Infiltration of polymorphonuclear cells into the post-ischaemic myocardium is dependent on beta2 and alpha4 integrins

Abstract

Polymorphonuclear cells (PMN) are believed to be important effector cells responsible the myocardial damage seen following ischaemia. However, the exact kinetics of their migration remains controversial. Isolated PMN (10 x 10(6) cells) labelled with (51)Cr were injected into four groups of Lewis rats: 0 h (T0h; n = 13), 2 h (T2h; n = 7), 4 h (T4h; n = 7) or 6 h following ischaemia (T6h; n = 4). In all recipients, a left thoracotomy and ligation of the left anterior descending coronary was performed. Control animals underwent sham thoracotomy (n = 10). All animals were killed at 24 h and the radioactivity in the tissue measured to estimate labelled PMN migration. Monoclonal antibody blockade was also performed in experimental animals to assess the contribution of beta2 and alpha4 integrins to the PMN migration (n = 32). Labelled PMN migration to the myocardium was similar in all experimental groups, T0-T6h (7.2-11 x 10(5) labelled PMN) and significantly higher than sham controls (2.2 x 10(5) labelled PMN; P = 0.03). In contrast PMN migration to dermal inflammatory sites was highest in T0h group, and reached background level in the T4h and T6h groups. beta2 integrin blockade inhibited labelled PMN migration by 32%. Blockade of alpha4 integrin inhibited PMN migration by 30% while the combined beta2 + alpha4 blockade resulted in 63% inhibition of labelled PMN migration compared to treatment with isotype control antibody (P = 0.035). PMN migration following myocardial ischaemia persists over several hours after myocardial infarction and does not follow similar migration kinetics to dermal inflammation. Our findings also suggest that PMN migration is dependent equally on beta2 and alpha4 integrins.

Figure 1

Effect of ischaemia on myocardial histology. Tissue specimens were fixed in 10% formalin, paraffin embedded, and 5 μm sections were cut and stained with standard H&E. Panel (a) prior to myocardial ischaemia, (b) 2 h, (c) 4 h and (d) 24 h after the onset of ischaemia. (Magnification 40×) Arrows indicate neutrophils.

Figure 2

MPO activity expressed Units/g of heart tissue between Sham, T0h, T2h, T4h and T6h groups. Each bar shows the mean ± SEM. *P < 0.05.

Figure 3

PMN infiltration in the ischaemic myocardium. ⁵¹Cr labelled blood neutrophils were injected i.v. at the time of coronary artery ligation, group T0h (n = 13), 2 h after the onset of ischaemia, group T2h (n = 7), 4 h after the onset of ischaemia, group T4h (n = 7), and 6 h after the onset of ischaemia, group T6h (n = 4). Sham operated control animals were injected with labelled cells at the time of surgery (n = 10). Twenty-four hour after surgery the accumulation of labelled neutrophils was determined by γ counting. Each bar shows the mean ± SEM. *P < 0.05.

Figure 4

PMN accumulation into dermal inflamation. ⁵¹Cr labelled blood neutrophils were injected i.v. at various times after the coronary artery ligation, into the same animals as shown in Figure 3. Each animal was injected intradermally with LPS (100 ng) and control diluent. Each bar shows the mean ± SEM. *P < 0.01.

Figure 5

Effect of ischaemia on myocardial ICAM-1. At the indicated times after onset of ischaemia, protein extracts of homogenized myocardium were prepared and the amount of ICAM-1 was determined by ELISA. Each bar shows the mean ± SEM.

Figure 6

Immunohistochemistry showing the effect of ischaemia on myocardial ICAM-1 expression. Tissue specimens were prepared as described in the Methods. Panel (a) prior to myocardial ischaemia, (b) 2 h, (c) 4 h and (d) 24 h after the onset of ischaemia. Positive ICAM-1 staining of myocardial cells and endothelial cells is shown by the arrows.

Figure 7

Effect of monoclonal antibody (mAb) blockade on PMN infiltration of the ischaemic myocardium. ⁵¹Cr labelled PMN were injected at the onset of ischaemia together with anti-β2 mAb (n = 8), anti-α4 (n = 8), anti-β2 + α4 (n = 6) or with an isotype matched mAb, B9 (n = 10). Infiltration was evaluated in myocardium and skin sites 24 h later. Each bar shows the mean ± SEM. *P < 0.05.

Figure 8

Effect of mAb blockade on PMN accumulation into dermal inflamation. ⁵¹Cr labelled blood PMN were injected i.v. at various times after the coronary artery ligation, into the same animals as shown in Figure 7. Each animal was injected intradermally with LPS (100 ng) and control diluent. Each bar shows the mean ± SEM. *P < 0.01.