Activated mast cells produce interleukin 13.

When mast cells are activated through their immunoglobulin (Ig)E receptors, release of low molecular weight mediators like histamine is followed by secretion of multiple cytokines, including interleukin (IL)-3, IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor. Here we report that stimulated mast cells also synthesize IL-13 mRNA and protein; secretion of this cytokine may be of particular importance because of its ability to stimulate IgE expression. IL-13 transcripts detected by a semiquantitative reverse transcriptase-mediated polymerase chain reaction assay were induced within 30 min after stimulation of mast cells by dinitrophenyl plus monoclonal IgE anti-dinitrophenyl, and peaked at about 1 h. Within 3 h of IgE stimulation, secreted IL-13 bioactivity, estimated by proliferation of an IL-13-dependent cell line, reached levels equivalent to 1-2 ng/ml of IL-13. When added to human B lymphocytes, the mast cell-derived IL-13 activity (like bone fide IL-13) induced Ig C epsilon transcripts, DNA recombination characteristic of the isotype switch to C epsilon, and the secretion of IgE protein. These results suggest a model of local positive feedback interactions between mast cells and B cells, which could play a role in the pathogenesis of atopy.

Clustering of cytokine genes on mouse chromosome 11.

The presence of positionally conserved amino acid residues suggests that the mouse proteins TCA3, P500, MIP1-alpha, MIP1-beta, and JE are members of a single gene family. These proteins are activation specific and can be expressed by both myeloid and lymphoid cells. MIP1-alpha/MIP1-beta and MCAF (the putative human homologue of JE) act as chemotactic and activating agents for neutrophils and macrophages, respectively. The functions of TCA3 and P500 are unknown. We have used interspecies somatic cell hybrids and recombinant inbred mouse strains to show that the genes encoding TCA3, MIP1-alpha, MIP1-beta, and JE (provisionally termed Tca3, Mip-1a, Mip-1b, and Sigje, respectively) map as a cluster on the distal portion of mouse chromosome 11 near the Hox-2 gene complex. DNA sequence analysis indicates that the P500 and TCA3 proteins are encoded by alternative splicing products of one genomic gene. Additionally, the genes encoding TCA3 and JE are found to be strikingly similar with respect to the positions of intron-exon boundaries. Together, these data support the model that the cytokines TCA3, P500, MIP1-alpha, MIP1-beta, and JE are encoded by a single cluster of related genes. The gene encoding IL-5 (Il-5), which acts as a T cell-replacing factor, a B cell growth factor, and an eosinophil differentiation factor, is also mapped to mouse chromosome 11.Il-5 maps approximately 25 cM proximal to the Tca-3 gene and appears tightly linked to a previously described gene cluster that includes Il-3, Il-4, and Csfgm. We discuss the potential relevance of the two cytokine gene clusters described here with particular attention to specific human hematologic malignancies associated with chromosomal aberrations at corresponding locations on human chromosomes 5 and 17.

Interleukin 3-dependent and -independent mast cells stimulated with IgE and antigen express multiple cytokines.

In response to IgE and specific multivalent antigen, mast cell lines (both growth factor-dependent and -independent) induce the transcription and/or secretion of a number of cytokines having a wide spectrum of activities. We have identified IL-1, IL-3, IL-5, IL-6, IFN-gamma, GM-CSF, JE, MIP1 alpha, MIP1 beta, and TCA3 RNA in at least two of four mast cell clones. The production of these products (except JE) is activation-associated and can be induced by IgE plus antigen. In selected instances cytokine expression can also be induced by activation with Con A or phorbol ester plus ionophore, albeit to levels less than those observed with IgE plus antigen. In addition, long-term mast cell clones and primary cultures of bone marrow-derived mast cells specifically release IL-1, IL-4, and/or IL-6 bioactivity after activation. These findings suggest that in addition to their inflammatory effector function mast cells may serve as a source of growth and regulatory factors. The relationship of mast cells to cells of the T lymphocyte lineage is discussed.

A novel mitogen-inducible gene product related to p50/p105-NF-kappa B participates in transactivation through a kappa B site.

A Rel-related, mitogen-inducible, kappa B-binding protein has been cloned as an immediate-early activation gene of human peripheral blood T cells. The cDNA has an open reading frame of 900 amino acids capable of encoding a 97-kDa protein. This protein is most similar to the 105-kDa precursor polypeptide of p50-NF-kappa B. Like the 105-kDa precursor, it contains an amino-terminal Rel-related domain of about 300 amino acids and a carboxy-terminal domain containing six full cell cycle or ankyrin repeats. In vitro-translated proteins, truncated downstream of the Rel domain and excluding the repeats, bind kappa B sites. We refer to the kappa B-binding, truncated protein as p50B by analogy with p50-NF-kappa B and to the full-length protein as p97. p50B is able to form heteromeric kappa B-binding complexes with RelB, as well as with p65 and p50, the two subunits of NF-kappa B. Transient-transfection experiments in embryonal carcinoma cells demonstrate a functional cooperation between p50B and RelB or p65 in transactivation of a reporter plasmid dependent on a kappa B site. The data imply the existence of a complex family of NF-kappa B-like transcription factors.

Human astrocytes inhibit HIV-1 expression in monocyte-derived macrophages by secreted factors.

OBJECTIVE: To determine the effects of primary human fetal and adult astrocytes on HIV-1 replication in monocyte-derived macrophages (MDM). DESIGN: HIV-1 can infect the brain in the early stage of systemic infection. The HIV-1-associated cognitive/motor complex develops later in the course of the disease, suggesting that brain cells may inhibit the early productive infection and the development of neurological disease. In this study, we established an in-vitro coculture system to determine whether astrocytes can modulate HIV-1 replication in MDM. METHODS: Elutriated human monocytes were differentiated in culture, then infected with monocyte tropic HIV-1. One day after infection, MDM were co-cultured with primary astrocytes. Reverse transcriptase (RT) activity was used to monitor virus replication. RT-polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and bioassay were used to assess cytokine production. RESULTS: Primary human astrocytes suppressed HIV-1 replication in MDM via the production of soluble factors. Cytokine inhibitors of HIV-1, such as IFN-gamma, IL-4, IL-10 and IL-13, were not detectable, whereas transforming growth factor beta (TGF-beta) was constitutively produced only in its latent form. Paraformaldehyde-fixed astrocytes, unable to secrete cytokines, failed to inhibit HIV-1. These cells caused enhanced virus replication, however, which correlated with an increase in macrophage colony stimulating factor (M-CSF) production. CONCLUSIONS: Human astrocytes can increase and decrease HIV-1 expression in MDM. An imbalance between the positive and negative effects of astrocytes may contribute to the expression of virus in the brain, and the development of HIV-1-associated cognitive/motor complex.

Differential effects of interleukin-12 treatment on gene expression by allostimulated T cells from young and aged mice.

Alloantigen stimulation was used to examine the effect of interleukin (IL-12) treatment of stimulated cells from young and aged mice on the expression of mRNAs for perforin and granzyme B, two proteins known to be intimately involved in an important lytic pathway used by CTL, and mRNA for interferon (IFN)-gamma, production of which is highly stimulated by IL-12 As reported previously, IL-12 augmented the lytic activity by cells from both young and aged mice, although the relative increase was greater for the latter. The mRNAs encoding perforin and granzyme B were both marginally enhanced at early time points (for cells from young mice) or throughout the stimulation (for cells from aged mice) following allo-stimulation in the presence of IL-12. The levels of augmentation of these mRNAs was consistent with the augmentation of lytic activity. In contrast, mRNA encoding IFN-gamma was markedly enhanced throughout stimulation in cells from animals of both age groups, corresponding to the more substantial increase in interferon protein in response to IL-12.

Thrombin is a regulator of astrocytic endothelin-1.

Endothelin-1, a potent vasoconstrictor of cerebral vessels, is produced by rat primary astrocytes and is subject to autostimulatory regulation in these cells. In this study we examined the effect of thrombin on astrocytic endothelins and report that endothelin-1 is released into the culture fluid in response to thrombin treatment. However, increased production of endothelin-1 is not accompanied by a concomitant increase in steady-state levels of endothelin-1 mRNA as assessed by reverse transcriptase-polymerase chain reaction, even though thrombin stimulation leads to increased inositolphospholipid turnover and activation of the nuclear factor AP1. Thus, astrocytic production of endothelin-1 may be mainly post-transcriptionally regulated in response to thrombin stimulation. In addition, two endothelin receptor genes (ET(A) and ETB) were found to be transcribed simultaneously in primary astrocyte cultures, and both thrombin and endothelin-1 stimulation result in a distinct temporary decrease in ET(A) mRNA. These studies suggest a role for thrombin in the regulation of brain perfusion through astrocytic endothelin-1 expression.

Disparate intracellular processing of human IL-12 preprotein subunits: atypical processing of the P35 signal peptide.

IL-12 is a heterodimeric cytokine produced by APC that critically regulates cell-mediated immunity. Because of its crucial function during immune responses, IL-12 production is stringently regulated, in part through transcriptional control of its p35 subunit, which requires the differentiative effects of IFN-gamma for expression. To determine whether post-transcriptional aspects of IL-12 production might be regulated, we examined intracellular protein processing of each subunit. We report here that p40 and p35 subunits are processed by disparate pathways. Whereas processing of p40 conforms to the cotranslational model of signal peptide removal concomitant with translocation into the endoplasmic reticulum (ER), processing of p35 does not. Translocation of the p35 preprotein into the ER was not accompanied by cleavage of the signal peptide; rather, removal of the p35 signal peptide occurred via two sequential cleavages. The first cleavage took place within the ER, and the cleavage site localized to the middle of the hydrophobic region of the signal peptide. Although the preprotein was glycosylated upon entry into the ER, its glycosylation status did not affect primary cleavage. Subsequently, the remaining portion of the p35 signal peptide was removed by a second cleavage, possibly involving a metalloprotease, concomitant with additional glycosylation and secretion. Secretion could be inhibited by mutation of the second cleavage site or by inhibition of glycosylation with tunicamycin. In contrast, p40 secretion was not affected by inhibition of glycosylation. Our findings demonstrate that IL-12 subunits are processed by disparate pathways and suggest new modalities for regulation of IL-12 production.

Mast cells as a source of multifunctional cytokines.

Mast cells have been implicated in the expression of a wide variety of biological responses, including immediate hypersensitivity reactions, host responses to parasites and neoplasms, immunologically non-specific inflammatory and fibrotic conditions, angiogenesis, and tissue remodeling and wound healing. However, the molecular basis for the action of the mast cell in many of these responses is obscure. In this review, John Gordon, Parris Burd and Stephen Galli suggest that the production of a broad panel of multifunctional cytokines may represent an important mechanism by which mast cells influence physiological, immunological and pathological processes.

IL-3-dependent mast cells attach to plate-bound vitronectin. Demonstration of augmented proliferation in response to signals transduced via cell surface vitronectin receptors.

The adhesive interactions of activated mast cells with the extracellular matrix play an important role in anchorage and cellular motility. In this report we demonstrate that IL-3-dependent bone marrow-derived mast cells adhere to plate-bound vitronectin with high affinity in a saturable and dose-dependent manner. This adhesion interaction is unique in that it does not require prior mast cell activation through Fc epsilon RI or after treatment with PMA. It is inhibited by divalent cation chelation and by competitive inhibition with a synthetic Arginine-Glycine-Aspartate-Serine tetrapeptide. Polyclonal antisera for alpha v beta 3, an integrin known to bind vitronectin, inhibits attachment to plate-bound vitronectin in a dose-dependent manner. Comparison of the adhesion interactions for vitronectin, fibronectin, and laminin indicate that adhesion to vitronectin is greater than that seen with either fibronectin or laminin, either in the presence or absence of PMA. FACS analysis using a monoclonal hamster anti-murine vitronectin receptor (alpha v) antibody followed by a fluorescein-conjugated rabbit anti-hamster IgG revealed no change in surface vitronectin receptor expression after Fc epsilon RI-mediated cell activation. Proliferation assays with correction for cell viability revealed a 25% increase in cell number above the maximal IL-3 response over a 24-h period of adhesion to a vitronectin-coated surface and a 41% increase over 96 h of adhesion to vitronectin. Binding to plate-bound vitronectin was not able to sustain cell viability in the absence of IL-3. Thus, IL-3-dependent bone marrow-derived mast cells adhere to vitronectin, an extracellular matrix protein present throughout connective tissues. This interaction generates a signal that results in the augmentation of the maximal IL-3-dependent mast cell proliferative response, thus demonstrating at least one way in which the interaction between mast cells and extracellular matrix alter the biologic responsiveness of the mast cell.

Interferon-gamma-dependent inducible expression of the human interleukin-12 p35 gene in monocytes initiates from a TATA-containing promoter distinct from the CpG-rich promoter active in Epstein-Barr virus-transformed lymphoblastoid cells.

Interleukin-12 (IL-12) production by human monocytes is stringently regulated through the inducibility of both subunits, p35 and p40, and expression of p35 mRNA is the limiting factor for the secretion of the bioactive IL-12 p70 heterodimer. Optimal induction of p35 mRNA requires priming of the monocytes by interferon-gamma (IFN-gamma), followed by brief exposure to lipopolysaccharide or other bacterial products. To investigate control of p35 gene expression, we isolated genomic clones containing the human p35 gene and determined the 5' end of the mRNA expressed in monocytes. We discovered that a unique p35 transcript is induced in monocytes that begins downstream of a consensus TATA box that lies within the 5' end of the cDNA originally cloned from Epstein-Barr virus (EBV)-transformed B cells. Analysis of p35 mRNA by Northern blotting showed that the message from monocytes is approximately 200 bases shorter than message derived from the EBV-transformed B-cell line VDS. The initiation sites downstream from the TATA box were confirmed by RNase protection and 5' RACE. The data indicate that p35 transcription can initiate from different sites depending on the cell type and that the shorter inducible transcript in monocytes is the one that accumulates after stimulation. Protein translation of these two forms may result in proteins of different sizes with potential implications for the regulation of IL-12 secretion and function.

Distribution of restriction enzyme sites in the bovine parvovirus genome and comparison to other autonomous parvoviruses.

A physical map has been constructed for bovine parvovirus (BPV) replicative form (RF) DNA synthesized in vitro. Analysis of restriction enzyme digestion products for 12 enzymes by neutral gel electrophoresis allowed the mapping of 26 cleavage sites between the 3' and 5' termini of the in vitro RF DNA. Cleavage sites for these enzymes were not detected within the 3'-terminal hairpin. HindIII, SalI, SmaI, SstII and XorII did not cleave BPV DNA, while 12 other enzymes cleaved the genome at one to four sites. Eleven additional enzymes were shown to cleave the genome at 8 to 20 sites. Comparison of the restriction site locations for BPV with those of other autonomous parvoviruses suggests that there may be conservation within the genome of certain restriction sites.

Passive cutaneous anaphylaxis in mouse skin is associated with local accumulation of interleukin-6 mRNA and immunoreactive interleukin-6 protein.

We used a BALB/c model of passive cutaneous anaphylaxis (PCA), an IgE-mediated, mast cell-dependent reaction, to demonstrate the early production of the proinflammatory cytokine interleukin-6 (IL-6) mRNA and protein product. Northern blot analysis detects IL-6 mRNA 1, and 2 hours after antigen challenge (dinitrophenyl30-40 human serum albumin [DNP30-40-HSA]) and in situ hybridization reveals that it is primarily cells with round-to-oval nuclei within the dermis (1 to 3 per high-power field) expressing IL-6 mRNA. Immunohistochemistry revealed perinuclear and cytoplasmic staining for immunoreactive IL-6 in mononuclear dermal cells and also cells within the basal keratinocyte layer. Injection of recombinant murine IL-6 (rmIL-6) either systemically or locally during antidinitrophenyl IgE skin sensitization resulted in increased vasopermeability at the PCA site after DNP30-40-HSA. However, this increased permeability was not associated with a change in the character of the cellular infiltrate at the PCA site 8 hours later. Although the specific role of IL-6 in the generation of the allergic response remains unknown, its detection during PCA unequivocally demonstrates that IL-6 be considered one of the mediators identified in inflammation that follows allergic reactions.

Cloning of a mitogen-inducible gene encoding a kappa B DNA-binding protein with homology to the rel oncogene and to cell-cycle motifs.

We have cloned and characterized a mitogen-inducible gene isolated from human T cells that predicts a protein of 968 amino acids. The amino-terminal domain has regions homologous to the oncogene rel and to the developmentally important gene dorsal of Drosophila. The carboxy-terminal domain contains repeat structures found in a variety of proteins that are involved in cell-cycle control of yeast and in tissue differentiation in Drosophila and Ceanorhabditis elegans, as well as in the putative human oncogene bcl-3 and in the ankyrin protein. A truncated form of the product of this gene translated in vitro is a DNA-binding protein which interacts specifically with the kappa B binding site found in many inducible genes, including the enhancer in human immunodeficiency virus. This gene is yet another in a growing list of important regulatory molecules whose expression is transcriptionally induced upon cellular activation.

The expression and regulation of a potential lymphokine gene (TCA3) in CD4 and CD8 T cell clones.

The TCA3 gene was originally isolated from a cDNA library derived from a TH1 (inflammatory) T cell clone. Expression of TCA3 RNA was limited to cells in the activated state. Based on its expression profile and the existence of a hydrophobic leader sequence with a predicted cleavage site, we proposed that TCA3 encodes a new lymphokine. In the present study, we examine the subset distribution, kinetics, and regulation of TCA3 RNA expression. We show that TCA3 is expressed in response to selected T cell-activating stimuli. TCA3 is transcribed to peak steady state levels by 4 h after stimulation in TH1, TH2 (helper), and CTL clones. Two intracellular signals, supplied in vitro by phorbol ester and one of several agents capable of increasing intracellular free calcium concentrations, are required for the initiation of TCA3 transcription. In addition, TCA3 transcription is blocked by anti-L3T4 mAb, suggesting that prior signaling through L3T4 can inhibit expression of TCA3 and other lymphokines. In contrast to IL-2, IL-4, and IFN-gamma TCA3 is uniformly expressed at high levels among all individual T cell clones examined, including four TH1, three TH2, and three CTL clones. Furthermore, activation-specific TCA3 expression can be dissociated from T cell proliferation.

Differential chemokine expression in tissues involved by Hodgkin's disease: direct correlation of eotaxin expression and tissue eosinophilia.

Hodgkin's disease (HD) is a lymphoid malignancy characterized by infrequent malignant cells surrounded by abundant inflammatory cells. In this study, we examined the potential contribution of chemokines to inflammatory cell recruitment in different subtypes of HD. Chemokines are small proteins that are active as chemoattractants and regulators of cell activation. We found that HD tissues generally express higher levels of interferon-gamma-inducible protein-10 (IP-10), Mig, RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha), and eotaxin, but not macrophage-derived chemotactic factor (MDC), than tissues from lymphoid hyperplasia (LH). Within HD subtypes, expression of IP-10 and Mig was highest in the mixed cellularity (MC) subtype, whereas expression of eotaxin and MDC was highest in the nodular sclerosis (NS) subtype. A significant direct correlation was detected between evidence of Epstein-Barr virus (EBV) infection in the neoplastic cells and levels of expression of IP-10, RANTES, and MIP-1alpha. Levels of eotaxin expression correlated directly with the extent of tissue eosinophilia. By immunohistochemistry, IP-10, Mig, and eotaxin proteins localized in the malignant Reed-Sternberg (RS) cells and their variants, and to some surrounding inflammatory cells. Eotaxin was also detected in fibroblasts and smooth muscle cells of vessels. These results provide evidence of high level chemokine expression in HD tissues and suggest that chemokines may play an important role in the recruitment of inflammatory cell infiltrates into tissues involved by HD.

Human immunodeficiency virus-1-infected macrophages induce inducible nitric oxide synthase and nitric oxide (NO) production in astrocytes: astrocytic NO as a possible mediator of neural damage in acquired immunodeficiency syndrome.

Nitric oxide (NO) plays an important role in normal neural cell function. Dysregulated or overexpression of NO contributes to neurologic damage associated with various pathologies, including human immunodeficiency virus (HIV)-associated neurological disease. Previous studies suggest that HIV-infected monocyte-derived macrophages (MDM) produce low levels of NO in vitro and that inducible nitric oxide synthase (iNOS) is expressed in the brain of patients with neurologic disease. However, the levels of NO could not account for the degree of neural toxicity observed. In this study, we found that induction of iNOS with concomitant production of NO occurred in primary human astrocytes, but not in MDM, when astrocytes were cocultured with HIV-1-infected MDM. This coincided with decreased HIV replication in infected MDM. Supernatants from cocultures of infected MDM and astrocytes also stimulated iNOS/NO expression in astrocytes, but cytokines known to induce iNOS expression (interferon-gamma, interleukin-1beta, and tumor necrosis factor-alpha) were not detected. In addition, the recombinant HIV-1 envelope protein gp41, but not rgp120, induced iNOS in cocultures of uninfected MDM and astrocytes. This suggests that astrocytes may be an important source of NO production due to dysregulated iNOS expression and may constitute one arm of the host response resulting in suppression of HIV-1 replication in the brain. It also leads us to speculate that neurologic damage observed in HIV disease may ensue from prolonged, high level production of NO.

Human NK cells express endothelial nitric oxide synthase, and nitric oxide protects them from activation-induced cell death by regulating expression of TNF-alpha.

Although NO appears important in rodent immune responses, its involvement in the human immune system is unclear. We report that human NK cells express constitutive endothelial NO synthase mRNA and protein, but not detectable levels of inducible NO synthase. They produce NO following activation by coculture with target cells or cross-linking with anti-CD16 mAb, and production is increased in the presence of IL-2. N-monomethyl-L-arginine (L-NMA), a NOS inhibitor, partially inhibited NK cell lysis of four different target cells (<40% inhibition at 500 microM L-NMA), but not granule release following coculture with target cells, or Fas ligand induction following cross-linking with anti-CD16 mAb. However, L-NMA augmented apoptosis of NK cells induced by activation through CD16 ligation or coculture with K562. An NO donor, S-nitroso-N-acetylpenicillamine (SNAP), suppressed apoptosis of NK cells induced by CD16 cross-linking or coculture with target cells, suggesting that endogenous NO production is involved in protection of NK cells from activation-induced apoptosis, thereby maintaining NK activity. SNAP also suppressed, and L-NMA enhanced, expression of TNF-alpha, reported to be involved in activation-induced NK cell death, in response to CD16 cross-linking. Suppression of anti-CD16-induced apoptosis by SNAP was reversed by the addition of rTNF-alpha. DNA-binding activity of the transcription factor, NF-AT, which is involved in TNF-alpha induction upon ligation of CD16, was inhibited by SNAP and enhanced by L-NMA. Our results suggest that down-regulation of TNF-alpha expression, possibly due to suppression of NF-AT activation, is a mechanism by which endogenous NO protects NK cells from activation-induced apoptosis, and maintains lytic capacity.

Two inflammatory mediator cytokine genes are closely linked and variably amplified on chromosome 17q.

Mitogenic stimulation of resting T cells results in the de novo transcription of a large number of genes including those encoding regulatory molecules such as lymphokines. The genomic organization of two newly described induced lymphokine genes, 464.1 and 744.1, has been determined. 464.1 and 744.1 appear to be the human homologues of the recently cloned murine macrophage inflammatory proteins, MIP-1 alpha and MIP-1 beta, respectively. The 464.1 and 744.1 genes share 55% amino acid homology and demonstrate parallel regulation of induced expression in T cells. It was therefore of interest to observe that these genes are closely linked in the human genome, separated by 14 kb, and are organized in a head to head fashion. Each of the genes is present in an additional nonallelic copy (referred to as 464.2 and 744.2) as part of an apparent amplification unit in the genome of many individuals. The 464.2 gene is expressed and potentially encodes a protein highly related to 464.1, varying in 5 of 92 amino acids. As expected, 464.2 and 744.2 are also closely linked to each other as determined by population linkage disequilibrium studies. Individuals bearing a chromosome with a third amplification event, involving a 464-related gene but not a 744-related gene, are also infrequently observed. These genes are all located on chromosome 17 in bands q11-q21, the region implicated in von Recklinghausen neurofibromatosis (NF1) and in acute promyelocytic leukemia (AML-M3).

Endothelins belong to the assortment of mast cell-derived and mast cell-bound cytokines.

Local accumulation of endothelins (ETs) as cytokine-like factors via autocrine/paracrine mechanisms seems to represent an important aspect of their pathophysiological action. This assumption prompted us to investigate mast cells as a possible source of these peptides. With the use of a combination of high-performance liquid chromatography and a radioimmunoassay specific for endothelin-1 (ET-1), 3-week-old cultures of primary murine bone marrow mast cells (BMMC) as well as various mast cell lines were shown to contain and secrete immunoreactive ET-1. The amounts of this peptide were constitutively high in cellular extracts of BMMC, while there was considerable variation in the basal cellular content among mast cell lines, ranging from high (C57) to undetectable (RBL) levels. Treatment of the cells with the combination of phorbol myristate acetate (PMA) and A23187 for 5 h led to induction of ET-1 production in all cases tested. In contrast to the rapid stimulation by PMA/A23187 of histamine release from BMMC or C57 cells, however, no ET-1 secretory response was noted as early as 30 min after this combined treatment. Moreover, stimulation of mast cells with crosslinked IgE for 30 min or 5 h did not affect ET-1 secretion, suggesting that mast cell ET-1 release is not directly related to mast cell degranulation. After exposure of the cells to crosslinked IgE for 20 h, however, there was a distinct increase in immunoreactive ET-1 in the medium, to approximate 10 times the basal level. Polymerase chain reaction (PCR) analysis of mRNA expression in mast cells revealed that the amount of ET-1 PCR product, which is low or undetectable under nonstimulated conditions, is enhanceable by both PMA/A23187 and crosslinked IgE. The IgE-mediated induction kinetics for ET-1 mRNA parallel the kinetics obtained with PMA/A23187, albeit at somewhat lower levels. With the use of fluorescent ligand binding/flow cytometry as a screening method and a radioreceptor assay as the confirming method, mast cells were found to express a single class of high affinity ET receptors with distinct selectivity for ET-1 and a pharmacological profile resembling that of the ETA type ET receptor. Stimulation of mast cell ET-1 receptors did not provoke histamine release, nor did it result in a mitogenic response of BMMC. In conclusion, mast cells synthesize and secrete ET-1 and have ET receptors, suggesting that ET-1 may participate in mediating mast cell-related long-term changes in the microenvironment, e.g., in smooth muscle tone or the proliferation rate of fibroblasts.