Sheri M. Skinner

Regulatory effects of IFN-β on production of osteopontin and IL-17 by CD4+ T Cells in MS

IFN‐β currently serves as one of the major treatments for MS. Its anti‐inflammatory mechanism has been reported as involving a shift in cytokine balance from Th1 to Th2 in the T‐cell response against elements of the myelin sheath. In addition to the Th1 and Th2 groups, two other important pro‐inflammatory cytokines, IL‐17 and osteopontin (OPN), are believed to play important roles in CNS inflammation in the pathogenesis of MS. In this study, we examined the potential effects of IFN‐β on the regulation of OPN and IL‐17 in MS patients. We found that IFN‐β used in vitro at 0.5–3 ng/mL significantly inhibited the production of OPN in primary T cells derived from PBMC. The inhibition of OPN was determined to occur at the CD4+ T‐cell level. In addition, IFN‐β inhibited the production of IL‐17 and IL‐21 in CD4+ T cells. It has been described that IFN‐β suppresses IL‐17 production through the inhibition of a monocytic cytokine, the intracellular translational isoform of OPN. Our further investigation demonstrated that IFN‐β also acted directly on the CD4+ T cells to regulate OPN and IL‐17 expression through the type I IFN receptor‐mediated activation of STAT1 and suppression of STAT3 activity. Administration of IFN‐β to EAE mice ameliorated the disease severity. Furthermore, spinal cord infiltration of OPN+ and IL‐17+ cells decreased in IFN‐β‐treated EAE mice along with decreases in serum levels of OPN and IL‐21. Importantly, decreased OPN production by IFN‐β treatment contributes to the reduced migratory activity of T cells. Taken together, the results from both in vitro and in vivo experiments indicate that IFN‐β treatment can down‐regulate the OPN and IL‐17 production in MS. This study provides new insights into the mechanism of action of IFN‐β in the treatment of MS.

Evaluating zona pellucida structure and function using antibodies to rabbit 55 kDa ZP protein expressed in baculovirus expression system.

A cDNA encoding the rabbit 55 kDa ZP protein was expressed using a baculovirus expression system and was evaluated for its ability to elicit antibodies which may interfere with sperm‐ZP interaction. The expressed glycosylated protein, BV55, was purified by wheat germ agglutinin lectin affinity chromatography. Antisera made in guinea pigs immunized with BV55 (GP‐α‐BV55) is specific for the 55 kDa rabbit ZP protein. Indirect immunofluorescence studies indicate that GP‐α‐BV55 localizes to a filamentous meshwork on the surface of the ZP of isolated rabbit eggs. Immunohistochemical analysis of rabbit ovaries demonstrated that this antigen is localized within the ZP of primary and more advanced stage ovarian follicles but is not detected in primordial follicles. In addition, the 55 kDa antigen was detected in the granulosa cells of secondary stage follicles but not in the oocyte. GP‐α‐BV55 effectively blocked the binding of rabbit sperm to rabbit eggs in vitro. However, Fab fragments generated from GP‐α‐BV55 failed to block sperm binding, suggesting that the inhibitory effect of GP‐α‐BV55 was due to stearic hindrance rather than specific blocking of a sperm receptor site. Although the Fab fragment did not inhibit sperm binding, additional studies demonstrated that biotinylated BV55 protein bound to rabbit sperm in the acrosomal region in a manner consistent with ligand activity in the sperm‐ZP interaction, and that BV55 bound to rabbit sperm in a dose‐dependent manner. These studies therefore demonstrate that antibodies against recombinant ZP proteins recognize the native intact ZP and inhibit sperm‐ZP interaction. They also provide evidence that the rabbit 55 kDa ZP protein, which is the homolog of the pig ZP3α sperm receptor protein, has sperm receptor activity.

Zona pellucida antigens: targets for contraceptive vaccines.

The mammalian ovary is unique among secretory organs in its ability both to produce the female gamete and to provide the necessary endocrine support for its orderly development. This set of functions utilizes complex feedback mechanisms involving the hypothalamus and the anterior pituitary over the reproductive life of the individual. The late stages of follicular development just prior to ovulation have been the focus of most ovarian studies. However, it is necessary to investigate the earliest stages of follicular development in order to begin to understand the foundations of immunologically based interference with ovarian function.

Regulation of differentiation and functional properties of monocytes and monocyte-derived dendritic cells by interferon beta in multiple sclerosis

Interferon beta (IFN beta) has complex immune regulatory properties that contribute to its treatment effect on multiple sclerosis (MS). In this study, we investigated the role of IFN beta in differentiation and functional properties of monocytes and monocyte-derived dendritic cells that are critical to the inflammatory process in MS. The results revealed that IFN beta inhibited intracellular production of interleukin (IL)-1b (PB/0.01) in both monocytes exposed toin vitro treatment of IFN beta and monocytes analysedex vivo from MS patients treated with IFN beta. IFN beta was shown to modulate differentiation of monocytes into dendritic cells in the presence of IL-4 and GM-CSF, which resulted in a delayed differentiation process. Furthermore, it characteristically altered the phenotypic features of differentiated dendritic cells by inhibiting the expression of CD1a, CD11b, CD11c, CD123 and CD209 while upregulating costimulatory molecules, such as CD86. The selective regulatory properties of IFN beta appeared to render the function of differentiated dendritic cells to produce an increased amount (PB/0.01) while their ability to secrete proinflammatory IL-12 and TGF beta was significantly reduced. The observed collective effects of IFN beta seemed to correlate with Th2 immune deviation. The study has provided new insights into the regulatory mechanisms of IFN beta in the treatment of MS.

Molecular Analysis of a Carbohydrate Antigen Involved in the Structure and Function of Zona Pellucida Glycoproteins

Abstract A lactosaminoglycan-associated antigen is associated with a carbohydrate moiety of all three zona pellucida (ZP) glycoproteins of pig and rabbit but is absent in the mouse and rat. A monoclonal antibody (PS1) recognizing this determinant was obtained by immunizing mice with a porcine ZP glycoprotein isoform purified by two-dimensional polyacrylamide gel electrophoresis. Conditions known to remove O-linked or sialic acid carbohydrate moieties (alkaline reduction; O-glycanase or neuraminidase enzymatic cleavage) did not remove the carbohydrate epitope. However, treatment with endo-β-glycosidase, endoglycosidase F, or combinations of neuraminidase plus β-galactosidase, totally removed the determinant, indicating that it is associated with a poly-N-acetyllactosaminoglycan structure present on an N-linked oligosaccharide. Molecular morphology studies using immunofluorescence and confocal microscopy techniques demonstrate that the PS1 antigen is localized at the surface of the ZP. Confirmation of this localization was obtained through studies that show that this antibody will inhibit homologous sperm binding to the pig ZP. Additional analyses using modular contrast microscopy and immunocytochemistry demonstrate that this carbohydrate-associated antigen is localized in discrete layers throughout the ZP matrix. These studies are the first to demonstrate the presence of a lactosaminoglycan type carbohydrate moiety in all three ZP proteins using a monoclonal antibody that appears to be involved in sperm recognition and structural organization.

Regulatory and pro-inflammatory phenotypes of myelin basic protein-autoreactive T cells in multiple sclerosis

MBP-specific autoreactive T cells are considered pro-inflammatory T cells and thought to play an important role in the pathogenesis of multiple sclerosis (MS). Here, we report that MBP(83-99)-specific T cells generated from MS patients (n = 7) were comprised of pro-inflammatory and regulatory subsets of distinct phenotypes. The pro-inflammatory phenotype was characterized by high production of IFN-gamma, IL-6, IL-21 and IL-17 and low expression of FOXP3, whereas the regulatory subset expressed high levels of FOXP3 and exhibited potent regulatory functions. The regulatory subset of MBP-specific T cells appeared to expand from the CD4(+)CD25(-) T-cell pool. Their FOXP3 expression was stable, independent of the activation state and it correlated with suppressive function and inversely with the production of IFN-gamma, IL-6, IL-21 and IL-17. In contrast, the phenotype and function of FOXP3(low) MBP-specific T cells were adaptive and dependent on IL-6. The higher frequency of FOXP3(high) MBP-specific T cells was observed when IL-6 was neutralized in the culture of PBMC with MBP. The study provides new evidence that MBP-specific T cells are susceptible to pro-inflammatory cytokine milieu and act as either pro-inflammatory or regulatory T cells.

Induction of Adaptive CD4+CD25+Foxp3+ Regulatory T Cell Response in Autoimmune Disease

CD4+CD25+Foxp3+ regulatory T cells (Tregs) represent one of the most critical regulatory components of the immune system. They are vitally important for homeostasis of the immune system and adequate immune responses. Tregs contain heterogeneous populations that share common markers and regulatory function but differ in the origin of differentiation or conversion. Deficiencies either in the number of Tregs or the expression of Foxp3 lead to various autoimmune pathologies in both humans and experimental animals. Here the role of adaptive Tregs that are converted from CD4+CD25– T cells in the periphery is reviewed in relationship to their induction by cytokines, such as gamma-interferon, and therapeutic modalities, including T cell vaccination and Copolymer-I currently being used or tested in multiple sclerosis. In particular, important issues related to the potential therapeutic application of adaptive Tregs in autoimmune disease are discussed.