Peripheral alveolar macrophages express more ACE2 immunoreactivity than in hilar area in human autopsied lung

Abstract

Objective

SARS-coronavirus 2 (SARS-CoV-2) is the cause of the coronavirus disease 19 (COVID-19). SARS-CoV-2 spike (S) glycoprotein binds to a cellular receptor angiotensin converting enzyme 2 (ACE2). COVID-19 pneumonia has a tendency to cause peripheral inflammation. To test the possibility that peripheral predominance of COVID-19 pneumonia is dependent on the peripheral expressions of ACE2, ACE2-positive macrophages and type 2 alveolar epithelial cells were immunohistochemically counted under the microscope, and compared between peripheral and hilar lung areas.

Results

Our result shows that peripheral alveolar macrophages express statistically significantly higher positivity in ACE2 than hilar alveolar macrophages. On the other hand, no statistically significant difference is shown in the expression of ACE2 in type 2 alveolar epithelia between peripheral and hilar alveoli. The higher expression of ACE2 immunoreactivity in peripheral alveolar macrophages may contribute to understanding the peripheral susceptibility of COVID-19 pneumonia.

Background

The novel coronavirus, SARS-coronavirus 2 (SARS-CoV-2) is the cause of the coronavirus disease 19 (COVID-19), the rapidly spreading pandemic. When SARS-CoV-2 enters the target cell, the spike (S) glycoprotein binds to a cellular receptor angiotensin converting enzyme 2 (ACE2) [1]. COVID-19 pneumonia has a tendency to cause peripheral inflammation [2, 3]. The reason for this tendency has not been elucidated. One possibility is that ACE2 could be more abundant in the peripheral region in the lung. ACE2 molecules are present in type2 alveolar epithelia and in alveolar macrophages [4]. CD68 molecules can be used as a marker for alveolar macrophages, whereas surfactant protein C (SFTPC) is produced in type2 alveolar cells, thus can be used as a marker. The purpose of this study is to clarify the mysterious susceptibility of COVID-19 pneumonia in the peripheral lung areas. For this purpose, we immunohistochemically counted cells positive for ACE2 and CD68 (alveolar macrophages), or for ACE2 and SFTPC (type2 alveolar epithelia) in human lung alveoli from autopsy specimens. Then, we compared the positivity in the hilar and peripheral areas to see if there is a difference in ACE2 expression dependent on the areas.

Materials and methods

Materials

The human lung tissues were obtained from forensic autopsies of our institution. The profile of autopsy cases were Asian and shown in detail in Table 1. At the time of autopsy, all cases were tested negative for nasal swab SARS-CoV-2 S protein antigen using ESPRINE SARS-CoV-2 (Fujirebio, Tokyo, Japan), and some cases were also negative for nasal swab SARS-CoV-2 N gene PCR using SARS-CoV-2 detection kit NCV-301_302 (Toyobo, Osaka, Japan).

Table 1.

Case profiles

Age	Gender	Cause of Death	Interval between Death and Autopsy (Days)
43	Male	Cervical spine injury, traumatic subarachnoid hemorrhage, and medullary contusion	3
64	Male	Congestive heart failure	5
68	Male	Ischemic heart disease	about 14
71	Male	Burn shock	5
54	Male	Unknown (suspect of drug intoxication)	6
86	Female	Acute subdural hematoma, and diffuse axonal injury	2
mid 50’s or above	Male	Death by hanging	9
6	Female	Hypoxic ischemic encephalopathy, and septicemia	2
83	Male	Unknown (possibility of fatal arrhythmia)	2
85	Female	Senility	2
72	Male	Aortic rupture	2
79	Female	Brain contusion, subdural hematoma	4
38	Male	Suspect of ketoacidosis	about 14
49	Male	Multiple injury	1
60–90	Male	Drowning, and cervical spine injury	2
36	Male	Brotizolam intoxicaion and choking	2
58	Male	Aortic rupture	2
28	Female	Death by hanging	2
56	Male	Acute ischemic heart disease	1
54	Male	Fatal arrhythmia	3
46	Male	Unknown (suspect of fatal arrhythmia, or heat stroke)	1
51	Male	Death by hanging	8
39	Male	Sertraline intoxication	4

The study was approved by Teikyo University Ethical Review Board for Medical and Health Research Involving Human Subjects (Approve No. 20-084-4). The profiles of human autopsy cases in this study are shown in Table 1.

Human autopsy samples were fixed in 16% formalin, embedded in paraffin, and sectioned. The immunohistochemistry was performed by the BOND RX automated immunostainer (Leica, Germany) using BOND polymer refine detection system [5]. The antibodies used were 50 times diluted anti-ACE2 (rabbit polyclonal, ab15348, Abcam, UK), 4 times diluted anti-CD68 for BOND (mouse polyclonal, PA0273, Leica, Germany), 3000 times diluted anti-surfactant protein C (SFTPC, rabbit polyclonal, 10774-1-AP, Proteintech, USA). The sections were pretreated for 20 min in BOND Epitope Retrieval Solution 1 for ACE2, and for 10 min in BOND Epitope Retrieval Solution 2 for CD68 and surfactant protein C. Double staining with ACE2 and either CD68 or SFTPC was performed. Diaminobenzidine (DAB) for ACE2, and Chrome Green for CD68 or SFTPC were used for staining, and hematoxylin was used as counterstain. The immunohistochemical positivity was measured morphometrically, using the cellSence software (Olympus, Tokyo, Japan) from the histological photography taken by the Olympus DP3CCD camera attached to the BX53 microscope as follows: the photographic pictures of the histological slides stained with hematoxylin and either CD68 or SFTPC were taken, and the hematoxylin- and CD68-positive alveolar cells were counted as the total number of alveolar macrophages, whereas hematoxylin- and SFTPC-positive cells were counted as the total number of type 2 alveolar epithelial cells. To count the cell numbers, the cellSence software picked up the cells with the positive stains morphometrically. Percentages of immunohistochemically positive cells for ACE2 in the total alveolar macrophages or total type 2 alveolar cells were calculated in the fields.

Statistical analysis

Graph drawings and the statistical analysis using paired t test were performedby Prism 7 (GraphPad Software, USA). The p values less than 0.01 were considered statistically significant.

Results

We have performed ACE2 immunohistochemistry on CD68-positive macrophages in peripheral (Fig. 1a), and hilar (Fig. 1b) areas. We have also performed ACE2 immunohistochemistry on SFPTC-positive type 2 alveolar epithelial cells in peripheral (Fig. 1c), and hilar (Fig. 1d) areas. Our results clearly show that CD68-positive alveolar macrophages present with the statistically significantly higher ACE2-positivity in the peripheral alveoli compared with hilar alveoli (Fig. 2a). On the other hand, SFTPC-positive type 2 alveolar epithelial cells failed to show significant differences in the ACE2-positivity in the peripheral alveoli compared with hilar alveoli (Fig. 2b).

Fig. 1.

Representative ACE2 (DAB, brown) immunohistochemistry of autopsied human lung for in alveolar macrophages (chrome green, CD68-positive) and type 2 alveolar epithelial cells (chrome green, SFTPC-positive).　Background brightness is enhanced for presentation. Some epithelial cells are detached due to postmortem changes in c and d. Inset bars show 50 micrometers. a: alveolar macrophages in peripheral lung. b: alveolar macrophages in hilar lung. c: type 2 epithelial cells in peripheral lung. d: type 2 epithelial cells in hilar lung

Fig. 2.

Percentages of ACE2 positive cells in peripheral and hilar lungs. a: alveolar macrophages in peripheral and hilar lung. b: type 2 epithelial cells in peripheral and hilar lung. *: statistically significant (p < 0.01)

Discussion

COVID-19 pneumonia has a tendency to cause peripheral inflammation [2, 3]. SARS-CoV-2 enters the target cells by S glycoprotein binding to ACE2. A previous report indicates the expression of ACE2 in alveolar epithelia is enhanced in smokers [6]. Alveolar macrophages have been immunohistochemically identified by CD68, and ACE2 is present in these cells [4]. The alveolar macrophage infected by SARS-CoV-2 may be a driver of the cytokine storm to cause damages in pulmonary tissues [4]. So far, no reports are found regarding the difference in the expression of ACE2 between hilar and peripheral lung region.

Our study indicates that, while type 2 alveolar epithelia show no statistically significant difference in ACE2 immunoreactivity between central and peripheral areas, there is a stronger expression of ACE2 immunohistochemistry in peripheral macrophages in human autopsy specimens, which may partially explain the tendency that COVID-19 pneumonia preferably involves peripheral areas in the lung [2, 3].

Our results may offer one possible explanation to the mysterious peripheral susceptibility of COVID-19 pneumonia.

Conclusion

Our results clearly show that CD68-positive alveolar macrophages present with a statistically significantly higher ACE2-positivity in the peripheral alveoli compared with hilar alveoli, which may partially explain the likelihood of peripheral inflammation in COVID-19 pneumonia.

Limitations: Since this is an autopsy tissue study, the functional aspects of ACE2 are not covered.

Abbreviations

SARS-CoV-2

SARS coronavirus-2

COVID-19

Coronavirus disease of 2019

ACE2

Angiotensin converting enzyme 2

SFTPC

Surfactant protein C

Author contributions

M.N. performed investigation, and wrote the main manuscript text. H.M-A. performed investigation, prepared figures and a table, and performed editing. H.T., Y.T., and T.A. performed investigation. T.N. performed investigation and editing. All authors reviewed the manuscript.

Funding

This study was supported in part by JSPS KAKENHI [20K10564 (M.N.) and 22K10620 (T.N.)].

Data availability

The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study was approved by Teikyo University Ethical Review Board for Medical and Health Research Involving Human Subjects (Approve No.20-084-3). Pursuant to the Chap. 4 of the ethical guidelines for medical health and health research involving human subjects by the Ministry of Health, Labor and Welfare, individual informed consent was not required. The requirement for informed consent was waived by Teikyo University Ethical Review Board for Medical and Health Research Involving Human Subjects because of the retrospective nature of the study. All experiments were performed in accordance with the Declaration of Helsinki.

Consent for publication

Not applied.

Competing interests

The authors declare no competing interests.