Nitrotyrosine is widely recognized as a surrogate marker of up-regulated inducible NO synthase expression at sites of inflammation. However, the potential immunogenicity of autologous proteins containing nitrotyrosine has not previously been investigated. Herein, we used the I-EK-restricted T cell epitope of pigeon/moth cytochrome c (PCC/MCC88–103) to assess the ability of T cells to recognize ligands containing nitrotyrosine. Substitution of the single tyrosine (Y97) in PCC/MCC88–103 with nitrotyrosine abrogates recognition by the MCC88–103-specific T cell hybridoma 2B4. CBA (H2K) mice immunized with MCC88–103 or nitrated MCC88–103 peptides produce T cell responses that are mutually exclusive. Transgenic mice that constitutively express PCC under the control of an MHC class I promoter are tolerant toward immunization with MCC88–103, but exhibited a robust immune response against nitrated MCC88–103. Analysis of T cell hybridomas specific for nitrated-MCC88–103 indicated that subtle differences in TCR VDJ gene usage are sufficient to allow nitrotyrosine-specific T cells to escape the processes of central tolerance.

Although K+ channels are essential for hepatocellular function, it is not known which channels are involved in the regulatory volume decrease (RVD) in these cells. We have used a combination of electrophysiological and molecular approaches to describe the potential candidates for these channels. The dialysis of short-term cultured rat hepatocytes with a hypotonic solution containing high K+ and low Cl- concentration caused the slow activation of an outward, time-independent current under whole-cell configuration of the patch electrode voltage clamp. The reversal potential of this current suggested that K+ was the primary charge carrier. The swelling-induced K+ current (IKvol) occurred in the absence of Ca2+ and was inhibited with 1 microM Ca2+ in the pipette solution. The activation of IKvol required both Mg2+ and ATP and an increasing concentration of Mg-ATP from 0.25 through 0.5 to 0.9 mM activated IKvol increasingly faster and to a larger extent. The KCNQ1 inhibitor chromanol 293B reversibly depressed IKvol with an IC50 of 26 microM. RT-PCR detected the expression of members of the KCNQ family from KCNQ1 to KCNQ5 and of the accessory proteins KCNE1 to KCNE3 in the rat hepatocytes, but not KCNQ2 and KCNE2 in human liver. Western blotting showed KCNE3 expression in a plasma membrane-enriched fraction from rat hepatocytes. The results suggest that KCNQ1, probably with KCNE2 or KCNE3 as its accessory unit, provides a significant fraction of IKvol in rat hepatocytes.

Although CD103(+) cells recently emerged as key regulatory cells in the gut, the role of CD103 ubiquitous expression in the lung and development of allergic airway disease has never been studied. To answer this important question, we evaluated the response of Cd103(-/-) mice in two separate well-described mouse models of asthma (ovalbumin and house dust mite extract). Pulmonary inflammation was assessed by analysis of bronchoalveolar lavage content, histology, and cytokine response. CD103 expression was analyzed on lung dendritic cells and T cell subsets by flow cytometry. Cd103(-/-) mice exposed to antigens developed exacerbated lung inflammation, characterized by increased eosinophilic infiltration, severe tissue inflammation, and altered cytokine response. In wild-type mice exposed to house dust mite, CD103(+) dendritic cells are increased in the lung and an important subset of CD4(+) T cells, CD8(+) T cells, and T regulatory cells express CD103. Importantly, Cd103(-/-) mice presented a deficiency in the resolution phase of inflammation, which supports an important role for this molecule in the control of inflammation severity. These results suggest an important role for CD103 in the control of airway inflammation in asthma.