Marco Tucci

Glomerular accumulation of plasmacytoid dendritic cells in active lupus nephritis: role of interleukin-18.

OBJECTIVE Defective circulating dendritic cells (DCs) have been described in systemic lupus erythematosus (SLE) and correlated with high levels of interferon-alpha (IFNalpha). DCs are differentiated as being either myeloid or plasmacytoid, according to chemokine expression and the tendency to migrate toward inflamed tissue. We investigated the potential role of interleukin-18 (IL-18) in driving the glomerular migration of DCs in lupus nephritis (LN) and in affecting the ability of DCs to induce an imbalance in the Th1:Th2 ratio. METHODS DC subsets were characterized by flow cytometry and defined as either myeloid or plasmacytoid according to the expression of CD11c/blood dendritic cell antigen 1 (BDCA-1) and CD123/BDCA-2, respectively. The serum Th1:Th2 profile was studied by enzyme-linked immunosorbent assay. IL-18 receptor (IL-18R) and other chemokine receptors were analyzed by flow cytometry. Glomerular levels of IL-18/IL-18R and the presence of plasmacytoid DCs and myeloid DCs were investigated by immunohistochemical analysis. RESULTS The number of peripheral plasmacytoid DCs was decreased in patients with SLE compared with control subjects, and this defect in the number of DCs was correlated with LN. Patients with LN showed a prevalent Th1 response, with high production of IL-18, IL-12 and IFNgamma. Only plasmacytoid DCs expressed IL-18R. Patients with severe LN showed a high accumulation of IL-18 within glomeruli in association with the presence of plasmacytoid DCs, whereas myeloid DCs were almost absent. CONCLUSION A deficient number of peripheral plasmacytoid DCs correlated with high levels of Th1 cytokines and was associated with LN. Both serum and glomerular IL-18 were increased in LN. It is suggested that the high level of expression of IL-18R by peripheral plasmacytoid DCs allows the DCs to relocate within glomeruli under IL-18 stimulation and triggers the resident T cells, thus promoting renal damage.

Up-regulation of IL-18 and predominance of a Th1 immune response is a hallmark of lupus nephritis

There is evidence that nephritis is dominated by a Th1 immune response in systemic lupus erythematosus. Since IL‐18 promotes polarization of the immune response toward Th1, we investigated the role of this cytokine in lupus nephritis (LN). A total of 133 lupus patients and 44 healthy subjects were enrolled. Demographic and clinical characteristics with renal biopsy data were recorded. IL‐18 along with IFN‐γ and IL‐4, two prototypical of Th1 and Th2 cytokines, were measured in serum by ELISA. Peripheral blood lymphocytes were analysed by flow cytometry for IFN‐γ and IL‐4. IL‐18 expression was determined by immunohistochemistry in 13 renal biopsy specimens from patients with LN and 2 controls. Serum IL‐18 was higher in lupus patients than in controls. Levels of IL‐18 correlated with urinary microalbumin and were increased in patients with LN when compared to those without LN. IL‐18 expression was also increased within the glomeruli of nephritic patients and was primarily detected within the mesangial matrix and in infiltrating mononuclear cells. Measurement of IFN‐γ and IL‐4 in either sera or peripheral blood lymphocytes showed high IFN‐γ along with low IL‐4 expression in LN patients compared to patients without nephritis. A positive correlation between serum IL‐18 and IFN‐γ levels was found. IL‐18 may play a prominent role in the pathogenesis of LN by promoting a cytokine imbalance towards a Th1 immune response. Measurement of IL‐18 may be helpful for the early identification of lupus patients with LN and may help gauge the response to treatment in patients with active LN undergoing treatment.

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.

Overexpression of interleukin-12 and T helper 1 predominance in lupus nephritis.

Imbalance of cytokine homeostasis is a prominent feature of both experimental and human systemic lupus erythematosus (SLE). Because interleukin (IL)‐12 promotes interferon (IFN)‐γ production leading to polarization of peripheral cells toward a T helper (Th) 1 phenotype, we investigated its role in lupus nephritis (LN). Soluble Th1 and Th2 cytokines were measured by enzyme‐linked immunosorbent assay (ELISA) in sera and urines of SLE patients and controls. Th1/Th2 peripheral lymphocyte polarization was determined by flow cytometry. Glomerular accumulation of IL‐12 was evaluated by immunohistochemistry, whereas urinary IL‐12 was evaluated by ELISA. Higher serum IL‐12 levels in SLE were associated with LN, whereas IL‐4 was unrelated to the renal damage. Peripheral cells from LN patients showed a Th1 phenotype with a high IFN‐γ expression that paralleled the severity of renal damage. IL‐12 was present within glomerular mononuclear cells in classes IV and V LN, and its accumulation was correlated strongly with urinary levels. IL‐12 overexpression in SLE may contribute to the development of LN. Both serum and urinary IL‐12 elevation reflect its glomerular production and parallel Th1 polarization of peripheral T cells and high IFN‐γ production. In SLE patients, IL‐12 measurement may thus be predictive of the development of LN.

Upregulation of osteoblast apoptosis by malignant plasma cells: a role in myeloma bone disease

Summary. Typical features of multiple myeloma (MM) are osteolytic lesions and severely affected bone regeneration. This study of 53 MM patients demonstrates an enhancement of osteoblast cytotoxicity by malignant myeloma cells via the upregulation of apoptogenic receptors, including Fas ligand (Fas‐L) and tumour‐necrosis‐factor‐related apoptosis inducing ligand (TRAIL). Both were significantly increased in the marrow myeloma cells of patients with extensive osteolytic lesions in a fashion similar to the highly malignant human myeloma cell line MCC‐2. Osteoblasts from these subjects over‐expressed Fas and death receptor (DR) 4/5 and underwent dramatic apoptosis when co‐cultured with either MCC‐2 or autologous myeloma cells. In osteoblast and myeloma cell co‐cultures, monocyte chemoattractant protein 1 (MCP‐1) mRNA was upregulated in osteoblasts from patients with severe bone disease in parallel with increased CC‐chemokine receptor R2 (CCR2) expression, the ligand of MCP‐1, in the myeloma cells. This chemokine was shown to activate malignant cell migration in vitro. An upregulation of ICAM‐1 expression occurred in osteoblasts from patients with active skeleton disease. This upregulation appeared to be an effect of malignant plasma cell contact, as MCC‐2 co‐culture greatly enhanced ICAM‐1 production by resting osteoblasts from patients without skeleton involvement. Our results suggest that osteoblasts in active myeloma are functionally exhausted and promptly undergo apoptosis in the presence of myeloma cells from patients with severe bone disease. It is suggested that this cytotoxic effect plays a pivotal role in the pathogenesis of defective bone repair.

Induction of apoptosis by the hydrocarbon oil pristane: implications for pristane-induced lupus.

Intraperitoneal injection of the hydrocarbon oil pristane into normal mice leads to a lupus-like autoimmune syndrome. Although advances in defining the roles of cellular and humoral mediators involved in this syndrome have been made, the mechanisms that initiate a break in tolerance leading to autoimmunity remain unknown. We describe in this study that pristane induces apoptosis both in vivo and in vitro. Pristane arrests cell growth and induces cell death by apoptosis via the mitochondrial pathway of caspase activation in a dose-dependent manner. Nuclear autoantigens created by pristane-induced apoptosis of lymphoid cells within the peritoneal cavity in the setting of a profoundly altered cytokine milieu may be the initiating event in the development of autoimmunity in this syndrome. These findings suggest that apoptosis may be a critical initial event in the pathogenesis of pristane-induced lupus and are of potential relevance for human systemic lupus erythematosus.

Interleukin-18 overexpression as a hallmark of the activity of autoimmune inflammatory myopathies.

The objective of this study was to explore the role of interleukin (IL)‐18 in patients with inflammatory myopathies (IM) such as dermatomyositis (DM) and polymyositis (PM) in relation to the possible predominance of a Th1 immune response in their pathogenesis. Serum concentrations of IL‐18, interferon (IFN)‐γ, IL‐4 and IL‐6 were measured in six patients by enzyme‐linked immunosorbent assay (ELISA). IL‐18 expression was evaluated by in situ hybridization (ISH), whereas CD68, CD8 and CD83 were investigated by immunohistochemistry (IHC) to define the main producers of IL‐18. Lastly, the expression of both IL‐18 receptor (IL‐18R) and monocyte chemoattractant protein (MCP)‐1 was also explored by IHC. High serum levels of IL‐18 and IFN‐γ, and conversely low titres of IL‐4 and IL‐6, were demonstrated in both diseases. In addition, IL‐18 was overexpressed in muscle biopsy specimens from patients with IM. Both macrophages and dendritic cells (DC) surrounding either perivascular and perimysium areas in DM or endomysium in PM were the main producers of IL‐18. Endothelial cells (EC), smooth muscle cells (SMC) and CD8+ T cells expressed a high content of IL‐18R. Vessel cells overexpressed MCP‐1 in parallel with IL‐18R. High concentrations of serum IL‐18 as well as muscular up‐regulation of IL‐18 and IL‐18R suggest that deregulation of the IL‐18/IL‐18R pathway is a pathogenetic mechanism in IM. Measurement of IL‐18 may thus predict the severity of both DM and PM.

Cytokine Overproduction, T-Cell Activation, and Defective T-Regulatory Functions Promote Nephritis in Systemic Lupus Erythematosus

Lupus nephritis (LN) occurs in more than one-third of patients with systemic lupus erythematosus. Its pathogenesis is mostly attributable to the glomerular deposition of immune complexes and overproduction of T helper- (Th-) 1 cytokines. In this context, the high glomerular expression of IL-12 and IL-18 exerts a major pathogenetic role. These cytokines are locally produced by both macrophages and dendritic cells (DCs) which attract other inflammatory cells leading to maintenance of the kidney inflammation. However, other populations including T-cells and B-cells are integral for the development and worsening of renal damage. T-cells include many pathogenetic subsets, and the activation of Th-17 in keeping with defective T-regulatory (Treg) cell function regards as further event contributing to the glomerular damage. These populations also activate B-cells to produce nephritogenic auto-antibodies. Thus, LN includes a complex pathogenetic mechanism that involves different players and the evaluation of their activity may provide an effective tool for monitoring the onset of the disease.

The Interplay of Chemokines and Dendritic Cells in the Pathogenesis of Lupus Nephritis

Abstract: Lupus nephritis (LN) occurs in more that one‐third of patients with systemic lupus erythematosus. Production of nephritogenic autoantibodies, glomerular immune complex deposition, and cytokine overproduction have been postulated to contribute to the pathogenesis of LN. However, overexpression of chemokines and imbalance of dendritic cell (DC) homeostasis may contribute to the development of nephritis in SLE. We present evidence that monocyte chemoattractant protein (MCP)‐1 promotes renal disease in experimental glomerulonephritis, while its increased urinary levels reflect the severity of the disease in humans. Although macrophages are the prevalent infiltrating population within the kidney, it has been recently proposed that several chemokines and related receptors expressed by DCs may divide this cell population into myeloid (mDC) and plasmacytoid (pDC) subsets. However, the chemokine receptors expressed by pDCs are not functional, and other molecules are involved in the chemoattraction of these cells. We found increased expression of interleukin (IL)‐18 in glomeruli of patients with active LN along with glomerular infiltration by pDCS. Since pDCs bear IL‐18 receptor (IL‐18R), it is conceivable that circulating pDCs may migrate toward glomeruli by IL‐18/IL‐18R interactions. Therefore, the relative depletion of circulating pDCs reflects the severity of inflammatory disease in LN.

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.