Claudio Pioli

Inhibition of IgG1 and IgE Production by Stimulation of the B Cell CTLA-4 Receptor

Although a large amount of information is available on the activity of CTLA-4 in T cells, the role of this receptor in B cells has not been previously characterized. Our results show that CD40 or LPS stimulation in the presence of IL-4 induces CTLA-4 expression in purified B cells; the maximum level is reached in both membrane and intracellular compartments after 48–72 h. Engagement of the B cell CTLA-4 by immobilized mAb inhibits IgG1 and IgE production and reduces the frequency of IgG1- and IgE-expressing B cells. Cε and Cγ1 germline mRNA expression as well as NF-κB and STAT6 activation, events required for isotype switching, are also inhibited by CTLA-4 engagement. Together these findings show the critical role of CTLA-4 in the control of IL-4-driven isotype switching and suggest new approaches for modulating immediate-type hypersensitivity responses.

Beyond DNA repair, the immunological role of PARP-1 and its siblings.

ADP‐ribosylation is the addition of one or more (up to some hundreds) ADP‐ribose moieties to acceptor proteins. There are two major families of enzymes that catalyse this reaction: extracellular ADP‐ribosyl‐transferases (ARTs), which are bound to the cell membrane by a glycosylphosphatidylinositol anchor or are secreted, and poly(ADP‐ribose)‐polymerases (PARPs), which are present in the cell nucleus and/or cytoplasm. Recent findings revealed a wide immunological role for ADP‐ribosylating enzymes. ARTs, by sensing extracellular NAD concentration, can act as danger detectors. PARP‐1, the prototypical representative of the PARP family, known to protect cells from genomic instability, is involved in the development of inflammatory responses and several forms of cell death. PARP‐1 also plays a role in adaptive immunity by modulating the ability of dendritic cells to stimulate T cells or by directly affecting the differentiation and functions of T and B cells. Both PARP‐1 and PARP‐14 (CoaSt6) knockout mice were described to display reduced T helper type 2 cell differentiation and allergic responses. Our recent findings showed that PARP‐1 is involved in the differentiation of Foxp3+ regulatory T (Treg) cells, suggesting a role for PARP‐1 in tolerance induction. Also ARTs regulate Treg cell homeostasis by promoting Treg cell apoptosis during inflammatory responses. PARP inhibitors ameliorate immune‐mediated diseases in several experimental models, including rheumatoid arthritis, colitis, experimental autoimmune encephalomyelitis and allergy. Together these findings show that ADP‐ribosylating enzymes, in particular PARP‐1, play a pivotal role in the regulation of immune responses and may represent a good target for new therapeutic approaches in immune‐mediated diseases.

Cardiopulmonary bypass in man: role of the intestine in a self-limiting inflammatory response with demonstrable bacterial translocation

BACKGROUND Cardiopulmonary bypass provokes a systemic inflammatory reaction that, in 1% to 2% of all cases, leads to multiorgan disfunction. The aim of this study was to evaluate the possible role of the intestine in the pathogenesis and development of this reaction. METHODS Eleven selected patients scheduled for elective coronary artery bypass graft surgery were enrolled in a open, prospective clinical study. Gastric tonometry, chromium-labeled test and double sugar intestinal absorption tests, polymerase chain reaction microbial DNA test, and measurement of cytokines and transcriptional factor (nuclear factor kappaB) activation were performed. RESULTS During the postoperative period, gastric pH remained stable (range,7.2 to 7.3). The partial pressure for carbon dioxide gradient between the gastric mucosa and arterial blood increased significantly (from 1 to 23 mm Hg), peaking in the sixth postoperative hour. Interleukin 6 increased significantly over basal levels, peaking 3 hours after cardiopulmonary bypass (96.3 versus 24 pg/mL). Nuclear factor kappaB never reached levels higher than those observed after lipopolysaccharide stimulation. Escherichia coli translocation was documented in 10 patients: in eight cases from removal of aortic cross-clamps and in two cases from the first postoperative hour. With respect to basal value (6.4%), the urine collection revealed a significant increase in excretion of the radioisotope during the first 24 hours after surgery (39.1%), although there were no significant variations with the double sugar test. CONCLUSIONS The results obtained showed a correlation between the damage of the gastrointestinal mucosa, subsequent increased permeability, E coli bacteremia, and the activation of a self-limited inflammatory response in the absence of significant macrocirculatory changes and postoperative complications.

IL-4 Modulation of CD4+CD25+ T Regulatory Cell-Mediated Suppression

Murine CD4+CD25+ T regulatory (Treg) cells were cocultured with CD4+CD25− Th cells and APCs or purified B cells and stimulated by anti-CD3 mAb. Replacement of APCs by B cells did not significantly affect the suppression of CD4+CD25− Th cells. When IL-4 was added to separate cell populations, this cytokine promoted CD4+CD25− Th and CD4+CD25+ Treg cell proliferation, whereas the suppressive competence of CD4+CD25+ Treg cells was preserved. Conversely, IL-4 added to coculture of APCs, CD4+CD25− Th cells, and CD4+CD25+ Treg cells inhibited the suppression of CD4+CD25− Th cells by favoring their survival through the induction of Bcl-2 expression. At variance, suppression was not affected by addition of IL-13, although this cytokine shares with IL-4 a receptor chain. When naive CD4+CD25− Th cells were replaced by Th1 and Th2 cells, cell proliferation of both subsets was equally suppressed, but suppression was less pronounced compared with that of CD4+CD25− Th cells. IL-4 production by Th2 cells was also inhibited. These results indicate that although CD4+CD25+ Treg cells inhibit IL-4 production, the addition of IL-4 counteracts CD4+CD25+ Treg cell-mediated suppression by promoting CD4+CD25− Th cell survival and proliferation.

Cyclic Adenosine 5′-Monophosphate and Calcium Induce CD152 (CTLA-4) Up-Regulation in Resting CD4+ T Lymphocytes

The CTLA-4 (CD152) molecule is up-regulated upon T cell activation and proliferation, and plays a critical role in the inhibition of immune responses. We show in this study that cAMP induces up-regulation of CD152 in human CD4+ T lymphocytes. This effect occurs in the absence of the up-regulation of CD69 and CD25 activation markers and T cell proliferation. In addition, we found that the Ca2+ ionophore ionomycin also up-regulates CD152, and that the combination of a cAMP analog or cAMP inducers with ionomycin further enhances the expression of CD152 in resting CD4+ T lymphocytes. However, cyclosporin A, which inhibits Ca2+/calcineurin signaling pathway, fully prevented the ionomycin- but not the cAMP-induced up-regulation of CD152. The effects of cAMP and ionomycin involve increase of both CD152 mRNA transcripts, coding for the membrane and the soluble forms of CD152. Furthermore, we show that CD152 molecules are translocated to the membrane and are functional, as their engagement by specific mAbs prevented NF-κB activation by anti-CD3/CD28 stimulation. These findings demonstrate that at least two novel signal pathways regulate CTLA-4 gene expression and CD152 molecule up-regulation in human CD4+ T lymphocytes, in the absence of full T cell activation.

Regulation of cytokine production in aging: use of recombinant cytokines to upregulate mitogen-stimulated spleen cells

We investigated the production of IL-2 and IFN-gamma (Th1 type) and IL-4 (Th2 type) cytokines by mitogen-activated spleen cells from young, adult and old mice. Cytokine production was evaluated in culture supernatants by CTLL proliferation (IL-2), ELISA (IFN-gamma), CT4.S proliferation (IL-4) and in mRNA extracted from activated CD4+ cells by RT-PCR (IL-2, IFN-gamma and IL-4). Results show that the production of IL-2, as protein and mRNA, is profoundly depressed by aging, whereas that of IFN-gamma, as protein and mRNA, firstly declines and then increases with age. The production of IL-4, as protein, monotonically declines with aging whereas, as mRNA, firstly decreases and then increases above the level in young mice. Spleen cells in culture were also incubated with mitogens and with a recombinant cytokine (IL-1 beta, IL-2, IL-3, IL-4, IL-12 or IFN-gamma) at various concentrations. It was found that recombinant cytokines by and large enhance cytokine production when the level induced by mitogens only is low. This conclusion applies to IL-2 and IFN-gamma production as protein and mRNA. The addition of recombinant cytokines also increases the production of IL-4 at the protein level in spleen cells from old mice but, at the mRNA level, only in spleen cells from young mice. This finding suggests age-related changes in IL-4-specific mRNA transcription rate and post-transcriptional half-life as well as translation kinetics.

Effects of In Vivo Exposure to GSM-Modulated 900 MHz Radiation on Mouse Peripheral Lymphocytes

Abstract Gatta, L., Pinto, R., Ubaldi, V., Pace, L., Galloni, P., Lovisolo, G. A., Marino, C. and Pioli, C. Effects of In Vivo Exposure to GSM-Modulated 900 MHz Radiation on Mouse Peripheral Lymphocytes. Radiat. Res. 160, 600–605 (2003). The aim of this study was to evaluate whether daily whole-body exposure to 900 MHz GSM-modulated radiation could affect spleen lymphocytes. C57BL/6 mice were exposed 2 h/day for 1, 2 or 4 weeks in a TEM cell to an SAR of 1 or 2 W/kg. Untreated and sham-exposed groups were also examined. At the end of the exposure, mice were killed humanely and spleen cells were collected. The number of spleen cells, the percentages of B and T cells, and the distribution of T-cell subpopulations (CD4 and CD8) were not altered by the exposure. T and B cells were also stimulated ex vivo using specific monoclonal antibodies or LPS to induce cell proliferation, cytokine production and expression of activation markers. The results did not show relevant differences in either T or B lymphocytes from mice exposed to an SAR of 1 or 2 W/kg and sham-exposed mice with few exceptions. After 1 week of exposure to 1 or 2 W/kg, an increase in IFN-γ (Ifng) production was observed that was not evident when the exposure was prolonged to 2 or 4 weeks. This suggests that the immune system might have adapted to RF radiation as it does with other stressing agents. All together, our in vivo data indicate that the T- and B-cell compartments were not substantially affected by exposure to RF radiation and that a clinically relevant effect of RF radiation on the immune system is unlikely to occur.

Increased Foxp3+ Regulatory T Cells in Poly(ADP-Ribose) Polymerase-1 Deficiency

Growing evidence is unveiling a role for poly(ADP-ribose) polymerase (PARP)-1 in the regulation of inflammatory/immune responses. In the current study, we investigated the effects of PARP-1 deficiency on regulatory T cell differentiation. Increased numbers of regulatory CD4+CD25+/Foxp3+ T cells were found in thymus, spleen, and lymph nodes of PARP-1 knockout (KO) mice compared with wild-type (WT) controls. The increased frequency of regulatory T cells in the periphery resulted in impaired CD4 cell proliferation and IL-2 production, which could be restored by CD25+ cell depletion. Phenotype and inhibitory functions of PARP-1 KO regulatory T cells were similar to WT cells, indicating that PARP-1 affects regulatory T cell differentiation rather than function. Purified naive CD4 cells from PARP-1 KO mice stimulated in vitro expressed forkhead box p3 mRNA at higher levels and generated a greater number of Foxp3+ cells (inducible regulatory T [iTreg] cells) than the WT counterpart. This finding was due to a higher rate of naive CD4 cell to Foxp3+ iTreg cell conversion rather than to higher resistance to apoptosis induction. Interestingly, PARP-1 deficiency did not affect retinoid-related orphan receptor-γt mRNA expression and differentiation of purified naive CD4 cells to Th17 cells. PARP-1 KO iTreg cells showed features similar to WT regulatory T cells, suggesting that modulation of PARP-1 during the immune response might be used to induce greater numbers of functional regulatory T cells. In conclusion, our findings represent the first evidence that PARP-1 can affect regulatory T cell differentiation and open new perspectives on potential targets for modulating immune responses.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) inhibits CD28-induced IκBα degradation and RelA activation

Purified CD4+ cells from the spleens of C57BL/6 mice were stimulated with anti‐CD3, anti‐CD28 and anti‐cytotoxic T lymphocyte antigen (CTLA)‐4 monoclonal antibodies. The results show that CTLA‐4 stimulation inhibits IL‐2 production induced by CD3‐CD28 co‐stimulation. Since CD3‐CD28 co‐stimulation induces IκBα degradation and consequently activates RelA, an NFκB family member relevant for the induction of IL‐2 mRNA transcription, we tested whether the inhibitory effect of CTLA‐4 stimulation interferes with this mechanism. CD3‐CD28 co‐stimulation was found to induce a drastic decrease in cytoplasmic IκBα and increase in nuclear RelA. CTLA‐4 stimulation abrogates this effect of co‐stimulation by increasing the level of cytoplasmic IκBα and decreasing the nuclear RelA level and DNA‐binding activity. In conclusion, our results indicate that the inhibitory effect of CTLA‐4 engagement on cytokine production correlates with prevention of IκBα degradation and inhibition of RelA nuclear translocation.

Role of mRNA stability in the different patterns of cytokine production by CD4+ cells from young and old mice

CD4+ cells from young (3 months) and old (19 months) mice were stimulated by plate‐bound anti‐CD3 monoclonal antibody (mAb) alone or also by soluble anti‐CD28 mAb. Supernatants were analysed by enzyme‐linked immunosorbent assay (ELISA) to determine cytokine concentrations. Total RNA was extracted from cells, reverse transcribed and the cDNA amplified by polymerase chain reaction (PCR) to evaluate the amount of specific mRNA. The results indicate that anti‐CD3 alone is not sufficient to induce interleukin‐2 (IL‐2) production in CD4+ cells from both young and old mice. However, anti‐CD28, together with anti‐CD3 mAb, induces a much higher production of IL‐2 in CD4+ cells from young as compared with old mice. Conversely, interferon‐γ (IFN‐γ) production is also induced by anti‐CD3 alone and is higher in CD4+ cells from old as compared with young mice. Upon addition of anti‐CD28 mAb, IFN‐γ production increases in both groups, but it remains much higher in old than in young mice. Also the production of IL‐4 and IL‐10 is induced by anti‐CD3 mAb but it is increased by the addition of anti‐CD28 mAb. CD4+ cells from old mice produce more IL‐4 and IL‐10 as compared with cells from young mice. The amounts of cytokine specific mRNA in CD4+ cells from young and old mice parallel the cytokine levels in culture supernatants. Results on the mRNA turnover indicate that when CD4+ cells are stimulated by anti‐CD3 or costimulated also by anti‐CD28 mAb, the IFN‐γ, IL‐4 and IL‐10 specific mRNAs are more stable in old than in young mice, suggesting that mRNA stability has a relevant role in the different patterns of cytokine production.