Faculty
Joshua A. Boyce, M.D.
Specialty: Allergy and Pediatric Pulmonary Medicine
Massachusetts General Hospital for Children
Smith Research Bldg., 5th floor
1 Jimmy Fund Way
Boston, MA 02115
Publications
The following is a list of recent publications for which this Partners Asthma Center physician has been cited as an author in PubMed databases. Study abstracts have been provided for your convenience.
Price, K. S., D. S. Friend, et al. (2003). "CC chemokine receptor 3 mobilizes to the surface of human mast cells and potentiates immunoglobulin E-dependent generation of interleukin 13." Am J Respir Cell Mol Biol 28(4): 420-7.
Eotaxins-1, -2, and -3 mediate the recruitment of blood-borne eosinophils to allergically inflamed tissues by binding CC chemokine receptor (CCR) 3. Mast cells (MCs) are resident tissue cells that also express CCR3. In the present study, we demonstrate that human (h) MCs in nasal polyps and cultured cord blood-derived hMCs express CCR3 not only on their surfaces but also in their secretory granules. Activation of hMCs mediated by the high-affinity Fc receptor for immunoglobulin (Ig)E (Fc epsilon RI) increased the surface presentation of CCR3 within 1 h, with a parallel decrease in intracellular CCR3 as determined by flow cytometry on saponin-permeabilized hMCs. Recombinant eotaxin-1 alone did not induce histamine release or cytokine generation, and did not significantly augment IgE-dependent histamine release by interleukin (IL)-4-primed hMCs. Nevertheless, stimulation of hMCs with eotaxin-1 2 h after Fc epsilon RI cross-linkage (concomitantly with maximal surface CCR3 expression) increased Fc epsilon RI-dependent IL-13 generation by hMCs, compared with their replicates stimulated simultaneously with both agonists. Thus, hMCs may store CCR3 and rapidly mobilize it to their surface with IgE-dependent activation, providing a novel potential mechanism for enhanced hMC effector function, including IL-13 production.
Boyce, J. A. (2003). "The role of mast cells in asthma." Prostaglandins Leukot Essent Fatty Acids 69(2-3): 195-205.
While the role of mast cells in allergic reactions is unequivocal, their precise functions in asthma remain controversial. Mast cells uniquely populate all vascularized organs and tissues, including the upper and lower respiratory tree, even in healthy individuals. Histologic evidence suggests that asthma is accompanied by a mast cell hyperplasia in the inflamed mucosal epithelium and the adjacent smooth muscle. The mechanisms responsible for constitutive mast cell development have been partly elucidated. Moreover, both in vitro studies and in vivo disease models indicate that mast cells have a remarkably flexible program of gene expression, and this program can be drastically altered by the T-cell-derived Th2 cytokines relevant to asthma. Moreover, the role of mast cells in innate immunity is now firmly established, and the capacity for numerous microbial pathogens to initiate their activation in vitro and in vivo suggest mechanisms by which microbes could initiate disease exacerbations.
Boyce, J. A. (2003). "Mast cells: beyond IgE." J Allergy Clin Immunol 111(1): 24-32; quiz 33.
Mast cells, historically known for their involvement in type I hypersensitivity, also serve critical protective and homeostatic functions. They directly recognize the products of bacterial infection through several surface receptor proteins, releasing proteases, cytokines, and eicosanoid mediators that recruit neutrophils, limit the spread of bacterial infection, and facilitate subsequent tissue repair. In vitro studies suggest that the spectrum of microbes capable of initiating mast cell activation is broad and extends to common respiratory viruses, mycoplasma, and even products of tissue injury, such as nucleotides. TH2-polarized inflammation elicits a reactive hyperplasia of mast cells at the involved mucosal surfaces in both mice and human subject. Several recombinant TH2 cytokines (IL-3, IL-4, IL-5, and IL-9) act synergistically with stem cell factor to facilitate proliferation of nontransformed human mast cells in vitro. IL-4 induces the expression of critical inflammation-associated genes by human mast cells, such as those encoding leukotriene C4 synthase, Fc(epsilon)RI, and several cytokines. Consequently, priming with IL-4 not only amplifies classical Fc(epsilon)RI-dependent mast cell activation but also dramatically alters the product profile of mast cells activated by innate signals and by chemical mediators of inflammation. Strikingly, IL-4 induces an activation response by mast cells to cysteinyl leukotrienes, which act through a receptor shared with uridine diphosphate to induce cytokine generation without exocytosis. It Is possible that alterations in mast cell phenotype by the TH2 milieu of allergy permits otherwise trivial infections or homeostatic chemical signals to initiate harmful inflammatory cascades and sustain tissue pathology. Drug development must take these nonclassical mast cell activation pathways into account without compromising the beneficial and protective functions of mast cells.
Wong, G. W., P. S. Foster, et al. (2002). "Biochemical and functional characterization of human transmembrane tryptase (TMT)/tryptase gamma. TMT is an exocytosed mast cell protease that induces airway hyperresponsiveness in vivo via an interleukin-13/interleukin-4 receptor alpha/signal transducer and activator of transcription (STAT) 6-dependent pathway." J Biol Chem 277(44): 41906-15.
Transmembrane tryptase (TMT)/tryptase gamma is a membrane-bound serine protease stored in the secretory granules of human and mouse lung mast cells (MCs). We now show that TMT reaches the external face of the plasma membrane when MCs are induced to degranulate. Analysis of purified recombinant TMT revealed that it is a two-chain neutral protease. Thus, TMT is the only MC protease identified so far which retains its 18-residue propeptide when proteolytically activated. The genes that encode TMT and tryptase betaI reside on human chromosome 16p13.3. However, substrate specificity studies revealed that TMT and tryptase betaI are functionally distinct even though they are approximately 50% identical. Although TMT is rapidly inactivated by the human plasma serpin alpha(1)-antitrypsin in vitro, administration of recombinant TMT (but not recombinant tryptase betaI) into the trachea of mice leads to airway hyperresponsiveness (AHR) and increased expression of interleukin (IL) 13. T cells also increase their expression of IL-13 mRNA when exposed to TMT in vitro. TMT is therefore a novel exocytosed surface mediator that can stimulate those cell types that are in close proximity. TMT induces AHR in normal mice but not in transgenic mice that lack signal transducer and activator of transcription (STAT) 6 or the alpha-chain of the cytokine receptor that recognizes both IL-4 and IL-13. Based on these data, we conclude that TMT is an exocytosed MC neutral protease that induces AHR in lungs primarily by activating an IL-13/IL-4Ralpha/STAT6-dependent pathway.
Mellor, E. A., K. F. Austen, et al. (2002). "Cysteinyl leukotrienes and uridine diphosphate induce cytokine generation by human mast cells through an interleukin 4-regulated pathway that is inhibited by leukotriene receptor antagonists." J Exp Med 195(5): 583-92.
We previously reported that interleukin (IL)-4 upregulates the expression of leukotriene C(4) synthase (LTC(4)S) by human cord blood--derived mast cells (hMCs), augments their high-affinity Fc receptor for IgE (Fc(epsilon)RI)-dependent generation of eicosanoids and cytokines, and induces a calcium flux in response to cysteinyl leukotrienes (cys-LTs) and uridine diphosphate (UDP) that is blocked by cys-LT receptor antagonists. We speculated that this IL-4-dependent, receptor-mediated response to the cys-LTs and UDP might induce cytokine generation by hMCs without concomitant exocytosis. Unlike hMCs maintained in cytoprotective stem cell factor (SCF) alone, hMCs primed for 5 d with IL-4 responded to UDP (1microM), LTC(4) (100 nM), and LTD(4) (100 nM) by producing IL-5, tumor necrosis factor (TNF)-alpha, and especially large quantities of macrophage inflammatory protein (MIP)-1beta de novo at 6 h, preceded by the induced expression of the corresponding mRNAs. Cys-LT- and UDP-mediated cytokine production by the primed hMCs occurred without histamine release or PGD(2) generation and was inhibited by the CysLT1 receptor antagonist MK571. Additionally, pretreatment of hMCs with MK571 or with the cys-LT biosynthetic inhibitor MK886 decreased IL-5 and TNF-alpha production in response to IgE receptor cross-linkage, implying a positive feedback by endogenously produced cys-LTs. Cys-LTs and UDP thus orchestrate a novel, IL-4-regulated, non-IgE-dependent hMC activation for cytokine gene induction that could be initiated by microbes, cellular injury, or neurogenic or inflammatory signals; and this pathobiologic event would not be recognized in tissue studies where hMC activation is classically defined by exocytosis.
Gurish, M. F., A. Humbles, et al. (2002). "CCR3 is required for tissue eosinophilia and larval cytotoxicity after infection with Trichinella spiralis." J Immunol 168(11): 5730-6.
The CCR3 binds at least seven different CC chemokines and is expressed on eosinophils, mast cells (MC), and a subset of Th cells (Th2) that generate cytokines implicated in mucosal immune responses. Using mice with a targeted disruption of CCR3 (CCR3(-/-)) and their +/+ littermates, we investigated the role of CCR3 in the amplification of tissue eosinophilia and MC hyperplasia in the mouse after infection with Trichinella spiralis. In CCR3(-/-) mice, eosinophils are not recruited to the jejunal mucosa after infection and are not present in the skeletal muscle adjacent to encysting larvae. In addition, the number of cysts in the skeletal muscle is increased and the frequency of encysted larvae exhibiting necrosis is reduced. The CCR3(-/-) mice exhibit the expected MC hyperplasia in the jejunum and caecum and reject the adult worms from the small intestine at a normal rate. This study is consistent with distinct functions for MC (adult worm expulsion) and eosinophils (toxicity to larvae) in immunity to a helminth, T. spiralis, and defines the essential requirement for CCR3 in eosinophil, but not MC recruitment to tissues.
Gurish, M. F. and J. A. Boyce (2002). "Mast cell growth, differentiation, and death." Clin Rev Allergy Immunol 22(2): 107-18.
Mast cells (MC) begin development in the bone marrow. Following initial lineage commitment, the cells move into the vasculature as a committed progenitor (MCp) that is poorly phenotypically defined, but appears to be an agranular cell lacking the high-affinity IgE receptor characteristic of the mature tissue-localized MC. Full maturation occurs after the cells move into the various tissues. In the mouse, MCp localizing in the connective tissues appear to differentiate into mature MC, whereas those localizing in the lung and mucosal compartment of the small intestine remain largely as committed MCp. Movement of the MCp into the small intestine is controlled by the alpha 4 beta 7 integrin, whereas the factors controlling movement into other tissues remain to be defined. Following an inflammatory stimulus, Th2-derived cytokines drive the maturation process of these MCps, leading to the mature mucosal MC hyperplasia associated with events such as an intestinal helminth infection and possibly human allergy such as asthma and rhinitis. The expanded MC number disappears as the stimulus resolves. Various routes are used in the resolution of the MC hyperplasia including apoptosis, shedding along with the villous epithelium, and recirculation back to the spleen for elimination. Unlike the reactive MC that appears in association with inflammation, the connective tissue-localized MC is a long-lived radiation-resistant cell, which appears to depend principally on the presence of stem cell factor (SCF) for its persistence.
Boyce, J. A., E. A. Mellor, et al. (2002). "Human mast cell progenitors use alpha4-integrin, VCAM-1, and PSGL-1 E-selectin for adhesive interactions with human vascular endothelium under flow conditions." Blood 99(8): 2890-6.
Mast cells (MCs) are central to asthma and other allergic diseases, and for responses to infection and tissue injuries. MCs arise from committed progenitors (PrMCs) that migrate from the circulation to tissues by incompletely characterized mechanisms, and differentiate in situ in perivascular connective tissues of multiple organs. PrMCs derived in vitro from human cord blood were examined for adhesion molecule expression and their ability to adhere to human umbilical vein endothelial cells (HUVECs) under conditions that mimic physiologic shear flow. The PrMCs expressed alpha(4)beta(1), low levels of beta7, and the beta2-integrins alphaLbeta2 and alphaMbeta2. The PrMCs also expressed PSGL-1, but not L-selectin. At low (0.5 dynes/cm(2)-1.0 dynes/cm(2)) shear stress, PrMCs attached and rolled on recombinant E-selectin and P-selectin and VCAM-1. An anti-PSGL-1 monoclonal antibody (mAb) blocked essentially all adhesion to P-selectin but reduced adhesion to E-selectin by only 40%, suggesting PrMCs express other ligands for E-selectin. PrMCs adhered strongly to tumor necrosis factor-alpha (TNF-alpha)-activated HUVECs, whereas adhesion to interleukin 4 (IL-4)-activated HUVECs was lower. PrMC adhesion to IL-4-activated HUVECs was totally alpha4-integrin- and VCAM-1-dependent. Adhesion to TNF-alpha-activated HUVECs was blocked by 50% by mAbs against alpha4-integrin, vascular cell adhesion molecule-1 (VCAM-1), E-selectin, or PSGL-1, whereas combinations of mAbs to alpha4-integrin plus PSGL-1, or VCAM-1 plus E-selectin, blocked adhesion by greater than 70%. Thus, PrMCs derived in vitro predominantly use alpha4-integrin, VCAM-1, PSGL-1, and other ligands that bind E-selectin for adhesion to cytokine-activated HUVEC monolayers. These observations may explain the abundance of MCs at sites of mucosal inflammation, where VCAM-1 and E-selectin are important inducible receptors.
Beller, T. C. and J. A. Boyce (2002). "Prolonged anticonvulsant hypersensitivity syndrome related to lamotrigine in a patient with human immunodeficiency virus." Allergy Asthma Proc 23(6): 415-9.
Anticonvulsant hypersensitivity syndrome (AHS) is a rare, potentially life-threatening allergic disorder, which is well described in relation to many aromatic anticonvulsants. Lamotrigine is a relatively new aromatic anticonvulsant agent that is thought to act on voltage-dependent sodium channels. Initially, it was licensed as add-on therapy for seizures inadequately controlled by other medications. However, its use has been broadened to other indications, including stand-alone therapy for seizures as well as for bipolar disorder. There is extensive experience with hypersensitivity syndromes related to phenytoin, carbomazepine, primidone, and phenobarbital, but fewer reactions have been reported to lamotrigine because of its relatively recent release. Patients with human immunodeficiency virus (HIV) have a higher rate of adverse reactions to many medications. It is unknown if they react more commonly to anticonvulsants such as lamotrigine. It is also unknown if the syndrome lias a tendency to be more severe or prolonged in such patients. The diagnosis of AHS may be particularly elusive in patients with HIV because its common features can easily be confused with an infectious etiology. We report the occurrence of a prolonged hypersensitivity syndrome likely related to lamotrigine in a 32-year-old female with HIV and review the literature regarding this condition.
Mellor, E. A., A. Maekawa, et al. (2001). "Cysteinyl leukotriene receptor 1 is also a pyrimidinergic receptor and is expressed by human mast cells." Proc Natl Acad Sci U S A 98(14): 7964-9.
The cysteinyl leukotrienes (cys-LTs) LTC(4), LTD(4), and LTE(4) are a class of peptide-conjugated lipids formed from arachidonic acid and released during activation of mast cells (MCs). We now report that human cord-blood-derived MCs (hMCs) express the CysLT1 receptor, which responds not only to inflammation-derived cys-LTs, but also to a pyrimidinergic ligand, UDP. hMCs express both CysLT1 protein and transcript, and respond to LTC(4), LTD(4), and UDP with concentration-dependent calcium fluxes, each of which is blocked by a competitive CysLT1 receptor antagonist, MK571. Stably transfected Chinese hamster ovary cells expressing the CysLT1 receptor also exhibit MK571-sensitive calcium flux to all three agonists. Both hMCs and CysLT1 transfectants stimulated with UDP are desensitized to LTC(4), but only partially to LTD(4). Priming of hMCs with IL-4 for 5 days enhances their sensitivity to each agonist, but preferentially lowers their threshold for activation by LTC(4) and UDP (approximately 3 log(10)-fold shifts in dose-response for each agonist) over LTD(4) (1.3 log(10)-fold shift), without altering CysLT1 receptor mRNA or surface protein expression, implying the likely induction of a second receptor with CysLT1-like dual ligand specificity. hMCs thus express the CysLT1 receptor, and possibly a closely related IL-4-inducible receptor, which mediate dual activation responses to cys-LTs and UDP, providing an apparent intersection linking the inflammatory and neurogenic elements of bronchial asthma.
Hsieh, F. H., B. K. Lam, et al. (2001). "T helper cell type 2 cytokines coordinately regulate immunoglobulin E-dependent cysteinyl leukotriene production by human cord blood-derived mast cells: profound induction of leukotriene C(4) synthase expression by interleukin 4." J Exp Med 193(1): 123-33.
Human mast cells (hMCs) derived in vitro from cord blood mononuclear cells exhibit stem cell factor (SCF)-dependent comitogenic responses to T helper cell type 2 (Th2) cytokines. As cysteinyl leukotriene (cys-LT) biosynthesis is a characteristic of immunoglobulin (Ig)E-activated mucosal hMCs, we speculated that Th2 cytokines might regulate eicosanoid generation by hMCs. After passive sensitization for 5 d with IgE in the presence of SCF, anti-IgE-stimulated hMCs elaborated minimal cys-LT (0.1 +/- 0.1 ng/10(6) hMCs) and abundant prostaglandin (PG)D(2) (16.2 +/- 10.3 ng/10(6) hMCs). Priming of hMCs by interleukin (IL)-4 with SCF during passive sensitization enhanced their anti-IgE-dependent histamine exocytosis and increased their generation of both cys-LT (by 27-fold) and PGD(2) (by 2. 5-fold). Although priming with IL-3 or IL-5 alone for 5 d with SCF minimally enhanced anti-IgE-mediated cys-LT generation, these cytokines induced further six- and fourfold increases, respectively, in IgE-dependent cys-LT generation when provided with IL-4 and SCF; this occurred without changes in PGD(2) generation or histamine exocytosis relative to hMCs primed with IL-4 alone. None of these cytokines, either alone or in combination, substantially altered the levels of cytosolic phospholipase A(2) (cPLA(2)), 5-lipoxygenase (5-LO), or 5-LO activating protein (FLAP) protein expression by hMCs. In contrast, IL-4 priming dramatically induced the steady-state expression of leukotriene C(4) synthase (LTC(4)S) mRNA within 6 h, and increased the expression of LTC(4)S protein and functional activity in a dose- and time-dependent manner, with plateaus at 10 ng/ml and 5 d, respectively. Priming by either IL-3 or IL-5, with or without IL-4, supported the localization of 5-LO to the nucleus of hMCs. Thus, different Th2-derived cytokines target distinct steps in the 5-LO/LTC(4)S biosynthetic pathway (induction of LTC(4)S expression and nuclear import of 5-LO, respectively), each of which is necessary for a full integrated functional response to IgE-dependent activation, thus modulating the effector phenotype of mature hMCs.
Bannert, N., M. Farzan, et al. (2001). "Human Mast cell progenitors can be infected by macrophagetropic human immunodeficiency virus type 1 and retain virus with maturation in vitro." J Virol 75(22): 10808-14.
Mast cells are critical components of innate and adaptive immunity that differentiate in tissues in situ from circulating committed progenitor cells. We now demonstrate that human cord blood-derived mast cell progenitors are susceptible to infection with macrophagetropic (M-tropic) and dualtropic human immunodeficiency virus type 1 (HIV-1) isolates but not with T-cell-tropic (T-tropic) strains. Mast cell progenitors (c-kit(+) CD13(+) cells with chloroacetate esterase activity) were purified from 4-week-old cultures of cord blood mononuclear cells maintained in stem cell factor, interleukin-6 (IL-6), and IL-10 using a CD14 depletion column. These progenitors expressed CCR3, CCR5, and CXCR4, as well as low levels of CD4. When infected in vitro with viruses pseudotyped with different HIV and simian immunodeficiency virus envelope glycoproteins, only M-tropic and dualtropic, but not T-tropic, viruses were able to enter mast cell progenitors. Both the CCR5-specific monoclonal antibody 2D7 and TAK-779, a nonpeptide inhibitor of CCR5-mediated viral entry, blocked HIV-1 strain ADA infection by >80%. Cultures infected with replication-competent virus produced progressively increasing amounts of virus for 21 days as indicated by p24 antigen detection. Mast cell progenitors that were exposed to an M-tropic, green fluorescent protein-expressing HIV-1 strain exhibited fluorescence indicative of viral entry and replication on a single-cell level and retained virus production during differentiation. The trafficking of mast cell progenitors to multiple tissues, combined with the long life span of mature mast cells, suggests that they could provide a widespread and persistent HIV reservoir in AIDS.
Austen, K. F. and J. A. Boyce (2001). "Mast cell lineage development and phenotypic regulation." Leuk Res 25(7): 511-8.
Three cornerstones of mast cell development are an absolute dependence on the presence of stem cell factor, T-cell-independent and T-cell-dependent tissue mast cell populations derived from a single lineage, and a diversity of phenotypes for mature tissue mast cells as defined by immunohistochemical and biochemical properties. The in vivo biology of the mast cell in the mouse has been deduced through the availability of mice with genetic and induced gene disruptions, whereas limited but compatible findings for the human have been acquired through the study of patients with systemic mastocytosis and T-cell deficiency. The characteristics of mast cells recognized from these in situ circumstances can be used to establish culture systems for obtaining mouse and human mast cells from progenitor cell sources. These cells allow studies of receptor-mediated gene regulation by cytokines derived from both stromal cells and T cells.
Ochi, H., N. H. De Jesus, et al. (2000). "IL-4 and -5 prime human mast cells for different profiles of IgE-dependent cytokine production." Proc Natl Acad Sci U S A 97(19): 10509-13.
Mast cells (MC) are stem cell factor-dependent tissue-based hematopoietic cells with substantial functional heterogeneity. Cord blood-derived human MC (hMC) express functional receptors for IL-5, and IL-5 mediates stem cell factor-dependent comitogenesis of hMC in vitro. Although IL-5 is not required for normal hMC development, we considered that it might prime hMC for their high-affinity Fc receptor for IgE (FcvarepsilonRI)-dependent generation of cytokines, as previously demonstrated for IL-4. Compared with hMC maintained in stem cell factor alone, hMC primed with IL-5 expressed 2- to 4-fold higher steady-state levels of TNF-alpha, IL-5, IL-13, macrophage inflammatory protein 1alpha, and granulocyte-macrophage colony-stimulating factor transcripts 2 h after FcvarepsilonRI crosslinking and secreted 2- to 5-fold greater quantities of the corresponding cytokines, except IL-13, at 6 h. Unlike IL-4, IL-5 priming did not enhance FcvarepsilonRI-dependent histamine release. Thus, IL-5 augments cytokine production by hMC by a mechanism distinct from that of IL-4 and with a different resultant profile of cytokine production. These observations suggest a potentially autocrine effect of IL-5 on hMC for amplification of allergic immune responses, in addition to its recognized paracrine effects on eosinophils, and implicate both IL-4 and IL-5 in the modulation of the hMC phenotype.
Ochi, H., W. M. Hirani, et al. (1999). "T helper cell type 2 cytokine-mediated comitogenic responses and CCR3 expression during differentiation of human mast cells in vitro." J Exp Med 190(2): 267-80.
Mast cells (MCs) arise in situ from circulating stem cell factor (SCF)-dependent committed progenitors (PrMCs) and accumulate at sites of allergic mucosal inflammation. We hypothesized that human (h)PrMCs and their mature counterparts might share overlapping patterns of chemokine and cytokine receptor utilization with eosinophils, basophils, and T helper type 2 (Th2) lymphocytes for their homing and allergy-associated hyperplasia. We have characterized committed hPrMCs and fully mature hMCs derived in vitro from cord blood for their functional responses to chemokine and cytokine agonists germane to allergic inflammation and for their maturation-related expression of the corresponding receptors. After 4 wk of culture in the presence of recombinant stem cell factor (SCF), interleukin (IL)-6, and IL-10, the cells were characterized as hPrMCs based upon their uniform surface expression of c-kit and CD13, low-level expression of FcinRIalpha, absence of CD14 and CD16 expression, and immunoreactivity for MC chymase in >80%, and about half were immunoreactive for tryptase and metachromatic with toluidine blue. By week 9, the cells had matured into hMCs, identified by higher levels of c-kit, continued expression of CD13 and low-level FcinRIalpha, uniform toluidine blue metachromasia, and uniform immunoreactivity for both tryptase and chymase. The 4-wk-old hPrMCs expressed four chemokine receptors (CXCR2, CCR3, CXCR4, and CCR5). Each receptor mediated transient rapid calcium fluxes in response to its respective ligand. Both recombinant human eotaxin and stromal cell-derived factor 1alpha elicited chemotaxis of hPrMCs. Only CCR3 was retained on the mature 9-wk-old hMCs from among these chemokine receptors, and hMCs responded to eotaxin with a sustained calcium flux but without chemotaxis. The Th2 cytokines IL-3, IL-5, IL-6, IL-9, and granulocyte/macrophage colony-stimulating factor each augmented the SCF-dependent proliferation of hPrMCs and hMCs. In contrast, the prototypical Th1 cytokine, interferon gamma, suppressed SCF-driven proliferation of both hPrMCs and hMCs. Thus, throughout their development in vitro, hMCs obey SCF-dependent, cytokine-driven mitogenic responses that reflect a Th2-type polarization characteristic of allergy and asthma. Furthermore, committed hPrMCs have a unique profile of chemokine receptor expression from among reported hematopoietic cells, including CCR3, which is shared with the other cells central to allergic inflammation (eosinophils, basophils, and Th2 lymphocytes).
Yuan, Q., M. F. Gurish, et al. (1998). "Generation of a novel stem cell factor-dependent mast cell progenitor." J Immunol 161(10): 5143-6.
Tissue mast cell development requires stem cell factor (SCF), whereas helminth-induced intestinal mucosal mast cell hyperplasia also requires T cell-derived factors such as IL-3. We generated progenitor mast cells (PrMC) from mouse bone marrow cells (BMC) in vitro with a triad of SCF, IL-6, and IL-10 that exhibit IL-3-mediated mitogenic and maturation responses. SCF/IL-6/IL-10 transiently elicited a cell subpopulation with the phenotype (c-kit(high)Thy-1(low)) of fetal blood promastocytes at 3 wk of culture that progressed within 1 wk to FcepsilonRI-bearing PrMC, designated PrMCTriad. PrMCTriad lacked mouse mast cell carboxypeptidase A (mMC-CPA) protein, required SCF for IL-3-driven thymidine incorporation, and responded to SCF plus IL-3 with strong mMc-CPA immunoreactivity, clarifying distinct sequential roles for SCF and IL-3 in mast cell development. PrMCTriad, arising from BMC through promastocytes, are metamastocytes that acquire microenvironmentally determined phenotypic features.
Yuan, Q., K. F. Austen, et al. (1997). "Human peripheral blood eosinophils express a functional c-kit receptor for stem cell factor that stimulates very late antigen 4 (VLA-4)-mediated cell adhesion to fibronectin and vascular cell adhesion molecule 1 (VCAM-1)." J Exp Med 186(2): 313-23.
We evaluated mature peripheral blood eosinophils for their expression of the surface tyrosine kinase, c-kit, the receptor for the stromal cell-derived cytokine, stem cell factor (SCF). Cytofluorographic analysis revealed that c-kit was expressed on the purified peripheral blood eosinophils from 8 of 8 donors (4 nonatopic and 4 atopic) (mean channel fluorescence intensity 2.0- 3. 6-fold, average 2.8 +/- 0.6-fold, greater than the negative control). The uniform and selective expression of c-kit by eosinophils was confirmed by immunohistochemical analysis of peripheral blood buffy coats. The functional integrity of c-kit was demonstrated by the capacity of 100 ng/ml (5 nM) of recombinant human (rh) SCF to increase eosinophil adhesion to 3, 10, and 30 microg/ml of immobilized FN40, a 40-kD chymotryptic fragment of plasma fibronectin, in 15 min by 7.7 +/- 1.4-, 5.3 +/- 3.3-, and 5.4 +/- 0. 2-fold, respectively, and their adhesion to 0.1, 0.5, and 1.0 microg/ml vascular cell adhesion molecule-1 (VCAM-1), by 12.7 +/- 9. 2-, 3.8 +/- 2.5-, and 1.7 +/- 0.6-fold, respectively. The SCF-stimulated adhesion occurred without concomitant changes in surface integrin expression, thereby indicating an avidity-based mechanism. rhSCF (100 ng/ml, 5 nM) was comparable to rh eotaxin (200 ng/ml, 24 nM) in stimulating adhesion. Cell adhesion to FN40 was completely inhibited with antibodies against the alpha4 and beta1 integrin subunits, revealing that the SCF/c-kit adhesion effect was mediated by a single integrin heterodimer, very late antigen 4 (VLA-4). Thus, SCF represents a newly recognized stromal ligand for the activation of eosinophils for VLA-4-mediated adhesion, which could contribute to the exit of these cells from the blood, their tissue localization, and their prominence in inflammatory lesions.
Boyce, J. A. (1997). "The pathobiology of eosinophilic inflammation." Allergy Asthma Proc 18(5): 293-300.
Eosinophils are bone-marrow-derived granulocytes that are involved in both allergic and nonallergic inflammation. They possess a diverse repertoire of functional responses and effector capabilities, including the release of preformed cytotoxic granule proteins, superoxide production, leukotriene biosynthesis, and cytokine production. Each of these functional capabilities is linked to the production of tissue damage and physiologic derangements that are characteristic of human diseases associated with eosinophil-dominated inflammation, such as asthma. This review concerns the biology of eosinophils as it pertains to the pathogenesis of allergic disease.
Boyce, J. A., B. K. Lam, et al. (1996). "Expression of LTC4 synthase during the development of eosinophils in vitro from cord blood progenitors." Blood 88(11): 4338-47.
The expression of leukotriene C4 synthase (LTC4S) was examined during the development of eosinophils in vitro from cord blood mononuclear cells. At 7 days, the cells contained mRNA and sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblot signals for cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO), and 5-lipoxygenase-activating protein (FLAP), but lacked LTC4S and did not generate cysteinyl leukotrienes when stimulated with 20 mumol/L calcium ionophore. At 14 days, 94% of the cells were of eosinophil lineage, both LTC4S mRNA transcript and protein were present, and ionophore stimulation resulted in the generation of 23.9 +/- 6.0 pmol cysteinyl leukotrienes/10(6) eosinophil-lineage cells (mean +/- SEM, n = 6). At 28 days, progressive eosinophil maturation was accompanied by further increments in 5-LO, FLAP, and LTC4S proteins, and by the ionophore-induced production of 94.6 +/- 9.0 pmol cysteinyl leukotrienes/10(6) eosinophil-lineage cells (n = 6). Cells selected for CD34 expression lacked detectable 5-LO/LTC4S pathway proteins, and with culture generally expressed immunodetectable cPLA2 and 5-LO proteins by 3 days, FLAP protein by 7 days, and LTC4S protein by 10 days. Thus, during the development of eosinophils in vitro, cPLA2, 5-LO, and FLAP are expressed before LTC4S. Once the lineage is established by morphologic criteria, the eosinophilopoietic cytokines mediate upregulation of FLAP and LTC4S, members of a newly recognized gene family, and of 5-LO, during ongoing cell maturation.
Boyce, J. A., D. Friend, et al. (1996). "Constitutive production of granulocyte/macrophage colony-stimulating factor by hypodense mononuclear eosinophils developed in vitro from hybrid eosinophil/basophil granulocytes." Proc Natl Acad Sci U S A 93(6): 2436-41.
We recently described the development in vitro of cells with granules characteristic of eosinophils and basophils (hybrid granulocytes) from normal human cord blood mononuclear cells cultured for 14 days with recombinant human (rh) interleukin (IL)-3, rhIL-5, and a soluble basement membrane, Matrigel. Hybrid granulocytes constitutively produced granulocyte/macrophage colony-stimulating factor (GM-CSF) and rapidly developed into eosinophils after the exogenous cytokines and Matrigel were removed. To characterize the developmental progression of hybrid granulocytes, cells were maintained for an additional 14 days in medium containing rhIL-3, rhIL-5, and Matrigel. After 28 days, 73% +/- 1% (mean +/- SEM; n = 6) of the nonadherent cells were mononuclear eosinophils, 13% +/- 3% were eosinophils with two or more nuclear lobes, 13% +/- 4% were hybrid granulocytes, and 0.2% +/- 0.1% were basophils. More than 90% of the mononuclear eosinophils were hypodense as determined by centrifugation through metrizamide gradients. After an additional 5 days of culture in medium without exogenous cytokines, 65% +/- 3% (n = 5) of the 28-day cells excluded trypan blue. In contrast, 2% +/- 1% of freshly isolated peripheral blood eosinophils survived 5 days of culture without exogenous cytokines (n = 5). Fifty percent conditioned medium from in vitro derived 28-day mononuclear eosinophils and 14-day hybrid granulocytes maintained the survival of 60% +/- 7% and 77% +/- 7%, respectively, of freshly isolated peripheral blood eosinophils for 72 h, compared with 20% +/- 8% survival in medium alone (n = 3). The eosinophil viability-sustaining activity of 50% mononuclear eosinophil-conditioned medium was neutralized with a GM-CSF antibody. A total of 88% of the 28-day cells exhibited immunochemical staining for GM-CSF. Thus, during eosinophilopoiesis, both hybrid eosinophil/basophil intermediates and immature mononuclear eosinophils exhibit autocrine regulation of viability due to constitutive production of GM-CSF.
Boyce, J. A., D. Friend, et al. (1995). "Differentiation in vitro of hybrid eosinophil/basophil granulocytes: autocrine function of an eosinophil developmental intermediate." J Exp Med 182(1): 49-57.
Granulocytes with the hybrid characteristics of eosinophils and basophils have been identified in the bone marrow and peripheral blood of humans with myeloid leukemias. We now describe a technique by which such hybrid granulocytes can be developed in vitro from normal cord blood precursors cultured in the presence of recombinant human interleukin (rhIL) 3 (350 pM) and rhIL-5 (200 pM) in a plastic vessel coated with Matrigel. After 14 d in culture, 90 +/- 3% (mean +/- standard error of the mean) of the nonadherent cells cultured in the Matrigel-coated flasks contained both eosinophil and basophil granules, as indicated by staining with Wright's and Giemsa stains. Of the nonadherent cells, 93 +/- 1% contained cyanide-resistant peroxidase, and 88 +/- 2% were toluidine blue-positive, characteristic of eosinophil and basophil granules, respectively. Transmission electron micrographs showed hybrid cells containing ultrastructurally distinct eosinophil granules with developing crystalline cores and basophil granules with reticular structures. These 14-d cord blood-derived cell cultures showed strong hybridization signals for eosinophil-derived neurotoxin by RNA blot analysis and contained 78 ng histamine per 10(6) cells. When the granulocytes were removed from cytokine-containing medium and suspended without Matrigel in RPMI 1640 medium containing 10% fetal calf serum (FCS), more than 80% of the granulocytes excluded trypan blue for as long as 5 d, and 93% had developed into eosinophils at 6 d. Conditioned medium prepared over 48 h from the 14-d cell cultures (hybrid granulocytes) sustained the 4-d viability in vitro of 78% of peripheral blood eosinophils from atopic donors. In comparison, 13% survived in RPMI 1640 containing 10% FCS alone. This viability-sustaining activity was nearly completely neutralized by an anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) antibody and was only minimally reduced by anti-IL-3 or IL-5. Thus, cells possessing both eosinophil and basophil granules by both histochemical and ultrastructural analysis can be developed from normal progenitors in vitro in response to eosinophilopoietic cytokines and Matrigel. Their subsequent spontaneous development into mature eosinophils suggests that hybrid granulocytes are part of a normal developmental sequence during eosinophilopoiesis. Furthermore, these hybrid granulocytes are capable of autoregulation through elaboration of GM-CSF, which sustains their viability.
