Intracellular IFN-gamma production and IL-12 serum levels in latent autoimmune diabetes of adults (LADA) and in type 2 diabetes.

Th1 cytokines, such as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), and Th1-inducing cytokines, such as IL-12, are involved in the pathogenesis of various organ-specific autoimmune diseases, including autoimmune diabetes. In this study, we investigated intracellular IFN-gamma release by T lymphocytes and IL-12 serum levels in 48 type 2 and 36 latent autoimmune diabetes of adults (LADA) diabetics and 25 control subjects in an attempt to evaluate their role in the pathogenesis of these clinical entities. Ionomycin (ION) and phorbol-12-myristate-13-acetate (PMA)-activated peripheral blood mononuclear cells (PBMCs) were stained with anti-CD4-FITC or anti-CD8-FITC and anti-IFN-gamma phycoerythrin (PE) monoclonal antibodies (mAbs) and analyzed by flow cytometry. IL-12 serum levels were determined by enzyme-linked immunosorbent assay (ELISA). In all study groups, IFN-gamma content of CD4(+) and CD8(+) lymphocytes was significantly upregulated by stimulation. Furthermore, it was observed that CD4(+) and CD8(+) lymphocytes from type 2 diabetics produced significantly lower levels of IFN-gamma compared with LADA patients and controls. However, the percentages of CD4(+)/IFN-gamma(+) and CD8(+)/IFN-gamma(+) cells from type 2 diabetics were significantly higher compared with controls. The flow cytometric picture of intracellular IFN-gamma release in LADA patients did not differ from that observed in controls. However, IL-12 serum levels in type 2 and LADA diabetics were lower than in controls. Because Th1 cytokines have been associated with the pathogenesis of autoimmune diabetes, these results preclude Th1 involvement in the autoimmune phenomena observed in LADA patients. In contrast, the low IFN-gamma levels observed in type 2 diabetics in combination with the low IL-12 serum levels might be a contributing factor in the frequently observed chronic complications in these patients.

Responsiveness of preactivated B cells to IL-2 and IL-6. Effect of cyclosporine and rapamycin.

In order to determine which drug may be more effective in clinical abnormalities associated with polyclonal B lymphocyte activation, we compared the in vitro effects of CsA and rapamycin on proliferation or differentiation of preactivated B cells. For that purpose, highly purified B lymphocytes were preactivated in the presence of formalinized Staphylococcus aureus bacteria and then recultured in the presence or in the absence of either rIL-2, rIL-6, or combination or rIL-2 and rIL-6. After 48 hr in culture, S. aureus bacteria upregulated significantly the binding of phycoerythrin-conjugated IL-2 and IL-6, respectively, by purified B lymphocytes, indicating generation and/or upregulation of receptors for these cytokines. Such preactivated B lymphocytes proliferated in response to optimal concentrations of rIL-2, whereas the addition of rIL-6 to preactivated cells was always accompanied by a decrease of the proliferation rate. CsA upregulated cell proliferation when it was added in the second culture period in the presence or in the absence of rIL-6, whereas rapamycin had no effect in these cases. A combination of rIL-2 plus rIL-6 upregulated significantly the proliferative responses of preactivated B cells. In such cultures both CsA and rapamycin had an inhibitory effect on the proliferative responses. IgM production was unaffected by the addition of rIL-6 to cultures of preactivated B cells, whereas addition of rIL-2 and of the IL-2/IL-6 combination enhanced considerably IgM production. Irrespective of cytokines added, CsA upregulated the production of IgM. In contrast, rapamycin inhibited IgM production in all cases. Our results indicate that, in this experimental system, rapamycin is an effective immunosuppressive agent and its use, at least in vitro, is not accompanied by an upregulation of either the proliferation or differentiation of B lymphocytes.

Binding of phycoerythrin-conjugated interleukin-6 to in vitro-activated human peripheral blood mononuclear cells--effect of immunosuppressive agents and of a calcium channel blocker.

We studied the effect of cyclosporine A, prednisolone, and the Ca2+ channel blocker verapamil on interleukin-6 binding to mitogen-activated peripheral blood mononuclear cells, using a flow cytometric technique and phycoerythrin-conjugated IL-6. All mitogenic stimuli up-regulated IL-6 binding to a variable degree. PHA alone or in combination with PMA was the most effective stimulant in up-regulating IL-6 binding in all the experiments performed. The main changes in IL-6 binding were seen in the large cell cluster, which consisted mainly of lymphoblasts. PHA and PHA/PMA, however, also up-regulated the mean fluorescence intensity on the small cell cluster, which consisted mainly of quiescent lymphocytes. The overall effect of the three pharmacological agents on mitogen-up-regulated IL-6 binding was minimal; most significant were a down-regulation by all three agents of IL-6 binding by small lymphocytes in PHA/PMA cultures, a down-regulation of IL-6 binding by CsA in PHA/PMA-induced large PBMC, and an up-regulation by verapamil of PMA-induced IL-6 binding in large PBMC. Measurements of IL-2 binding and of IL-6 production in the same cultures showed a different pattern than that seen with IL-6 binding, as well as different CsA, prednisolone, and verapamil action. In conclusion, by using a new flow cytometric technique providing information both about the quantity of bound cytokine and about the proportion of IL-6-binding cells, we have demonstrated that IL-6 receptor expression in vitro by PBMC can be up-regulated by the use of stimulants differing in the signal transduction pathways they activate. In addition, by using different pharmacological agents and stimuli to dissect different activation pathways of the in vitro immune response, we conclude that IL-6R generation is regulated differently from IL-6 production. Furthermore, since CsA and prednisolone are known inhibitors of in vitro IL-2 production, our results indicate that IL-6R generation does not rely exclusively on the presence of IL-2.

Mixed lymphocyte reaction-induced release of soluble IL-2 receptor.

We studied the release of soluble interleukin 2 receptor (sIL-2R) by PBMC in mixed lymphocyte reaction (MLR) in order to clarify the significance of high plasma levels of sIL-2R in transplant patients undergoing rejection. Levels of sIL-2R were shown to increase progressively after the first day of the MLR and reached their peak on day 5. This pattern of sIL-2R correlated with the incorporation of [3H]thymidine. CsA and prednisolone (PRED) were added at the beginning of the MLR and were shown to inhibit the release of sIL-2R. This inhibition correlated with an inhibition of the [3H]thymidine incorporation. When CsA and PRED were added 24 hr after the initiation of the MLR, a similar inhibition of sIL-2R release was observed, but when they were added 48 hr after the initiation or in the last day of the MLR little or no effect was observed. Incubation of responder or stimulator-responder cells with either CsA or PRED before the initiation of MLR showed that only CsA preincubation was accompanied by decreased [3H]thymidine incorporation. Preincubation with CsA inhibited the release of sIL-2R, whereas PRED had a variable effect. Recombinant IL-2 was shown to augment the release of sIL-2R even at very low doses, but it did not alter significantly MLR-induced [3H]thymidine incorporation. The addition of rIL-2 at the initiation of the MLR was also shown to reverse completely the PRED inhibition of the MLR-induced release of sIL-2R and of the [3H]thymidine incorporation. Addition of rIL-2 reversed only partially CsA-induced inhibition. Addition of different concentrations of sIL-2R at the initiation of the MLR were not shown to affect incorporation of [3H]thymidine. We conclude that the release of sIL-2R in response to alloantigens is an IL-2-dependent phenomenon, and determination of its levels might be a useful indicator of either in vitro or in vivo alloantigen responses and of the effectiveness of immunosuppressive treatment.

In vitro studies on lymphocyte Fc gamma receptor blocking factors in human renal transplantation.

In this study we have confirmed our earlier observation that the presence in pre-transplant serum of a high-molecular-weight lymphocyte Fc gamma receptor blocking factor correlates with improved human renal allograft survival. This factor was found to bind preferentially to B cells and to impair B cell function in vitro.

Flow cytometric detection of intracellular IL-12 release: in vitro effect of widely used immunosuppressants.

Interleukin 12 (IL-12) is a potent regulator of the Th1/Th2 pathway, enhancing alloantigen-specific immune functions. In the present study, we developed a flow cytometric assay detecting intracellular IL-12 production by human CD14+ monocytes in order to assess the in vitro effects of widely used immunosuppressants, such as cyclosporine (CsA), sirolimus (SRL) and dexamethasone (DXM). For the purpose of the study, a two-step activation procedure was developed involving the preactivation of peripheral blood mononuclear cells (PBMC) with interferon-gamma (IFN-gamma) and reactivation with IFN-gamma and lipopolysaccharide (LPS). All immunosuppressive agents were added at the initiation of the preactivation or the reactivation step. Following this activation protocol, a fourfold to fivefold up-regulation of the percentage of CD14+/IL-12+ cells and of the mean fluorescence intensity was observed. CsA did not significantly affect the intracellular IL-12 release by CD14+ cells, independent of the time point of the addition. SRL exerted an up-regulatory effect when added at the initiation of the IFN-gamma pre-incubation, and this was manifested as a significant increase in the percentage of CD14+/IL-12+ cells. In contrast, DXM effectively repressed both the percentage and the fluorescence intensity of IL-12-producing CD14+ cells when added at the initiation of the reactivation step. Since only the steroid preparation was shown to down-regulate the intracellular release of IL-12, it is tempting to assume that steroid addition in immunosuppressive schemes is beneficial for the suppression of Th1-inducing cytokine production, as well as for the compensation of possible up-regulation induced by other immunosuppressive agents administered concurrently.

In vitro effects of retinoids on mitogen-induced peripheral blood leucocyte responses.

The aim of the present study was to investigate the influence of three different retinoids, isotretinoin, etretinate and acitretin, on the mitogenic response of peripheral blood mononuclear cells (PBMCs) to PHA and PMA in vitro. All three retinoids at high concentrations (10(-4)M) significantly inhibited the mitogenic response of PBMCs to these mitogens. At lower concentrations (10(-5) M and 10(-6) M) none of the three retinoids had any effect on PBMC proliferation in response to PHA. Interestingly, isotretinoin and etretinate significantly enhanced the PMA-induced stimulation of proliferation, whereas acitretin at 10(-5) M failed to influence the mitogenic response to PMA but enhanced it at 10(-6) M. The enhancing effects of retinoids on the proliferative response of PMA-stimulated PBMCs were reversed by rapamycin (10 ng/ml), an immunosuppressant known to inhibit the phorbol ester-induced protein kinase C pathway of lymphocyte activation. In conclusion, our study indicates that retinoids directly trigger the PMA-induced protein kinase C pathway of lymphocyte activation in a concentration-dependent manner. This observation could explain the findings of previous studies showing an in vivo immunopotentiating effect of retinoids on certain immune functions.

Comparative performance evaluation of a donor-specific bead-based crossmatch technique for the detection of donor-specific anti-HLA antibodies in heart transplantation.

INTRODUCTION: Recently new human leukocyte antigen (HLA) antibody screening methods have been shown to significantly improve thoracic transplantation outcomes. The best combination of assays for both allocation and monitoring is still under investigation. Herein, we evaluated the correlation and clinical relevance of three methodologies to detect donor-specific anti-HLA antibodies (DSA). METHODS: For the same donor-recipient combinations we compared the results of the Luminex donor-specific crossmatch (DSA-LX), using donor-isolated HLA antigens coated onto microbeads, with those of a flow-cytometric crossmatch (FCXM) and of the Luminex single-antigen bead methodology (SA-LX), wherein recombinant HLA molecules are coated onto microbeads. We compared 46 pre- (n = 27) and post-transplant (n = 19) serum samples using DSA-LX and SA-LX, while 27 pretransplant samples were additionally analyzed by FCXM. RESULTS: Among the pretransplant sera, the 3 methods were in agreement for class I DSA in 22/27 (81.5%) samples and for class II DSA in 17/27 (63.0%) of tested samples. When the results of the 2 bead-based methodologies were compared with the FCXM methodology, the SA-LX results better correlated than the DSA-LX results both for class I and class II antibodies. Furthermore, the SA-LX results showed greater clinical relevance. The reproducibility scores for both the SA-LX and FCXM were 100%, whereas for the DSA-LX it was 85.5%. CONCLUSIONS: DSA-LX did not correlate with FCXM and SA-LX to detect DSA, particularly for class II antibodies. Furthermore, in comparison to FCXM and SA-LX, DSA-LX showed lower reproducibility rendering the method not eligible for prediction, allocation, or monitoring purposes.

Heart transplantation in a low-organ-donation environment: a single center experience.

OBJECTIVE: Heart transplantation is the "gold standard" for treating patients in end-stage heart failure who satisfy strict selection criteria. However, infrequent transplant performance, eg, less than nine per year, may be associated with suboptimal results. METHODS: We reviewed our 13-year clinical experience (1996-2008) with 73 orthotopic heart transplants performed under strict selection criteria and followed closely thereafter at the only accredited center in Greece, a country with an annual rate of only seven donors per million population. RESULTS: Low perioperative (5.47%) and long-term (7.5%) mortality rates were responsible for a 94% survival rate in the first year, 92% at five years, and 70% at ten years-similar to those reported worldwide-along with excellent functional recovery. CONCLUSION: Strict recipient and donor selection criteria, combined with a rigorous multidisciplinary follow-up, yield excellent results despite the existing shortage of available grafts.

Correlation between intracellular interferon-gamma (IFN-gamma) production by CD4+ and CD8+ lymphocytes and IFN-gamma gene polymorphism in patients with type 2 diabetes mellitus and latent autoimmune diabetes of adults (LADA).

IFN-gamma is considered to be involved in the pathogenesis of diabetes mellitus. In this study, the presence of T/A mutation at position -874 in IFN-gamma gene was assessed in patients with latent autoimmune diabetes of adults (LADA), in patients with type 2 diabetes and in healthy individuals. Subsequently, an attempt was made to correlate the presence of this mutation with the ability of CD4+ or CD8+ lymphocytes from these individuals to release IFN-gamma following mitogenic stimulation. There were no significant differences in the distribution of genotypes and haplotypes in the three study groups. However, the frequency of the low IFN-gamma production allele (IFN-gamma 874( *)A) was significantly higher in type 2 diabetics compared to controls. CD4+ and CD8+ cells obtained from type 2 diabetics released significantly lower amounts of IFN-gamma in the intracellular space, compared to those released by cells obtained from LADA patients and healthy volunteers. Furthermore, even CD4+ and CD8+ from type 2 diabetics bearing the TT genotype (high producers) released significantly lower amounts of IFN-gamma than LADA patients carrying the same genotype, probably due to the activity of molecules directly or indirectly inhibiting IFN-gamma production. The results of this study indicate that IFN-gamma may contribute to the development of type 2 diabetes, based on a combination of molecular and immunological observations.

Up-regulation by cyclosporine (CsA) of the in vitro release of soluble CD23 (sCD23) and of the in vitro production of IL-6 and IgM.

In the present study, we examined the effect of CsA on the in vitro production of Ig and on the in vitro production of molecules known to have B-cell growth and differentiation activities, such as IL-6 and sCD23. For the purpose of this study, we developed an experimental in vitro system closely resembling an in vivo model of ongoing B-cell activation. Pre-activated B cells proliferated and produced IgM optimally when they were re-cultured in the presence of IL-2/IL-6. CsA down-regulated the IL-2/IL-6-induced proliferative responses of pre-activated B cells by at least 50%, but it up-regulated IgM production in the same experiments. This up-regulating effect was not cytokine-related since it was also seen when cells were re-cultured in the absence of any cytokines. Optimal release of sCD23 was observed when SAC-pre-activated B cells were re-cultured in the presence of IL-4 or IL-4 plus IL-2 and CsA up-regulated significantly the release of this molecule in these cultures. Finally, CsA was shown to inhibit PHA-induced cell proliferation of PBMC and to up-regulate IL-6 production in the same cultures. We conclude that CsA can amplify in vitro both the production of Ig and the release of sCD23 by pre-activated B cells. This finding, in combination with the CsA-induced up-regulation of lectin-induced IL-6 production, may have clinical implications in disease states with an ongoing immune activation, where prolonged administration of CsA might be anticipated.

Rapamycin inhibits the in vitro release of soluble interleukin-2 receptor by activated peripheral blood mononuclear cells (PBMC) independently of the mode of activation.

In the present study we compared the effect of rapamycin to that of CsA on the in vitro responses of lectin (PHA), phorbol-ester (PMA) and CA2+ ionophore (ionomycin)-activated peripheral blood mononuclear cells by measuring the release of soluble IL-2R (sIL-2R), high levels of which have been detected in clinical syndromes characterized by an ongoing immune activation. PHA was the stimulant associated with high sIL-2R release, whereas ionomycin-induced sIL-2R release only exceeded the background response and the sensitivity of the ELISA kit. The highest sIL-2R release, however, was obtained when PMA was used in combination with either PHA or ionomycin. Rapamycin inhibited the release of sIL-2R in response to all activators, whereas CsA only abolished the ionomycin-induced sIL-2R release. In parallel experiments rapamycin inhibited cell proliferation in response to all stimulants with the exception of PMA/ionomycin, whereas CsA inhibited all proliferation. Our study clearly shows that for optimal sIL-2R release both Ca+ and protein kinase C-triggered signals are required and that rapamycin has a distinct advantage over CsA in inhibiting the release of sIL-2R, which has been shown to be a reliable marker of lymphocyte activation either in vivo or in vitro.