Pro-Inflammatory Cytokines in Nasopharyngeal Aspirate From Hospitalized Children With Respiratory Syncytial Virus Infection With or Without Rhinovirus Bronchiolitis, and Use of the Cytokines as Predictors of Illness Severity.

Respiratory syncytial virus (RSV) and human rhinovirus (HRV) respiratory infection in children induce production of inflammatory interleukins (ILs) in the respiratory epithelium. As IL(s) determine the severity of illness, the purpose of this study was to identify the pro-inflammatory IL(s) that could be predictor(s) of clinical severity. One hundred and fifteen patients <2 years old with bronchiolitis due to RSV and /or HRV and 38 controls were selected from a hospital and an outpatient clinic. Clinical data of all patients were recorded. Severity was defined by the number of days with oxygen need. Nasopharyngeal aspirates (NPA) were collected to perform viral diagnosis by quantitative reverse transcription and polymerase chain reaction (qRT-PCR) and to quantify ILs: TNF-α, IL-10, IL-6, IL-1ß, and IL-8, by flow cytometry. Simple and multiple regression and receiver operating characteristic (ROC) curves were used for statistical analysis. Of the patients selected 60 were single RSV, 28 RSV associated to HRV, and 27 single HRV. All patients (115) showed significantly higher IL levels when compared with controls. Levels of IL-6, IL-1ß, and IL-8 detected in NPA from RSV single and associated to HRV were significantly higher than HRV infected and positively associated with days requiring O2.Levels of IL-6, IL-1ß, and IL-8 detected in NPA from patients infected with RSV only or with both RSV and HRV are increased, and any of those 3 cytokines may have a predictive value for the number of days with need of supplemental oxygen.

Notch signalling regulates cytokine production by CD8+ and CD4+ T cells.

The Notch signalling pathway regulates several aspects of cellular differentiation such as T lineage commitment and effector functions on peripheral T cells; however, there is limited information regarding Notch receptor expression on different T cell subsets and the putative role of the different receptors on T cell effector function. Here, we studied the protein expression of Notch receptors on murine T cells in vitro and in vivo and analysed the role of the Notch pathway in cytokine production by CD4+ and CD8+ T cells. We found that resting CD4+ and CD8+ T cells do not express Notch receptors, but they upregulate Notch 1 and Notch 2 shortly after in vitro and in vivo activation. Using a γ-secretase inhibitor, which blocks Notch signalling through all Notch receptors, we demonstrated that the Notch pathway regulates IL-10 production by CD4+ T cells and IFN-γ and IL-17 production by CD8+ T cells. These results suggest that Notch 1 and 2 are expressed by CD4+ and CD8+ T cells and represent the putative Notch receptors that regulate effector functions and cytokine production by these cells.

Retinoic acid generates regulatory T cells in experimental transplantation.

Regulatory T cells play a key role to inhibit effector lymphocytes, avoid, autoimmunity, and restrain allogeneic immunity. Retinoic acid is an important cofactor that stimulates the generation and expansion of regulatory T cells. Naive T cells, coincubated with allogeneic antigen-presenting cells and retinoic acid, in conjunction with transforming growth factor (TGF) ß and interleukin (IL) 2, generated allogeneic regulatory T cells de novo. These cells were able to inhibit skin rejection in adoptive transfer experiments. The generation of regulatory T cells ex vivo with retinoic acid, TGF-ß, and IL-2 represents a new step toward specific regulation of allogeneic immune responses.

Delivery of alloantigens via apoptotic cells generates dendritic cells with an immature tolerogenic phenotype.

BACKGROUND: Dendritic cells (DCs) are professional antigen-presenting cells able to induce immunity or tolerance. The interactions of immature DCs with naive T lymphocytes induce peripheral tolerance through mechanisms that include anergy or deletion of lymphocytes or the generation of regulatory T cells. Because of the central role of DCs in the immune response, they are potential targets for the induction of experimental tolerance. Thus, the generation of immature (tolerogenic) DCs able to capture and present alloantigens to T cells represents an important aim in our efforts to achieve better transplant acceptance. METHODS: In this work, we generated immature DCs by using vitamin D(3) (VD3) during the process of DC differentiation. RESULTS: The VD3-DCs showed an immature phenotype characterized by a low expression of major histocompatibility complex antigens of class II, CD86, and CD80 molecules and the secretion of a tolerogenic cytokine pattern. Furthermore, we showed that VD3-DCs phagocytose apoptotic allogeneic cells efficiently without inducing DC maturation or activation. Most important, our experiments demonstrated that mice treated with VD3 produce immature DCs in vivo, and that DCs from VD3-treated mice immunized with allogeneic apoptotic cells maintained their tolerogenic phenotype. CONCLUSION: Our results show that allogeneic apoptotic cells in combination with VD3 generate DCs with tolerogenic characteristics that could be used to induce tolerance towards alloantigens.

Differential regulation of Notch ligands in dendritic cells upon interaction with T helper cells.

The Notch signalling pathway has recently been linked to T helper 1 (Th1)/T helper 2 (Th2) cell polarization via a mechanism involving differential expression of Notch ligands, Delta-like and Jagged, in antigen-presenting cells. However, whether stimuli other than pathogen-derived factors are involved in the regulation of Notch ligand expression in dendritic cells (DCs) remains unknown. Here, we address the effect of T helper cells (Th1 and Th2) on Delta-like 4 and Jagged 2 expression in bone marrow-derived DCs. We demonstrate that both Th1 and Th2 cells induce Delta-like 4 mRNA expression in DCs, in a process that is, in part, mediated by CD40 signalling. In contrast, only Th2 cells induce a significant increase in Jagged 2 mRNA levels in DCs. Additionally, we show that IL-4, a hallmark Th2 cytokine, plays a role in Jagged 2 expression, as evidenced by the fact that cholera toxin, a Th2-promoting stimulus, induces Jagged 2 mRNA expression in DCs only in the presence of IL-4. Finally, we demonstrate that DCs also express Notch 1 and that this expression is downregulated by IL-4. These data suggest that Notch ligands are differentially regulated in DCs: Delta-like 4 is regulated by T helper cells and by pathogen-derived Th1 stimuli, whereas Jagged 2 is regulated by Th2 cells and pathogen-derived Th2-promoting stimuli. Based on our results, we propose that the positive feedback loop that Th2 cells exert on T cell polarization may involve the induction of Jagged 2 expression in DCs.

Dendritic cells and B cells cooperate in the generation of CD4(+)CD25(+)FOXP3(+) allogeneic T cells.

BACKGROUND: CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) play an essential role in immune tolerance, suppressing responses against self-antigens. Additionally, Treg play an important role in maintaining immunosuppression to alloantigens as well as to other antigens. It is well known that in the gut, a subset of dendritic cells produces retinoic acid (RA), which together with transforming growth factor (TGF-beta) is able to differentiate naïve T cells into Treg. The aim of this study was to establish the role of antigen-presenting cells (APC) in the differentiation of allogeneic Tregs under the effect of RA and TGF-beta. METHODS: Splenic CD4(+)CD25(-) naïve T cells from C57BL/6 mice were co-cultured with splenic CD11c-enriched APC from Balb/c mice in the presence of TGF-beta, RA, and interleukin (IL-2). After 6 days of culture, cells were analyzed for the expression of Foxp3 by flow cytometry. Additionally, we investigated the role of B cells and dendritic cells (DCs) and their stimulatory capacity in the generation of Tregs. RESULTS: Our results showed that co-culture of naive T cells with the appropriate level of stimulation by APC in the presence of TGF-beta, RA, and IL-2 provided a new powerful approach to generate allogeneic Treg cells. We demonstrated that although B cells and DCs can generate Tregs by themselves, a mixure of both APC improved their capacity to efficiently generate Tregs. Also, we observed that although the addition of IL-2 to the cultures was not crucial to generate Tregs, it was required to optimize their expansion and cell survival.

Transforming growth factor-beta and all-trans retinoic acid generate ex vivo transgenic regulatory T cells with intestinal homing receptors.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) mediate immunologic self-tolerance and suppress immune responses. In the gut, a subset of dendritic cells is specialized to induce Treg in a transforming growth factor-beta (TGF-beta)- and retinoic acid (RA)-dependent manner. The aim of this study was to establish if RA synergizing with TGF-beta induced antigen specific CD4(+) CD25(high) Foxp3(+) Treg portraying gut homing receptors. Splenic CD4(+)CD25(-) Foxp3(-) naïve T cells from DO11.10 mice were cocultured with splenic CD11c(+) dendritic cells from Balb/c mice in the presence of TGF-beta, RA, and low levels of an antigenic peptide. After 5 days of culture, cells were analyzed for the expression of Foxp3 and the gut homing receptors CCR9 and alpha4beta7. The number of Foxp3(+) T cells generated with TGF-beta and RA was at least 3 times higher than in the cultures with TGF-beta alone and 15 times higher than in controls without exogenous cytokines. Also, supplementation of the cultures with RA induced the expression of the intestinal homing receptors CCR9 and alpha4beta7. Our results showed that coculture of naïve T cells with antigen-presenting cells in the presence of TGF-beta and RA represents a powerful approach to generate Treg with specific homing receptors.

Alemtuzumab induction in kidney transplantation: clinical results and impact on T-regulatory cells.

Alemtuzumab (ALT), a humanized monoclonal anti-CD52 antibody, was introduced in solid organ transplantation as an induction agent. ALT associated with anticalcineurins has provided a low incidence of acute rejection episodes (ARE) and potential tolerogenic properties. We analyzed the clinical outcomes and effects on peripheral Treg of renal transplant recipients treated with ALT. Six-month data on kidney alone or kidney combined with pancreas or liver patients treated with ALT and tacrolimus (TAC) in standard doses were compared with those on renal transplant recipients of similar demography who were not treated with ALT. We evaluated patient and graft survivals, ARE incidence, hematological parameters, renal function, adverse events, and CD4+CD25+FoxP3+ T cells in peripheral blood. Demographics of recipients, donors, and transplants were similar in both groups. Mean HLA mismatch was slightly greater among ALT-treated patients (3.5 vs 2.5). No combined transplantation was performed in the ALT-untreated group. Patient and graft survivals were 100% without rejection or serious infections in both groups. ALT-treated recipients showed anemia and leukopenia in 3 patients as well as severe lymphopenia in 5 recipients, who partially recovered on day 90. Final mean plasma creatinine was 1.4 mg/dL, while calculated creatinine clearance was approximately 65 mL/min in both groups. Mean Treg cell percentage was higher among ALT-treated recipients than the comparative group or healthy controls (P < .05). In conclusion, renal transplantation results obtained using ALT with rigorous immunosuppressive therapy were excellent; serious adverse events and acute rejection were absent. The effect of the increased proportion of Treg cells must be evaluated with longer observation.

Generation of dendritic cells with regulatory properties.

Dendritic cells (DCs) are professional antigen presenting cells with the ability to induce and regulate an immune response. DCs that capture and present antigen under noninflammatory conditions maintain an immature phenotype and acquire tolerogenic properties. These DCs generate regulatory T lymphocytes that potentiate tolerogenic responses. Here we developed a method for the generation of immature murine DCs able to process and present a specific antigen in a tolerogenic context. Immature DCs were prepared from bone marrow precursors after differentiation with granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence of vitamin D(3) and characterized by their low expression of major histocompatibility complex class (MHC) II and CD86 molecules. Purified phagosomes containing either MHC II molecules or ovalbumin were used to deliver antigens to immature DCs. More than 80% of the DCs captured the phagosomes, while maintaining a low expression of maturation markers and showing basal levels of secretion of activating cytokines such as interleukin (IL)-2 and IL-12. Treatment of the immature DCs with lipopolysaccharides (LPS) increased IL-10 secretion, in agreement with their anti-inflammatory and immune regulatory properties. Cocultures of transgenic OT-II T lymphocytes with the immature DCs carrying OVA-phagosomes succeeded in generating a subpopulation of regulatory T lymphocytes characterized by the expression of CD4, CD25, CD62L, and Foxp3. Taken together, our results suggest that vitamin D(3) generates immune tolerance through the modulation of DC phenotype and could be useful to induce tolerance to allotransplants.

Interleukin-4 selectively inhibits interleukin-2 secretion by lipopolysaccharide-activated dendritic cells.

Dendritic cells (DCs) generated in vitro from bone marrow precursors using granulocyte-macrophage colony-stimulating factor (GM-CSF) secrete interleukin-2 (IL-2) upon activation, an event probably associated to the initiation of adaptive immune responses. Additionally, they produce IL-12, a cytokine related to T-cell polarization. To analyse the effect of IL-4 on DC differentiation and function, we assessed the capacity of murine bone marrow dendritic cells (BMDCs) differentiated with GM-CSF in the presence or absence of IL-4 to produce IL-2 and IL-12 upon lipopolysaccharide (LPS) activation. We found that although IL-4 enhanced DC IL-12p70 production, it strongly impaired IL-2 secretion by BMDCs. This inhibition, which depends on the presence of IL-4 during LPS activation, is DC specific, as IL-4 did not affect IL-2 secretion by T cells. Interestingly, inhibition of DC IL-2 production did not prevent DC priming of T lymphocytes. These results illustrate a new putative role for IL-4 on the regulation of the immune response and should help clarify the controversial reports on the effect of IL-4 on DCs.

Cyclosporine preconditions dendritic cells during differentiation and reduces IL-2 and IL-12 production following activation: a potential tolerogenic effect.

The mode of action of cyclosporine (CsA) has been ascribed to its capacity to inhibit IL-2 and IFNgamma production by T cells, two cytokines implicated in allograft rejection. Recently, it has been reported that upon activation, dendritic cells (DCs) exhibit transient production of IL-2, a property that appears to be related to their capacity to initiate immune responses. On the other hand, DCs can generate signals determining Th1/Th2 polarizing effects, an effect that can drastically influence the outcome of organ transplant. The purpose of the present study was to investigate the effect of CsA on cytokine production by immature and mature DCs. DC precursors from mouse bone marrow were induced to differentiate by incubation with GM-CSF for 5 days followed by activation with LPS for 4 hours. CsA was added at different times during this process. Our results show that when CsA is added during the differentiation period following activation with LPS, IL-2 and IL-12 secretion are significantly reduced without affecting the evolution of the DC. Conversely, CsA had no effect when added during the LPS activation period. These results show that CsA affects DCs before they receive the final activation stimulus, preconditioning them to antigen stimulation. This preconditioning of DCs by calcineurin-inhibiting drugs conceptually integrates the mode of action of CsA with the tolerogenic and T-cell polarization function ascribed to DCs. These results may be especially meaningful for the future design of immunosuppressive protocols.

Expression of the human SRY protein during development in normal male gonadal and sex-reversed tissues.

Sex determination in mammals is controlled by the SRY gene located on the Y chromosome. It encodes a protein containing a DNA-binding and DNA-bending domain. In spite of recent advances in the identification of the mechanisms that regulate male sex determination in mammals, the expression profile of the SRY protein in normal and sex-reversed human tissues is not well established. In order to localize the SRY protein and determine its cellular distribution and expression at different stages of development, we prepared monoclonal antibodies (mAb) against the recombinant SRY protein. One of these antibodies, LSRY1.1, recognizes a protein of 27 kDa in total lysates of HeLa SRYB3, a human cell line transfected with the SRY gene under the control of the SV40 promoter. Immunocytochemical analysis in the cell lines shows nuclear localization of the SRY protein. We have studied SRY protein expression in human tissues at different stage of fetal development until adult life and have demonstrated that the SRY protein is located in the nuclei of somatic cells and germ cells in the genital ridge during testis development. After testis determination, it can be detected until the adult stage in both germ cells and Sertoli cells. The presence of the SRY protein was also analyzed in biopsies of gonadal tissues of sex-reversal patients such as SRY-positive 46,XX males or SRY-positive 46,XX true hermaphrodites. SRY protein is detected in the nuclei of Sertoli cells of the testis and in the nuclei of granulosa cells in the ovotestis in these patients and in the nuclei of germ cells of both tissue types. These results suggest a common cellular origin for both Sertoli cells and granulosa cells.

Increased expression of c-rel, from the NF-kappaB/Rel family, in T cells from patients with systemic lupus erythematosus.

OBJECTIVE: To explore the role of the NF-kappaB/Rel transcription factor family in autoimmunity, we investigated whether peripheral blood mononuclear cells (PBMC) and T cells from the blood of patients with systemic lupus erythematosus (SLE) exhibit abnormal expression of c-rel, both when recently isolated and/or during in vitro activation. METHODS: Total RNA and protein extracts were prepared from PBMC and T cells isolated by immunoadsorption with magnetic beads. The relative concentrations of c-rel mRNA and of c-Rel protein were determined by semiquantitative assays of competitive reverse transcriptase-polymerase chain reaction and chemiluminescent immunoblots, respectively. Activity of NF-kappaB/Rel was studied by electrophoretic mobility shift assay of nuclear extracts. RESULTS: Significantly increased levels of c-rel mRNA were found (1) in PBMC from SLE patients (n = 48; p<0.0000001), even during inactive disease (n = 11; p<0.001), compared to controls (n = 54), and (2) in T cells isolated from a subgroup of these patients (n = 11; p<0.00002) and controls (n = 12). c-Rel protein was found increased in the cytosol but not in the nucleus of PBMC of patients with SLE (n = 12; p<0.02) compared to controls (n = 12). No evidence of NF-kappaB/Rel nuclear activity was detected. In vitro stimulation of T cells by incubating PBMC with concanavalin A showed that less c-Rel entered the nucleus in lupus cells than healthy cells, correlating with lower interleukin 2 production. However, the same stimulating conditions provoked an increase in c-rel mRNA to higher levels in lupus cells from 2 patients compared with 2 controls. Increased levels of both IkappaB alpha and IkappaB beta could account for c-Rel cytosolic retention. CONCLUSION: Our data suggest that T cells from patients with SLE possess altered regulatory mechanisms of c-rel expression and nuclear import that might potentially determine conditions for developing autoimmunity. Other cells present in the PBMC could also be affected.

The human SRY protein is present in fetal and adult Sertoli cells and germ cells.

Sex determination in mammals is controlled by the Y chromosome located SRY gene. Despite recent advances towards understanding the mechanisms that regulate sex determination in mammals, the expression profile of the SRY protein in human tissues is unknown. To localize the SRY protein and determine its cellular distribution, we prepared monoclonal antibodies (mAb) against the recombinant SRY protein. One antibody, LSRY1.1, recognizes a SRY-specific epitope and was used to localize the protein in different cells and tissues. The mAb recognizes a protein of 27 kDa in total lysates of HeLa SRYB3 cells. Immunocytochemical staining showed a nuclear localization of the protein. Immunohistochemical studies performed on gonadal tissue of a fetus, a one month-old boy and an adult man, demonstrated the presence of SRY protein in the nucleus of Sertoli and germ cells. In addition two 46,XX SRY(+) males had the SRY protein in their gonadal tissues. All other samples were negative, including all female tissue studied and the testis of a 46,XX SRY(-) male. The presence of SRY protein in fetal and adult gonadal tissues including germ cells suggests that SRY may have other male-specific functions in addition to sex determinism.

A flow cytometric procedure for the quantification of cell adhesion in complex mixtures of cells.

We present a simple non-radioactive cytometry-based assay that permits the simultaneous quantitation of cell adhesion of distinct subsets of cells contained in a mixture without any previous fractionation. The procedure is simple and highly reproducible and has the advantage of confining the quantitation of cell adhesion to live cells only. This new approach is based on counting the absolute number of cells. This is done by adding known numbers of distinguishable beads to the cell suspension and counting beads and cells in a cytometer. Quantitation of adhesion is accomplished by counting each subpopulation of cells before and after the adhesive process. To illustrate this methodology we determined adhesion of Ramos cells to monolayers of endothelial cells and its inhibition by specific antibodies. Also, we determined adhesion to endothelial cells of B lymphocytes and subsets of T lymphocytes present in a preparation of unfractionated human mononuclear cells. The results presented here demonstrate that the new assay has the required properties to be used in the quantitation of cell adhesion.