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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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Characterization of  local valency in promoting  LCL adhesion via integrins.  (A) LCL or M21 cells were  assessed for attachment to  wells coated with either recombinant adenovirus penton base (PB) protein (10  nM), a pentamer, or a monomeric PB construct (100 nM)  containing the integrin-binding domain, and phase– contrast images captured  at 200× magnification. (B)  LCL adhesion to multimeric  PB was assessed in the presence of function-blocking integrin-specific monoclonal  antibodies specific to β1  (P4C10), β2 (TS1/18), αvβ3  (LM609), αvβ5 (P1F6),  α4β1/β7 (P4C2) (25 μg/ml),  or soluble antagonists of integrin adhesion including a  cyclic peptide antagonist (cyclo Arg-Gly-Asp-dPhe-Val)  (5 μM), control antagonist (cyclo Arg-βAla-Asp-dPhe-Val) (5 μM), EDTA (10 μM), and both monomeric and multimeric forms of PB  in solution (10 μM). (C) The adhesion of LCL to pentameric PB was determined by attachment assay as described above, as a function  of increasing coating concentration of pentavalent or monovalent PB. To rescue attachment to monomeric PB, LCL were treated with  PMA (20 ng/ml) immediately before assay. (D) The density of native integrin-binding sites on BSA-blocked, substrate-coated adhesion  assay plates was assessed on monomer and pentamer coated wells by the specific binding of radio-iodinated mAb DAV-1 (0.5 μg),  which recognizes the Ile-Arg-Gly-Asp-Thr-Phe-Ala-Thr sequence found in the integrin-binding domain of PB. Data is expressed as the  mean ± SE of triplicate determinations from one of three separate experiments.
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Figure 3: Characterization of local valency in promoting LCL adhesion via integrins. (A) LCL or M21 cells were assessed for attachment to wells coated with either recombinant adenovirus penton base (PB) protein (10 nM), a pentamer, or a monomeric PB construct (100 nM) containing the integrin-binding domain, and phase– contrast images captured at 200× magnification. (B) LCL adhesion to multimeric PB was assessed in the presence of function-blocking integrin-specific monoclonal antibodies specific to β1 (P4C10), β2 (TS1/18), αvβ3 (LM609), αvβ5 (P1F6), α4β1/β7 (P4C2) (25 μg/ml), or soluble antagonists of integrin adhesion including a cyclic peptide antagonist (cyclo Arg-Gly-Asp-dPhe-Val) (5 μM), control antagonist (cyclo Arg-βAla-Asp-dPhe-Val) (5 μM), EDTA (10 μM), and both monomeric and multimeric forms of PB in solution (10 μM). (C) The adhesion of LCL to pentameric PB was determined by attachment assay as described above, as a function of increasing coating concentration of pentavalent or monovalent PB. To rescue attachment to monomeric PB, LCL were treated with PMA (20 ng/ml) immediately before assay. (D) The density of native integrin-binding sites on BSA-blocked, substrate-coated adhesion assay plates was assessed on monomer and pentamer coated wells by the specific binding of radio-iodinated mAb DAV-1 (0.5 μg), which recognizes the Ile-Arg-Gly-Asp-Thr-Phe-Ala-Thr sequence found in the integrin-binding domain of PB. Data is expressed as the mean ± SE of triplicate determinations from one of three separate experiments.

Mentions: To further establish the structural basis of lymphoid-mediated integrin function, LCL were allowed to attach to one of two structurally-defined αvβ3 ligands. In this case, microtiter wells were coated with a substrate comprised of either a monovalent or pentavalent form of the adenovirus PB protein (Mathias et al., 1994). The pentavalent form of PB contains five available αvβ3-binding sites equally separated by ∼60 Å (Stewart et al., 1997), whereas the monomeric construct contains only a single αvβ3-binding site (Mathias et al., 1994). As shown in Fig. 3 A, the adherent M21 cell line attached to either form of PB, whereas LCL selectively attached to the pentamer. Adhesion to multimeric PB was entirely dependent on integrin αvβ3, since either LM609 or a peptide antagonist of this integrin completely blocked adhesion while antibodies to other integrins showed no effect (Fig. 3 B). The addition of soluble monomeric or multimeric PB also disrupted adhesion (Fig. 3 B), indicating that either form of PB was an effective soluble inhibitor of integrin αvβ3.


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)

Characterization of  local valency in promoting  LCL adhesion via integrins.  (A) LCL or M21 cells were  assessed for attachment to  wells coated with either recombinant adenovirus penton base (PB) protein (10  nM), a pentamer, or a monomeric PB construct (100 nM)  containing the integrin-binding domain, and phase– contrast images captured  at 200× magnification. (B)  LCL adhesion to multimeric  PB was assessed in the presence of function-blocking integrin-specific monoclonal  antibodies specific to β1  (P4C10), β2 (TS1/18), αvβ3  (LM609), αvβ5 (P1F6),  α4β1/β7 (P4C2) (25 μg/ml),  or soluble antagonists of integrin adhesion including a  cyclic peptide antagonist (cyclo Arg-Gly-Asp-dPhe-Val)  (5 μM), control antagonist (cyclo Arg-βAla-Asp-dPhe-Val) (5 μM), EDTA (10 μM), and both monomeric and multimeric forms of PB  in solution (10 μM). (C) The adhesion of LCL to pentameric PB was determined by attachment assay as described above, as a function  of increasing coating concentration of pentavalent or monovalent PB. To rescue attachment to monomeric PB, LCL were treated with  PMA (20 ng/ml) immediately before assay. (D) The density of native integrin-binding sites on BSA-blocked, substrate-coated adhesion  assay plates was assessed on monomer and pentamer coated wells by the specific binding of radio-iodinated mAb DAV-1 (0.5 μg),  which recognizes the Ile-Arg-Gly-Asp-Thr-Phe-Ala-Thr sequence found in the integrin-binding domain of PB. Data is expressed as the  mean ± SE of triplicate determinations from one of three separate experiments.
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Figure 3: Characterization of local valency in promoting LCL adhesion via integrins. (A) LCL or M21 cells were assessed for attachment to wells coated with either recombinant adenovirus penton base (PB) protein (10 nM), a pentamer, or a monomeric PB construct (100 nM) containing the integrin-binding domain, and phase– contrast images captured at 200× magnification. (B) LCL adhesion to multimeric PB was assessed in the presence of function-blocking integrin-specific monoclonal antibodies specific to β1 (P4C10), β2 (TS1/18), αvβ3 (LM609), αvβ5 (P1F6), α4β1/β7 (P4C2) (25 μg/ml), or soluble antagonists of integrin adhesion including a cyclic peptide antagonist (cyclo Arg-Gly-Asp-dPhe-Val) (5 μM), control antagonist (cyclo Arg-βAla-Asp-dPhe-Val) (5 μM), EDTA (10 μM), and both monomeric and multimeric forms of PB in solution (10 μM). (C) The adhesion of LCL to pentameric PB was determined by attachment assay as described above, as a function of increasing coating concentration of pentavalent or monovalent PB. To rescue attachment to monomeric PB, LCL were treated with PMA (20 ng/ml) immediately before assay. (D) The density of native integrin-binding sites on BSA-blocked, substrate-coated adhesion assay plates was assessed on monomer and pentamer coated wells by the specific binding of radio-iodinated mAb DAV-1 (0.5 μg), which recognizes the Ile-Arg-Gly-Asp-Thr-Phe-Ala-Thr sequence found in the integrin-binding domain of PB. Data is expressed as the mean ± SE of triplicate determinations from one of three separate experiments.
Mentions: To further establish the structural basis of lymphoid-mediated integrin function, LCL were allowed to attach to one of two structurally-defined αvβ3 ligands. In this case, microtiter wells were coated with a substrate comprised of either a monovalent or pentavalent form of the adenovirus PB protein (Mathias et al., 1994). The pentavalent form of PB contains five available αvβ3-binding sites equally separated by ∼60 Å (Stewart et al., 1997), whereas the monomeric construct contains only a single αvβ3-binding site (Mathias et al., 1994). As shown in Fig. 3 A, the adherent M21 cell line attached to either form of PB, whereas LCL selectively attached to the pentamer. Adhesion to multimeric PB was entirely dependent on integrin αvβ3, since either LM609 or a peptide antagonist of this integrin completely blocked adhesion while antibodies to other integrins showed no effect (Fig. 3 B). The addition of soluble monomeric or multimeric PB also disrupted adhesion (Fig. 3 B), indicating that either form of PB was an effective soluble inhibitor of integrin αvβ3.

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