The "missing link" in autoimmunity and autism: extracellular mitochondrial components secreted from activated live mast cells.

Autoimmune diseases continue to increase, but the reason(s) remain obscure and infections have not proven to be major contributors. Mast cells are tissue immune cells responsible for allergies, but have been increasingly shown to be involved in innate and acquired immunity, as well as inflammation. This involvement is possible because of their ability to release multiple mediators in response to a great variety of triggers. We recently published that activation of mast cells is accompanied by mitochondrial fission and translocation to the cell surface from where they secrete at least ATP and DNA outside the cell without cell damage. These extracellular mitochondrial components are misconstrued by the body as "innate pathogens" leading to powerful autocrine and paracrine auto-immune/auto-inflammatory responses. We also showed that mitochondrial DNA is increased in the serum of young children with autism spectrum disorders (ASD), a condition that could involve "focal brain allergy/encephalitits". Blocking the secretion of extracellular mitochondrial components could present unique possibilities for the therapy of ASD and other autoimmune diseases. Unique formulation of the flavonoid luteolin offers unique advantages.

Focal brain inflammation and autism.

Increasing evidence indicates that brain inflammation is involved in the pathogenesis of neuropsychiatric diseases. Autism spectrum disorders (ASD) are characterized by social and learning disabilities that affect as many as 1/80 children in the USA. There is still no definitive pathogenesis or reliable biomarkers for ASD, thus significantly curtailing the development of effective therapies. Many children with ASD regress at about age 3 years, often after a specific event such as reaction to vaccination, infection, stress or trauma implying some epigenetic triggers, and may constitute a distinct phenotype. ASD children respond disproportionally to stress and are also affected by food and skin allergies. Corticotropin-releasing hormone (CRH) is secreted under stress and together with neurotensin (NT) stimulates mast cells and microglia resulting in focal brain inflammation and neurotoxicity. NT is significantly increased in serum of ASD children along with mitochondrial DNA (mtDNA). NT stimulates mast cell secretion of mtDNA that is misconstrued as an innate pathogen triggering an auto-inflammatory response. The phosphatase and tensin homolog (PTEN) gene mutation, associated with the higher risk of ASD, which leads to hyper-active mammalian target of rapamycin (mTOR) signalling that is crucial for cellular homeostasis. CRH, NT and environmental triggers could hyperstimulate the already activated mTOR, as well as stimulate mast cell and microglia activation and proliferation. The natural flavonoid luteolin inhibits mTOR, mast cells and microglia and could have a significant benefit in ASD.

Neurotensin and CRH interactions augment human mast cell activation.

Stress affects immunity, but the mechanism is not known. Neurotensin (NT) and corticotropin-releasing hormone (CRH) are secreted under stress in various tissues, and have immunomodulatory actions. We had previously shown that NT augments the ability of CRH to increase mast cell-dependent skin vascular permeability in rodents. Here we show that NT triggered human mast cell degranulation and significantly augmented CRH-induced vascular endothelial growth factor (VEGF) release. Investigation of various signaling molecules indicated that only NF-κB activation was involved. These effects were blocked by pretreatment with the NTR antagonist SR48692. NT induced expression of CRH receptor-1 (CRHR-1), as shown by Western blot and FACS analysis. Interestingly, CRH also induced NTR gene and protein expression. These results indicate unique interactions among NT, CRH, and mast cells that may contribute to auto-immune and inflammatory diseases that worsen with stress.

Perinatal stress, brain inflammation and risk of autism-review and proposal.

BACKGROUND: Autism Spectrum Disorders (ASD) are neurodevelopmental disorders characterized by varying deficits in social interactions, communication, and learning, as well as stereotypic behaviors. Despite the significant increase in ASD, there are few if any clues for its pathogenesis, hampering early detection or treatment. Premature babies are also more vulnerable to infections and inflammation leading to neurodevelopmental problems and higher risk of developing ASD. Many autism "susceptibility" genes have been identified, but "environmental" factors appear to play a significant role. Increasing evidence suggests that there are different ASD endophenotypes. DISCUSSION: We review relevant literature suggesting in utero inflammation can lead to preterm labor, while insufficient development of the gut-blood-brain barriers could permit exposure to potential neurotoxins. This risk apparently may increase in parents with "allergic" or autoimmune problems during gestation, or if they had been exposed to stressors. The presence of circulating auto-antibodies against fetal brain proteins in mothers is associated with higher risk of autism and suggests disruption of the blood-brain-barrier (BBB). A number of papers have reported increased brain expression or cerebrospinal fluid (CSF) levels of pro-inflammatory cytokines, especially TNF, which is preformed in mast cells. Recent evidence also indicates increased serum levels of the pro-inflammatory mast cell trigger neurotensin (NT), and of extracellular mitochondrial DNA (mtDNA), which is immunogenic. Gene mutations of phosphatase and tensin homolog (PTEN), the negative regulator of the mammalian target of rapamycin (mTOR), have been linked to higher risk of autism, but also to increased proliferation and function of mast cells. SUMMARY: Premature birth and susceptibility genes may make infants more vulnerable to allergic, environmental, infectious, or stress-related triggers that could stimulate mast cell release of pro-inflammatory and neurotoxic molecules, thus contributing to brain inflammation and ASD pathogenesis, at least in an endophenotype of ASD patients.

Contribution of stress to asthma worsening through mast cell activation.

OBJECTIVE: To review the available evidence linking stress to asthma and to investigate whether mast cells contribute to the effect of stress through activation by corticotropin-releasing hormone (CRH). DATA SOURCE: The PubMed database was searched for articles (1998-2011) using the keywords anxiety, asthma, exacerbation, inflammation, mast cells, socioeconomic status, stress, violence, and worsening. STUDY SELECTION: Articles were selected based on their relevance to the topic, with emphasis on clinical or epidemiologic data linking stress to asthma and studies that offered possible explanations for how stress may affect asthma. RESULTS: Many articles point to an association between stress (socioeconomic status, interpersonal conflicts, emotional distress, terrorism) and asthma exacerbations but without any distinct pathogenetic mechanism. A few articles have reported reduced circulating cortisol and/or sensitivity to corticosteroids. We propose that mast cells, known to be involved in the pathophysiology of asthma, can be activated by CRH, which is secreted under stress in the lungs, leading to selective release of proinflammatory mediators. This effect may be augmented by neuropeptides or cytokines. CRH also reduces T-regulatory cell production of interleukin 10, which in known to inhibit allergic mast cell activation. CONCLUSION: More studies are required to investigate lung levels of CRH and selective mast cell mediators. Reducing stress and using CRH receptor antagonists and/or mast cell blockers may serve as possible new therapeutic approaches for asthma.

Corticotropin-releasing hormone and extracellular mitochondria augment IgE-stimulated human mast-cell vascular endothelial growth factor release, which is inhibited by luteolin.

BACKGROUND: Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by varying degrees of dysfunctional social abilities, learning deficits, and stereotypic behaviors. Many patients with ASDs have 'allergy-like' symptoms and respond disproportionally to stress. We have previously shown that the peptide neurotensin (NT) is increased in the serum of young children with autism and that can stimulate extracellular secretion of mitochondrial (mt)DNA which was also increased in the serum of these children. METHODS: Human mast cells were stimulated by corticotropin-releasing hormone (CRH), mitochondrial DNA, IgE/anti-IgE, either for 24 hours to measure vascular endothelial growth factor (VEGF) release by ELISA or for 6 hours or quantitative PCR. RESULTS: CRH augmented IgE/anti-IgE-induced human mast-cell release of VEGF and it also induced the expression of IgE receptor (FcεRI) on mast cells. Moreover, sonicated mitochondria also augmented VEGF release, and this effect was blocked by the natural flavone luteolin. CONCLUSION: These results indicate that stress and infection-mimicking extracellular mitochondrial components augment allergic inflammation that may be involved in the early pathogenesis of ASDs. Moreover, luteolin inhibits these processes and may be helpful in the treatment of ASDs.

Irritable bowel syndrome and the elusive mast cells.

Irritable bowel syndrome (IBS) is the most common condition seen by gastroenterologists. It presents with alternating symptoms of bowel dysfunction that often worsens with stress. The cause of these symptoms eludes investigators and many attempts have been made to discover an underlying pathology. This is a daunting task since symptoms come and go, and change characteristics. Furthermore, the pathology of IBS is unlikely to be identical in all patients. In addition, all symptoms and all features studied thus far have a strong overlap with healthy volunteers. Elsewhere in this issue, Braak et al. report a well-designed clinical investigation in patients with IBS and come to the conclusion that IBS is not characterized by mast cell or other immune cell proliferation, but by immune dysregulation in the colon. Is this the final answer?

Quercetin is more effective than cromolyn in blocking human mast cell cytokine release and inhibits contact dermatitis and photosensitivity in humans.

Mast cells are immune cells critical in the pathogenesis of allergic, but also inflammatory and autoimmune diseases through release of many pro-inflammatory cytokines such as IL-8 and TNF. Contact dermatitis and photosensitivity are skin conditions that involve non-immune triggers such as substance P (SP), and do not respond to conventional treatment. Inhibition of mast cell cytokine release could be effective therapy for such diseases. Unfortunately, disodium cromoglycate (cromolyn), the only compound marketed as a mast cell "stabilizer", is not particularly effective in blocking human mast cells. Instead, flavonoids are potent anti-oxidant and anti-inflammatory compounds with mast cell inhibitory actions. Here, we first compared the flavonoid quercetin (Que) and cromolyn on cultured human mast cells. Que and cromolyn (100 µM) can effectively inhibit secretion of histamine and PGD(2). Que and cromolyn also inhibit histamine, leukotrienes and PGD(2) from primary human cord blood-derived cultured mast cells (hCBMCs) stimulated by IgE/Anti-IgE. However, Que is more effective than cromolyn in inhibiting IL-8 and TNF release from LAD2 mast cells stimulated by SP. Moreover, Que reduces IL-6 release from hCBMCs in a dose-dependent manner. Que inhibits cytosolic calcium level increase and NF-kappa B activation. Interestingly, Que is effective prophylactically, while cromolyn must be added together with the trigger or it rapidly loses its effect. In two pilot, open-label, clinical trials, Que significantly decreased contact dermatitis and photosensitivity, skin conditions that do not respond to conventional treatment. In summary, Que is a promising candidate as an effective mast cell inhibitor for allergic and inflammatory diseases, especially in formulations that permit more sufficient oral absorption.

Stimulated human mast cells secrete mitochondrial components that have autocrine and paracrine inflammatory actions.

Mast cells are hematopoietically-derived tissue immune cells that participate in acquired and innate immunity, as well as in inflammation through release of many chemokines and cytokines, especially in response to the pro-inflammatory peptide substance P (SP). Inflammation is critical in the pathogenesis of many diseases, but the trigger(s) is often unknown. We investigated if mast cell stimulation leads to secretion of mitochondrial components and whether these could elicit autocrine and/or paracrine inflammatory effects. Here we show that human LAD2 mast cells stimulated by IgE/anti-IgE or by the SP led to secretion of mitochondrial particles, mitochondrial (mt) mtDNA and ATP without cell death. Mitochondria purified from LAD2 cells and, when mitochondria added to mast cells trigger degranulation and release of histamine, PGD(2), IL-8, TNF, and IL-1ß. This stimulatory effect is partially inhibited by an ATP receptor antagonist and by DNAse. These results suggest that the mitochondrial protein fraction may also contribute. Purified mitochondria also stimulate IL-8 and vascular endothelial growth factor (VEGF) release from cultured human keratinocytes, and VEGF release from primary human microvascular endothelial cells. In order to investigate if mitochondrial components could be secreted in vivo, we injected rats intraperiotoneally (ip) with compound 48/80, which mimicks the action of SP. Peritoneal mast cells degranulated and mitochondrial particles were documented by transimission electron microscopy outside the cells. We also wished to investigate if mitochondrial components secreted locally could reach the systemic circulation. Administration ip of mtDNA isolated from LAD2 cells in rats was detected in their serum within 4 hr, indicating that extravascular mtDNA could enter the systemic circulation. Secretion of mitochondrial components from stimulated live mast cells may act as "autopathogens" contributing to the pathogenesis of inflammatory diseases and may be used as targets for novel treatments.

Substance P (SP) induces expression of functional corticotropin-releasing hormone receptor-1 (CRHR-1) in human mast cells.

Corticotropin-releasing hormone (CRH) is secreted under stress and regulates the hypothalamic-pituitary-adrenal axis. However, CRH is also secreted outside the brain where it exerts proinflammatory effects through activation of mast cells, which are increasingly implicated in immunity and inflammation. Substance P (SP) is also involved in inflammatory diseases. Human LAD2 leukemic mast cells express only CRHR-1 mRNA weakly. Treatment of LAD2 cells with SP (0.5-2 µM) for 6 hours significantly increases corticotropin-releasing hormone receptor-1 (CRHR-1) mRNA and protein expression. Addition of CRH (1 µM) to LAD2 cells, which are "primed" with SP for 48 hours and then washed, induces synthesis and release of IL-8, tumor necrosis factor (TNF), and vascular endothelial growth factor (VEGF) 24 hours later. These effects are blocked by pretreatment with an NK-1 receptor antagonist. Treatment of LAD2 cells with CRH (1 µM) for 6 hours induces gene expression of NK-1 as compared with controls. However, repeated stimulation of mast cells with CRH (1 µM) leads to downregulation of CRHR-1 and upregulation in NK-1 gene expression. These results indicate that SP can stimulate mast cells and also increase expression of functional CRHR-1, whereas CRH induces NK-1 gene expression. These results may explain CRHR-1 and NK-1 expression in lesional skin of psoriatic patients.

Mast cells and inflammation.

Mast cells are well known for their role in allergic and anaphylactic reactions, as well as their involvement in acquired and innate immunity. Increasing evidence now implicates mast cells in inflammatory diseases where they are activated by non-allergic triggers, such as neuropeptides and cytokines, often exerting synergistic effects as in the case of IL-33 and neurotensin. Mast cells can also release pro-inflammatory mediators selectively without degranulation. In particular, IL-1 induces selective release of IL-6, while corticotropin-releasing hormone secreted under stress induces the release of vascular endothelial growth factor. Many inflammatory diseases involve mast cells in cross-talk with T cells, such as atopic dermatitis, psoriasis and multiple sclerosis, which all worsen by stress. How mast cell differential responses are regulated is still unresolved. Preliminary evidence suggests that mitochondrial function and dynamics control mast cell degranulation, but not selective release. Recent findings also indicate that mast cells have immunomodulatory properties. Understanding selective release of mediators could explain how mast cells participate in numerous diverse biologic processes, and how they exert both immunostimulatory and immunosuppressive actions. Unraveling selective mast cell secretion could also help develop unique mast cell inhibitors with novel therapeutic applications. This article is part of a Special Issue entitled: Mast cells in inflammation.

Mast cell activation and autism.

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by varying degrees of dysfunctional communication and social interactions, repetitive and stereotypic behaviors, as well as learning and sensory deficits. Despite the impressive rise in the prevalence of autism during the last two decades, there are few if any clues for its pathogenesis, early detection or treatment. Increasing evidence indicates high brain expression of pro-inflammatory cytokines and the presence of circulating antibodies against brain proteins. A number of papers, mostly based on parental reporting on their children's health problems, suggest that ASD children may present with "allergic-like" problems in the absence of elevated serum IgE and chronic urticaria. These findings suggest non-allergic mast cell activation, probably in response to environmental and stress triggers that could contribute to inflammation. In utero inflammation can lead to preterm labor and has itself been strongly associated with adverse neurodevelopmental outcomes. Premature babies have about four times higher risk of developing ASD and are also more vulnerable to infections, while delayed development of their gut-blood-brain barriers makes exposure to potential neurotoxins likely. Perinatal mast cell activation by infectious, stress-related, environmental or allergic triggers can lead to release of pro-inflammatory and neurotoxic molecules, thus contributing to brain inflammation and ASD pathogenesis, at least in a subgroup of ASD patients. This article is part of a Special Issue entitled: Mast cells in inflammation.

Human mast cell degranulation and preformed TNF secretion require mitochondrial translocation to exocytosis sites: relevance to atopic dermatitis.

BACKGROUND: Mast cells derive from hematopoietic cell precursors and participate in tissue allergic, immune, and inflammatory processes. They secrete many mediators, including preformed TNF, in response to allergic, neuropeptide, and environmental triggers. However, regulation of mast cell degranulation is not well understood. OBJECTIVE: We investigated the role of mitochondrial dynamics in degranulation of human cultured mast cells. METHODS: Human umbilical cord blood-derived mast cells (hCBMCs) and Laboratory of Allergic Diseases 2 (LAD2) mast cells were examined by confocal and differential interference contrast microscopy during activation by IgE/antigen and substance P (SP). Mast cells in control and atopic dermatitis (AD) skin were evaluated by transmission electron microscopy. LAD2 cells were pretreated with mitochondrial division inhibitor, a dynamin-related protein 1 (Drp1) inhibitor, and small interfering RNA for Drp1, which is necessary for mitochondrial fission and translocation. Calcineurin and Drp1 gene expression was analyzed in stimulated LAD2 cells and AD skin biopsies. RESULTS: Stimulation of hCBMCs with IgE/antigen or LAD2 cells with SP leads to rapid (30 minutes) secretion of preformed TNF. Degranulation is accompanied by mitochondrial translocation from a perinuclear location to exocytosis sites. Extracellular calcium depletion prevents these effects, indicating calcium requirement. The calcium-dependent calcineurin and Drp1 are activated 30 minutes after SP stimulation. Reduction of Drp1 activity by mitochondrial division inhibitor and decrease of Drp1 expression using small interfering RNA inhibit mitochondrial translocation, degranulation, and TNF secretion. Mitochondrial translocation is also evident by transmission electron microscopy in skin mast cells from AD biopsies, in which gene expression of calcineurin, Drp1, and SP is higher than in normal skin. CONCLUSION: Human mast cell degranulation requires mitochondrial dynamics, also implicated in AD.

Brief report: "allergic symptoms" in children with Autism Spectrum Disorders. More than meets the eye?

Many children with Autism Spectrum Disorders (ASD) have either family and/or personal history of "allergic symptomatology", often in the absence of positive skin or RAST tests. These symptoms may suggest mast cell activation by non-allergic triggers. Moreover, children with mastocytosis or mast cell activation syndrome (MCAS), a spectrum of rare diseases characterized by increased number of activated mast cells in many organs, appear to have ASD at a rate tenfold higher (1/10 children) than that of the general population (1/100 children). Mast cell activation by allergic, infectious, environmental and stress-related triggers, especially perinatally, would release pro-inflammatory and neurotoxic molecules. We speculate these could disrupt the gut-blood-brain barriers, thus contributing to brain inflammation and ASD pathogenesis. Increased mast cell responsiveness may define at least a subgroup of ASD subjects, who could benefit from inhibition of mast cell activation.

Mitochondrial DNA and anti-mitochondrial antibodies in serum of autistic children.

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by difficulties in communication, cognitive and learning deficits, as well as stereotypic behaviors. For the majority of cases there are no reliable biomarkers or distinct pathogenesis. However, increasing evidence indicates ASD may be associated with some immune dysregulation, and may have a neuroimmune component. We recently showed that the peptide neurotensin (NT) is increased in autistic children. We now show that NT induces release of extracellular mitochondrial DNA (mtDNA) that could act as "autoimmune" trigger. We further show that serum from young autistic patients contains mtDNA (n = 20; cytochrome B, p = 0.0002 and 7S, p = 0.006), and anti-mitochondrial antibody Type 2 (n = 14; p = 0.001) as compared to normally developing, unrelated controls (n = 12). Extracellular blood mtDNA and other components may characterize an autistic endophenotype and may contribute to its pathogenesis by activating autoimmune responses.

Mercury induces inflammatory mediator release from human mast cells.

BACKGROUND: Mercury is known to be neurotoxic, but its effects on the immune system are less well known. Mast cells are involved in allergic reactions, but also in innate and acquired immunity, as well as in inflammation. Many patients with Autism Spectrum Disorders (ASD) have "allergic" symptoms; moreover, the prevalence of ASD in patients with mastocytosis, characterized by numerous hyperactive mast cells in most tissues, is 10-fold higher than the general population suggesting mast cell involvement. We, therefore, investigated the effect of mercuric chloride (HgCl2) on human mast cell activation. METHODS: Human leukemic cultured LAD2 mast cells and normal human umbilical cord blood-derived cultured mast cells (hCBMCs) were stimulated by HgCl2 (0.1-10 microM) for either 10 min for beta-hexosaminidase release or 24 hr for measuring vascular endothelial growth factor (VEGF) and IL-6 release by ELISA. RESULTS: HgCl2 induced a 2-fold increase in beta-hexosaminidase release, and also significant VEGF release at 0.1 and 1 microM (311 +/- 32 pg/106 cells and 443 +/- 143 pg/106 cells, respectively) from LAD2 mast cells compared to control cells (227 +/- 17 pg/106 cells, n = 5, p < 0.05). Addition of HgCl2 (0.1 microM) to the proinflammatory neuropeptide substance P (SP, 0.1 microM) had synergestic action in inducing VEGF from LAD2 mast cells. HgCl2 also stimulated significant VEGF release (360 +/- 100 pg/106 cells at 1 microM, n = 5, p < 0.05) from hCBMCs compared to control cells (182 +/- 57 pg/106 cells), and IL-6 release (466 +/- 57 pg/106 cells at 0.1 microM) compared to untreated cells (13 +/- 25 pg/106 cells, n = 5, p < 0.05). Addition of HgCl2 (0.1 microM) to SP (5 microM) further increased IL-6 release. CONCLUSIONS: HgCl2 stimulates VEGF and IL-6 release from human mast cells. This phenomenon could disrupt the blood-brain-barrier and permit brain inflammation. As a result, the findings of the present study provide a biological mechanism for how low levels of mercury may contribute to ASD pathogenesis.

IL-33 augments substance P-induced VEGF secretion from human mast cells and is increased in psoriatic skin.

The peptide substance P (SP) has been implicated in inflammatory conditions, such as psoriasis, where mast cells and VEGF are increased. A relationship between SP and VEGF has not been well studied, nor has any interaction with the proinflammatory cytokines, especially IL-33. Here we report that SP (0.1-10 microM) induces gene expression and secretion of VEGF from human LAD2 mast cells and human umbilical core blood-derived cultured mast cells (hCBMCs). This effect is significantly increased by coadministration of IL-33 (5-100 ng/mL) in both cell types. The effect of SP on VEGF release is inhibited by treatment with the NK-1 receptor antagonist 733,060. SP rapidly increases cytosolic calcium, and so does IL-33 to a smaller extent; the addition of IL-33 augments the calcium increase. SP-induced VEGF production involves calcium-dependent PKC isoforms, as well as the ERK and JNK MAPKs. Gene expression of IL-33 and histidine decarboxylase (HDC), an indicator of mast cell presence/activation, is significantly increased in affected and unaffected (at least 15 cm away from the lesion) psoriatic skin, as compared with normal control skin. Immunohistochemistry indicates that IL-33 is associated with endothelial cells in both the unaffected and affected sites, but is stronger and also associated with immune cells in the affected site. These results imply that functional interactions among SP, IL-33, and mast cells leading to VEGF release contribute to inflammatory conditions, such as the psoriasis, a nonallergic hyperproliferative skin inflammatory disorder with a neurogenic component.