Biomedical subjects

K F Austen

Publications and source records attributed to K F Austen.

At least 37 records · Page 2Linked to original sources

PubMed2000

Mast-cell responses in the development of asthma.

Many cells participate in the pathogenesis of asthmatic inflammation. The mast cell is localized at the interface of the internal and external environment within the lung where it may respond to allergens and other exogenous stimuli. The activation of mast cells leads to the release of mediators that contribute to the early phase of asthmatic inflammation. Mast-cell-derived products may also contribute to the late-phase asthmatic response. This review summarizes the developmental biologic features of the mast cell, its receptor-mediated activation, and its range of preformed, newly synthesized, and induced mediators that contribute to asthmatic inflammation.

Animals↗

PubMed1999

Low molecular weight group IIA and group V phospholipase A(2) enzymes have different intracellular locations in mouse bone marrow-derived mast cells.

The subcellular location of the enzymes of eicosanoid biosynthesis is critical for their co-ordinate action in the generation of leukotrienes and prostaglandins. This activity is thought to occur predominantly at a perinuclear location. Whereas the subcellular locations of cytosolic phospholipase (PL) A(2) and each of the pathway enzymes of eicosanoid generation have been defined, the distribution of the low molecular weight species of PLA(2) has remained elusive because of the lack of antibodies that distinguish among homologous family members. We have prepared affinity-purified rabbit antipeptide IgG antibodies that distinguish mouse group IIA PLA(2) and group V PLA(2). Immunofluorescence staining and immunogold electron microscopy reveal different subcellular locations for the enzymes. Group IIA(2) PLA(2) is present in the secretory granules of mouse bone marrow-derived mast cells, consistent with its putative role in facilitating secretory granule exocytosis and its consequent extracellular action. In contrast, group V PLA(2) is associated with various membranous organelles including the Golgi apparatus, nuclear envelope, and plasma membrane. The perinuclear location of group V PLA(2) is consistent with a putative interaction with translocated cytosolic PLA(2) in supplying arachidonic acid for generation of eicosanoid products, while the location in Golgi cisternae may also reflect its action as a secreted enzyme. The spatial segregation of group IIA PLA(2) and group V PLA(2) implies that these enzymes are not functionally redundant.

Amino Acid Sequence↗

PubMed1999

T helper cell type 2 cytokine-mediated comitogenic responses and CCR3 expression during differentiation of human mast cells in vitro.

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).

Cell Differentiation↗

PubMed1999

Cytosolic phospholipase A2 is essential for both the immediate and the delayed phases of eicosanoid generation in mouse bone marrow-derived mast cells.

We have used mice in which the gene for cytosolic phospholipase A2 (cPLA2) has been disrupted to demonstrate the absolute requirement for cPLA2 in both the immediate and the delayed phases of eicosanoid generation by bone marrow-derived mast cells. For the immediate phase, quantitative analysis of the products of the 5-lipoxygenase pathway showed that gene disruption of cPLA2 prevented the provision of arachidonic acid substrate for biosynthesis of proximal intermediates. By analogy, we conclude that arachidonic acid substrate was also not available to prostaglandin endoperoxide synthase 1 in the immediate phase of prostaglandin (PG) D2 generation. These defects occurred with two distinct stimuli, stem cell factor and IgE/antigen, which were, however, sufficient for signal transduction defined by exocytosis of beta-hexosaminidase. Whereas cPLA2 is essential for immediate eicosanoid generation by providing arachidonic acid, its role in delayed-phase PGD2 generation is more complex and involves the activation-dependent induction of prostaglandin endoperoxide synthase 2 and the supply of arachidonic acid for metabolism to PGD2.

Animals↗

PubMed1999

gp49B1 inhibits IgE-initiated mast cell activation through both immunoreceptor tyrosine-based inhibitory motifs, recruitment of src homology 2 domain-containing phosphatase-1, and suppression of early and late calcium mobilization.

We define by molecular, pharmacologic, and physiologic approaches the proximal mechanism by which the immunoglobulin superfamily member gp49B1 inhibits mast cell activation mediated by the high affinity Fc receptor for IgE (FcepsilonRI). In rat basophilic leukemia-2H3 cells expressing transfected mouse gp49B1, mutation of tyrosine to phenylalanine in either of the two immunoreceptor tyrosine-based inhibitory motifs of the gp49B1 cytoplasmic domain partially suppressed gp49B1-mediated inhibition of exocytosis, whereas mutation of both abolished inhibitory capacity. Sodium pervanadate elicited tyrosine phosphorylation of native gp49B1 and association of the tyrosine phosphatases src homology 2 domain-containing phosphatase-1 (SHP-1) and SHP-2 in mouse bone marrow-derived mast cells (mBMMCs). SHP-1 associated transiently with gp49B1 within 1 min after coligation of gp49B1 with cross-linked FcepsilonRI in mBMMCs. SHP-1-deficient mBMMCs exhibited a partial loss of gp49B1-mediated inhibition of FcepsilonRI-induced exocytosis at concentrations of IgE providing optimal exocytosis, revealing a central, but not exclusive, SHP-1 requirement in the counter-regulatory pathway. Coligation of gp49B1 with cross-linked FcepsilonRI on mBMMCs inhibited early release of calcium from intracellular stores and subsequent influx of extracellular calcium, consistent with SHP-1 participation. Because exocytosis is complete within 2 min in mBMMCs, our studies establish a role for SHP-1 in the initial counter-regulatory cellular responses whereby gp49B1 immunoreceptor tyrosine-based inhibition motifs rapidly transmit inhibition of FcepsilonRI-mediated exocytosis.

Amino Acid Sequence↗

PubMed1999

The gp49A gene has extensive sequence conservation with the gp49B gene and provides gp49A protein, a unique member of a large family of activating and inhibitory receptors of the immunoglobulin superfamily.

Members of the gp49-related family of mouse and human immunoglobulin (Ig) superfamily receptors have significant amino acid sequence homology in their C2-type, Ig-like domains and include the killer cell Ig-like receptors (KIRs) for major histocompatibility complex class I molecules. We now report the cloning, complete sequence, and organization of the mouse gp49A gene that encodes the only member of this newly-appreciated family without either of two mutually exclusive functional motifs, namely, immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or a charged transmembrane amino acid for heterodimerization with activation molecules. The gp49A and gp49B genes are 94% identical over 5.6 kilobases, the 5' flanking regions are 94% identical over 1900 nucleotides, and the 3' flanking regions are 97% identical for 121 nucleotides and then diverge completely; the gp49B gene encodes gp49B1 bearing two ITIMs. As measured by flow cytometry with specific antibody, gp49A is expressed on immature bone-marrow-derived mast cells, mature serosal mast cells, and several mouse mast cell lines. The substantial sequence identity of the introns of the gp49A and gp49B genes is comparable to that of the exons, establishing the gene pair as the most homologous of the gp49-related family and suggesting that the gp49A and gp49B genes arose by duplication with relatively little subsequent mutation. The findings also represent the first demonstration that gp49A is expressed on mast cells in tandem with inhibitory gp49B1, and establish that the gp49A gene is not a pseudogene, but rather encodes a protein product with characteristics different from the other family members.

Amino Acid Sequence↗

PubMed1998

Impaired mast cell development and innate immunity in Mac-1 (CD11b/CD18, CR3)-deficient mice.

Mac-1 (CD11b/CD18, CR3), a beta2 integrin expressed on leukocytes, is important in leukocyte migration. We demonstrate that Mac-1 is also expressed on peritoneal mast cells and LPS stimulated bone marrow-derived cultured mast cells, and that Mac-1-deficient mice, which lack this receptor, have significant reductions in the numbers of mast cells resident in the peritoneal cavity, peritoneal wall, and dorsal skin. The reduced numbers of mast cells in Mac-1-deficient mice may have important functional consequences, in that Mac-1-deficient mice exhibit significantly increased mortality after cecal ligation and puncture, a model of acute septic peritonitis in which host resistance has been shown to be dependent on both mast cells and complement. These findings demonstrate that Mac-1 is required for the expression of normal levels of mast cells in the peritoneal cavity, peritoneal wall, and certain areas of the skin, as well as for maintaining adequate mast cell-dependent host defense against bacterial infection.

Acute Disease↗

PubMed1998

Generation of a novel stem cell factor-dependent mast cell progenitor.

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.

Animals↗

PubMed1998

Reversible expression of tryptases and chymases in the jejunal mast cells of mice infected with Trichinella spiralis.

It is has been established that mouse mast cells (MCs) can reversibly alter their expression of serglycin proteoglycans and the homologous granule chymases that have been designated mouse MC protease (mMCP)-1, mMCP-2, and mMCP-5 in vivo. Nevertheless, it remained to be determined whether these immune cells could modify their expression of other chymases and the granule tryptases mMCP-6 and mMCP-7. As assessed immunohistochemically, we now show that MCs reversibly change their expression of the recently described chymase mMCP-9 and both tryptases as these cells traverse the jejunum during the amplification and regression stages of the reactive MC hyperplasia. In noninfected mice, most jejunal MCs reside in the submucosa and express mMCP-6 and mMCP-7, but not mMCP-9 or the chymase mMCP-2. During the inductive phase of the helminth-induced inflammation, when the jejunal MCs move from the submucosa to the tips of the villus, the MCs briefly express mMCP-9, cease expressing mMCP-6 and mMCP-7, and then express mMCP-2. During the recovery phase of the inflammation, jejunal MCs cease expressing mMCP-2 and then express varied combinations of mMCP-6, mMCP-7, and mMCP-9 as they move from the tips of the villus back toward the submucosa. In other model systems, mMCP-6 elicits neutrophil extravasation, and mMCP-7 regulates fibrin deposition and fibrinogen-mediated signaling events. Thus, the ability of a jejunal MC to reversibly alter its tryptase expression during an inflammatory event has important functional implications.

Animals↗

PubMed1998

Cyclo-oxygenase.

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Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1997

Mouse mast cell protease 9, a novel member of the chromosome 14 family of serine proteases that is selectively expressed in uterine mast cells.

Mouse mast cell protease (mMCP) 1, mMCP-2, mMCP-4, and mMCP-5 are members of a family of related serine proteases whose genes reside within an approximately 850 kilobase (kb) complex on chromosome 14 that does not readily undergo crossover events. While mapping the mMCP-1 gene, we isolated a novel gene that encodes a homologous serine protease designated mMCP-9. The mMCP-9 and mMCP-1 genes are only approximately 7 kb apart on the chromosome and are oriented back to back. The proximity of the mMCP-1 and mMCP-9 genes now suggests that the low recombination frequency of the complex is due to the closeness of some of its genes. The mMCP-9 transcript and protein were observed in the jejunal submucosa of Trichinella spiralis-infected BALB/c mice. However, in normal BALB/c mice, mMCP-9 transcript and protein were found only in those mast cells that reside in the uterus. Thus, the expression of mMCP-9 differs from that of all other chymases. The observation that BALB/c mouse bone marrow-derived mast cells developed with interleukin (IL) 10 and c-kit ligand contain mMCP-9 transcript, whereas those developed with IL-3 do not, indicates that the expression of this particular chymase is regulated by the cytokine microenvironment. Comparative protein structure modeling revealed that mMCP-9 is the only known granule protease with three positively charged regions on its surface. This property may allow mMCP-9 to form multimeric complexes with serglycin proteoglycans and other negatively charged proteins inside the granule. Although mMCP-9 exhibits a >50% overall amino acid sequence identity with its homologous chymases, it has a unique substrate-binding cleft. This finding suggests that each member of the chromosome 14 family of serine proteases evolved to degrade a distinct group of proteins.

Amino Acid Sequence↗

PubMed1997

Molecular cloning of the gene for mouse leukotriene-C4 synthase.

Leukotriene C4 (LTC4) synthase (LTC4S), an integral membrane protein, catalyzes the conjugation of leukotriene A4 with reduced glutathione to form LTC4, the biosynthetic parent of the additional cysteinyl leukotriene metabolites. An XmnI-digested fragment of a P1 clone from a 129 mouse ES library contained the full-length gene of 2.01 kb for mouse LTC4S. The mouse LTC4S gene is comprised of 5 exons of 122, 100, 71, 82 and 241 nucleotides, with intron sizes that range from 76 nucleotides to 937 nucleotides. The intron/exon boundaries are identical to those of the human genes for LTC4S and 5-lipoxygenase-activating protein (FLAP). Primer extension demonstrated a single transcription-initiation site 64 bp 5' of the ATG translation-start site. Nucleotide sequencing of 1.2 kb of the 5' flanking region revealed multiple putative sites for activating protein-2, CCAAT/enhancer-binding protein, and polyoma virus enhancer-3. Fluorescent in situ hybridization mapped the mouse LTC4S gene to mouse chromosome 11, in a region containing the genes for interleukin 13 and granulocyte/macrophage-colony-stimulating factor, and orthologous to the chromosomal location of 5q35 for the human LTC4S gene. Thus, the mouse LTC4S gene is similar in size, intron/exon organization and chromosomal localization to the human LTC4S gene. Recent mutagenic analysis of the conjugation function of human LTC4S has identified R51 and Y93 as critical for acid and base catalysis of LTA4 and reduced glutathione, respectively. A comparison across species for proteins that possess LTC4S activity reveals conservation of both of these residues, whereas R51 is absent in the FLAP molecules. Thus, within the glutathione S-transferase superfamily of genes, alignment of specific residues allows the separation of LTC4S family members from their most structurally similar counterparts, the FLAP molecules.

Amino Acid Sequence↗

PubMed1997

Molecular identification of a novel family of human Ig superfamily members that possess immunoreceptor tyrosine-based inhibition motifs and homology to the mouse gp49B1 inhibitory receptor.

The co-cross-linking of gp49B1, a member of the Ig superfamily containing immunoreceptor tyrosine-based inhibition motifs, with the high affinity Fc receptor for IgE on mouse bone marrow culture-derived mast cells inhibits IgE-dependent exocytosis and lipid mediator generation. We now describe the cloning of human cDNAs homologous to the mouse gp49 family. A human monocyte cDNA library was probed with the mouse gp49A cDNA, which is 97% identical with mouse gp49B1, to obtain a homologous partial cDNA that was then used to identify and clone full-length cDNAs from monocyte and human lung cDNA libraries. The 1.6-kbp cDNA, HM18, predicts a 49-kDa type 1 integral membrane protein that, like mouse gp49B1, contains two extracellular C2 type Ig superfamily domains and two consensus immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic domain. ALIGN analysis of the amino acid sequence of the extracellular domains showed that HM18 belongs to a family that includes mouse gp49, the bovine Fc receptor for IgG2, the human myeloid Fc receptor for IgA, and the human NK cell inhibitory receptors. The gene encoding HM18, in common with the genes for the human Fc receptor for IgA and the human NK cell inhibitory receptors, was localized to chromosome 19q13.4. Two other closely related cDNAs, each with four C2 Ig superfamily domains, were characterized. Transcripts for these novel Ig superfamily members were identified in peripheral blood monocytes, the THP-1 monocytic cell line, human lung, human lung mast cells, and NK cells. The data suggest that HM18 is a novel mononuclear cell inhibitory receptor homologous to mouse gp49B1.

Amino Acid Sequence↗

PubMed1997

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).

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.

Cell Adhesion↗

PubMed1997

A newly recognized pathway for the negative regulation of mast cell-dependent hypersensitivity and inflammation mediated by an endogenous cell surface receptor of the gp49 family.

Mast cells are constitutively present at the portals between self and nonself, and contain a large and diverse complement of proinflammatory mediators. These characteristics suggest that the activation of mast cells must be carefully regulated in vivo. Regulation of pathologic and physiologic mast cell activation has been traditionally associated simply with the presence or absence of an activating signal. We examine here evidence supporting a new paradigm: mast cell homeostasis may result from inhibition of activation mediated by receptors on the surface of mast cells, typified by a member of the gp49 family.

Amino Acid Sequence↗

PubMed1997

Site-directed mutagenesis of human leukotriene C4 synthase.

The functional characteristics of leukotriene C4 synthase (LTC4S), which specifically conjugates leukotriene A4 with GSH, were assessed by mutagenic analysis. Human LTC4S and the 5-lipoxygenase-activating protein share substantial amino acid identity and predicted secondary structure. The mutation of Arg-51 of LTC4S to Thr or Ile abolishes the enzyme function, whereas the mutation of Arg-51 to His or Lys provides a fully active recombinant protein. The mutations Y59F, Y97F, Y93F, N55A, V49F, and A52S increase the Km of the recombinant microsomal enzyme for GSH. The mutation Y93F also markedly reduces enzyme function and increases the optimum for pH-dependent activity. The deletion of the third hydrophobic domain with the carboxyl terminus abolishes the enzyme activity, and function is restored by the substitution of the third hydrophobic domain and carboxyl terminus of 5-lipoxygenase-activating protein for that of LTC4S. Mutations of C56S and C82V alone or together and the deletion of Lys-2 and Asp-3 of LTC4S do not alter enzyme function. The direct linkage of two LTC4S monomers by a 12-amino acid bridge provides an active dimer, and the same bridging of inactive R51I with a wild-type monomer creates an active pseudo-dimer with function similar to that of the wild-type enzyme. These results suggest that in the catalytic function of LTC4S, Arg-51 probably opens the epoxide ring and Tyr-93 provides the thiolate anion of GSH. Furthermore, the monomer has independent conjugation activity, and dimerization of LTC4S maintains the proper protein structure.

5-Lipoxygenase-Activating Proteins↗

PubMed1996

Expression of LTC4 synthase during the development of eosinophils in vitro from cord blood progenitors.

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.

5-Lipoxygenase-Activating Proteins↗

PubMed1996

A heparin-sensitive phospholipase A2 and prostaglandin endoperoxide synthase-2 are functionally linked in the delayed phase of prostaglandin D2 generation in mouse bone marrow-derived mast cells.

BALB/cJ mouse bone marrow-derived mast cells (BMMC) developed with interleukin (IL)-3 can be stimulated by c-kit ligand (KL) in the presence of IL-10 and IL-1beta for sequential immediate and delayed generation of prostaglandin (PG) D2 through utilization of constitutive prostaglandin endoperoxide synthase (PGHS) -1 and induced PGHS-2, respectively (Murakami, M., Matsumoto, R., Austen, K. F., and Arm, J. P. (1994) J. Biol. Chem. 269, 22269-22275). We now report that BALB/cJ BMMC stimulated with KL + IL-10 + IL-1beta also exhibit the biphasic release of [3H]arachidonic acid with an immediate phase over the first 10 min followed by a delayed phase from 2 to 7 h. The delayed phase of arachidonic acid release and of PGD2 generation was inhibited by heparin, which concomitantly released a phospholipase (PL) A2 from the cells into the supernatant. Both dexamethasone and a type II PLA2 inhibitor, 12-epi-scalaradial, suppressed delayed-phase PGD2 generation at concentrations that did not affect immediate eicosanoid generation. Transcripts for type IIA PLA2, as assessed by reverse transcription-polymerase chain reaction, were progressively induced in BALB/cJ BMMC treated for 2 to 7 h with KL + IL-10 + IL-1beta; the induction of these transcripts was down-regulated by 10(-6) M dexamethasone. The expression of steady-state transcripts and protein for cytosolic PLA2 (cPLA2) did not change. PGHS-2-dependent delayed-phase PGD2 generation elicited by IgE-dependent activation of BALB/cJ BMMC primed with KL + IL-10 was also accompanied by the induction of type IIA PLA2 transcripts and was suppressed by heparin, with concomitant release of PLA2 into the supernatant. However, both the direct, cytokine-stimulated and the cytokine-primed, IgE-dependent, delayed-phase PGD2 generation occurred in BMMC from C57BL/6J mice, which have a natural disruption of the type IIA PLA2 gene. Thus, kinetic, pharmacologic, and genetic analyses suggest that an inducible, heparin-sensitive PLA2, rather than cPLA2, provides arachidonic acid to concomitantly induced PGHS-2 for delayed-phase PGD2 biosynthesis in activated BMMC. Furthermore, this heparin-sensitive PLA2 likely represents a novel PLA2 or a new function for a known low molecular weight PLA2.

Animals↗