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Transcriptional regulation of Munc13-4 expression in cytotoxic lymphocytes is disrupted by an intronic mutation associated with a primary immunodeficiency.

Cichocki F, Schlums H, Li H, Stache V, Holmes T, Lenvik TR, Chiang SC, Miller JS, Meeths M, Anderson SK, Bryceson YT - J. Exp. Med. (2014)

Bottom Line: The mechanisms regulating Munc13-4 expression are unknown.This mutation impairs UNC13D intron 1 recruitment of STAT4 and the chromatin remodeling complex component BRG1, diminishing active histone modifications at the locus.Thus, mutations associated with primary immunodeficiencies may cause disease by disrupting transcription factor binding.

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Affiliation: Centre for Infectious Medicine, Department of Medicine; Clinical Genetics Unit, Department of Molecular Medicine and Surgery, and Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden Division of Hematology, Oncology and Transplantation, University of Minnesota Cancer Center, Minneapolis, MN 55455.

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Munc13-4 expression levels correlate with lymphocyte cytotoxicity. (a) Western blot analysis of Munc13-4, Stx11, and Munc18-2 expression in eight different isolated subsets of human peripheral blood lymphocytes from a healthy peripheral blood donor. All cell types were isolated by negative magnetic bead selection except for CD56bright and CD56dim NK cells, which were FACS sorted. Munc13-4 was run on a separate Western blot gel from Stx11 and Munc18-2. Munc13-4 and β-actin for CD8+ T cell and NK cell subsets (rows 1 and 2 in columns 2 and 3) were run on the same Western blot gel as those shown for STAT4 in Figure 5 a (columns 2 and 3) and BRG1 in Figure 5 j (columns 2 and 3), accounting for the shared β-actin image. (b) Cumulative Munc13-4, Stx11, and Munc18-2 fold expression values relative to B cells, naive CD8+ T cells, or CD56bright NK cells from four healthy donors in three independent experiments. All values are normalized to β-actin. Fold expression differences with P ≤ 0.05 are marked with an asterisk.
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fig1: Munc13-4 expression levels correlate with lymphocyte cytotoxicity. (a) Western blot analysis of Munc13-4, Stx11, and Munc18-2 expression in eight different isolated subsets of human peripheral blood lymphocytes from a healthy peripheral blood donor. All cell types were isolated by negative magnetic bead selection except for CD56bright and CD56dim NK cells, which were FACS sorted. Munc13-4 was run on a separate Western blot gel from Stx11 and Munc18-2. Munc13-4 and β-actin for CD8+ T cell and NK cell subsets (rows 1 and 2 in columns 2 and 3) were run on the same Western blot gel as those shown for STAT4 in Figure 5 a (columns 2 and 3) and BRG1 in Figure 5 j (columns 2 and 3), accounting for the shared β-actin image. (b) Cumulative Munc13-4, Stx11, and Munc18-2 fold expression values relative to B cells, naive CD8+ T cells, or CD56bright NK cells from four healthy donors in three independent experiments. All values are normalized to β-actin. Fold expression differences with P ≤ 0.05 are marked with an asterisk.

Mentions: Understanding how Munc13-4 expression is controlled in distinct lymphocyte subsets is of interest considering the pivotal role of this protein for lymphocyte cytotoxicity (Feldmann et al., 2003). A ubiquitous Munc13-4 expression pattern was initially reported by Koch and colleagues (Koch et al., 2000), but a more detailed analysis has not since been performed. To compare Munc13-4 expression levels between cytotoxic and noncytotoxic lymphocytes, we isolated lymphocyte subsets from the peripheral blood of healthy donors and performed Western blots for Munc13-4. Relative to CD19+ B cells, CD3−CD56+ NK cells expressed very high levels of Munc13-4. Moreover, CD4+ T cells expressed low levels of Munc13-4, whereas total CD8+ T cells expressed intermediate levels. To determine whether Munc13-4 is concomitantly induced along with the acquisition of lytic granules, we compared expression in CD56bright and CD56dim NK cells as well as in naive and effector CD8+ T cells. In both NK cells and CD8+ T cells, maturation and acquisition of lytic granules was associated with a marked increase in Munc13-4 expression, indicating that Munc13-4 is up-regulated during differentiation of cytotoxic lymphocyte subsets (Fig. 1, a and b). High levels of Munc13-4 expression thus correlate with a strong propensity to degranulate in response to engagement of activating receptors on cytotoxic T cells or NK cells (Chiang, et al., 2013). Notably, expression levels of syntaxin-11 and Munc18-2, also encoded by genes associated with FHL, were less correlated with cytotoxicity in lymphocyte subsets (Fig. 1, a and b). Thus, the data suggest an important role for induction of Munc13-4 expression in regulating the cytotoxic capacity of lymphocytes.


Transcriptional regulation of Munc13-4 expression in cytotoxic lymphocytes is disrupted by an intronic mutation associated with a primary immunodeficiency.

Cichocki F, Schlums H, Li H, Stache V, Holmes T, Lenvik TR, Chiang SC, Miller JS, Meeths M, Anderson SK, Bryceson YT - J. Exp. Med. (2014)

Munc13-4 expression levels correlate with lymphocyte cytotoxicity. (a) Western blot analysis of Munc13-4, Stx11, and Munc18-2 expression in eight different isolated subsets of human peripheral blood lymphocytes from a healthy peripheral blood donor. All cell types were isolated by negative magnetic bead selection except for CD56bright and CD56dim NK cells, which were FACS sorted. Munc13-4 was run on a separate Western blot gel from Stx11 and Munc18-2. Munc13-4 and β-actin for CD8+ T cell and NK cell subsets (rows 1 and 2 in columns 2 and 3) were run on the same Western blot gel as those shown for STAT4 in Figure 5 a (columns 2 and 3) and BRG1 in Figure 5 j (columns 2 and 3), accounting for the shared β-actin image. (b) Cumulative Munc13-4, Stx11, and Munc18-2 fold expression values relative to B cells, naive CD8+ T cells, or CD56bright NK cells from four healthy donors in three independent experiments. All values are normalized to β-actin. Fold expression differences with P ≤ 0.05 are marked with an asterisk.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4042637&req=5

fig1: Munc13-4 expression levels correlate with lymphocyte cytotoxicity. (a) Western blot analysis of Munc13-4, Stx11, and Munc18-2 expression in eight different isolated subsets of human peripheral blood lymphocytes from a healthy peripheral blood donor. All cell types were isolated by negative magnetic bead selection except for CD56bright and CD56dim NK cells, which were FACS sorted. Munc13-4 was run on a separate Western blot gel from Stx11 and Munc18-2. Munc13-4 and β-actin for CD8+ T cell and NK cell subsets (rows 1 and 2 in columns 2 and 3) were run on the same Western blot gel as those shown for STAT4 in Figure 5 a (columns 2 and 3) and BRG1 in Figure 5 j (columns 2 and 3), accounting for the shared β-actin image. (b) Cumulative Munc13-4, Stx11, and Munc18-2 fold expression values relative to B cells, naive CD8+ T cells, or CD56bright NK cells from four healthy donors in three independent experiments. All values are normalized to β-actin. Fold expression differences with P ≤ 0.05 are marked with an asterisk.
Mentions: Understanding how Munc13-4 expression is controlled in distinct lymphocyte subsets is of interest considering the pivotal role of this protein for lymphocyte cytotoxicity (Feldmann et al., 2003). A ubiquitous Munc13-4 expression pattern was initially reported by Koch and colleagues (Koch et al., 2000), but a more detailed analysis has not since been performed. To compare Munc13-4 expression levels between cytotoxic and noncytotoxic lymphocytes, we isolated lymphocyte subsets from the peripheral blood of healthy donors and performed Western blots for Munc13-4. Relative to CD19+ B cells, CD3−CD56+ NK cells expressed very high levels of Munc13-4. Moreover, CD4+ T cells expressed low levels of Munc13-4, whereas total CD8+ T cells expressed intermediate levels. To determine whether Munc13-4 is concomitantly induced along with the acquisition of lytic granules, we compared expression in CD56bright and CD56dim NK cells as well as in naive and effector CD8+ T cells. In both NK cells and CD8+ T cells, maturation and acquisition of lytic granules was associated with a marked increase in Munc13-4 expression, indicating that Munc13-4 is up-regulated during differentiation of cytotoxic lymphocyte subsets (Fig. 1, a and b). High levels of Munc13-4 expression thus correlate with a strong propensity to degranulate in response to engagement of activating receptors on cytotoxic T cells or NK cells (Chiang, et al., 2013). Notably, expression levels of syntaxin-11 and Munc18-2, also encoded by genes associated with FHL, were less correlated with cytotoxicity in lymphocyte subsets (Fig. 1, a and b). Thus, the data suggest an important role for induction of Munc13-4 expression in regulating the cytotoxic capacity of lymphocytes.

Bottom Line: The mechanisms regulating Munc13-4 expression are unknown.This mutation impairs UNC13D intron 1 recruitment of STAT4 and the chromatin remodeling complex component BRG1, diminishing active histone modifications at the locus.Thus, mutations associated with primary immunodeficiencies may cause disease by disrupting transcription factor binding.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Infectious Medicine, Department of Medicine; Clinical Genetics Unit, Department of Molecular Medicine and Surgery, and Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden Division of Hematology, Oncology and Transplantation, University of Minnesota Cancer Center, Minneapolis, MN 55455.

Show MeSH
Related in: MedlinePlus