Mast Cells In Patients Research Articles

Detection of Activated STAT5 in Neoplastic Mast Cells in Patients with Systemic Mastocytosis: Role of c-kit D816V.

Recent data suggest that activated STAT5 contributes to growth and differentiation of mast cells (MC) and that STAT5-knock out mice are MC-deficient. We have recently shown that constitutively activated STAT5 acts as a potent oncogenic signaling molecule in hematopoietic progenitor cells (Cancer Cell 2005; 7:87–99). In the present study, we examined the expression of activated STAT5 in neoplastic MC in systemic mastocytosis (SM) and asked whether the SM-related oncogene c-kit D816V is involved in STAT5-activation. For the immunohistochemical detection of activated tyrosine phosphorylated STAT5 (P-Y-STAT5), we used the specific monoclonal antibody AX1 (Advantex) which does not react with inactive STAT5. In all patients with SM tested (indolent SM, n=11; smouldering SM, n=2; aggressive SM, n=1; mast cell leukemia, n=1; all exhibiting c-kit D816V), MC were found to display P-Y-STAT5. Expression of activated STAT5 was also demonstrable in the c-kit D816V-positive mast cell leukemia-derived cell line HMC-1. The reactivity of HMC-1 cells with AX1 antibody was abrogated by a STAT5-specific blocking-peptide. To define the role of c-kit D816V in STAT5-activation, Ba/F3 cells with doxycycline-inducible expression of c-kit D816V (Ton.kit) were employed. In these cells, induction of c-kit D816V was followed by a massive increase in phosphorylated STAT5 as determined by a specific DNA-binding assay, whereas the total amounts of STAT5-mRNA and of the STAT5-protein showed only a slight increase or remained unchanged. In summary, these data show that neoplastic MC in SM express activated STAT5 (P-Y-STAT5), and that the transforming c-kit mutation D816V leads to persistent activation of STAT5 in these cells.

Nature of airway inflammation and remodeling in chronic cough

Chronic cough may be a result of asthma and non-asthma causes, but it is unclear whether there are specific inflammatory or remodeling changes. We determined airway mucosal changes in patients presenting with asthmatic cough and cough associated with non-asthmatic causes. Patients with chronic cough of non-asthmatic (n=33; postnasal drip/rhinitis in 6, gastroesophageal reflux in 5, bronchiectasis in 3, and idiopathic in 19) and asthmatic (n=14) causes and 15 healthy controls underwent fiberoptic bronchoscopy. Morphometry of bronchial biopsies and capsaicin cough sensitivity were assessed. Compared with controls, submucosal eosinophils and neutrophils were increased in patients with asthmatic cough (P<.005) and submucosal mast cells in patients with non-asthmatic cough (P=.01). Sub-basement membrane thickness, goblet cell area, vascularity, and vessel size were also increased in both groups. Smooth muscle area was higher only in patients with non-asthmatic cough (P=.0007 vs control and P=.019 vs asthmatic cough). None of the pathologic changes were related to the duration of coughing. Cough sensitivity was heightened in patients with non-asthmatic cough compared with controls and patients with asthmatic cough. The degree of goblet cell hyperplasia and epithelial shedding positively correlated with cough sensitivity in patients with non-asthmatic cough (r=0.43; P=.01; and r=0.40; P=.02, respectively). Features of airway wall remodeling are prominent in the airways with non-asthmatic as well as asthmatic cough. These are linked to chronic cough rather than to asthma. Mast cell hyperplasia rather than eosinophilia is distinctive for non-asthmatic cough.

Analysis of the lineage relationship between mast cells and basophils using the c- kit D816V mutation as a biologic signature

Mast cells and basophils share similar morphologic and functional properties; however, it is not known whether they are derived from a bilineage (basophil/mast cell)-restricted progenitor. To assess whether basophils and mast cells are derived from common committed progenitors using the c-kit D816V mutation as a biologic signature. The D816V c-kit mutation found in mast cells of patients with systemic mastocytosis is used as a trackable genetic marker to assess the lineage relationship between mast cells and basophils. Blood and bone marrow aspirates were collected from 33 consecutive patients with mastocytosis with different disease severity. Peripheral blood basophils, monocytes and neutrophils were sorted by immunomagnetic beads. Presence of the D816V c-kit mutation was analyzed by restriction fragment length polymorphism in the genomic DNA and mRNA from sorted cells in all patients and in the genomic DNA of individual basophils of 1 patient. The c-kit D816V mutation was detectable in basophils of 5 patients (15%). All 5 patients had the c-kit mutation also detectable in monocytes and thus had multilineage involvement. Single cell analysis of the genomic DNA in 1 patient showed a similar degree of clonal expansion in basophils, monocytes, and neutrophils. Mutated c-kit was expressed at the mRNA level in all 5 patients. There was no difference in surface Kit expression levels in basophils. Basophils carrying the D816V c-kit mutation in mastocytosis were detected only in the context of a multilineage involvement. These results argue against the presence of a bilineage-restricted committed progenitor for mast cells and basophils.

Systemic Mastocytosis

Systemic mastocytosis is a clonal disorder of the mast cell and its progenitor. The symptoms of systemic mastocytosis are due to the pathologic accumulation and activation of mast cells in various tissues such as bone marrow, skin, gastrointestinal tract, liver, and spleen. Recent studies revealed striking differences between the molecular and cellular biology of mast cells in patients with mastocytosis and those of healthy individuals. These findings are being used in formulating diagnostic criteria as well as designing novel treatment approaches to the disease.

Alterations of enteric mucosal mast cells in patients with irritable bowel syndrome

Alterations of enteric mucosal mast cells in patients with irritable bowel syndrome

Effects of tyrosine kinase inhibitor STI571 on human mast cells bearing wild-type or mutated c-kit.

STI571 is a tyrosine kinase inhibitor which inhibits the kinase activity of kit, the receptor for stem cell factor (SCF). Because activating mutations of c-kit affecting codon 816 are associated with human mast cell neoplasms, we determined whether STI571 exerted a similar cytotoxic effect on neoplastic and normal human mast cells. We investigated the effect of addition of STI571 in increasing concentrations (0.01 to 10 micromolar) to two HMC-1 human mast cell leukemia cell lines carrying two different activating c-kit mutations in codons 816 or 560, as well as the effect of the drug on short-term bone marrow cultures obtained from patients who carry a mutated codon 816 or wild-type c-kit. STI571 failed to inhibit the growth of HMC-1(560,816) cells bearing a codon 816 mutation but effectively suppressed the proliferation of HMC-1(560) carrying c-kit with the wild-type codon 816. STI571 did not induce preferential killing of neoplastic bone marrow mast cells in short-term cultures from patients bearing a codon 816 c-kit mutation. In contrast, STI571 caused a dramatic reduction in mast cells in patients without codon 816 c-kit mutations. These results suggest that STI571, while effectively killing mast cells with wild-type c-kit, did not show preferential cytotoxicity to neoplastic human mast cells and thus may not be effective in the treatment of human systemic mastocytosis associated with codon 816 c-kit mutations.

Seasonal intestinal inflammation in patients with birch pollen allergy

Background: The pathophysiologic interactions of inflammatory reactions between the mucosa of the respiratory tract and that of the gastrointestinal tract in individuals with allergy are poorly studied, despite the fact that allergic symptoms in the airways and the gastrointestinal tract might arise in the same patient. Objective: The objective of this study was to examine the inflammatory response histologically by enumerating eosinophils, IgE+ cells, and T cells in duodenal biopsy specimens in adult patients with IgE-mediated birch pollen allergy during the birch pollen season and off-season. Methods: Nine patients with birch pollen allergy verified by skin prick test and serum IgE antibodies were investigated toward the end of the birch pollen season and again 6 months later (off-season). Duodenal biopsy specimens were obtained and studied by immunostaining for the eosinophil major basic protein (MBP), IgE, and CD3+ T cells. Results: Oral allergy syndrome to birch-associated foods was present in all patients as indicated by questionnaire. Duodenal increases of MBP+ eosinophils and IgE-bearing cells were found in the patients during the birch pollen season as compared with off-season. No seasonal differences in the T-cell numbers in these patients were seen. Off-season, there was no significant difference between the patients and the control subjects regarding the intestinal frequencies of MBP+ eosinophils, mucosal IgE+ cells, and T cells. Conclusion: Birch pollen exposure triggered a local inflammation with an increase in duodenal eosinophils and IgE-carrying mast cells in patients with allergy. Our study gives evidence for the interplay between immunologically active cells in the airways and the gut. (J Allergy Clin Immunol 2003;112:45-51.)

Differential expression of CD2 on neoplastic mast cells in patients with systemic mast cell disease with and without an associated clonal haematological disorder.

Recently, aberrant coexpression of CD2 and CD25 has been reported to reliably distinguish neoplastic mast cells from normal or so-called reactive mast cells. Such expression is included in the consensus diagnostic criteria for systemic mast cell disease (SMCD). In our study of patients with SMCD, we found CD2 expression to be more prevalent on mast cells from patients without an associated haematological disorder (P = 0.04). Furthermore, no correlation was found between mast cell CD2 expression and other clinicopathological features in these patients.

Overexpression of complement receptors and related antigens on the surface of bone marrow mast cells in patients with systemic mastocytosis.

Depending on their stage of maturation and other factors, mast cell (MC) subsets differ from each other in terms of the expression of complement-associated antigens. This study analysed the expression of various complement-related cell surface antigens (CD11b/CR3, CD11c/CR4, CD35/CR1, CD55/DAF, CD59/MIRL, CD88/C5aR) on bone marrow mast cells (BMMC) in patients suffering from systemic mastocytosis (SM), other haematological diseases and non-haematological disorders (control groups). Expression of complement-associated cell surface antigens was analysed by flow cytometry. There were clear immunophenotypic differences between BMMC obtained from patients with SM and those from the control subjects: the percentage of patients expressing surface CD11c, CD35 and CD88 was significantly higher in patients with SM (76%, 100%, 54%) than in the control subjects (58%, 11%, 18%) (P < 0.05). In addition, the levels of CD11c, CD35 and CD88 expressed per MC (sites per cell) were significantly higher (P < 0.05) in SM than in the control group. Expression of the complement regulatory molecules CD55 and CD59 was detected in BMMC in all patients analysed. However, the levels of CD59 per BMMC were higher in patients with SM as compared with the control subjects, which could help to explain the formation of BMMC aggregates in the former group of individuals. Together, our results showed that BMMC in systemic mastocytosis overexpressed the cell surface membrane receptors involved in binding of complement components and complement-mediated cell activation. Whether this pathological expression of complement receptors is of pathophysiological significance remains to be determined.

Evidence for the involvement of a hematopoietic progenitor cell in systemic mastocytosis from single-cell analysis of mutations in the c-kit gene

AbstractMast cells are derived from multipotential hematopoietic progenitors and are clonally increased in systemic mastocytosis, a disease associated with point mutations of codon 816 (most commonly Asp816Val) of c-kit. To study the lineage relationship and the extent of expansion of cells derived from the mutated clone, we examined the occurrence of the Asp816Val c-kit mutation in genomic DNA of individual sorted peripheral blood T cells, B cells, and monocytes in patients with indolent systemic mastocytosis. The mutation was detected in varying frequencies in the genomic DNA of individual B cells and monocytes and bone marrow mast cells in patients with extensive disease. In B cells, the immunoglobulin repertoire was polyclonal, indicating that the mutation occurred before VH/(D)/JH recombination. These results show that mastocytosis is a disorder of a pluripotential hematopoietic progenitor cell that gives rise to B cells and monocytes in addition to mast cells and that the affected clone shows variable expansion in these lineages in the peripheral blood of patients with systemic mastocytosis.

Skin nerve fibres and their contacts with mast cells in patients with palmoplantar pustulosis.

Patients with palmoplantar pustulosis (PPP) frequently report that stress worsens their condition. A study was therefore made of the distribution and number of nerve fibres positive for protein gene product (PGP) 9.5 (a general nerve marker) and nerve fibres with substance P- and calcitonin gene-related peptide-like immunoreactivity in involved skin from patients with PPP and in skin from healthy controls. The number of mast cells in the papillary dermis was larger (P = 0.0003) in lesional palmar PPP skin than in control skin, and the number of contacts between mast cells and nerve fibres was significantly larger (P = 0.02) in PPP skin than in control skin. Image analysis of the nerve fibres around the sweat glands showed that the positively stained area as a percentage of the total area of the sweat gland (coil + surrounding nerves) was significantly lower in PPP skin (P = 0.0006). Furthermore, the nerves seemed to be fragmented. Neutrophils within and below the pustules and in the papillary dermis showed positive substance P staining. The increased number of contacts between nerves and mast cells in PPP skin and the intense substance P-like immunoreactivity of the neutrophils indicate that neuromediation may influence the inflammation in PPP, whereas the destruction of the nerve fibres around the sweat glands might be a result of the inflammation.

Tumor Necrosis Factor-Alpha in Ileal Mast Cells in Patients with Crohn’s Disease

Background: Reports that both intestinal and extraintestinal Crohn’s disease (CD) had healed successfully after treatment with anti-tumor necrosis factor-alpha (TNF-α) antibody have strengthened the hypothesis that it has a role in the treatment of CD. The macrophage is one source of TNF-α. Intestinal mast cells are also thought to have a role in CD, but it is not known if human ileal mast cells express TNF-α. Aim: To find out whether TNF-α is expressed by mast cells in the ileal wall in CD patients and controls. Methods: TNF-α was sought immunohistochemically in full thickness specimens of ileal wall from patients with CD (histologically normal, n = 9; inflamed, n = 6) and controls (patients with colonic cancer, n = 8). Mast cells were identified by metachromasia and anti-mast cell tryptase immunoreactivity. Results: In all layers of the ileal wall, and in every specimen investigated, mast cells were the main cell type that expressed TNF-α immunoreactivity out of the TNF-α-labelled cells. The number of TNF-α- labelled mast cells was greater in the muscularis propria in patients compared with controls, both in uninflamed (1.7-fold, p < 0.05) and in inflamed bowel (4.6-fold, p < 0.002); greater in the submucosa in inflamed compared with uninflamed CD (1.6-fold, p < 0.01), and less in the lamina propria in inflamed compared with uninflamed CD (0.4-fold, p < 0.05). Conclusion: Mast cells are an important source of TNF-α in all layers of the ileal wall, and the increased density of TNF-α-positive mast cells in the submucosa and muscularis propria may contribute to the tissue changes and symptoms in CD.

Increased adenoid mast cells in patients with otitis media with effusion

Objective: Previous studies have been suggested the possible role of adenoid mast cells in the pathogenesis of otitis media with effusion (OME). The aim of the present study was to evaluate the possible relationship of adenoid mast cells and hearing loss in patients with chronic OME. Methods: Twenty patients with combined chronic OME and chronic adenoiditis (OME-A) and 20 patients with isolated chronic adenoiditis were studied. Hearing thresholds were determined by pure tone audiometry in both groups. All subjects underwent adenoidectomy and adenoid mast cells were counted in each specimen. Number of adenoid mast cells were determined in both groups. Possible relationship of adenoid mast cells and hearing thresholds in OME-A patients was evaluated by comparing the hearing thresholds of OME-A patients with mast cell count above the mean of OME-A group and hearing thresholds of OME-A patients with mast cell count below the mean of OME-A group. Results: All isolated chronic adenoiditis patients had normal hearing thresholds. OME-A patients had hearing thresholds ranging from 12–52 dB. The number of adenoid mast cells in OME-A group (median: 80) was significantly greater than isolated chronic adenoiditis group (median: 38) ( P<0.05). OME-A patients with adenoid mast cell count above the mean of OME-A group had significantly higher hearing thresholds compared to OME-A patients with mast cell count below the mean of OME-A group ( P<0.05). Conclusions: Patients with OME-A have greater number of adenoid mast cells than patients with isolated chronic adenoiditis. OME-A patients with adenoid mast cell count above the mean of the OME-A group had higher hearing thresholds than OME-A patients with adenoid mast cell count below the mean of the OME-A group. Increased number of adenoid mast cells may contribute to the pathogenesis of higher hearing thresholds in some OME-A patients.

Expression of the C5a receptor (CD88) on synovial mast cells in patients with rheumatoid arthritis.

To analyze the immunophenotype and functional properties of synovial mast cells (SyMC) in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Synovial tissue was obtained from 25 patients with RA and 17 patients with OA. Tissue was dispersed by enzymatic digestion using collagenase. Surface receptor expression on SyMC was analyzed by monoclonal antibodies (MAb) and indirect immunofluorescence staining. Histamine release experiments were performed using the MC agonist recombinant human (rHu) stem cell factor (SCF), the anaphylatoxin rHuC5a, and an anti-IgE antibody. In both groups of patients (RA and OA), SyMC were found to react with MAb to IgE, SCF receptor (c-kit, CD117), as well as CD antigens likewise expressed in lung MC (CD9, CD29, CD33, CD43, CD44, CD45). However, a significantly increased proportion of SyMC from RA patients reacted with MAb against C5a receptor (C5aR; CD88), compared with SyMC from OA (mean +/- SD percentage of SyMC reacting with CD88 MAb S5/1 in RA 27.5 +/- 8.6% versus 0.0% in OA, and with CD88 MAb W17/1 in RA 58.3 +/- 15.2% versus 12.5 +/- 15.0% in OA; P < 0.05). Furthermore, in RA, significant histamine release from SyMC above control was induced by rHuC5a, anti-IgE, and rHuSCF, whereas SyMC in OA released histamine after stimulation with anti-IgE and rHuSCF, but not rHuC5a. SyMC exhibit phenotypic and functional properties similar to MC in other tissues. In patients with RA, but not OA, SyMC express significant amounts of C5aR (CD88) and release histamine in response to rHuC5a. These results indicate a role for SyMC and C5a/C5aR in the pathogenesis of RA.

Expression of the C5a receptor (CD88) on synovial mast cells in patients with rheumatoid arthritis

Expression of the C5a receptor (CD88) on synovial mast cells in patients with rheumatoid arthritis

Dermal IgG Deposits and Increase of Mast Cells in Patients with Fibromyalgia - Relevant Findings or Epiphenomena?

Skin biopsies from 25 patients with fibromyalgia, 5 healthy controls, 8 patients with rheumatoid arthritis, and 9 patients with local chronic pain after whiplash injury, were examined for the occurrence of IgG deposits and collagen types, using direct and indirect immunofluorescence, and for dermal connective tissue mast cells, using semithin Epon sections. Fibromyalgia skin biopsies had significantly higher values of IgG deposits in the dermis and vessel walls and showed a higher reactivity for collagen III. They also had a higher mean number of mast cells. There was a correlation between the percentage of damaged/degranulated mast cells and the individual IgG immunofluorescence scores. These findings support the hypothesis of neurogenic inflammation involvement in fibromyalgia.

Mast cells in aural polyps: a preliminary report

Mast cells are a rich source of potent biologically active mediators and are found in connective tissue, associated with blood vessels in many varied inflammatory conditions. Mast cells have been described in nasal polyps and turbinates and in adenoidal tissue in the upper aerodigestive tract. As the middle ear lining is contiguous with the nose and the nasopharynx, the presence of mast cells in aural polyps is interesting. This preliminary study investigated the presence of mast cells in inflammatory aural polyps using light microscopy. All patients presenting to the department in one year were included. Patients with previous ear disease or surgery and in whom cholesteatoma was suspected were excluded. Except for one patient mast cells were seen in all aural polyps. The implications of these findings is discussed. Further work is needed using electron microscopy.

Abnormal cutaneous neurosensitivity in atopic skin.

Having found an inability of patients with atopic eczema to distinguish different levels of iontophoretically applied histamine concentrations, as shown by their diminished vascular reactions and itch responses, and reviewing this result in the light of our new findings of smaller flare reactions and weaker itch sensations following different concentrations of intradermally injected substance P, we have concluded that unmyelinated afferent skin nerve fibres in these patients seem to be affected by the pathophysiological mechanism of atopic eczema. We therefore suspect that a down-regulation of histamine receptors at nerve endings compensates for elevated histamine release from cutaneous mast cells in patients with atopic eczema.

Detection of human IgE to sulfamethoxazole by skin testing with sulfamethoxazoyl-poly- l-tyrosine

Adverse reactions to sulfamethoxazole (SMX) occur in 4% to 6% of normal individuals. Many of these reactions resemble immunopathologic reactions, but skin test or in vitro evidence of a role for IgE is limited. Earlier RAST studies in our laboratory provided evidence that the N 4-SMX hapten was a major determinant in immediate hypersensitivity reactions to SMX. We tested the hypothesis that IgE to this hapten is present on the mast cells of patients who have experienced immediate hypersensitivity reactions temporally related to exposure to SMX. A multivalent skin test reagent, SMX 168-poly- l-tyrosine, and a univalent hapten, SMX-tyrosine, were synthesized. Forty-four patients with histories of allergic reactions to SMX and six subjects who had been exposed to the drug, but who had not reacted, were skin tested. Twenty-seven percent of the history-positive patients were skin test positive. None of the control individuals was positive. The immunologic responses to SMX in three patients who had experienced allergic reactions during SMX/trimethoprim therapy were analyzed in serial skin test and RAST assessments. One to three years after the clinical reactions, IgE to SMX could be demonstrated by skin testing in all three patients with a SMX-poly- l-tyrosine skin test reagent. Skin test reactions were inhibited by the monovalent reagent, SMX-tyrosine, in a dose-dependent manner. SMX-specific IgE antibodies could also be detected by RAST in serum obtained within days of the reactions from two of the three individuals. These IgE antibodies could not be detected in samples taken several years after the clinical reaction. RAST-inhibition experiments confirmed the immunologic specificity of the univalent hapten inhibition. The results of these experiments support the concept that the N 4-SMX determinant is a major determinant in SMX allergy. These data also demonstrate that IgE to this SMX determinant can be detected by skin testing with a multivalent hapten carrier conjugate.

IgE-Positive Duodenal Mast Cells in Patients with Food-Related Diarrhea

Thirty-seven consecutive adult patients with a history of adverse reactions to foods, manifested mainly as diarrhea, were investigated with skin prick test (SPT), IgE levels in serum, and the presence of IgE bound to mast cells in duodenal biopsy specimens. Nineteen percent had increased serum IgE levels indicating the presence of atopic disease and in 35% positive SPTs for one or several allergens related to their gastrointestinal symptoms were found. In 92% of the patients with positive SPTs to food, IgE-positive mast cells in duodenal biopsy specimens were found, twice as many as in patients with negative SPTs (47%). Forty-two percent of normal individuals without any adverse reactions to foods had IgE-bearing mast cells in their intestinal mucosa. The results showed that 'arming' of mast cells with IgE locally was relatively prominent but not consistent in the gut of patients with food-related diarrhea suggested to be allergic by positive SPT. Intestinal IgE-mediated hypersensitivity reactions could be of pathogenetic significance in most of these patients. However, since also half of the normal individuals were found to have IgE-positive intestinal mast cells, this phenomenon presumably also reflects a normal physiological defence mechanism.