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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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(a) Sequential immunoprecipitations of α6, β1, or β4  chains from HT29 colon carcinoma cell line after anti-β1 or anti-β4 immunoprecipitations (IP). (Left) IP with anti-β1 mAb K20,  anti-α6 mAb S3-41, anti-α6 mAb GOH3, and anti-β4 mAb  3E1F6 were performed on total cell lysates. (Middle) To deplete  lysates of β1 integrins, a first IP with the anti-β1 mAb K20 was  performed on cell lysates and a second IP was then carried out on  the supernatants using the same mAb. Finally, a third immunoprecipitation was carried out on the supernatants with the indicated mAbs. (Right) To deplete lysates of α6β4, a first IP with the  anti-β4 mAb 3E1F6 was performed on cell lysates and a second  IP was then performed on the supernatants using the same mAb.  Finally, a third immunoprecipitation was performed on the supernatants with the indicated mAbs. Controls showed that the  material recovered was efficiently depleted in β1 (middle panel,  lane 1) or β4 chains (right panel, lane 4), respectively. Immunoprecipitated proteins were separated on SDS-PAGE 7.5%, under  reducing conditions. Results were revealed by autoradiography.  (b) Immunoblots with the anti-α6 mAb S3-41 were performed on  immunoprecipitates obtained with mAbs K20 (anti-β1), 3E1F6  (anti-β4) and GOH3 (anti-α6) on lysates from (left) HT29 cells  that express α6β4 at higher density than α6β1 or (right) K562 cells  that express α6β1 at higher density than α6β4. Immunoprecipitated proteins were separated on SDS-PAGE 7.5%, under reducing conditions before blotting. Immunolabeling was revealed by  chemiluminescence (ECL) after reaction with goat anti–mouse  immunoglobulins conjugated with HRP.
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Figure 7: (a) Sequential immunoprecipitations of α6, β1, or β4 chains from HT29 colon carcinoma cell line after anti-β1 or anti-β4 immunoprecipitations (IP). (Left) IP with anti-β1 mAb K20, anti-α6 mAb S3-41, anti-α6 mAb GOH3, and anti-β4 mAb 3E1F6 were performed on total cell lysates. (Middle) To deplete lysates of β1 integrins, a first IP with the anti-β1 mAb K20 was performed on cell lysates and a second IP was then carried out on the supernatants using the same mAb. Finally, a third immunoprecipitation was carried out on the supernatants with the indicated mAbs. (Right) To deplete lysates of α6β4, a first IP with the anti-β4 mAb 3E1F6 was performed on cell lysates and a second IP was then performed on the supernatants using the same mAb. Finally, a third immunoprecipitation was performed on the supernatants with the indicated mAbs. Controls showed that the material recovered was efficiently depleted in β1 (middle panel, lane 1) or β4 chains (right panel, lane 4), respectively. Immunoprecipitated proteins were separated on SDS-PAGE 7.5%, under reducing conditions. Results were revealed by autoradiography. (b) Immunoblots with the anti-α6 mAb S3-41 were performed on immunoprecipitates obtained with mAbs K20 (anti-β1), 3E1F6 (anti-β4) and GOH3 (anti-α6) on lysates from (left) HT29 cells that express α6β4 at higher density than α6β1 or (right) K562 cells that express α6β1 at higher density than α6β4. Immunoprecipitated proteins were separated on SDS-PAGE 7.5%, under reducing conditions before blotting. Immunolabeling was revealed by chemiluminescence (ECL) after reaction with goat anti–mouse immunoglobulins conjugated with HRP.

Mentions: To confirm that S3-41 would react only with the α6 chains linked to β4, we performed sequential immunoprecipitations using the colon carcinoma cell line HT29, which expresses high densities of both α6β1 and α6β4. Cells were lysed with 1% NP-40 and lysates were depleted of β1 or β4 integrins by two successive immunoprecipitations either with the anti-β1 mAb K20 or the anti-β4 mAb 3E1F6. Depleted lysates were then immunoprecipitated with either the anti-α6 mAbs S3-41 or GOH3 (Fig. 7 a). It can be seen that GOH3 was still able to immunoprecipitate residual material when lysates were depleted of either β1 or β4 integrins (Fig. 7 a, middle, lane 3; right, lane 3). In contrast, S3-41 did not precipitate any residual material when lysates were depleted of β4 integrins (Fig. 7 a, right, lane 2) whereas it remained fully reactive when lysates were depleted of β1 integrins (Fig. 7 a, middle, lane 2). Thus, if GOH3 immunoprecipitates α6 chains associated with either β1 or β4 chains, S3-41 recognizes only α6 chains paired with β4. These results were confirmed by immunoblotting experiments with S3-41. Cell lysates were first immunoprecipitated either with the anti-β1 mAb K20 or the anti-β4 3E1F6. Fig. 7 b shows that S3-41 recognized only the α6 chains that coimmunoprecipitated with β4. Note that these experiments were performed on two distinct cell lines that expressed different relative amounts of α6β1 and α6β4 (Fig. 7 b, left and right). Taken together, these experiments demonstrate that the anti-α6 mAb S3-41 reacts with an epitope present only when α6 chains are linked to β4 chains.


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)

(a) Sequential immunoprecipitations of α6, β1, or β4  chains from HT29 colon carcinoma cell line after anti-β1 or anti-β4 immunoprecipitations (IP). (Left) IP with anti-β1 mAb K20,  anti-α6 mAb S3-41, anti-α6 mAb GOH3, and anti-β4 mAb  3E1F6 were performed on total cell lysates. (Middle) To deplete  lysates of β1 integrins, a first IP with the anti-β1 mAb K20 was  performed on cell lysates and a second IP was then carried out on  the supernatants using the same mAb. Finally, a third immunoprecipitation was carried out on the supernatants with the indicated mAbs. (Right) To deplete lysates of α6β4, a first IP with the  anti-β4 mAb 3E1F6 was performed on cell lysates and a second  IP was then performed on the supernatants using the same mAb.  Finally, a third immunoprecipitation was performed on the supernatants with the indicated mAbs. Controls showed that the  material recovered was efficiently depleted in β1 (middle panel,  lane 1) or β4 chains (right panel, lane 4), respectively. Immunoprecipitated proteins were separated on SDS-PAGE 7.5%, under  reducing conditions. Results were revealed by autoradiography.  (b) Immunoblots with the anti-α6 mAb S3-41 were performed on  immunoprecipitates obtained with mAbs K20 (anti-β1), 3E1F6  (anti-β4) and GOH3 (anti-α6) on lysates from (left) HT29 cells  that express α6β4 at higher density than α6β1 or (right) K562 cells  that express α6β1 at higher density than α6β4. Immunoprecipitated proteins were separated on SDS-PAGE 7.5%, under reducing conditions before blotting. Immunolabeling was revealed by  chemiluminescence (ECL) after reaction with goat anti–mouse  immunoglobulins conjugated with HRP.
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Figure 7: (a) Sequential immunoprecipitations of α6, β1, or β4 chains from HT29 colon carcinoma cell line after anti-β1 or anti-β4 immunoprecipitations (IP). (Left) IP with anti-β1 mAb K20, anti-α6 mAb S3-41, anti-α6 mAb GOH3, and anti-β4 mAb 3E1F6 were performed on total cell lysates. (Middle) To deplete lysates of β1 integrins, a first IP with the anti-β1 mAb K20 was performed on cell lysates and a second IP was then carried out on the supernatants using the same mAb. Finally, a third immunoprecipitation was carried out on the supernatants with the indicated mAbs. (Right) To deplete lysates of α6β4, a first IP with the anti-β4 mAb 3E1F6 was performed on cell lysates and a second IP was then performed on the supernatants using the same mAb. Finally, a third immunoprecipitation was performed on the supernatants with the indicated mAbs. Controls showed that the material recovered was efficiently depleted in β1 (middle panel, lane 1) or β4 chains (right panel, lane 4), respectively. Immunoprecipitated proteins were separated on SDS-PAGE 7.5%, under reducing conditions. Results were revealed by autoradiography. (b) Immunoblots with the anti-α6 mAb S3-41 were performed on immunoprecipitates obtained with mAbs K20 (anti-β1), 3E1F6 (anti-β4) and GOH3 (anti-α6) on lysates from (left) HT29 cells that express α6β4 at higher density than α6β1 or (right) K562 cells that express α6β1 at higher density than α6β4. Immunoprecipitated proteins were separated on SDS-PAGE 7.5%, under reducing conditions before blotting. Immunolabeling was revealed by chemiluminescence (ECL) after reaction with goat anti–mouse immunoglobulins conjugated with HRP.
Mentions: To confirm that S3-41 would react only with the α6 chains linked to β4, we performed sequential immunoprecipitations using the colon carcinoma cell line HT29, which expresses high densities of both α6β1 and α6β4. Cells were lysed with 1% NP-40 and lysates were depleted of β1 or β4 integrins by two successive immunoprecipitations either with the anti-β1 mAb K20 or the anti-β4 mAb 3E1F6. Depleted lysates were then immunoprecipitated with either the anti-α6 mAbs S3-41 or GOH3 (Fig. 7 a). It can be seen that GOH3 was still able to immunoprecipitate residual material when lysates were depleted of either β1 or β4 integrins (Fig. 7 a, middle, lane 3; right, lane 3). In contrast, S3-41 did not precipitate any residual material when lysates were depleted of β4 integrins (Fig. 7 a, right, lane 2) whereas it remained fully reactive when lysates were depleted of β1 integrins (Fig. 7 a, middle, lane 2). Thus, if GOH3 immunoprecipitates α6 chains associated with either β1 or β4 chains, S3-41 recognizes only α6 chains paired with β4. These results were confirmed by immunoblotting experiments with S3-41. Cell lysates were first immunoprecipitated either with the anti-β1 mAb K20 or the anti-β4 3E1F6. Fig. 7 b shows that S3-41 recognized only the α6 chains that coimmunoprecipitated with β4. Note that these experiments were performed on two distinct cell lines that expressed different relative amounts of α6β1 and α6β4 (Fig. 7 b, left and right). Taken together, these experiments demonstrate that the anti-α6 mAb S3-41 reacts with an epitope present only when α6 chains are linked to β4 chains.

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