Identification of a bone marrow-derived CD8αα+ dendritic cell-like population in inflamed autoimmune target tissue with capability of inducing T cell apoptosis.

DCs play critical roles in promotion of autoimmunity or immune tolerance as potent APCs. In our anti-GBM GN model, WKY rats develop severe T cell-mediated glomerular inflammation followed by fibrosis. A DC-like cell population (CD8αα(+)CD11c(+)MHC-II(+)ED1(-)) was identified in the inflamed glomeruli. Chimera experiments demonstrated that the CD8αα(+) cells were derived from BM. The CD8αα(+) cells infiltrated glomeruli at a late stage (Days 28-35), coincident with a rapid decline in glomerular inflammation before fibrosis. The CD8αα(+) cells isolated from inflamed glomeruli were able to migrate rapidly from the bloodstream into inflamed glomeruli but not into normal glomeruli, suggesting that the migration was triggered by local inflammation. Despite high-level expression of surface and cellular MHC class II molecules, in vitro experiments showed that this CD8αα(+) DC-like cell induced apoptosis but not proliferation in antigen-specific CD4(+) T cells from T cell lines or freshly isolated from lymph nodes; they were not able to do so in the absence of antigens, suggesting induction of apoptosis was antigen-specific. Furthermore, apoptotic T cells were detected in a large number in the glomeruli at Day 32, coincident with the infiltration of the cells into glomeruli, suggesting that the cells may also induce T cell apoptosis in vivo. A potential role of this CD8αα(+) DC-like population in peripheral immune tolerance and/or termination of autoimmune inflammation was discussed.

A Rubredoxin based system for screening of protein expression conditions and on-line monitoring of the purification process.

Rubredoxin (Rub) from Thermotoga maritima, a 6.1-kDa red protein containing an Fe(III)-cysteine(4) center, was evaluated for its usefulness as a colored fusion tag for expression of recombinant proteins in E. coli. Here, we describe the Rub features relevant to accelerating screening for optimal high yield soluble expression conditions and automating the ensuing purification process. Spectroscopic properties and the yield of Rub fused to a typical target protein were compared to analogous GFP and Flavodoxin constructs, showing Rub absorption to be sufficient for structural genomics purposes while being produced at much higher soluble levels than GFP constructs. Based entirely on Rub absorption at 380 nm, both generic and affinity purification of crude cell lysate were performed: thus guided anion exchange purification of a Rub fusion construct as well as automated Ni-NTA purification resulted in pure protein. Rub is stable over a wide range of pH, temperature, and buffer environments, enabling robust purification protocols. Across a variety of fusion constructs, including N- and C-terminal Rub, quantitation via the Rub signal was shown to reliably correlate with analytical HPLC data obtained at 220 nm. We propose the "RubyTag" as an alternative to conventional protein fusion tags, as it combines a specific absorption signal with convenient biochemical and biological properties. Further, it allows direct on-line readout on conventional chromatography systems, holding promise for automated multi-step chromatography.

Requirements for alpha d in IgG immune complex-induced rat lung injury.

Alpha d is a newly cloned adhesion molecule that forms a heterodimer with CD18. The requirement for alpha d in IgG immune complex-induced lung injury in rats has been evaluated by the use of blocking polyclonal and monoclonal antibodies to rat alpha d. Using whole lung extracts, Northern and Western blot analyses have revealed up-regulation of mRNA and alpha d protein in inflamed lungs. Immunostaining has revealed the presence of alpha d in lung tissue and in alveolar macrophages as early as 1 h after initiation of the inflammatory reaction. When polyclonal rabbit Ab to rat alpha d was coinstilled into lung together with Ab to BSA, lung injury (as determined by leakage of [125I]albumin into lung parenchyma) was significantly diminished. In parallel, there was reduced accumulation of neutrophils recoverable in bronchoalveolar lavage (BAL) fluids. These findings were associated with reduced levels of TNF-alpha as well as NO2-/NO3- in BAL fluids. A hamster mAb to rat alpha d was also protective in this lung injury model. Anti-alpha d inhibited in vitro production of NO2-/NO3- by rat alveolar macrophages (stimulated with LPS and IFN-gamma) by approximately 60%. These data suggest that, in the lung inflammatory model employed, alpha d up-regulation occurs in lung macrophages and is necessary for expression of TNF-alpha, recruitment of neutrophils, and full development of lung injury.