Uncovering the mechanism of IL-4-mediated T cell survival

Although IL-4 is a well-characterized multi-functional cytokine, its role as a survival factor in T cells is not completely understood. In an attempt to uncover IL-4-mediated survival, we studied caspase activation in primary mouse T lymphocytes undergoing death by neglect, activation-induced or steroid-induced cell death. Here, we identify the executioner caspases-3, and -6, and, to a lesser extent, caspase-4 to be involved in the apoptotic process of T cells in our model, as demonstrated by a peptide inhibition assay. Furthermore, we show that IL-4 blocks caspase-3 activation in T cells undergoing all three forms of apoptosis. Western blot and flow cytometric analysis showed a substantial decrease in dexamethasoneinduced and death by neglect-induced caspase-3 activation upon IL-4 treatment, indicating IL-4 to be an important modulator of caspase-3 activity. Since a major pathway leading to caspase activation involves mitochondrial disruption and/or the release of pro-apoptotic proteins, we analyzed mitochondrial membrane potential (ΔΨₘ) in T cells undergoing various forms of apoptosis in an attempt to define the mechanism(s) by which IL-4 blocks caspase-3 activation. IL-4 treatment resulted in a reduction of mitochondrial disruption, as defined by ΔΨₘ. Moreover, in T cells derived from B ax-deficient mice, it appeared that IL-4-mediated survival involved a mechanism that does not include the modulation of the pro-apototic Bax protein. These data suggest that IL-4-mediated T cell survival involves the blockage of caspase-3 activation through a mechanism that relies on the maintenance of ΔΨₘ

WARP interacts with collagen VI-containing microfibrils in the pericellular matrix of human chondrocytes.

Collagen VI and WARP are extracellular structural macromolecules present in cartilage and associated with BM suprastructures in non-skeletal tissues. We have previously shown that in WARP-deficient mice, collagen VI is specifically reduced in regions of the peripheral nerve ECM where WARP is expressed, suggesting that both macromolecules are part of the same suprastructure. The object of this study was to conduct a detailed analysis of WARP-collagen VI interactions in vitro in cartilage, a tissue rich in WARP and collagen VI. Immunohistochemical analysis of mouse and human articular cartilage showed that WARP and collagen VI co-localize in the pericellular matrix of superficial zone articular chondrocytes. EM analysis on extracts of human articular cartilage showed that WARP associates closely with collagen VI-containing suprastructures. Additional evidence of an interaction is provided by immunogold EM and immunoblot analysis showing that WARP was present in collagen VI-containing networks isolated from cartilage. Further characterization were done by solid phase binding studies and reconstitution experiments using purified recombinant WARP and isolated collagen VI. Collagen VI binds to WARP with an apparent K(d) of approximately 22 nM and the binding site(s) for WARP resides within the triple helical domain since WARP binds to both intact collagen VI tetramers and pepsinized collagen VI. Together, these data confirm and extend our previous findings by demonstrating that WARP and collagen VI form high affinity associations in vivo in cartilage. We conclude that WARP is ideally placed to function as an adapter protein in the cartilage pericellular matrix

Localization of WARP and collagen VI using electron microscopy.

<p>EM analysis on native supramolecular fragments isolated from human articular cartilage. Sheep antisera against WARP (18-nm gold particles; black arrow heads) (panels a–c) and a rabbit polyclonal anti-collagen VI (panels a and b) or monoclonal antibody against collagen VI (panel c) (12-nm gold particles; white arrowheads) was used. A secondary antibodies only control is shown in panel d. Scale bars: 100 nm.</p

Solid phase analysis of WARP/collagen VI interaction.

<p>a, WARP binding to collagen VI tetramers. Coated collagen VI was incubated with recombinant WARP. Data are means of triplicate determinations +/− SE. <i>Inset</i>, Scatchard analysis of binding data reveals an apparent kDa of 22 nM. b, Reciprocal solid phase binding experiment showing collagen VI binding to coated WARP dimer but not WARP multimers. Representative curves are shown. c, WARP binds to both pepsinized collagen VI tetramers (black diamonds) and pepsinized intact collagen VI (white squares).</p

Collagen VI suprastructure analysis.

<p>A, Collagen VI suprastructures were isolated from human articular cartilage extracts using superparamagnetic immunobeads coupled to collagen VI antibodies. The isolated suprastructures were then doubly immuno-labeled with sheep anti-WARP antiserum (18-nm gold particles; black arrowheads) and rabbit anti-collagen VI antibody (small gold particles; white arrowheads). WARP is present in collagen VI suprastructures. Scale bar is 100 nm.</p

WARP binds to collagen VI <i>in vitro</i>.

<p>Isolated collagen VI microfibrils (shown in a) were mixed with recombinant WARP. Globular structures representing WARP are marked with arrowheads and visualized by negative staining (shown in b). Biotinylated recombinant WARP was visualized by gold labeled streptavidin as shown in panel c (5-nm gold particles). WARP is present on the collagen VI microfibrils (panel d) visible as structures close to the globular domains of the collagen VI microfibrils (bound WARP; black arrowheads). These structures are absent in the control experiments where WARP is omitted (see a). Biotinylated recombinant WARP (5-nm gold particles) bound near the junction between helical and globular domains of collagen VI microfibrils (panels e–g). Scale bars: 100 nm in a, b and d, 50 nm in e, 25 nm in c and f, and 10 nm in g.</p

Representative ultrathin sections of articular cartilage of adult mice co-stained for WARP (18-nm gold particles; black arrow heads) and collagen VI (12-nm gold particles; white arrowheads).

<p>Scale bars: 100 nm.</p

Analysis of collagen VI, WARP and perlecan in human articular cartilage.

<p>Immuno-gold EM was conducted on native supramolecular fragments isolated from human articular cartilage for WARP (18-nm gold particles; black arrowheads), perlecan (12-nm gold particles; white arrowheads) and collagen VI (6-nm gold particles, arrows). All three components are part of complexes at the suprastructural level (shown in a). The magnified image (shown in b) shows these complexes in close contact to banded collagen fibrils. Scale bars: 100 nm (panel a) and 200 nm (panel b).</p

Immunohistochemical localization of WARP and collagen VI in articular cartilage.

<p>A, Superficial zone human articular cartilage was stained for collagen VI (panels a, d and g) and WARP (panels b, e and h). The merged images show clear co-localization of collagen VI and WARP in the pericellular environment (panels c, f and i). The scale bar shown in panel (a) is 100 µm. B, Mouse tibial articular cartilage stained with collagen VI (panels a and d) and WARP antisera (panels b and e). Merged images show co-localization of collagen VI and WARP in the chondrocyte pericellular matrix (panels c and f). Scale bar in panel (a) is 200 µm and in (d) 50 µm.</p