Lucia Gatta

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.

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.

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.

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.

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.

Increased levels of NF-κB inhibitors (IκBα and IκBγ) in the intestinal mucosa of Crohn's disease patients during infliximab treatment

The treatment with infliximab is employed successfully in Crohns disease (CD) but predictors of efficacy are lacking. Activation of the transcription factor NF-kB has been demonstrated in CD and its inhibition is one of the mechanisms by which anti-inflammatory agents exert their effects. We evaluated the production of TNFα by peripheral blood mononuclear cells (PBMC) and the levels of NF-κB family molecules in the intestinal mucosa during infliximab therapy in 12 patients. TNFα was assayed on supernatants of PBMC culture stimulated with PHA or LPS. Immunohistochemistry was also done on intestinal biopsies. In six patients, Western blot analysis of the NF-kB subunit Rel-A, and its inhibitors IκBα and IκBγ was performed on intestinal biopsies and PBMC. The TNFα production by LPS stimulated PBMC showed mild changes, while it was increased by PHA–stimulated PBMC after treatment. The number of inflammatory cells in the intestinal mucosa was reduced (p<0.002) by the treatment. In five out of six cases we detected an increase of the IκBα and IκBγ inhibitor levels in intestinal biopsies after treatment. An increase of IκB inhibitors levels could be one of the mechanisms by which infliximab decreases NF-κB activity and exerts its anti-inflammatory effects.

CTLA-4 Engagement Inhibits Th2 but not Th1 Cell Polarisation

CTLA-4 deficient mice show severe lymphoproliferative disorders with T helper sub-population skewed toward the Th2 phenotype. In the present work, we investigated the role of CTLA-4 in T helper cell subset differentiation. Naïve CD4+ cells were stimulated with anti-CD3 and anti-CD28 mAbs in the presence of either IL-12 or IL-4 to induce polarisation to Th1 or Th2 cells, respectively. Under these two polarising conditions cells express comparable levels of CTLA-4. CTLA-4 was stimulated by plastic-bound mAb. The frequency of IFN-γ- and IL-4-producing cells were estimated by FACS analysis. In parallel cultures, polarised Th1 and Th2 cells were re-stimulated with anti-CD3 and anti-CD28 mAbs for 48 h and their culture supernatants analysed by ELISA. Results show that CTLA-4 engagement during differentiation inhibits polarisation of naïve CD4+ cells to the Th2 but not the Th1 cell subset. At variance, once cells are polarised, CTLA-4 engagement inhibits cytokine production in both effector Th2 and Th1 cells. Altogether these data indicate that CTLA-4 may interfere not only in the signalling involved in acute transcriptional activation of both Th1 and Th2 cells but also in the development of one of the Th cell subsets.

Changes in the amount and level of phosphorylation of p56lck in PBL from aging humans

The effects of aging on the activation of the cytoplasmic tyrosine protein kinase p56(lck) have been investigated in PBL from adult and elderly subjects upon activation with mitogens or different co-stimuli. Results show that the amount and phosphorylation of p56(lck) are reduced in PBL from elderly as compared to adult subjects. This finding suggests that alterations in p56(lck) may contribute to the age-associated loss of some T cell functions, such as proliferation and IL-2 production, which are found decreased in PBL from old individuals. However, p56(lck) seems irrelevant to the production of IFN-gamma and IL-4 which were both found increased in the PBL from old subjects, as expected from the relative expansion of memory versus naive T cell subpopulations in aging.

Cytotoxic T lymphocyte-associated antigen-4 inhibits integrin-mediated stimulation.

The negative role exerted by cytotoxic T lymphocyte‐associated antigen‐4 (CTLA‐4) in the regulation of T‐cell activity, as induced by T‐cell receptor (TCR)/CD3 and CD28 costimulation, has been widely described. In the present work we investigated the role of CTLA‐4 in the control of cell activation, as induced by costimulation of the adhesion molecule lymphocyte function‐associated antigen‐1 (LFA‐1) in murine CD4+ T cells. Results show that CTLA‐4 engagement inhibits interleukin‐2 (IL‐2) production, not only when induced by CD3/CD28 costimulation, but also when CD4+ T cells are costimulated by anti‐CD3 and anti‐LFA‐1 monoclonal antibodies (mAbs). LFA‐1 has been described to induce Ca2+ mobilization also in the absence of TCR engagement. Moreover, we found that CTLA‐4 engagement negatively affects Ca2+ mobilization and NF‐AT activation, as induced by LFA‐1 engagement alone. PLCγ1 phosphorylation was also dampened within minutes after CTLA‐4 engagement. Altogether these data indicate that through the control of signals induced by different receptors, CTLA‐4 could be a global attenuator of T‐cell activation.