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D D Metcalfe

Publications and source records attributed to D D Metcalfe.

At least 37 records · Page 2Linked to original sources
PubMed2001

Characterization of alpha 5-integrin-dependent mast cell adhesion following Fc epsilon RI aggregation.

BACKGROUND: Integrin receptors are engaged in the upregulation of mast cell adhesion to extracellular matrix components upon stimulation with cytokines and antigen. Fibronectin receptor containing the alpha 5-integrin subunit is critical for mast cell interaction with the extracellular matrix protein fibronectin (FN). METHODS: The murine MCP5/L mast cell line was employed to investigate the process of Fc epsilon RI-mediated mast cell adhesion to FN. RT-PCR and cytofluorimetric analysis were used to assess the expression of alpha 5 integrin in MCP5/L mast cells. Radiolabelled mast cells were sensitized with monoclonal IgE and used in adhesion assays. Anti-alpha 5-integrin antibody (Ab), monovalent hapten and metabolic inhibitors were used to characterize antigen-mediated mast cell adhesion to FN. RESULTS: Addition of antigen to IgE-sensitized cells resulted in transient upregulation of mast cell adhesion to FN with a maximum adhesion following 30 min of incubation. Mast cell adhesion was inhibited with anti-alpha 5-integrin monoclonal antibodies blocking FN receptor or with excess monovalent hapten preventing antigen-mediated IgE cross-linking. The presence of the protein kinase C (PKC) inhibitor staurosporine also inhibited mast cell adhesion in a dose-dependent fashion. The process of Fc epsilon RI-mediated upregulation of mast cell adhesion to FN was not associated with an increase in surface expression of mast cell FN receptors. CONCLUSION: The major FN receptor on MCP5/L mast cell surface, an integrin containing the alpha 5 subunit mediates a transient change in mast cell adhesiveness following IgE cross-linking. Fc epsilon RI-derived signals engage PKC and upregulate mast cell adhesion in a process which might involve changes in integrin avidity rather than integrin expression.

Animals↗
PubMed2001

Secretion of interleukin-1 receptor antagonist from human mast cells after immunoglobulin E-mediated activation and after segmental antigen challenge.

Mast cells produce substances with antiinflammatory properties in addition to their capacity to release proinflammatory mediators. To further probe the antiinflammatory aspect of mast-cell function we investigated the ability of human mast cells (huMCs) to produce interleukin (IL)-1 receptor antagonist (IL-1ra) in response to high-affinity Fc receptor for immunoglobulin E (Fcalpha RI) aggregation, and examined IL-1ra in bronchoalveolar lavage fluid (BALF) to determine whether it might be of mast-cell origin. Using a ribonuclease protection assay, flow cytometry, and enzyme-linked immunosorbent assay (ELISA), IL-1ra message and protein were found to be constitutively expressed in cultured huMCs. Upon stimulation through Fcalpha RI, IL-1ra message was upregulated in huMCs and IL-1ra protein secreted from cultured huMCs and isolated human lung mast cells. By immunoblot analysis, huMCs were found to produce the 17-kD form of IL-1ra and the presence of IL-1ra in human lung mast cells was confirmed by immunohistochemistry. In BALF obtained from allergic asthmatic subjects, IL-1ra production increased after specific antigen challenge, with the 17-kD isoform of IL-1ra predominating. These findings demonstrate that huMCs produce and release IL-1ra after Fcalpha RI aggregation, which may contribute to a local inhibition of IL-1-dependent effects on inflammation in the lung.

Allergens↗
PubMed2000

Soluble stem cell factor receptor (CD117) and IL-2 receptor alpha chain (CD25) levels in the plasma of patients with mastocytosis: relationships to disease severity and bone marrow pathology.

Systemic mastocytosis is a disease of mast cell proliferation that may be associated with hematologic disorders. There are no features on examination that allow the diagnosis of systemic disease, and mast cell-derived mediators, which may be elevated in urine or blood, may also be elevated in individuals with severe allergic disorders. Thus, the diagnosis usually depends on results of bone marrow biopsy. To facilitate evaluation, surrogate markers of the extent and severity of the disease are needed. Because of the association of mastocytosis with hematologic disease, plasma levels were measured for soluble KIT (sKIT) and soluble interleukin-2 receptor alpha chain (sCD25), which are known to be cleaved in part from the mast cell surface and are elevated in some hematologic malignancies. Results revealed that levels of both soluble receptors are increased in systemic mastocytosis. Median plasma sKIT concentrations as expressed by AU/mL (1 AU = 1.4 ng/mL) were as follows: controls, 176 (n = 60); urticaria pigmentosa without systemic involvement, 194 (n = 8); systemic indolent mastocytosis, 511 (n = 30); systemic mastocytosis with an associated hematologic disorder, 1320 (n = 7); aggressive mastocytosis, 3390 (n = 3). Plasma sCD25 levels were elevated in systemic mastocytosis; the highest levels were associated with extensive bone marrow involvement. Levels of sKIT correlated with total tryptase levels, sCD25 levels, and bone marrow pathology. These results demonstrate that sKIT and sCD25 are useful surrogate markers of disease severity in patients with mastocytosis and should aid in diagnosis, in the selection of those needing a bone marrow biopsy, and in the documentation of disease progression. (Blood. 2000;96:1267-1273)

Adolescent↗
PubMed2000

Expression of a functional high-affinity IgG receptor, Fc gamma RI, on human mast cells: Up-regulation by IFN-gamma.

Biologically relevant activation of human mast cells through Fc receptors is believed to occur primarily through the high-affinity IgE receptor Fc epsilon RI. However, the demonstration in animal models that allergic reactions do not necessarily require Ag-specific IgE, nor the presence of a functional IgE receptor, and the clinical occurrence of some allergic reactions in situations where Ag-specific IgE appears to be lacking, led us to examine the hypothesis that human mast cells might express the high-affinity IgG receptor Fc gamma RI and in turn be activated through aggregation of this receptor. We thus first determined by RT-PCR that resting human mast cells exhibit minimal message for Fc gamma RI. We next found that IFN-gamma up-regulated the expression of Fc gamma RI. This was confirmed by flow cytometry, where Fc gamma RI expression on human mast cells was increased from approximately 2 to 44% by IFN-gamma exposure. Fc epsilon RI, Fc gamma RII, and Fc gamma RIII expression was not affected. Scatchard plots were consisted with these data where the average binding sites for monomeric IgG1 (Ka = 4-5 x 108 M-1) increased from approximately 2,400 to 12,100-17,300 per cell. Aggregation of Fc gamma RI on human mast cells, and only after IFN-gamma exposure, led to significant degranulation as evidenced by histamine release (24.5 +/- 4.4%): and up-regulation of mRNA expression for specific cytokines including TNF-alpha, GM-CSF, IL-3 and IL-13. These findings thus suggest another mechanism by which human mast cells may be recruited into the inflammatory processes associated with some immunologic and infectious diseases.

Antibody Affinity↗
PubMed2000

Mast cells migrate, but do not degranulate, in response to fractalkine, a membrane-bound chemokine expressed constitutively in diverse cells of the skin.

Mast cells (MC) are anatomically located near nerves and blood vessels in skin and the gastrointestinal tract and tend to localize within certain cutaneous tumors such as neurofibromas. However, the molecular mechanisms by which MC home to these sites are not well characterized. Fractalkine (FK) is a membrane-bound CX3C chemokine that displays constitutive expression in dendritic cells as well as in non-hematopoietic tissues including mammalian brain. Here we show that FK is constitutively expressed by skin endothelial cells, dermal dendrocytes and cells within neurofibromas. By reverse transcription-PCR, FK receptor, CX3CR1, is expressed by cultured murine bone marrow-derived MC (BMMC) of both connective tissue and mucosal phenotypes. Non-activated human dermal MC isolated from neonatal foreskin similarly demonstrated CX3CR1 expression. In chemotaxis assays, FK attracted MC with maximal migration occurring between 25 - 125 ng / ml. BMMC were not stimulated to release proinflammatory mediators in the presence of FK as measured by granule-associated beta-hexosaminidase release. Thus, CX3CR1 is expressed by MC and effectively mediates chemotaxis without inducing degranulation. We propose that the constitutive expression of FK on certain cells in the skin may be a factor in the tissue-specific homing of MC.

Animals↗
PubMed2000

Human mast cells express the hyaluronic-acid-binding isoform of CD44 and adhere to hyaluronic acid.

CD44 is expressed in various isoforms on multiple cell lineages including those of hematopoietic origin and is believed in part to mediate cell adhesion to hyaluronic acid. Elevated levels of soluble CD44 (sCD44) have been identified in the serum of some patients with specific neoplasms. We thus sought to determine whether human mast cells express functional CD44 and whether sCD44 might be associated with systemic mast cell disease. Using a standard assay, CD34(+)-derived cultured human mast cells were first demonstrated to adhere to hyaluronic-acid-coated surfaces. Human mast cells were then found by flow cytometry to express CD44S, but not the v5, v6, v7, and v8 isoforms, and to shed CD44S following activation induced by PMA or aggregation of FcvarepsilonRI. However, CD44S was not found to be consistently elevated in serum obtained from patients with mastocytosis or individuals experiencing anaphylaxis. Thus, human cultured mast cells express and shed CD44S, which appears to mediate the attachment of these cells to hyaluronic acid.

Anaphylaxis↗
PubMed2000

Mastocytosis complicating pregnancy.

OBJECTIVE: To review the experience of women who conceived after developing mastocytosis and who were observed at the National Institutes of Health. METHODS: We reviewed our patient database for the years 1984-1998 to identify women with mastocytosis who had conceived. We then reviewed each woman's record, asked each woman to complete a questionnaire, and with permission wrote outside hospitals to obtain records of each labor and delivery. RESULTS: We identified eight women who had become pregnant. These women delivered a total of 11 live infants. In approximately a third of the pregnancies, patients experienced worsening of symptoms. They often used fewer medications during pregnancy because of safety concerns, and no greater incidence of adverse reactions was noted. Antihistamines were used most commonly, followed by oral prednisone. Medications used during delivery were well tolerated and included epidural analgesics. Neonates were generally healthy. None to date have developed urticaria pigmentosa or systemic mastocytosis. CONCLUSION: A subset of women with mastocytosis might have had exacerbated mastocytosis during and after pregnancy, but labor and delivery progressed normally. Infants were born generally healthy and were without mastocytosis. Thus there appears to be no absolute contraindication to pregnancy for women with mastocytosis, although women should be aware that the choice to have a child is not without some added risk.

Adult↗
PubMed2000

Analysis of the surface expression of c-kit and occurrence of the c-kit Asp816Val activating mutation in T cells, B cells, and myelomonocytic cells in patients with mastocytosis.

OBJECTIVE: The Asp816Val c-kit activating mutation is detectable in the peripheral blood cells of some patients with mastocytosis and in lesional skin biopsies obtained from adult patients with urticaria pigmentosa. These observations led to the conclusion that this mutation is present in mast cells and mast cell precursors that express c-kit. However, the distribution of the Asp816Val mutation among hematopoietic lineages is unknown. To determine the distribution of the Asp816Val mutation among hematopoietic lineages and to explore its relationship to clinical disease, we examined cells bearing differentiation markers for myelomonocytic cells as well as T and B lymphocytes, in both peripheral blood and bone marrow obtained from patients with mastocytosis. MATERIALS AND METHODS: The presence of Asp816Val c-kit mutation in cells magnetically sorted from peripheral blood or bone marrow according to surface differentiation markers was studied by reverse transcriptase polymerase chain reaction (RT-PCR) restriction fragment length polymorphism (RFLP) analysis. The surface expression of c-kit was determined by flow cytometry. RESULTS: The mutation was detectable by RT-PCR in at least one cell lineage in the bone marrow in 7 of 7 patients examined and in the peripheral blood of 11 of 11 adult patients with urticaria pigmentosa and indolent disease. The mutation was identified most frequently in B cells and myeloid cells. Flow cytometric analysis demonstrated that the differentiated cells expressing mutated c-kit were negative for surface KIT. CONCLUSION: These results are consistent with the conclusion that the c-kit Asp816Val mutation occurs in an early progenitor cell and is carried by myelomonocytic cells, T cells, and B cells in addition to mast cells. However, unlike mast cells, these myelomonocytic cells, T cells, and B cells do not concomitantly express surface c-kit and thus may be less susceptible to the effects of this mutation.

Adult↗
PubMed2000

Kit signal transduction.

The current understanding of kit signaling is that a limited number of signaling proteins interact to build multiple interacting networks that allow diverse cellular responses. Cytoplasmic signaling proteins are increasingly seen to form networks directed through converging and interacting pathways rather than following a simple linear model. There are also numerous cross-connections between signaling proteins more distal to the receptor. Ras thus binds PI3 kinase and potentiates its activation, whereas the Rac-dependent protein kinase PAK phosphorylates MEK and thereby stabilizes its association with Raf. A signaling network with multiple intersecting pathways can obtain a single, coherent response from numerous, potentially conflicting signals. There is still limited information about the effect of activating mutations on various aspects of kit signaling. There is, however, mounting evidence that an activating mutation may enhance kit signaling and also induce factor-independent activation of kit. For instance, this activation could occur through degradation of SHP-1, the protein tyrosine phosphatase that negatively regulates kit signaling. There is also emerging evidence that inherent inhibitory factors may exist in the juxtamembrane of kit and may be suppressed as a result of a mutation in that region. Understanding the impact of these activating mutations on kit signaling is important, not only in contributing to the understanding of the pathogenesis of mastocytosis but ultimately in forming the basis for more effective therapeutic intervention in this disease.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Pediatric mastocytosis.

Pediatric mastocytosis presents with heterogeneous cutaneous lesions and symptoms that are caused by increased numbers of tissue mast cells. In contrast with adult patients with mastocytosis, the course of pediatric patients is usually transient. Therefore, it has long been speculated that pediatric and adult mastocytosis may be based on different pathogenetic mechanisms. Indeed, new genetic findings now indicate differences in the pathogenesis. Adult patients usually express activating mutations of the growth factor receptor c-kit. Most children lack these mutations but sometimes carry other inactivating mutations of c-kit. Only children with progressive mastocytosis seem to express the activating mutations seen in adults. Causal treatment is not yet available, but H1 and H2 antihistamines may provide relief of symptoms. It is important to counsel patients and their parents carefully to avoid triggers that induce systemic mast cell degranulation.

Adolescent↗
PubMed2000

A proposed classification of mastocytosis incorporating molecular genetics.

As an understanding of the molecular genetic causes of different forms of mastocytosis is developed, the therapy of choice may depend on the specific genetic abnormalities expressed by a patient's neoplastic mast cells. The authors propose a new classification system for mastocytosis that incorporates both molecular-genetic and clinical data. This system provides a theoretic framework for mast cell researchers and helps practicing physicians in estimating prognosis and determining therapeutic options for individual patients.

Adult↗
PubMed2000

Mast cells in innate immunity.

Mast cells are known to be the main effector cells in the elicitation of the IgE-mediated allergic response. The specific location of mast cells within tissues that interface the external environment, and the extent of their functional capacity, including the ability to phagocytose and to produce and secrete a wide spectrum of mediators, have led investigators to propose a potential role for mast cells in innate immune responses. Certain microorganisms have been found to interact either directly or indirectly with mast cells. This interaction results in mast cell activation and mediator release which elicit an inflammatory response or direct killing leading to bacterial clearance. The in vivo relevance of these in vitro observations has been demonstrated by the use of complement-deficient and/or mast cell-deficient and mast cell-reconstituted mice. In thus has been shown that both C3 and mast cell- and tumor necrosis factor-alpha-dependent recruitment of circulating leukocytes with bactericidal properties are crucial to a full response in certain models of acute infection. Modulation of mast cell numbers in vivo was also found to affect the host response against bacterial infection. Thus, mast cells do have a role in innate immunity in defined animal models of bacterial infection. Whether mast cells participate in innate immune responses in the protection of the human host against bacteria remains to be determined.

Animals↗
PubMed2000

Demonstration that platelet-activating factor is capable of activating mast cells and inducing a chemotactic response.

Platelet-activating factor (PAF) is generated in a variety of inflammatory conditions in which mast cells accumulate. However, little is known about the ability of PAF to influence mast cell function directly. In this study we examine the ability of PAF to activate mast cells and regulate mast cell chemotaxis. PAF was found to induce intracellular calcium mobilization and chemotactic responses in both murine and human mast cells. PAF induced transient increases in intracellular Ca2+ concentrations with a 50% effective dose of 1 nM and induced significant migratory responses at PAF concentrations of 1 nM to 1 microM in the human leukaemia mast cell line (HMC-1). Using signal transduction inhibitors, both PAF-induced calcium mobilization and migration of mast cells were shown to require activation of pertussis toxin-sensitive G proteins. PAF-induced calcium and chemotactic responses were cross-desensitized by C5a. Together, these data demonstrate that PAF is capable of activating distinct signalling pathways in mast cells associated with calcium mobilization and cell migration; and that PAF may thus contribute to the regulation of mast cell responses and hyperplasia at sites of inflammation.

Animals↗
PubMed1999

Demonstration that human mast cells arise from a progenitor cell population that is CD34(+), c-kit(+), and expresses aminopeptidase N (CD13).

Human mast cells are known to arise from a CD34(+)/c-kit(+) progenitor cell population that also gives rise to neutrophils, eosinophils, basophils, and monocytes. To further characterize cells within the CD34(+)/c-kit(+) population that yield mast cells, this progenitor was additionally sorted for CD13, a myeloid marker known to appear early on rodent mast cells and cultured human mast cells, but not expressed or expressed at low levels on human tissue mast cells; and cultured in recombinant human (rh) stem cell factor (rhSCF), rh interleukin-3 (rhIL-3; first week only), and rhIL-6. Initial sorts revealed that although the majority of cells in culture arose from the CD34(+)/c-kit(+)/CD13(-) cell population, mast cells arose from a CD34(+)/c-kit(+)/CD13(+) progenitor cell that also gave rise to a population of monocytes. Sequential sorting confirmed that CD34(+)/c-kit(+)/CD13(+) cells in CD34(+)/c-kit(+)/CD13(-) sorts gave rise to the few mast cells observed in CD13(-) sorted cells. CD34(+)/c-kit(+)/CD13(+) cells plated as single cells in the presence of various cytokine combinations gave rise to pure mast cell, monocyte, or mixed mast cell/monocyte progeny. Addition of either rh granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or rhIL-5 to the CD34(+)/c-kit(+)/CD13(+) progenitor cell population cultured in rhSCF, rhIL-3, and rhIL-6 did increase the number of total cells cultured and in the case of rhIL-5, did increase total mast cell numbers. Neither rhGM-CSF or rhIL-5 led to additional cell populations, ie, even with the addition of rhGM-CSF or rhIL-5, only mast cells and monocytes grew from CD34(+)/c-kit(+)/CD13(+) cells. Thus, human mast cells and a population of monocytes arise from precursor cells that express CD34, c-kit, and CD13; and within which, are mast cell, monocyte, and mast/monocyte (bipotential) precursors.

Antigens, CD↗
PubMed1999

Receptor-mediated modulation of murine mast cell function by alpha-melanocyte stimulating hormone.

The proopiomelanocortin (POMC)-derived neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) is known to modulate some aspects of inflammation through direct effects on T cells, B cells, and monocytes. To determine whether alpha-MSH might similarly influence mast cell responsiveness, mast cells were examined to see if they expressed the receptor for alpha-MSH, melanocortin-1 (MC-1), and whether alpha-MSH altered mast cell function. We thus first identified MC-1 on bone marrow cultured murine mast cells (BMCMC) and a murine mast cell line (MCP-5) employing flow cytometry and through detection of specific binding. Subsequent treatment of mast cells with alpha-MSH increased the cAMP concentration in a characteristic biphasic pattern, demonstrating that alpha-MSH could affect intracellular processes. We next examined the effect of alpha-MSH on mediator release and cytokine expression. IgE/DNP-human serum albumin-stimulated histamine release from mast cells was inhibited by approximately 60% in the presence of alpha-MSH. Although activation of BMCMC induced the expression of mRNAs for the inflammatory cytokines IL-1beta, IL-4, IL-6, TNF-alpha, and the chemokine lymphotactin, mRNAs for IL-1beta, TNF-alpha, and lymphotactin were down-modulated in the presence of alpha-MSH. Finally, IL-3-dependent proliferative activity of BMCMC was slightly but significantly augmented by alpha-MSH. Taken together, these observations suggest that alpha-MSH may exert an inhibitory effect on the mast cell-dependent component of a specific inflammatory response.

Animals↗
PubMed1999

Eotaxin potentiates antigen-dependent basophil IL-4 production.

Basophils are a major source of IL-4, which is a critical factor in the generation of allergic inflammation. Eotaxin induces chemotaxis mediated through the CC chemokine receptor 3 (CCR3) present on basophils as well as eosinophils and Th2 cells, thereby promoting cell recruitment. To determine whether eotaxin has other proinflammatory activity, we examined the effect of eotaxin on basophil IL-4 expression by flow cytometry. Eotaxin alone had no effect on basophil IL-4 production, but further increased allergen-stimulated IL-4 expression. Eotaxin also enhanced IL-4 release from purified basophils 2- to 4-fold, as determined by ELISA (p < 0.01). Addition of eotaxin to cultures resulted in a 40-fold left shift in the dose response to Ag. This effect was obtained with physiologic concentrations of eotaxin (10 ng/ml), was abrogated by an Ab to the CCR3 receptor, and was noted with other chemokine ligands of CCR3. Additionally, eotaxin augmented IL-3 priming of basophil IL-4 production in a synergistic manner (p < 0.01). In contrast, no priming was observed with either IL-5 or GM-CSF. These results establish a novel function for eotaxin and other chemokine ligands of CCR3: the potentiation of Ag-mediated IL-4 production in basophils, and suggest a potential nonchemotactic role for CC chemokines in the pathogenesis and amplification of inflammation.

Adjuvants, Immunologic↗