Reciprocal regulation of permeability through a cultured keratinocyte sheet by IFN-gamma and IL-4

The T cell cytokines profoundly modify the phenotypic and functional characteristics of keratinocytes. Until now, no study has focused on the effect of Th1 and Th2 cytokines on keratinocyte permeability. Using a two-layer well culturing system, permeability was assessed through cultured keratinocyte sheet in the presence or absence of various concentrations of IFN-gamma and IL-4. Transepithelial electrical resistance (TER) and the flux of 40 kDa FITC-dextrans were measured across the cultured keratinocyte sheet. IFN-gamma significantly increased the TER in a dose- and time-dependent manner, suggesting that IFN-gamma profoundly inhibited the permeability of ions through the keratinocyte sheet. In contrast, IL-4 did not affect the TER. When compared to medium control, the flux of FITC-dextran of the IFN-gamma group was significantly decreased in a dose-dependent fashion. In sharp contrast, the flux of FITC-dextran was significantly and dose-dependently increased in the presence of IL-4. A significant increase in TER and a significant decrease in the flux of dextran suggested that IFN-gamma clearly reduced the permeability of both ions and high molecular weight material through the keratinocyte sheet. Although IL-4 did not affect the permeability of the ions, it significantly enhanced the permeability of high molecular weight material. A flow cytometric assay revealed that the expression of desmoglein-3 was suppressed by IL-4, but was enhanced by IFN-gamma. The reciprocal regulation of permeability of the cultured keratinocyte sheet by IFN-gamma and IL-4 may be partly related to the modification of intercellular adhesion molecules.     

The role of IL-12, IL-23 and IFN-gamma in immunity to viruses

IL-12, IL-23 and IFN-gamma form a loop and have been thought to play a crucial role against infectious viruses, which are the prototype of "intracellular" pathogens. In the last 10 years, the generation of knock-out (KO) mice for genes that control IL-12/IL-23-dependent IFN-gamma-dependent mediated immunity (STAT1, IFN-gammaR1, IFNgammaR2, IL-12p40 and IL-12Rbeta1) and the identification of patients with spontaneous germline mutations in these genes has led to a re-examination of the role of these cytokines in anti-viral immunity. We here review viral infections in mice and humans with genetic defects in the IL-12/IL-23-IFN-gamma axis. A comparison of the phenotypes observed in KO mice and deficient patients suggests that the human IL-12/IL-23-IFN-gamma axis plays a redundant role in immunity to most viruses, whereas its mouse counterparts play a more important role against several viruses.     

CNOT7/hCAF1 is involved in ICAM-1 and IL-8 regulation by tristetraprolin

Tristetraprolin (TTP) is an RNA-binding protein which can bind to the AU-rich elements (AREs) at the 3′-untranslated region (3′-UTR) of target mRNA and promote mRNA deadenylation and degradation. We have shown in a previous study that TTP regulates tumor necrosis factor-α (TNF-α)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8), both of whose mRNAs have AREs in the 3′-UTR, in human pulmonary microvascular endothelial cells (HPMEC) through destabilizing target mRNAs, nevertheless, the mechanism by which TTP promotes mRNA decay remains unclear. Observations have indicated that TTP can interact with CAF1 (CNOT7/hCAF1 in human), a subunit of the CCR4-NOT complex with deadenylase activity. Another study illustrated that TTP can directly bind to CNOT1, the scaffold subunit of the CCR4-NOT complex. The present study showed that TTP bound to the AREs of ICAM-1 and IL-8 mRNAs and was coimmunoprecipitated with intracellular ICAM-1 and IL-8 mRNAs. TTP, CNOT7 and CNOT1 were coimmunoprecipitated in HPMEC. CNOT7 silencing stabilized ICAM-1 and IL-8 mRNAs and increased ICAM-1 and IL-8 production following TNF-α stimulation. These results, together with our previous study, suggest that CNOT7/hCAF1 is involved in ICAM-1 and IL-8 regulation by TTP in HPMEC.     

IL-12 induces monocyte IL-18 binding protein expression via IFN-gamma

IL-18 is a Th1 cytokine that synergizes with IL-12 and IL-2 in the stimulation of lymphocyte IFN-gamma production. IL-18 binding protein (IL-18BP) is a recently discovered inhibitor of IL-18 that is distinct from the IL-1 and IL-18 receptor families. In this report we show that IL-18BPa, the IL-18BP isoform with the highest affinity for IL-18, was strongly induced by IL-12 in human PBMC. Other Th1 cytokines, including IFN-gamma, IL-2, IL-15, and IL-18, were also capable of augmenting IL-18BPa expression. In contrast, IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma-inducible protein-10, and Th2 cytokines such as IL-4 and IL-10 did not induce IL-18BPa. Although monocytes were found to be the primary source of IL-18BPa, the induction of IL-18BPa by IL-12 was mediated through IFN-gamma derived predominantly from NK cells. IL-18BPa production was observed in cancer patients receiving recombinant human IL-12 and correlated with the magnitude of IFN-gamma production. The IFN-gamma/IL-18BPa negative feedback loop identified in this study may be capable of broadly controlling immune activation by cytokines that synergize with IL-18 to induce IFN-gamma and probably plays a key role in the modulation of both innate and adaptive immunity.     

Homeostatic regulation of Salmonella-induced mucosal inflammation and injury by IL-23

IL-12 and IL-23 regulate innate and adaptive immunity to microbial pathogens through influencing the expression of IFN-γ, IL-17, and IL-22. Herein we define the roles of IL-12 and IL-23 in regulating host resistance and intestinal inflammation during acute Salmonella infection. We find that IL-23 alone is dispensable for protection against systemic spread of bacteria, but synergizes with IL-12 for optimal protection. IL-12 promotes the production of IFN-γ by NK cells, which is required for resistance against Salmonella and also for induction of intestinal inflammation and epithelial injury. In contrast, IL-23 controls the severity of inflammation by inhibiting IL-12A expression, reducing IFN-γ and preventing excessive mucosal injury. Our studies demonstrate that IL-23 is a homeostatic regulator of IL-12-dependent, IFN-γ-mediated intestinal inflammation.     

Posttranscriptional regulation of angiotensin II type 1 receptor expression by glyceraldehyde 3-phosphate dehydrogenase

Regulation of angiotensin II type 1 receptor (AT1R) has a pathophysiological role in hypertension, atherosclerosis and heart failure. We started from an observation that the 3′-untranslated region (3′-UTR) of AT1R mRNA suppressed AT1R translation. Using affinity purification for the separation of 3′-UTR-binding proteins and mass spectrometry for their identification, we describe glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an AT1R 3′-UTR-binding protein. RNA electrophoretic mobility shift analysis with purified GAPDH further demonstrated a direct interaction with the 3′-UTR while GAPDH immunoprecipitation confirmed this interaction with endogenous AT1R mRNA. GAPDH-binding site was mapped to 1–100 of 3′-UTR. GAPDH-bound target mRNAs were identified by expression array hybridization. Analysis of secondary structures shared among GAPDH targets led to the identification of a RNA motif rich in adenines and uracils. Silencing of GAPDH increased the expression of both endogenous and transfected AT1R. Similarly, a decrease in GAPDH expression by H₂O₂ led to an increased level of AT1R expression. Consistent with GAPDH having a central role in H₂O₂-mediated AT1R regulation, both the deletion of GAPDH-binding site and GAPDH overexpression attenuated the effect of H₂O₂ on AT1R mRNA. Taken together, GAPDH is a translational suppressor of AT1R and mediates the effect of H₂O₂ on AT1R mRNA.     

Differential regulation of class II MHC determinants on macrophages by IFN-gamma and IL-4

Initiation of an immune response depends upon expression of class II MHC determinants on plasma membranes of APC. Murine peritoneal macrophages treated with either rIFN-gamma or rIL-4 display significantly more class II MHC determinants than untreated control cells. Analysis of the induction of macrophage Ia Ag by these cytokines showed considerable quantitative and qualitative differences. Maximal levels of Ia Ag induced in macrophages and detected by ELISA after IL-4 treatment at 48 h was about 80% of that induced by IFN-gamma. However, the frequency of Ia+ cells in replicate macrophage populations cultured for 48 h in excess concentrations of cytokine was 60 to 80% with IFN-gamma, 30 to 40% with IL-4, and 5% with medium alone. Thus, the subpopulation of macrophages able to respond to IL-4 for induction of Ia Ag expression was less than that able to respond to IFN-gamma. Expression of Ia Ag on macrophages continuously exposed to IFN-gamma was maximal at 48 h and remained at this high level through 6 days. Maximal Ia Ag expression for IL-4-treated cells was also detected at 48 h, but was not sustained with time in culture, and returned to base line by 4 days. A similar time course for levels of Ia-specific message in macrophages at various times after IFN-gamma and IL-4 treatment was detected by Northern dot blot analysis. Loss of Ia mRNA and Ag with time in culture in the IL-4 treated cells was not due to macrophage cell death, depletion of active cytokine, or presence of fluid-phase inhibitors. IL-4 unresponsive cells were fully capable of maximal response to IFN-gamma for Ia Ag induction. These findings suggest that IL-4 and IFN-gamma induce class II MHC determinants through different mechanisms which may provide discrete regulatory control of APC function.     

IL-21 inhibits IFN-gamma production in developing Th1 cells through the repression of Eomesodermin expression

Exposure of naive Th cell precursors (Thp) to IL-21 inhibits IFN-gamma production from developing Th1 cells. The inhibition of IFN-gamma seen in IL-21-treated Thp cells is specific as the expression of other Th1 cytokines is unaffected. Recently, it has been reported that Eomesodermin (Eomes), a member of the T-box gene family, is expressed in developing CD8+ T cells and plays an important role in regulating IFN-gamma production and cytolytic effector function. In this study, we show that Eomes mRNA and protein are also expressed in developing Th1 cells, and exposure of naive Thp cells to IL-21 results in a decrease in Eomes expression. Moreover, the repression of Eomes expression by IL-21 is not due to an indirect effect of IL-21 on the expression of IFN-gamma or STAT4 and is independent of STAT1 and T-bet expression. Finally, we show that ectopic expression of Eomes prevents the inhibition of IFN-gamma production from IL-21-treated Thp cells. Taken together, these results demonstrate that Eomes plays a role in regulating IFN-gamma production in CD4+ T cells and IL-21 inhibits IFN-gamma production in developing Th1 cells through the repression of Eomes expression.