SUMMARYDecay-accelerating factor (DAF) is a membrane regulator of C3 activation that protects self cells from autologous complement attack. In humans, DAF is uniformly expressed as a glycosylphosphatidylinositol (GPI)-anchored molecule. In mice, both GPI-anchored and transmembraneanchored DAF proteins are produced, each of which can be derived from two different genes (Daf1 and Daf 2). In this report, we describe a Daf1 gene knock-out mouse arising as the ®rst product of a strategy for targeting one or both Daf genes. As part of the work, we characterize recently described monoclonal antibodies against murine DAF protein using deletion mutants synthesized in yeast, and then employ the monoclonal antibodies in conjunction with wild-type and the Daf1 knock-out mice to determine the tissue distribution of the mouse Daf1 and Daf 2 gene products. To enhance the immunohistochemical detection of murine DAF protein, we utilized the sensitive tyramide¯uores-cence method. In wild-type mice, we found strong DAF labelling of glomeruli, airway and gut epithelium, the spleen, vascular endothelium throughout all tissues, and seminiferous tubules of the testis. In Daf1 knock-out mice, DAF labelling was ablated in most tissues, but strong labelling of the testis and splenic dendritic cells remained. In both sites, reverse transcription-polymerase chain reaction analyses identi®ed both GPI and transmembrane forms of Daf 2 gene-derived protein. The results have relevance for studies of in vivo murine DAF function and of murine DAF structure.

Osteoarthritis is characterized by articular cartilage breakdown, and emerging evidence suggests that dysregulated innate immunity is likely involved. Here, we performed proteomic, transcriptomic, and electron microscopic analyses to demonstrate that mast cells are aberrantly activated in human and murine osteoarthritic joint tissues. Using genetic models of mast cell deficiency, we demonstrate that lack of mast cells attenuates osteoarthritis in mice. Using genetic and pharmacologic approaches, we show that the IgE/FcεRI/Syk signaling axis is critical for the development of osteoarthritis. We find that mast cell-derived tryptase induces inflammation, chondrocyte apoptosis, and cartilage breakdown. Our findings demonstrate a central role for IgE-dependent mast cell activation in the pathogenesis of osteoarthritis, suggesting that targeting mast cells could provide therapeutic benefit in human osteoarthritis.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

Tryptase (α and β) levels in serum are used to assess mast cell involvement in human disease. Using cultured cells, the current study examines the hypothesis that protryptase(s) are spontaneously secreted by mast cells at rest, whereas mature tryptase(s) are stored in secretory granules until their release by activated cells. HMC-1 cells have only β-tryptase genes and the corresponding mRNA. Mono-Mac-6 cells have both α- and β-tryptase genes but preferentially express α-tryptase. Mono-Mac-6 cells spontaneously secrete most of their tryptase, which consists of α-protryptase, whereas mature tryptase is retained inside these cells. HMC-1 cells also spontaneously secrete most of their tryptase, identified as β-protryptase, and retain mature tryptase. Skin-derived mast cells retain most of their tryptase, which is mature, and spontaneously secrete protryptase(s). Total tryptase levels in plasma are detectable but no different in healthy subjects with and without the gene for α-tryptase, consistent with pro forms of both α- and β-tryptase being spontaneously secreted. Thus, protryptase(s) are spontaneously secreted by resting mast cells, whereas mature tryptase is retained by mast cells until they are activated to degranulate.

The expression of FcγR by human skin-derived mast cells of the MCTC type was determined in the current study. Expression of mRNA was analyzed with microarray gene chips and RT-PCR; protein by Western blotting and flow cytometry; function by release of β-hexosaminidase, PGD2, leukotriene C4 (LTC4), IL-5, IL-6, IL-13, GM-CSF, and TNF-α. FcγRIIa was consistently detected along with FcεRI at the mRNA and protein levels; FcγRIIc was sometimes detected only by RT-PCR; but FcγRIIb, FcγRI, and FcγRIII mRNA and protein were not detected. FcγRIIa-specific mAb caused skin MCTC cells to degranulate and secrete PGD2, LTC4, GM-CSF, IL-5, IL-6, IL-13, and TNF-α in a dose-dependent fashion. FcεRI-specific mAb caused similar amounts of each mediator to be released with the exception of LTC4, which was not released by this agonist. Simultaneous but independent cross-linking of FcεRI and FcγRIIa did not substantially alter mediator release above or below levels observed with each agent alone. Skin MCTC cells sensitized with dust-mite-specific IgE and IgG, when coaggregated by Der p2, exhibited enhanced degranulation compared with sensitization with either IgE or IgG alone. These results extend the known capabilities of human skin mast cells to respond to IgG as well as IgE-mediated signals.

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