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alpha(4)-integrin mediates neutrophil-induced free radical injury to cardiac myocytes.

Poon BY, Ward CA, Cooper CB, Giles WR, Burns AR, Kubes P - J. Cell Biol. (2001)

Bottom Line: Myocyte shortening was decreased by 30-50% and rates of contraction and relaxation were reduced by 30% within the first 10 min.These findings demonstrate that profound molecular changes occur within PMNs as they emigrate, such that CD18 and associated intracellular signaling pathways leading to oxidant production are uncoupled and newly expressed alpha(4)-integrin functions as the ligand that signals oxidant production.The results also provide pathological relevance as the emigrated PMNs have the capacity to injure cardiac myocytes through the alpha(4)-integrin-coupled NADPH oxidase pathway that can be inhibited by extracellular, but not intracellular SOD.

View Article: PubMed Central - PubMed

Affiliation: Immunology Research Group, University of Calgary, Calgary, Alberta T2N 1N4, Canada.

ABSTRACT
Previous work has demonstrated that circulating neutrophils (polymorphonuclear leukocytes [PMNs]) adhere to cardiac myocytes via beta(2)-integrins and cause cellular injury via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme system. Since PMNs induced to leave the vasculature (emigrated PMNs) express the alpha(4)-integrin, we asked whether (a) these PMNs also induce myocyte injury via NADPH oxidase; (b) beta(2)-integrins (CD18) still signal oxidant production, or if this process is now coupled to the alpha(4)-integrin; and (c) dysfunction is superoxide dependent within the myocyte or at the myocyte-PMN interface. Emigrated PMNs exposed to cardiac myocytes quickly induced significant changes in myocyte function. Myocyte shortening was decreased by 30-50% and rates of contraction and relaxation were reduced by 30% within the first 10 min. Both alpha(4)-integrin antibody (Ab)-treated PMNs and NADPH oxidase-deficient PMNs were unable to reduce myocyte shortening. An increased level of oxidative stress was detected in myocytes within 5 min of PMN adhesion. Addition of an anti-alpha(4)-integrin Ab, but not an anti-CD18 Ab, prevented oxidant production, suggesting that in emigrated PMNs the NADPH oxidase system is uncoupled from CD18 and can be activated via the alpha(4)-integrin. Addition of exogenous superoxide dismutase (SOD) inhibited all parameters of dysfunction measured, whereas overexpression of intracellular SOD within the myocytes did not inhibit the oxidative stress or the myocyte dysfunction caused by the emigrated PMNs. These findings demonstrate that profound molecular changes occur within PMNs as they emigrate, such that CD18 and associated intracellular signaling pathways leading to oxidant production are uncoupled and newly expressed alpha(4)-integrin functions as the ligand that signals oxidant production. The results also provide pathological relevance as the emigrated PMNs have the capacity to injure cardiac myocytes through the alpha(4)-integrin-coupled NADPH oxidase pathway that can be inhibited by extracellular, but not intracellular SOD.

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(A) Superoxide production in PMNs from WT (n = 4) and NADPH oxidase KO mice (n = 2). (B) Cumulative unloaded cell shortening in control (no PMNs, n = 6; WT PMNs, n = 7; and NADPH oxidase KO PMNs, n = 6) at 5 min. +, P < 0.05 between indicated groups.
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Figure 2: (A) Superoxide production in PMNs from WT (n = 4) and NADPH oxidase KO mice (n = 2). (B) Cumulative unloaded cell shortening in control (no PMNs, n = 6; WT PMNs, n = 7; and NADPH oxidase KO PMNs, n = 6) at 5 min. +, P < 0.05 between indicated groups.

Mentions: Emigrated PMNs from WT mice generated O2− levels of 1.46 ± 0.12 fM/cell/min (n = 4). This level is approximately half of the levels produced by maximally stimulated human circulating PMNs (West et al. 1983). Emigrated PMNs from NADPH oxidase–deficient mice did not produce free radicals. Results showed O2− levels below the detectable limits of the assay in these cells (n = 2) (Fig. 2 A). Cumulative cell shortening data are shown in Fig. 2 B. Unloaded cell shortening remains at baseline at 5 min in controls (n = 6). The addition of emigrated PMNs to the myocyte caused ∼40% reduction in cell shortening within 5 min (n = 7, P < 0.05). This result was observed regardless of whether one or many PMNs adhered to the myocyte. In contrast, PMNs from NADPH oxidase–deficient mice did not alter cell shortening patterns (96.8 ± 8.9% of baseline, n = 6, P < 0.05 compared with PMN-only group). There was no difference in adhesion efficiency to cardiac myocytes between WT and NADPH oxidase–deficient PMNs, as shown in the inset. Leaving the NADPH oxidase–deficient PMNs bound to the myocytes for even 10 min still revealed no impairment (data not shown).


alpha(4)-integrin mediates neutrophil-induced free radical injury to cardiac myocytes.

Poon BY, Ward CA, Cooper CB, Giles WR, Burns AR, Kubes P - J. Cell Biol. (2001)

(A) Superoxide production in PMNs from WT (n = 4) and NADPH oxidase KO mice (n = 2). (B) Cumulative unloaded cell shortening in control (no PMNs, n = 6; WT PMNs, n = 7; and NADPH oxidase KO PMNs, n = 6) at 5 min. +, P < 0.05 between indicated groups.
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Figure 2: (A) Superoxide production in PMNs from WT (n = 4) and NADPH oxidase KO mice (n = 2). (B) Cumulative unloaded cell shortening in control (no PMNs, n = 6; WT PMNs, n = 7; and NADPH oxidase KO PMNs, n = 6) at 5 min. +, P < 0.05 between indicated groups.
Mentions: Emigrated PMNs from WT mice generated O2− levels of 1.46 ± 0.12 fM/cell/min (n = 4). This level is approximately half of the levels produced by maximally stimulated human circulating PMNs (West et al. 1983). Emigrated PMNs from NADPH oxidase–deficient mice did not produce free radicals. Results showed O2− levels below the detectable limits of the assay in these cells (n = 2) (Fig. 2 A). Cumulative cell shortening data are shown in Fig. 2 B. Unloaded cell shortening remains at baseline at 5 min in controls (n = 6). The addition of emigrated PMNs to the myocyte caused ∼40% reduction in cell shortening within 5 min (n = 7, P < 0.05). This result was observed regardless of whether one or many PMNs adhered to the myocyte. In contrast, PMNs from NADPH oxidase–deficient mice did not alter cell shortening patterns (96.8 ± 8.9% of baseline, n = 6, P < 0.05 compared with PMN-only group). There was no difference in adhesion efficiency to cardiac myocytes between WT and NADPH oxidase–deficient PMNs, as shown in the inset. Leaving the NADPH oxidase–deficient PMNs bound to the myocytes for even 10 min still revealed no impairment (data not shown).

Bottom Line: Myocyte shortening was decreased by 30-50% and rates of contraction and relaxation were reduced by 30% within the first 10 min.These findings demonstrate that profound molecular changes occur within PMNs as they emigrate, such that CD18 and associated intracellular signaling pathways leading to oxidant production are uncoupled and newly expressed alpha(4)-integrin functions as the ligand that signals oxidant production.The results also provide pathological relevance as the emigrated PMNs have the capacity to injure cardiac myocytes through the alpha(4)-integrin-coupled NADPH oxidase pathway that can be inhibited by extracellular, but not intracellular SOD.

View Article: PubMed Central - PubMed

Affiliation: Immunology Research Group, University of Calgary, Calgary, Alberta T2N 1N4, Canada.

ABSTRACT
Previous work has demonstrated that circulating neutrophils (polymorphonuclear leukocytes [PMNs]) adhere to cardiac myocytes via beta(2)-integrins and cause cellular injury via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme system. Since PMNs induced to leave the vasculature (emigrated PMNs) express the alpha(4)-integrin, we asked whether (a) these PMNs also induce myocyte injury via NADPH oxidase; (b) beta(2)-integrins (CD18) still signal oxidant production, or if this process is now coupled to the alpha(4)-integrin; and (c) dysfunction is superoxide dependent within the myocyte or at the myocyte-PMN interface. Emigrated PMNs exposed to cardiac myocytes quickly induced significant changes in myocyte function. Myocyte shortening was decreased by 30-50% and rates of contraction and relaxation were reduced by 30% within the first 10 min. Both alpha(4)-integrin antibody (Ab)-treated PMNs and NADPH oxidase-deficient PMNs were unable to reduce myocyte shortening. An increased level of oxidative stress was detected in myocytes within 5 min of PMN adhesion. Addition of an anti-alpha(4)-integrin Ab, but not an anti-CD18 Ab, prevented oxidant production, suggesting that in emigrated PMNs the NADPH oxidase system is uncoupled from CD18 and can be activated via the alpha(4)-integrin. Addition of exogenous superoxide dismutase (SOD) inhibited all parameters of dysfunction measured, whereas overexpression of intracellular SOD within the myocytes did not inhibit the oxidative stress or the myocyte dysfunction caused by the emigrated PMNs. These findings demonstrate that profound molecular changes occur within PMNs as they emigrate, such that CD18 and associated intracellular signaling pathways leading to oxidant production are uncoupled and newly expressed alpha(4)-integrin functions as the ligand that signals oxidant production. The results also provide pathological relevance as the emigrated PMNs have the capacity to injure cardiac myocytes through the alpha(4)-integrin-coupled NADPH oxidase pathway that can be inhibited by extracellular, but not intracellular SOD.

Show MeSH
Related in: MedlinePlus