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Ann Thorac Surg 1999;68:1920-1923
© 1999 The Society of Thoracic Surgeons

I. Pathophysiology of Ischemic Reperfusion Injury

Role of the ß2-integrins and immunoglobulin superfamily members in myocardial ischemia-reperfusion

Allan M. Lefer, PhDa

a Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

Address reprint requests to Dr Lefer, Department of Physiology, Jefferson Medical College, 1020 Locust St, Philadelphia, PA 19107;
e-mail: allan.m.lefer{at}mail.tju.edu

Presented at the International Symposium on Myocardial Protection From Surgical Ischemic-Reperfusion Injury, Asheville, NC, Sep 21–24, 1997.

Abstract

Leukocytes play a key role in the inflammatory processes such as ischemia-reperfusion of the coronary vasculature. Their interaction with the endothelium is closely regulated. The first step in the process is the rolling of leukocytes (eg, neutrophils) along the microvascular endothelium. This is regulated by the selectin family of cell adhesion molecules, primarily P-selectin. The next step in the inflammatory cascade is the firm adhesion of these neutrophils to the activated or dysfunctional endothelium. This process is governed by the ß2-integrins on the leukocytes (eg, CD11/CD18) and by ICAM-1 on the activated endothelium. CD11/CD18 is a ß2-integrin, and ICAM-1 is a member of the immunoglobulin superfamily of adhesion glycoproteins. By their interaction, neutrophils flatten out and adhere to the vascular endothelium. Many of these adhered neutrophils are then able to transmigrate across the endothelium to the site of the inflammation (ie, the focus of the ischemia-reperfusion). This transmigration is primarily stimulated by PECAM-1, another member of the immunoglobulin superfamily. These processes are discussed in this brief review.

Reperfusion of the previously ischemic myocardium usually leads to enhanced injury of the myocardium manifesting as a greater degree of cardiac necrosis than would occur in response to ischemia alone [1, 2]. Much of the reperfusion injury is caused by the infiltration of neutrophils into the reperfused myocardium. Either depletion of circulatory leukocytes or perfusion in the absence of neutrophils markedly attenuate the degree of the reperfusion injury [3, 4]. Previous studies have also shown that the major predisposing factor which allows leukocytes to play a role in reperfusion injury is endothelial dysfunction [1, 5]. Endothelial dysfunction occurs in the first 2.5 to 5 minutes of reperfusion [1, 5] and is characterized by a marked reduction in endothelial release of nitric oxide (NO).

The reduced NO release by the dysfunctional coronary vascular endothelium occurs at the microvascular and macrovascular level [1, 5] and leads to increased leukocyte-endothelium interaction [6]. Many investigators have therefore focused on the three critical steps known to occur in leukocyte-endothelium interaction in inflammatory states such as that occurring in myocardial ischemia-reperfusion (MI-R) [7]. This well-characterized sequence of events in leukocyte-endothelium interaction occurs primarily in venules. The first step in the process is termed "leukocyte rolling." In this phase, leukocytes slow their velocity through the axial stream of the flow, and "roll" or "hop" along the vascular endothelium. This process is largely regulated by the selectin family of adhesion glycoproteins [8]. These leukocytes slow down and make contact with the dysfunctional or activated endothelium, and these "tethered leukocytes" can proceed to the second step which is "firm adherence" to the endothelium. This key process is regulated by a pair of receptors, one on the surface of the neutrophil (ie, CD11/CD18) a member of the ß2-integrins which interacts with ICAM-1, a member of the immunoglobulin superfamily of adhesion molecules, which is upregulated on the surface of the endothelial cell [9, 10]. Many of these adhered neutrophils became activated during this adhesion process. Some of these adhered neutrophils then migrate between endothelial cells. This "transendothelial migration" is largely regulated by PECAM-1, another immunoglobulin family member [11, 12]. PECAM-1 is present in significant quantities at the junction of endothelial cells [11] as well as in platelets and leukocytes.

The purpose of this report, therefore, is to review effects of the ß2-integrins and the relevant immunoglobulin superfamily members on leukocyte-endothelial interactions in MI-R. Most of the data on this subject has been obtained in a well-characterized model of MI-R in the cat in which the left anterior descending (LAD) coronary artery is occluded for 90 minutes followed by 270 minutes of reperfusion. The relevant antibodies directed against specific cell adhesion molecules are given intravenously 10 minutes prior to reperfusion. This model of MI-R allows for the analysis of myocardial necrosis, assessment of coronary endothelial integrity, and the infiltration of polymorphonuclear leukocytes (PMNs) into the ischemic reperfused myocardium.

The first study undertaken was to test a monoclonal antibody to CD18 (the common ß-chain of the ß2-integrins located on PMNs [13]. CD18 forms a dimer with CD11a (Mac-1) to form the primary neutrophil adhesion glycoprotein. In this study, as in all the studies employing this model of MI-R, the monoclonal antibody was administered intravenously, 10 minutes prior to reperfusion (ie, 80 minutes into the ischemic period). This monoclonal antibody directed against CD18 (ie, R15.7) was found to markedly attenuate PMN adherence to cat coronary endothelial sheets in vitro to formyl-methonyl-leucyl-phenylalanine (fMLP) stimulated neutrophils [13]. Flow cytometry showed that R15.7 cross-reacted with cat neutrophils. R15.7 was compared to an isotype matched MAb which does not neutralize CD18 in the cat. Figure 1 summarizes the effects of R15.7 compared with its control antibody MAb R3.1 on cardiac necrosis at the end of the 90 minutes of ischemia and 270 minutes of reperfusion. Clearly, R15.7, given at a dose of 1 mg/kg intravenously, markedly protected against reperfusion injury to the cat myocardium. Moreover, R15.7, but not R3.1, dramatically preserved the ability of the LAD coronary endothelium to release NO to endothelium-dependent dilators (ie, acetylcholine and A23187). Furthermore, R15.7, but not R3.1, clearly attenuated neutrophil infiltration into the ischemic-reperfused myocardium, as evidenced by dramatically lower cardiac myloperoxidase (MPO) activity in the R15.7-treated hearts. As expected, R15.7 did not exert any systemic hemodynamic effect (ie, no change in blood pressure, heart rate, or the pressure-rate index). This was also reported in primates subjected to MI-R [14]. These were the first reports that blockade of CD18 could preserve the ischemic-reperfused myocardium. Both of these studies followed the pioneering work of Simpson and coworkers [15], who showed a CD11b MAb preserved the canine heart in MI-R.



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  		Fig 1. Cardioprotective effect of a monoclonal antibody (MAb) directed against CD18 on cardiac necrosis following 90 minutes of ischemia and 270 minutes of reperfusion in cats. Bar heights are means, brackets indicate ± SEM, and numbers in bars indicate numbers of cats in each group. The anti-CD18 MAb markedly attenuated the degree of cardiac necrosis indexed to the Area-At-Risk (p < 0.001). (AAR = Area-At-Risk, LV = left ventricle; MI/R = myocardial ischemia/reperfusion; NS = not significant.)

 
This study was followed up by another study in the same feline model of MI-R, employing a monoclonal antibody directed against ICAM-1. This antibody RR1/1 was given at a dose of 2 mg/kg intravenously 10 minutes prior to reperfusion. RR1/1 was shown to cross-react with cat coronary endothelial cells by means of immunohistochemistry but did not react with cat neutrophils. As with R15.7, RR1/1, but not a control antibody, significantly attenuated the degree of reperfusion injury (ie, postreperfusion cardiac necrosis) (Fig 2) [16]. Figure 2 clearly shows that the anti-ICAM-1 monoclonal antibody markedly attenuated cardiac necrosis, but not quite as effectively as the anti-CD18 antibody. As with the anti-CD18 MAb, RR1/1 did not exert any significant effect on systemic hemodynamics (Table 1). Similarly, RR1/1 also significantly preserved the LAD coronary artery endothelium against loss of NO release by acetylcholine and A23187, thus providing strong evidence that prevention of PMN adherence to the endothelium preserves endothelium-derived nitric oxide (EDNO). In addition, the anti-ICAM-1 MAb also significantly attenuated neutrophil infiltration into the area-at-risk (ie, the ischemic region; Table 1). These results are consistent with the anti-CD18 data and extend these findings to include one of the major relevant endothelial cell adhesion molecules. These results followed the pioneering work of Smith and associates [17], who showed that once PMNs adhere to the canine coronary endothelium and migrate across it, these activated PMNs adhere to cardiac myocytes by an ICAM-1-dependent mechanism. Thus, an anti-ICAM-1 monoclonal antibody is quite effective in treating MI-R in the cat and dog, and may exert a dual effect limiting neutrophil adherence to the endothelium, and retarding migrated neutrophil adherence to cardiac myocytes.



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  		Fig 2. Cardioprotective effect of a monoclonal antibody (MAb) directed against ICAM-1 on cardiac necrosis following 90 minutes of ischemia and 270 minutes of reperfusion in cats. Bar heights are means, brackets indicate ± SEM, and numbers in bars indicate numbers of cats in each group. The anti-ICAM-1 MAb markedly attenuated the degree of cardiac necrosis indexed to the Area-At-Risk (p < 0.01). (AAR = Area-At-Risk, LV = left ventricle; MI/R = myocardial ischemia/reperfusion; NS = not significant.)

 

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  		Table 1. Effects of Antibodies Directed Against Cell Adhesion Molecules in Feline Myocardial Ischemia-Reperfusion

 
More recently, an antibody has become available against PECAM-1 [18]. This antibody was found to cross-react with cat neutrophils by flow cytometry, and to cross-react with cat coronary endothelial cells by immunohistochemistry. This PECAM-1Ab was also given to cats at a dose of 1 mg/kg 10 minutes prior to reperfusion just as in the other feline studies mentioned above. This PECAM-1Ab significantly protected the ischemic-reperfused feline myocardium to a comparable degree as the ICAM-Ab [19]. However, the mechanism of this cardioprotection is slightly different, since the PECAM-1Ab only slightly preserved the coronary endothelium and did not attenuate MPO activity (ie, neutrophil accumulation) in the ischemic-reperfused myocardium [19]. In contrast, the PECAM-1Ab dramatically reduced the number of PMNs undergoing transendothelial migration in cats (Table 1). Thus, the PECAM-1Ab, acted on the third step in leukocyte-endothelial interaction, namely the migration of PMNs across the endothelium. This explains why the MPO activity was still elevated in these PECAM-Ab treated MI-R cats. It is because many of these PMNs are firmly adhered to the coronary vascular endothelium; the MPO assay of homogenized or minced cardiac tissue does not allow one to distinguish between neutrophils merely adhering to the endothelium in the inflammatory region from those PMNs actually migrating through the endothelium and either being trapped in the subendothelium or adhering to cardiac myocytes. As with all the other studies with anti-CAM antibodies, there was no significant effect of the antibody on systemic hemodynamics. Moreover, the antibody did not alter the number of circulating neutrophils in any way (ie, no precipitation or mobilization of PMNs by the antibody). Recently, these results were confirmed in rats subjected to MI-R and given a PECAM-1Ab [20].

Taken together, all these findings support the concept of a well-orchestrated time-dependent sequence of events occurring between the leukocytes and the endothelium in a region of local inflammation such as that occurring in ischemia-reperfusion of the heart. This is also consistent with recent data by Weyrich and colleagues [21], who determined the time course of expression of a variety of endothelial cell-adhesion molecules in the ischemic-reperfused feline coronary microcirculation via immunohistochemistry. P-selectin upregulation occurs early, followed by ICAM-1 expression about 2 hours later, whereas E-selectin does not seem to play any significant role in MI-R in the first 5 hours of reperfusion. The ß2-integrins and the immunoglobulin superfamily cell adhesion molecules play a very key role in neutrophil-endothelial interaction, and are important players in the pathogenesis of ischemia-reperfusion injury.

Acknowledgments

This study was supported by research grant GM45434 from the National Institute of General Medical Science of the National Institutes of Health.

References

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