TGF-ß-mediated sustained ERK1/2 activity promotes the inhibition of intracellular growth of Mycobacterium avium in epithelioid cells surrogates.

Transforming growth factor beta (TGF-ß) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-ß production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-ß and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-ß produced by ECs are sufficient to induce a self-sustaining autocrine TGF-ß signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-ß to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-ß receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-ß produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-ß receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-ß production. In summary, our findings indicate that the amplitude of TGF-ß signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth.

Leukotriene B4 creates a favorable microenvironment for murine melanoma growth.

Chronic inflammation has long been associated with neoplastic progression. Our group had recently shown that the addition of a large number of apoptotic tumor cells to the tumor microenvironment induces a potent acute inflammatory reaction capable of promoting melanoma growth; however, primarily necrotizing cells do not cause such a reaction. Here, we show that potent inflammatory agents, such as lipopolysaccharide (LPS) and carrageenan, also promote growth of subtumorigenic doses of melanoma cells, having no effect on melanoma proliferation in vitro. Inhibition of 5-lipoxygenase (5-LOX) seems to have a pivotal role in this model because caffeic acid and MK886, a FLAP (5-LOX-activating protein) inhibitor, partially hindered tumor growth induced by apoptotic cells or LPS. Other enzymes of the arachidonic acid pathway, cyclooxygenase-1 and cyclooxygenase-2, seem to have no participation in this tumor promoter effect, as the inhibitor of both enzymes (indomethacin) did not alter melanoma growth. Leukotriene B4 (LTB4), the main product of the 5-LOX pathway, was able to induce growth of subtumorigenic inocula of melanoma cells, and a LTB4 receptor antagonist inhibited acute inflammation-associated tumor growth. Addition to the tumor inflammatory microenvironment of eicosapentaenoic acid, an omega3-polyunsaturated fatty acid with anti-inflammatory properties, or leukotriene B5, an eicosapentaenoic acid-derived leukotriene, significantly inhibited tumor development. These results give new insights to the mechanisms through which inflammation may contribute to tumor progression and suggest that LOX has an important role in tumor progression associated with an inflammatory state in the presence of apoptosis, which may be a consideration for apoptosis-inducing treatments, such as chemotherapy and radiotherapy.

Effects of transforming growth factor-beta in the development of inflammatory pseudotumour-like lesions in a murine model.

Alterations in transforming growth factor (TGF)-beta signalling have been frequently implicated in human cancer, and an important mechanism underlying its pro-oncogenic nature is suppression of the host antitumour immune response. Considering the immunosuppressive effect of TGF-beta, we asked whether human tumour cells, known to secrete TGF-beta in culture, would survive and grow when implanted into the peritoneal cavity of immunocompetent mice. Therefore, we developed a xenogeneic model where mice were intraperitoneally (i.p.) injected with a TGF-beta-secreting human colorectal adenocarcinoma cell line, LISP-A10. Although animals did not develop macroscopic tumours, the recovery and isolation of human tumour cells was achieved when an inflammatory environment was locally induced by the administration of complete Freund's adjuvant (CFA). This procedure significantly increased TGF-beta concentrations in the peritoneal fluid and was accompanied by impaired activation of the host-specific immune response against LISP-A10 cells. Furthermore, inflammatory lesions resembling human inflammatory pseudotumours (IPTs) were observed on the surface of i.p. organs. These lesions could be induced by either injection of LISP-A10 cells, cells-conditioned medium or recombinant TGF-beta but only after administration of CFA. In addition, host cyclooxygenase-2 and kinin receptors played an important role in the induction of TGF-beta-mediated IPT-like lesions in our experimental model.

Effect of electroacupuncture on bronchial asthma induced by ovalbumin in rats.

INTRODUCTION: Asthma is a worldwide disabling chronic inflammatory airway disease characterized by an intense eosinophilic inflammatory infiltrate on bronchial mucous membranes. Among the complementary therapeutic approaches to asthma, acupuncture has been widely used. OBJECTIVE: Here we used a rat pulmonary hypersensitivity experimental model that mimics human asthma in order to address whether electroacupuncture (EA) treatment could reduce the inflammatory process. MATERIALS AND METHODS: Experimental animals were divided in four groups: control (C), immobilized (I), sham-acupuncture (SA), and acupuncture (A). All rats were sensitized with heat-solidified hen egg white implant. Using clinical acupuncture points, EA treatment began 2 days after antigen priming and was repeated on alternate days for 2 weeks. Subsequently, animals were challenged by inhalation with aggregated ovalbumin and sacrificed 24 hours later when blood samples, bronchoalveolar lavage (BAL), and lungs were collected. RESULTS: Histopathologic analyses showed that peribronchial and perivascular inflammatory cell infiltrates were significantly lower in group A compared to groups SA and I (shown to be similar to group C). Furthermore, BAL total cell count and percentage of polymorphonuclears (as well as the differential counts of neutrophils and eosinophils) were significantly reduced in group A compared to group I. Corsticosterone plasma levels were similar in all groups. CONCLUSIONS: Taken together these results show that EA efficiently diminishes the bronchial immune-mediated inflammation induced in rats and that this effect is dependent on the choice of specific acupoints.

Murine macrophages cultured with IL-4 acquire a phenotype similar to that of epithelioid cells from granulomatous inflammation.

Epithelioid cells (ECs) found in granulomas are thought to derive from mononuclear phagocytes. Although GM-CSF and/or IL-4 are known to promote cell differentiation their role in the development of ECs has never been demonstrated. Here we showed that mouse macrophages treated exclusively with recombinant IL-4 (rIL-4) differentiate into epithelioid-like cells. Macrophages cultivated with rIL-4 presented a fried-egg shape, and ultrastructural studies revealed membrane interdigitations, cytoplasmic vesicles, prominent Golgi complex, and rough endoplasmic reticulum. Compared with controls, rIL-4 treated cells displayed increased expression of MHC class II molecules and of Migration Inhibitory Factor-Related Protein-14. Whereas mannose receptor-mediated phagocytosis was increased, Fcgamma-receptor mediated phagocytosis and the production of nitric oxide were decreased in treated cultures. All these features overlap those reported for ECs from granulomatous lesions. In conclusion, treatment of mouse peritoneal macrophages with rIL-4 drives their in vitro differentiation to an epithelioid phenotype and provides a tool to investigate the biology of ECs.