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To investigate the role of mast cells in hematopoietic cell transplantation, we assessed graft-versus-host disease (GVHD) in C57BL/6-KitW-sh/W-shrecipients, which virtually lack mast cells, compared with C57BL/6 WT recipients. GVHD was severely exacerbated in C57BL/6-KitW-sh/W-shmice (median survival time = 13 vs 60 days in wild-type [WT] mice; P< .0001). The increased mortality risk in C57BL/6-KitW-sh/W-shhosts correlated with increased T-cell numbers in lymph nodes, liver, and gastrointestinal tract sites, as indicated by bioluminescence imaging (P< .001). We did not detect any deficit in the number or function of CD4+CD25+regulatory T cells (Tregs) in C57BL/6-KitW-sh/W-shmice. Furthermore, Tregs were equally effective at reducing GVHD in C57BL/6-KitW-sh/W-shrecipients compared with WT recipients containing mast cells. Furthermore, we found that survival of C57BL/6-KitW-sh/W-shmice during GVHD was significantly improved if the mice were engrafted with bone marrow–derived cultured mast cells from WT C57BL/6 mice but not from interleukin (IL)-10–deficient C57BL/6 mice. These data indicate that the presence of mast cells can significantly reduce GVHD independently of Tregs, by decreasing conventional T-cell proliferation in a mechanism involving IL-10. These experiments support the conclusion that mast cells can mediate a novel immunoregulatory role during hematopoietic cell transplantation.

Both mast cells and IL-17 can contribute to host defense and pathology in part by orchestrating neutrophil recruitment, but the possible role of mast cells in IL-17–induced inflammation remains to be defined. We found that mast cells and IL-17, but neither IFN-γ nor FcRγ signaling, contributed significantly to the antigen (Ag)–dependent airway neutrophilia elicited in ovalbumin-specific T-cell receptor (TCR)–expressing C57BL/6-OTII mice, and that IFN-γ significantly suppressed IL-17–dependent airway neutrophilia in this setting. IL-18, IL-1β, and TNF each contributed significantly to the development of Ag- and T helper 17 (Th17 cell)–mediated airway neutrophilia. Moreover, IL-17 enhanced mast cell TNF production in vitro, and mast cell–associated TNF contributed significantly to Ag- and Th17 cell–mediated airway neutrophilia in vivo. By contrast, we detected no significant role for the candidate mediators histamine, PGD2, LTB4, CXCL10, or IL-16, each of which can be produced by mast cells and other cell types, in the neutrophil infiltration elicited in this model. These findings establish that mast cells and mast cell–derived TNF can significantly enhance, by FcRγ-independent mechanisms, the Ag- and Th17 cell–dependent development of a neutrophil-rich inflammatory response at a site of Ag challenge.

The in vivo functions of interleukin-3 (IL-3) were investigated by generating IL-3–deficient mice. Although hematopoiesis was unimpaired in homozygous mutant animals, contact hypersensitivity reactions were compromised. IL-3 was required for efficient priming of hapten-specific contact hypersensitivity responses, but was dispensable for T-cell–dependent sensitization to tumor cells. These findings reveal a critical role for IL-3 in some forms of delayed-type hypersensitivity.

The normal skin and other tissues of adult genetically mast cell-deficient WBB6F1-W/Wvor WCB6F1-Sl/Sldmice contain less than 1.0% the number of mast cells present in the corresponding tissues of the congenic normal (+ / +) mice. We previously reported that mature dermal mast cells developed locally in the skin of W/Wv, but not Sl/Sld, mice at sites of chronic idiopathic dermatitis. We now report that the repeated application of phorbol 12-myristate 13-acetate (PMA) to the ear skin of either W/Wvor + / + mice induces both dermatitis and a striking and dose-dependent increase in the number of dermal mast cells. The number of dermal mast cells at sites treated for 6 weeks with 5 μg PMA, three times per week, was 39 ± 7/mm2and 305 ± 34/mm2for W/Wvand +/+ mice, respectively; the corresponding values for vehicle-treated skin were 1.5 ± 1.0/mm2and 145 ± 8/mm2, respectively. The PMA-induced dermal mast cells in W/Wvmice appeared mature by morphology, stained with the heparin-binding fluorescent dye, berberine sulfate, and were competent to express IgE-dependent passive cutaneous anaphylaxis responses. The development of mast cells was a local, not systemic, effect of PMA treatment. PMA treatment also induced dermatitis in both WCB6F1-Sl/Sldand +/+ mice, but was associated with increased numbers of dermal mast cells only in the WCB6F,- + / + mice. PMA treatment had no detectable effect on the ability of bone marrow-derived cultured mast cells to survive in the skin of Sl/Sldmice. These findings establish a convenient model system for analyzing factors associated with the development of endogenous populations of mast cells in genetically mast cell-deficient W/Wvmice.

The normal skin and other tissues of adult mast cell-deficient WBB6F1-W/WVor WCBeF1-SI/SIdmice contain <1.0% the number of mast cells present in the corresponding tissues of the congenic normal (+/ +) mice. As a result, genetically mast cell-deficient WBB6F1-W/Wvor WCB6F1-Sl/Sldmice are widely used for studies of mast cell differentiation and function. We found that mast cells developed at sites of idiopathic chronic dermatitis in WBB6F1-W/WVmice and that the number of mast cells present in the skin of WBB6F1-W/WVmice was proportional to the severity of the dermatitis (in ear skin, there were 33 ± 4 mast cells/mm2of dermis at sites of severe dermatitis v9± 3 at sites of mild dermatitis, 0.8 ± 0.3 in skin without dermatitis, and 100 ± 7 in the normal skin of congenic WBB6F1-+ / + mice; in back skin, the corresponding values were 2.0 ± 0.6, 1.1 ± 0.9, 0.025 ± 0.025, and 26.2 ± 3.2). The development of mast cells was a local, not systemic, consequence of the dermatitis. Thus, WBB6F1-W/Wv mice with severe dermatitis lacked mast cells in skin not showing signs of dermatitis and also in the peritoneal cavity, stomach, cecum, and tongue. Idiopathic chronic dermatitis was not associated with the local development of mast cells in WCB6F1-SI/SId mice, a mutant whose mast cell deficiency is due to a mechanism distinct from that of WBB6F1-W/WV mice. These findings may have implications for understanding the nature of the mast cell defi-ciency in WBB6F1-W/WV and WCB6F1-SI/Sld mice and for the use of these mutants to analyze mast cell differentiation and function.

We used light and electron microscopy to analyze the eyelid inflammation that develops in transgenic mice that overex-press interleukin-4 (IL-4; Tepper et al. Cell62:457, 1990). Analysis of alkaline Giemsa-stained plastic sections examined by light microscopy (Dvorak et al, J Exp Med132:558, 1970), as well as by routine transmission electron microscopy, indicated that the mast cells in the inflammatory eyelid lesions were undergoing piecemeal degranulation, a form of secretion in which the cells' cytoplasmic granules exhibit characteristic morphologic changes that are thought to be associated with the prolonged, vesicle-mediated release of the granules' constituents. Moreover, by using a newly reported enzyme affinity-gold method, which stains histamine based on binding to diamine oxidase-gold (Dvorak et al, J Histochem Cytochem41:787, 1993), we show that these activated mast cells had released much of their histamine content. The eyelid lesions also exhibited increased numbers of mast cells; interstitial fibrosis, particularly around cutaneous nerves and blood vessels; activated fibroblasts; focal axonal damage; venules with endothelial cells containing numerous vesiculo-vacuolar organelles; and infiltrates of neutrophils and eosinophils. Our findings illustrate that overexpression of the IL-4 gene in vivo can result in eyelid lesions associated with piecemeal degranulation of mast cells, as well as tissue fibrosis and a variety of other pathologic changes. These results also represent the first direct morphologic evidence for histamine secretion by mast cells in vivo.

We have established the identity of the basophilic leukocyte in the mouse. Previously undefined by electron microscopy, mouse basophils ultrastructurally resemble those of other mammalian species and are readily distin guishable from other granulocytes, lymphocytes, macrophages, and mast cells.

Mouse peritoneal mast cells (PMC) express a connective tissue-type mast cell (CTMC) phenotype, including reactivity with the heparin-binding fluorescent dye berberine sulfate and incorporation of [35S] sulfate predominantly into heparin proteoglycans. When PMC purified to >99% purity were cultured in methylcellulose with IL-3 and IL-4, -25% of the PMC formed colonies, all of which contained both berberine sulfate-positive and berberine sulfate-negative mast cells. When these mast cells were transferred to suspension culture, they generated populations that were 100% berberine sulfate-negative, a characteristic similar to that of mucosal mast cells (MMC), and that synthesized predominantly chondroitin sulfate [35S] proteoglycans. When “MMC-like” cultured mast cells derived from WBB6F1- + / + PMC were injected into the peritoneal cavities of mast cell-deficient WBB6F1-W/Wv mice, the adoptively transferred mast cell population became 100% berberine sulfate-positive. In methylcellulose culture, these “second generation PMC” formed clonal colonies containing both berberine sulfate-positive and berberine sulfate-negative cells, but exhibited significantly less proliferative ability than did normal + / + PMC. Thus, clonal mast cell populations initially derived from single PMC exhibited multiple and bidirectional alterations between CTMC-like and MMC-like phenotypes. However, this process was associated with a progressive diminution of the mast cells’ proliferative ability1988 by Grune & Stratton, Inc. 0006-4971/88/7203-0033$3.00/0.

The effects of rat stem-cell factor (SCF) and interleukin-3 (IL-3), alone or in combination, on the in vitro growth and serine proteinase expression of rat serosal/connective-tissue mast cells (CTMC) or bone marrow-derived mast cells (BMMC) were examined. Rat SCF stimulated the growth of both CTMC and BMMC. IL-3 stimulated BMMC growth to a lesser extent than did SCF, whereas CTMC numbers did not increase in IL-3. However, SCF and IL-3 had synergistic effects on the growth of both BMMC and CTMC. SCF favoured the maintenance of rat mast cell proteinase-l (RMCP-I) in CTMC, but did not induce detectable production of RMCP-I in BMMC. In contrast, when IL-3 or lymph node-conditioned medium (LNCM) was added to SCF, a subpopulation of CTMC expressed and stored the soluble proteinase RMCP-II. In BMMC, the RMCP-II content of cells maintained in SCF was significantly less than that of cells maintained in IL-3 or LNCM. RMCP-II also appeared in the supernatants of BMMC, especially when BMMC numbers were increasing rapidly in SCF with or without IL-3 or LNCM. Thus, SCF and IL-3 can regulate the growth of rat BMMC and CTMC, as well as influence their production and release of proteinases.