Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition by soluble anti-α6 mAb S3-41 of  early kinases phosphorylation events activated via the  CD3–TCR complex. Jurkat cells at 20 × 106/ml in  RPMI 5% FCS were incubated with Abs at 37°C for 2.5  min, solubilized, and lysates were immunoprecipitated  with (a) a pAb anti–Zap 70, (b) a pAb anti-lck, (c) a  pAb anti-fyn. After SDS-PAGE separation and electrotransfer onto Immobilon P membrane, proteins  were incubated with HRP conjugated anti-phosphotyrosine Ab (APY) followed by ECL. For reprobing, the  membranes were submerged in stripping buffer,  blocked, and immunodetected with (a) pAb anti–Zap  70, (b) pAb anti-lck, (c) pAb anti-fyn was performed.  Reactions were revealed by incubating membranes  with GAR HRP followed by ECL. For densitometric  analysis of APY immunoblotting, control values were reduced to 1 in order to compare signals after activation. Values are mentioned  under each lane. Anti-integrin antibodies were added in soluble form at 10 μg/ml.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132916&req=5

Figure 10: Inhibition by soluble anti-α6 mAb S3-41 of early kinases phosphorylation events activated via the CD3–TCR complex. Jurkat cells at 20 × 106/ml in RPMI 5% FCS were incubated with Abs at 37°C for 2.5 min, solubilized, and lysates were immunoprecipitated with (a) a pAb anti–Zap 70, (b) a pAb anti-lck, (c) a pAb anti-fyn. After SDS-PAGE separation and electrotransfer onto Immobilon P membrane, proteins were incubated with HRP conjugated anti-phosphotyrosine Ab (APY) followed by ECL. For reprobing, the membranes were submerged in stripping buffer, blocked, and immunodetected with (a) pAb anti–Zap 70, (b) pAb anti-lck, (c) pAb anti-fyn was performed. Reactions were revealed by incubating membranes with GAR HRP followed by ECL. For densitometric analysis of APY immunoblotting, control values were reduced to 1 in order to compare signals after activation. Values are mentioned under each lane. Anti-integrin antibodies were added in soluble form at 10 μg/ml.

Mentions: We tested the effects of S3-41 and P1B5 mAbs, used in soluble form, on early kinase phosphorylation events (Fig. 10). Jurkat cells expressing α6β4 in similar density as CD3high thymocytes were activated with a CD3 mAb (×3, 10 μg/ml) during 2.5 min at 37°C, with or without soluble mAbs. Immunoprecipitation of Zap 70, p56lck, or p59fyn was carried out on activated cell lysates. Precipitated proteins were transferred onto Immobilon, then membranes were incubated with an anti-phosphotyrosine antibody coupled to peroxidase. Results were revealed by ECL. We observed that the addition of S3-41 (10 μg/ml) induced the same pattern of inhibition as soluble laminin 5 with inhibition of Zap 70 and p59fyn tyrosine phosphorylation (Fig. 10, a and c, respectively) but no inhibition of overall p56lck (Fig. 10 b). In contrast, addition of soluble P1B5 (10 μg/ml) did not induce this particular pattern of reactivity, supporting the hypothesis that α6β4 is the laminin 5 receptor involved.


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)

Inhibition by soluble anti-α6 mAb S3-41 of  early kinases phosphorylation events activated via the  CD3–TCR complex. Jurkat cells at 20 × 106/ml in  RPMI 5% FCS were incubated with Abs at 37°C for 2.5  min, solubilized, and lysates were immunoprecipitated  with (a) a pAb anti–Zap 70, (b) a pAb anti-lck, (c) a  pAb anti-fyn. After SDS-PAGE separation and electrotransfer onto Immobilon P membrane, proteins  were incubated with HRP conjugated anti-phosphotyrosine Ab (APY) followed by ECL. For reprobing, the  membranes were submerged in stripping buffer,  blocked, and immunodetected with (a) pAb anti–Zap  70, (b) pAb anti-lck, (c) pAb anti-fyn was performed.  Reactions were revealed by incubating membranes  with GAR HRP followed by ECL. For densitometric  analysis of APY immunoblotting, control values were reduced to 1 in order to compare signals after activation. Values are mentioned  under each lane. Anti-integrin antibodies were added in soluble form at 10 μg/ml.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Inhibition by soluble anti-α6 mAb S3-41 of early kinases phosphorylation events activated via the CD3–TCR complex. Jurkat cells at 20 × 106/ml in RPMI 5% FCS were incubated with Abs at 37°C for 2.5 min, solubilized, and lysates were immunoprecipitated with (a) a pAb anti–Zap 70, (b) a pAb anti-lck, (c) a pAb anti-fyn. After SDS-PAGE separation and electrotransfer onto Immobilon P membrane, proteins were incubated with HRP conjugated anti-phosphotyrosine Ab (APY) followed by ECL. For reprobing, the membranes were submerged in stripping buffer, blocked, and immunodetected with (a) pAb anti–Zap 70, (b) pAb anti-lck, (c) pAb anti-fyn was performed. Reactions were revealed by incubating membranes with GAR HRP followed by ECL. For densitometric analysis of APY immunoblotting, control values were reduced to 1 in order to compare signals after activation. Values are mentioned under each lane. Anti-integrin antibodies were added in soluble form at 10 μg/ml.
Mentions: We tested the effects of S3-41 and P1B5 mAbs, used in soluble form, on early kinase phosphorylation events (Fig. 10). Jurkat cells expressing α6β4 in similar density as CD3high thymocytes were activated with a CD3 mAb (×3, 10 μg/ml) during 2.5 min at 37°C, with or without soluble mAbs. Immunoprecipitation of Zap 70, p56lck, or p59fyn was carried out on activated cell lysates. Precipitated proteins were transferred onto Immobilon, then membranes were incubated with an anti-phosphotyrosine antibody coupled to peroxidase. Results were revealed by ECL. We observed that the addition of S3-41 (10 μg/ml) induced the same pattern of inhibition as soluble laminin 5 with inhibition of Zap 70 and p59fyn tyrosine phosphorylation (Fig. 10, a and c, respectively) but no inhibition of overall p56lck (Fig. 10 b). In contrast, addition of soluble P1B5 (10 μg/ml) did not induce this particular pattern of reactivity, supporting the hypothesis that α6β4 is the laminin 5 receptor involved.

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.

Show MeSH
Related in: MedlinePlus