The type I interferons (IFNs) are pleiotropic cytokines that regulate many different cellular functions. The major signaling pathway activated by type I IFNs involves sequential phosphorylation of the tyrosine residues of the Janus kinase (JAK) and signal transducers and activators of transcription (STAT) proteins, providing the primary mechanism through which gene expression is induced. Recent work has shown that the responses are quite complex, as shown by different responses to specific subtypes of type I IFN, activation of kinases in addition to JAKs, patterns of activation of all seven STATs in different cells, and activation of transcription factors other than STATs. The type I IFNs use this complexity to regulate many different biological functions in different types of cells, by activating different specific signals and patterns of gene expression.

Summary:Objectives: A common experimental model of status epilepticus (SE) utilizes intraperitoneal administration of the cholinergic agonist pilocarpine preceded by methylscopolamine treatment. Currently, activation of cholinergic neurons is recognized as the only factor triggering pilocarpine SE. However, cholinergic receptors are also widely distributed systemically and pretreatment with methyl-scopolamine may not be sufficient to counteract the effects of systemically injected pilocarpine. The extent of such peripheral events and the contribution to SE are unknown and the possibility that pilocarpine also induces SE by peripheral actions is yet untested.Methods: We measured in vivo at onset of SE: brain and blood pilocarpine levels, blood-brain barrier (BBB) permeability, Tlymphocyte activation and serum levels of IL-1β and TNF-α. The effects of pilocarpine on neuronal excitability was assessed in vitro on hippocampal slices or whole guinea pig brain preparations in presence of physiologic or elevated [K + ] out .Results: Pilocarpine blood and brain levels at SE were 1400 ± 200 µM and 200 ± 80 µM, respectively. In vivo, after pilocarpine injection, increased serum IL-1β, decreased CD4:CD8 T-lymphocyte ratios and focal BBB leakage were observed. In vitro, pilocarpine failed to exert significant synchronized epileptiform activity when applied at concentrations identical or higher to levels measured in vivo. Intense electrographic seizure-like events occurred only in the copresence of levels of K + (6 mM) mimicking BBB leakage.Conclusions: Early systemic events increasing BBB permeability may promote entry of cofactors (e. g. K + ) into the brain leading to pilocarpine-induced SE. Disturbance of brain homeostasis represents an etiological factor contributing to pilocarpine seizures.

Treatment of cell lines with type I IFNs activates the formation of ISGF3 (STAT1/STAT2/IRF9), which induces the expression of many genes. To study this response in primary cells, we treated fresh human blood with IFN-β and used flow cytometry to analyze phosphorylated STATs1, 3 and 5 in CD4+ and CD8+ T cells, B cells, and monocytes. The activation of STAT1 was remarkably different among these leukocyte subsets. In contrast to monocytes, CD4+ and CD8+ T cells, few B cells activated STAT1 in response to IFN-β, a finding that could not be explained by decreased levels of IFNAR2 or STAT1 or enhanced levels of SOCS1 or relevant protein tyrosine phosphatases in B cells. Micro-array and real-time PCR analyses revealed the induction of STAT1-dependent pro-apoptotic mRNAs in monocytes but not in B cells. These data show that ISGF3 or STAT1 homodimers are not the main activators of gene expression in primary B cells of healthy humans. Notably, in B cells and especially in CD4+ T cells, IFN-β activated STAT5 in addition to STAT3, with biological effects often opposite from those driven by activated STAT1. These data help to explain why IFN-β increases the survival of primary human B cells and CD4+ T cells, but enhances the apoptosis of monocytes, and also to understand how leukocyte subsets are differentially affected by endogenous type I IFNs during viral or bacterial infections, and by type I IFN treatment of patients with multiple sclerosis, hepatitis or cancer.

The mechanism of IFN-β therapy in relapsing-remitting multiple sclerosis (RRMS) is not well understood, but induction of apoptosis in specific leukocyte subsets is likely to be important. Enhanced expression of TNFSF10 or TNF-related apoptosis-inducing ligand (TRAIL) mRNA in unseparated leukocytes has been put forward as a therapeutic response marker, but it is unclear which leukocyte subsets express TRAIL. We investigated the basis of TRAIL expression in response to IFN-β by studying activation of STATs 1, 3, and 5, p38 MAPK, and NF-κB in different leukocyte subsets of patients with RRMS. Monocytes, B cells, and T cells showed substantial differences in the activation of p38 and the STATs in response to i.m. injection of IFN-β1a or stimulation in vitro. Induction of cellsurface TRAIL, analyzed in nine leukocyte subsets, was observed only on monocytes and granulocytes and correlated with the activation of p38 and/or NF-κB in these subsets only, in agreement with previous work in fibroblasts showing that the induction of TRAIL in response to IFN-β depends on the activation of p38 and NF-κB as well as STATs 1 and 2. We propose that, in myeloid cells, the differential activation of p38 and NF-κB and induction of TRAIL, which sensitizes cells to apoptosis, can help to explain differences in responsiveness to IFN-β therapy among patients with RRMS and, furthermore, that such differential patterns of activation and expression may also be important in understanding the therapeutic responses to IFN-α/β in hepatitis and cancer.humans | phosphoproteins | signal transduction | flow cytometry

In this study we analyzed fibroblasts derived from an MHC class II deficiency patient (type III bare lymphocyte syndrome). Northern blot analysis showed that upon induction with IFN-gamma these fibroblasts did not express HLA class II genes and displayed a strongly reduced level of HLA class I gene expression when compared with fibroblasts of a healthy individual. However, when analyzed by RT-polymerase chain reaction (PCR), residual expression could be detected for HLA-DRA, DPB, and DQA, but not for HLA-DRB, DPA, and DQB. The lack of HLA-DRB transcripts in the patient fibroblasts and the high degree of sequence polymorphism of HLA-DRB were exploited in the further analysis of these fibroblasts. Thus far, at least three, and probably four, complementation groups have been defined among patient-derived and experimentally-derived MHC class II-negative cell lines. Transient heterokaryons between the patient fibroblasts and representative B-lymphoblastoid cell lines from each of the complementation groups were analyzed by RT-PCR and Southern blotting, using HLA-DRB-specific primers and biotin-labeled sequence specific oligonucleotides, respectively. These analyses showed that the fibroblasts of this particular patient belonged to a novel complementation group in MHC class II deficiency.