Autoantibodies to IgE can induce the release of proinflammatory and vasoactive mediators from human cardiac mast cells

Mast cells are multifunctional immune cells with complex roles in tissue homeostasis and disease. Cardiac mast cells (HCMCs) are strategically located within the human myocardium, in atherosclerotic plaques, in proximity to nerves, and in the aortic valve. HCMCs express the high-affinity receptor (FcεRI) for IgE and can be activated by anti-IgE and anti-FcεRI. Autoantibodies to IgE and/or FcεRI have been found in the serum of patients with a variety of immune disorders. We have compared the effects of different preparations of IgG anti-IgE obtained from patients with atopic dermatitis (AD) with rabbit IgG anti-IgE on the release of preformed (histamine and tryptase) and lipid mediators [prostaglandin D2 (PGD2) and cysteinyl leukotriene C4 (LTC4)] from HCMCs. Functional human IgG anti-IgE from one out of six AD donors and rabbit IgG anti-IgE induced the release of preformed (histamine, tryptase) and de novo synthesized mediators (PGD2 and LTC4) from HCMCs. Human IgG anti-IgE was more potent than rabbit IgG anti-IgE in inducing proinflammatory mediators from HCMCs. Human monoclonal IgE was a competitive antagonist of both human and rabbit IgG anti-IgE. Although functional anti-IgE autoantibodies rarely occur in patients with AD, when present, they can powerfully activate the release of proinflammatory and vasoactive mediators from HCMCs.

Recent epidemiological studies have reported an increased risk of coronary artery disease and/or heart failure in patients with IgE-mediated allergic disorders [50][51][52]. Moreover, increased IgE levels are associated with atherosclerosis [53], and the IgE-FcεRI network has been implicated in pathological cardiac remodeling and dysfunction [54].
In this study, we first examined the effects of functional and non-functional human IgG anti-IgE obtained from patients with atopic dermatitis (AD) on the release of preformed and de novo synthesized mediators from HCMCs. Second, we compared the effects of functional human IgG anti-IgE and rabbit IgG anti-IgE on the release of histamine and lipid mediators from cardiac mast cells. Finally, we evaluated whether human monoclonal IgE can antagonize the activating properties of human and rabbit IgG anti-IgE.

Atopic dermatitis patients
This study was approved by the Ethics Committee of the University of Naples Federico II, School of Medicine (Protocol N. 198/18), and informed consent was obtained from participants prior to the collection of blood specimens according to recommendations from the Declaration of Helsinki. Peripheral blood was obtained from six AD patients (aged 5-17 years) with similar clinical pictures (e.g., chronic pruritic skin erythema, papules, or lichenification of flexural areas of the extremities, face and neck) [79]. Blood samples were obtained from these patients not taking any drug for at least 1 week.

Purification of human IgG anti-IgE antibody
Serum from six AD patients and comparable high levels of IgG antibodies to anti-IgE were passed through the immunosorbent Sepharose column coated with purified IgE (ADZ). Immunosorbent-bound IgG with anti-IgE activity were collected. IgE content was less than 0.05 U/ml [59].

Purification of human monoclonal IgE and polyclonal IgG
IgE myeloma protein was purified from a myeloma patient (ADZ) as previously described [80][81][82]. No IgG, IgM, or IgA contamination was detected by immunoassays [83]. Human polyclonal IgG were purified from the serum of five healthy donors and two patients with AD as previously described [81,84].

Isolation of human cardiac mast cells
This study was approved by the Ethics Committee of the University of Naples Federico II, School of Medicine (Protocol N. 7/19). Heart tissue was obtained from patients undergoing heart transplantation as previously described [31,74]. The explanted heart was finely minced into 2-5 mm fragments and subjected to enzymatic dispersion [31]. HCMCs were partially purified by flotation through a discontinuous Percoll gradient yielding a population of mast cell purity ranging from 0.3% to 26%. HCMCs were further purified using a CD117 MicroBead kit sorting system (Miltenyi Biotec, Bologna, Italy). Mast cell purity using these techniques ranged from 29 to 61% as assessed by Alcian blue staining [85].

Statistical analysis
Data were analyzed with the GraphPad Prism 8 software package (GraphPad Software, La Jolla, CA, USA). Values are expressed as mean ± SEM (standard error of the mean). Statistical analysis was performed using Student's t-test or one-way analysis of variance [92]. Correlations between two variables were assessed by Spearman's rank correlation analysis and reported as coefficient of correlation (r). Values of p ≤ 0.05 were considered significant.

Effects of human and rabbit IgG anti-IgE on histamine release from HCMCs
We first compared the effects of increasing concentrations of IgG anti-IgE purified from the sera of six patients with AD, and rabbit IgG anti-IgE on the release of histamine from seven different preparations of HCMCs. Figure 1 shows that one preparation of human IgG anti-IgE (10 -2 to 1 μg/ml) isolated from AD patient [58] triggered histamine release from HCMCs from seven different donors. By contrast, five preparations of IgG anti-IgE isolated from different AD patients did not induce histamine release from HCMCs. In the same experiments, we also evaluated the effects of rabbit IgG anti-IgE (3 × 10 -2 to 3 μg/ml), which also induced a concentration-dependent release of histamine (Fig. 1). The maximal percent histamine release (HR MAX ) of HCMC response to functional human anti-IgE (26.6% ± 1.15%) was similar to mast cell reactivity to rabbit anti-IgE (26.0% ± 1.11%) ( Table 1). By contrast, the threshold sensitivity (HR SENS ) [i.e., the secretagogue concentration inducing half-maximal histamine release (EC 50 )] induced by functional human anti-IgE (4.2 × 10 -2 ± 5 × 10 -3 μg/ml) was lower than HR SENS caused by rabbit anti-IgE (4.6 × 10 -1 ± 5 × 10 -2 μg/ ml) (p < 0.005) ( Table 2). These results indicate that a 1 3 preparation of human IgG anti-IgE (hereafter referred to as "human anti-IgE") is more potent than rabbit IgG anti-IgE in inducing histamine release from HCMCs.

Effects of human and rabbit IgG on lipid mediators from HCMCs
Cysteinyl leukotrienes (LTC 4 , LTD 4 , and LTE 4 ) are derived from arachidonic acid (AA) through the 5-lipoxygenase (5-LO) pathway [41]. Upon cell activation, cytosolic phospholipase A 2 (cPLA 2 ) cleaves phospholipids at the outer nuclear membrane to generate free AA. 5-LO then oxidizes AA in the presence of 5-LO activating protein to generate leukotriene A 4 (LTA 4 ), which is subsequently converted to LTC 4 by LTC 4 synthase [41,93]. Activated HCMCs metabolize AA through the 5-LO to form LTC 4 and through the cyclo-oxygenase to form PGD 2 [31,74]. In seven experiments, we compared the effects of human anti-IgE (3 × 10 -2 to 1 μg/ml) and rabbit anti-IgE (3 × 10 -2 to 3 μg/ml) on the de novo synthesis of LTC 4 and PGD 2 from HCMCs. Figure 3A shows that both human and rabbit anti-IgE caused a concentration-dependent release of LTC 4 from HCMCs. The maximal LTC 4 release induced by human anti-IgE was similar to that caused by rabbit anti-IgE (Table 1). However, the LTC 4 release induced by each concentration of human anti-IgE tested was significantly higher than that caused by rabbit anti-IgE (Fig. 3A). Accordingly, the EC 50 for LTC 4 release was significantly lower for human anti-IgE (6.7 × 10 -2 ± 1.4 × 10 -2 μg/ml) compared to rabbit anti-IgE (4.7 × 10 -1 ± 3.6 × 10 -2 μg/ml) (p < 0.0002) ( Table 2). Similar results were obtained when we compared the effects of increasing concentrations of human and rabbit anti-IgE on PGD 2 release from HCMCs (Fig. 3B, Tables 1 and 2).

Effects of human polyclonal IgG on mediator release from HCMCs
In these experiments, we evaluated the effects of increasing concentrations of human polyclonal IgG purified from five healthy donors on the release of preformed (histamine, tryptase) and de novo synthesized mediators (LTC 4 , PGD 2 ) from HCMCs. Figure 4 shows the results of these experiments indicating that a wide spectrum of concentrations (10 -2 to 10 μg/ml) of human polyclonal IgG failed to induce the release of proinflammatory and vasoactive mediators from HCMCs. Similar results were obtained when human polyclonal IgG purified from AD patients were incubated with HCMCs (data not shown). Figure 5A shows that there was a positive correlation between the release of two mediators (histamine and tryptase), which specifically reside in the secretory granules of human mast cells, induced by human anti-IgE from HCMCs (r = 0.79; p < 0.0001). These results suggest that these cells are a source of both mediators in the supernatants of anti-IgE-activated HCMCs. Similarly, there was a positive correlation between the release of histamine and LTC 4 (r = 0.89; p < 0.0001) (Fig. 5B), histamine and PGD 2 (r = 0.83; p < 0.0001) (Fig. 5C), and LTC 4 and PGD 2 (r = 0.83; p < 0.0001) (Fig. 5D). Comparable results were obtained when we examined the correlations between the release of different mediators induced by rabbit anti-IgE from HCMCs (Fig. 6).

Effects of human monoclonal IgE on human or rabbit anti-IgE-induced histamine release from HCMCs
The previous results are compatible with the hypothesis that human and rabbit anti-IgE induce the release of To support this hypothesis, we examined whether human monoclonal IgE purified from a myeloma patient (ADZ) [81] interfere with the activating properties of human and rabbit IgG anti-IgE. HCMCs were preincubated with increasing concentrations of monoclonal IgE and then incubated in the presence of graded concentrations of human or rabbit IgG anti-IgE. Figure 7 shows the results of a typical experiment showing that increasing concentrations of human monoclonal IgE shifted to the right the activating properties of both human (Fig. 7A) and rabbit anti-IgE (Fig. 7B) in a concentration-dependent manner without affecting the maximal release. The parallel shift to the right of the concentration-response curve induced by increasing concentrations of IgE on both human and rabbit anti-IgE was compatible with the hypothesis that human monoclonal IgE acted as a competitive inhibitor of both stimuli. Preincubation of HCMCs with higher concentrations (10 -1 and 1 μg/ml) of human polyclonal IgG did not modify the activating capacity of either human or rabbit anti-IgE to induce histamine release from mast cells (data not shown).
Autoantibodies to IgE and/or FcεRI can occur in patients with different inflammatory disorders such as CSU [56, 61-63, 65, 105-107] and AD [57][58][59][60]67]. In the vast majority of patients with CSU [56,65,105] and AD [58,62,108] autoantibodies to IgE and/or FcεRI lacked the capacity to activate mediator release from human basophils. To the best of our knowledge, we provide the first evidence that a functional preparation of human IgG anti-IgE can induce the release of preformed and de novo synthesized mediators from HCMCs.
Although the role of naturally occurring autoantibodies to IgE and/or FcεRI in inflammatory disorders is still a fascinating and unsettled issue [109], several investigators have documented their presence in CSU [55, 61-64, 105-107, 110], asthma [57,65,111], and in AD patients [57][58][59][60]67]. The vast majority of these studies have evaluated the effects of autoantibodies to IgE/FcεRI only on histamine release from human basophils [55, 56, 61-63, 105, 107, 110]. In most cases these autoantibodies lack the capacity to activate human basophils [56,58,62,65,105]. The above results did not rule out the hypothesis that naturally occurring autoantibodies to IgE and/or FcεRI can activate human mast cells to produce proinflammatory arachidonic acid metabolites.
Our results provide preliminary information on the prevalence of anti-IgE autoantibodies in AD patients. In this study, only one preparation of human IgG anti-IgE out of six patients with AD triggered the release of mediators from mast cells isolated from human cardiac tissue. The apparent low frequency of functional anti-IgE autoantibodies might explain the controversial results on the presence of functional autoantibodies in different types of AD patients [57-59, 62, 67]. Further studies with larger cohorts of AD patients will be necessary to estimate the prevalence of functional and non-functional anti-IgE and anti-FcɛRI autoantibodies in this heterogeneous immunologic disorder.
Our results also provide some insight into the potency of naturally occurring IgG autoantibodies anti-IgE. Although the HCMC reactivity to human IgG anti-IgE was similar to that of rabbit IgG anti-IgE, the potency of functional human anti-IgE was consistently higher than that of rabbit anti-IgE in inducing the release of preformed and de novo synthesized mediators from HCMCs.
Our results also demonstrate that when the human anti-IgE is functionally active, it can work as a complete Fig. 6 A Correlation between the percent histamine release and tryptase secretion induced by rabbit anti-IgE from human cardiac mast cells (HCMCs). B Correlation between the percent histamine and LTC 4 release induced by rabbit anti-IgE from HCMCs. C Correlation between histamine and PGD 2 release induced by rabbit anti-IgE from HCMCs. D Correlation between LTC 4 and PGD 2 release induced by rabbit anti-IgE from HCMCs secretagogue inducing the release of a wide spectrum of proinflammatory, vasoactive, and immunomodulatory mediators from HCMCs. For instance, histamine exerts profound cardiovascular effects in humans [70,71] and tryptase, the most abundant secretory granule protein in human mast cells [112], stimulates collagen production by fibroblasts [113]. LTC 4 is detrimental for atherosclerosis and myocardial infarction [75] and dilated cardiomyopathies [10]. PGD 2 is also detrimental for the cardiovascular and respiratory systems [69,76].
The presence of spontaneously occurring autoantibodies to IgE/FcεRI has been described by several investigators since the mid-80s, but the relevance of these observations continues to generate some controversies in the field [56,109]. We still do not know the prevalence and functional relevance of IgG autoantibodies to IgE and FcεRI in different inflammatory disorders [114]. To the best of our knowledge, autoantibodies to IgE have not yet been investigated in patients with cardiovascular diseases. Interestingly, serum IgE concentrations are increased in patients with myocardial infarction [101,102], coronary artery disease [100], and heart failure [54]. Moreover, epidemiological studies have found an increased risk of coronary artery disease and/or heart failure in patients with IgE-mediated allergic disorders [50][51][52]. Increased IgE levels are associated with atherosclerosis [53], and the IgE-FcεRI network has been implicated in pathological cardiac remodeling and dysfunction [54]. Finally, it has been reported that serum IgE concentrations were higher in a preclinical model of heart failure [54]. Omalizumab, a monoclonal antibody (mAb) anti-IgE, is highly effective in patients with CSU [115] and severe asthma with high levels of IgE [116][117][118]. Future studies should investigate the effects of omalizumab in preclinical models of heart failure associated with high serum IgE [54]. Further clinical and experimental studies are needed to investigate the presence and functional activity of autoantibodies to IgE and/or FcεRI in patients with different cardiovascular diseases.
Increasing concentrations of human monoclonal IgE concentration-dependently shifted to the right the activating properties of both human and rabbit anti-IgE. These results are compatible with the hypothesis that soluble monoclonal IgE was a competitive antagonist of both human and rabbit antibodies to IgE. The specificity of this observation was supported by the finding that human polyclonal IgG did not interfere with the capacity of either human and rabbit anti-IgE to induce histamine secretion from HCMCs.
Our study has several limitations that should be pointed out. The experiments were performed using primary mast cells isolated from myocardial tissue obtained from patients undergoing heart transplantation. These mast cells might have biochemical characteristics different from those of cells obtained from healthy donors. In the past we addressed this issue by comparing the release of mediators from mast cells isolated from failing hearts and from subjects who died without cardiovascular diseases [31]. In this study, we found quantitative, but not qualitative differences in the release of mediators from "normal" cardiac mast cells when compared with those from explanted hearts. In addition, our experiments were performed with partially purified (29-61%) HCMCs. There is the possibility that subsets of contaminating cells expressing (e.g., macrophages, monocytes, dendritic cells, basophils) or non-expressing FcεRI (e.g., fibroblasts, cardiomyocytes) may have directly or indirectly affected some of our results. However, the excellent correlations between the release of histamine and tryptase, exclusively present in mast cells, and other mediators (i.e., LTC 4 , PGD 2 ) induced by human and rabbit anti-IgE, suggest that HCMCs are the targets of these antibodies.
Our results might have translational relevance in several cardiovascular disorders. Cardiac mast cells seem to play a role in experimental myocarditis [98,119,120], myocardial infarction [30], and post-ischemic myocardial remodeling [30,121]. In humans, cardiac mast cells might play a role in human dilated cardiomyopathies [31], aortic valve stenosis [122], different phases of atherosclerosis [123], and autoimmune myocarditis [124]. Moreover, cardiac mast cell activation has been suggested to be implicated in the severe clinical presentations of anaphylaxis (e.g., hypotension, arrhythmias, ventricular dysfunction) [125,126]. Our results indicating that IgG autoantibodies to IgE from some patients with allergic disorders potently activate HCMCs might explain, at least in part, the cardiovascular involvement in patients with anaphylaxis or severe asthma [125,127,128].
In conclusion, although functional autoantibodies to IgE rarely occur in patients with AD, when these antibodies are present, they are able to trigger the release of proinflammatory and vasoactive mediators from HCMCs.
Acknowledgements The authors would like to thank the administrative staff (Dr. Roberto Bifulco, Dr. Anna Ferraro and Dr. Maria Cristina Fucci) of CISI without whom it would not be possible to work as a team.
Funding Open access funding provided by Università degli Studi di Napoli Federico II within the CRUI-CARE Agreement. This work was supported in part by grants from the CISI-Lab Project (University of Naples Federico II) and TIMING Project and Campania Bioscience (Regione Campania).

Data availability
The data presented in this study are available on request from the corresponding authors.

Conflict of interest
The authors declare that they have no conflict of interest.

Consent for publication
Patients signed informed consent regarding publishing their data.

Ethics approval
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee) of the University of Naples Federico II (protocols N.198/18 and 7/19) for studies involving humans.
Informed consent Informed consent was obtained from all individual participants included in the study.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.