No abstract

Progesterone Receptor Membrane Component-1 (PGRMC1) is present in both the cytoplasm and nucleus of spontaneously immortalized granulosa cells (SIGCs). PGRMC1 is detected as a monomer in the cytoplasm and a DTT-resistant PGRMC1 dimer in the nucleus. Transfected PGRMC1-GFP localizes mainly to the cytoplasm and does not form a DTT-resistant dimer. Moreover, forced expression of PGRMC1-GFP increases the sensitivity of the SIGCs to progesterone (P4) 's antiapoptotic action, indicating that the PGRMC1 monomer is functional. However, when endogenous PGRMC1 is depleted by siRNA treatment and replaced with PGRMC1-GFP, P4 responsiveness is not enhanced, although overall levels of PGRMC1 are increased. P4's anti-apoptotic action is also attenuated by actinomycin D, an inhibitor of RNA synthesis, and P4 activation of PGRMC1 suppresses Bad and increases Bcl2a1d expression. Taken together, the present studies suggest a genomic component to PGRMC1's anti-apoptotic mechanism of action, which requires the presence of the PGRMC1 dimer.

Sensitized mice acutely challenged with inhaled ovalbumin (OVA) develop allergic airway inflammation, characterized by OVA-specific IgE production, airway eosinophilia, increased pulmonary B and T lymphocytes, and airway hyperreactivity. In this study, a chronic exposure model was developed and two distinct patterns of response were observed. Discontinuous inhalational exposure to OVA (6 weeks) produced airway inflammation and hyperreactivity that were similar to acute (10 days) responses. Continuous inhalational exposure to OVA (6 or 11 weeks) resulted in attenuation of airway eosinophilia and hyperresponsiveness without reduction of OVA-specific IgE and IgG 1 levels. The inhibition of airway inflammation was dependent on continuous exposure to antigen, because continuously exposed mice with attenuated inflammatory responses redeveloped allergic airway disease if the OVA aerosols were interrupted and then restarted (11-week-discontinuous). Inhalational tolerance induced by continuous OVA exposure demonstrated bystander suppression of cockroach allergen-mediated airway eosinophilia. These findings may be attributed to changes in production of the anti-inflammatory cytokine interleukin-10 during continuous OVA aerosol exposure. The symptomatic and asymptomatic allergic responses in human asthmatics could be explained by similar variable or discontinuous exposures to aeroantigens. Throughout the past 40 years, the prevalence of allergic disease, including atopic dermatitis, hay fever, and asthma, has risen dramatically in the developed world. This disturbing trend is documented best for asthma. 1,2 A wealth of clinical and experimental data suggests that allergic asthma is due to an aberrant lung immune response mediated through T-helper type 2 cells (Th2 cells) and associated cytokine-signaling pathways. The normal lung is able to distinguish between airborne antigens associated with infectious agents, to which an immunological response is generated, and harmless inhaled antigens, which are usually ignored. In the asthmatic lung, some of these normally harmless antigens activate specific Th2 cells and elicit an inflammatory response characterized by Th2 cytokine production, eosinophilic airway inflammation, airway hyperresponsiveness, and bronchoconstriction. These pulmonary responses may be accompanied by systemic allergic sensitization, manifested by elevated titers of antigen-specific IgE. The mechanisms that control CD4 ϩ T lymphocyte polarization to allergenic Th2 phenotypes are incompletely understood but seem to involve genetic predispositions, local factors such as pre-existing cytokine concentrations and inflammation, and antigenic factors (ie, potency, dose, and duration of exposure).Several investigators have used mouse models to investigate the mechanisms of inhalational tolerance to antigens 3,4 or of allergic airway sensitization. [5][6][7][8][9][10] However, these responses have typically been assessed in isolation from each other. We have recently demonstrated that C57BL/6J mice undergo a biphasic...

Bromelain attenuated development of AAD while altering CD4+ to CD8+ T lymphocyte populations. The reduction in AAD outcomes suggests that bromelain may have similar effects in the treatment of human asthma and hypersensitivity disorders.

Progesterone (P4) inhibits apoptosis of rat granulosa cells and spontaneously immortalized granulosa cells (SIGCs), which were derived from rat granulosa cells. Defining the mechanism through which P4 mediates its action has been difficult because these cells do not express the classic nuclear P4 receptor. Previous studies have shown that a P4 receptor antibody, C-262, detects a 60-kDa protein that is involved in regulating P4's antiapoptotic action. Using a C-262 affinity column, this 60-kDa protein was isolated and sequenced by mass spectrometry. This analysis revealed that the C-262-detectable protein is an unnamed protein referred to as RDA288. This protein has several putative hyaluronic acid binding sites. Further hyaluronic acid antagonizes (3)H-P4 binding to SIGCs and mimics P4's action, whereas exogenous hyaluronic acid binding protein attenuates P4's actions. RT-PCR demonstrated that RDA288 mRNA was present in SIGCs, immature rat ovary, lung, and skeletal muscle but was not present in several other organs. Forced expression of RDA288 increased the capacity of SIGCs to bind and respond to P4. An antibody was also developed against RDA288. Using this antibody in a Western blot protocol, RDA288 expression was confirmed in both SIGCs and granulosa cells. An immunohistochemical study detected RDA288 in the cytoplasm and plasma membrane components of granulosa cells of antral follicles. Immunocytochemical studies on living nonpermeabilized SIGCs revealed that RDA288 was present on the extracellular surface of the plasma membrane. Finally, pretreatment with the RDA288 antibody blocked P4's antiapoptotic actions. Taken together, these data suggest that RDA288 plays a significant role in mediating P4's antiapoptotic action in granulosa cells.

Background Natural Killer (NK) cells have been implicated in the development of allergic airway inflammation. However the in vivo role of NK cells has not been firmly established due to the lack of animal models with selective deficiencies in NK cells. Objective To determine the specific contribution of NK cells in a murine model of allergic airway disease (AAD). Methods The role of NK cells in AAD was studied using NK-deficient (NKD) mice, perforin−/− mice, and mice depleted of Ly49A/D/G+ NK cell subsets in an ovalbumin-induced model of allergic airway disease (OVA-AAD). Results Induction of OVA-AAD in C57BL/6 wild-type (WT) mice resulted in the expansion of airway NK cells and the development of pronounced airway eosinophilia. In the absence of NK cells or specific subsets of NK cells, either in NKD mice, or after the depletion of Ly49A/D/G+ NK cells, the development of OVA-AAD was significantly impaired as seen by decreased airway inflammation and eosinophilia, decreased secretion of the Th2 cytokines IL-4, IL-5 and IL-13 and diminished OVA-specific antibody production. Furthermore, while OVA-exposure induced a dramatic expansion of dendritic cells (DCs) in WT mice, their induction was significantly attenuated in NKD mice. Development of OVA-AAD in perforin−/− mice suggested that the proinflammatory role of NK cells is not dependent on perforin-mediated cytotoxicity. Lastly, induction of allergic disease by OVA-specific CD4 T cells from WT but not NK-depleted or NKD mice in RAG−/− recipients, demonstrates that NK cells are essential for T cell priming. Conclusions and Clinical Relevance Our data demonstrate that conventional NK cells play an important and distinct role in the development of AAD. The presence of activated NK cells has been noted in patients with asthma. Understanding the mechanisms by which NK cells regulate allergic disease is therefore an important component of treatment approaches.

Concomitant infection of murine CMV (MCMV), an opportunistic respiratory pathogen, altered Th1/Th2 cytokine expression, decreased bronchoalveolar lavage (BAL) fluid eosinophilia, and increased mucus production in a murine model of OVA-induced allergic airway disease. Although no change in the total number of leukocytes infiltrating the lung was observed between challenged and MCMV/challenged mice, the cellular profile differed dramatically. After 10 days of OVA-aerosol challenge, eosinophils comprised 64% of the total leukocyte population in BAL fluid from challenged mice compared with 11% in MCMV/challenged mice. Lymphocytes increased from 11% in challenged mice to 30% in MCMV/challenged mice, and this increase corresponded with an increase in the ratio of CD8+ to CD4+TCRαβ lymphocytes. The decline in BAL fluid eosinophilia was associated with a change in local Th1/Th2 cytokine profiles. Enhanced levels of IL-4, IL-5, IL-10, and IL-13 were detected in lung tissue from challenged mice by RNase protection assays. In contrast, MCMV/challenged mice transiently expressed elevated levels of IFN-γ and IL-10 mRNAs, as well as decreased levels of IL-4, IL-5, and IL-13 mRNAs. Elevated levels of IFN-γ and reduced levels of IL-5 were also demonstrated in BAL fluid from MCMV/challenged mice. Histological evaluation of lung sections revealed extensive mucus plugging and epithelial cell hypertrophy/hyperplasia only in MCMV/challenged mice. Interestingly, the development of airway hyperresponsiveness was observed in challenged mice, not MCMV/challenged mice. Thus, MCMV infection can modulate allergic airway inflammation, and these findings suggest that enhanced mucus production may occur independently of BAL fluid eosinophilia.

Objective To determine the relationship between Progesterone Receptor Membrane Component-1 (PGRMC1) expression and the outcome of in vitro fertilization treatment. Design A prospective study in which PGRMC1 mRNA levels, methylation status of the Pgrmc1 promoter, and the presence of point mutations within Pgrmc1 were obtained from granulosa/luteal cells of women undergoing controlled ovarian hyperstimulation (COH). Setting Fertility center/Basic science laboratory Patients Eighty-five IVF patients and 10 women, who were undergoing COH for the purpose of oocyte donation, were included in this study. Interventions None. Main Outcome Measures PGRMC1 measurements were correlated with clinical outcomes, such as number of follicles, number of retrieved oocytes and ongoing pregnancy rates. Results PGRMC1 mRNA levels within granulosa/luteal cells of 18% of IVF patients were > 2.25 fold higher than those of oocyte donors. Individuals with elevated PGRMC1 mRNA levels had 30% fewer large follicles and fewer oocytes retrieved. The elevated PGRMC1 mRNA levels were associated with an increase in the methylation of Pgrmc1 promoter. Conclusion In patients with elevated PGRMC1 mRNA levels, gonadotropin-induced follicle development is attenuated, although sufficient numbers of follicles develop to allow for embryo transfer and subsequent pregnancy.