IntroductionMyeloid cell leukemia sequence 1 (Mcl-1) 1 has been identified as an intracellular antiapoptotic factor in a variety of hematopoietic cells, both in vitro and in vivo. [2][3][4][5][6] Human mast cells express 7,8 and Mcl-1 can promote the survival of some populations of human neoplastic mast cells in vitro. 7 Basophils, granulocytes with many characteristics and functions that partially overlap with those of tissue mast cells, [9][10][11][12] can also express Mcl-1. 13 However, it is not clear to what extent Mcl-1 is important in the development and/or survival of mast cells or basophils in vivo.Opferman et al showed that the genetic manipulation of Mcl-1 can be used to delete individual hematopoietic cell populations in mice. 4 We therefore used this approach to examine the effects of reducing expression of Mcl-1 in the mast cell lineage in vivo. To attempt to delete Mcl-1 selectively in mast cells, we used the promoter for the peptidase carboxypeptidase A3 (CPA3; originally named mast cell carboxypeptidase A 14 ). CPA3 is highly expressed in mast cells, 15 but is also expressed in basophils 16 and can be expressed in some populations of T-cell progenitors and thymic T cells 17,18 and in certain hematopoietic progenitor cells. 19 We generated C57BL/6 mice in which a segment of the Cpa3 promoter drives expression of Cre recombinase, and then mated these Cpa3-Cre transgenic mice to mice bearing a floxed allele of Mcl-1. 4 We found that C57BL/6-Cpa3-Cre; Mcl-1 fl/fl mice are severely deficient in mast cells and have a marked deficiency in basophils, and also exhibit striking impairment in mast cell-or basophil-and IgE-dependent biologic responses. Methods MiceAll animal experiments were carried out following protocols approved by the Stanford University Administrative Panel on Laboratory Animal Care. B6-Tg(Cpa3-cre)3Glli (Cpa3-Cre-transgenic mice) were generated by microinjecting the Cpa3-Cre transgene into embryonic stem cells in the B6 background (Stanford University). Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo /J(mT/mG) mice, obtained from The Jackson Laboratory, were crossed to Cpa3-Cre mice for Cre expression analysis. Mcl-1 ϩ/fl (B6;129-Mcl1 tm3sjk J) animals were obtained from The Jackson Laboratory. Mcl-1 ϩ/fl mice were bred to progeny from 2 Cpa3-Cre founder lines (founder lines #4 and #5) to obtain Cpa3-Cre; Mcl-1 ϩ/ϩ , Cpa3-Cre; Mcl-1 ϩ/fl , and Cpa3-Cre; Mcl-1 fl/fl animals, but only the Cpa3-Cre; Mcl-1 fl/fl mice derived from founder line #4 exhibited a substantial mast cell deficiency. Therefore, the mice used were derived from crosses between founder line #4 (subsequently referred to as Cpa3-Cre mice) and Mcl-1 fl animals, and these mice had been intercrossed a minimum of 6 generations into the C57BL/6 background. Heterozygous Cpa3-Cre mice were determined to have 5 copies of the Cpa3-Cre transgene by real-time PCR. To emphasize that Cpa3-Cre; Mcl-1 fl/fl mice have deficiencies in mast cells and basophils that are independent of mutations affecting Kit, we call them informally in our labo...

Deposits of Ig and complement are abundant in affected joints of patients with rheumatoid arthritis (RA) and in animal models of RA in which antibodies are demonstrably pathogenic. To identify molecular targets of the Igs deposited in arthritic joints, which may activate local inflammation, we used a combination of mass spectrometry (MS) and protein microarrays. Immune complexes were affinity-purified from surgically removed joint tissues of 26 RA and osteoarthritis (OA) patients. Proteins complexed with IgG were identified by proteomic analysis using tandem MS. A striking diversity of components of the extracellular matrix, and some intracellular components, copurified specifically with IgG from RA and OA tissues. A smaller set of autoantigens was observed only in RA eluates. In complementary experiments, IgG fractions purified from joint immune complexes were tested on protein microarrays against a range of candidate autoantigens. These Igs bound a diverse subset of proteins and peptides from synovium and cartilage, different from that bound by normal serum Ig. One type of intracellular protein detected specifically in RA joints (histones H2A/B) was validated by immunohistology and found to be deposited on the cartilage surface of RA but not OA joints. Thus, autoantibodies to many determinants (whether deposited as ''neoantigens'' or normal constituents of the extracellular matrix) have the potential to contribute to arthritic inflammation.autoantibodies ͉ histones ͉ mass spectrometry ͉ proteomics

IntroductionBasophils are the least prevalent of the granulocytes, generally representing less than 1% of leukocytes in the peripheral blood. Basophil studies have been hampered by the rarity of these cells and, until recently, the lack of tools such as basophil-deficient mice with which to assess their roles in vivo. However, recent studies have unveiled evidence for several previously unrecognized roles for basophils that are distinct from those of mast cells. [1][2][3][4][5][6][7][8][9][10][11] In addition to hampering investigations of basophil function, the small numbers of basophils and the paucity of tools for their analysis have made studies of basophil development challenging and therefore there have been few studies of this process. Arinobu et al showed that basophil lineage-restricted progenitors (BaPs) are identifiable in the BM and that the transcription factor CCAAT/ enhancer-binding protein-␣ (C/EBP␣) is important for the fate decision to develop into terminally differentiated basophils. 12 Ohmori et al reported that the IL-3-STAT5 axis is important for differentiating granulocyte-monocyte progenitors to BaPs, 13 and Siracusa et al showed that thymic stromal lymphopoietin (TSLP) can facilitate the development of BaPs into mature basophils. 8 Despite such progress, many of the details of the basophil differentiation pathway remain to be determined. For example, it is known that IL-3-deficient, 8,14,15 TSLP receptor (TSLPR)-deficient, 8 and IL-3/TSLPR double-deficient 8 mice have normal baseline numbers of basophils, indicating that other factors are more important in maintaining basophil levels at baseline. Moreover, C/EBP␣-deficient mice die within 8 hours of birth 16 and STAT5-deficient mice die in utero, 17 limiting the ability to use these animals to evaluate factors that might regulate basophil development at baseline in adult mice in vivo.Runt-related transcription factor (Runx) proteins are a family of transcription factors 18,19 that have crucial roles during the development of many tissues and the immune system. Each of the 3 kinds of Runx proteins, Runx1, Runx2, and Runx3,19,20 has distinct roles in development, with Runx1 being required for hematopoiesis, 18 Runx2 for osteogenesis, 21,22 and Runx3 for neurogenesis, thymopoiesis, and the control of gastric epithelial-cell proliferation. [23][24][25] Although a constitutive deficiency in Runx1 is embryonically lethal, studies of conditional Runx1-knockout mice have indicated that Runx1 can regulate the differentiation of hematopoietic stem cells (HSCs), B lymphocytes, natural killer T (NKT) cells, and T lymphocytes. 18,[26][27][28][29][30] Mx-Cre Runx1-knockout mice, which have an inducible Runx1 inactivation system, exhibit normal numbers of HSCs, a normal myeloid-cell (neutrophil) compartment, a severe reduction in megakaryocyte differentiation and platelet formation, and defects in B and T lymphocytes. 31 All 3 Runx genes can be transcribed from the distal (P1) or proximal (P2) promoters, 32 and P1-and P2-derived Runx1 variants differ i...

Systemic sclerosis (SSc) is an autoimmune disease in which the tyrosine kinases platelet-derived growth factor receptor (PDGFR) and Abl are hypothesized to contribute to the fibrosis and vasculopathy of the skin and internal organs. Herein we describe 2 patients with early diffuse cutaneous SSc (dcSSc) who experienced reductions in cutaneous sclerosis in response to therapy with the tyrosine kinase inhibitor imatinib mesylate. Immunohistochemical analyses of skin biopsy specimens demonstrated reductions of phosphorylated PDGFR␤ and Abl with imatinib therapy. By gene expression profiling, an imatinib-responsive signature specific to dcSSc was identified (P < 10 ؊8 ). The response of these patients and the findings of the analyses suggest that PDGFR␤ and Abl play critical, synergistic roles in the pathogenesis of SSc, and that imatinib targets a gene expression program that is frequently dysregulated in dcSSc.

Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the presence of anticitrullinated protein Abs, although the exact targets and role of anticitrullinated protein autoimmunity in the pathogenesis of RA remain to be defined. Fibrinogen, which can be citrullinated, has recently emerged as a candidate autoantigen. To determine whether autoimmunity against fibrinogen can mediate inflammatory arthritis, we immunized a variety of common mouse strains with fibrinogen and found that DBA/1 and SJL mice developed an inflammatory and erosive arthritis. Mice with fibrinogen-induced arthritis (FIA) possess fibrinogen-reactive T cells that produce the proinflammatory cytokines IL-6, IL-17, TNF-α, and IFN-γ. FIA can be adoptively transferred with either plasma or fibrinogen-specific T cells from diseased mice. Mice with FIA possess rheumatoid factor, circulating immune complexes, and anticyclic citrullinated peptide Abs, all of which are characteristic of human RA. These observations demonstrate that fibrinogen is arthritogenic in mice and that the pathogenesis of FIA is mediated by both autoantibodies and fibrinogen-reactive T cells.