IL-5 overexpression induced blood eosinophilia associated progression of cardiac abnormalities

Abstract

Eosinophilia may be responsible for cardiac injuries of widely varying severity, from acute myocarditis to endomyocardial fibrosis. In this manuscript, we present both the molecular and physiological evidence that promotes eosinophils accumulation in mice heart epicardium and myocardium region of allergen or transgene-insertion of overexpressed IL-15, eotaxin-deficient CD2 promoter driven IL-5 transgenic mice shows abnormal physiological function. Numerous etiologies can lead to severe eosinophilia, but these are mainly represented by hypersensitivity reactions, rheumatological diseases and hypereosinophilic syndrome. Because cardiac involvement may be extremely severe; therefore, we even present echocardiography analysis that indicates IL-15 overexpressed mice showed induced blood eosinophilia associated progression of cardiac abnormalities.

Introduction

Eosinophils associated cardiac disorder ca is rare medical condition and was first reported in 1936 by Wilhelm Loffler, who called it ‘fibroplastic parietal endocarditis with blood eosinophilia’ [1]. Cardiac eosinophilia is reported to be the cause of promoting acute myocarditis to eosinophils associated endomyocardial fibrosis. Eosinophils are normally found in very few numbers in blood and home manly in gastrointestinal tract in healthy state [2] that are involved in normal antimicrobial immunity [3]. Eosinophils possess the ability to elaborate substances that are toxic to a wide variety of parasites that are too large to phagocytose [4]. In the disease state eosinophils accumulate in the respiratory tract and skin. Eosinophils are 12–15 μm in diameter and are characterized by a bilobed nucleus and numerous eosin-staining specific mainly represented by systemic diseases, malignancies and hypereosinophilic syndrome (HES). The early phase of cardiac eosinophilia is characterized by an eosinophilic endomyocarditis with eosinophil and lymphocyte infiltration [17-18]. When infiltrating cardiac tissues, eosinophils degranulate and release toxic cationic proteins, thus inducing necrosis and apoptosis [19]. Clinical and in vivo recognition of eosinophilic myocarditis is infrequent, whereas it accounts for up to 0.5% of unselected myocarditis autopsy series [20]. In this manuscript, we present the evidence that heathy heart is devoid of eosinophils and both allergen-induced IL-5, IL-5 transgene-induced and eotaxin-deficient IL-5 overexpressed mice showed high accumulation of cardiac eosinophils and cardiac functional abnormalities in mice.

Materials and Methods

Mice:

Specific pathogen-free Balb/c mice 10 weeksold were obtained from the Jackson laboratory (Bar Harbor, ME, USA) and used as a wild type (WT) mice. IL-5 (CD2) transgenic (originally obtained from C. Sanderson Institute for Child Health Research, Perth, Australia) were obtained from the Cincinnati Children’s Hospital and Medical Center (CCHMC) and brought to Tulane from Dr. Marc Rothernberg’s laboratory. Eotaxin-1-deficient IL-5 overexpressed mice will be genenrated in the laborator by breedind eotaxin-deficient mice with CD2-IL-5 transgenic mice. The Institutional Animal Care and Use Committee (IACUC) approved the animal protocol in accordance with National Institutes of Health (NIH) guidelines. All experiments were performed according to animal ethics rules and regulations.

OVA Challenge Protocol:

Mice were sensitized with 20 μg ovalbumin (OVA) and 4 mg aluminium hydroxide (alum) (both Nacalai Tesque, Inc.) in 100 μl PBS (21). Starting on day 21, the mice were challenged with 1 % OVA aerosol for 20 min/day for 7 consecutive days by using an ultrasonic nebulizer (Mabist mist; Mabist DMI Healthcare, Illinois, CA, USA) as described. The animals in the normal group were sensitized and challenged with normal saline at the same time intervals. Finally, 24 h following the final challenge, the mice were euthanized with i.p pentobarbital (200 mg/kg) and samples were collected for analyses.

Immunohistochemical analysis:

Paraffin embedded 5-μm esophageal biopsy sections of non-EoE and EoE patients were immunostained with antiserum against MBP protein using IHC staining methods previously described. [22-23] In brief, endogenous peroxidase in the tissues was quenched with 0.3% hydrogen peroxide in methanol followed by non-specific protein blocking using 3% normal goat serum. Tissue sections were then incubated with primary antibodies anti-MBP followed by a 1:250 dilution of biotinylated goat anti-rat IgG or goat anti-mouse IgG secondary antibody and avidin-peroxidase complex (Vector Laboratories, Burlingame, CA) for 30 minutes each. Slides were further developed with nickel diaminobenzidine-cobalt chloride solution to form a black precipitate, and counterstained with nuclear fast red. The positive-stained cells were quantified by counting in each tissue section with the assistance of digital morphometry using the Metamorph Imaging System (Universal Imaging Corp, West Chester, PA) and expressed as number of positive cells/mm² tissue area as described earlier.

mRNA analysis of ANF and alpha-SA:

Total ribonucleic acid (RNA) was extracted from the mouse heart using Trizol reagent (GIBCO-BRL, Grand-Island, NY) following the manufacturer’s protocol. Twenty micrograms of total RNA from each sample were electrophoresed on formaldehyde-1.5% agarose gel and transferred to GeneScreen hybridization membrane (DuPont/NEN) with 10X toxic granules in their cytoplasm [4] that contain high concentrations of hydrolases and cationic and basic proteins [5], [6]. Additionally, some drugs (anticonvulsants, non-steroidal anti-inflammatory drugs, antimicrobial agents, sulfonamides) are well known to trigger an abnormal production of eosinophils. Eosinophilia may be associated to drug-induced hypersensitivity reaction and following withdrawal usually eosinophil count normalized within 7–10 days [7-13]. Drug-related eosinophilia is associated with a severe tissue damage and can be observed as drug rash with eosinophilia [14]. Smallpox or diphtheria/tetanus vaccination is reported to induce eosinophilic myocarditis [15], [16]. Other etiologies are SSC. The membrane was UV crosslinked and prehybridized at 42C for one hour in a 50% formamide buffer (pH7.5), containing 10% dextran sulfate, 5X SSC, 1X Denhardt’s solution, 1% (wt/vol) SDS, 100 ug/ml of herring sperm DNA, and 20 mM Tris. The mouse 32p cDNA probes were prepared for ANF and alpha-SA following previously described methods (32) and hybridized overnight at 42C using 1-2X10⁶ dpm/ml of the respective probes. The membrane was washed for 20 min at 42C, 20 min at 50C, 20 min at 60C in 2X SSC-0.1% SDS and 20 min in 0.1X SSC-0.1% SDS.

Statistical analysis

All data were analyzed using GraphPad Prism 5.0 software (GraphPad, San Diego, CA) and the statistical analysis performed by the nonparametric Mann–Whitney U-test was employed for comparison of data between two groups. Parametric data were compared using t -tests analysis. Values are reported as mean ± S.D. P - values < 0.05 were considered statistically significant.

Results

The IL-5 transgene induces peripheral blood eosinophilia and moderate eosinophil trafficking to the heart.

We were interested in establishing a mouse model that induces eosinophil trafficking to the heart. Therefore, as an initial analysis, we investigated eosinophil levels in the heart of mice transgenic for IL-5 under the control of the T cell promoter CD2 (24) (since the mice had massively elevated peripheral blood eosinophilia). These transgenic mice were found to have a modest accumulation of eosinophils compared to wild-type (Figures 1 and 3). The number of eosinophils in the blood of IL-5 transgenic and wild-type control mice were measured in parallel and IL-5 transgenic mice exhibited an approximately 20-fold increase in the blood.

Figure 1.

A representative photomicrograph of eosinophil infiltration in the heart of IL-5 transgenic mice, (a) shows eosinophils in the epicardial tissue of the right atrium (RA). and (b) shows eosinophils in the myocardium of the left ventricle (LV). The black dots represent eosinophils immunostained with anti-MBP antibodies.

Figure 3.

Eosinophil levels in 2 different regions of the heart of IL-5 transgenic and eotaxin-deficient IL-5 transgenic mice.

Allergen challenge increases peripheral blood eosinophilia and eosinophil trafficking into the heart of mice.

We were next interested in developing a model of allergen-induced pulmonary inflammation that would increase blood eosinophilia and allow the analysis of eosinophil recruitment to the heart during inflammatory events. In order to induce peripheral blood eosinophilia, wild-type and eotaxin gene-deficient mice were repeatedly challenged with intranasal inoculations of ovalbumin (OVA) antigen using a well-established protocol (21). We hypothesized that intranasal OVA delivery to sensitized mice would result in a strong systemic Th2 response and induce peripheral blood eosinophilia and that this might promote eosinophil trafficking to the heart as occurs in IL-5 transgenic mice. The sensitized mice challenged with saline did not show any inflammation in the heart or lung. However, the OVA challenged mice developed reproducible peripheral eosinophilia, pulmonary (Figure 2) and cardiac eosinophilia in eotaxin-deficient mice. Eotaxin gene-deficient mice following OVA challenge have approximately 0.2±0.07 eosinophil/mm² in epicardial and 0.12±0.05 eosinophil/mm² in myocardial tissue in comparison to undetectable eosinophil in OVA challenged wild-type mice.

Figure 2.

OVA-Challenged mice show an increased blood and lung eosinophilia.

The healthy heart is devoid of eosinophils; however, eotaxin gene-deficient IL-5 transgenic mice have a high accumulation of eosinophils in the heart.

We have evaluated the level of eosinophils in the hearts of wild type mice (Balb/c anSvEv129) at baseline using anti-MBP immunohistochemistry. In contrast to the blood compared to IL-5 transgenic mice that had wild-type eotaxin. Collectively, these data suggest that the elevation in circulating eosinophils even in baseline eotaxin is sufficient to induce cardiac eosinophilia.

Eotaxin gene-deficient IL-5 transgenic mice develop cardiac hypertrophy.

In order, to further evaluate eosinophil-induced alterations of heart function in our transgenic mouse model, we performed working heart analysis (Lan gendorff heart analysis) in IL-5 transgenic and eotaxin-deficient IL-5 transgenic mice as per the protocol described without early diagnosis and appropriate treatment [26-30]. In the current study, we show cardiac abnormalities and diffuse ST-segment elevation in allergic or IL-5 transgenic mice have left ventricular systolic dysfunction with wall motion abnormalities following ECG analysis. This endocardial inflammation will develop delayed-enhancement sequences by extensive eosinophilic infiltrates. Eosinophilic myocarditis resulting from a earlier (55-57). We observed that eotaxin-deficient IL-5 transgenic mice had larger cardiac hypertrophy The early phase of cardia eosinophilia is characterized by an eosinophilic endomyocarditis with eosinophil [19], in which eosinophils degranulate and release toxic cationic proteins and promotes tissue necrosis. Clinical reports and presented in vivo experimental data indicates eosinophilic myocarditis is infrequent during induction of IL-5 by allergic reaction or genetically manumulation of transgene [25]. In the present study we present the evidence of supraventricular tachycardia, non-specific ST-segment anomalies or conduction delays by performing Electrocardiography (ECG) on mice that overexpressed IL-5. Echocardiography reveals and increased cardiac left ventricular function (dPt/dt max) in comparison to IL-5 transgenic mice alone (Table 1). Furthermore, cardiac hypertrophy in IL-5 transgenic and eotaxin-deficient IL-5 transgenic mice was confirmed by performing echocardiography. Our presented data confirmed that eotaxin-deficient IL-5 transgenic mice had significantly increased left ventricle mass (cardiac hypertrophy) (Figure 4), whereas both transgenic mice showed reduced left ventricular chamber dimensions during both systolic and diastolic functioning (data not shown). This may be associated with induced blood eosinophilia in IL- and gastrointestinal tract (6), eosinophils were barely detected in the heart, only it is found induced when tissue specific IL-15 is induced in CD2-IL-5 transgenic mice. Further when we examined, the relationship between IL-5 and eotaxin in regulating eosinophils in the heart. In eosinophils chemoattraction gene to address the role of eotaxin in the accumulation of eosinophils in cardiac tissue, we observed that IL-5 transgenic mice deficient in eotaxin in the heart. We were surprised to learn that eotaxin-deficient IL-5 transgenic mice had high eosinophil accumulation in each compartment of the heart compared to IL-5 transgenic mice. These data suggest that eotaxin deficiency plays a critical role in the increased trafficking of eosinophils to the heart (Figure 3). In contrast to the heart, eotaxin-deficient IL-5 transgenic mice have a significantly reduced level of eosinophils within the gastrointestinal tract in comparison to IL-5 transgenic mice that contained wild-type eotaxin (6). Interestingly, the IL-5 transgenic mice that were deficient in eotaxin had a >40-fold higher level of circulating eosinophils compared to wild-type and a >2-fold increase in 5 transgenic and eotaxin-deficient IL-5.This indicates blood eosinophilia directly affect the cardiac function. Most importantly, we present the evidence that of induced cardiac development genes like atrial natriuretic factor (ANF), which is a natriuretic peptide hormone secreted from the cardiac atria and alpha-SA involved physiological hypertrophy, positive inotropy, ischemic preconditioning, and protection from cell death (Figure 5). Taken together, the induction of both genes indicates eosinophils associated damage in the cardia tissue. Data represented as Mean ± SEM (Standard Error of the Mean).

Table 1:

Working heart analysis of cardiac left ventricular function of WT and IL-5 transgenic mice (n = 8)

Parameters	Wild-type (WT)	CD2-IL5 Tg	% Change from WT	CD2-IL5T g/ EOTAXN-1 −/−	% Change from WT	% Change from IL5 Tg
Heart rate (beats per min)	413 ± 2	416 ± 3	Paced	406 ± 1	Paced (−2*)	Paced (−2**)
-dP/dt (mmHg/sec)	5083 ± 183	4612 ± 213	−9	5915 ± 154	+16**	+28***
tau (msec)	28 ± 1	51 ± 13	+82*	22 ± 1	−21	−57**
Langendorff Heart	(N=8, 30)	(N=8,30)		(N=8, 30)
heart rate (beats per min)	404 ± 1	417 ± 3	Paced (+3***)	406 ± 1	Paced	Paced (−3***)
-dP/dt (mmHg/sec)	2038 ± 41	2065 ± 49	+1	2758 ± 25	+35***	+34***

Figure 4.

Left ventricle mass of Wild-type, IL-5 transgenic and eotaxin deficient IL-5 transgenic mice.

Figure 5.

Northern blot analysis of ANF and alpha-SA (cardiac developmental genes) in the heart of wld-type. IL-5 tg and eotaxin-deficient IL-5 transgenic mice.

Intranasal OVA increases heart-body ratio in eotaxin gene-deficient mice.

Using our model of OVA induced Th2-associated peripheral blood eosinophilia; we followed eosinophil trafficking to the heart. In this experiment, we used both wild-type and eotaxin gene-deficient mice to study eosinophil-induced alterations in the heart of mice. Our hypothesis was that eotaxin gene-deficient mice would induce cardiac hypertrophy, following intranasal OVA inoculation in OVA-alum-sensitized mice. Therefore, we first examined peripheral blood eosinophil levels and pulmonary inflammation in OVA- or saline-challenged wild-type and eotaxin gene-deficient mice and second, examined the heart-body ratio. Our preliminary data shows increased peripheral blood eosinophilia (Figure 6a) and increased heart-body ratio in OVA-challenged eotaxin gene-deficient mice. Interestingly, no significant change was observed in the heart-body ratio of allergen-challenged wild-type mice (Figure 6b).

Figure 6.

(A) OVA-induced increase of blood eosinophilia in eotaxin-deficient mice; (B) Ova-induced alterations in the Heart-Body ratio of wild-type and eotaxin gene-deficient mice.

Discussion

an increased left ventricular wall thickness because of interstitial myocardial oedema (Table 1). Histological analysis indicates the induction of eosinophilic myocarditis in IL-5 overexpressed mice. Indeed, histological sections show eosinophilic infiltration of the endocardium and subendocardial interstitial myocardial necrosis and sometimes eosinophilic granulomas (Figure 1). Reports indicated that acute narcotizing eosinophilic myocarditis represents the most severe form of acute eosinophilic heart disease and may be rapidly fatal hypersensitivity mechanism due to elevated blood eosinophil counts. This will be also reported earlier even in patients with hypereosinophilia at the time of disease induction that results from the migration of circulating eosinophils [31-33]. Mechanisms that explain why, in sustained eosinophilia, the heart is particularly targeted by eosinophils are not well understood. Nonetheless, a profound eosinophilic infiltration of the interstitial compartment is very deleterious to cardiac tissues. Thus, based on previous clinical reports and current experimental findings that white blood count examinations are important in case of myocarditis. Corticosteroid therapy inhibits the degranulation of eosinophils; therefore, it may be the first-line of treatment for eosinophilic myocarditis in order to limit myocardial necrosis.

In conclusion, we propose that in the absence of etiology, the first-line of treatment may be antihelminthic therapy, corticosteroid therapy and anticoagulant therapy for eosinophils associated cardiac myopathy. The goal of these therapeutics is to rapidly lower the eosinophil count and prevent thromboembolic events. Eosinophilic coronary periarteritis is a rare isolated eosinophilic injury localized to epicardial coronary arteries, which is responsible for vasospastic angina.