Adriano Fontana

Melanoma cell expression of Fas(Apo-1/CD95) ligand : Implications for tumor Immune escape

Malignant melanoma accounts for most of the increasing mortality from skin cancer. Melanoma cells were found to express Fas (also called Apo-1 or CD95) ligand (FasL). In metastatic lesions, Fas-expressing T cell infiltrates were proximal to FasL+ tumor cells. In vitro, apoptosis of Fas-sensitive target cells occurred upon incubation with melanoma tumor cells; and in vivo, injection of FasL+ mouse melanoma cells in mice led to rapid tumor formation. In contrast, tumorigenesis was delayed in Fas-deficient lpr mutant mice in which immune effector cells cannot be killed by FasL. Thus, FasL may contribute to the immune privilege of tumors.

RORγt drives production of the cytokine GM-CSF in helper T cells, which is essential for the effector phase of autoimmune neuroinflammation

Although the role of the TH1 and TH17 subsets of helper T cells as disease mediators in autoimmune neuroinflammation remains a subject of some debate, none of their signature cytokines are essential for disease development. Here we report that interleukin 23 (IL-23) and the transcription factor RORγt drove expression of the cytokine GM-CSF in helper T cells, whereas IL-12, interferon-γ (IFN-γ) and IL-27 acted as negative regulators. Autoreactive helper T cells specifically lacking GM-CSF failed to initiate neuroinflammation despite expression of IL-17A or IFN-γ, whereas GM-CSF secretion by Ifng−/−Il17a−/− helper T cells was sufficient to induce experimental autoimmune encephalomyelitis (EAE). During the disease effector phase, GM-CSF sustained neuroinflammation via myeloid cells that infiltrated the central nervous system. Thus, in contrast to all other known helper T cell–derived cytokines, GM-CSF serves a nonredundant function in the initiation of autoimmune inflammation regardless of helper T cell polarization.

Increased Number of Islet-Associated Macrophages in Type 2 Diabetes

Activation of the innate immune system in obesity is a risk factor for the development of type 2 diabetes. The aim of the current study was to investigate the notion that increased numbers of macrophages exist in the islets of type 2 diabetes patients and that this may be explained by a dysregulation of islet-derived inflammatory factors. Increased islet-associated immune cells were observed in human type 2 diabetic patients, high-fat–fed C57BL/6J mice, the GK rat, and the db/db mouse. When cultured islets were exposed to a type 2 diabetic milieu or when islets were isolated from high-fat–fed mice, increased islet-derived inflammatory factors were produced and released, including interleukin (IL)-6, IL-8, chemokine KC, granulocyte colony-stimulating factor, and macrophage inflammatory protein 1α. The specificity of this response was investigated by direct comparison to nonislet pancreatic tissue and β-cell lines and was not mimicked by the induction of islet cell death. Further, this inflammatory response was found to be biologically functional, as conditioned medium from human islets exposed to a type 2 diabetic milieu could induce increased migration of monocytes and neutrophils. This migration was blocked by IL-8 neutralization, and IL-8 was localized to the human pancreatic α-cell. Therefore, islet-derived inflammatory factors are regulated by a type 2 diabetic milieu and may contribute to the macrophage infiltration of pancreatic islets that we observe in type 2 diabetes.

Murine brain macrophages induce NMDA receptor mediated neurotoxicity in vitro by secreting glutamate

Supernatants (SN) of brain macrophages in culture induce death of cerebellar granule cells in vitro, while those of astrocytes and endothelial cells do not. This toxicity can be prevented by N-methyl-D-aspartate (NMDA) receptor antagonists. Macrophage SN contain high concentrations of glutamate. Reducing the glutamate level of macrophage SN, either by exposure to astrocytes or by enzymatic degradation abolished the toxic effect. Thus, macrophage neurotoxicity is mediated by glutamate acting on NMDA receptors, and might play a role in vivo in traumatic and cerebrovascular brain lesions.

Complementary DNA for human glioblastoma-derived T cell suppressor factor, a novel member of the transforming growth factor-beta gene family.

Human glioblastoma cells secrete a peptide, termed glioblastoma‐derived T cell suppressor factor (G‐TsF), which has suppressive effects on interleukin‐2‐dependent T cell growth. As shown here, complementary DNA for G‐TsF reveals that G‐TsF shares 71% amino acid homology with transforming growth factor‐beta (TGF‐beta). In analogy to TGF‐beta it is apparently synthesized as the carboxy‐terminal end of a precursor polypeptide which undergoes proteolytic cleavage to yield the 112 amino‐acid‐long mature form of G‐TsF. Comparison of the amino‐terminal sequence of G‐TsF with that of porcine TGF‐beta 2 and bovine cartilage‐inducing factor B shows complete homology, which indicates that we have cloned the human analogue of these factors. It is tempting to consider a role for G‐TsF in tumor growth where it may enhance tumor cell proliferation in an autocrine way and/or reduce immunosurveillance of tumor development.

Anti-Fas/APO-1 antibody-mediated apoptosis of cultured human glioma cells. Induction and modulation of sensitivity by cytokines.

Fas/APO-1 is a transmembrane protein of the nerve growth factor/TNF alpha receptor family which signals apoptotic cell death in susceptible target cells. We have investigated the susceptibility of seven human malignant glioma cell lines to Fas/APO-1-dependent apoptosis. Sensitivity to Fas/APO-1 antibody-mediated cell killing correlated with cell surface expression of Fas/APO-1. Expression of Fas/APO-1 as well as Fas/APO-1-dependent cytotoxicity were augmented by preexposure of human malignant glioma cells to IFN gamma and TNF alpha. Further, pretreatment with TGF beta 2, IL1 and IL8 enhanced Fas/APO-1 antibody-induced glioma cell apoptosis whereas other cytokines including TNF beta, IL6, macrophage colony-stimulating factor, IL10 and IL13 had no such effect. None of the human malignant glioma cell lines was susceptible to TNF alpha-induced cytotoxicity. Fas/APO-1 antibody-sensitive glioma cell lines (n = 5), but not Fas/APO-1 antibody-resistant glioma cell lines (n = 2), became sensitive to TNF alpha when co-treated with inhibitors of RNA and protein synthesis. Resistance of human glioma cells to Fas/APO-1 antibody-mediated apoptosis was mainly related to low level expression of Fas/APO-1 and appeared not to be linked to overexpression of the anti-apoptotic protooncogene, bcl-2. Given the resistance of human malignant glioma to surgery, irradiation, chemotherapy and immunotherapy, we propose that Fas/APO-1 may be a promising target for a novel locoregionary approach to human malignant glioma. This strategy gains support from the demonstration of Fas/APO-1 expression in ex vivo human malignant glioma specimens and from the absence of Fas/APO-1 in normal human brain parenchyma.

T cell suppressor factor from human glioblastoma cells is a 12.5-kd protein closely related to transforming growth factor-beta.

T cell suppressor factor produced by human glioblastoma cells inhibits T cell proliferation in vitro and more specifically interferes with interleukin‐2 (IL‐2)‐dependent T cell growth. Here we report the purification of this factor from conditioned medium of the human glioblastoma cell line 308. Amino‐terminal sequence analysis of the 12.5‐kd protein demonstrates that eight out of the first 20 amino acids are identical to human transforming growth factor‐beta. Purified glioblastoma‐derived T cell suppressor factor and transforming growth factor‐beta from porcine platelets inhibit both IL‐2‐induced proliferation of ovalbumin‐specific T helper cells and lectin‐induced thymocyte proliferation with similar specific activities. If released by glioblastoma cells in vivo, the factor may contribute to impaired immunosurveillance and to the cellular immunodeficiency state detected in the patients.

Interleukin 1 Activity in the Synovial Fluid of Patients with Rheumatoid Arthritis

SummaryThe synovial fluids (SF) of patients with rheumatoid arthritis (RA) were investigated for their effects on thymocytes of C3H/HeJ mice. Of the 20 SF tested, 17 (85%) showed an augmentation of the phytohaemagglutinin (PHA) induced thymocyte stimulation. Out of 16 SF of patients with osteoarthrosis, such an activity was detected in only one (6.25%). Further characterisation of the amplification factor revealed that (1) the SF of RA patients augmented both the PHA and the Concanavalin A response of the thymocytes (2) in the absence of mitogens, SF-treated thymocytes showed an increased uptake of 3H-thymidine, (3) the SF did not propagate the growth of an interleukin 2 dependent ovalbumin specific T cell clone, but (4) the SF were found to be required for optimal interleukin 2 release by spleen cells stimulated with suboptimal doses of lectin. Based on these biological effects the factor in the SF of RA patients is suggested to represent an interleukin 1 (IL-1). IL-1 produced in cultures by activated macrophages has been shown to stimulate T and B cell functions and to induce the production of collagenase and prostaglandins by cultured synovial cells. Both properties of IL-1 could be relevant in the pathogenesis of RA.

IL-6-deficient mice resist myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is induced by immunization with myelin components including myelin oligodendrocyte glycoprotein (MOG). Myelin‐specific Th1 cells enter the central nervous system (CNS) via binding of very late antigen 4 (VLA‐4) to the endothelial vascular cell adhesion molecule 1 (VCAM‐1). In the present study, mice with a homologous disruption of the gene encoding IL‐6 are found to be resistant to MOG‐induced EAE as evidenced by absence of clinical symptoms, minimal infiltration of CD3+ T cells and monocytes into the CNS and lack of demyelination. The failure to induce EAE in IL‐6− / − mice is not due to the absence of priming, since lymphocytes of immunized IL‐6− / − mice proliferate in response to MOG and produce pro‐inflammatory cytokines including IL‐2 and IFN‐γ. However, in MOG‐immunized IL‐6− / − mice, serum anti‐MOG antibody titers were found to be drastically reduced. This observation is unlikely to be responsible for resistance to EAE, because B cell‐deficient (μMT) mice proved to be fully susceptible to the disease. A striking difference between MOG‐immunized wild‐type (wt) and IL‐6− / − mice was the expression of endothelial VCAM‐1 and ICAM‐1, which were dramatically up‐regulated in the CNS in wt but not in IL‐6− / − mice. Taking into account recent studies on the role of VCAM‐1 in the entry of Th1 cells into the CNS, the absence of VCAM‐1 on endothelial cells in IL‐6− / − mice may explain their resistance to EAE.

Protooncogene bcl-2 gene transfer abrogates Fas/APO-1 antibody-mediated apoptosis of human malignant glioma cells and confers resistance to chemotherapeutic drugs and therapeutic irradiation.

The majority of human malignant glioma cells express Fas/APO-1 and are susceptible to Fas/APO-1 antibody-mediated apoptosis in vitro. The sensitivity of Fas/APO-1-positive glioma cell lines to Fas/APO-1 antibody-mediated killing correlates inversely with the constitutive expression of the antiapoptotic protooncogene bcl-2. Here we report that BCL-2 protein expression of human glial tumors in vivo correlates with malignant transformation in that BCL-2 immunoreactive glioma cells were more abundant in WHO grade III/IV gliomas than in grade I/II gliomas. Fas/APO-1 antibody-sensitive human glioma cell lines stably transfected with a murine bcl-2 cDNA acquired resistance to Fas/APO-1 antibody-mediated apoptosis. Forced expression of bcl-2 also attenuated TNF alpha-mediated cytotoxicity of glioma cell lines in the presence of actinomycin D and cycloheximide and conferred partial protection from irradiation and the cancer chemotherapy drugs, cisplatin and BCNU. Preexposure of the glioma cell lines to the cytokines, IFN gamma and TNF alpha, which sensitize for Fas/APO-1-dependent killing, partially overcame bcl-2-mediated rescue from apoptosis, suggesting that multimodality immunotherapy involving cytokines and Fas/APO-1 targeting might eventually provide a promising approach to the treatment of human malignant gliomas.