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Inflammasomes are multiprotein complexes that assemble in response to microbial and other danger signals and regulate the secretion of biologically active IL-1β and IL-18. Although they are important in protective immunity against bacterial, viral and parasitic infections, aberrant inflammasome activity promotes chronic inflammation associated with autoimmune disease. Inflammasomes have been described in many immune cells, but the majority of studies have focused on their activity in macrophages. Here we discuss an important role for mast cell-inflammasome activity in EAE, the rodent model of multiple sclerosis, a CNS demyelinating disease. We review our evidence that mast cells in the meninges, tissues that surround the brain and spinal cord, interact with infiltrating myelin-specific T cells in early disease. This interaction elicits IL-1β expression by mast cells, which in turn, promotes GM-CSF expression by T cells. In view of the essential role that GM-CSF plays in T cell encephalitogenicity, we propose this mast cell-T cell crosstalk in the meninges is critical for EAE disease development., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis, are autoimmune CNS inflammatory diseases. As a result of a breakdown in the relatively impermeable blood-brain barrier (BBB) in affected individuals, myelin-specific CD4 + and CD8 + T cells gain entry into the immune privileged CNS and initiate myelin, oligodendrocyte, and nerve axon destruction. However, despite the absolute requirement for T cells, there is increasing evidence that innate immune cells also play critical amplifying roles in disease pathogenesis. By modulating the character and magnitude of the myelin-reactive T cell response and regulating BBB integrity, innate cells affect both disease initiation and progression. Two classes of innate cells, mast cells and innate lymphoid cells (ILCs), have been best studied in models of allergic and gastrointestinal inflammatory diseases. Yet, there is emerging evidence that these cell types also exert a profound influence in CNS inflammatory disease. Both cell types are residents within the meninges and can be activated early in disease to express a wide variety of disease-modifying cytokines and chemokines. In this review, we discuss how mast cells and ILCs can have either disease-promoting or -protecting effects on MS and other CNS inflammatory diseases and how sex hormones may influence this outcome. These observations suggest that targeting these cells and their unique mediators can be exploited therapeutically.

Like many of us who had the great fortune to work with Bill Paul, my science life was immeasurably altered by my interactions with him. Although intimidating at first because of his stature in the immunology world, it was soon clear that he not only truly cared about the specific research we were doing together, but he wished to convey to his trainees an approach to science that was open, always questioning, and infinitely fun. His enthusiasm was infectious and after my training with him, despite stresses due to funding and publishing hurdles, I never regretted the path I took. My research took a sharp turn from the studies of adaptive immunity I had planned on pursuing after my fellowship with Bill to a life long quest to understand the wonders of the mast cell, a relatively rare innate immune cell. This came about because Bill's curiosity and expectation of the unexpected allowed him to view, in retrospect, a rather mundane observation we made together involving a non-physiological transformed mast cell line as something that might be really interesting. I have never forgotten that lesson: Look at the data with an eye on the big picture. Sometimes the unexpected is more interesting than predicted results. His example in this regard was incredibly important when as an independent investigator a mistake in mouse sex determination led to unexpected and very confusing data. Yet, these data ultimately revealed a role for mast cells in male-specific protection in experimental autoimmune encephalomyelitis, the mouse model of multiple sclerosis. Bill's influence in immunology is far-reaching and will continue to be felt as those of us who train our own students and post-doctoral fellows pass on his wisdom and approach to scientific research.

The cellular and molecular basis of sex-dimorphic autoimmune diseases, such as the CNS demyelinating disease multiple sclerosis (MS), remains unclear. Our studies in the SJL mouse model of MS, experimental autoimmune encephalomyelitis (EAE), reveal that sex-determined differences in Il33 expression by innate immune cells in response to myelin peptide immunization regulate EAE susceptibility. IL-33 is selectively induced in PLP 139-151 -immunized males and activates type 2 innate lymphoid cells (ILC2s), cells that promote and sustain a nonpathogenic Th2 myelin-specific response. Without this attenuating IL-33 response, females generate an encephalitogenic Th17-dominant response, which can be reversed by IL-33 treatment. Mast cells are one source of IL-33 and we provide evidence that testosterone directly induces Il33 gene expression and also exerts effects on the potential for Il33 gene expression during mast cell development. Thus, in contrast to their pathogenic role in allergy, we propose a sex-specific role for both mast cells and ILC2s as attenuators of the pathogenic Th response in CNS inflammatory disease., Competing Interests: The authors declare no conflict of interest.

GM-CSF is a cytokine produced by T helper (Th) cells that plays an essential role in orchestrating neuroinflammation in experimental autoimmune encephalomyelitis, a rodent model of multiple sclerosis. Yet where and how Th cells acquire GM-CSF expression is unknown. In this study we identify mast cells in the meninges, tripartite tissues surrounding the brain and spinal cord, as important contributors to antigen-specific Th cell accumulation and GM-CSF expression. In the absence of mast cells, Th cells do not accumulate in the meninges nor produce GM-CSF. Mast cell-T cell co-culture experiments and selective mast cell reconstitution of the meninges of mast cell-deficient mice reveal that resident meningeal mast cells are an early source of caspase-1-dependent IL-1β that licenses Th cells to produce GM-CSF and become encephalitogenic. We also provide evidence of mast cell-T cell co-localization in the meninges and CNS of recently diagnosed acute MS patients indicating similar interactions may occur in human demyelinating disease., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The proinflammatory cytokine IL-17 has an important role in pathogenesis of several inflammatory diseases. In immune-mediated joint diseases, IL-17 can induce secretion of other proinflammatory cytokines such as IL-1, IL-6 and TNF, as well as matrix metalloproteinase enzymes, leading to inflammation, cartilage breakdown, osteoclastogenesis and bone erosion. In animal models of inflammatory arthritis, mice deficient in IL-17 are less susceptible to development of disease. The list of IL-17-secreting cells is rapidly growing, and mast cells have been suggested to be a dominant source of IL-17 in inflammatory joint disease. However, many other innate sources of IL-17 have been described in both inflammatory and autoinflammatory conditions, raising questions as to the role of mast cells in orchestrating joint inflammation. This article will critically assess the contribution of mast cells and other cell types to IL-17 production in the inflammatory milieu associated with inflammatory arthritis, understanding of which could facilitate targeted therapeutic approaches.

The meninges are often considered inert tissues that house the CSF and provide protection for the brain and spinal cord. Yet emerging data demonstrates that they are also active sites of immune responses. Furthermore, the blood-CSF barrier surrounding meningeal blood vessels, together with the blood-brain barrier (BBB), is postulated to serve as a gateway for the pathological infiltration of immune cells into the CNS in multiple sclerosis (MS). Our previous studies using mast cell-deficient (Kit(W/Wv)) mice demonstrated that mast cells resident in the dura mater and pia mater exacerbate experimental autoimmune encephalomyelitis (EAE), a rodent model of MS, by facilitating CNS inflammatory cell influx. Here we examined the underlying mechanisms that mediate these effects. We demonstrate that there are dramatic alterations in immune associated gene expression in the meninges in pre-clinical disease, including those associated with mast cell and neutrophil function. Meningeal mast cells are activated within 24 h of disease induction, but do not directly compromise CNS vascular integrity. Rather, through production of TNF, mast cells elicit an early influx of neutrophils, cells known to alter vascular permeability, into the meninges. These data add to the growing evidence that inflammation in the meninges precedes CNS immune cell infiltration and establish that mast cells are among the earliest participants in these disease-initiating events. We hypothesize that mast cell-dependent neutrophil recruitment and activation in the meninges promotes early breakdown of the local BBB and CSF-blood barrier allowing initial immune cell access to the CNS., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Mast cells are classically considered innate immune cells that act as first responders in many microbial infections and have long been appreciated as potent contributors to allergic reactions. However, recent advances in the realm of autoimmunity have made it clear that these cells are also involved in the pathogenic responses that exacerbate disease. In the murine models of multiple sclerosis, rheumatoid arthritis and bullous pemphigoid, both the pathogenic role of mast cells and some of their mechanisms of action are shared. Similar to their role in infection and a subset of allergic responses, mast cells are required for the efficient recruitment of neutrophils to sites of inflammation. Although this mast cell-dependent neutrophil response is protective in infection settings, it is postulated that neutrophils promote local vascular permeability and facilitate the entry of inflammatory cells that enhance tissue destruction at target sites. However, there is still much to learn. There is little information regarding mechanisms of mast cell activation in disease. Nor is it known how many mast cell-derived mediators are relevant and whether interactions with other cells are implicated in these diseases including T cells, B cells and astrocytes. Here we review the current state of knowledge about mast cells in autoimmune disease. We also discuss findings regarding newly discovered mast cell actions and factors that modulate mast cell function. We speculate that much of this new information will ultimately contribute to a greater understanding of the full range of mast cell actions in autoimmunity. This article is part of a Special Issue entitled: Mast cells in inflammation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Mast cells (MCs) exert a significant pathologic influence on disease severity in C57BL/6 (B6) strain-dependent experimental allergic encephalomyelitis (EAE), a model of primary progressive multiple sclerosis (MS). However, relapsing-remitting MS, which is modeled in SJL mice, is the more prevalent form. Given genetically determined heterogeneity in numbers and responsiveness of MCs from various strains of mice, we asked whether these cells also influence this more clinically relevant MS model using SJL-Kit(W/W-v) mice. Similar to the commercially available WBB6F(1)-Kit(W/W-v) mice, SJL-Kit(W/W-v) mice are MC-deficient, anemic, and neutropenic and have normal T cell compartments. They exhibit significantly reduced disease severity, but retain the relapsing-remitting course, a phenotype reversed by selective MC reconstitution. These data confirm that MC influence is not confined to an isolated model of EAE and reveal a new system to study the effects of MC heterogeneity on relapsing-remitting EAE and other SJL strain-specific diseases.

Mast cells contribute to the pathogenesis of experimental autoimmune encephalomyelitis, a rodent model of the human demyelinating disease multiple sclerosis. Yet their site and mode of action is unknown. In both diseases, myelin-specific T cells are initially activated in peripheral lymphoid organs. However, for disease to occur, these cells must enter the immunologically privileged CNS through a breach in the relatively impermeable blood-brain barrier. In this study, we demonstrate that a dense population of resident mast cells in the meninges, structures surrounding the brain and spinal cord, regulate basal CNS barrier function, facilitating initial T cell CNS entry. Through the expression of TNF, mast cells recruit an early wave of neutrophils to the CNS. We propose that neutrophils in turn promote the blood-brain barrier breach and together with T cells lead to further inflammatory cell influx and myelin damage. These findings provide specific targets for intervention in multiple sclerosis as well as other immune-mediated CNS diseases.

Mast cells were discovered more than 100 years ago and until recently, have been considered renegades of the host with the sole purpose of perpetuating allergy. The discovery of mast cell-deficient mice that could be reconstituted with mast cells (the so called "mast cell knock-in" mice) has allowed the study of the in vivo functions of mast cells and revealed several new facets of these cells. It is now evident that mast cells have a much broader impact on many physiological and pathologic processes. Mast cells, particularly through their dynamic interaction with the nervous system, have been implicated in wound healing, tissue remodeling, and homeostasis. Perhaps the most progress has been made in our understanding of the role of mast cells in immunity outside the realm of allergy, and host defense. Mast cells play critical roles in both innate and adaptive immunity, including immune tolerance. Greater insight into mast cell biology has prompted studies probing the additional consequences of mast cell dysfunction, which reveal a central role for mast cells in the pathogenesis of autoimmune disorders, cardiovascular disorders, and cancer. Here, we review recent developments in the study of mast cells, which present a complex picture of mast cell functions.

Background: The idea that the innate and adaptive immune systems are not separate entities is no longer new. In fact, it is surprising that this paradigm was accepted without question for so long. Many innate cells express cell surface molecules and soluble mediators that are essential for the development and activation of T cells and B cells. Yet among the innate cell populations, mast cells may play the major role in regulating adaptive immune cell function., Discussion: This role first came to light in studies of mast cells and their involvement in the autoimmune disease experimental allergic encephalomyelitis, the major rodent model of multiple sclerosis and has subsequently been verified in many in vitro and in vivo model systems.

There are many parallels between allergic and autoimmune responses. Both are considered hypersensitivity responses: pathologies that are elicited by an exuberant reaction to antigens that do not pose any inherent danger to the organism. Although mast cells have long been recognized as central players in allergy, only recently has their role in autoimmunity become apparent. Because of the commonalities of these responses, much of what we have learned about the underlying mast cell-dependent mechanisms of inflammatory damage in allergy and asthma can be used to understand autoimmunity. Here we review mast cell biology in the context of autoimmune disease. We discuss the huge diversity in mast cell responses that can exert either proinflammatory or antiinflammatory activity. We also consider the myriad factors that cause one response to predominate over another in a particular immune setting.

Among the potential outcomes of an aberrantly functioning immune system are allergic disease and autoimmunity. Although it has been assumed that the underlying mechanisms mediating these conditions are completely different, recent evidence shows that mast cells provide a common link. Mast cells reside in most tissues, are particularly prevalent at sites of Ag entry, and act as sentinel cells of the immune system. They express many inflammatory mediators that affect both innate and adaptive cellular function. They contribute to pathologic allergic inflammation but also serve an important protective role in bacterial and parasite infections. Given the proinflammatory nature of autoimmune responses, it is not surprising that studies using murine models of autoimmunity clearly implicate mast cells in the initiation and/or progression of autoimmune disease. In this review, we discuss the defined and hypothesized mechanisms of mast cell influence on autoimmune diseases, including their surprising and newly discovered role as anti-inflammatory cells.

Although mast cells have long been considered the integral effector cell in allergy and atopic disease, the paradigm of mast cell function is now evolving to incorporate data showing that mast cells make innumerable contributions to both protective and pathologic immune responses. Mast cells express cell surface molecules with costimulatory or co-inhibitory activity and produce a multitude of mediators that can direct dendritic cell (DC) or T-cell differentiation and function. In addition, mast cells exhibit a widespread distribution and are in close proximity to DCs and T cells at several critical sites. While there has been amazing progress in characterizing mast cell populations in vitro, only recently has the ability to monitor their in vivo effects become a reality. In this review, we discuss the evolution of our understanding of mast cell biology with an emphasis on their established and hypothesized roles in influencing T-cell differentiation and function. The fact that T-cell and mast cell interactions exist and are a normal component of most adaptive immune responses is one of the best illustrations of the now established concept that innate and adaptive immunity are not completely independent entities.

When exposed to a pathogen, a naive CD4(+) T cell is forced to make a cell fate decision that leads to a polarized population of Th1 IFN-gamma- or Th2 IL-4- producing cells. Although IL-4 has traditionally been considered a factor that promotes Th2 cell differentiation, recent evidence has demonstrated that the site and timing of IL-4 expression in an immune response determines its ultimate effects on CD4(+) T cell fate. Using a mast cell (MC) reconstitution model, we demonstrate that MC-derived IL-4 promoted Th1 responses in vivo. Furthermore, MCs from genetically disparate mouse strains varied in their potential for IL-4 expression. Independent of the activation mode, MCs from Th1-prone C57BL/6 mice exhibited a more robust Il4 response than did the Th2-prone strain Balb/c. The hierarchy of IL-4 expression potential was directly associated with the degree of basal chromatin accessibility at cis-regulatory elements conserved noncoding sequence-1 and V(A) enhancer within the Th2 locus. GATA1/2 and Ikaros, factors with opposing roles in chromatin remodeling, acted at these sites. We propose that GATA and Ikaros proteins coordinately fine-tune accessibility at the Il4 locus during development to variably regulate IL-4 expression. These events likely contribute to the genetically determined heterogeneity in Th1 responses that underlie susceptibility to many diseases.

As in the fashion industry, trends in a particular area of scientific investigation often are fleeting but then return with renewed and enthusiastic interest. Studies of mast cell biology are good examples of this. Although dogma once relegated mast cells almost exclusively to roles in pathological inflammation associated with allergic disease, these cells are emerging as important players in a number of other physiological processes. Consequently, they are quickly becoming the newest "trendy" cell, both within and outside the field of immunology. As sources of a large array of pro- and anti-inflammatory mediators, mast cells also express cell surface molecules with defined functions in lymphocyte activation and trafficking. Here, we provide an overview of the traditional and newly appreciated contributions of mast cells to both innate and adaptive immune responses.

Once considered to be of sole importance in allergy and parasitic infections, the role of mast cells in other pathologic and protective immune responses is becoming increasingly evident. We previously demonstrated that mast cells contribute to the severity of EAE, the rodent model of multiple sclerosis. Here we show that one mode of mast cell action is through effects on the autoreactive T cell response. Early indices of both peripheral CD4 and CD8 T cell activation, including IFN-gamma production and increases in CD44 and CD11a expression, are attenuated in mast cell-deficient (W/Wv) mice after myelin oligodendrocyte glycoprotein(35-55) priming when compared to WT animals. Reduced infiltrates of activated T cells in the central nervous system are also observed. Importantly, selective repletion of the mast cell compartment restores most T cell responses in the lymph nodes and the central nervous system, correlating with reconstitution of severe disease. The adoptive transfer of WT-derived encephalitogenic T cells results in significantly less severe disease in W/Wv recipients, indicating that mast cells also exert potent effects after the initial T cell response is generated. Our data provide the first in vivo evidence that mast cells can significantly influence T cell responses and suggest that mast cells exacerbate disease during both the inductive and effector phases.

Several GATA-binding sites have been identified in regions both distal to and within the murine IL-4 gene locus, yet their relative role in IL-4 expression is unknown. Chromatin immunoprecipitation assays were used to demonstrate that GATA-1 and GATA-2 are associated with a regulatory element within the second intron of the IL-4 gene in murine mast cells in vivo. Furthermore, although expression from a stably integrated wild-type IL-4 minigene parallels endogenous IL-4 gene expression, mutation of the GATA-binding element, but not an SP-1-binding site, virtually abolishes transcription in mast cells, an observation that correlates with the local loss of H3 and H4 histone acetylation in the intron. Treatment with the chromatin remodeling agents 5 azacytidine and trichostatin A can restore this defect in transcription. These results define an essential site of GATA influence on IL-4 expression in mast cells and directly support the idea that GATA factors have a profound impact on locus accessibility.

Mast cells are established participants in allergic disease and in protection against extracellular parasites. Recently, it has become apparent that mast cells exert many profound effects on a variety of both innate and adaptive immune responses. Using mast cell-deficient WBB6F1/J-kitW/kitWv (W/Wv) mice, we have demonstrated that mast cells are critical for severe disease in a murine model of multiple sclerosis, experimental allergic encephalomyelitis (EAE). Reconstitution of the mast cell population in the periphery, but not the CNS, restores EAE severity. Mast cells exert their effects at both the inductive and effector phases of disease. EAE is mediated by autoreactive T cells that enter the CNS and initiate inflammatory responses, leading to demyelination within the spinal cord and brain. Although there are no intrinsic defects in W/Wv-derived T cells, both CD4+ and CD8+ autoreactive T cell responses are attenuated during early disease in W/Wv mice. Thus mast cells are essential for the optimal priming of autoreactive T cells. The entry of encephalitogenic T cells into the CNS is compromised in these mice as well. The effects on early T cell responses are due, in part, to the reduced percentage of activated dendritic cells in the lymph nodes of W/Wv mice after disease induction compared with wild-type mice. The finding that mast cells can alter T cell responses in EAE has much broader implications for understanding the impact of these cells on all T cell-mediated responses.

Previous studies using mast cell-deficient mice (W/W(v)) revealed that mast cells influence disease onset and severity of experimental allergic/autoimmune encephalomyelitis (EAE), the murine model for multiple sclerosis. The mast cell populations of these mice can be restored by transferring bone marrow-derived mast cells (BMMCs). Studies using the W/W(v) reconstitution model have lead to major advances in our understanding of mast cell roles in vivo. However, despite its common use, details regarding the sites and kinetics of mast cell repopulation have remained largely uncharacterized. In this study, we examined the kinetics and tissue distribution of green fluorescent protein(+) BMMCs in reconstituted W/W(v) mice to identify sites of mast cell influence in EAE. Reconstitution of naive animals with BMMCs does not restore mast cell populations to all organs, notably the brain, spinal cord, lymph nodes, and heart. Despite the absence of mast cells in the CNS, reconstituted mice exhibit an EAE disease course equivalent to that induced in wild-type mice. Mast cells are found adjacent to T cell-rich areas of the spleen and can migrate to the draining lymph node after disease induction. These data reveal that mast cells can act outside the CNS to influence EAE, perhaps by affecting the function of autoreactive lymphocytes.

Murine nuclear factor of activated T cells (NFAT)2.alpha/beta differ by 42 and 28 unique amino-terminal amino acids and are differentially expressed. Both isoforms share conserved domains that regulate DNA-binding and subcellular localization. A genetic "one-hybrid" assay was used to define two distinct transactivation (TA) domains: in addition to a conserved TAD present in both isoforms, a second, novel TAD exists within the beta-specific amino terminus. Pharmacologic inhibitors Gö6976 and rottlerin demonstrate that both conventional and novel protein kinase C (PKC) family members regulate endogenous mast cell NFAT activity, and NFAT2 TA. Overexpression of dominant active PKC (which has been implicated in immune receptor signaling) induces NFAT2.alpha/beta TA. Mutations within the smallest PKC-responsive transactivation domain demonstrate that the PKC effect is at least partially indirect. Significantly, the beta-specific domain confers greater ability to TA in response to treatment with phorbol 12-myristate 13-acetate/ionomycin or lipopolysaccharide, and unique sensitivity to FcepsilonRI signaling. Accordingly, overexpression of NFAT2.beta results in significantly greater NFAT- and interleukin-4 reporter activity than NFAT2.alpha. These results suggest that whereas NFAT2 isoforms may share redundant DNA-binding preferences, there are specialized functional consequences of their isoform-specific domains.

Mast cell-deficient mice (W/W(v)) exhibit significantly reduced severity of experimental allergic encephalomyelitis (EAE), a murine model of multiple sclerosis. In this study, the contribution of FcR-mediated mast cell activation to disease was examined. W/W(v) mice were reconstituted i.v. with bone marrow-derived mast cells (BMMCs) from wild-type mice or those lacking functional FcRs. Eight weeks later, EAE was induced by immunization with the myelin oligodendrocyte glycoprotein 35-55 peptide. Disease scores were analyzed in reconstituted mice and compared with age-matched W/W(v) mice and wild-type littermates. Mice reconstituted with FcRgamma(-/-) BMMCs or FcgammaRIII(-/-) BMMCs exhibited less severe clinical symptoms similar to W/W(v) controls, while reconstitution with FcRIIB(-/-) BMMCs resulted in disease significantly more severe than wild-type controls. Notably, mice reconstituted with FcgammaRIII(-/-) BMMC exhibit a relapsing-remitting course of disease. These data demonstrate that both activating and inhibitory FcRs expressed on mast cells influence the course of EAE.

IL-4 is a potent, pleiotropic cytokine that, in general, directs cellular activation, differentiation, and rescue from apoptosis. However, in mast cells, IL-4 induces the down-regulation of activation receptors and promotes cell death. Mast cells have been shown to transduce IL-4 signals through a unique C-terminally truncated isoform of STAT6. In this study, we examine the mechanism through which STAT6 is processed to generate this isoform. We demonstrate that STAT6 processing in mast cells is initiated by IL-4-induced phosphorylation and nuclear translocation of full-length STAT6 and subsequent cleavage by a nuclear serine-family protease. The location of the protease in the nucleus ensures that the truncated STAT6 has preferential access to bind DNA. IL-4-responsive target genes in mast cells are identified by chromatin immunoprecipitation of STAT6, including the IL-4 gene itself. These results suggest a molecular explanation for the suppressive effects of IL-4 on STAT6-regulated genes in mast cells.

It is well established that CD4(+) T cells are of central importance in mediating the autoimmune destruction associated with the neurological demyelinating disease Multiple sclerosis (MS) and the rodent model of MS, EAE (experimental allergic encephalomyelitis). However, other cells also play a critical role in the inflammatory events that lead to the varying degrees of myelin and axonal damage observed in this disease syndrome. In this review, we present evidence that mast cells, best studied in the context of allergic disease, contribute to EAE disease pathology. Using mast cell-deficient mice, we demonstrate that mast cells are necessary for the full manifestation of MOG-induced EAE disease and show that cross-linking of Fc receptors is one mechanism of mast cell activation in disease. In addition, we provide evidence that mast cells exert influences outside the CNS, perhaps through the effects on the generation of the anti-MOG T cell response.

The role of interleukin (IL)-4 as an important immunomodulatory cytokine is well established. IL-4 exhibits a highly restricted pattern of expression by cells of distinct lineages. The cell types that produce IL-4 are located in anatomically distinct locations (e.g. circulating T cells vs. fixed tissue mast cells) and thus have access to different IL-4-responsive target cells. In addition, these cells appear to regulate IL-4 expression in cell-type-specific ways. These findings suggest that an understanding of IL-4 gene regulation in T and mast cells could provide the means to specifically control IL-4 release in a lineage- and site-specific manner. In this article we review the current knowledge regarding the cell-type specific regulation of IL-4 gene expression in mast cells and compare this to what has been defined in T cells. We show that there are distinct yet parallel events that control developmentally determined chromatin modifications, allowing accessibility of the locus, and provide the potential for transcription. In differentiated cells, a subset of unique cell activation signals initiates the cascade of events that lead to transcriptional activation of the IL-4 gene.

The cell type-specific expression of a gene is dependent on developmentally regulated modifications in chromatin structure that allow accessibility of basal and inducible transcription factors. In this study, we demonstrate that a cis-acting element in the second intron of the murine IL-4 gene has a dual function in regulating transcription in mast cells as well as chromatin accessibility of the IL-4 gene locus through its influence on the methylation state of the gene. Previous studies have shown that mast cell-restricted transcription factors GATA-1/2 and PU.1 associate with the intron element and regulate its activity. In this study, we use DNase I footprinting and mutational analyses to identify two additional sites that contribute to the element's ability to enhance transcription. One of these sites associates preferentially with STAT5a and STAT5b. We also demonstrate that deletion of the element or mutation of the GATA binding site in the context of a stably integrated IL-4 genomic construct prevents maintenance of a demethylated locus in IL-4-producing mast cells. These data indicate that, analogous to Ig and TCR intron regulatory elements, the intron enhancer has an essential role in maintaining developmentally regulated demethylation at the IL-4 gene locus. In addition, they indicate that members of the GATA family of transcription factors likely play an important role in these processes.

In addition to their well characterized role in allergic inflammation, recent data confirm that mast cells play a more extensive role in a variety of immune responses. However, their contribution to autoimmune and neurologic disease processes has not been investigated. Experimental allergic encephalomyelitis (EAE) and its human disease counterpart, multiple sclerosis, are considered to be CD4(+) T cell-mediated autoimmune diseases affecting the central nervous system. Several lines of indirect evidence suggest that mast cells could also play a role in the pathogenesis of both the human and murine disease. Using a myelin oligodendrocyte glycoprotein (MOG)-induced model of acute EAE, we show that mast cell-deficient W/W(v) mice exhibit significantly reduced disease incidence, delayed disease onset, and decreased mean clinical scores when compared with their wild-type congenic littermates. No differences were observed in MOG-specific T and B cell responses between the two groups, indicating that a global T or B cell defect is not present in W/W(v) animals. Reconstitution of the mast cell population in W/W(v) mice restores induction of early and severe disease to wild-type levels, suggesting that mast cells are critical for the full manifestation of disease. These data provide a new mechanism for immune destruction in EAE and indicate that mast cells play a broader role in neurologic inflammation.

The c-maf transcription factor is selectively expressed in IL-4-producing Th2 cells. It has been implicated in IL-4 gene transcription based on its ability to directly activate the IL-4 gene in nonexpressing B cells and to promote IL-4-induced Th2 differentiation. However, it has not been definitively shown that IL-4 production by other cells is dependent on the presence of c-maf. Here, we show that IL-4-producing mast cells do not express the c-maf factor. Furthermore, mutation of a defined c-maf binding site within the proximal IL-4 promoter, which profoundly affects transcription in T cells, has no effect on expression of a reporter gene driven by the IL-4 promoter in mast cells. These results demonstrate that c-maf and its target binding site are not required for IL-4 production in all cell types and delineate additional cis- and trans-acting elements that contribute to the cell-type specific transcriptional regulation of IL-4.

The acquisition of an IL-4-producing phenotype in Th2 cells requires IL-4 signaling through the STAT6 pathway during T cell differentiation. In this study we demonstrate that, unlike in naive T cells, IL-4 is not necessary for the development of an IL-4-producing phenotype in mast cells. Bone marrow-derived mast cell precursors from STAT6-/- mice can differentiate into mature cells that express IL-4 levels comparable to those of wild-type mast cells. In differentiated mast cells, activation in the presence of neutralizing anti-IL-4 antibodies or mutation of the consensus STAT6 sites does not diminish IL-4 promoter activity, indicating that IL-4 is not required for active transcription. Taken together, these data suggest that mast cell IL-4 production is not STAT6 dependent, providing evidence that these cells could generate IL-4 needed for the initiation and amplification of an effective Th2 immune response.

NF of activated T cells (NF-AT) denotes a family of transcription factors that regulate the activation-dependent expression of many immunologically important proteins. At least four distinct genes encode the various family members, and several isoforms of these have been identified as well. The overlapping expression patterns and similar in vitro binding and trans-activation activities on various promoter elements of NF-AT-regulated genes suggest some redundancy in the function of these proteins. However, the phenotypic analysis of NF-AT-deficient mice supports the idea that there are tissue- and gene-specific functions as well. In this study we have characterized the expression of NF-AT cDNAs in murine mast cells. The majority of clones identified correspond to two NF-ATc isoforms that differ only in their amino-terminal sequence. Despite minimal discrepancies in the coding region, there are striking tissue- and cell type-specific differences in isoform expression patterns. Detection of NF-ATc.alpha mRNA is strictly dependent on cell activation signals in both T and mast cell lines. In contrast, the beta isoform is expressed at very low constitutive levels in both cell types but is only up-regulated in response to mast cell activation signals delivered through the FcepsilonRI or via calcium ionophores. These results demonstrate another level of regulation within the NF-AT family that can contribute to cell type-specific gene expression.

IL-4 is a pleiotropic cytokine that signals through STAT6 to direct the transactivation of multiple gene targets. In this study, we demonstrate that mast cells express a distinct STAT6 isoform. This "mast cell STAT" is a product of the STAT6 gene, but is only 65 kDa in size and appears to lack the defined C-terminal transactivation domain. Despite the presence of the conventional 94-kDa STAT6 molecule, it is the smaller isoform that associates with a consensus STAT6 binding site in extracts from IL-4-treated mast cells. This is the first evidence that STAT6 isoforms can be preferentially activated and bind to DNA in a cell-specific manner. These results imply that an additional level of specificity in the IL-4R signaling mechanism exists and may partially explain the diverse effects that IL-4 exerts on different cell types.

IL-4 has been called the "prototypic immunoregulatory cytokine." Like many cytokines, it can affect a variety of target cells in multiple ways. IL-4 has an important role in regulating antibody production, hematopoiesis and inflammation, and the development of effector T-cell responses. It is produced only by a subset of activated hematopoietic cells, including T cells and Fc epsilon R1+ mast cells and basophils. Based on the different tissue distribution and access to distinct target cells, IL-4 derived from T and Fc epsilon R1+ cells may have quite different effects on these immunological processes. In view of this, as well as the clear correlation of aberrant expression with disease, it is of interest to understand the signals that regulate IL-4 expression in a cell-specific manner. Recently, progress has been made in defining the T-cell- and Fc epsilon R1-receptor-mediated signals that stimulate IL-4 gene expression. These studies have demonstrated that there are common and cell-specific signaling pathways that regulate production of this cytokine. In this review, we summarize the activities of IL-4 defined both in vitro and in vivo and compare the signals leading to IL-4 expression in cells of both T- and mast-cell lineage.

Interleukin 4 (IL-4), an immunoregulatory cytokine, is produced only by a subset of activated T cells and cells of the mast cell-basophil lineage. The production of IL-4 by mast cells likely represents a significant source of this protein in local immune-inflammatory responses in the skin, brain, gastrointestinal, and respiratory tracts, in which mast cells are prevalent. In the present study, the cis- and trans-acting elements that control inducible mast cell IL-4 gene transcription were examined and compared with those that function in T cells. We demonstrate that, as in T cells, sequences between bp -87 and -70 are critical for protein association and activation-dependent gene transcription and that this region (termed the activation-responsive element region) is the target of an inducible, cyclosporin A-sensitive, DNA-protein interaction. When assessed by electrophoretic mobility shift assays and UV cross-linking analyses, multiple proteins in both T- and mast cell nuclear extracts associate with the activation-responsive element in vitro, and some of these appear identical. However, distinct proteins are associated with each of the complexes as well. AP-1 family members are unique to the T-cell-stimulation-dependent complex, whereas mast cell complexes contain factors that are reactive with anti-nuclear factor of activated T cells p (NF-ATp) and anti-NF-ATc antibodies but have distinct molecular masses compared with those of T-cell-derived NF-AT. Furthermore, an anti-NF-ATp-reactive factor with a molecular mass of approximately 41 kDa is present in the nuclei of unstimulated cells and binds independently of cell activation, unlike the previously described NF-AT family members. These data support the idea that there are uniquely regulated, cell lineage-specific transcription factors related to T-cell-derived NF-AT that mediate inducible IL-4 transcription in mast cells. These differences likely reflect the distinct cell surface signaling requirements for IL-4 production in T and mast cells.

Interleukin 4 (IL-4), a critical immunoregulatory cytokine, is produced by a subset of T lymphocytes and cells of the mast cell/basophil lineage. There are cell-specific differences in the regulatory elements that control IL-4 transcription in these two cell types. A 683-bp Bgl II fragment, located within the second intron of the murine IL-4 gene, was previously shown to exhibit mast cell-specific enhancer activity. To define critical cis-acting elements that regulate this enhancer, a series of deletions from the 5' and 3' ends of the Bgl II fragment were generated. Their effect on enhancer activity was assessed in IL-4-producing mast cell lines in transient transfection assays. Two functionally independent subregions, E1 and E2, were defined in this analysis. Both are required for full enhancer activity. Sequences identical to previously defined DNA-binding sites for SP1 and GATA are present within E1, and an ets binding site is located within E2. Although mutation of the SP1 sites had no effect on enhancer function, alteration of either the GATA or ets site reduced enhancer activity by 50-60%. Proteins that associate with the IL-4 intronic GATA and ets sites were detected in mast cell nuclear extracts by mobility-shift assays. Specific antibodies identified these factors as GATA-1 and GATA-2 and the ets family member PU.1. GATA-1, GATA-2, and PU.1 exhibit cell-specific expression, suggesting that these proteins play a critical role in the lineage-restricted activity of the IL-4 intronic enhancer in mast cells.

There is increasing evidence that the neurologic system is capable of modulating a wide range of immunologic responses, including certain inflammatory processes in the lung, gastrointestinal tract, and skin. It has been proposed that secreted neuropeptides such as substance P (SP) may mediate these neuroinflammatory interactions by binding to and stimulating immune cells such as mast cells and lymphoid cells. SP is secreted in a variety of tissues by an extensive network of neurosensory C and A5 fibers in response to a wide range of noxious stimuli and injury. Previous studies to examine the effect of SP on mast cells have focused on its role in triggering histamine release and mediating immediate hypersensitivity responses. Recently it was demonstrated that mast cells are also capable of secreting multiple cytokines including TNF-alpha, IL-1, IL-3, IL-4, IL-6, and GM-CSF. In this study we tested the possibility that SP may also influence mast cell-mediated late inflammatory events by modulating the production of one or several of these cytokines. Our results indicate that SP induces TNF-alpha mRNA expression and TNF-alpha secretion in a dose-dependent manner in a murine mast cell line, CFTL12. Likewise, SP stimulates TNF-alpha secretion in freshly isolated murine peritoneal mast cells. The induction of mast cell TNF-alpha is selective, since SP does not stimulate the production of IL-1, IL-3, IL-4, IL-6, or GM-CSF in these cells. The CFTL 12 mast cell line constitutively expresses high levels of SP receptor mRNA which is not modulated by PMA/cycloheximide treatment or SP. These results further support the concept that the neurologic system modulates inflammatory events by neuropeptide-mediated mast cell cytokine release.

IL-4 is a potent immunoregulatory cytokine that exhibits extremely diverse effects on a number of target cells. Although IL-4 was originally described as a T cell-derived product, it is evident that cells of the basophil/mast cell lineage are also an important source of this cytokine. Based on their different tissue distribution, mast cell and T cell-derived IL-4 may have distinct effects on local immune responses. The physiologic production of IL-4 appears to be tightly regulated because most T and mast cells require activation to express significant levels of IL-4. In contrast, a majority of murine transformed mast cell lines constitutively express relatively high levels of IL-4. In this study, transformed mast cell lines were used as models to define cis acting sequences that regulate mast cell IL-4 transcription. Chloramphenicol acetyltransferase reporter gene constructs containing 6.3 kb of 5' IL-4 flanking sequence direct relatively low chloramphenicol acetyltransferase expression in these cells. These results indicated that additional sequences may be important in stimulating transcriptional activity of the IL-4 gene. Using DNAse I hypersensitive site analysis to define other potential IL-4 transcriptional regulatory regions, two sites were identified in the murine IL-4 gene that appear to be unique to IL-4 expressing transformed mast cells. One site defines an intronic sequence that exhibits prototypic enhancer activity in several independently derived transformed mast cell lines. This enhancer is also active in stimulated, non-transformed mast cells but not stimulated EL-4 T cells. Taken together, these data indicate that the IL-4 intronic sequence contains a mast cell specific enhancer that plays an essential role in the unregulated expression of IL-4 in transformed mast cells and may also be important in the inducible expression of IL-4 in normal mast cells.

Macrophage inflammatory protein-1 (MIP) is a recently cloned cytokine that causes neutrophilic infiltration and induces an inflammatory response. We studied the effect of MIP-1 alpha on histamine secretion from basophils and mast cells. Leukocytes from allergic and normal subjects were studied. MIP-1 alpha caused dose-dependent release of histamine from basophils of 14 of 20 allergic donors at concentrations of 10(-9)-10(-7) M, and the mean release was 13.50 +/- 2.9% at the highest concentration. In the same experiments, the mean histamine release by anti-immunoglobulin E and monocyte chemotactic and activating factor (MCAF) (10(-7) M) was 32 +/- 7% and 31 +/- 3%, respectively. The cells from only 2 of 10 normal subjects released histamine in response to MIP-1 alpha. Histamine release by MIP-1 alpha was rapid, and almost complete within the first 3 min. MIP-1 alpha-induced degranulation was a calcium-dependent noncytotoxic process. MIP-1 alpha showed chemotactic activity for purified basophils that was comparable to MCAF. Both MIP-1 alpha and MCAF at 10(-7) M concentration elicited a chemotactic response that was 40% of the maximal response to C5a (1 microgram/ml). Murine MIP-1 alpha induced histamine release from mouse peritoneal mast cells in a dose-dependent manner. Thus, we have established that MIP-1 alpha is a novel activator of basophils and mast cells.

BSF-1/interleukin-4, a product of activated T cells, has multiple biological activities that affect cells of most hematopoietic lineages. Among these is the ability of BSF-1 to costimulate the growth of mast cells and regulate the production of IgE. We demonstrate here that BSF-1 mRNA is expressed by a majority of transformed mast cell lines and by 5 IL-3-dependent non-transformed mast cell lines. BSF-1 activity, including the ability to enhance the growth of IL-3-dependent mast cells, was detected in the supernatants of transformed mast cells. The role of BSF-1 as a mast cell growth factor, its constitutive production by transformed mast cells, and the lack of IL-3 production by most of these cells raise the possibility that BSF-1 may act as an autocrine growth factor for some transformed mast cells. Furthermore, production of BSF-1 mRNA by nontransformed cells indicates that mast cells may be an important physiologic source of this factor.