Interferon-beta differentially regulates expression of the IL-12 family members p35, p40, p19 and EBI3 in activated human dendritic cells.

Interferon-beta is thought to provide clinical improvement to multiple sclerosis (MS) patients, in part, through its ability to suppress the generation of IL-12-dependent autoimmune T helper type 1 (Th1) cells by monocyte-derived dendritic cells (DC). We now describe how pre-incubation with 1000 U/ml of IFN-beta differentially regulates expression of multiple IL-12 family members in activated, immature human DC, inhibiting CD40/IFN-gamma-induced p35 and p40 message levels, while enhancing p19 and Epstein-Barr virus-induced gene 3 (EBI3) levels. IFN-beta-mediated inhibition of p40 mRNA and augmentation of p19 mRNA both require de novo protein synthesis. These findings indicate that IFN-beta will be found to have contrasting effects on DC secretion of the various IL-12 family homo- and heterodimers.

The effect of type I and II interferons on human fetal retinal pigment epithelium-induced apoptosis in Jurkat T cells.

PURPOSE: To examine the regulatory effects of interferon (IFN)-alpha, IFN-gamma, transforming growth factor (TGF)-beta, and tumor necrosis factor (TNF)-alpha on human fetal retinal pigment epithelial (HFRPE) cell-induced apoptosis of human Jurkat T (Jkt) cells. METHODS: Pure cultures of HFRPE cells were isolated. The cells were precultured with medium alone or with addition of IFN-alpha, IFN-gamma, TNF-alpha, or TGF-beta for 72 hours. Thereafter, HFRPE cells were extensively washed before they were cocultured jointly with Jkt cells (standard) or cultured alone for another 48 hours to accumulate conditioned medium that is collected and added as cell-free conditioned medium to Jkt cell cultures (supernatant). Jkt cells were cocultured under the two culture conditions for 48, 72, and 96 hours. The rate of apoptosis in Jkt T cells was determined with annexin V staining and flow cytometry. RESULTS: Both IFN-alpha and -gamma upregulated HFRPE-induced apoptosis in Jkt T cells. However, the apoptosis induced by IFN-alpha-activated HFRPE cells was significant only in the absence of cell-cell contact (supernatant). The supernatant induced a higher rate of apoptosis in Jkt T cells when compared to the direct coculture of the cells. TGF-beta and TNF-alpha did not upregulate HFRPE-induced apoptosis in Jkt T cells. CONCLUSIONS: These results indicate that type I and type II IFNs can upregulated HFRPE-induced apoptosis in Jkt T cells, IFN-gamma being the more effective cytokine. Neither, TGF-beta nor TNF-alpha upregulated the HFRPE-induced apoptosis in Jkt T cells. Although HFRPE-induced apoptosis was mediated in a cell-cell-contact-independent pathway, HFRPE cells may also express membrane-bound antiapoptotic molecules. These findings may help us to understand better the modulatory effects of pro- and anti-inflammatory cytokines on immune suppressive characteristics of RPE cells.

Human helper T cell activation and differentiation is suppressed by porcine small intestinal submucosa.

A cell-free biomaterial derived from porcine small intestinal submucosa (SIS) has been used successfully in many models as a xenogeneic scaffolding material without generating immune-mediated inflammatory reactions. We investigated whether this absence of inflammation is due to the presence of porcine transforming growth factor beta (TGF-beta) activity found in SIS that may have immunosuppressive properties on helper T (Th) cell subset activation and differentiation. We used in vitro models for the generation of human Th1 and Th2 cells to investigate the influence of SIS. We found that SIS partially suppressed Th1 cell expansion and secretion of interleukin 12 (IL-12) and interferon gamma (IFN-gamma) in a TGF-beta-dependent manner, but Th1 cell expansion and IFN-gamma secretion could be fully overcome by addition of recombinant IL-12. The suppression by SIS of Th cell activation also involved the induction of Th cell apoptosis. In addition, SIS completely abolished the generation of Th2 cells in vitro, but this effect of SIS was not reversed by neutralizing TGF-beta antibodies. Our results indicate the presence in SIS of factors that can suppress Th cell activation through both the inhibition of IL-12 secretion and the induction of Th cell apoptosis. We established further that these factors include TGF-beta and at least one other factor.

Indocyanine green induces apoptosis in human retinal pigment epithelial cells.

PURPOSE: To examine whether indocyanine green (ICG) dye induces apoptosis in human retinal pigment epithelial (RPE) cells. DESIGN: Laboratory investigation. METHODS: Pure cultures of human RPE cells were isolated. Retinal pigment epithelial cells were incubated with different concentrations of ICG dye (1 mg/ml, 5 mg/ml, or 20 mg/ml) for 30 minutes. The rate of RPE cell apoptosis was assessed with Annexin V-FITC staining and propidium iodide (PI) by flow cytometry. RESULTS: Retinal pigment epithelial cells maintained their monolayer morphology after incubation with ICG dye. However, ICG induced a statistically significant amount of apoptosis in RPE cells at all the concentrations (1 mg/ml, 5 mg/ml, and 20 mg/ml) after 30 minutes of incubation (P <.05). The solvent solution alone (without the ICG dye) did not induce any significant apoptosis in RPE cells, when compared with culture medium. CONCLUSIONS: The incubation of RPE cells with ICG dye increased the number of apoptotic RPE cells in vitro. Our findings indicate that the decision over the intravitreal application of ICG dye needs to be made with caution.

Human fetal retinal pigment epithelium induces apoptosis in human T-cell line Jurkat which is independent from its expression of TRAIL.

PURPOSE: To evaluate whether human fetal retinal pigment epithelial (HFRPE) cells express TRAIL (tumor necrosis factor related apoptosis inducing ligand). The role of TRAIL in HFRPE induced apoptosis was evaluated. METHODS: Pure cultures of HFRPE cells were isolated. The expression of TRAIL protein and mRNA in non-activated and IFN-gamma activated HFRPE cells was evaluated with RT-PCR. The role of TRAIL in HFRPE induced apoptosis was assessed by incubating HFRPE cells with human T-cell leukemia line Jurkat (Jkt) in the presence or absence of neutralizing TRAIL antibodies. Cultures were pulsed with [(3)H]-thymidine to measure Jkt cell proliferation. The role of TRAIL was further examined by western blott evaluating the cleavage of caspases 8 and 10 in Jkt cells after their incubation with HFRPE cells. RESULTS: HFRPE cells expressed TRAIL mRNA. The expression of TRAIL mRNA and protein was up-regulated by IFN-gamma activation. However, anti-TRAIL antibodies were not able to prevent the HFRPE induced suppression of Jkt cell proliferation. The caspases 8 and 10 were also not cleaved in Jkt cells after their incubation with IFN-gamma activated HFRPE cells. CONCLUSIONS: Although HFRPE cells express TRAIL and its expression is upregulated by IFN-gamma activation, TRAIL is not involved in HFRPE induced apoptosis in Jkt cells. Currently the role of TRAIL in HFRPE cells is under investigation.

Blockade of TLR9 agonist-induced type I interferons promotes inflammatory cytokine IFN-gamma and IL-17 secretion by activated human PBMC.

Type I interferons (IFN) (IFN-alpha/beta) are recognized as both inhibitors and effectors of autoimmune disease. In multiple sclerosis, IFN-beta therapy appears beneficial, in part, due to its suppression of autoimmune inflammatory Th cell responses. In contrast, in systemic lupus erythematosus (SLE) triggering of plasmacytoid DC (pDC) Toll-like receptors (TLRs) by autoimmune complexes (autoICs) results in circulating type I IFN that appear to promote disease by driving autoantigen presentation and autoantibody production. To investigate how pDC-derived type I IFN might regulate Th cells in SLE, we examined a model in which sustained pDC stimulation by autoICs is mimicked by pretreating normal human PBMC with TLR9 agonist, CpG-A. Subsequently, PBMC Th cells are activated with superantigen, and APC are activated with CD40L. The role of CpG-A/TLR9-induced type I IFN in regulating PBMC is determined by blocking with virus-derived soluble type I IFN receptor, B18R. In summary, pretreatment with either rhIFN-alpha/beta or CpG-A inhibits PBMC secretion of superantigen-induced IFN-gamma and IL-17, and CD40L-induced IL-12p70 and IL-23. B18R prevents these effects. Data indicate that CpG-A-induced type I IFN inhibit IL-12p70-dependent PBMC IFN-gamma secretion by enhancing IL-10. Our results suggest that in SLE, circulating type I IFN may potentially act to inhibit inflammatory cytokine secretion.

Timing of IFN-beta exposure during human dendritic cell maturation and naive Th cell stimulation has contrasting effects on Th1 subset generation: a role for IFN-beta-mediated regulation of IL-12 family cytokines and IL-18 in naive Th cell differentiation.

Type I IFNs, IFN-alpha and IFN-beta, are early effectors of innate immune responses against microbes that can also regulate subsequent adaptive immunity by promoting antimicrobial Th1-type responses. In contrast, the ability of IFN-beta to inhibit autoimmune Th1 responses is thought to account for some of the beneficial effects of IFN-beta therapy in the treatment of relapsing remitting multiple sclerosis. To understand the basis of the paradoxical effects of IFN-beta on the expression of Th1-type immune responses, we developed an in vitro model of monocyte-derived dendritic cell (DC)-dependent, human naive Th cell differentiation, in which one can observe both positive and negative effects of IFN-beta on the generation of Th1 cells. In this model we found that the timing of IFN-beta exposure determines whether IFN-beta will have a positive or a negative effect on naive Th cell differentiation into Th1 cells. Specifically, the presence of IFN-beta during TNF-alpha-induced DC maturation strongly augments the capacity of DC to promote the generation of IFN-gamma-secreting Th1 cells. In contrast, exposure to IFN-beta during mature DC-mediated primary stimulation of naive Th cells has the opposite effect, in that it inhibits Th1 cell polarization and promotes the generation of an IL-10-secreting T cell subset. Studies with blocking mAbs and recombinant cytokines indicate that the mechanism by which IFN-beta mediates these contrasting effects on Th1 cell generation is at least in part by differentially regulating DC expression of IL-12 family cytokines (IL-12 and/or IL-23, and IL-27) and IL-18.