Desensitization of delayed-type hypersensitivity in mice: suppressive environment

The systemic injection of high doses of antigen into a preimmunized animal results in transient unresponsiveness of cell-mediated immune responses. This phenomenon is known as desensitization. Serum interleukin 2 (IL-2) activity was found transiently in desensitized mice at 3 h after the antigen challenge. These mice could not reveal antigen nonspecific delayed-type hypersensitivity (DTH) 1 d after the challenge. Specific suppression of DTH was observed at later stages. Sera from 3 h desensitized mice showed suppressive effects on DTH in preo immunized mice. Administration of recombinant IL-2 into preimmunized mice led to the failure of development of DTH to antigens. These observations suggest that IL-2 plays an important role in the suppressive environment

Identification of SCAN domain zinc-finger gene ZNF449 as a novel factor of chondrogenesis.

Transcription factors SOX9, SOX5 and SOX6 are indispensable for generation and differentiation of chondrocytes. However, molecular mechanisms to induce the SOX genes are poorly understood. To address this issue, we previously determined the human embryonic enhancer of SOX6 by 5'RACE analysis, and identified the 46-bp core enhancer region (CES6). We initially performed yeast one-hybrid assay for screening other chondrogenic factors using CES6 as bait, and identified a zinc finger protein ZNF449. ZNF449 and Zfp449, a counterpart in mouse, transactivated enhancers or promoters of SOX6, SOX9 and COL2A1. Zfp449 was expressed in mesenchyme-derived tissues including cartilage, calvaria, muscle and tendon, as well as in other tissues including brain, lung and kidney. In limb cartilage of mouse embryo, Zfp449 protein was abundantly located in periarticular chondrocytes, and decreased in accordance with the differentiation. Zfp449 protein was also detected in articular cartilage of an adult mouse. During chondrogenic differentiation of human mesenchymal stem cells, ZNF449 was increased at an early stage, and its overexpression enhanced SOX9 and SOX6 only at the initial stage of the differentiation. We further generated Zfp449 knockout mice to examine the in vivo roles; however, no obvious abnormality was observed in skeletal development or articular cartilage homeostasis. ZNF449 may regulate chondrogenic differentiation from mesenchymal progenitor cells, although the underlying mechanisms are still unknown

Histological analyses of <i>Zfp449</i> knockout mice.

<p>(A) Safranin-O staining and immunofluorescence of Sox9, Sox6, Col2a1 and Zfp449 in proximal tibia of <i>Zfp449</i> null (−/−) and WT littermate embryos (+/+) (E18.5). Enlarged immunofluorescence figures on the left are from the areas indicated in the yellow inset box on Safranin-O staining. The further magnified views on the right are from the areas indicated in the blue inset boxes. Scale bars, 100 µm (Safranin-O stainings and immunofluorescence images), 10 µm (maginified views of immunofluorescence). (B) mRNA levels of <i>Sox9</i>, <i>Sox6</i>, <i>Col2a1</i> and <i>Zfp449</i> in primary chondrocytes from <i>Zfp449</i> null and WT littermate mice (6 d). Data are expressed as means (bars) ± SDs (error bars) for three wells/group. ^#<i>P</i><0.01 versus WT. (C) Safranin-O staining of knee joints in <i>Zfp449</i> null mice and WT littermates 8 weeks after the surgical induction of OA. Scale bars, 100 µm. (D) Safranin-O staining of knee joints in 17-month-old <i>Zfp449</i> null mice and WT littermates. Scale bars, 100 µm. (E) Immunofluorescence of GFP and Zfp449 in elbow joints of <i>Zfp449</i> heterozygous mutant. Scale bars, 200 µm.</p

Enhancement of chondrogenic differentiation of hMSCs by ZNF449.

<p>(A) mRNA levels of <i>ZNF449, SOX9, SOX6 and COL2A1</i> during chondrogenic differentiation of hMSCs. Data are expressed as means (bars) ± SDs (error bars) for three wells/group. *<i>P</i><0.05, ^#<i>P</i><0.01 versus 0 d. (B) mRNA levels of <i>SOX9, SOX6 and COL2A1</i> during chondrogenic differentiation of hMSCs transduced with GFP or ZNF449 by adenovirus. Adenoviral transduction was performed 2 days before the differentiation. Data are expressed as means (bars) ± SDs (error bars) for three wells/group. *<i>P</i><0.05 versus GFP. (C) Protein levels of the transduced ZNF449 (HA) and Actin during the differentiation of hMSC.</p

Expression pattern of Zfp449 in mouse tissues and cartilage.

<p>(A) mRNA level of <i>Zfp449</i> in mouse tissues. Data are expressed as means (bars) ± SDs (error bars) for three wells/group. (B) Immunofluorescence of Zfp449 in limb cartilage of mouse embryo. Inset boxes in the top panel indicate the regions of the bottom three rows representing periarticular (Per) zone, proliferative (Pro) zone, and hypertrophic (Hyp) zone. Scale bars, 100 µm (top), 20 µm (bottom). (C) Immunofluorescence of Zfp449 in articular cartilage of 8-week-old mouse knee joint. Inset box in middle panel indicates the regions of the bottom three rows. Scale bars, 100 µm (top and middle), 10 µm (bottom).</p

Generation of <i>Zfp449</i> knockout mice.

<p>(A) Schematic representation of the <i>Zfp449</i> locus and engineering of a <i>Zfp449</i> null allele by knocking in EGFP. UTR, untranslated region. CDS, coding sequence. NeoR, neomycin resistance gene. (B) Gross appearance of <i>Zfp449</i> null mice and WT littermate embryo (E18.5, female). Scale bars, 1 mm. (C) Double staining with Alizarin red and Alcian blue of the whole skeleton (left), upper extremities (right-top), and lower extremities (right-bottom) of <i>Zfp449</i> null mice and WT littermate embryo (E18.5, female). Scale bars, 1 mm. (D) Growth curves of <i>Zfp449</i> knockout littermates (+ and − for male, and +/+, +/−, and −/− for female). Height indicates nose-anus length. *<i>P</i><0.05 versus GFP.</p

Transactivation of proximal promoters and/or enhancers of chondrocyte marker genes by ZNF449 (human) and Zfp449 (mouse).

<p>(A) Luciferase activities by the transfections of GFP, ZNF449 and Zfp449 into HeLa and SW1353 cells with a reporter construct containing the indicated fragment. Each luciferase activity is presented as fold increase relative to GFP control. Data are expressed as means (bars) ± SDs (error bars) for three wells/group. *<i>P</i><0.05, ^#<i>P</i><0.01 versus GFP. (B) ChIP assay with cell lysates of SW1353 cells transfected with HA-tagged ZNF449. PCR was performed with a primer set spanning the CES6 (−177 to −86 bp) or not spanning the CES6 (−2,629 to −2,505 bp) before (INPUT) and after immunoprecipitation with anti-HA (HA) or non-immune IgG (IgG).</p