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Laminin 5 in the human thymus: control of T cell proliferation via alpha6beta4 integrins.

Vivinus-Nebot M, Ticchioni M, Mary F, Hofman P, Quaranta V, Rousselle P, Bernard A - J. Cell Biol. (1999)

Bottom Line: In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures.We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect.This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck).

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Sant¿e et de la Recherche M¿edicale, U343, Nice 06202, France.

ABSTRACT
Laminin 5 (alpha3beta3gamma2) distribution in the human thymus was investigated by immunofluorescence on frozen sections with anti-alpha3, -beta3, and -gamma2 mAbs. In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures. We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect. By contrast, soluble laminin 5 inhibits thymocyte proliferation induced by a TCR signal. This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck). Using a mAb specific for alpha6beta4 integrins, we observed that while alpha3beta1 are known to be uniformly present on all thymocytes, alpha6beta4 expression parallels thymocyte maturation; thus a correspondence exists between laminin 5 in the thymic medulla and alpha6beta4 on mature thymocytes. Moreover, the soluble Ab against alpha6beta4 inhibits thymocyte proliferation and reproduces the same pattern of tyrosine kinase phosphorylation suggesting that alpha6beta4 is involved in laminin 5-induced modulation of T cell activation.

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Frozen human thymus section stained with anti-laminin 5 α3 chain mAb  BM165. (a and b) BM165  staining on vessels; staining  was revealed by FITC conjugated goat anti–mouse  F(ab)′2 fragments and analysis was performed by laser  scanning confocal microscopy. Cell nuclei were  stained with propidium iodide. (c and d) BM165 staining on stellate thymic epithelial cells; double staining  obtained with mAb BM165  revealed by rabbit anti– mouse IgG1 F(ab)′2 fragments followed by PE conjugated goat anti–rabbit  F(ab)′2 fragments, and the  anti-keratin CK19 revealed  by FITC conjugated goat  anti–mouse IgM Ab. Controls made with secondary  Abs gave no staining (not  shown).
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Figure 2: Frozen human thymus section stained with anti-laminin 5 α3 chain mAb BM165. (a and b) BM165 staining on vessels; staining was revealed by FITC conjugated goat anti–mouse F(ab)′2 fragments and analysis was performed by laser scanning confocal microscopy. Cell nuclei were stained with propidium iodide. (c and d) BM165 staining on stellate thymic epithelial cells; double staining obtained with mAb BM165 revealed by rabbit anti– mouse IgG1 F(ab)′2 fragments followed by PE conjugated goat anti–rabbit F(ab)′2 fragments, and the anti-keratin CK19 revealed by FITC conjugated goat anti–mouse IgM Ab. Controls made with secondary Abs gave no staining (not shown).

Mentions: We investigated laminin 5 (α3β3γ2) expression in the human thymus by immunofluorescence, using mAbs specific for the α3 chain (BM165 [49]; Fig. 1, a and b), for the β3 chain (6F12 [33]; Fig. 1 c), or for the γ2 chain (GB3, [35]; Fig. 1 d). Staining of laminin 5 was performed together with a nuclear counter-staining with propidium iodide (Fig. 1, a–d; Fig. 2, a and b) in order to recognize the cortical (C) and the medullary (M) regions. All three mAbs stained strongly the medullary area of parenchyma and the basal laminae from subcapsular cortex, consistent with the presence of laminin 5 in these areas. More precisely, laminin 5 was localized in stellate keratin positive epithelial cells (Fig. 2, c and d) and in the basal laminae of vessels, including small vessels and capillary structures, together with endothelial cells of the larger vessels (Fig. 1 b, arrows; Fig. 2, a and b). Of note, a more intense staining at the corticomedullary junction was only visible with the anti-α3 mAb BM165, suggesting that laminin 6 (α3β1γ1) and/or 7 (α3β2γ1) could be associated with laminin 5 in this area. As to the cortex, mAbs stained very scattered structures that corresponded for the most part to vascular structures.


Laminin 5 in the human thymus: control of T cell proliferation via alpha6beta4 integrins.

Vivinus-Nebot M, Ticchioni M, Mary F, Hofman P, Quaranta V, Rousselle P, Bernard A - J. Cell Biol. (1999)

Frozen human thymus section stained with anti-laminin 5 α3 chain mAb  BM165. (a and b) BM165  staining on vessels; staining  was revealed by FITC conjugated goat anti–mouse  F(ab)′2 fragments and analysis was performed by laser  scanning confocal microscopy. Cell nuclei were  stained with propidium iodide. (c and d) BM165 staining on stellate thymic epithelial cells; double staining  obtained with mAb BM165  revealed by rabbit anti– mouse IgG1 F(ab)′2 fragments followed by PE conjugated goat anti–rabbit  F(ab)′2 fragments, and the  anti-keratin CK19 revealed  by FITC conjugated goat  anti–mouse IgM Ab. Controls made with secondary  Abs gave no staining (not  shown).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132916&req=5

Figure 2: Frozen human thymus section stained with anti-laminin 5 α3 chain mAb BM165. (a and b) BM165 staining on vessels; staining was revealed by FITC conjugated goat anti–mouse F(ab)′2 fragments and analysis was performed by laser scanning confocal microscopy. Cell nuclei were stained with propidium iodide. (c and d) BM165 staining on stellate thymic epithelial cells; double staining obtained with mAb BM165 revealed by rabbit anti– mouse IgG1 F(ab)′2 fragments followed by PE conjugated goat anti–rabbit F(ab)′2 fragments, and the anti-keratin CK19 revealed by FITC conjugated goat anti–mouse IgM Ab. Controls made with secondary Abs gave no staining (not shown).
Mentions: We investigated laminin 5 (α3β3γ2) expression in the human thymus by immunofluorescence, using mAbs specific for the α3 chain (BM165 [49]; Fig. 1, a and b), for the β3 chain (6F12 [33]; Fig. 1 c), or for the γ2 chain (GB3, [35]; Fig. 1 d). Staining of laminin 5 was performed together with a nuclear counter-staining with propidium iodide (Fig. 1, a–d; Fig. 2, a and b) in order to recognize the cortical (C) and the medullary (M) regions. All three mAbs stained strongly the medullary area of parenchyma and the basal laminae from subcapsular cortex, consistent with the presence of laminin 5 in these areas. More precisely, laminin 5 was localized in stellate keratin positive epithelial cells (Fig. 2, c and d) and in the basal laminae of vessels, including small vessels and capillary structures, together with endothelial cells of the larger vessels (Fig. 1 b, arrows; Fig. 2, a and b). Of note, a more intense staining at the corticomedullary junction was only visible with the anti-α3 mAb BM165, suggesting that laminin 6 (α3β1γ1) and/or 7 (α3β2γ1) could be associated with laminin 5 in this area. As to the cortex, mAbs stained very scattered structures that corresponded for the most part to vascular structures.

Bottom Line: In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures.We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect.This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck).

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Sant¿e et de la Recherche M¿edicale, U343, Nice 06202, France.

ABSTRACT
Laminin 5 (alpha3beta3gamma2) distribution in the human thymus was investigated by immunofluorescence on frozen sections with anti-alpha3, -beta3, and -gamma2 mAbs. In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures. We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect. By contrast, soluble laminin 5 inhibits thymocyte proliferation induced by a TCR signal. This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck). Using a mAb specific for alpha6beta4 integrins, we observed that while alpha3beta1 are known to be uniformly present on all thymocytes, alpha6beta4 expression parallels thymocyte maturation; thus a correspondence exists between laminin 5 in the thymic medulla and alpha6beta4 on mature thymocytes. Moreover, the soluble Ab against alpha6beta4 inhibits thymocyte proliferation and reproduces the same pattern of tyrosine kinase phosphorylation suggesting that alpha6beta4 is involved in laminin 5-induced modulation of T cell activation.

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