Nonpolarizing Conditions Research Articles

APRIL (TNFSF13) regulates collagen‐induced arthritis, IL‐17 production and Th2 response

A proliferation-inducing ligand (APRIL or TNFSF13) shares receptors with B-cell activation factor of the TNF family (BAFF) on B and T cells. Although much is known about the function of APRIL in B cells, its role in T cells remains unclear. Blocking both BAFF and APRIL suggested that BAFF and/or APRIL contributed to collagen-induced arthritis (CIA); however, the role of APRIL alone in CIA remained unresolved. We show here that, in vitro, our newly generated APRIL(-/-) mice exhibited increased T-cell proliferation, enhanced Th2 cytokine production under non-polarizing conditions, and augmented IL-13 and IL-17 production under Th2 polarizing conditions. Upon immunization with OVA and aluminum potassium sulfate, APRIL(-/-) mice responded with an increased antigen-specific IgG1 response. We also show that in APRIL(-/-) mice, the incidence of CIA was significantly reduced compared with WT mice in parallel with diminished levels of antigen-specific IgG2a autoantibody and IL-17 production. Our data indicate that APRIL plays an important role in the regulation of cytokine production and that APRIL-triggered signals contribute to arthritis. Blockade of APRIL thus may be a valuable adjunct in the treatment of rheumatoid arthritis.

Blimp-1 Attenuates Th1 Differentiation by Repression of ifng, tbx21, and bcl6 Gene Expression

T cell-specific deletion of Blimp-1 causes abnormal T cell homeostasis and function, leading to spontaneous, fatal colitis in mice. Herein we explore the role of Blimp-1 in Th1/Th2 differentiation. Blimp-1 mRNA and protein are more highly expressed in Th2 cells compared with Th1 cells, and Blimp-1 attenuates IFN-gamma production in CD4 cells activated under nonpolarizing conditions. Although Blimp-1-deficient T cells differentiate normally to Th2 cytokines in vitro, Blimp-1 is required in vivo for normal Th2 humoral responses to NP-KLH (4-hydroxy-3-nitrophenylacetyl/keyhole lymphocyte hemocyanin) immunization. Lack of Blimp-1 in CD4 T cells causes increased IFN-gamma, T-bet, and Bcl-6 mRNA. By chromatin immunoprecipitation we show that Blimp-1 binds directly to a distal regulatory region in the ifng gene and at multiple sites in tbx21 and bcl6 genes. Our data provide evidence that Blimp-1 functions in Th2 cells to reinforce Th2 differentiation by repressing critical Th1 genes.

Evidence for PI-3K-dependent migration of Th17-polarized cells in response to CCR2 and CCR6 agonists

IL-17-producing Th cells (Th17) are a distinct subset of effector cells that bridge the innate and adaptive immune system and are implicated in autoimmune disease processes. CD4(+) splenocytes from DO11.10 mice were activated with OVA peptide(323-339) and maintained under Th17 polarization conditions, resulting in significantly higher proportions of IL-17(+) T cells compared with nonpolarized (Th0) cells. Th17-polarizing conditions significantly increased the proportion of cells expressing the chemokine receptors CCR2, CCR6, and CCR9 when compared with Th0 cells. In contrast, there was a significant decrease in the proportion of cells expressing CXCR3 under Th17-polarizing conditions compared with nonpolarizing conditions. The respective chemokine agonists for CCR2 (CCL2 and CCL12), CCR6 (CCL20), and CCR9 (CCL25) elicited migration and PI-3K-dependent signaling events in Th17-polarized cells, thus indicating that all three receptors were functionally and biochemically responsive. Furthermore, postmigration phenotypic analysis demonstrated that the agonists for CCR2 and CCR6, but not CCR9, stimulated a modest enrichment of IL-17(+) cells compared with the premigration population. Pan-isoform inhibitors of PI-3K/Akt signaling prevented CCR2- and CCR6-mediated, polarized Th17 cell migration in a concentration-dependent manner. The unique chemokine receptor expression pattern of Th17 cells and their corresponding PI-3K-dependent migratory responses are important for understanding the pathogenesis of autoimmune diseases and may provide opportunities for the application of CCR2 and CCR6 antagonists and PI-3K isoform-selective inhibitors in defined inflammatory settings.

Failure of cAMP agonists to activate rescued ΔF508 CFTR in CFBE41o– airway epithelial monolayers

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated chloride channel. Mutations in the CFTR gene result in cystic fibrosis (CF). The most common mutation, deltaF508, results in endoplasmic reticulum-associated degradation (ERAD) of CFTR. DeltaF508 CFTR has been described as a temperature-sensitive mutation that can be rescued following growth at 27 degrees C. In order to study the processing and function of wild-type and rescued deltaF508 CFTR at the cell surface under non-polarized and polarized conditions, we developed stable cell lines expressing deltaF508 or wild-type CFTR. CFBE41o- is a human airway epithelial cell line capable of forming high resistance, polarized monolayers when cultured on permeable supports, while HeLa cells are normally grown under non-polarizing conditions. Immunoprecipitation, cell surface biotinylation, immunofluorescence, and functional assays confirmed the presence of deltaF508 CFTR at the cell surface in both cell lines after incubating the cells for 48 h at 27 degrees C. However, stimulators of wild-type CFTR such as forskolin, beta2-adrenergic or A2B-adenosine receptor agonists failed to activate rescued deltaF508 CFTR in CFBE41o- monolayers. Rescued deltaF508 CFTR could be stimulated with genistein independent of pretreatment with cAMP signalling agonists. Interestingly, rescued deltaF508 CFTR in HeLa cells could be efficiently stimulated with either forskolin or genistein to promote Cl- transport. These results indicate that deltaF508 CFTR, when rescued in CFBE41o- human airway epithelial cells, is poorly responsive to signalling pathways known to regulate wild-type CFTR. Furthermore, the differences in rescue and activation of deltaF508 CFTR in the two cell lines suggest that cell-type specific differences in deltaF508 CFTR processing are likely to complicate efforts to identify potentiators and/or correctors of the deltaF508 defect.

Sustained NFAT signaling promotes a Th1-like pattern of gene expression in primary murine CD4+ T cells.

T cell activation is known to be critically regulated by the extent and duration of TCR-induced signaling pathways. The NFAT family of transcription factors is believed to play an important role in coupling these quantitative differences in TCR-induced signaling events into changes in gene expression. In this study we have specifically investigated the effects of sustained NFAT signaling on T cell activation by introducing a constitutively active mutant version of NFATc1 (caNFATc1) into primary murine CD4(+) T cells and examining its effects on gene expression. We now report that ectopic expression of caNFATc1 partially mimics TCR signaling, resulting in enhanced expression of CD25 and CD40 ligand and down-regulation of CD62L. More importantly, we find that expression of caNFATc1 in T cells maintained under either nonpolarizing or Th1-skewing conditions leads to a marked selective increase in the number of cells expressing the prototypical Th1 cytokine, IFN-gamma. Furthermore, when expressed in Th2-skewed cells, caNFATc1 appears to attenuate Th2 differentiation by decreasing production of IL-4 and promoting the expression of IFN-gamma. Finally, we find that caNFATc1 enhances expression of functional P-selectin glycoprotein ligand-1, up-regulates Fas ligand expression, and increases susceptibility to activation-induced cell death, cellular traits that are preferentially associated with Th1 effector cells. Taken together, these results suggest that sustained NFAT signaling, mediated by ectopic expression of caNFATc1, acts to promote a Th1-like pattern of gene expression and thereby serves to highlight the important relationship between the degree of NFAT signaling and the qualitative pattern of gene expression induced during T cell activation.

Morphological and functional characterization of bovine oviductal epithelial cell monolayers cultured on polarizing membranes.

Several characteristics of oviductal cells, cultured under either polarizing or nonpolarizing conditions, were studied. In vitro produced bovine embryos tested the embryotrophic abilities of the respective conditioned media. Conditioned medium from the apical face of polarized cell monolayers supported higher rates of development to blastocyst and expanded blastocysts. In contrast, conditioned medium from the basal face supported embryo development only to the 8-16 cell stage; however, these embryos were able to continue development to the morula stage when cultured in medium from the apical and basal faces, indicating total cell confluence and a clear functional polarization. At the ultrastructural level, cells cultured in polarizing conditions displayed characteristics nearer to the same cells in vivo and signs of a metabolic activity higher than that in cells cultured under non-polarizing conditions. It can be concluded that cell-polarization, in our culture conditions, is beneficial to embryo development.

Artifacts associated with the use of thiocyanate and valinomycin/K+ as permeant ions in oxidant pulse experiments on denitrifying bacteria

By means of15N-tracer and oxidant pulse methods and with nitrate-grownParacoccus denitrificans it was found that KSCN completely inhibited reduction of N2O and nitrate in the 1 to 10 mM range, but had little or no effect on reduction of O2 or nitrite at 150 mM. These observations confirm a previous report. Potassium thiocyanate was insufficiently permeant across the cytoplasmic membrane ofParacoccus denitrificans andPseudomonas denitrificans even at 150 mM to prevent membrane polarization when oxidant pulses were large. Polarization and inhibition artifacts due to KSCN have created some confusion in the literature. Whereas valinomycin had little direct effect on reduction of nitrite, N2O, and O2 individually byPa. denitrificans, it caused a temporary nitrite-dependent inhibition of N2O reduction. Under nonpolarizing conditions the →H+/2e− ratios for O2, N2O, and nitrite (8.0, 4.3, and 4.5, respectively) confirmed those reported previously from this laboratory. The present results largely but not entirely agree with data from another laboratory.