Modulation of the free sphingosine levels in Epstein Barr virus transformed human B lymphocytes by phorbol dibutyrate.

The amounts of free sphingosine in Epstein Barr virus transformed B lymphocytes (EBV-B) treated with sphingosine and phorbol-12,13-dibutyrate (PD) has been quantified by high performance liquid chromatography (HPLC). PD treatment did not affect intracellular sphingosine level, while it seems to lessen the removal of this long chain base in sphingosine-treated EBV-B cells. The previous results relative to sphingosine-dependent changes in choline-metabolite levels have to be interpreted on the basis of these results.

Modulation of human lymphoblastoid B cell line by phorbol ester and sphingosine. A 31P-NMR study.

Changes in phospholipid and energy metabolism in Epstein-Barr Virus transformed B lymphocytes (EBV-B), induced by phorbol 12,13-dibutyrate (PD) and sphingosine (an inhibitor of protein kinase C), have been evaluated by 31P-NMR spectroscopy. The effects of PD and sphingosine on [3H]thymidine incorporation have also been studied. An increase in phosphorylcholine (PCho) levels has been observed in sphingosine and sphingosine + PD treated cells after 30 min of incubation, whereas no change was observed in lymphocytes incubated with PD during the same period. Extracellular choline levels increased in sphingosine treated cells but decreased in PD treated cells. Hence, a sphingosine-dependent hydrolysis of choline-linked phospholipids is suggested. A time-dependent reduction of PCho observed after 120 min PD incubation is consistent with an increase of the synthesis of choline-linked phospholipids.

Dexamethasone-dependent modulation of human lymphoblastoid B cell line through sphingosine production.

The relationship between dexamethasone-dependent changes in intracellular sphingosine levels, energy and phospholipid metabolism have been investigated by 31P-NMR spectroscopy and high-performance liquid chromatography. The cellular functions have been evaluated by cellular growth and immunoglobulin M secretion (IgM). Significant increases in intracellular phosphorylcholine (PCho), extracellular choline (Cho), and endogenous sphingosine levels were observed only at 30 min incubation with dexamethasone. These results confirmed a sphingosine-dependent hydrolysis of choline-linked phospholipids (Miccheli, A., Ricciolini, R., Piccolella, E., Delfini, M. and Conti, F. (1991) Biochim. Biophys. Acta 1093, 29-35). Furthermore, no significant variations were evidenced at hours 1, 2, 6 and 18 of incubation. Dexamethasone causes an inhibition of cellular growth and IgM secretion as well as the sphingosine treatment. The results suggest that the effect of dexamethasone may be mediated by endogenous sphingosine production in Epstein-Barr virus transformed B lymphocytes.

Dexamethasone-dependent modulation of cholesterol levels in human lymphoblastoid B cell line through sphingosine production.

The effect of dexamethasone on lipid composition of Epstein-Barr virus transformed human B lymphocytes have been investigated by 31P- and 1H-NMR spectroscopy and compared to the effects due to exogenous sphingosine treatment. Furthermore, the effects of dexamethasone and sphingosine on membrane structure was evaluated by fluorimetry. No significant changes were evidenced in phospholipid composition and in the ratio of unsaturated to total fatty-acid chains. A significant increase in total cholesterol levels was evident at 30 min incubation with dexamethasone or sphingosine; a parallel increase in DPH polarization at 30 min was also demonstrated. TMA-DPH intensity measurements suggest a slowing of vesicular intracellular traffic due to the treatment. The results suggest a dexamethasone- and sphingosine-dependent inhibition of intracellular cholesterol transport.

Dexamethasone induces apoptosis in human T cell clones expressing low levels of Bcl-2.

Previous results of ours have demonstrated that the same clonotype can express both a sensitive and a resistant phenotype to Dex-mediated PCD induction depending on its cell cycle phase. In particular, we demonstrated that human T lymphocytes, arrested in the G0/G1 phase of the cell cycle, are susceptible, while proliferating T cells are resistant to Dex-mediated apoptosis. In this paper, we have further characterized the sensitive and resistant phenotypes and investigated whether a different expression of the apoptotic genes Fas, FasL, Bcl-2, Bcl-x and Bax is involved in the regulation of Dex-mediated apoptosis. The results show that the amount of Bcl-2 expression, that changes during cell cycle phases, determines susceptibility or resistance to apoptosis induced by Dex. In fact, undetectable expression of Bcl-2 in sensitive cells favors Dex-mediated apoptosis while high expression of Bcl-2 in proliferating cells counterbalances apoptosis induction. Moreover, the addition of exogenous IL-2, in the presence of Dex, fails to up-regulate Bcl-2 expression and to revert Dex-mediated apoptotic phenomena.

CD4+ T cells orchestrate both amplification and deletion of CD8+ T cells.

This review focuses on the role of CD4+ T cells in regulating immune responses, orchestrating both the amplification and deletion of immune cells, particularly CD8+ T cells. These two functions, which represent only an apparent contradiction, appear to be two faces of the same process of regulation. In fact, because the immune response, once activated, needs to be carefully controlled or switched off when the antigenic stimulus is eliminated, the immune system has developed several strategies either to regulate clonal amplification or to avoid useless expansion of activated cells. In particular, we have reported many data demonstrating that CD4+ T cells may be indicated as the regulatory element in the activation as well as the deletion of CD8+ T cells. New data are also reported on the ability of anergic CD4+ T cells to suppress CD8+ T-cell activation through induction of apoptosis, and on the need for CD8+ T cells for antigen recognition in inducing cell death in CD4+ T cells. Moreover, the central role of CD4+ T cells in the maintenance of peripheral tolerance has been widely described.

Gene activating and proapoptotic potential are independent properties of different CD4 epitopes.

CD4 engagement triggers an early signaling cascade which initiates late events such as transcription factor activation. The outcome of CD4 engagement is T-cell commitment to alternative, dramatically different fates, such as activation and apoptosis. We have tested a panel of anti-CD4 mAbs specific for different CD4 epitopes, as well as HIV-1 gp120, for the capacity to activate crucial early events such as enhancement of p56(lck) kinase activity and Shc phosphorylation. The same CD4 epitopes were characterized for their capacity both to deliver a gene activating signal and to program T-cells to activation dependent death. No correlation could be found between capacity of specific CD4 epitopes to deliver a gene activating signal and capacity to prime T-cells to apoptosis, suggesting that gene activating and proapoptotic potential are independent functions of CD4 epitopes. Furthermore, while triggering of the calcium pathway appears critical in NF-AT activation, optimal p56(lck) activation and Shc phosphorylation might be required for initiation of the apoptotic pathway.

Effect of caffeic acid on tert-butyl hydroperoxide-induced oxidative stress in U937.

Nonvitamin phenolic compounds are ubiquitous in food plants and therefore potentially present in human plasma in a diet-dependent concentration. The aim of this study was to evaluate the ability of caffeic acid, a phenolic acid with antioxidant activity, to affect cellular response in U937 human monocytic cells to t-butyl hydroperoxide-induced oxidative stress. In our experimental conditions caffeic acid was incorporated into cells without any cytotoxic effect. Caffeic acid-treated cells showed an increased resistance to oxidative challenge, as revealed by an higher percent of survival and the maintenance of an higher proliferative capacity in respect to control cells. This effect seems to be due to the ability of caffeic acid to reduce glutathione depletion and to inhibit lipid peroxidation during tBOOH treatment. It can be concluded that caffeic acid exerts an antioxidant action inside the cell, responsible for the observed modulation of the cellular response to oxidative challenge. Due to its presence in the diet, therefore, caffeic acid may play a role in the modulation of oxidative processes in vivo.

Peptide analogues as a strategy to induce tolerance in T cells with indirect allospecificity.

BACKGROUND: It has been demonstrated that indirect recognition of allogeneic MHC molecules might play an important role in provoking graft rejection. Although direct recognition of allogeneic molecules on antigen presenting cells of the graft may induce a state of tolerance, the continuous presentation of processed alloantigens by specialized antigen presenting cells does not allow the same phenomenon to occur. Tolerance to interleukin-2 secreting T cells can be achieved in different ways, among these is the exposure to mutants of the wild type allopeptide. We have investigated whether peptide analogues of the allopeptide can induce tolerance in T cells with indirect allospecificity. METHODS: T cell clones with indirect anti-HLA-A2-specificity generated from a HLA-A2-DRB1*1502+ patient who chronically rejected a HLA-A2-expressing kidney allograft were used for this study. Nine peptide analogues of HLA-A2 (residues: 103-120) were produced with single amino acid substitutions at the putative T cell receptor for antigen contact positions. Their effect on the proliferation of a panel of T cell clones was evaluated. RESULTS: Peptide analogues and wild type peptide had similar capacity to bind to the restriction molecule HLA-DRB1*1502. Co-presentation of the peptide analogues 111R/A, H, K and 114H/K, with the wild type peptide inhibited T cell responses, indicative of antagonism. In addition, one analogue 112G/S induced unresponsiveness in the T cells to subsequent culture with the wild type peptide. CONCLUSIONS: The data presented here suggest that using reagents such as altered peptides may represent a strategy to prevent the activation of T cells with indirect alloreactivity and allograft rejection in vivo.

Different regions of the N-terminal domains of HLA-DR1 influence recognition of individual peptide-DR1 complexes.

The contributions of individual amino acids in the polymorphic beta chain and the conserved alpha chain of HLA-DR1 to influenza HA-specific DR1-restricted and anti-DR1 allospecific T-cell recognition were analyzed. The genes encoding HLA-DR1 were subjected to site-directed mutagenesis in order to introduce single amino acid substitutions at 12 positions in the beta 1 domain and 11 positions in the alpha 1 domain. The beta 1-domain substitutions were all at polymorphic positions and introduced residues that are found in DR4 alleles. The amino acids introduced into the DR alpha 1 domain were based on the sequences of other human and mouse class II alpha chains. The responses of 12 DR1-restricted T-cell clones specific for two peptides of HA and seven anti-DR1 allospecific clones were studied. Substitutions at positions that point up from and into the peptide-binding site in the third variable region of the beta 1-domain alpha-helix caused substantial reduction in the responses of all of the clones. Substitutions at multiple positions in the beta 1-domain floor and in the alpha 1 domain influenced the anti-DR1 responses of the alloreactive and of the HA100-115-specific T-cell clones. In contrast, very few changes outside of the beta 1 domain third variable region affected the responses of the HA306-324-specific DR1-restricted T-cell clones. These results suggest that a surprisingly limited region of the HLA-DR1 molecule is critically involved in T-cell recognition of HA306-324 by DR1-restricted T cells. However, the susceptibility of the HA100-115-specific and the anti-DR1 allospecific T-cell clones to substitutions at multiple positions in both N-terminal domains shows that the response to DR1-HA306-324 is unusual and may reflect the promiscuity with which this peptide binds to HLA-DR molecules.

Differential effect of alpha-tocopherol and ascorbate on oxidative injury induced in immune cells by thermal stress.

As immune cells are often subjected to hyperthermia that can easily occur either after intense and/or prolonged exercise or during defense against pathogens, in this paper we analysed whether superoxide anion production occurred in lymphocytes exposed to high temperature and, consequently, if antioxidants could exert any protective function. The results demonstrated that an increase of superoxide anion was induced in rabbit lymphocytes exposed to 42 degrees C for 1h, although cell viability was no affected. However, suppression of either Pokeweed mitogen (PWM)-driven cell proliferation, or immunoglobulin production or IL-2 synthesis was observed. To evaluate the capacity of antioxidants to restore the immune suppressed responses, two vitamins, alpha-tocopherol and ascorbic acid, were added to PWM-stimulated cultures following heat treatment. The data demonstrated that alpha-tocopherol was able to totally abrogate the inhibitory effects mediated by thermal stress, while ascorbic acid did not give any protective results.

Induction and regulation of cytotoxic T cells by microbial antigens and recombinant interleukin 2.

The proliferation and development of cytotoxic T cells was investigated in human peripheral blood mononuclear cell (PBMC) cultures stimulated with an antigenic extract from Candida albicans (MPPS), or with the purified protein derivative from Mycobacterium tuberculosis (PPD), or with human recombinant interleukin 2 (rIL-2). Microbial antigen- and rIL-2-induced cytotoxic T cells were able to lyse both natural killer (NK) sensitive and resistant targets. No correlation was observed between the development of T cell cytotoxicity and interferon (IFN) production in vitro. The addition of anti-class II monoclonal antibodies at the beginning of MPPS/PPD-stimulated cultures inhibited the cell proliferation, IFN production and T cell cytotoxicity, while all these cellular activities were not inhibited by anti-class II antibodies in rIL-2-stimulated cultures. Finally, antibodies to class I determinants inhibit T cell cytotoxicity, suggesting a role of such determinants in the development of the non-adaptive immunity to microbial infections.

Influence of thermal and dietary stress on immune response of rabbits.

With the aim of analyzing the effects of prolonged thermal stress or food intake reduction on lymphoid cell proliferation and antibody synthesis, New Zealand White (NZW) male rabbits, both immunized and nonimmunized with Mycobacterium tuberculosis, were kept in individual cages for 24 d at controlled climatic conditions. Both immunized and nonimmunized rabbits were divided into two experimental groups and one control group. The thermal-stressed rabbits (TS) were exposed to a room temperature of 33.5 degrees C, and dietary restricted rabbits (DR) were pair-fed on the basis of the average feed intake of the TS groups and maintained at a room temperature of 18.0 degrees C. The control group (Ctr) was maintained at 18.0 degrees C and was given ad libitum access to feed. All rabbits were maintained at relative humidity 62 +/- 5%. Sera and peripheral blood mononuclear cells (PBMC) were isolated from blood samples collected on d 0, 6, 12, and 24. Sera were used for determining total proteins and immunoglobulins (Ig) specific or not to mycobacterial antigens. Antibodies to heat-shock protein (HSP) were also determined. The PBMC were used to measure cell proliferation and in vitro Ig synthesis. Both experiments in vivo and in vitro suggest that thermal stress and dietary restriction severely affect the immune cell functions. In fact, both stress treatments decreased the capacity of PBMC to proliferate and inhibited the differentiation of B lymphocytes in antibody-secreting cells. However, a recovery of immune cell functions was only observed in vivo after 12 d of treatment, suggesting that other defensive mechanisms may come into play in vivo. Sera collected from both TS and DR rabbits after 24 d presented antibodies to HSP70, suggesting that the analysis of anti-HSP antibodies could represent a useful indicator to reveal the effects of different stress effectors regardless of the nature of the stress.

Identification of a specific Mycobacterium tuberculosis protein able to activate T lymphocytes.

The recent advances in the field of immunology and molecular biology allow to consider not distant the identification of protective antigens to be used in new prophilactic and diagnostic tools. In our laboratory we have analysed a M. tuberculosis expression library by murine monoclonal antibodies, human sera and human T lymphocytes. We have identified a 35 Kd protein present only on the M. tuberculosis complex (M. tuberculosis, M. bovis, M. africanum). This 35 Kd protein is also detected, by immunohistological analysis, inside the alveolar macrophage of pulmonary tuberculosis patients. Both recombinant beta-galactosidase and MS2 polymerase-fused proteins have been produced and used to perform western blotting and T cell proliferation assay. As the cloned fragment contains an internal EcoRI restriction site, it was possible to identify two fragments of 1.7 and 2.2 kb respectively. Southern blot analysis showed that both the fragments hibridized with the genomic DNA from M. tuberculosis complex but not with the DNA from other mycobacterial isolates from 12 pulmonary tuberculous patients hibridized with the 1.7 kb fragment. The possible use of this insert for the molecular diagnosis of tuberculosis is under investigation.

Immune complexed (IC) hepatitis C virus (HCV) in chronically and acutely HCV-infected patients.

In infected individuals, hepatitis C virus (HCV) exists in various forms of circulating particles which role in virus persistence and in HCV resistance to IFN therapy is still debated. Here, the proportion of HCV bound to immunoglobulin was determined in plasma of 107 chronically infected patients harbouring different HCV genotypes and, for comparison, of six patients with acute HCV infection. The results showed that, in spite of wide individual variability, chronically HCV-infected patients exhibited an extremely high proportion of immune complexed (IC) virus regardless of plasma HCV load and infecting genotype. Moreover, no significant association was found between baseline proportion of IC HCV and response to IFN treatment. Plasma samples collected within 2 weeks of treatment from 20 patients revealed a significant decline of mean IC HCV values relative to baseline that clearly paralleled the decay of total HCV load. In acutely infected patients, circulating HCV was not IC or IC at very low levels only in patients developing chronic HCV infection. Collectively, these findings strengthen the possibility that IC virus could play a critical role in the pathogenesis of HCV infection.

Early impairment of hepatitis C virus specific T cell proliferation during acute infection leads to failure of viral clearance.

BACKGROUND AND AIMS: Cellular mediated immunity (CMI) is thought to play a key role in resolution of primary hepatitis C virus (HCV) infection. However, CD4+ and CD8+ T cell responses are also generated during acute infection in individuals who become chronic, suggesting that they developed a defective CMI. The aim of this study was to verify if and when such immune dysfunction is established by measuring the breadth, magnitude, function, and duration of CMI in a large cohort of subjects during the natural course of acute HCV infection. METHODS: CMI was comprehensively studied by prospective sampling of 31 HCV acutely infected subjects enrolled at the onset of infection and followed for a median period of one year. RESULTS: Our results indicated that while at the onset of acute HCV infection a measurable CMI with effector function was detected in the majority of subjects, after approximately six months less than 10% of chronically infected individuals displayed significant CMI compared with 70% of subjects who cleared the virus. We showed that progressive disappearance of HCV specific T cells from the peripheral blood of chronic patients was due to an impaired ability to proliferate that could be rescued in vitro by concomitant exposure to interleukin 2 and the antigen. CONCLUSION: Our data provide evidence of strong and multispecific T cell responses with a sustained ability to proliferate in response to antigen stimulation as reliable pharmacodynamic measures of a protective CMI during acute infection, and suggest that early impairment of proliferation may contribute to loss of T cell response and chronic HCV persistence.

Human lymphocyte-activating properties of a purified polysaccharide from Candida albicans: B and T cell cooperation in the mitogenic response.

Purified human T and B lymphocytes were tested for their ability to show a mitogenic response to a purified polysaccharide (MPPS) extracted from C. albicans. It was found that neither T nor B cells alone could respond to MPPS. When a mixture of these two types of cells was tested, a normal response (comparable to the one of unfractionated peripheral blood lymphocytes) was observed. This response was not affected by prior depletion of macrophages. Results obtained with normal B or T cells mixed with mitomycin C-treated T or B cells indicated that a reciprocal helper effect exists between these two types of cells in their response to MPPS. This response could be inhibited by antisera against HL-A and Ia-like antigens.