Francesca Quintieri

Cholera toxin B subunit promotes the induction of regulatory T cells by preventing human dendritic cell maturation

Cholera toxin B subunit (CTB) is an efficient mucosal carrier molecule for the generation of immune responses to linked antigens. There is also good evidence that CTB acts as an immunosuppressant, as it is able to down‐modulate human monocyte/macrophage cell line activation and to suppress Th1‐type responses. In the present study, we examined the possibility that recombinant CTB (rCTB) may affect human dendritic cell (DC) functions in response to LPS stimulation and may induce the generation of DC with the capacity to generate CD4+ regulatory T cells (Tregs). Our findings show that rCTB partially prevents the LPS‐induced maturation process of monocyte‐derived DC (MDDC) and decreases their IL‐12 production with no relevant effect on IL‐10 production. LPS‐stimulated MDDC pretreated with rCTB are able to promote the induction of low proliferating T cells, which show an enhanced IL‐10 production associated with a reduced IFN‐γ production and the same high levels of TGF‐β as the control. These T cells suppress proliferation of activated autologous T cells. Transwell experiments and blockade of IL‐10R and TGF‐β showed that the immunomodulatory effect is mediated by soluble factors. Thus, T cells induced by rCTB‐conditioned MDDC acquire a regulatory phenotype and activity similar to those described for type 1 Tregs.

Diltiazem impairs maturation and functions of human dendritic cells

The aim of this study was to define the effects of diltiazem, a calcium antagonist drug used in cardiology and in clinical transplantation, on the differentiation and maturation of human dendritic cells (DC). Herein, we demonstrate that diltiazem, in association with granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), induces monocytes to differentiate into cells with many of the characteristic of DC. However, diltiazem-induced DC express high levels of mannose receptor and Fc gamma RII and, consequently, manifest a higher endocytic activity compared with GM-CSF+IL-4-induced DC. Importantly, diltiazem-induced DCs have an impaired responsiveness to lipopolysaccharide and CD40 ligand because they produce decreased levels of IL-12 and reveal a reduced ability to stimulate alloreactive T-cell responses as well as in inducing interferon-gamma producing Th1 cells. These effects may contribute to a decreased DC-dependent T-cell activation and may help to explain the immunoregulatory function of diltiazem and its effectiveness in preventing transplant rejection.

Diltiazem modulates monokine production in human mixed lymphocyte culture

BACKGROUND Calcium channel blockers are widely used in transplantation. Their immunosuppressive activity is well known and has been demonstrated both in vitro and in vivo. Nevertheless, their effect on cytokine production has never been reported. METHODS One-way mixed lymphocyte cultures (MLCs) have been obtained from healthy human subjects. Cytokine production has been assessed by three different methods: by enzyme-linked immunosorbent assay on supernatants of MLC, by enzyme-linked immunospot method on MLC cells for measuring cytokine-producing cells, and by the reverse transcription polymerase chain reaction technique on MLC cells for measuring cytokine mRNAs. RESULTS An interesting effect on proinflammatory monokines was observed: in this study, we demonstrate that the calcium antagonist diltiazem enhances interleukin-1beta and slightly reduces interleukin-6 production in MLC, but it has no effect on tumor necrosis factor-alpha levels. CONCLUSION For the first time, a modulation of monokine production by diltiazem can be demonstrated. This evidence suggests that calcium antagonist drugs may exert effects on monocytes and possibly on other antigen-presenting cells.

A Limiting-Dilution Analysis of Activated Circulating B Cells in Crohn's Disease

In the present study the spontaneousin vitro production of immunoglobulins G, A, and M by peripheral mononuclear cells was evaluated, in patients with Crohns disease, in relation to the state of B-cell activation and further characterized by limiting-dilution analysis. A total of 25 patients with Crohns disease and 10 healthy controls was studied. The proportion of the transferrin receptor-bearing cells in the B7+ subset was higher in active Crohns disease patients than in either those with quiescent disease or controls. There was a significant rise in thein vitro IgG, IgM, and IgA production in patients with untreated active Crohns disease compared to either those with untreated quiescent disease or controls. When patients were followed up from the active phase to clinical remission, a significant decrease in the production of IgG and IgM was observed. IgA levels also showed a decrease, although not reaching statistical significance. When the Ig production was analyzed by limiting dilution, no difference was observed between patients and controls in terms of either precursor frequency of Ig-producing cells or patterns of frequency distribution. In both patients and controls a biphasic limiting-dilution profile was observed. This study shows that a significant B-cell activation is present in Crohns disease patients, which is accompanied by an increase in immunoglobulin production. This study also indicates that in Crohns disease the increased immunoglobulin production is related to an augmented B-cell clone size rather than to an increased precursor frequency.

Further evaluation of autoreactive T cells in hydatid patients

We evaluated the peripheral autoreactive response in patients with Echinococcus granulosus who showed a negative humoral response as compared to seropositive patients and healthy controls. For this purpose, a limiting dilution analysis (LDA) of autologous mixed lymphocyte cultures was established to both estimate frequency of autoreactive T cells and, by analysing the shape of the curves, to clarify the mechanisms that underlie the autoreactive response. Different LDA curves were observed between healthy controls and patients, suggesting that different cell interactions are involved in the two populations. More interestingly, all hydatid patients, independent of their humoral response, showed a higher number of autoreactive T cells than controls. Precisely, subjects with a negative humoral response showed a range of values for autoreactive T cells exactly between the value ranges observed in seropositive and normal subjects. The present data also show that the increase of autoreactive T cells in hydatid patients correlates with the production of specific antibodies.

Detection of anti-extractable nuclear antigens in connective tissue diseases: Comparison between passive hemagglutination, counterimmunoelectrophoresis and double immunodiffusion

SummaryAntibodies to the three major components of the complex called soluble extractable nuclear antigen (ENA) were detected by passive hemagglutination (HA), counterimmunoelectrophoresis (CIE) and double immunodiffusion (DI) in 256 patients with connective tissue diseases. Anti-ENA antibodies were demonstrated by all the three employed methods in only 44.9% of the cases. These methods were not able to detect all antibodies to these antigens or any single specificity; CIE was however the most sensitive method for anti-RNP and HA for anti-Sm antibodies, while DI was the most suitable technique for serum samples with multiple anti-ENA specificities. Only in less than 50% of the cases the specificity detected by HA was comparable with that given by CIE or DI. Hence, for detecting anti-ENA antibodies a combination of these methods should be maintained, at least until more precise and reliable methods will become available.

Dilitiazem reduces nitric oxide production by human immune cells

NITRIC OXIDE (NO) is known to exist in tissues as the smallest biological product of mammalian cells and is gaining recognition as an important biological mediator. Besides its antimicrobial effect and its role in septic and inflammatory responses, this molecule acts as a potent inhibitory agent of proliferation and cytolytic T-cell induction. NO is synthesized from the amino acid L-arginine by a family of enzymes, the nitric oxide synthase (NOS). The inducible form of NOS (iNOS) can be expressed in many cell types and in part mediates the cytostatic and cytotoxic effect of activated macrophages. Although NO is produced mainly by macrophages, dendritic cells were shown to represent an additional source. Interestingly, NO is increased in allograft rejection, and conventional immunosuppressive agents have been shown to decrease its production. The calcium antagonist drug diltiazem is often used in preventing graft rejection just after kidney transplants because of its immunosuppressive effect and its capability in reducing the undesirable side effects due to the use of cyclosporine and corticosteroids. We investigated in vitro NO production by human immune cells, both by mixed lymphocyte cultures and isolated cell populations in presence of the drug, and discovered an additional effect that makes it suitable for more frequent use in clinical transplantation.

Modulation of Human Peripheral Blood Mononuclear Cell Proliferative Response by Diltiazem : In Vitro Comparison in Younger versus Older Subjects

BackgroundThe gradual aging of population has increased the number of elderly patients receiving kidney transplants. In elderly transplant recipients, careful immunosuppression has to be maintained to avoid both rejection and adverse effects. Clinical protocols after kidney transplantation include use of the calcium channel antagonist diltiazem to ameliorate the hypertensive effect and nephrotoxicity of the immunosuppressant agent ciclosporin (cyclosporine).ObjectiveSince immune response can be impaired by senescence, we evaluated the influence of diltiazem on lymphocyte proliferation both alone and in the presence of ciclosporin in younger versus older subjects.MethodsPeripheral blood mononuclear cells (PBMC) from younger healthy donors (aged 19–24 years) and older subjects (aged 59–65 years) were isolated and stimulated with mitogens, recombinant human interleukin-2 (IL-2), purified protein derivative (PPD) antigen from Mycobacterium tuberculosis, and anti-CD3 monoclonal antibody (αCD3 moAb) in the presence or absence of 10−4, 10−5, 10−6, 10−7 mol/L concentrations of diltiazem. In some experiments, lymphocytes from younger and older subjects were used as responder cells in an allogeneic mixed lymphocyte reaction (MLR) in the presence of different concentrations of diltiazem and 10 ng/mL of ciclosporin.ResultsWe found that PBMC from older subjects were more susceptible to immunosuppression induced by low concentrations of diltiazem when mitogens were used to stimulate cells. In particular, when pokeweed mitogen was used, diltiazem 10−7 mol/L was associated with statistically significant immunosuppression in older subjects compared with younger subjects. This effect was not observed when IL-2, PPD antigen and αCD3 moAb were used as stimulators. Moreover, in the allogeneic MLR, we found no differences between younger and older subjects when the 10−5, 10−6 and 10−7 mol/L concentrations of diltiazem were used alone or in the presence of ciclosporin. Only addition of the suprather-apeutic 10−4 mol/L concentration of diltiazem to ciclosporin was associated with statistically significant immunosuppression in older versus younger subjects.DiscussionOur results show that PBMC from older subjects are no more susceptible than PBMC from younger subjects to therapeutic doses of diltiazem when T-cell receptors are directly or indirectly involved. On the contrary, when PBMC activation was not mediated by T-cell receptor involvement, as in the case of pokeweed mitogen, susceptibility to a therapeutic concentration of diltiazem in older subjects was enhanced. Moreover, co-administration of therapeutic doses of diltiazem and ciclosporin in an MLR showed no significant differences between younger and older subjects in an in vitro model of lymphocyte response to allogeneic transplantation.ConclusionSince we found no variations in immunosuppression between older and younger subjects when therapeutic doses of diltiazem were added to ciclosporin, our data do not discourage the use of diltiazem in older kidney transplant recipients receiving ciclosporin therapy.

Diltiazem induces regulatory T cells in vitro by modulating human dendritic cell maturation

Diltiazem is a calcium channel antagonist that has been commonly associated with currently used immunosuppressants to prevent acute graft rejection in humans. In this study, we examined the possibility that diltiazem may affect human dendritic cell (DC) functions in response to lipopolysaccharide (LPS) stimulation and may induce the generation of DC with the capacity to generate CD4+ regulatory T cells (Tregs). Blood monocytes were cultured in the presence of diltiazem at the beginning of their differentiation process into DC. Monocyte‐derived DCs were stimulated with LPS, and DCs differentiated in the presence of diltiazem showed a decreased interleukin (IL)‐12 production and an enhanced IL‐10 production. When cultured with CD4+CD45RA+ they were able to enhance the CD4+Foxp3+ T‐cell population and to induce slowly proliferating T cells, which showed a significant increase of transforming growth factor (TGF)‐β production. These T cells suppress proliferation of activated autologous T cells, and we show that this effect is attributable to soluble factors, primarily to TGF‐β. Blockade of TGF‐β by specific monoclonal antibodies reversed this inhibitory effect. Herein, we provide new evidence that diltiazem‐conditioned monocyte‐derived DC induce T cells which acquire a regulatory phenotype and activity similar to those described for Tregs.