Epigallocatechin-3-gallate inhibits doxorubicin-induced inflammation on human ovarian tissue.

Chemotherapy protocol can destroy the reproductive potential of young cancer patients. Doxorubicin (DOX) is a potent anthracycline commonly used in the treatment of numerous malignancies. The purpose of the study was to evaluate the ovarian toxicity of DOX via inflammation and the possible protective effect of the green tea polyphenol epigallocatechin-3-gallate (EGCG). Ovarian tissue of three patients was cultured with 1 µg/ml DOX and/or 10 µg/ml EGCG for 24 and 48 h. Levels of inflammatory factors were determined by quantitative Real-Time PCR, western blot, zimography, and multiplex bead-based immunoassay. Morphological evaluation, damaged follicle count and TUNEL assay were also performed. DOX influenced inflammatory responses by inducing a significant increase in the expression of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and cyclooxigenase-2 (COX-2), of inflammatory interleukins (IL), such as interleukin-6 (IL-6) and interleukin-8 (IL-8), and the inflammatory proteins mediators metalloproteinase-2 and metalloproteinase-9 (MMP2 and MMP9). IL-8 secretion in the culture supernatants and MMP9 activity also significantly raised after DOX treatment. Moreover, a histological evaluation of the ovarian tissue showed morphological damage to follicles and stroma after DOX exposure. EGCG significantly reduced DOX-induced inflammatory responses and improved the preservation of follicles. DOX-induced inflammation could be responsible for the ovarian function impairment of chemotherapy. EGCG could have a protective role in reducing DOX-mediated inflammatory responses in human ovarian tissue.

Potential pathogenetic role of Th17, Th0, and Th2 cells in erosive and reticular oral lichen planus.

OBJECTIVES: The role of Th17 cells and associated cytokines was investigated in oral lichen planus. MATERIAL AND METHODS: 14 consecutive patients with oral lichen planus were investigated. For biological studies, tissues were taken from reticular or erosive lesions and from normal oral mucosa (controls) of the same patient. mRNA expression for IL-17F, IL-17A, MCP-1, IL-13, IL-2, IL-10, IL-1ß, RANTES, IL-4, IL-12B, IL-8, IFN-γ, TNF-α, IL-1α, IL-18, TGF-ß1, IL-23R, IL-7, IL-15, IL-6, MIG, IP-10, LTB, VEGF, IL-5, IL-27, IL-23A, GAPDH, PPIB, Foxp3, GATA3, and RORC was measured using the QuantiGene 2.0. RESULTS: Results showed that Th17-type and Th0-type molecules' mRNAs, when compared with results obtained from tissue controls, were increased in biopsies of erosive lesions, whereas Th2-type molecules' mRNAs were increased in reticular lesions. When the CD4+ T-cell clones, derived from oral lichen planus tissues and tissue controls, were analyzed, a higher prevalence of Th17 (confirmed by an increased CD161 expression) and Th0 CD4+ T clones was found in erosive lesions, whereas a prevalence of Th2 clones was observed in reticular lesions. CONCLUSIONS: Our data suggest that Th17, Th0, and Th2 cells, respectively, may have a role in the pathogenesis of erosive and reticular oral lichen planus.

Cytokines and chemokines in follicular fluids and potential of the corresponding embryo: the role of granulocyte colony-stimulating factor.

BACKGROUND: The cytokine/chemokine levels of individual follicular fluids (FFs) were measured to determine whether a biomarker could be linked to the developmental potential of the derived embryo. METHODS: Fluid was collected from 132 individual FFs that were the source of oocytes subsequently fertilized and transferred. In each, a bead-based multiplex sandwich immunoassay (Luminex) was used to measure 28 cytokines and chemokines simultaneously. RESULTS: Significantly higher levels of interleukin (IL-2) and interferon (IFN-gamma) were detected in FF for embryos that underwent early cleavage. IL-12 was significantly higher in FF corresponding to highly fragmented embryos and the chemokine CCL5 was significantly higher in FF related to the best quality (Top) embryos. The level of granulocyte colony-stimulating factor (G-CSF) in individual FF samples was correlated with the implantation potential of the corresponding embryo. The area under the receiver operating characteristics curve, which distinguished the embryos that definitely led to delivery from those that did not, was 0.84 (0.75-0.90) (P = 0.0001) for FF G-CSF. FF G-CSF was significantly lower in patients older than 36 years compared with those <30-year old. When the FF G-CSF was 20 pg/ml or higher, the ratio between Top and non-Top embryos was significantly higher than for the group with FF G-CSF below 20 pg/ml (45 versus 20.45%, P = 0.007). CONCLUSIONS: Individual FF composition is related to the development of the corresponding in vitro generated embryo and its potential of implantation. Individual FF G-CSF may provide a non-invasive biomarker of implantation that needs to be evaluated together with in vitro observation to select the oocyte, and hence the embryo, to transfer.

Defective production of LIF, M-CSF and Th2-type cytokines by T cells at fetomaternal interface is associated with pregnancy loss.

Development of CD4+ helper T (Th) cells into type 1 (Th1) or type 2 (Th2) effectors can be influenced by hormones enhanced during pregnancy. Progesterone, at concentrations comparable to those found at fetomaternal interface, promotes the production of IL-4 and IL-5, whereas relaxin promotes the production of IFN-gamma by T cells. Furthermore, Th1-type cytokines promote allograft rejection and, therefore, may compromise pregnancy, whereas Th2-type cytokines, which inhibit Th1 responses, may allow allograft tolerance. In addition, T cell production of Leukemia Inhibitory Factor (LIF) and macrophage-stimulating factor (M-CSF), which are essential for embryo implantation and development, are up-regulated by IL-4 and progesterone. Finally, a direct cause-and-effect relationship between the defective production of LIF, M-CSF and Th2-type cytokines by T cells present at feto maternal interface and the pregnancy loss has been observed.

Microchimerism and systemic sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease characterized by progressive fibrosis of the skin and internal organs. SSc is an immunologically mediated disease. A prominent immunological abnormality in SSc patients is the presence of circulating autoantibodies against a variety of nuclear proteins. Furthermore, SSc is characterized by the presence of increased numbers of activated T cells, with the prevalence of CD4+ cells, present in the periphery of skin lesions as well as in other organs in the early stages of the disease. We have recently shown the existence of a predominant activation of IL-4-producing Th2-like T cells in patients with SSc, which may account for the major alterations which occur in this disease. SSc has clinical and serological similarities to chronic graft versus host disease (cGVHD), although there are some important differences. T cells, which orchestrate the tissue damage, are present in great amounts in the inflammatory infiltrates in SSc- and cGVHD-affected tissues. More importantly, T cells from cGVHD tissues produce Th2-like cytokines, thus showing a pathogenetic similarity with SSc. SSc has been postulated as a type of cGVHD resulting from the transplacental transfer of cells between mother and fetus. Very recently, we have shown that in SSc, the microchimeric T cells react with the maternal MHC antigens and are able to produce Th2-type cytokines. Both features are characteristics of cGVHD, supporting the hypothesis that SSc is a disease similar to cGVHD.

Production of IL-4 and leukemia inhibitory factor by T cells of the cumulus oophorus: a favorable microenvironment for pre-implantation embryo development.

The nature and the functional activity of immunocytes present in the cumulus oophorus, a mass of cells surrounding the oocyte, were examined here for the first time. The cumuli oophorus were obtained from women who had taken part in an in vitro fertilization program and were suffering from blocked fallopian tubes. Both macrophages and CD4(+) T cells were detected in all cumuli. CD4(+) T cell clones, generated from T cells of these cumuli, showed higher potential to produce IL-4 and leukemia inhibitory factor (LIF) than CD4(+) T cell clones generated from peripheral blood or ovary specimens from the same women. More importantly, IL-4 and LIF, but not IFN-gamma mRNA was found to be constitutively expressed in vivo by cumulus oophorus cells. Progesterone is highly produced by the cumulus oophorus/oocyte complex. We recently showed that progesterone up-regulates the production of LIF by T cells and that the progesterone-induced LIF production is mediated by IL-4. Progesterone produced by cumulus granulosa cells may favor IL-4 production by T cells, which in turn can produce LIF. As the treatment with LIF enhances the in vitro growth and development of mammalian embryos, our data suggest that T cells present in the cumulus oophorus produce cytokines that may provide a microenvironment suitable for pre-implantation development of the mammalian embryo.

Oligoclonal T cell repertoire in cerebrospinal fluid of patients with inflammatory diseases of the nervous system.

OBJECTIVE: To evaluate the T cell receptor beta chain variable region (TCRBV) gene usage ex vivo in CSF cells and peripheral blood mononuclear cells (PBMCs) collected from patients with autoimmune and inflammatory diseases of the nervous system. METHODS: A novel sensitive seminestedpolymerase chain reaction coupled with heteroduplex analysis was developed. RESULTS: Under these experimental conditions, the minimal number of cells required for the analysis of the whole T cell repertoire was established at 2.5x10(4)-sufficient to evaluate most of the samples collected during diagnostic lumbar punctures. In the 21 patients examined, restrictions in TCRBV gene family usage were not seen. However, using heteroduplex analysis, oligoclonal T cell expansions were found in the CSF of 13 patients and monoclonal expansions in five patients. The T cell abnormalities found did not correlate with intrathecal IgG production or with any clinical variable considered. CONCLUSION: T cell clonal expansions, useful for further characterisation of pathogenetic T cells, can be found during the course of nervous system inflammations, but this abnormality is probably not disease specific.

Role of hormone-controlled Th1- and Th2-type cytokines in successful pregnancy.

Development of CD4+ helper T (Th) cells into type 1 (Th1) or type 2 (Th2) effectors, as characterized by their opposite pattern of cytokine production, can be influenced by several factors, including hormones. Progesterone promotes the production of IL-4 and IL-5, whereas relaxin promotes the production of IFN-gamma by T cells. Leukemia inhibitory factor (LIF), essential for embryo implantation, is up-regulated by IL-4 and progesterone. Moreover, the production of LIF and/or Th2 cytokines by decidual T cells contributes to the maintenance of pregnancy. Our results suggest that relaxin and progesterone may contribute to the regulation of the immune homeostasis during pregnancy.

Role of hormone-controlled T-cell cytokines in the maintenance of pregnancy.

Human CD4 T helper lymphocytes can be subdivided into at least three distinct functional subsets on the basis of their cytokine secretion profiles. One type of CD4+ lymphocyte, T helper 1 (Th1), produces interferon (IFN)-gamma and tumour necrosis factor beta, a second type (Th2) produces interleukin (IL)-4 and IL-5 and a third type (Th0) produces both Th1 and Th2 cytokines. The apparent paradox that embryos are not rejected by the maternal immune system despite the presence of paternal MHC histocompatibility antigens has been explained in mice by a Th2 switch at the level of the materno-fetal interface. We showed that some hormones enhanced during pregnancy can affect the development of Th1 and Th2 responses. Indeed, we found that progesterone promotes the production of IL-4 and IL-5, whereas relaxin promotes the production of IFN-gamma by T-cells. In addition, we showed that leukaemia inhibitory factor (LIF), which is essential for embryo implantation, associates with Th2 cells and is upregulated by IL-4 and progesterone. We also showed that LIF is down-regulated by Th1 inducers [IL-12, IFN-gamma and IFN-alpha]. Furthermore, we found a decreased production of LIF, IL-4 and IL-10 by decidual T-cells in women with unexplained recurrent abortions in comparison with women with normal gestation at the moment of voluntary abortion. The decreased production of LIF, IL-4 and IL-10 was not found in peripheral-blood T-cells. These results suggest that the local production of LIF and/or Th2 cytokines may contribute to the maintenance of pregnancy.

Th1/Th2 cells, their associated molecules and role in pathophysiology.

Cytokines, chemokines, and/or chemokine receptors associated with type 1 T helper (Th1) or Th2 cells play a role in different physiological conditions, such as T lymphopoiesis and pregnancy, as well as in pathological conditions, such as unexplained recurrent abortions, proliferative glomerulonephritis, and control of angiogenesis.

Environmental factors favoring the allergen-specific Th2 response in allergic subjects.

Allergen-reactive type 2 helper T cells (Th2) play a triggering role in the activation and/or recruitment of IgE antibody-producing B cells, mast cells, and eosinophils, i.e., the cellular triad involved in the allergic inflammation. Interleukin (IL)-4 production at the time of antigen presentation to the Th cell is critical for the development of Th2 cells. Other cytokines, such as IL-1 and IL-10, and hormones, such as calcitriol and progesterone, also play a positive role. In contrast, cytokines such as interferon (IFN)-alpha, IFN-gamma, IL-12, and transforming growth factor (TGF)-beta, and relaxin play a negative regulatory role on the development of Th2 cells. However, the mechanisms underlying the preferential activation by environmental allergens of Th2 cells in atopic individuals still remain obscure. Some gene products selectively expressed in Th2 cells or selectively controlling the expression of IL-4 have recently been described. Moreover, cytokines and other gene products that dampen the production of IL-4, as well as the development and/or the function of Th2 cells, have been identified. These findings allow us to suggest that the upregulation of genes controlling IL-4 expression and/or abnormalities of regulatory mechanisms of Th2 development and/or function may be responsible for Th2 responses against common environmental allergens in atopic subjects.

Relaxin favors the development of activated human T cells into Th1-like effectors.

Differentiation of naive CD4+ helper T (Th) cells into Th1 or Th2 effectors, as characterized by their opposite pattern of cytokine production, can be influenced by several factors, including hormones. In this study, we demonstrate that porcine relaxin, at concentrations ranging from 10(-10) to 10(-6) M, favors the in vitro development of human antigen-specific T cells into Th1-like effectors and enhances both IFN-gamma mRNA expression and IFN-gamma production by established human T cell clones. The promoting effect of relaxin on the development of IFN-gamma-producing cells was not due to a relaxin-induced release of IL-12 and/or IFN-alpha by antigen-presenting cells. These results suggest that relaxin may contribute to the regulation of the immune homeostasis during pregnancy and may also play some role in counteracting Th2-dominated disorders.

Defective production of both leukemia inhibitory factor and type 2 T-helper cytokines by decidual T cells in unexplained recurrent abortions.

Leukemia inhibitory factor is essential for embryo implantation, and a shift from type 1 T-helper to type 2 T-helper response at the fetal-maternal interface may contribute to successful pregnancy. We show that LIF production is associated with type 2 T-helper cells, is upregulated by IL-4 and progesterone and is downregulated by IL-12, IFN-gamma and IFN-alpha. We also show a decreased production of LIF, IL-4 and IL-10 by decidual T cells of women with unexplained recurrent abortions in comparison with that of women with normal gestation. The defective production of LIF and/or type 2 T-helper cytokines may contribute to the development of unexplained recurrent abortions.

An update on human Th1 and Th2 cells.

The existence of functionally polarized human T cell responses based on their profile of cytokine secretion in both the CD4+ T helper (Th) and the CD8+ T cytotoxic cell subset has been established. Human Th1 and Th2 cells not only produce a different set of cytokines but also exhibit distinct functional properties and preferential expression of some activation markers, such as LAG-3 and CD30, respectively. Several factors are involved in the Th cell differentiation into the polarized Th1 or Th2 pathway. They include the cytokine profile of 'natural immunity' evoked by different offending agents, the nature of the peptide ligand, as well as the activity of some costimulatory molecules and microenvironmentally secreted hormones, in the context of different host genetic backgrounds. Polarized Th1-type and Th2-type responses play different roles in protection, Th1 being effective in the defense against intracellular pathogens and Th2 against intestinal nematodes. Moreover, they are responsible for different types of immunopathological reactions. Th1 responses predominate in organ-specific autoimmune disorders, acute allograft rejection, unexplained recurrent abortions, and in some chronic inflammatory disorders of unknown etiology. In contrast, Th2 responses predominate in Omenn's syndrome, transplantation tolerance, chronic graft versus host disease, systemic sclerosis; moreover allergen-reactive Th2 cells are involved in the triggering of atopic disorders.

Abnormal production of T helper 2 cytokines interleukin-4 and interleukin-5 by T cells from newborns with atopic parents.

T cell clones were generated from umbelical cord blood lymphocytes (UCBL) of nine newborns with atopic or nonatopic parents and their cytokine secretion profile was assessed. Both phytohemagglutinin-induced and Dermatophagoides pteronyssinus-specific T cell clones from newborns with atopic parents exhibited an enhanced ability to produce the Th2 cytokines interleukin (IL)-4 and IL-5, compared to T cell clones from newborns with nonatopic parents. In contrast, the ability to produce interferon-gamma by UCBL from the two groups of newborns was not different. Of the five children who could be followed up to 3 years after birth, four with atopic parents developed clinical and/or biological atopic manifestations, whereas one without atopic parents did not. Thus, the pronounced production of IL-4 and IL-5 by UCBL not only appears to be related to the atopic status of parents, but also associates with the subsequent development of atopy in childhood.

Regulation of fetal allograft survival by a hormone-controlled Th1- and Th2-type cytokines.

There is clear evidence to suggest that the maternal immune system during pregnancy can enhance or inhibit the development of the fetoplacental unit. Recent data support the view that some cytokines produced by both T cells and non-T cells (IL-3, GM-CSF, TGF-beta, IL-4, IL-10), favor fetal survival and growth. In contrast, other cytokines, such as IFN-gamma, TNF-beta and TNF-alpha, can rather compromise pregnancy. Accordingly, we show here that T-cell clones generated from the decidua of women with unexplained recurrent abortion produced significantly lower concentrations of IL-4 than clones derived from the decidua of voluntary abortions or the endometrium of nonpregnant women. Thus, despite the complexity of the cytokine network, it appears that cytokines favoring the maintenance of fetal survival mainly belong to the Th2 pathway, whereas the failure of pregnancy rather associates with the predominance of Th1-type cytokines and/or the absence of Th2-type cytokines. Interestingly, we also found that, at least in vitro, progesterone promotes the preferential development of Th2-like cells and induces transient IL-4 production by established Th1 cells, whereas relaxin, another corpus luteum-derived hormone, mainly promotes the development of Th1-like cells. These data provide an excellent basis for investigating the relationship between the endocrine and the immune system in the regulation of the maternal-fetal interaction.

No relationship between skin-infiltrating TH2-like cells and allergen-specific IgE response in atopic dermatitis.

More than 500 CD4+ T-cell clones (TCCs) derived from the skin of eight patients with atopic dermatitis (AD), two patients with nonatopic dermatologic disorders, two patients with allergic rhinitis, and one healthy nonatopic donor were analyzed for both their pattern of cytokine production and their antigen specificity. The proportions of TCCs from patients with AD producing interleukin-4 in response to stimulation with phorbol 12-myristate 13-acetate plus anti-CD3 antibody were higher, whereas the proportions of interferon-gamma--producing TCCs were lower than those of control subjects. In two patients with AD, the majority of TCCs had a TH2/TH0-like phenotype, whereas in six patients with AD a TH1/TH0-like phenotype was prevalent. TCCs with a TH2/TH0-like phenotype were also isolated from the healthy skin of two patients with allergic rhinitis and one nonatopic donor. In contrast, no TH2-like TCCs were derived from the skin of the two patients with dermatologic disorders of nonallergic origin. No unambiguous correlations was found between the proportions of TCCs producing interleukin-4 or interferon-gamma (or of TCCs with TH2- or TH1-like profile) and the level of total serum IgE, suggesting that CD4+ T cells infiltrating the atopic skin do not play a major role in the production of serum IgE antibodies. When TCCs from five patients with AD were examined for their specificity, the proportions of allergen-specific (Dermatophagoides pteronyssinus and Lol p 1) clones were consistently 6% or lower even in patients with high titers of ryegrassor D. pteronyssinus-specific IgE antibodies. Because similar percentages of allergen-specific TCCs were found in skin from two healthy control subjects, the role of aeroallergens in favoring and maintaining skin lesions in patients with AD remains unclear.

Progesterone favors the development of human T helper cells producing Th2-type cytokines and promotes both IL-4 production and membrane CD30 expression in established Th1 cell clones.

The effect of progesterone (P) on the cytokine production profile of Ag-specific human CD4+ T cell lines and clones was investigated. T cell lines specific for purified protein derivative or streptokinase (SK) derived in the presence of P exhibited significant increased ability to produce IL-5 in comparison with T cell lines derived in the absence of P. Moreover, IL-4 was significantly increased in SK-specific T cell lines derived in the presence of P in comparison with SK-specific T cell lines derived in the absence of this hormone. In addition, SK-specific T cell lines generated in the presence of P developed into T cell clones showing a Th0-, instead of Th1-like, cytokine profile. Furthermore, SK-specific T cell clones with an established Th1 profile of cytokine secretion did express mRNA for, and produced detectable amounts of, IL-4 when stimulated with P in combination with insoluble anti-CD3 mAb. Combined stimulation with P and insoluble anti-CD3 mAb also enabled Th1 clones to express CD30 on their surface membrane. These results indicate that P can favor the development of Th cells producing Th2-type cytokines and is an inducer of both transient IL-4 production and CD30 expression in established Th1 cells. Thus, P production at the placental level may be responsible, at least in part, for increased production of Th2-type cytokines which have been implied in fetal allograft survival and maintenance of successful pregnancy.

CD30 expression by CD8+ T cells producing type 2 helper cytokines. Evidence for large numbers of CD8+CD30+ T cell clones in human immunodeficiency virus infection.

A large panel of CD8+ T cell clones generated from peripheral blood lymphocytes (PBL) of healthy donors or human immunodeficiency virus (HIV)-infected individuals were assessed for both cytokine secretion profile and CD30 expression and release. The great majority of CD8+ T cell clones generated from healthy individuals showed the ability to produce interferon gamma (IFN-gamma), but not interleukin 4 (IL-4), and none of them either expressed membrane CD30 or released substantial amounts of soluble CD30 (sCD30) in their supernatant. In contrast, high numbers of CD8+ T cell clones generated from HIV-infected individuals, which produced IL-4 (and IL-5) in addition to IFN-gamma or IL-4 (and IL-5) alone, expressed membrane CD30 and released detectable amounts of sCD30 in their supernatants. Indeed, CD30 expression appeared to be positively correlated with the ability of CD8+ T cell clones to produce IL-4 and IL-5 and inversely correlated with their ability to produce IFN-gamma, whereas no correlation between CD30 expression and production of IL-10 was observed. These data suggest that CD30 is a marker for CD8+ T cells that have switched to the production of type 2 helper cytokines.