice implanted with the same tumour. The ratio between fibroblast- and epithelial-like cells of TS/A adenocarcinoma is subjected to marked modifications depending on the substrate on which the two cell lines are grown. IL4 release per cell unit is increased by collagen I as is the number of CD54 positive cells, suggesting that, at least in part, the in vivo rejection of TS/A-IL4 tumor might be ascribed to the stimulatory effect of the tissue on the IL4 release by tumor cells. The overall result is that gene modified TS/A-IL4 line shows marked changes of behaviour, most of them depending on the substrate on which tumor cells are growing.

Human neutrophils specifically interact with human monocyte-derived macrophage monolayers.

Neutrophils and macrophages express on their membrane molecules which may, in principle, interact with each other, promote specific cell to cell adhesion, affect cell function and finally, as a consequence, modulate the progression of the inflammatory process. We tested therefore if human neutrophils specifically adhere to human monocyte-derived macrophage monolayer (MDMM). Our findings show that neutrophils significantly adhere to 4-day old MDMM and that the extent of adhesion is increased by LPS-activation of MDMM. The specificity of the interaction was shown by the very low extent of adhesion of neutrophils either to freshly prepared monocyte or other types of cell monolayers and by the low percent of adhesion showed by eosinophils exposed to 7-day old MDMM. A role for beta2 integrins, CD31 and PAF-receptor in the mechanism of neutrophil-MDMM interaction is suggested by specific antagonists. We suggest that the adhesion between the two cell types could lead to an increase in concentration of neutrophil- or macrophage released factors in the interaction site and in a mutual modulation of phagocyte functions.

Paracrine effects of IL-4 transfection on TS/A adenocarcinoma cells mediate reduced in vivo growth.

The in vitro/in vivo growth capacity and phenotype of TS/A and the IL4-transfected TS/A-IL4 cell lines were studied by cell cycle analysis, expression of ICAM-1/CD54, transferrin receptor/CD71 and E-cadherin and by histology of the primary tumors. TS/A-IL4, unlike the TS/A line, shows in vitro a marked increase in the fibroblastoid cell type and a decreased E-cadherin expression. Administration of conditioned medium containing IL4 obtained from the TS/A-IL4 cell line, stimulates CD54 expression in the TS/A cell line. TS/A-IL4 tumors grow more slowly in vivo and are ultimately rejected. These processes are accompanied by a marked increase in collagen and extracellular matrix proteins and increased recruitment and degranulation of mast cells. The paracrine effect of IL4, released by the transfected tumor cells, might be responsible for the reduced in vivo growth of the TS/A cell line in the presence of TS/A-IL4 cells.

Low density lipoprotein-apheresis decreases oxidized low density lipoproteins and monocyte adhesion to endothelial cells.

The mutual interaction between monocytes and low density lipoprotein (LDL) in atherogenesis prompted a test of the hypothesis that LDL-apheresis could reduce the adhesive properties of monocytes to endothelium; and therefore interfere with a key mechanism in atheroma formation. Five patients affected by heterozygous familial hypercholesterolemia were studied. All patients received LDL-apheresis treatment with selective adsorption of LDL-cholesterol on dextran-sulphate columns. Low density lipoprotein particles were isolated by sequential preparative ultracentrifugation and subfractionated by ion exchange high performance liquid chromatography. Thiobarbituric acid reacting products of lipid peroxidation were measured fluorometrically. Vitamin E was estimated by high performance liquid chromatographic technique. Monocytes were isolated from patients blood before and 1 day after LDL-apheresis by Percoll gradient. The blood samples for monocyte adhesion were drawn from control subjects for 2 consecutive days. The adhesion of monocytes to an endothelial monolayer was evaluated by assaying the peroxidase content of the adherent monocytes. Low density lipoprotein-apheresis reduced total cholesterol (-65%; p < 0.01), LDL-cholesterol (-75%; p < 0.01), triglycerides (-51%; p < 0.05), and fibrinogen (-28%; p < 0.01). With LDL-apheresis treatment, a reduction of 54% in oxidized LDLs was observed; vitamin E concentration significantly increased in LDLs (+ 14.2%; p < 0.05). The monocyte adhesion decreased by approximately 61% after apheresis; the variation became statistically significant (-65%; p < 0.01) when endothelial cells were stimulated by lipopolysaccaride.

Human eosinophil peroxidase enhances tumor necrosis factor and hydrogen peroxide release by human monocyte-derived macrophages.

Inhibition of growth or eradication of experimentally induced tumors has been shown to be accompanied by infiltration of eosinophils and macrophages into the tumor mass. Since macrophages are important mediators of host antitumor activity, the possibility arises that a collaboration may exist between these two cell types in the control of tumor growth. In this study, we report the effect of eosinophil peroxidase (EPO), a basic protein contained in eosinophils that binds to several cell types including macrophages, on tumor necrosis factor (TNF) production and hydrogen peroxide release by human monocyte-derived macrophages. After incubation with EPO, the macrophages produced large amounts of TNF and displayed an enhanced phorbol 12-myristate 13-acetate-triggered hydrogen peroxide release. These effects were accompanied by an increased cell protein content and by morphologic changes leading the large, round macrophages of the control cultures to become elongated, pear-like or spindle shaped cells after treatment with EPO. The stimulatory effect of EPO on hydrogen peroxide release was insensitive to addition of exogenous catalase, a H2O2-degrading enzyme, suggesting that an extracellular catalytic activity of EPO was not involved. In addition, myeloperoxidase, the homologous peroxidase of neutrophils with a catalytic activity similar to that of EPO, was ineffective. The EPO-induced effects differed in several aspects from the effects of lipopolysaccaride and interferon-gamma, two well-known macrophage activators. These findings provide supportive evidence for a functional interrelationship between eosinophils and macrophages that may be physiologically relevant in the tumoricidal activity of macrophages.

Glycosylation improves the priming effect exerted by recombinant human granulocyte colony-stimulating factor (lenograstim) on human neutrophil superoxide production.

The role of glycosylation in modulating the activity of recombinant human granulocyte colony-stimulating factor (rHuG-CSF) on polymorphonuclear leukocytes (PMNs) was investigated. We addressed this study by comparing the effects of lenograstim (glycosylated rHuG-CSF) and its deglycosylated counterpart on superoxide production by PMNs on fibronectin. When the triggering activity of the cytokine was evaluated, no O2- release was elicited from neutrophils treated with either preparation of rHuG-CSF. Instead, a clear potentiation of both fMLP- and TNF-induced respiratory burst was produced by preincubating the cells with rHuG-CSF. Such effect was found to be significantly increased when glycosylated versus deglycosylated preparation was used, leading to the conclusion that the sugar moiety of the molecule could be of importance in improving the priming activity exerted by rHuG-CSF on PMN metabolic response.

Expression of lacto-N-fucopentaose III (CD15)- and sialyl-Lewis X-bearing molecules and their functional properties in eosinophils from patients with the idiopathic hypereosinophilic syndrome.

As the carbohydrate lacto-N-fucopentaose III (CD15 antigen or X-determinant) and its sialylated derivative sialyl-Lewis X are involved in the adhesion of cells rolling along the surface of endothelial cells, experiments were done to study the presence of these molecules on human eosinophils from patients with the idiopathic hypereosinophilic syndrome. Normal-density eosinophils from some patients showed higher levels of expression for lacto-N-fucopentaose III than light-density eosinophils. In contrast, sialyl-Lewis X was highly expressed by light-density eosinophils. Activation of normal-density eosinophils with calcium ionophore A23187 resulted in increased expression of these molecules for a short time. Monoclonal antibodies to these carbohydrates stimulated eosinophils to secrete eosinophil cationic protein, but not eosinophil peroxidase, and acted as costimulatory signals for C3b-induced degranulation of eosinophil cationic protein. It was suggested that CD15 and sialyl-Lewis X might contribute to eosinophil-mediated tissue injury in patients with the idiopathic hypereosinophilic syndrome.

A new, one-step assay on whole cell suspensions for peroxidase secretion by human neutrophils and eosinophils.

The degranulation of neutrophils and eosinophils is frequently monitored by assaying myeloperoxidase (MPO) and eosinophil peroxidase (EPO) activity in the cell-free supernatant of degranulating cells, after removal of the cells by centrifugation. This procedure leads to underestimation of the extent of degranulation, since both peroxidases tend to stick to cell surfaces, to test tube walls, and to particulate stimuli used to elicit degranulation, because of their highly cationic nature. In this paper we describe a method for assaying MPO and EPO secretion in whole cell suspensions that avoids separation of the cells from the incubation medium. The least toxic and thus safest among the sensitive peroxidase substrates, 3,3',5,5'-tetramethylbenzidine (TMB), was employed for peroxidase assay. The method we describe here is applied to the detection of peroxidase release by neutrophil and eosinophil cell suspensions incubated in either polypropylene test tubes or flat-bottomed microtiter plate wells. Because of the omission of the centrifugation step, the TMB method offers two major advantages over the currently used techniques: (1) higher estimates of degranulation, which permits the use of a smaller number of cells (in the microassay version, 150,000 neutrophils and 50,000 eosinophils) and smaller amounts of the secretagogues, and (2) rapidity, since the degranulation assay can be performed immediately on completion of the cell incubation with the secretagogue.

A simple and rapid method for isolation of eosinophilic granulocytes from human blood.

A simple and rapid method for the purification of morphologically and functionally intact eosinophils from human blood of both normal and eosinophilic subjects is described. The method is based on a single centrifugation of total blood leukocytes suspended in Percoll with specific gravity 1.0853 g/ml, after erythrocyte removal by dextran sedimentation. The peculiarity of this isolation technique is the maintenance of strictly physiological values of pH and osmolality throughout the entire procedure. Moreover, the cells are not subjected to measures aimed at changing the physical properties of either neutrophils or eosinophils. Because of such characteristics, this isolation method could be usefully exploited for comparative studies of normal and eosinophilic normodense eosinophils and of neutrophils and eosinophils from the same noneosinophilic subject.

Observations on the antimetastatic action of lysozyme in mice bearing Lewis lung carcinoma.

The effects of the oral administration of 100 mg/kg/day of lysozyme chloride on lung metastasis development were studied in mice bearing Lewis lung carcinoma. Lysozyme was administered to mice by supplying the daily amount of lysozyme with the powdered food. Lysozyme treatment reduces lung metastasis development, by significantly reducing the number of metastases of large dimension (diameters greater than 2mm) and by causing a significant increase of the percentage of animals free of large metastases, as compared with untreated controls. Correspondingly, the same animals show a pronounced increase of the number of multinuclear giant cells in the spleen; this parameter appears to be inversely correlated with the antimetastatic effect. These effects support the hypothesis that the antimetastatic effect of orally administered lysozyme depends upon spleen activation and perhaps upon induction of multinuclear giant cells of macrophage origin. This effect is consistent with previous findings indicating the occurrence of host-mediated effects in the antitumor action of lysozyme administered through the oral route.

Eosinophilic granuloma of the bone in Hand-Schüller-Christian disease: extensive in vivo eosinophil degranulation and subsequent binding of released eosinophil peroxidase (EPO) to other inflammatory cells.

The eosinophils from bone granuloma, bone marrow, and peripheral blood of a patient with Hand-Schüller-Christian disease (HSCD) were studied by electron microscopy and cytochemistry. Impressive eosinophil degranulation was observed. Extracellular release of eosinophil peroxidase (EPO) and EPO binding to surrounding cells were seen in the granuloma and bone marrow. Cells with peroxidase-positive plasma membrane were also observed in peripheral blood. The pattern of eosinophil degranulation showed quite different features from those described so far. In the granuloma, the process begins with intracytoplasmic release of the granule matrix content, as revealed by both extensive extragranular accumulation of EPO and progressive decrease of the matrix electron density. Core dissolution follows thereafter, leading to complete disappearance of the granules. At the end of the process, the cells show rupture of the plasma membrane and release of their content into the surrounding environment. This pattern of secretion was also observed in blood and marrow eosinophils of the patient. In view of the previously reported findings that EPO binding to inflammatory cells influences their functions, EPO release and binding to surrounding cells in HSCD may play a role in the evolution of the inflammatory lesion in the disease.

Evidence for host-mediated antitumor effects of lysozyme in mice bearing the MCa mammary carcinoma.

The host-mediated effects of lysozyme on primary tumor growth and on the formation of pulmonary metastases were investigated in mice bearing the MCa mammary carcinoma. The oral administration of lysozyme to CBA mice for 7 consecutive days before i.v. inoculation of MCa mammary carcinoma cells causes a significant reduction in the formation of lung tumors. The growth of s.c. tumors and the development of lung metastases is also significantly lowered in mice inoculated with tumor cells previously kept at 37 degrees C for 30 min in the presence of peritoneal resident cells or whole plasma samples obtained from normal mice treated with 25-100 mg/kg/day lysozyme for 7 consecutive days. The lysozyme concentration in plasma samples of the treated mice remains undetectable even at daily dosages up to 400 mg/kg, ruling out the hypothesis of a direct effect of the ingested lysozyme. These data seem to suggest a role for host immune reactivity in the antineoplastic effects of lysozyme. The results are consistent with previously reported data and further stress the interesting antitumor properties of the oral administration of lysozyme in mice bearing solid metastasizing tumors.

Phagocytosis of Campylobacter jejuni and C. coli by peritoneal macrophages.

Guinea-pig resident peritoneal macrophages had no activity against freshly isolated Campylobacter jejuni, whilst C. coli was phagocytosed and killed. The number of bacteria killed by macrophages always exceeded the number of those ingested, suggesting an extracellular mechanism of killing.