Regulation of murine dendritic cell functions in vitro by taurine chloramine, a major product of the neutrophil myeloperoxidase-halide system

Taurine chloramine (TauCl) is a major chloramine generated in activated neutrophils as a result of the reaction of highly toxic hypochlorous acid and taurine, the most abundant free amino acid in cytosol. In this study we have tested the influence of TauCl on the properties of murine dendritic cells (DC), the major cell population involved in the initiation of an adaptive immune response against pathogenic organisms. N418+, MHC II+, B7-2+ dendritic cells, generated from the mouse bone marrow cells cultured in the presence of granulocyte-macrophage colony-stimulating factor, were stimulated by interferon-gamma and lipopolysaccharide to produce nitric oxide, reactive oxygen species, interleukin-6 (IL-6), tumour necrosis factor-alpha, and IL-12, in the presence of different doses of TauCl. TauCl differently inhibited the generation of these inflammatory mediators in a dose-dependent manner. Furthermore, TauCl selectively modulated the ability of DC to induce the release IL-2 and IL-10 from T cells. These results suggest that neutrophil-derived mediators, such as TauCl, at a site of inflammation, may affect the functions of sentinel DC and macrophages, and play a role in maintaining the balance between the inflammatory response and the induction of an antigen-specific immune response.     

Placentally derived prostaglandin E2 acts via the EP4 receptor to inhibit IL-2-dependent proliferation of CTLL-2 T cells

A number of immunomodulatory molecules are present in the placenta, including cytokines, prostaglandins, progesterone and indoleamine 2,3-dioxygenase. An undefined factor capable of down-regulating T-cell activity has recently been reported [1] as being produced by short-term cultures of placental fragments. By careful repetition of these studies we have confirmed that chorionic villi isolated from term placenta produce a low molecular weight, heat stable factor capable of inhibiting the IL-2-dependent proliferation of mouse CTLL-2 cells. This activity was not due, however, to a previously unknown immunosuppressive molecule, but rather to prostaglandin E2 (PGE2). Expression of cyclooxygenase (COX)-2 was detected in the syncytiotrophoblast of chorionic villi explants using immunohistochemistry. Culture of the explants in the presence of the COX-1/COX--2 inhibitors indomethacin and diclofenac, or with the COX-2-selective inhibitor DFP, blocked the production of the immunosuppressive factor. The immunosuppressive activity was restored by adding PGE2 to the supernatants obtained from diclofenac-inhibited explants. A number of different receptors are involved in mediating the biological effects of prostaglandins. By utilizing selective antagonists of individual receptors, we have established that the immunosuppressive effect of PGE2 on CTLL-2 cells is exerted via the EP4 receptor. Thus, addition of an EP4-selective antagonist, but not of EP1 or EP3 antagonists, abolished the immunosuppressive effect of PGE2 on CTLL-2 cells. This may have implications for attempts to selectively manipulate T-cell responses.     

The antigen-presenting environment in normal and human papillomavirus (HPV)-related premalignant cervical epithelium

The activation of HPV-specific T cells within the cervical microenvironment is likely to play an important part in the natural history of cervical intraepithelial neoplasia (CIN). The extent and the type of T cell activation will depend critically on the expression of MHC, costimulatory cell surface molecules and cytokines by keratinocytes and Langerhans cells within the cervical lesion. Expression of MHC class II (HLA-A-DR and -DQ), costimulatory/adhesion molecules (CD11a/18, CD50, CD54, CD58 and CD86) and cytokines (tumour necrosis factor-alpha (TNF-alpha) and IL-10) was therefore investigated by immunohistochemistry in normal squamous epithelium (n = 12), low-grade (n = 23) and high-grade (n = 18) squamous intraepithelial lesions of the cervix. CIN progression was associated with de novo expression of HLA-DR and CD54, and increased expression of CD58 by keratinocytes. However, significantly, there was no expression of any adhesion/costimulation molecule by epithelial Langerhans cells in any cervical biopsy studied. Furthermore, TNF-alpha, a potent activator of Langerhans cells, was expressed constitutively by basal keratinocytes in normal cervix (12+/12). but expression of this cytokine was absent in a number of CIN samples (20+/23 for low-grade, 12+/18 for high-grade CIN). Conversely, the suppressive cytokine IL-10 was absent in normal epithelium (0+/12), but was up-regulated in a number of CIN lesions (12+/23 for low-grade; 8+/18 for high-grade CIN). The restricted expression of costimulation/adhesion molecules and the nature of the cytokine microenvironment within the epithelium may act to limit effective immune responses in some CIN lesions.     

Real-time PCR method for the quantitative analysis of human T-cell receptor gamma and beta gene rearrangements

Analyzing the status of T-cell receptor (TCR) gene rearrangements has been an essential part of deciphering the stages of thymocyte development, understanding the β vs. γδ lineage decision, and characterizing T-cell leukemias. Methods such as PCR and quantitative Southern blotting provide useful information, but also have significant shortcomings such as lack of quantitation in the case of PCR and technical challenges in the case of Southern blotting. Here we describe a real-time PCR method that overcomes many of these shortcomings. This new method shows comparable results for the fraction of unrearranged TCRγ and TCRβ genes in human thymocytes and peripheral blood T cells as Southern blotting, and has the advantages of being simple to perform, highly quantitative, and requiring nanogram quantities of DNA. We also describe a real-time PCR method to quantitate T-cell receptor excision circles formed during TCRβ rearrangements.     

Oxidative modification of type II collagen differentially affects its arthritogenic and tolerogenic capacity in experimental arthritis

INTRODUCTION: Oxidative modification of proteins affects their biological properties. Previously we have shown that hypochlorite (HOCl), the product of activated neutrophils, enhances protein immunogenecity. Collagen type II, a primary component of cartilage, is commonly used in the induction of arthritis in animals (CIA). The aim of this study was to examine whether HOCl may affect immunogenic, tolerogenic, and arthritogenic properties of collagen. MATERIALS AND METHODS: DBA/J mice were injected with either native (CNAT) or chlorinated collagen (CHOCl) to induce arthritis. The effect of chlorination on collagen properties was measured by evaluation of incidence and severity of CIA. Moreover, the concentration of serum anti-collagen IgG antibodies and myeloperoxidase (MPO) activity in inflamed joints was determined. RESULTS: Mice immunized with CNAT in adjuvant developed arthritis (CIA) with an incidence of 69%. CNAT also exerted tolerogenic properties when injected intravenously either before or shortly after primary immunization, resulting in decreased incidence and severity of CIA, reduced MPO activity in inflamed joints, and lowered serum levels of anti-CNAT IgG anti-bodies. Chlorination of collagen significantly diminished its ability to induce CIA and to trigger generation of anti-CNAT IgG antibodies. Interestingly, chlorination did not affect tolerogenic properties of collagen administered prior to primary immunization with CNAT. CONCLUSIONS: These results suggest that chlorination of collagen may selectively affect functional epitopes of collagen. It is likely that in inflamed joints, neutrophil derived HOCl, in some circumstances, will destroy arthritogenic and immunogenic B cell epitopes, while regulatory T cell epitopes will be preserved.     

Steady-state distribution of cycloleucine and alpha-aminoisobutyric acid between plasma and cerebrospinal fluid

Estimates of the steady-state distribution ratios of two nonmetabolizable amino acids, alpha-aminoisobutyric acid and aminocyclopentane carboxylic acid (cycloleucine), between plasma and cerebrospinal fluid were made with a view to establishing whether or not the low values found with metabolizable amino acids, such as glycine or leucine, could be accounted for by uptake and metabolism by the brain. The estimates, based on the ratios found after i.p. injections either in bolus form or by implantation of "osmotic pumps" containing the labeled amino acids, were comparable with those found for metabolizable amino acids.