Lorella Lanza

Prednisone increases apoptosis in in vitro activated human peripheral blood T lymphocytes.

Glucocorticoid hormones (GCH) regulate, through the apoptotic process, the negative selection of immature T cells in the thymus. Because apoptosis seems to occur also in the maintenance of peripheral tolerance, we have investigated whether GCH may induce apoptosis in human mature lymphocytes. Peripheral blood lymphocytes (PBL) or peripheral CD4+ and CD8+ T cell subsets were cultured in the presence of phytohaemaglutinin (PHA) or PHA and prednisone (PDN) at 10−3‐10−12M concentrations for 72, 96 and 120h. Cell cycle and membrane antigen expression were evaluated by flow cytometry and DNA degradation was detected by agarose gel electrophoresis. PDN blocks PBL growth in the G1 phase of cell cycle and inhibits both IL‐2 receptor (IL‐2R) expression and IL‐2 secretion. Apoptosis is clearly increased by PDN in PHA‐activated human PBL, and the apoptotic effect of PDN is stronger on CD8+ than on CD4+ T lymphocytes. All these effects are dose‐ and time‐dependent. The addition of exogenous IL‐2 did not rescue lymphocytes from PDN‐increased apoptosis. These results show that PDN increases apoptosis in mature activated human peripheral blood lymphocytes, suggesting a possible role of GCH in the maintenance of immune tolerance at post‐thymic level.

Increased level of serum HLA class I antigens in HIV infection correlation with disease progression

Analysis of (sHLA-I) antigens in a large number of HIV-positive subjects found a significant increase of their level, but did not detect any change in their molecular profile. Monitoring at yearly intervals for four years of the sHLA-I antigen level in 14 HIV-positive subjects with a normal sHLA-I antigen level at study entry showed a significant correlation between progressive increase of sHLA-I antigen level and disease progression. Furthermore, a Kaplan-Meier plot of the frequency of development of AIDS in 34 patients whose cases were followed for 7 years showed that sHLA-I antigen level is a strong predictor of progression to AIDS. Its predictive value is comparable to that of serum beta 2-mu level, greater than that of serum neopterin, and lower than that of CD4+ T-cell percentage. The predictive value of sHLA-I antigen level in combination with serum beta 2-mu level, neopterin level, or CD4+ T-cell percentage is greater than that of each individual variable. These results suggest that measurement of the sHLA-I antigen level may provide useful prognostic information in HIV-positive subjects.

SOLUBLE HLA CLASS I AND CLASS II MOLECULE LEVELS IN SERUM AND CEREBROSPINAL FLUID OF MULTIPLE SCLEROSIS PATIENTS

Abstract Increased concentrations of soluble HLA class I and class II molecules (sHLA-I and sHLA-II) have been observed in infectious, inflammatory, and autoimmune diseases. Because autoimmune mechanisms are considered to play a role in the pathogenesis of multiple sclerosis (MS), we decided to dose sHLA-I and sHLA-II in serum and cerebrospinal fluid (CSF) of MS patients comparing their concentrations with those observed in serum and CSF of patients with other neurologic diseases (OND) without evidence of neuroradiologic involvement of central nervous system (CNS) and in serum of healthy donors. The serum concentrations of sHLA-I were higher in both MS and OND patients than in healthy donors (P < 0.05) whereas sHLA-II serum concentrations were lower in MS patients than in both OND patients and healthy donors (P < 0.01). Detectable amounts of sHLA-II were observed in the CSF of 45% of MS patients and in CSF of only 6% of OND patients (P < 0.001). In MS patients a significant correlation between sHLA-I serum and CSF concentrations was observed (P < 0.01), whereas sHLA-II serum and CSF levels did not correlate. In conclusion, alterations of sHLA-I and sHLA-II serum and CSF concentrations are present in MS patients and could be involved in the induction of enhanced susceptibility to develop MS or in MS pathogenesis.

IL‐15/IL‐15Rα Intracellular Trafficking in Human Cells and Protection from Apoptosisaa

ABSTRACT: IL‐15 is an immunostimulatory cytokine sharing with IL‐2 the IL‐2Rβγ complex. In vivo, IL‐15 detection in synovial fluids has been associated with the development of rheumatoid arthritis. A debate exists as to whether IL‐15 has the potential to be secreted in meaningful amounts or to act as a pericellular cytokine. Our data show (1) the presence of two IL‐15 isoforms displaying signal peptides of different length and the capacity to be secreted restricted to the isoform bearing the longer one; (2) in cells expressing the two isoforms, the existence of different nuclear localization and intracellular trafficking of IL‐15 and IL‐15Rα and (3) an intercellular microcirculation of IL‐15, not detectable with ELISA kits, but displaying a role as an anti‐apoptotic factor able to induce the deflection of the TNFR associated factor 2 (TRAF) to IL‐15Rα. Our data point to a juxtacrine mechanism of action of IL‐15 and suggest a role for IL‐15/IL‐15Rα in the regulation of apoptosis.

Immune regulatory properties of corticosteroids: prednisone induces apoptosis of human T lymphocytes following the CD3 down-regulation.

ABSTRACT: Glucocorticoid hormones (GCH) induce apoptosis in PHA‐primed peripheral blood T lymphocytes (PBL) and down‐regulate membrane‐bound proteins involved in the immune response. We have analyzed whether GCH are able to affect the expression of the TCR‐associated molecules CD3, CD4, and CD8 on PBL‐PHA, and whether the modulation of those receptors is related to the GCH‐driven apoptosis of the PBL‐PHA. Lymphocytes were cultured with PHA or with PHA plus prednisone (PDN) 10−3, 10−6, and 10−9 M. Then expression of CD2, CD3, CD4, CD8, and CD56 antigens was studied by cytofluorimetric assay using propidium iodide (PI) staining and annexin procedure, and by gel electrophoresis of low molecular weight DNA. PDN, at a pharmacological concentration (10−6 M), was able to inhibit the CD3 expression on T cells. The kinetics of CD3 decrement and of apoptosis show that the down‐regulation of CD3 molecules precedes DNA fragmentation and that the cells lacking CD3 are those prone to PDN‐induced apoptosis. The inhibition of CD3 is not related to a transcriptional or posttranscriptional phenomenon, because both PBL‐PHA and PBL‐PHA‐PDN expressed the same amount of intracytoplasmic CD3 molecule. PDN also induced a down‐regulation of the CD4 and CD8 molecules that resulted sooner in more intense CD8. In vitro PDN is able to induce apoptosis in PBL‐PHA through a down‐regulation of CD3 molecules.

Double‐stranded deoxyribonucleic acid binds to HLA class II molecules and inhibits HLA class II‐mediated antigen presentation

CD4+ T cells proliferating in response to purified double‐stranded deoxyribonucleic acid (dsDNA) have been recently demonstrated in peripheral blood mononuclear cells of patients with systemic lupus erythematosus. Their activation was inhibited by anti‐HLA class II (HLA‐II) monoclonal antibodies; thus, the existence of a molecular interaction between dsDNA and HLA‐II is conceivable. In this report we show that dsDNA specifically bind to HLA‐II. After preincubating cells with purified dsDNA or synthetic oligonucleotides, dsDNA was detected on the cell membrane and in the lysates of HLA‐II+ but not of isogenic HLA‐II− cell lines. We demonstrate that dsDNA binding inhibits that of a specific peptide to HLA‐II. Mixed lymphocyte reaction and antigen‐specific T cell proliferation were inhibited by the preincubation of stimulator cells or antigen‐presenting cells with dsDNA. These results suggest the existence of a novel mechanism of down‐modulation of the CD4+ T cell function generated by lack of stimulation due to the HLA‐II presenting molecules being “n;‐occupied” by dsDNA.

Interferons up-regulate with different potency HLA class I antigen expression in M14 human melanoma cell line. Possible interaction with glucocorticoid hormones

The relative potency of interferon α (IFNα), interferon β (IFNβ), and interferon γ (IFNγ) in inducing the expression of HLA class I antigens, as well as their capacity to counteract the inhibition induced by glucocorticoid hormones on HLA class I antigen expression, were analysed in the human melanoma cell line M14, both at membrane and at mRNA level. The data obtained indicate that (a) IFN enhance with different potency (IFNγ>IFNβ>IFNα) the expression of HLA class I antigens in M14 cells, (b) prednisone inhibits HLA class I antigen expresion, (c) glucocorticoid hormones, when associated with IFNα or IFNγ, inhibit the HLA class I enhancement induced by IFN alone, and ffinally, (c) the association between 1 μM prednisone or 1 μM deflazacort and IFNβ seems to potentiate the enhancing capacity of IFN on the expression of HLA class I molecules at the mRNA level. These findings, if confirmed, might indicate that IFN and glucocorticoid hormones are not mutually exclusive in the management of human melanoma.

Comparison of two glucocorticoid preparations (deflazacort and prednisone) in the treatment of immune-mediated diseases

SummaryDifferent glucocorticoid preparations modify the immune reaction in different ways. In this paper, the therapeutic efficacy of two glucocorticoids, deflazacort (DFZ) and prednisone (PDN), are discussed in relation to a group of 30 patients with systemic lupus erythematosus (n = 12) or rheumatoid arthritis (n = 18). The disease subgroups were divided into two arms, one of which was treated with DFZ and one with PDN in a double-blind protocol.The results of this study indicate that DFZ and PDN induced a clinical remission within 1 month which was maintained until the 6th month. Nevertheless, certain immunological modifications, including a significant reduction of the circulating T lymphocyte level and of the CD4/CD8 ratio, which was between 1 and 1,5 during the DFC treatment and between 1 and 2 during the PDN treatment, are more pronounced and more stable with DFZ than with PDN. Moreover, DFZ has a smaller effect on calcium and glucose metabolism than PDN since the serum glucose and calcium level of patients treated with PDN increased respectively from 90 up to 130 mg/dl and from 9,5 to 11,5 mg/dl whereas those of patients treated with DFC remained within the normal range. These findings indicate that DFZ may have advantages over PDN in the treatment of immune-mediated diseases.

S-adenosil-L-methionine is able to reverse the immunosuppressive effects of chenodeoxycholic acid in vitro

The study was conceived to evaluate if S-adenosil-L-methionine, a substance commonly used in the treatment of cholestasis in patients with cirrhosis and chronic hepatitis, exerts any immunological effect and of it is able to counterbalance bile acid-mediated immunosuppression. Proliferation and interleukin 2 and interferon-gamma secretion of human lymphocytes, collected from healthy subjects and exposed to mitogenic stimuli (phytohemagglutinin, pokeweed and anti-CD3 monoclonal antibodies), were analysed in the basal condition or after exposure to S-adenosil-L-methionine and/or chenodeoxycholic acid. Chenodeoxycholic acid inhibited phytohemagglutinin-induced lymphocyte proliferation and interferon-gamma secretion, and phytohemagglutinin and pokeweed-mediated interleukin 2 secretion. S-adenosil-L-methionine did not affect lymphocyte proliferation while it reduced interleukin 2 secretion upon phytohemagglutinin and pokeweed stimulation and interferon-gamma secretion upon all stimuli tested. Moreover, S-adenosil-L-methionine counteracted chenodeoxycholic acid-mediated inhibition of lymphocyte proliferation and interleukin 2 secretion. The results of our study confirm the immunosuppressive role of chenodeoxycholic acid on both secretive and proliferative lymphocyte functions and provide evidence of immunomodulatory activities of S-adenosil-L-methionine and its capacity to antagonize chenodeoxycholic acid-mediated inhibition of lymphocyte proliferation and interleukin 2 secretion.

CD4+ Th0 cell clones, isolated from a metastatic lymph node of a melanoma patient, possess cytolytic function

In the present study T lymphocytes isolated from a metastatic lymph node (T-LNL) of a melanoma patient have been cloned. In the attempt to verify whether T-LNL may acquire in vitro functional activities in the absence of tumour-associated antigens, they were cloned utilizing allogeneic lymphocytes as feeder cells. Nineteen clones generated from T-LNL proved to be CD4+ and, among these, five were able to kill autologous and allogeneic human melanoma cells in HLA-class-II-restricted way. On the basis of their cytokine production, these CD4+ cytolytic T-LNL clones were shown to belong to the Th0 subset and three of them expersseed the Vβ17 chain of the T cell receptor. These results suggest the presence of melanomaspecific but functionally inactive lymphocytes with T cell receptor oligoclonality in the lymph node environment. These specific T cells may acquire in vitro the capacity to kill autologous and allogeneic tumours without any induction by autologous melanoma cells.