Development of inflammation in proteoglycan-induced arthritis is dependent on Fc gamma R regulation of the cytokine/chemokine environment

FcgammaRs are specialized cell surface receptors that coordinately regulate immune responses. Although FcgammaR expression is a prerequisite for the development of several immune complex-mediated diseases, the mechanism responsible for FcgammaR-dependent regulation in autoimmunity remains unclear. Therefore, we assessed FcgammaR-dependent regulation of inflammation in proteoglycan-induced arthritis (PGIA) using FcgammaR(-/-) mice. FcgammaRIIb(-/-) mice developed arthritis at an earlier time point and with a greater severity than wild-type (WT) mice. In gamma-chain(-/-) (FcgammaRI(-/-) and FcgammaRIII(-/-)) mice, no clinical or histological evidence of inflammation was observed. Exacerbation of arthritis in FcgammaRIIb(-/-) mice correlated with enhanced PG-specific Ab production, but did not significantly affect PG-specific T cell priming. In gamma-chain(-/-) mice, the absence of arthritis did not correlate with serum Ab responses, as PG-specific Ab production was normal. Although PG-specific T cell proliferation was diminished, spleen cells from gamma-chain(-/-) mice successfully adoptively transferred arthritis into SCID mice. Our studies indicated that the mechanism responsible for FcgammaR regulation of PGIA development was at the level of inflammatory cytokine and beta-chemokine expression within the joint. FcgammaRIIb regulated the development of PGIA by controlling the initiation of cytokine and chemokine expression within the joint before the onset of arthritis, whereas the expression of FcgammaRI and or FcgammaRIII controlled cytokine and chemokine expression late in the development of PGIA during the onset of disease. These results suggest that FcgammaRs are critical for the development of inflammation during PGIA, possibly by maintaining or enhancing inflammatory cytokine and beta-chemokine production.     

Antigen-driven oligoclonal expansion of tumor-infiltrating B cells in infiltrating ductal carcinoma of the breast

The objective of this study was to determine whether tumor-infiltrating B cells (TIL-B) of infiltrating ductal carcinoma (IDC) of the breast represent a tumor-specific humoral immune response. Immunohistochemical analysis of three Her-2/neu-negative IDC tumors from geriatric patients showed that TIL-B cluster in structures similar to germinal centers containing CD20(+) B lymphocyte and CD3(+) T lymphocyte zones with interdigitating CD21(+) follicular dendritic cells, suggesting an in situ immune response. A total of 29, 31, and 58 IgG1 H chain clones was sequenced from the three IDC tumors, respectively. Intratumoral oligoclonal expansion of TIL-B was demonstrated by a preponderance (45-68%) of clonal B cells. In contrast, only 7% of tumor-draining lymph node and 0% of healthy donor PBL IgG H chains were clonal, consistent with the larger repertoires of node and peripheral populations. Patterns and levels of TIL-B IgG H chain somatic hypermutation suggested affinity maturation in intratumoral germinal centers. To examine the specificity of TIL-B Ig, a phage-displayed Fab library was generated from the TIL-B of one IDC tumor. Panning with an allogeneic breast cancer cell line enriched Fab binding to breast cancer cells, but not nonmalignant cell lines tested. However, panning with autologous tumor tissue lysate increased binding of Fab to both tumor tissue lysate and healthy breast tissue lysate. These data suggest an in situ Ag-driven oligoclonal B cell response to a variety of tumor- and breast-associated Ags.     

Distinct mechanisms for cross-protection of the upper versus lower respiratory tract through intestinal priming

A main feature of the common mucosal immune system is that lymphocytes primed in one mucosal inductive site may home to distant mucosal effector sites. However, the mechanisms responsible for such cross-protection remain elusive. To address these we have used a model of local mucosal infection of mice with reovirus. In immunocompetent mice local duodenal priming protected against subsequent respiratory challenge. In the upper respiratory tract this protection appeared to be mainly mediated by specific IgA- and IgG2a-producing B cells, whereas ex vivo active effector memory CTL were found in the lower respiratory tract. In accordance with these findings, clearance of reovirus from the lower respiratory tract, but not from the upper respiratory tract, of infected SCID mice upon transfer of gut-primed lymphocytes depended on the presence of T cells. Taken together this study reveals that intestinal priming leads to protection of both the upper and lower respiratory tracts, however through distinct mechanisms. We suggest that cross-protection in the common mucosal immune system is mediated by trafficking of B cells and effector memory CTL.     

Expression,purification and characterisation of full-length histidine protein kinase RegB from Rhodobacter sphaeroides

The global redox switch between aerobic and anaerobic growth in Rhodobacter sphaeroides is controlled by the RegA/RegB two-component system, in which RegB is the integral membrane histidine protein kinase, and RegA is the cytosolic response regulator. Despite the global regulatory importance of this system and its many homologues, there have been no reported examples to date of heterologous expression of full-length RegB or any histidine protein kinases. Here, we report the amplified expression of full-length functional His-tagged RegB in Escherichia coli, its purification, and characterisation of its properties. Both the membrane-bound and purified solubilised RegB protein demonstrate autophosphorylation activity, and the purified protein autophosphorylates at the same rate under both aerobic and anaerobic conditions confirming that an additional regulator is required to control/inhibit autophosphorylation. The intact protein has similar activity to previously characterised soluble forms, but is dephosphorylated more rapidly than the soluble form (half-life ca 30 minutes) demonstrating that the transmembrane segment present in the full-length RegB may be an important regulator of RegB activity. Phosphotransfer from RegB to RegA (overexpressed and purified from E. coli) by RegB is very rapid, as has been reported for the soluble domain. Dephosphorylation of active RegA by full-length RegB has a rate similar to that observed previously for soluble RegB.     

Differential accumulation of dimethylallyl diphosphate in leaves and needles of isoprene- and methylbutenol-emitting and nonemitting species

The biosynthesis and emission of volatile plant terpenoids, such as isoprene and methylbutenol (MBO), depend on the chloroplastic production of dimethylallyl diphosphate (DMAPP). To date, it has been difficult to study the relationship of cellular DMAPP levels to emission of these volatiles because of the lack of a sensitive assay for DMAPP in plant tissues. Using a recent DMAPP assay developed in our laboratories, we report that species with the highest potential for isoprene and MBO production also exhibit elevated light-dependent DMAPP production, ranging from 110% to 1,063%. Even species that do not produce significant amounts of volatile terpenoids, however, exhibit some potential for light-dependent production of DMAPP. We used a nonaqueous fractionation technique to determine the intracellular distribution of DMAPP in isoprene-emitting cottonwood (Populus deltoides) leaves; approximately 65% to 70% of the DMAPP recovered at midday occurred in the chloroplasts, indicating that most of the light-dependent production of DMAPP was chloroplastic in origin. The midday concentration of chloroplastic DMAPP in cottonwood leaves is estimated to be 0.13 to 3.0 mM, which is consistent with the relatively high K(m)s that have been reported for isoprene synthases (0.5-8 mM). The results provide support for the hypothesis that the light dependence of isoprene and MBO emissions is in part due to controls over DMAPP production.     

The priming of amylose synthesis in Arabidopsis leaves

We investigated the mechanism of amylose synthesis in Arabidopsis leaves using (14)C-labeling techniques. First, we tested the hypothesis that short malto-oligosaccharides (MOS) may act as primers for granule-bound starch synthase I. We found increased amylose synthesis in isolated starch granules supplied with ADP[(14)C]glucose (ADP[(14)C]Glc) and MOS compared with granules supplied with ADP[(14)C]Glc but no MOS. Furthermore, using a MOS-accumulating mutant (dpe1), we found that more amylose was synthesized than in the wild type, correlating with the amount of MOS in vivo. When wild-type and mutant plants were tested in conditions where both lines had similar MOS contents, no difference in amylose synthesis was observed. We also tested the hypothesis that branches of amylopectin might serve as the primers for granule-bound starch synthase I. In this model, elongated branches of amylopectin are subsequently cleaved to form amylose. We conducted pulse-chase experiments, supplying a pulse of ADP[(14)C]Glc to isolated starch granules or (14)CO(2) to intact plants, followed by a chase period in unlabeled substrate. We detected no transfer of label from the amylopectin fraction to the amylose fraction of starch either in isolated starch granules or in intact leaves, despite varying the time course of the experiments and using a mutant line (sex4) in which high-amylose starch is synthesized. We therefore find no evidence for amylopectin-primed amylose synthesis in Arabidopsis. We propose that MOS are the primers for amylose synthesis in Arabidopsis leaves.