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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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Rescue of integrin-mediated attachment to  monomeric PB. (A) The attachment of LCL to monomeric PB substrate (200 nM)  was assessed in untreated  cells or in the presence of  PMA (20 ng/ml) or manganese chloride (5 mM). (B)  Manganese-treated LCL with  rescued adhesion were tested  for relative expression of the  LIBS-1 neoepitope, an indicator of conformational  changes in β3 integrin associated with ligand binding. Increased FITC–LIBS-1 binding (indicating integrin conformational change) was detected by FACS™ in manganese-treated LCL (black histogram, upper panel) relative to untreated controls (light gray histogram, top) in the absence of ligand.  PMA-activated LCL (black histogram, bottom) were also compared with unactivated LCL (light gray histogram, bottom). Dark gray  indicates regions where activated or unactivated histograms overlap. (C) The influence of PMA on LCL integrin affinity was examined  using soluble ligand binding as a relative indicator. PMA-treated LCL, untreated LCL, or adherent M21 melanoma cells were assessed  for their capacity to bind soluble PB monomer as a function of ligand concentration using FACS™ analysis of FITC–LIBS-1 binding.  The relative shift in mean fluorescence intensity is plotted for each ligand concentration as a percentage of the maximum shift observed.  A representative experiment from three experiments is shown.
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Figure 4: Rescue of integrin-mediated attachment to monomeric PB. (A) The attachment of LCL to monomeric PB substrate (200 nM) was assessed in untreated cells or in the presence of PMA (20 ng/ml) or manganese chloride (5 mM). (B) Manganese-treated LCL with rescued adhesion were tested for relative expression of the LIBS-1 neoepitope, an indicator of conformational changes in β3 integrin associated with ligand binding. Increased FITC–LIBS-1 binding (indicating integrin conformational change) was detected by FACS™ in manganese-treated LCL (black histogram, upper panel) relative to untreated controls (light gray histogram, top) in the absence of ligand. PMA-activated LCL (black histogram, bottom) were also compared with unactivated LCL (light gray histogram, bottom). Dark gray indicates regions where activated or unactivated histograms overlap. (C) The influence of PMA on LCL integrin affinity was examined using soluble ligand binding as a relative indicator. PMA-treated LCL, untreated LCL, or adherent M21 melanoma cells were assessed for their capacity to bind soluble PB monomer as a function of ligand concentration using FACS™ analysis of FITC–LIBS-1 binding. The relative shift in mean fluorescence intensity is plotted for each ligand concentration as a percentage of the maximum shift observed. A representative experiment from three experiments is shown.

Mentions: Integrin organization on the cell surface may be an important factor contributing to adhesion, since the activation of LCL attachment to monomeric PB (Fig. 3 B) did not influence αvβ3 expression (our unpublished data). Lymphoid integrins can be activated by conformational (affinity) changes by a variety of reagents, including manganese (Bazzoni and Hemler, 1998). Manganese activated the attachment of LCL to monomeric PB similarly to PMA (Fig. 4 A), and caused conformational changes in integrin αvβ3 which were detected with monoclonal antibody ligand-induced binding site (LIBS)-1 (Fig. 4 B) (Frelinger et al., 1990). Although LIBS-1 detects the ligand-occupied form of αvβ3, the presence of manganese elicited conformational changes which exposed this epitope on LCL even in the absence of ligand (Fig. 4 B, top). In contrast, LCL activated with PMA did not differ from basal LIBS-1 expression (Fig. 4 B, bottom). Further, PMA did not stabilize integrin binding of soluble ligand (a relative measurement of affinity) (Frelinger et al., 1990; Filardo et al., 1995). Nonactivated or PMA-activated LCL were allowed to bind PB monomer, and LIBS-1 was used to detect ligand binding as a function of ligand concentration. No difference in half-maximal ligand occupancy was evident between PMA-activated and untreated LCL for soluble monomeric PB (∼1.5 μM) (Fig. 4 C). We observed that LCL displayed the same relative affinity for ligand as determined for M21 cells (Fig. 4 C) and other adherent cells (Filardo et al., 1995). Thus, activation of LCL by PMA leading to stable adhesion on monomer does not appear to result from preexisting affinity changes.


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)

Rescue of integrin-mediated attachment to  monomeric PB. (A) The attachment of LCL to monomeric PB substrate (200 nM)  was assessed in untreated  cells or in the presence of  PMA (20 ng/ml) or manganese chloride (5 mM). (B)  Manganese-treated LCL with  rescued adhesion were tested  for relative expression of the  LIBS-1 neoepitope, an indicator of conformational  changes in β3 integrin associated with ligand binding. Increased FITC–LIBS-1 binding (indicating integrin conformational change) was detected by FACS™ in manganese-treated LCL (black histogram, upper panel) relative to untreated controls (light gray histogram, top) in the absence of ligand.  PMA-activated LCL (black histogram, bottom) were also compared with unactivated LCL (light gray histogram, bottom). Dark gray  indicates regions where activated or unactivated histograms overlap. (C) The influence of PMA on LCL integrin affinity was examined  using soluble ligand binding as a relative indicator. PMA-treated LCL, untreated LCL, or adherent M21 melanoma cells were assessed  for their capacity to bind soluble PB monomer as a function of ligand concentration using FACS™ analysis of FITC–LIBS-1 binding.  The relative shift in mean fluorescence intensity is plotted for each ligand concentration as a percentage of the maximum shift observed.  A representative experiment from three experiments is shown.
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Related In: Results  -  Collection

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Figure 4: Rescue of integrin-mediated attachment to monomeric PB. (A) The attachment of LCL to monomeric PB substrate (200 nM) was assessed in untreated cells or in the presence of PMA (20 ng/ml) or manganese chloride (5 mM). (B) Manganese-treated LCL with rescued adhesion were tested for relative expression of the LIBS-1 neoepitope, an indicator of conformational changes in β3 integrin associated with ligand binding. Increased FITC–LIBS-1 binding (indicating integrin conformational change) was detected by FACS™ in manganese-treated LCL (black histogram, upper panel) relative to untreated controls (light gray histogram, top) in the absence of ligand. PMA-activated LCL (black histogram, bottom) were also compared with unactivated LCL (light gray histogram, bottom). Dark gray indicates regions where activated or unactivated histograms overlap. (C) The influence of PMA on LCL integrin affinity was examined using soluble ligand binding as a relative indicator. PMA-treated LCL, untreated LCL, or adherent M21 melanoma cells were assessed for their capacity to bind soluble PB monomer as a function of ligand concentration using FACS™ analysis of FITC–LIBS-1 binding. The relative shift in mean fluorescence intensity is plotted for each ligand concentration as a percentage of the maximum shift observed. A representative experiment from three experiments is shown.
Mentions: Integrin organization on the cell surface may be an important factor contributing to adhesion, since the activation of LCL attachment to monomeric PB (Fig. 3 B) did not influence αvβ3 expression (our unpublished data). Lymphoid integrins can be activated by conformational (affinity) changes by a variety of reagents, including manganese (Bazzoni and Hemler, 1998). Manganese activated the attachment of LCL to monomeric PB similarly to PMA (Fig. 4 A), and caused conformational changes in integrin αvβ3 which were detected with monoclonal antibody ligand-induced binding site (LIBS)-1 (Fig. 4 B) (Frelinger et al., 1990). Although LIBS-1 detects the ligand-occupied form of αvβ3, the presence of manganese elicited conformational changes which exposed this epitope on LCL even in the absence of ligand (Fig. 4 B, top). In contrast, LCL activated with PMA did not differ from basal LIBS-1 expression (Fig. 4 B, bottom). Further, PMA did not stabilize integrin binding of soluble ligand (a relative measurement of affinity) (Frelinger et al., 1990; Filardo et al., 1995). Nonactivated or PMA-activated LCL were allowed to bind PB monomer, and LIBS-1 was used to detect ligand binding as a function of ligand concentration. No difference in half-maximal ligand occupancy was evident between PMA-activated and untreated LCL for soluble monomeric PB (∼1.5 μM) (Fig. 4 C). We observed that LCL displayed the same relative affinity for ligand as determined for M21 cells (Fig. 4 C) and other adherent cells (Filardo et al., 1995). Thus, activation of LCL by PMA leading to stable adhesion on monomer does not appear to result from preexisting affinity changes.

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