Mast Cells and Disease Processes
Mast cells are known for their involvement in allergic reactions, but we were the first to show that they are also necessary to inititate inflammation, thus participating in inflammatory diseases that worsen by stress, such as autism, cancer, chronic fatigue syndrome, interstitial cystitis, migraine headaches, psoriasis and multiple sclerosis. We have developed in vivo and in vitro models for these diseases and we are studying neurohormonal activation of mast cells. The only plausible way to explain how mast cells can participate in so many diverse processes is their ability to secrete distinct chemicals relative for different pathophysiological settings.
Figure 1. Pathways of mast cell activation and secretion of key pro-inflammatory mediators. 
Figure 2. The American Academy of Allergy, Asthma and Immunology funded the production of a DVD by the
Mastocytosis Society
to sensitize health professionals about the involvement of mast cells in many disorders other than allergies, as shown in the daisy above.
Molecular Events Involved in Mast Cell Stimulus-Secretion Coupling
Mast cells are typically activated by immunoglobulin E and antigen, a process that leads to an explosive release of over 20 biologically active molecules (some of which are presorted in some 500 secretory grqnules, wile others are synthesized during activation) through a process called degranulation or exocytosis. However, we were the first to show that mast cells can also respond to non-allergic triggers and release mediators selectively, without degranulation. We specifically reported that the inflammatory cytokine IL-1 can induce selective release of the also inflammatory cytokine IL-6, that the stress hormone corticotropin-releasing hormone (CRH) can induce selective release of vascular endothelial growth factor (VEGF), and IL-33 augments substance P-induced VEGF release. We are currently studying the regulation of CRH receptor expression and function on human cultured mast cells and the effects of their respective activation on allergic stimulation.
Figure 3. Differential release of mast cell mediators and the pathogenesis of inflammation (left) ; ultrastructural cryo-immunocytochemistry of VEGF molecule release from a secretory vesicle, 1/10th the diameter of that of secretory granules, of a human mast cell following stimulation with CRH (right) .
Mast Cell - T Cell Interaction
Mast cells have the ability to superstimulate activated T cells; moreover, the mast cell mediators IL-6 and TGFβ participate in the maturation of Th17 cells, which are increasingly imlplicated in autoimmune diseases such as multiple sclerosis and psoriasis. Par of this process requires cell-to-cell contact, but also TNF secretion. We are investigating how IL-33 and SP (see above) could drive mast cells to participate in Th1-type of diseases and how such interactions may be interrupted for therapeutic purposes.
Figure 4. SP and IL-33 have synergistic action in inducing (A) VEGF protein secretion and (B) VEGF mRNA expression over 6 hr from LAD2 cells with IL-33 (100ng/ml)or together with SP (1μM) or (C) with SP (0.1 or 0.2μM) .
Figure 5. Mast cells and SP enhance Jurkat T cell activation. T cells were activated with anti-CD3/anti-CD28 (1 µg/ml each) and then incubated for 48 hours with SP and equal number of hCBMCs in 96 well tissue culture plates. After 48 hours, IL-2 levels in the supernatant fluid was measured by ELISA (n=3, *p<0.05, **p<0.05 comparing conditions as shown by numbers) .
Mast Cell Secretion and the Mitochondrial Uncoupling Protein UCP2
We showed for the first time that there is an inverse relationship between UCP2 expression and mast cell secretion. We also showed for the firtst time that mast cell degranulation, but not selective release of tumor necrosis factor (TNF), is associated with mitochondrial fission and relocation from perinuclear localization to cell-widespread distribution.
Figure 6. (Top) UCP2-deficient mast cells have enhanced degranulation. Wild-type and Ucp2-/- BMMCs. were (A) sensitized overnight with anti-DNP IgE (100 ng/ml) and challenged for 20 min with DNP-HSA (10 ng/ml); (B) stimulated for 10 min with SP (10 μg/ml) or C48/80 (30 μg/ml) or ionomycin (1 μg/ml), after which histamine was measured in the supernatant and pellet. *P <0.05 (n=6). (Bottom) Enhanced vascular permeability in Ucp2-/- mouse skin. (A) Evans blue extravasation following intradermal injection of either normal saline and SP (50 pmol) for 10 min. (B) Evan’s blue extravasation following passive sensitization with IgE and local skin challenge with DNP-HSA for 10 min. (C) Evans blue extravasation after intradermal challenge with histamine (10 μg) for 10 min. Evans blue was extracted from the injection sites in N,N-dimethylformamide overnight, quantified by absorbance at 620 nm and normalized to tissue weight. All results represent net extravasation after subtraction of extravasation from control saline injections. *P<0.05.
Neonatal Mast Cell Activation in the Pathogenesis of Autism
We reported that the prevalence of autism is ten times higher (1/10 children) in patients with mastocytosis than the general population (1/100 children). What makes this finding even more impressive is that mastocytosis is a rare disease occurring in 1/4000 children and is characterized by many hypersensitive mast cells in many tissues; once would, therefore, expect that the combined prevalence would have been 1/100x1/4,000=1/400,000 and not 1/10 children! We also found that the neuropeptide neurotensin, which is a pogtent mast cxell trigger, is statistically much higher in the serum of autistic patients. We are investigating the presence of additional biomarkers in the serum of autistic patients, the effect of stress (CRH) and environmental (mercury) triggers on the action of neurotensin on mast cells, as well as the potential benefit of a flavonoid formulation for the treatment of autism.
Figure 7. Schematic depiction of the proposed role of mast cell activation in brain inflammation and ASD. Allergic and non-allergic triggers could derive from the GI tract and stimulate the release of mast cell-derived vasoactive, inflammatory and neurotoxic mediators, which could increase gut-blood-brain-barrier permeability. These substances could disrupt the blood–brain-barrier and stimulate brain mast cells to further release molecules that increase BBB permeability and contribute to brain inflammation, and autism .
Antisecretory and Antiproliferative Function of the 78 kD Mast Cell Phosphoprotein
We sequenced this protein and showed that, in its phosphorylated state, it inhibits mast cell secretion and tumor growth. We are investigating the regulation of its expression, identification of the phosphorylated sites and molecules that could increase and sustain the phosphorylated state of this protein.
Figure 8. Cancer cells secrete chemoattractants that recruit mast cells to its vicinity. Mast cells are then activated either by direct contact or by cancercell– derived triggers to release “procancer” mediators selectively. These mediators induce angiogenesis, promote tumor proliferation, inhibit immune responses, and break down the surrounding stroma to permit metastases .
Figure 9. Moesin immunohistochemistry on human mast cells. Cytospin smears were prepared from (A) leukemic HMC-1 cells and (B) human umbilical cord blood-derived cultured mast cells(hCBMCs) >12 weeks old; they were fixed with acetone and methanol (1:1) for 90 sec at room temperature. They were washed in TBS once and incubated with mouse anti-moesin monoclonal antibody overnight at 4°C. Presence of brown staining indicates moesin.
Anti-allergic and Anti-inflammatory Actions of Some Natural Polyphenolic Flavonoids
We are investigating the mechanism of action of the select flavnoids luteolin and quercetin, their formulation in ways that increases their absorption, as well as their blood concentrations in order to achieve maximal effective levels. Such compounds could serve as anti-allergic, anti-inflammatory and anticancer drugs.
Figure 10. Inhibition of human mast cell activation by quercetin. Human mast cells were pretreated with quercetin, (0.1 mM) for 15 min and then stimulated with Neurotesin (100 nM, n=6, *<0.05) .
Learn more about the Theoharides lab here. 