Maria Antonia Frassanito

Th1 polarization of the immune response in Behçet's disease: a putative pathogenetic role of interleukin-12.

OBJECTIVE To investigate whether immunologic abnormalities in patients with Behçets disease (BD) are related to abnormalities of the Th1/Th2 ratio. METHODS Th1/Th2 cytokine production by peripheral blood lymphocytes (PBL) from 31 patients with BD, 11 patients with inflammatory arthritis, and 10 healthy blood donors was evaluated by intracellular immunofluorescence staining. Serum interleukin-12 (IL-12) levels were measured using an enzyme amplified-sensitivity immunoassay. The effect of recombinant IL-12 (rIL-12) on spontaneous and Fas-mediated apoptosis of phytohemagglutinin (PHA)-stimulated PBL was evaluated by flow cytometry using propidium iodide (PI) staining and a bromodeoxyuridine (BrdU)/PI procedure. RESULTS Intracellular immunofluorescence staining of IL-2, IL-4, and interferon-gamma (IFNgamma) in CD3+ lymphocytes from BD patients demonstrated a strong polarization of the immune response toward the Th1 pathway that correlated with the progression of BD. Peripheral Th1 cells were significantly increased in patients with active disease (n = 14) as compared with those in patients in complete remission (n = 17), patients with inflammatory arthritis, and normal donors. In addition, serum IL-12 levels were correlated with peripheral Th1 lymphocytes and disease progression. Apoptotic analysis revealed that PHA-activated PBL from patients with active disease were highly sensitive to spontaneous and Fas-mediated activation-induced cell death. However, addition of rIL-12 to complete medium prevented this spontaneous and Fas-induced apoptosis and enhanced the proliferation of Th1 lymphocytes. CONCLUSION Taken together, these results indicate that a strong Th1 immune response occurs in active BD and suggest that IL-12 plays a substantial part in the pathogenesis of BD. By preventing spontaneous and Fas-induced cell death, in fact, it results in an abnormal growth of autoreactive Th1 lymphocytes that could contribute to the prolonged inflammatory autoimmune condition of BD.

Overexpression of Fas antigen on T cells in advanced HIV-1 infection: Differential ligation constantly induces apoptosis

Objectives:To investigate Fas in peripheral lymphocytes from HIV-1-positive patients at different disease stages with respect to the extent of apoptosis. Design:The study included analysis of Fas involvement in T-cell apoptosis observed during HIV-1 infection. Because ligation of Fas can result in costimulation of proliferation or the induction of apoptosis in uninfected cells, we evaluated the effect on T cells of Fas activation by monoclonal antibodies (MAb) of different specificity from both UB2 and CH11 clones and activation by the Fas ligand (Fas-L). Methods:Fas was measured by FACS in peripheral blood and in phytohaemagglutinin (PHA)-driven cultures derived from 59 HIV-1-positive individuals with different Centers for Disease Control and Prevention stages. The percentage of apoptotic cells was detected by propidium iodide cell staining. The effect of Fas ligation was assessed in peripheral T cells from patients and healthy controls by a proliferative test measuring the 3H-thymidine uptake. Results:FACS analysis revealed that Fas was predominantly expressed in advanced disease, although it was promptly exposed in PHA cultures from asymptomatic individuals. In several instances, Fas overexpression was associated with substantial subdiploid DNA content in cells from severely lymphopenic patients. The proliferative assay showed a significant inhibition of 3H-thymidine uptake in T cells from all patients following Fas ligation by the immunoglobulin (Ig) G1 MAb from the UB2 clone. This was in contrast to the apparent cell activation detected in controls and the weak suppression observed in Fas-positive cell lines. In addition, the IgM anti-Fas and recombinant Fas-L concentrations inducing a moderate inhibition of fresh T cells from controls strongly depressed the proliferative rate of cells from patients. Conclusions:Our data suggest that Fas overexpression parallels the progression of the disease and that the increased susceptibility of T cells from HIV-1-infected individuals to undergo apoptosis may include a Fas pathway. Functionally exhausted T cells in advanced HIV-1 infection are primed to apoptosis because of their high sensitivity to Fas stimulation even using the IgG1 MAb, which is unreactive to the death domain of Fas. This suggests that the increased sensitivity of Fas is apparently unrelated to its trimeric ligation and supports the hypothesis that Fas pathway plays a role in increasing the lymphocyte apoptosis during the disease.

Deregulated cytokine network and defective Th1 immune response in multiple myeloma

Intracellular cytokine production by peripheral blood mononuclear cells (PBMC) was analysed in 51 patients with multiple myeloma (MM), 22 with monoclonal gammopathy of undetermined significance (MGUS) and 20 healthy subjects, as a parameter of immunological dysfunction in MM. An increased proportion of T cells and HLA‐DR+ cells producing IL‐6 was observed in MM patients with active disease (at diagnosis and relapsing) compared with patients in remission and with MGUS, whereas no difference of IFN‐γ+, IL‐2+ PBMC between patients and controls was evident. Determination of serum cytokine levels demonstrated that the imbalanced IL‐6 production by T cells and the defective anti‐tumour Th1 cell activity were related to elevated levels of IL‐6 and IL‐12. In vitro studies of PHA‐ and anti‐CD3/anti‐CD28 MoAbs stimulation of PBMC demonstrated the ability of lymphocytes from MM patients to differentiate towards the Th1 subset in the presence of rIL‐12. By contrast, addition of exogenous rIL‐6 impaired IFN‐γ production by rIL‐12‐prompted T cells. Inhibition of Th1 polarization of the immune response by IL‐6 was direct on T cells and not mediated by dendritic cells (DC). Evaluation of the ability of MM‐derived DC to stimulate cell proliferation of allogenic T lymphocytes and produce IL‐12 in vitro, in fact, suggested that MM‐derived DC were functionally active. Taken as a whole, these results indicate that a deregulated cytokine network occurs in active MM. They also suggest that increased IL‐6 production by peripheral T lymphocytes contributes to the immune dysfunction observed in MM, and enables tumour cells to escape immune surveillance by preventing the anti‐tumour Th1 immune response.

Antibody Production and In Vitro Behavior of CD27-Defined B-Cell Subsets: Persistent Hepatitis C Virus Infection Changes the Rules

ABSTRACT There is growing interest in the tendency of B cells to change their functional program in response to overwhelming antigen loading, perhaps by regulating specific parameters, such as efficiency of activation, proliferation rate, differentiation to antibody-secreting cells (ASC), and rate of cell death in culture. We show that individuals persistently infected with hepatitis C virus (HCV) carry high levels of circulating immunoglobulin G (IgG) and IgG-secreting cells (IgG-ASC). Thus, generalized polyclonal activation of B-cell functions may be supposed. While IgGs include virus-related and unrelated antibodies, IgG-ASC do not include HCV-specific plasma cells. Despite signs of widespread activation, B cells do not accumulate and memory B cells seem to be reduced in the blood of HCV-infected individuals. This apparent discrepancy may reflect the unconventional activation kinetics and functional responsiveness of the CD27+ B-cell subset in vitro. Following stimulation with T-cell-derived signals in the absence of B-cell receptor (BCR) engagement, CD27+ B cells do not expand but rapidly differentiate to secrete Ig and then undergo apoptosis. We propose that their enhanced sensitivity to BCR-independent noncognate T-cell help maintains a constant level of nonspecific serum antibodies and ASC and serves as a backup mechanism of feedback inhibition to prevent exaggerated B-cell responses that could be the cause of significant immunopathology.

Bone marrow fibroblasts parallel multiple myeloma progression in patients and mice: in vitro and in vivo studies

The role of cancer-associated fibroblasts (CAFs) has not been previously studied in multiple myeloma (MM). Here, cytofluorimetric analysis revealed higher proportions of bone marrow (BM) CAFs in patients with active MM (both at diagnosis and relapse) compared with patients in remission or those with monoclonal gammopathy of undetermined significance or deficiency anemia (controls). CAFs from MM patients produced increased levels of transforming growth factor-β, interleukin-6, stromal cell-derived factor-1α, insulin-like growth factor-1, vascular endothelial growth factor and fibroblast growth factor-2 and displayed an activated and heterogeneous phenotype, which supported their origin from resident fibroblasts, endothelial cells and hematopoietic stem and progenitor cells via the endothelial–mesenchymal transition as well as mesenchymal stem cells via the mesenchymal transition, as both of these processes are induced by MM cells and CAFs. Active MM CAFs fostered chemotaxis, adhesion, proliferation and apoptosis resistance in MM cells through cytokine signals and cell-to-cell contact, which were inhibited by blocking CXCR4, several integrins and fibronectin. MM cells also induced the CAFs proliferation. In syngeneic 5T33MM and xenograft mouse models, MM cells induced the expansion of CAFs, which, in turn, promoted MM initiation and progression as well as angiogenesis. In BM biopsies from patients and mice, nests of CAFs were found in close contact with MM cells, suggesting a supportive niche. Therefore, the targeting of CAFs in MM patients may be envisaged as a novel therapeutic strategy.

Deregulated expression of monocyte chemoattractant protein-1 (MCP-1) in arterial hypertension: role in endothelial inflammation and atheromasia.

Objective Arterial hypertension is recurrently associated with inflammation of the endothelium as an effect of the upregulation of functional molecules, including cytokines, adhesion molecules and chemokines. However, the role of monocyte chemoattractant protein-1 (MCP-1) in maintaining the inflammatory state of endothelial cells (EC) that leads to the progressive cardiovascular damage is unclear. Design Here, we investigated the expression of MCP-1, its major cell source as well as recurrence of a defined polymorphism (−2518 MCP-1) apparently linked to endothelial damage in several diseases. Methods Serum MCP-1 was measured by enzyme-linked immunosorbent assay (ELISA) in 740 hypertensive patients, subdivided according to their individual organ damage. Expression of both MCP-1 and its receptor CCR2 was evaluated in circulating ECs and macrophages by flow cytometry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), while gene variants of MCP-1 were revealed by PCR. Results Soluble MCP-1 was significantly elevated in patients with diffuse atheromasia. Furthermore, it was overexpressed by ECs activated to attract macrophages via the MCP-1/CCR2 pathway, whereas the −2518 MCP-1 polymorphism was correlated with atherosclerosis in most patients. Conclusions. Overexpression of MCP-1 is predominant in hypertensive patients with atheromasia in the form of a defined polymorphism. Measurement of MCP-1 may thus reflect the degree of endothelial damage, while early detection of such a polymorphism may acquire a prognostic value in the development of atherosclerosis.

Fas/Fas ligand (FasL)-deregulated apoptosis and IL-6 insensitivity in highly malignant myeloma cells

IL‐6 is a growth factor which interferes in the apoptosis of malignant plasma cells. Here we explore its role in the spontaneous and Fas/FasL‐regulated apoptosis of seven myeloma cell clones (MCC). MCC‐2 and ‐7 were constitutively defective in Fas antigen in the presence of large membrane exposure of FasL, and showed a high rate of cell proliferation irrespective of the presence of IL‐6. Cytofluorimetric analysis following propidium iodide (PI) staining revealed a minimal extent of spontaneous apoptosis, as in other IL‐6‐insensitive, though Fas‐positive MCC, namely MCC‐3 and ‐5. By contrast, a regular amplitude of apoptosis occurred in the remaining IL‐6‐dependent clones. Their propensity to cell death, as well as their FasL membrane expression, were promptly down‐modulated by the cytokine, whereas no substantial effect was detected in IL‐6‐independent MCC. Furthermore, we investigated the quantitative secretion of FasL. Both [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5diphenyl tetrazolium bromide] (MTT) cytotoxicity assay and PI staining of WC8 lymphoblasts from a Fas‐transfected mouse lymphoma, incubated with supernatants from MCC, showed a variable cytocidal property, thus confirming the cellular release of FasL. However, a significant elevation of FasL secretion occurred in both Fas− MCC, whereas molecular cloning and sequencing of Fas revealed the presence of a splicing variant, namely Fas Exo4,6Del, in the cDNA from both MCC‐3 and ‐5, which were previously demonstrated to be unresponsive to Fas stimulation. Taken together, these data provide evidence that concurrence of IL‐6 insensitivity and deregulation of apoptosis in myeloma cells reflects a high malignancy grade. It is suggested that the secretion of Fas splicing variants in Fas+ plasma cells, as well as the over‐production of FasL in Fas− myelomas, are differential mechanisms by which myeloma cells escape host immune surveillance.

Multiple myeloma exosomes establish a favourable bone marrow microenvironment with enhanced angiogenesis and immunosuppression

Multiple myeloma (MM) pathogenesis and progression largely rely on the cells and extracellular factors in the bone marrow (BM) microenvironment. Compelling studies have identified tumour exosomes as key regulators in the maintenance and education of the BM microenvironment by targeting stromal cells, immune cells, and vascular cells. However, the role of MM exosomes in the modification of the BM microenvironment and MM progression remains unclear. Here, we explored the functions of MM exosomes in angiogenesis and immunosuppression in vitro and in vivo. Murine MM exosomes carrying multiple angiogenesis‐related proteins enhanced angiogenesis and directly promoted endothelial cell growth. Several pathways such as signal transducer and activator of transcription 3 (STAT3), c‐Jun N‐terminal kinase, and p53 were modulated by the exosomes in endothelial and BM stromal cells. These exosomes promoted the growth of myeloid‐derived suppressor cells (MDSCs) in naive mice through activation of the STAT3 pathway and changed their subsets to similar phenotypes to those seen in MM‐bearing mice. Moreover, MM exosomes up‐regulated inducible nitric oxide synthase and enhanced the immunosuppressive capacity of BM MDSCs in vivo. Our data show that MM exosomes modulate the BM microenvironment through enhancement of angiogenesis and immunosuppression, which will further facilitate MM progression. Copyright

Alterations in the antigen processing-presenting machinery of transformed plasma cells are associated with reduced recognition by CD8+ T cells and characterize the progression of MGUS to multiple myeloma.

We hypothesized that progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) reflects the escape of transformed plasma cells from T-cell recognition because of impaired antigen processing-presenting machinery (APM). We studied plasma cells and CD8(+) T cells from bone marrow of 20 MGUS patients, 20 MM patients, and 10 control patients. Immunofluorescence and flow cytometry revealed significantly different patterns of APM component expression in plasma cells from the 3 groups. Compared with control patients, MM samples had lower expression of proteasome subunits and peptide transporters and greater expression of chaperones, considering both percentages of stained cells and molecular equivalents of soluble fluorochrome. MGUS samples had intermediate percentages of stained cells but molecular equivalents of soluble fluorochrome similar to control patients. Real-time polymerase chain reaction documented that APM changes occurred at the transcriptional level. Cytotoxicity assays demonstrated that MGUS CD8(+) T cells lysed autologous transformed plasma cells more than MM CD8(+) T cells did. MGUS progression correlated directly with calnexin, calreticulin, and tapasin and indirectly with delta, LMP2, and LMP10 expression levels; MM disease status did not correlate with APM levels. APM changes may allow transformed plasma cells to elude immunesurveillance in the MGUS-MM pathogenetic sequence.

Cancer associated fibroblasts and tumor growth: focus on multiple myeloma.

Cancer associated fibroblasts (CAFs) comprise a heterogeneous population that resides within the tumor microenvironment. They actively participate in tumor growth and metastasis by production of cytokines and chemokines, and the release of pro-inflammatory and pro-angiogenic factors, creating a more supportive microenvironment. The aim of the current review is to summarize the origin and characteristics of CAFs, and to describe the role of CAFs in tumor progression and metastasis. Furthermore, we focus on the presence of CAFs in hypoxic conditions in relation to multiple myeloma disease.