Mast Cells Contain Spleen-type Prostaglandin D Synthetase”

Prostaglandin D synthetase activity in the cytosol (100,000 g, supernatant) fraction of peritoneal mast cells of adult rats


Prostaglandin
D synthetase activity in the cytosol (100,000 x g, l-h supernatant) fraction of peritoneal mast cells of adult rats (105.0 nmol/min/mg protein) was the highest among such activities in various rat tissues and cells. As judged by the absolute requirement for glutathione for the reaction (K, = 300 KM), the K, value for prostaglandin Hz (200 wM), and insensitivity of the activity to 1 RIM l-chloro-2,4-dinitrobenzene, the enzyme in mast cells was similar to rat spleen prostaglandin D synthetase and differed from rat brain prostaglandin D synthetase or glutathione S-transferase, all of which catalyze the isomerase reaction from prostaglandin Hz to prostaglandin Dz. In immunotitration analyses, the activity in mast cells showed a titration curve exactly identical with that of the purified spleen-type enzyme and almost completely absorbed by an excess amount of antibody against this enzyme, but it remained unchanged after incubation with antibodies against the brain-type enzyme and glutathione Stransferase isozymes thus far purified. In Western blot after two-dimensional electrophoresis of crude extracts of mast cells, a single immunoreactive spot was observed with antibody against the spleen-type enzyme at the same position as that of the purified enzyme (Mr = 26,000, p1 = 5.2). Furthermore, the immunoreactive protein obtained from mast cells showed the same peptide fingerprints as those of the purified spleen-type enzyme, after partial digestion with Staphylococcus aureus VS protease or trypsin. In immunoperoxidase staining, the immunoreactivity of. the spleen-type enzyme was found in the cytosol of tissue mast cells in various organs such as thymus, intestine, stomach, and skin of adult rats. These findings indicate that prostaglandin Dz is produced by the spleen-type synthetase in mast cells of various tissues.
Prostaglandin (PG)' Dz is a major cyclooxygenase product formed in mast cells obtained from rats and humans (l-3). It shows various pharmacological activities such as inhibition of human platelet aggregation (4), peripheral vasodilation and  (5), and bronchoconstriction in dogs (5) and humans (6). PGD, is produced in mast cells during pharmacological or physiological stimulation with calcium ionophore or anti-IgE antibody (3,(7)(8)(9). In addition to these in vitro findings, Roberts et al. (10) observed overproduction of PGD2 in several patients with systemic mastocytosis as evidenced by their elevated levels of urinary metabolites of PGD2. They also showed that chronic aspirin therapy reduced overproduction of PGD, in those patients as well as suppressed their life-threatening hypotensive episodes (10). Therefore, PGDz produced in mast cells is thought to be significantly involved in the anaphylactic process.
The enzymic conversion of PGH2 to PGDz is observed in the soluble fraction of rat peritoneal mast cells (ll), but quantitative measurement of the activity and identification of the enzyme remain to be elucidated. Two different types of PGD synthetase (prostaglandin HP D-isomerase, EC 5.3.99.2) have been purified from rats; one is glutathione (GSH)independent enzyme from brain (12) and the other is GSHrequiring enzyme from spleen (13,14). These two PGD synthetases are similar acidic proteins with the same M, of 26,000 but differ from one another in terms of their amino acid compositions, catalytic properties, antigenic determinants (14), and mRNA sequences (15). Furthermore, we also showed that several isozymes of GSH S-transferase (EC 2.5.1.18) purified from various rat tissues catalyze the conversion of PGH2 to produce PGDz (16).
In this study, we reportkhat PGD synthetase activity in rat peritoneal mast cells is the highest among such activities in various rat tissues so far determined and demonstrate that the enzyme in these cells is biochemically and immunologically the same enzyme as that purified from rat spleen. We also examine the immunocytochemical localization of the spleen-type enzyme in tissue mast cells in various organs, indicating that the enzyme is responsible for biosynthesis of PGDz in this type of cells. In some experiments, an adjacent section was also stained with 0.01% toluidine blue for 4 min to identify mast cells in the tissue by their metachromasia.
For immunoelectron microscopy, the immunoperoxidase-stained sections were post-fixed with 1% osmium tetroxide, dehydrated through an ethanol series, and embedded in polyethylene capsules of epoxy resin. Ultrathin sections were then cut and observed under a Hitachi-300 electron microscope without electron optical counter staining.

Biochemical Characterization of PGD Synthetase Activity in Peritoneal
Mast Cells-When the mast cell homogenate was incubated with 40 PM PGH2 in the presence of 1 mM GSH, concentration-dependent conversion of PGH, to PGD, was observed (29.6 + 3.1 nmoI/min/107cells; mean f SE. of three determinations).
No enzymic conversion of PGHP to PGE, or PGFZo, was found during the incubation under our standard assay conditions. The concentration-dependent formation of PGDz was abolished after a heat treatment of the homogenate at 100 "C for 5 min. On the other hand, no significant PGD synthetase activity was detected in homogenates of macrophages, lymphocytes, or polymorphonuclear leukocytes (co.3 nmol/min/107cells).
When the homogenate of peritoneal mast cells was mixed with those of the other types of cells, neither inhibition nor activation of the PGD synthetase activity was observed.
About 98% of PGD synthetase activity in homogenates of peritoneal mast cells was recovered in the 100,000 X g supernatant fraction. The specific activity (105.0 + 10.0 nmol/min/ mg protein) was about 2-fold higher than that of rat spleen (57.1 nmol/min/mg protein), making it the highest activity among various rat tissues (26).
The PGD synthetase from mast cells absolutely required GSH for the reaction and was virtually inactive with other sulfhydryl compounds such as 1 mM /3-mercaptoethanol or 0.5 mM dithiothreitol. The K, value for GSH was calculated to be about 300 FM. In the presence of 1 mM GSH, the synthetase activity increased almost linearly with the concentration of PGHz up to 100 PM, and the K,,, value for PGH2 was calculated to be around 200 PM. These catalytic properties are similar to those of rat spleen PGD synthetase and clearly different from those of rat brain PGD synthetase (14). Furthermore, the synthetase from mast cells showed almost full activity in the presence of 1 mM l-chloro-2,4dinitrobenzene, a reagent which inhibits rat brain PGD synthetase activity completely and GSH S-transferase-associated PGD synthetase activity partially (26), but not rat spleen PGD synthetase activity (14).
Immunological and Molecular Properties of PGD Synthetase in Peritoneal Mast Cells-In an immunotitration analysis with antibody against rat spleen PGD synthetase (Fig. l), the activity from peritoneal mast cells decreased dose-dependently and showed a titration curve identical with that of the purified spleen-type PGD synthetase, indicating that the enzyme in these cells is immunologically the same as that purified from rat spleen. After incubation with an excess amount of the antibody, the synthetase activity from those cells decreased to less than 5% of the initial activity before incubation.
On the other hand, the activity remained unchanged after incubation with excess amounts of antibodies against rat brain PGD synthetase and GSH S-transferase Symbols represent antibodies specific for each rat spleen PGD synthetase (O), rat brain PGD synthetase (A) GSH S-transferase isozymes 1-2 (A.) 3-4 (A), 6-6 (+), and 7-7 (O), and nonimmunized rabbit IgG (W). The residual enzyme activities are expressed as percentages of those before incubation (25.2 nmol/ min). The titration curve of the purified rat spleen PGD synthetase with antibody against this enzyme (0) is also shown.
In a Western blot after two-dimensional PAGE of crude extracts of peritoneal mast cells (Fig. 2), an immunoreactive spot was intensively stained with antibody against the spleentype PGD synthetase at the same position as that of the purified enzyme (Mr = 26,000, p1 = 5.2) (12). Although weakly stained proteins were observed at regions of more acidic p1 and smaller M, than those of the major immunoreactive protein, all of the positions were clearly different from that of rat brain PGD synthetase (27) or of various rat GSH Stransferase isozymes characterized thus far. When antibody against the brain-type PGD synthetase was used for immunostain, no positive spot was detected in crude extracts of mast cells. After partial digestions with S. aureus V8 protease or trypsin, the spleen-type PGD synthetase-like immunoreactive protein recovered from peritoneal mast cells showed peptide finger prints identical to those of the purified spleentype enzyme (Fig. 3). The immunoreactive proteins of smaller M, (-12,000 and 14,000) in mast cells do not change in response to V8 protease but are degraded by trypsin. Note that a major proteolyic product of PGD synthetase by V8 protease migrates to the same position as that of the immunoreactive protein of M, 14,000. These immunoreactive peptides were not cross-reactive with antibody against the braintype PGD synthetase. Furthermore, both fragmentation profiles and the proteolytic sensitivity of the brain-type enzyme were quite different from those of PGD synthetase in spleen and mast cells.
These results both confirmed the specificity of the antibody and showed that PGD synthetase of the spleen-type, but neither the brain-type enzyme nor GSH S-transferases, is responsible for biosynthesis of PGD2 in peritoneal mast cells.
Immunocytochemical Localization of Spleen-type PGD Synthetase in Tissue Mast Cells of Various Rat Organs-When immunoperoxidase staining was carried out with antibody against spleen-type PGD synthetase on sections of various tissues of adult rats, significant positive staining was observed in several mast cells located in the connective tissues, such as First dimension, isoelectric focusing (IEF) in 5% polyacrylamide gel containing 2% Ampholine, pH 3.5-10; second dimension, SDS-PAGE (10.0% gel). Proteins (10 rg) separated in the gel were transferred to a nylon membrane and then immunostained as described under "Experimental Procedures." Arrowheads indicate the positions of purified spleen-type PGD synthetase in IEF (bottom) and SDS-PAGE (right).
The positions of biotinylated M, marker proteins (Bio-Rad) (arrow) and their ikf, values in thousands are indicated on the right: phosphorylase b (97,400), bovine serum albumin (66,200), ovalbumin (42,700), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500), and lysozyme (14,400). the septum of thymus (Fig. 4a), the submucosal layer of stomach (Fig. 4c) and intestine, dermis (Fig. 4e), and subcutaneous tissue. The positive staining was inhibited by preabsorption of the antibody with the purified rat spleen PGD synthetase ( Fig. 4, b, d, and f) and was not detected with nonimmunized IgG or anti-rat brain PGD synthetase IgG. The immunoreactivity of spleen-type PGD synthetase was undetectable in brain, spinal cord, kidney, and testis, where either brain-type PGD synthetase or GSH S-transferase is responsible for biosynthesis of PGD, in homogenates of such tissues (16,26).
Immunoelectron microscopy confirmed the localization of the immunoreactivity of this enzyme in mast cells (Fig. 5). Fine granular reaction products were widely distributed in the cytoplasm of mast cells, in agreement with the observation that the PGD synthetase activity in these cells was nearly completely recovered in the cytosolic (100,000 X g supernatant) fraction.

DISCUSSION
In this study, we show that the spleen-type PGD synthetase, and not the brain-type enzyme or the known isozymes of GSH S-transferase, is the major enzyme responsible for the biosynthesis of PGDz in peritoneal mast cells, as judged by the kinetic (K, (GSH) = 300 pM, Km (PGHZ) = 200 PM), immunological (Figs. 1 and 2), and molecular (MI = 26,000, p1 = 5.2, Fig. 3) properties of the enzyme. By using immunoperoxidase staining with antibody specific for the spleen-type enzyme (Figs. 4 and 5), we also demonstrate that this enzyme S. aureus V8 protease (

Prostaglandin D Synthetase in Mast Cells
The immunoreactivity of spleen-type PGD synthetase was observed in many, but not all, mast cells of the connective tissues of various organs (Figs. 4 and 5), but it was not significantly detected in mast cells of the parenchymal portions of spleen, thymus, and digestive tract. In those areas, so-called "mucosal mast cells," but not "tissue mast cells," are known to be distributed.
Such heterogeneity of the distribution of the immunoreactivity in mast cells may reflect the functional heterogeneity of both types of mast cells (32).
The specific activity of PGD synthetase in almost pure preparations of mast cells (105.0 nmol/min/mg protein) was shown to be the highest among any such activities found in various tissues or organs of adult rats so far examined (26), yet it was only 2-and g-fold higher than those in spleen and thymus, respectively, suggesting that mast cells are not only one type of cells bearing the PGD synthetase activity in these tissues. In fact, in parenchymal portions of those organs, the immunoreactivity of the spleen-type PGD synthetase was found in cells other than mast cells, such as Langerhans cells in epidermis (33-35) and histiocytes and dendritic cells in spleen, thymus, and digestive tract (36).
The presence of PGD synthetase in non-mast cells is also suggested by the study using w/wV mutant mice deficient in tissue mast cells (37), in which PGD, content and the PGD synthetase activity in various tissues of these mice were shown to be almost the same as those of congenic normal mice. On the other hand, PGD* is also produced in the central nervous system of rats by brain-type PGD synthetase (12). Previously we have shown that the brain-type enzyme is localized in neurons and oligodendrocytes of rat brain (27), ganglion cells in the retina (38, 39), and hair cells in the cochlea (40). Therefore, although mast cells are likely to be the active source, they are not the only type of cell that produces PGDZ.
Since the physiological significance of PGDz in mast cells and the mechanism(s) of action of PGD2 on modification of the anaphylactic process are still a matter of speculation, characterization of catalytic properties of spleen-type PGD synthetase (14) and screening of its inhibitors (41,42) should prove useful in addressing these problems.