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Upregulation of ICAM-1 on cardiomyocytes in jeopardized human myocardium during infarction

H.W.M. Niessen, W.K. Lagrand, C.A. Visser, C.J.L.M. Meijer, C.E. Hack
DOI: http://dx.doi.org/10.1016/S0008-6363(98)00236-3 603-610 First published online: 1 March 1999

Abstract

Objective: Impaired perfusion of the myocardium induces a local inflammatory response. In animal models, there is ample evidence that polymorphonuclear leucocytes (PMNs) infiltrating infarcted myocardium contribute significantly to infarct size. Methods: To explore a possible role for PMNs in the tissue damage of human myocardial infarction, we investigated localization of intercellular adhesion molecule-1 (ICAM-1) and CD66b (previously clustered as CD67), a marker of degranulation of human PMNs, in relation to deposition of complement in tissue specimens of infarcted and healthy parts of the heart obtained from 20 patients, who had died following acute myocardial infarction. Results: ICAM-1 was transiently expressed by endothelium and for a longer period (few days) on myofibers of infarcted myocardium. This expression only occurred in parts that stained positive for complement. PMN infiltration exclusively occurred in areas with ICAM-1 expression, but not every ICAM-1-positive area contained PMN infiltrates. CD66b was found in PMNs but was also fixed to the plasma membrane of myofibers that stained positive for complement and ICAM-1. Conclusion: These findings indicate that, in infarcted human myocardium, PMNs are degranulated, possibly upon interaction with ICAM-1 and activated complement.

Keywords
  • Infarction
  • Complement activation
  • Inflammation
  • Leukocytes
  • Human

Time for primary review 17 days.

1 Introduction

Impaired perfusion of the myocardium induces a local inflammatory response [1, 2]comprising a complicated interaction between ischemic myocardial and inflammatory cells, cytokines, complement factors and acute phase proteins. Although animal studies have shown that local inflammatory reactions may contribute significantly (up to 65%) to infarct size [1–3], the significance of inflammation on the extent of tissue damage following myocardial ischemia and infarction in humans is poorly understood. Recently, we have shown that acute phase reactant C-reactive protein (CRP) localizes in infarcted myocardium, in conjunction with activated complement, suggesting that CRP promotes local complement activation and, hence, tissue damage in acute myocardial infarction (AMI) [4].

Several studies in animals have shown that tissue damage in myocardial infarction is mediated by polymorphonuclear granulocytes (PMNs) adhering to cardiomyocytes via upregulated CD11b/CD18 (in PMNs) and intercellular adhesion molecule-1 (ICAM-1) (in cardiomyocytes). Adhering PMNs directly jeopardize cardiomyocytes via products of the respiratory burst of PMNs. In these studies, ICAM-1 was found to be upregulated only in ischemic but viable cardiomyocytes [5–9]. In concordance with these results, Hartman et al. [10]have shown in canines that inhibition of ICAM-1 upregulation on the myocytes significantly reduced neutrophil activity in vulnerable myocardium. However, whether this PMN-dependent inflammatory damage is relevant in human myocardial infarction, is unknown.

To assess a possible role for PMNs in human myocardial infarction, we investigated ICAM-1 upregulation in human infarcted heart tissue, in relation to degranulation of PMNs. For this, immunohistochemical studies were performed using tissue specimens obtained from 20 patients who died following AMI.

2 Methods

2.1 Patients

Patients, referred to the Department of Pathology for autopsy within 24 h after death, were included in this study when, at autopsy, they showed signs of a recently developed AMI, i.e. on histochemical examination they had decreased lactate dehydrogenase (LD) staining (decolouration) of the affected myocardium. Autopsies were performed as soon as possible after death, and not later than 24 h after death. To check for involvement of reperfusion of jeopardized myocardium with respect to the expression of ICAM-1 and localization of complement and CD66b, each patient was scored for receiving reperfusion therapy, recurrent myocardial infarction and macroscopical signs of reperfusion, as noticed at autopsy (i.e. absence of a thrombus in the infarct-related coronary artery). The study was approved by the ethics committee of the Free University Hospital Amsterdam. The investigation conforms with the principles outlined in the Declaration of Helsinki.

2.2 Processing of tissue specimens

Myocardial tissue specimens were obtained from the infarcted as well as from adjacent sites. These latter sites showed normal LD staining patterns and were studied as internal controls. Before being prepared as cryo-sections, the tissue specimens were stored at −196°C (liquid N2). The glass slides used for microscopy were pretreated with 0.1% poly-l-lysine (Sigma, St. Louis, MO, USA) to enhance adherence of the frozen tissue sections.

2.3 Antibodies

The monoclonal antibody (mAb; C4-4) against complement factor C4 used in this study is directed against C4d and has been used previously for immunohistochemical studies [4]. An antibody against CD66b [previously clustered as CD67 (B13.9)] was obtained from Dr. Verhoeven, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB) [11]. An antibody against ICAM-1 (RR-1) was kindly provided by Dr. Jaspars, Department of Pathology, Free University [12]. The mAbs (all belonging to the IgG-1 subclass) were stored at 1 mg/ml in phosphate-buffered saline (PBS).

2.4 Immunohistochemistry

Frozen sections (4 μm thick) were mounted onto glass slides, dried for 1 h by exposure to air and fixed in acetone (‘Baker analyzed reagent’, Mallinckrodt Baker, Deventer, Netherlands). The slides were incubated at room temperature for 10 min with normal rabbit serum (Dakopatts, Glostrup, Denmark), diluted 1 to 50 in PBS containing 1% (w/v) bovine serum albumin (PBS–BSA), (BSA from Boehringer Mannheim, Germany) after a rinse in PBS. Incubation of the slides with specific antibody solutions was performed for 60 min (CD66b and C4-4 were diluted 1 to 1000 in PBS–BSA; RR-1 was diluted 1 to 250). In control experiments, similar incubations were performed with two irrelevant mouse monoclonal antibodies: IgG-1 (antiphospholipase-A2, kindly provided by Dr. F.B. Taylor, Jr., Oklahoma Medical Research Foundation, Oklahoma, OK, USA) and mouse myeloma protein, MOPC (Cappel, Organon Teknika, Turnhout, Belgium).

The slides were washed for 30 min with PBS and incubated with horseradish peroxidase conjugated rabbit-anti-mouse immunoglobulins (RaM–HRP, Dakopatts), diluted 1 to 25 in PBS–BSA. Thereafter, the slides were washed again in PBS and incubated for 4 min in 0.5 mg/ml 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma) in PBS, pH 7.4, containing 0.01% (v/v) H2O2, washed again, counterstained with hematoxylin for 40 s, dehydrated, cleared and finally mounted.

Microscopic criteria [13, 14]were used to estimate infarct duration in all myocardial tissue specimens. In all cases, histologically assessed infarct age corresponded with the clinical course. Distinction between jeopardized parts, normal myocardium and the border zone were assessed in frozen sections. However, as morphological judgement is more reliable in paraffin slides, corresponding paraffin slides were also made, to confirm the determination of jeopardized versus non-jeopardized tissue. In both frozen and paraffin sections jeopardized myocardium was characterized by the intensity of eosinophilic staining of involved myofibers, loss of nuclei and cross striation, PMNs and lymphocyte infiltration and fibrosis. Reperfusion of ischemic myocardium was assessed using microscopic (interlesional hemorrhages and contraction band necrosis [14]) and macroscopic (presence of a thrombus in the infarct related coronary artery) criteria. The percentages of C4d, ICAM-1 and CD66b positivity of cardiomyocytes, and the amount of PMNs, was related to the area of decreased LD staining of affected myocardium.

Two independent investigators (W.K.L. and H.W.M.N.) each judged and scored all slides for infarct age and anatomical localization of specific antibody, as visualized by immunohistochemical staining. The anatomical localizations that were examined were myofiber (membrane, cytoplasm, cross striations) blood vessel elements (endothelium and surrounding tissue), and PMNs. For the final scoring results, consensus was achieved by the two investigators.

3 Results

3.1 Patients

Myocardial tissue specimens were obtained from 20 patients who had died after AMI, as proven by autopsy (Table 1). Most of these patients participated in earlier studies on the involvement of CRP and complement in infarcted human myocardium [4]. Reperfusion therapy was received by seven of these patients (Fig. 1) [streptokinase, coronary surgery and percutaneous transluminal coronary angioplasty (PTCA)]. Three patients had a history of previous AMI. Most patients had a posterior infarction, but other locations occurred as well (Table 1). Specimens were obtained from the infarcted myocardium as well as from normal appearing myocardial tissue. The infarct age, assessed by microscopical criteria [13, 14], varied from less than 12 h up to two weeks (Table 1). Furthermore, one patient with an infarct age of more than one year was included as a control (Table 1).

View this table:
Table 1

Patients’ characteristics

NumberPatientReason for admittanceCause of deathHistory of AMIaSites of recent AMIbReperfusion therapy for recent AMIDuration of recent AMI
M/Fage
1M51AMI/pneumoniaAMI/ARPW<12 h
2M76AMIAMI/respiratory failurePW<12 h
3M51AMIAMI/PFAW/LW/septal+, PTCAc<12 h
4F83AMIAMI/AR+AW/PW/LW<12 h
5F75AMIAMI/ARAW/LW/septal+, PTCAc/SKd<12 h
6M73CABGeAMI/AR+PW/AW+, CABGe<12 h
7M74resection upper jawAMI/PFPW/AW12–24 h
8M60AMIAMI/PFPW/LW/AW/septal+, SKd1–3 days
9F69AMIAMI/PFPW/RV/septal1–3 days
10F58AMIAMI/AVSf/PFAW/septal+, SKd1–3 days
11F62CABGeAMI/PFAW/PW/septal3–5 days
12M75AMIAMI/tamponadeAW/PW/septal3–5 days
13M75AMIAMI/ARPW3–5 days
14M63AMIAMI/AAAAg/AR+AW5–9 days
15M70AMIAMI/ARPW/RV+, SKd5–9 days
16F82AMI/AVRhAMI/PFseptal5–9 days
17M63AMIAMI/CVAiLW/PW9–14 days
18M66AMIAMI/PFAW/PW/septal9–14 days
19M53AMI due to aortic dissection type AAMI due to aortic dissection type A/PFPW/RV/septal+, aortic surgery (CABGe)9–14 days
20F83Gastric surgeryAMI/sepsisPW/LW1 year
  • AR: arrhythmia.

    PF: pump failure.

    a−=no AMI in history, +=AMI in history.

    bPW=posterior wall, AW=anterior wall, LW=lateral wall, RV=right ventricle.

    cPercutaneous transluminal coronary angioplasty.

    dStreptokinase.

    eCoronary artery bypass grafting.

    fAortic valve stenosis.

    gAcute abdominal aortic aneurysm.

    hAortic valve replacement.

    iCerebrovascular accident.

3.2 Localization of complement C4d, ICAM-1 and CD66b

By immunohistochemistry, C4d was found to be localized in 14 of the 20 infarcts studied, while ICAM-1 stained in 13 of the 20 infarctions (Fig. 1). The area containing deposits of C4d was larger than that with ICAM-1. The staining pattern for C4d was also more intense than that for ICAM-1 (Fig. 2). Most intense staining for ICAM-1 was found in the border zone of infarcted myocardium. This, however, was only observed in larger infarction sites. On the other hand, ICAM-1 was not found in C4d-negative fibers. This suggested that ICAM-1 upregulation exclusively appeared in jeopardized fibers that had fixed complement. Furthermore, except for one patient that received double reperfusion therapy, we did not find any differences in C4d and ICAM-1 staining between patients who either had or not had received reperfusion therapy (Fig. 1). Notably, in only four of the twelve patients that had not received reperfusion therapy, an obstructing thrombus was found at autopsy, whereas this was the case in two of the seven patients that had received reperfusion therapy. However, we also found no significant difference for ICAM-1 expression or C4d deposition in patients with or without the presence of a coronary thrombus.

Fig. 1

Immunohistochemical localization of C4d, ICAM-1 and CD66b on infarcted cardiomyocytes (*: patient number from Table 1; **: reinfarction. Intensity of immunostaining). −: negative immunostaining; ±: weak, positive immunostaining; +: moderate, positive immunostaining; ++: strong, positive immunostaining).

In ICAM-1-positive areas, the plasma membrane, cytoplasm (Fig. 3) and cross-striation of the myofibers each stained for both C4d and ICAM-1 (C4d more than ICAM-1).

Positive staining of the myofibers for C4d and ICAM-1 did not occur in infarctions of shorter duration than one day, except for one tissue sample of an infarction of less than 12 h. In this sample, only a weak staining for C4d and less for ICAM-1 was found. Notably, this patient had received percutaneous transluminal coronary angioplasty as well as streptokinase therapy.

Staining of the myofibers for C4d was maximal in infarcts of one–five days, whereas ICAM-1 expression was most pronounced in infarcts of three–five days (Fig. 1). In infarctions of five to nine days old, the intensity of positive immunostaining for both C4d and ICAM-1 was less than that in infarctions of three to five days old. However, in three infarctions of more than nine days old, again strong positive immunostaining of the myofibers for C4d and ICAM-1 was found, which was accompanied by a more intense infiltration of PMNs (Fig. 1). In each of these three cases, clinical as well as histological parameters were indicative for reinfarction (one–five days old).

The endothelium of the heart had already stained positive for ICAM-1 (Fig. 4) (and C4d), in infarctions that were less than 12 h old, when PMNs were not yet found extravascularly. In older infarctions, ICAM-1 staining of endothelium decreased. Extravascular migration of PMNs was found after one–three days (Fig. 1), in older infarctions, the number of infiltrating PMNs had also decreased.

All PMNs infiltrating the myocardium stained positive for CD66b in the cytoplasm and partly at the plasma membrane. Furthermore, in 11 of 20 tissue specimens, PMNs were seen adhering to the membrane of the myofibers (Fig. 5).

Notably, the presence of CD66b was not restricted to PMNs but was also found on cardiomyocytes. In most places, CD66b on cardiomyocytes occurred at sites where PMNs adhered. However, at some sites, cardiomyocyte membranes stained positive for CD66b in the absence of adhering PMNs. With increasing infarct age, fewer PMNs were found adhering to cardiomyocytes.

Adhering PMNs and positive staining of myofibers for CD66b were found exclusively in C4d- and ICAM-1-positive cardiomyocytes. In myocardium that stained negative for ICAM-1 and positive for C4d, sometimes PMNs were seen in between myofibers and, thus, at extravascular sites, but these PMNs did not adhere to myofibers. In these cases, myofibers were always negative for CD66b.

Fig. 2

Immunohistochemical localization of C4d (mAb C4-4) (A) and ICAM-1 (mAb RR-1) (B) in infarcted myocardium (J=jeopardized, N=normal). Tissue specimens were from patient number 11 (Table 1). The frozen tissue sections shown were from the same myocardial infarction site. (magnification ×100).

Fig. 3

Positive staining of myofiber elements (sarcolemma and cytoplasm) in infarcted myocardium for ICAM-1 (magnification ×400). The tissue specimen was from patient number 13 (Table 1).

Fig. 4

Positive staining of endothelium with ICAM-1 (arrow) (magnification ×400). The tissue specimen was from patient number 2 (Table 1).

Staining of myocardial tissue specimens with control antibodies yielded negative results. In addition, internal controls, i.e., specimens taken from non-infarcted sites of the myocardium (taken from the left as well as the right ventricle) of the same patient, did not show any staining for C4d, CD66b or ICAM-1. Furthermore, myocardial tissue specimens from an immature child that died intrauterinely at an amenorrhoea duration of 22 weeks, which were regarded to represent a pure, non-ischemic myocardial control, also did not show any staining for ICAM-1, C4d or CD66b. Finally, staining of an old infarction (>one year) for C4d, ICAM-1 and CD66b was also negative.

4 Discussion

Complement activation leading to the formation of membrane attack complex has been documented in infarcted human myocardium [1, 2, 4, 15], as evidenced by deposition of C4d in the present study. In animal models, this activation is a key event mediating the deleterious effects of the local inflammatory response occurring in the infarcted myocardium amongst others by mobilizing PMNs [1, 2, 4, 16, 17]. Here we report that infiltration of PMNs in ischemic myocardium in humans is restricted to the areas containing complement deposits. Moreover, cardiomyocytes in most of these areas were found to express ICAM-1. To the best of our knowledge, this is the first report that ICAM-1 is also upregulated in human cardiomyocytes after infarction.

Studies of infarcted myocardium in humans are limited to studies of material obtained during autopsy. These investigations have the inherent risk that the observed phenomena are due to post-mortem changes. Regarding our findings, we consider this to be unlikely since no ICAM-1 expression, complement deposition or PMN degranulation was found in non-infarcted areas (taken from both the left as well as the right ventricle in most patients). These non-infarcted areas of the heart were not supplied by the infarct-related coronary vessel and had no morphological signs of ischemia, as was supported by electron microscopy studies (not shown). Results for these non-infarcted areas were similar to those for the apparently normal areas of the affected ventricle. Hence, we do not believe that the immunohistochemical results obtained with the normal parts of the infarcted sites were in some way influenced by hypoperfusion, at least regarding the parameters investigated. Moreover, in control patients that had died from non-cardiac disease, neither detectable ICAM-1 upregulation or complement deposition was found, which shows that the observed upregulation of ICAM-1 was not simply caused by post-mortem delay.

Fig. 5

Localization of CD66b in extravascular, non-adherent PMNs (A) (magnification ×250), in PMNs adhering to myocardium (B) (magnification ×400), and in myofibers that were largely unrelated to (adherent) PMNs (C) (magnification ×250). Tissue specimens were from patient number 8 (A), patient number 17 (B) and patient number 17 (C) (Table 1).

Animal studies have shown that ICAM-1 expression in ischemic myocardium in particular occurs upon reperfusion [5, 7, 8]. We did not find differences in ICAM-1 expression in patients who had or had not received reperfusion therapy (Fig. 1). However, in the majority of the patients that either had been treated or were not treated with reperfusion therapy, we did not find thrombi obstructing the coronary vessels, suggesting that reperfusion had occurred in most patients. However, microscopic evidence for successful reperfusion [14]was absent in all tissue samples. Therefore, the role of reperfusion in ICAM-1 expression in ischemic human myocardium remains to be elucidated.

ICAM-1 expression occurred in endothelium, especially in infarctions of less than 12 h old, thus, before infiltrating PMNs appeared, suggesting that ICAM-1 was upregulated by the endothelium before infiltration of the ischemic heart by PMNs, in concordance with a study by Kukielka et al. [7]. In older infarctions, staining of endothelium for ICAM-1 decreased. This can be explained by down-regulation of ICAM-1 on endothelium or, alternatively, by proteolytic cleavage by activated PMNs. The relation of this down-regulation of ICAM-1 with the increased serum levels of soluble ICAM-1 [18, 19]remains to be established. Also, complement staining of endothelium occurred before PMN infiltration, consistent with the notion that most of the chemotactic activity had originated from activated complement [20].

ICAM-1 expression also occurred, even more abundantly, on cardiomyocytes. Entman et al. [6]found that, in animals, PMNs adhered to cardiomyocytes via expression of CD11b–CD18 on the former, and upregulated ICAM-1 on the latter. Consistent herewith, in human infarcted myocardium, we found that areas containing ICAM-1-positive cardiomyocytes were infiltrated by PMNs, for the most part adhering to these ICAM-1-positive cardiomyocytes. In ICAM-1-negative, but complement-positive parts, no adhesion of PMNs to cardiomyocytes could be found. Thus, this is in line with a role for ICAM-1 in the adhesion process between PMNs and ischemic cardiomyocytes. Although iC3b deposited on the cardiomyocytes also may contribute to the adhesion of PMNs, it is likely that this mechanism was only of minor importance since PMNs were found not to adhere to cardiomyocytes in areas that were negative for ICAM-1, but positive for complement.

An important question is the mechanism of upregulation of ICAM-1 on cardiomyocytes. In animal studies, it was suggested that interleukin 6 (IL-6) in particular might induce this upregulation [21, 22]. Circulating IL-6 indeed increases following myocardial infarction in patients [23, 24]. Remarkably, in our study, ICAM-1 was only expressed by cardiomyocytes that also stained positive for complement. Complement depositions occurred before upregulation of ICAM-1, as was concluded by comparing the staining results obtained from specimens of infarctions of varying age. Although co-localization does not always prove a causal relationship, our results suggested that complement also plays a role in upregulating ICAM-1 on the myocyte. In line with this hypothesis, it has been shown that ICAM-1 can be upregulated on the endothelium by complement [25–27].

In animal models, PMNs have been shown to contribute to the tissue damage caused during myocardial infarction [6, 28, 29]. In our study, ICAM-1-positive cardiomyocytes also stained positive for CD66b (a marker of degranulation of PMNs), in particular, at the plasma membrane. Thus, in addition to the generation of products of the respiratory burst [6], the release of granule constituents may also contribute to jeopardizing cardiomyocytes.

Thus, apparently during myocardial infarction in humans, binding of complement (presumably triggered by CRP [4]) to cardiomyocytes precedes ICAM-1 expression and PMN infiltration. In the majority of ICAM-1-positive myocardial areas, PMNs were found not only to adhere to cardiomyocytes, but also to be degranulated to some extent (CD66b positivity of the cardiomyocytes). We postulate that activation and degranulation of PMNs had resulted from the concerted action of ICAM-1 and deposited complement.

Upregulation of ICAM-1 and deposition of complement on myofibers occurs from 12 h on, subsequent to infarction, and increases for up to five days. Hence, therapeutical manoeuvres aimed at reduction of this inflammatory damage of myofibers should be continued for a minimum of five days subsequent to myocardial infarction. Moreover, in three patients, we observed the reinduction of inflammation at a myocardial infarction aged nine–fourteen days. In all three of these patients, however, reinfarction (less than five days) had occurred, which was more intense than the primary infarction, suggesting some kind of a priming effect of the inflammatory response in the infarcted area.

In conclusion, this study demonstrates complement- and PMN-dependent inflammatory reactions in the human heart during acute myocardial infarction, suggesting that these changes may contribute to myocardial damage and, hence, may constitute a target for intervention therapy.

Acknowledgements

We wish to thank Thea Tadema for expert technical and immunohistochemical assistance. Furthermore, we wish to thank Dr. Verhoeven of the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, for providing the CD66b antibody, and Dr. Jaspars, Department of Pathology, Free University, for providing the ICAM-1 antibody. This study was supported financially by the Netherlands Heart Foundation, grant number 93-119.

References

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