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The opioid peptide dynorphin A induces leukocyte responses via integrin Mac-1 (αMβ2, CD11b/CD18).

Podolnikova NP, Brothwell JA, Ugarova TP - Mol Pain (2015)

Bottom Line: In this study, we have synthesized the peptide library spanning the sequence of dynorphin AB, containing dynorphin A and B, and showed that the peptides bound recombinant αMI-domain, the ligand binding region of Mac-1.Further focusing on dynorphin A, we showed that its interaction with the αMI-domain was activation independent as both the α7 helix-truncated (active conformation) and helix-extended (nonactive conformation) αMI-domains efficiently bound dynorphin A.Together, the results identify dynorphins A and B as novel ligands for Mac-1 and suggest a role for the Dynorphin A-Mac-1 interactions in the induction of nonopiod receptor-dependent effects in leukocytes.

View Article: PubMed Central - PubMed

Affiliation: From the Center for Metabolic and Vascular Biology, School of Life Sciences, Arizona State University, Tempe, AZ, 85287, USA. Nataly.Podolnikova@asu.edu.

ABSTRACT

Background: Opioid peptides, including dynorphin A, besides their analgesic action in the nervous system, exert a broad spectrum of effects on cells of the immune system, including leukocyte migration, degranulation and cytokine production. The mechanisms whereby opioid peptides induce leukocyte responses are poorly understood. The integrin Mac-1 (αMβ2, CD11b/CD18) is a multiligand receptor which mediates numerous reactions of neutrophils and monocyte/macrophages during the immune-inflammatory response. Our recent elucidation of the ligand recognition specificity of Mac-1 suggested that dynorphin A and dynorphin B contain Mac-1 recognition motifs and can potentially interact with this receptor.

Results: In this study, we have synthesized the peptide library spanning the sequence of dynorphin AB, containing dynorphin A and B, and showed that the peptides bound recombinant αMI-domain, the ligand binding region of Mac-1. In addition, immobilized dynorphins A and B supported adhesion of the Mac-1-expressing cells. In binding to dynorphins A and B, Mac-1 cooperated with cell surface proteoglycans since both anti-Mac-1 function-blocking reagents and heparin were required to block adhesion. Further focusing on dynorphin A, we showed that its interaction with the αMI-domain was activation independent as both the α7 helix-truncated (active conformation) and helix-extended (nonactive conformation) αMI-domains efficiently bound dynorphin A. Dynorphin A induced a potent migratory response of Mac-1-expressing, but not Mac-1-deficient leukocytes, and enhanced Mac-1-mediated phagocytosis of latex beads by murine IC-21 macrophages.

Conclusions: Together, the results identify dynorphins A and B as novel ligands for Mac-1 and suggest a role for the Dynorphin A-Mac-1 interactions in the induction of nonopiod receptor-dependent effects in leukocytes.

No MeSH data available.


Related in: MedlinePlus

Chemotactic response of U937 cells to Dyn A. a, schematic representation of the experimental format. U937 cells (5x104) were placed in the center of the coverslip between two agar gels, one containing Dyn A and control gel without Dyn A. After 2 h at 37 °C, a series of photographs was taken in the direction of migration at 1-mm intervals to count the number of migrating cells. b, representative images after 2 h of migration show the cells within the starting point (marked with a cross; right panels) and at the border of the Dyn A-containing agar drop (left panels). Some cells were incubated with mAb 44a (20 μg/ml) or naloxone (10 μM) for 30 min before the initiation of chemotaxis assays. Two bottom panels show the experimental condition with no Dyn A gradient. c, quantification of U937 cells migrated from the initial point after 2 h based on the results shown in A (right panel). Migration of control untreated U937 cells that completely migrated from the initial point of cell placement was assigned a value of 100 %. In contrast, there was no cell migration without a Dyn A gradient (0 %). Migration of cells preincubated with mAb 44a and naloxone was blocked by ~80 % and ~20 %, respectively. A representative experiment is shown. d, distribution of U937 cells between two gels: Dyn A-containing (on the left) and without Dyn A (on the right). “0” on abscissa corresponds to the initial point where cells were loaded and the numbers show the distance that cells migrated in both directions from the starting point. All cells in each of 10 consecutive fields towards the Dyn A-containing and empty gels were counted and plotted as a distance from the starting point
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Fig7: Chemotactic response of U937 cells to Dyn A. a, schematic representation of the experimental format. U937 cells (5x104) were placed in the center of the coverslip between two agar gels, one containing Dyn A and control gel without Dyn A. After 2 h at 37 °C, a series of photographs was taken in the direction of migration at 1-mm intervals to count the number of migrating cells. b, representative images after 2 h of migration show the cells within the starting point (marked with a cross; right panels) and at the border of the Dyn A-containing agar drop (left panels). Some cells were incubated with mAb 44a (20 μg/ml) or naloxone (10 μM) for 30 min before the initiation of chemotaxis assays. Two bottom panels show the experimental condition with no Dyn A gradient. c, quantification of U937 cells migrated from the initial point after 2 h based on the results shown in A (right panel). Migration of control untreated U937 cells that completely migrated from the initial point of cell placement was assigned a value of 100 %. In contrast, there was no cell migration without a Dyn A gradient (0 %). Migration of cells preincubated with mAb 44a and naloxone was blocked by ~80 % and ~20 %, respectively. A representative experiment is shown. d, distribution of U937 cells between two gels: Dyn A-containing (on the left) and without Dyn A (on the right). “0” on abscissa corresponds to the initial point where cells were loaded and the numbers show the distance that cells migrated in both directions from the starting point. All cells in each of 10 consecutive fields towards the Dyn A-containing and empty gels were counted and plotted as a distance from the starting point

Mentions: The ability of Dyn A to support Mac-1-mediated cell migration was further confirmed using a chemotaxis migration system in which cells migrate to agar gels impregnated with a chemotactic agent (Fig. 7a). In these experiments, Mac-1-expressing U937 monocytic cells efficiently migrated along the gradient of Dyn A with many cells arriving at the edge of the gel (Fig. 7b, upper panel). In contrast, no directional migration was observed when Dyn A was absent (Fig. 7b, bottom panel); cells remained stationary and were evenly distributed at their initial position on the coverslip. Preincubation of cells with mAb 44a resulted in inhibition of their migration compared to nontreated control cells with only few cells arriving to the Dyn A-containing drop (Fig. 7b). As shown in Fig. 7c, only ~ 15 % of cells moved from the initial field in the presence of mAb 44a, implicating Mac-1 in mediating the chemotactic response of U937 cells to Dyn A.Fig. 7


The opioid peptide dynorphin A induces leukocyte responses via integrin Mac-1 (αMβ2, CD11b/CD18).

Podolnikova NP, Brothwell JA, Ugarova TP - Mol Pain (2015)

Chemotactic response of U937 cells to Dyn A. a, schematic representation of the experimental format. U937 cells (5x104) were placed in the center of the coverslip between two agar gels, one containing Dyn A and control gel without Dyn A. After 2 h at 37 °C, a series of photographs was taken in the direction of migration at 1-mm intervals to count the number of migrating cells. b, representative images after 2 h of migration show the cells within the starting point (marked with a cross; right panels) and at the border of the Dyn A-containing agar drop (left panels). Some cells were incubated with mAb 44a (20 μg/ml) or naloxone (10 μM) for 30 min before the initiation of chemotaxis assays. Two bottom panels show the experimental condition with no Dyn A gradient. c, quantification of U937 cells migrated from the initial point after 2 h based on the results shown in A (right panel). Migration of control untreated U937 cells that completely migrated from the initial point of cell placement was assigned a value of 100 %. In contrast, there was no cell migration without a Dyn A gradient (0 %). Migration of cells preincubated with mAb 44a and naloxone was blocked by ~80 % and ~20 %, respectively. A representative experiment is shown. d, distribution of U937 cells between two gels: Dyn A-containing (on the left) and without Dyn A (on the right). “0” on abscissa corresponds to the initial point where cells were loaded and the numbers show the distance that cells migrated in both directions from the starting point. All cells in each of 10 consecutive fields towards the Dyn A-containing and empty gels were counted and plotted as a distance from the starting point
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4481117&req=5

Fig7: Chemotactic response of U937 cells to Dyn A. a, schematic representation of the experimental format. U937 cells (5x104) were placed in the center of the coverslip between two agar gels, one containing Dyn A and control gel without Dyn A. After 2 h at 37 °C, a series of photographs was taken in the direction of migration at 1-mm intervals to count the number of migrating cells. b, representative images after 2 h of migration show the cells within the starting point (marked with a cross; right panels) and at the border of the Dyn A-containing agar drop (left panels). Some cells were incubated with mAb 44a (20 μg/ml) or naloxone (10 μM) for 30 min before the initiation of chemotaxis assays. Two bottom panels show the experimental condition with no Dyn A gradient. c, quantification of U937 cells migrated from the initial point after 2 h based on the results shown in A (right panel). Migration of control untreated U937 cells that completely migrated from the initial point of cell placement was assigned a value of 100 %. In contrast, there was no cell migration without a Dyn A gradient (0 %). Migration of cells preincubated with mAb 44a and naloxone was blocked by ~80 % and ~20 %, respectively. A representative experiment is shown. d, distribution of U937 cells between two gels: Dyn A-containing (on the left) and without Dyn A (on the right). “0” on abscissa corresponds to the initial point where cells were loaded and the numbers show the distance that cells migrated in both directions from the starting point. All cells in each of 10 consecutive fields towards the Dyn A-containing and empty gels were counted and plotted as a distance from the starting point
Mentions: The ability of Dyn A to support Mac-1-mediated cell migration was further confirmed using a chemotaxis migration system in which cells migrate to agar gels impregnated with a chemotactic agent (Fig. 7a). In these experiments, Mac-1-expressing U937 monocytic cells efficiently migrated along the gradient of Dyn A with many cells arriving at the edge of the gel (Fig. 7b, upper panel). In contrast, no directional migration was observed when Dyn A was absent (Fig. 7b, bottom panel); cells remained stationary and were evenly distributed at their initial position on the coverslip. Preincubation of cells with mAb 44a resulted in inhibition of their migration compared to nontreated control cells with only few cells arriving to the Dyn A-containing drop (Fig. 7b). As shown in Fig. 7c, only ~ 15 % of cells moved from the initial field in the presence of mAb 44a, implicating Mac-1 in mediating the chemotactic response of U937 cells to Dyn A.Fig. 7

Bottom Line: In this study, we have synthesized the peptide library spanning the sequence of dynorphin AB, containing dynorphin A and B, and showed that the peptides bound recombinant αMI-domain, the ligand binding region of Mac-1.Further focusing on dynorphin A, we showed that its interaction with the αMI-domain was activation independent as both the α7 helix-truncated (active conformation) and helix-extended (nonactive conformation) αMI-domains efficiently bound dynorphin A.Together, the results identify dynorphins A and B as novel ligands for Mac-1 and suggest a role for the Dynorphin A-Mac-1 interactions in the induction of nonopiod receptor-dependent effects in leukocytes.

View Article: PubMed Central - PubMed

Affiliation: From the Center for Metabolic and Vascular Biology, School of Life Sciences, Arizona State University, Tempe, AZ, 85287, USA. Nataly.Podolnikova@asu.edu.

ABSTRACT

Background: Opioid peptides, including dynorphin A, besides their analgesic action in the nervous system, exert a broad spectrum of effects on cells of the immune system, including leukocyte migration, degranulation and cytokine production. The mechanisms whereby opioid peptides induce leukocyte responses are poorly understood. The integrin Mac-1 (αMβ2, CD11b/CD18) is a multiligand receptor which mediates numerous reactions of neutrophils and monocyte/macrophages during the immune-inflammatory response. Our recent elucidation of the ligand recognition specificity of Mac-1 suggested that dynorphin A and dynorphin B contain Mac-1 recognition motifs and can potentially interact with this receptor.

Results: In this study, we have synthesized the peptide library spanning the sequence of dynorphin AB, containing dynorphin A and B, and showed that the peptides bound recombinant αMI-domain, the ligand binding region of Mac-1. In addition, immobilized dynorphins A and B supported adhesion of the Mac-1-expressing cells. In binding to dynorphins A and B, Mac-1 cooperated with cell surface proteoglycans since both anti-Mac-1 function-blocking reagents and heparin were required to block adhesion. Further focusing on dynorphin A, we showed that its interaction with the αMI-domain was activation independent as both the α7 helix-truncated (active conformation) and helix-extended (nonactive conformation) αMI-domains efficiently bound dynorphin A. Dynorphin A induced a potent migratory response of Mac-1-expressing, but not Mac-1-deficient leukocytes, and enhanced Mac-1-mediated phagocytosis of latex beads by murine IC-21 macrophages.

Conclusions: Together, the results identify dynorphins A and B as novel ligands for Mac-1 and suggest a role for the Dynorphin A-Mac-1 interactions in the induction of nonopiod receptor-dependent effects in leukocytes.

No MeSH data available.


Related in: MedlinePlus