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.

The genetic basis of susceptibility to pulmonary fibrosis is largely unknown. Initially, in this study, loci regulating the response of bleomycin-induced pulmonary fibrosis were mapped using a set of recombinant congenic strains bred from pulmonary fibrosis-resistant A/J and susceptible C57BL/6J (B6) mice. Linkage was identified (logarithm of the odds score = 4.9) on chromosome 9, and other suggestive loci were detected. The putative loci included alleles from both the B6 and A/J strains as increasing the fibrosis response of congenic mice. Gene expression analysis with microarrays revealed 3,304 genes or expressed sequence tags to be differentially expressed (P < 0.01) in lung tissue between bleomycin-treated B6 and A/J mice, and 246 of these genes mapped to potential susceptibility loci. Pulmonary genes differentially expressed between bleomycin-treated B6 and A/J mice included those of heparin binding and extracellular matrix deposition pathways. A review of available genomic sequences revealed 809 (43% of total) genes in the linkage intervals to have variations predicted to alter the encoded proteins or their regulation, 68 (8.4%) of which were also differentially expressed. Genomic approaches were combined to produce a set of candidate genes that may influence susceptibility to bleomycin-induced pulmonary fibrosis in the A/J:B6 mouse model.

Responsiveness of CD4 + T cells to the IFN-+ -inducing cytokines IL-12 and IL-18 is generally thought to be acquired only after stimulation via the TCR. We report herein that stimulation of naive CD4 + T cells with high-dose IL-2 (1000 U/ml) renders these cells responsive to IL-12 and/or IL-18 without a requirement for TCR ligation. Naive CD4 + CD62L + T cells from normal C57BL/6 mice or from DO11.10/Rag2 -/-OVA-specific TCR-transgenic mice secreted substantial amounts of IFN-+ when stimulated concurrently with high-dose IL-2 plus IL-12 or IL-18. mRNA encoding both chains of the IL-12 and the IL-18 receptors was expressed by CD4 + T cells after stimulation with high-dose IL-2. Furthermore, anti-CD3-induced IL-12/IL-18 responsiveness was fully abrogated in the presence of cyclosporin A whereas IL-2-induced IL-12/IL-18 responsiveness was not, reminiscent of the previously reported IL-12+IL-18 innate pathway of T cell activation. Lastly, after stimulation with IL-2+IL-12, naive CD4 + T cells from DO11.10/Rag2 -/-mice exhibited polarization towards a Th1 phenotype (high IFN-+ but no IL-4) during secondary stimulation with immobilized anti-CD3. We have coined the term 'bystander polarization' to describe this phenomenon and we speculate that bystander polarization of naive CD4 + T cells may occur in vivo during strong antigen-specific immune responses.

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Pulmonary fibrosis is a disease of significant morbidity, with an incompletely defined genetic basis. Here, we combine linkage and association studies to identify genetic variations associated with pulmonary fibrosis in mice. Mice were treated with bleomycin by osmotic minipump, and pulmonary fibrosis was histologically assessed 6 weeks later. Fibrosis was mapped in C57BL6/J (fibrosis-susceptible) × A/J (fibrosis-resistant) F2 mice, and the major identified linkage intervals were evaluated in consomic mice. Genome-wide and linkage-interval genes were assessed for their association with fibrosis, using phenotypic data from 23 inbred strains and the murine single-nucleotide polymorphism map. Susceptibility to pulmonary fibrosis mapped to a locus on chromosome 17, which was verified with consomic mice, and to three additional suggestive loci that may interact with alleles on chromosome 17 to affect the trait in F2 mice. Two of the loci, including the region on chromosome 17, are homologous to previously mapped loci of human idiopathic fibrosis. Of the 23 phenotyped murine strains, four developed significant fibrosis, and the majority presented minimal disease. Genome-wide and linkage region-specific association studies revealed 11 pulmonary expressed genes (including the autophagy gene Cep55, and Masp2, which is a complement component) to contain polymorphisms significantly associated with bleomycin-induced fibrotic lung disease. In conclusion, genomic approaches were used to identify linkage intervals and specific genetic variations associated with pulmonary fibrosis in mice. The common loci and similarities in phenotype suggest these findings to be of relevance to clinical pulmonary fibrosis.

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