Superantigenic Activation of Human Cardiac Mast Cells

B cell superantigens, also called immunoglobulin superantigens, bind to the variable regions of either the heavy or light chain of immunoglobulins mirroring the lymphocyte-activating properties of classical T cell superantigens. Protein A of Staphylococcus aureus, protein L of Peptostreptococcus magnus, and gp120 of HIV are typical immunoglobulin superantigens. Mast cells are immune cells expressing the high-affinity receptor for IgE (FcεRI) and are strategically located in the human heart, where they play a role in several cardiometabolic diseases. Here, we investigated whether immunoglobulin superantigens induced the activation of human heart mast cells (HHMCs). Protein A induced the de novo synthesis of cysteinyl leukotriene C4 (LTC4) from HHMCs through the interaction with IgE VH3+ bound to FcεRI. Protein L stimulated the production of prostaglandin D2 (PGD2) from HHMCs through the interaction with κ light chains of IgE. HIV glycoprotein gp120 induced the release of preformed (histamine) and de novo synthesized mediators, such as cysteinyl leukotriene C4 (LTC4), angiogenic (VEGF-A), and lymphangiogenic (VEGF-C) factors by interacting with the VH3 region of IgE. Collectively, our data indicate that bacterial and viral immunoglobulin superantigens can interact with different regions of IgE bound to FcεRI to induce the release of proinflammatory, angiogenic, and lymphangiogenic factors from human cardiac mast cells.


Introduction
The term "superantigen" (SAg) refers to several proteins synthesized by a variety of bacteria and viruses that not only mimic, but also exceed the activity of conventional antigens in activating T and B cells [1][2][3][4][5]. Typical antigens are processed by antigen-presenting cells (APCs) into small peptides that bind a distal groove in the molecules of the major histocompatibility complex (MHC) [6]. The peptide: MHC (p:MHC) complex on the APC surface acts as a ligand of both T cell receptor (TCR) α and TCR β variable domains on a few specific T cell clones. By contrast, SAgs bind directly to the lateral surfaces of the MHC class II molecules and to the Vβ domain of the TCR and thus bypass the processing and presentation of conventional antigens by APCs [7][8][9][10]. As a result, conventional antigens stimulate less than 1 in 10,000-100,000 T cells, while SAgs can stimulate up to 20% of all T cells [1,3]. A wide range of diseases from autoimmune and allergic disorders, neoplasia, and immunodeficiencies can be associated with SAgs [11][12][13][14][15].
In addition to classical T cell Sags, there are also B cell SAgs endowed with immunoglobulin (Ig)-binding capacity. In contrast to conventional antigens, which bind to both the heavy and light chain variable (V)-domains of Igs, B cell SAgs bind to the conserved sides of either the heavy (H)-or light (L)-chain [16][17][18], resulting in a massive proliferation of B cells. Staphylococcus aureus (S. aureus) is a source of several T cell SAgs (S. aureus enterotoxins: SE) [19]. Two staphylococcal B cell SAgs, S. aureus protein A and SEA, bind specifically to V H 3 domain of human Igs, whereas SED, which is also a T cell SAg, binds to V H 4 [11]. V H 3 is the largest of human Ig germline V H families; thereby, protein A can stimulate almost half of the B cells in the circulation [17]. Protein A is the archetypal B cell SAg and contains five homologous repeated domains, each of which can bind to all or most of the V H 3 + Igs. S. aureus is a common pathogen causing toxic shock syndrome and endocarditis [20,21]. Most of clinical isolates of S.aureus synthesize protein A, which can be released from the cell wall [22]. Protein A has two binding sites for human Igs: the classical site binds Fcγ, a constant region of IgG [23] and an alternative site that binds the Fab portion of 15% to 50% of human polyclonal IgG, IgM, IgA, and IgE [24].
Similarly, glycoprotein 120 (gp120) of HIV-1 is a viral B cell SAg, because it interacts with Ig V H 3 + [25,26]. The entry of HIV into host cells is mediated the interaction of viral glycoprotein [27] gp120 with CD4 [28] and chemokine receptors on the cell surface [29,30]. HIV gp120 is a member of the Ig SAg family [31][32][33]. Emergence of cardiovascular disease has become a leading concern for patients with HIV infection [34,35].
Protein L is a cell wall protein synthesized by Peptostreptococcus magnus (P. magnus) [36]. Protein L is a multi-domain protein that binds to some κ light chain variable domain without interfering with the antigen-binding site [37,38]. Protein L binds to the V domain of the κ light chains of Igs [39][40][41]. In particular, protein L binds with high affinity (~10 10 M −1 ) only human Vk I, Vk III and Vk IV subtypes, but does not interact with Vk II subtype [42].
This study has been undertaken to evaluate whether bacterial (protein A and protein L) and viral (gp120) superantigens induce the release of proinflammatory, angiogenic, and lymphangiogenic factors from human cardiac mast cells.

Effect of Human IgG Anti-IgE on Mediator Release from HHMCs
We have previously reported that IgG anti-IgE purified from the serum of a small percentage of atopic dermatitis patients can induce histamine and LTC 4 release from human basophils [86]. The activating property of human IgG anti-IgE (H-aIgE) is mediated by the interaction with membrane-bound IgE on human basophils. Therefore, we used this human autoantibody to activate human heart mast cells (HHMCs) in vitro. Figure 1 shows that H-aIgE (10 −2 to 3 µg/mL) induced a concentration-dependent histamine release from five different preparations of HHMCs. Four preparations of IgG (10 −2 to 3 µg/mL) purified from the serum of normal donors did not cause histamine release (data not shown). These results suggest that mast cells isolated from human heart express IgE bound to FcεRI. Vascular endothelial growth factors (VEGFs) are involved in new vessel formation and play a central role in cardiac pathophysiology [87]. Therefore, we evaluated the release of angiogenic (VEGF-A) and lymphangiogenic factors (VEGF-C) induced by H-aIgE from HHMCs. Figure 2 shows that H-aIgE induced a concentration-dependent release of both VEGF-A and VEGF-C from four different preparations of HHMCs.  Figure 3A shows that protein A induced a concentration-dependent release of LTC 4 from four different preparations of HHMC. To evaluate the mechanism by which protein A activates HHMCs, it was preincubated with human monoclonal IgM possessing different V H domains. Figure 3B shows that human monoclonal IgM V H 3 + dose-dependently inhibited the LTC 4 -releasing activity of protein A. By contrast, human monoclonal IgM V H 6 + had no inhibitory effect. These findings are compatible with the hypothesis that protein A activates HHMCs through the binding to IgE V H 3 + bound on FcεRI. We have previously found that P. magnus and protein L activate human basophils and mast cells [39,41]. Figure 4A shows that increasing concentrations of protein L induced de novo synthesis of PGD 2 from HHMCs. The activating property of protein L (100 nM) was inhibited by preincubation with increasing concentrations (0.1 to 3 µg/mL) of human monoclonal IgE k, but not by two human monoclonal IgE λ ( Figure 4B). These results are compatible with the hypothesis that protein L activates HHMCs through the interaction with the k light chain of IgE on cardiac mast cells.  Figure 5 shows the results of four independent experiments in which we incubated HHMCs with recombinant gp120. These experiments demonstrated that increasing concentrations of gp120 stimulated the release of histamine ( Figure 5A) and the de novo synthesis of LTC 4 from HHMCs ( Figure 5B). Preincubation of gp120 (30 nM) with increasing concentrations (0.1 to 3 µg/mL) of human monoclonal IgE V H 3 + inhibited the releasing activity of gp120 (data not shown). These results indicate that gp120 activates HHMCs by interacting with IgE V H 3 + bound to FcεRI. We then cultured HHMCs with increasing concentrations of recombinant gp120 (10 to 60 nM) for 6 h at 37 • C. At the end of this incubation the release of VEGF-A and VEGF-C was assayed in the supernatants of mast cells. Figure 6 shows the results of three preparations of HHMCs, indicating that gp120 induced the release of angiogenic (VEGF-A) and lymphangiogenic (VEGF-C) factors from HHMCs.

Discussion
This study shows that primary mast cells isolated from human myocardial tissue can be activated by a human IgG anti-IgE isolated from the serum of a patient with atopic dermatitis. These results are compatible with the hypothesis that HHMCs bind IgE, which has a role not only in allergic diseases [53,88] but also in several cardiovascular disorders [89][90][91]. Bacterial (protein A and protein L) and viral (gp120) superantigens can activate HHMCs to release a variety of proinflammatory (histamine, LTC 4 , PGD 2 ), angiogenic (VEGF-A), and lymphangiogenic (VEGF-C) mediators. The releasing activity of protein A and gp120 appears to be mediated by interaction with the V H 3 region of IgE on HHMCs. By contrast, protein L of P. magnus activates HHMCs by interaction with the κ light chains of IgE on cardiac mast cells. Our findings provide evidence, to our knowledge for the first time, that the immunologic (human IgG anti-IgE) and superantigenic activation of human myocardial mast cells can induce the release of angiogenic and lymphangiogenic factors.
Serum IgE levels are elevated in patients with myocardial infarction [90,91] and coronary artery disease compared to controls [89]. Moreover, IgE and FcεRI are overexpressed in human atherosclerotic lesions. These findings suggest that mast cells and perhaps other immune cells expressing FcεRI (e.g., dendritic cells, macrophages, basophils, platelets) [89,99,100] could play a role in the pathogenesis of human atherosclerosis. Previous studies have demonstrated that autoantibodies anti-IgE and anti-FcεRI can occur in several immunologic disorders [86,[101][102][103][104]. In this study we found that a human IgG anti-IgE induced the release of histamine, VEGF-A, and VEGF-C from HHMCs. To our knowledge this is the first evidence that cross-linking of IgE on human myocardial mast cells can induce the release of angiogenic factors. Angiogenesis, the process by which new capillaries develop from the pre-existing vasculature [105], plays a central role in cardiac pathophysiology [87,106]. VEGF-A is a pivotal mediator in angiogenesis and is synthesized by several immune cells [61,79,81,82,[107][108][109][110].
The possibility that human cardiac mast cells can contribute to myocardial angiogenesis, a process of major relevance in cardiac pathophysiology [106], requires further investigations.
The mammalian heart is rich of lymphatic vessels [111,112] and their number is increased in human heart following myocardial infarction, in atherosclerosis lesions, and in endocarditis [113,114]. The involvement of VEGF-C in salt-sensitive hypertension [115,116] and in coronary artery development [117] further add to the implications of lymphangiogenic factors in cardiovascular diseases [112]. Our results provide the first indication to our knowledge that immunologic and superantigenic activation of HHMCs leads to the production of VEGF-C, a major selective mediator of lymphangiogenesis [112].
S. aureus is an important human pathogen implicated in sepsis and endocarditis [118], and sepsis is a risk factor for cardiac arrhythmias [119]. This study demonstrates that protein A induces the release of LTC 4 from HHMCs through the interaction of the V H 3 region of IgE. These results extend on the previous observation that protein A induces the in vitro release of histamine from HHMCs [40]. Recently, it has been reported that in vivo challenge with protein A resulted in fatal anaphylaxis involving V H 3 + immunoglobulin interaction on mast cells and basophils [120]. Given the relevance of histamine and cysteinyl leukotrienes in heart pathophysiology [121][122][123], our results might explain, at least in part, how S. aureus can cause heart damage in patients with sepsis.
Protein L synthesized by P. magnus induces the de novo synthesis of PGD 2 from HHMCs, by interacting with the κ light chains of IgE on HHMCs. These results extend previous findings indicating that protein L induces the release of preformed histamine from HHMC [40]. Therefore, protein L is a complete secretagogue capable of releasing preformed and de novo synthesized mediators implicated in cardiovascular pathophysiology [121][122][123][124].
Our results provide the first indication that HIV gp120 activates HHMCs, thus acting as Ig SAg. Previous studies from our group have shown that gp120 induces the release of cytokines (IL-4 and IL-13) from human basophils [26]. Collectively, these findings support the hypothesis that virus-bound or shed gp120 [125] can function as a viral superantigen activating HMMCs and basophils to release proinflammatory mediators (histamine, LTC 4 ), cytokines (IL-4 and IL-13), and angiogenic/lymphangiogenic factors (VEGF-A and VEGF-C), thus contributing to the dysregulation of immune system in HIV infection. The successful rollout of anti-viral therapy ensured that HIV infection is managed as a chronic condition. Persistent inflammation and immune dysregulation associated with HIV leads to accelerated aging and cardiovascular diseases [34,35,126,127]. HIV-positive persons are, therefore, exhibiting increasing cardiovascular complications [34,35]. Our results, indicating that gp120 can induce the release of potent proinflammatory (histamine and LTC 4 ) mediators that exert cardiovascular effects [121][122][123] from myocardial mast cells, might explain, at least in part, how HIV can cause heart damage.
In this study we have identified several immunological, bacterial, and viral products that activate human cardiac mast cells through the interaction with IgE bound to FcεRI. However, mast cells can be activated by non-IgE-mediated stimuli such as cytokines (e.g., IL-33, SCF) [65,77,128], TLR ligands [60,129], and neuropeptides [52,130]. Additional studies are necessary to evaluate the effects of non-IgE-mediated stimuli on the release of proinflammatory mediators, angiogenic and lymphangiogenic factors from human cardiac mast cells.
Our study has a limitation which has to be pointed out. It was performed using primary mast cells isolated from myocardial tissue obtained from patients undergoing heart transplantation. Thus, these mast cells might have different characteristics from cells obtained from healthy donors. We have previously had the opportunity to address this important issue by comparing the release of mediators from mast cells isolated from failing hearts and from subjects who died in accidents without cardiovascular diseases [77]. 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 conclusion, our results demonstrate that bacterial and viral immunoglobulin superantigens can activate primary human cardiac mast cells to release vasoactive and proinflammatory mediators and angiogenic and lymphangiogenic factors. . Human IgG anti-IgE was purified from the serum of a patient with atopic dermatitis as described elsewhere [86].

Buffers
The Pipes (P) buffer used in these experiments was a mixture of 25 mM Pipes, 110 mM NaCl, 5 mM KCl, pH 7.37, referred to as P. P2CG, contains, in addition to P, 2 mM CaCl 2 and 1 g/L dextrose [49]; pH was titrated to 7.4 with sodium bicarbonate. PGMD contains 0.25 g/L MgCl 2 ·6H 2 O, 10 mg/L DNase, and 1 g/L gelatin in addition to P, pH 7.37.

Human Monoclonal IgM and IgE and Human Polyclonal IgG
Monoclonal IgM were purified from the serum of patients with Waldenström's macroglobulinemia as described elsewhere [77]. IgE myeloma proteins were purified from the serum of three patients described elsewhere [131,132]. Variable regions of these monoclonal IgM were determined using a panel of primary sequence-dependent V H and V K family specific reagents that identify framework regions [133]. Human polyclonal IgG were purified from the serum of healthy donors as described elsewhere [132].

Isolation of HHMCs
The study was approved by the Ethics Committee of the University of Naples Federico II (Protocol: Human MC No 7/19, 05/03/2009). The heart tissue was obtained from patients undergoing heart transplantation at the Deutsches Herzzentrum, Berlin, mostly for cardiomyopathy [77]. The explanted heart was immediately immersed in cold (4 • C) cardioplegic solution and was processed within 5 to 18 h of removal. Fat tissue, large vessels, and pericardium were removed. The tissue was finely minced into 2-5 mm fragments, suspended in P buffer (10 mL/g of wet tissue), and washed by centrifugation 3 times. After each centrifugation, the heart fragments were filtered through a 150 µm pore Nytex cloth (Tetko, Elmsford, NY, USA). Fragments were incubated (15 min, 37 • C) under constant stirring in P buffer containing 10 mg collagenase/g of wet tissue. At the end of the incubation the cell suspension was filtered through a 150 µm pore Nytex cloth and three additional cycles of enzymatic digestion were performed. After the last procedure, the cells were centrifuged (150× g, 22 • C, 8 min) and filtered through a 60 µm pore Nytex cloth to remove large particles and large cells (mostly myocytes). Lastly, cells were washed twice in PGMD by centrifugation (150× g, 22 • C, 8 min). Cell pellets were resuspended in P buffer containing 2% BSA and centrifuged (25× g, 22 • C, 2 min) to remove sedimented myocytes (>100 µm long). Supernatants containing endothelial cells, fibroblasts, and mast cells were then collected and centrifuged (150× g, 22 • C, 8 min). HHMC were partially purified by flotation through a discontinuous Percoll gradient [77]. The purity of these populations ranged from 0.1% to 18%. The enzymatic dispersion tissue yields ≈ 5 × 10 4 mast cells per gram of heart tissue. HHMCs were further purified using a CD117 MicroBead kit sorting system (Miltenyi Biotec, Bologna, Italy). Mast cell purities using these techniques ranged from 26% to 58% and was assessed by toluidine blue staining.

Histamine Release Assay
HHMCs (≈ 3 × 10 4 mast cells per tube) were resuspended in P2CG, and 0.3 mL of the cell suspension were placed in 12 × 75 mm polyethylene tubes and warmed to 37 • C; 0.2 mL of each prewarmed releasing stimulus was added, and incubation was continued at 37 • C for 45 min [39]. The reaction was stopped by centrifugation (1000× g, 22 • C, 2 min), and the supernatants were stored at −80 • C for subsequent assay of histamine, LTC 4 , and PGD 2 content. The cell-free supernatants were assayed for histamine with an automated fluorometric technique [134]. To calculate histamine release as a percentage of total cellular histamine, the "spontaneous" release from mast cells was subtracted from both numerator and denominator. All values are based on means of duplicate determinations which differed by less than 10%.

Immunoassay of LTC 4 and PGD 2
LTC 4 and PGD 2 were measured in duplicate determinations by radioimmunoassay [39,135]. The anti-LTC 4 and anti-PGD 2 antibodies are highly selective, with less than 1% cross-reactivity to other eicosanoids [39,135].

VEGF-A and VEGF-C Release
HHMCs (≈ 4 × 10 4 mast cells/per tube) were incubated (37 • C, 6 h) in RPMI 1640 containing 5% FCS, 2 mM L-glutamine, and 1% antibiotic-antimycotic solution, and activated with various concentrations of stimuli. At the end of incubation, cells were centrifuged (1000× g, 4 • C, 5 min) and the supernatants were stored at −80 • C for subsequent determination of mediator release. VEGF-A and VEGF-C were measured in duplicate determinations using commercially available ELISA kits (R&D System, Minneapolis, MN, USA) as previously described [136]. The ELISA sensitivity is 31.1-2000 pg/mL for VEGF-A and 62-4000 pg/mL for VEGF-C.

Statistical Analysis
Values were expressed as means ± SEM (standard error of the mean). The one-way repeated measures analysis of variance (ANOVA) with Greenhouse-Geisser corrections was used to examine the variations of continuous variables at different experimental conditions. Results were analyzed with GraphPad Prism software (version 8.01; GraphPad Software, La Jolla, CA, USA), and p values of less than 0.05 were considered significant.