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Matrix valency regulates integrin-mediated lymphoid adhesion via Syk kinase.

Stupack DG, Li E, Silletti SA, Kehler JA, Geahlen RL, Hahn K, Nemerow GR, Cheresh DA - J. Cell Biol. (1999)

Bottom Line: Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation.Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes.In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Lymphocytes accumulate within the extracellular matrix (ECM) of tumor, wound, or inflammatory tissues. These tissues are largely comprised of polymerized adhesion proteins such as fibrin and fibronectin or their fragments. Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation. This adhesion event depends on the appropriate spacing of integrin adhesion sites. Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes. In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments. These results reveal a cooperative interaction between signals emanating from integrins and antigen receptors that can serve to regulate stable lymphoid cell adhesion and retention within a remodeling ECM.

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Preferential attachment of unactivated LCL to multimeric ECM components. (A) Nonadherent LCL or adherent M21  cells were allowed to attach to wells coated overnight with various concentrations of either fibrinogen or fibrin. Cells remaining  attached after washing were stained with crystal violet and quantitated by reconstituting cell-bound dye with methanol and reading absorbance at 600 nm. The mean ± SE of triplicate determinations from one of three similar experiments is shown. (B)  Attachment to polymerized fibrin was assessed in the presence of  monoclonal antibodies specific for integrin α4 (P4C2), β2 (TS1/ 18), or αvβ3 (LM609) (25 μg/ml) or EDTA (10 mM) added to  cell suspensions immediately before adhesion assay. (C) Relative  affinity of the ligands for receptor was determined by binding of  soluble, biotinylated integrin to fibrin or fibrinogen-coated wells  that was detected by the addition of horseradish peroxidase–conjugated secondary anti-biotin monoclonal antibody as described  in Materials and Methods. (D) Multivalent ECM ligands, including fibrin, superfibronectin (SuperFN), pronectin, and multimeric vitronectin (Multimeric VN) or unpolymerized/proteolyzed  forms of these molecules forms including fibronectin fragments  of 110 (FN110) and 15 kD (FN15), fibrinogen, and monomeric  vitronectin (Vitronectin) were coated on adhesion assay wells  overnight (0.5–5 μg/ml) and assessed for their capacity to support  LCL adhesion (left). To determine if activation could rescue  LCL adhesion to unpolymerized substrates, LCL were treated  with PMA (20 ng/ml) immediately before adhesion assay on  plates coated as described above (right). Representative results  (mean ± SE of triplicate determinations) are shown; each substrate was tested three or more times.
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Figure 2: Preferential attachment of unactivated LCL to multimeric ECM components. (A) Nonadherent LCL or adherent M21 cells were allowed to attach to wells coated overnight with various concentrations of either fibrinogen or fibrin. Cells remaining attached after washing were stained with crystal violet and quantitated by reconstituting cell-bound dye with methanol and reading absorbance at 600 nm. The mean ± SE of triplicate determinations from one of three similar experiments is shown. (B) Attachment to polymerized fibrin was assessed in the presence of monoclonal antibodies specific for integrin α4 (P4C2), β2 (TS1/ 18), or αvβ3 (LM609) (25 μg/ml) or EDTA (10 mM) added to cell suspensions immediately before adhesion assay. (C) Relative affinity of the ligands for receptor was determined by binding of soluble, biotinylated integrin to fibrin or fibrinogen-coated wells that was detected by the addition of horseradish peroxidase–conjugated secondary anti-biotin monoclonal antibody as described in Materials and Methods. (D) Multivalent ECM ligands, including fibrin, superfibronectin (SuperFN), pronectin, and multimeric vitronectin (Multimeric VN) or unpolymerized/proteolyzed forms of these molecules forms including fibronectin fragments of 110 (FN110) and 15 kD (FN15), fibrinogen, and monomeric vitronectin (Vitronectin) were coated on adhesion assay wells overnight (0.5–5 μg/ml) and assessed for their capacity to support LCL adhesion (left). To determine if activation could rescue LCL adhesion to unpolymerized substrates, LCL were treated with PMA (20 ng/ml) immediately before adhesion assay on plates coated as described above (right). Representative results (mean ± SE of triplicate determinations) are shown; each substrate was tested three or more times.

Mentions: To characterize this interaction more carefully, LCL were allowed to attach to microtiter wells coated with either fibrin or fibrinogen. Although LCL attached to immobilized fibrin in a concentration-dependent manner, they failed to attach to fibrinogen at all concentrations tested (Fig. 2 A). The adhesion to fibrin, in vitro, was primarily dependent upon both αvβ3 and β2 integrins, as determined using function-blocking mAbs LM609 and TS1/ 18, respectively (Fig. 2 B). In contrast, adherent M21 melanoma cells readily attached to fibrinogen or its polymeric form, fibrin, in a dose-dependent manner (Fig. 2 A) and this was blocked by mAb LM609 (data not shown), as previously reported (Felding-Habermann et al., 1992).


Matrix valency regulates integrin-mediated lymphoid adhesion via Syk kinase.

Stupack DG, Li E, Silletti SA, Kehler JA, Geahlen RL, Hahn K, Nemerow GR, Cheresh DA - J. Cell Biol. (1999)

Preferential attachment of unactivated LCL to multimeric ECM components. (A) Nonadherent LCL or adherent M21  cells were allowed to attach to wells coated overnight with various concentrations of either fibrinogen or fibrin. Cells remaining  attached after washing were stained with crystal violet and quantitated by reconstituting cell-bound dye with methanol and reading absorbance at 600 nm. The mean ± SE of triplicate determinations from one of three similar experiments is shown. (B)  Attachment to polymerized fibrin was assessed in the presence of  monoclonal antibodies specific for integrin α4 (P4C2), β2 (TS1/ 18), or αvβ3 (LM609) (25 μg/ml) or EDTA (10 mM) added to  cell suspensions immediately before adhesion assay. (C) Relative  affinity of the ligands for receptor was determined by binding of  soluble, biotinylated integrin to fibrin or fibrinogen-coated wells  that was detected by the addition of horseradish peroxidase–conjugated secondary anti-biotin monoclonal antibody as described  in Materials and Methods. (D) Multivalent ECM ligands, including fibrin, superfibronectin (SuperFN), pronectin, and multimeric vitronectin (Multimeric VN) or unpolymerized/proteolyzed  forms of these molecules forms including fibronectin fragments  of 110 (FN110) and 15 kD (FN15), fibrinogen, and monomeric  vitronectin (Vitronectin) were coated on adhesion assay wells  overnight (0.5–5 μg/ml) and assessed for their capacity to support  LCL adhesion (left). To determine if activation could rescue  LCL adhesion to unpolymerized substrates, LCL were treated  with PMA (20 ng/ml) immediately before adhesion assay on  plates coated as described above (right). Representative results  (mean ± SE of triplicate determinations) are shown; each substrate was tested three or more times.
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Figure 2: Preferential attachment of unactivated LCL to multimeric ECM components. (A) Nonadherent LCL or adherent M21 cells were allowed to attach to wells coated overnight with various concentrations of either fibrinogen or fibrin. Cells remaining attached after washing were stained with crystal violet and quantitated by reconstituting cell-bound dye with methanol and reading absorbance at 600 nm. The mean ± SE of triplicate determinations from one of three similar experiments is shown. (B) Attachment to polymerized fibrin was assessed in the presence of monoclonal antibodies specific for integrin α4 (P4C2), β2 (TS1/ 18), or αvβ3 (LM609) (25 μg/ml) or EDTA (10 mM) added to cell suspensions immediately before adhesion assay. (C) Relative affinity of the ligands for receptor was determined by binding of soluble, biotinylated integrin to fibrin or fibrinogen-coated wells that was detected by the addition of horseradish peroxidase–conjugated secondary anti-biotin monoclonal antibody as described in Materials and Methods. (D) Multivalent ECM ligands, including fibrin, superfibronectin (SuperFN), pronectin, and multimeric vitronectin (Multimeric VN) or unpolymerized/proteolyzed forms of these molecules forms including fibronectin fragments of 110 (FN110) and 15 kD (FN15), fibrinogen, and monomeric vitronectin (Vitronectin) were coated on adhesion assay wells overnight (0.5–5 μg/ml) and assessed for their capacity to support LCL adhesion (left). To determine if activation could rescue LCL adhesion to unpolymerized substrates, LCL were treated with PMA (20 ng/ml) immediately before adhesion assay on plates coated as described above (right). Representative results (mean ± SE of triplicate determinations) are shown; each substrate was tested three or more times.
Mentions: To characterize this interaction more carefully, LCL were allowed to attach to microtiter wells coated with either fibrin or fibrinogen. Although LCL attached to immobilized fibrin in a concentration-dependent manner, they failed to attach to fibrinogen at all concentrations tested (Fig. 2 A). The adhesion to fibrin, in vitro, was primarily dependent upon both αvβ3 and β2 integrins, as determined using function-blocking mAbs LM609 and TS1/ 18, respectively (Fig. 2 B). In contrast, adherent M21 melanoma cells readily attached to fibrinogen or its polymeric form, fibrin, in a dose-dependent manner (Fig. 2 A) and this was blocked by mAb LM609 (data not shown), as previously reported (Felding-Habermann et al., 1992).

Bottom Line: Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation.Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes.In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Lymphocytes accumulate within the extracellular matrix (ECM) of tumor, wound, or inflammatory tissues. These tissues are largely comprised of polymerized adhesion proteins such as fibrin and fibronectin or their fragments. Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation. This adhesion event depends on the appropriate spacing of integrin adhesion sites. Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes. In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments. These results reveal a cooperative interaction between signals emanating from integrins and antigen receptors that can serve to regulate stable lymphoid cell adhesion and retention within a remodeling ECM.

Show MeSH
Related in: MedlinePlus