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Am J Physiol Heart Circ Physiol 283: H861-H870, 2002. First published April 11, 2002; doi:10.1152/ajpheart.00740.2001
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Vol. 283, Issue 3, H861-H870, September 2002

Stabilization of mast cells by heme oxygenase-1: an anti-inflammatory role

Rina Takamiya2, Makoto Murakami3, Mayumi Kajimura1, Nobuhito Goda1, Nobuya Makino1, Yoshihiro Takamiya1, Tokio Yamaguchi4, Yuzuru Ishimura1, Nobumichi Hozumi2, and Makoto Suematsu1

1 Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, Tokyo 160-8582; 2 Department of Biotechnology, Research Institute for Biological Sciences, Science University of Tokyo, Chiba 278-0022; 3 Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo 142-8555; and 4 Department of Biochemical Genetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8549, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study examined the role of bilirubin in heme oxygenase (HO)-1-mediated amelioration of mast cell (MC)-elicited inflammatory responses. Pretreatment of rats with an intraperitoneal injection of hemin, an inducer of HO-1, evolved a marked induction of the enzyme in MCs. Intravital videomicroscopy revealed that hemin pretreatment attenuated compound 48/80-elicited degranulation of MCs and resultant leukocyte adhesion in venules. Superfusion with biliverdin or bilirubin, but not with carbon monoxide (CO), another product of the HO reaction, mimicked suppressive actions of the HO-1 induction on both the cell degranulation and leukocyte adhesion elicited by the stimulus, suggesting a requirement of the enzyme reaction to generate bilirubin in the inhibitory mechanisms. Such MC-desensitizing actions of bilirubin were observed in primary-cultured MCs and reproduced irrespective of the choice of stimuli, such as compound 48/80, calcium ionophore, and anti-IgE serum. Furthermore, MC-stabilizing effects of HO-1 were reproduced by the gene transfection of the enzyme into mastocytoma cell line RBL2H3. These results suggest that bilirubin generated through HO-1 serves as an anti-inflammatory substance that desensitizes MCs and ameliorates leukocyte recruitment.

inflammation; bilirubin; biliverdin; leukocyte adhesion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE DEGRADATION of protoheme IX to biliverdin-IXalpha , divalent iron, and carbon monoxide (CO) involves the action of heme oxygenase (HO; EC1.14.99.3) (18). Biliverdin-IXalpha undergoes the reaction of biliverdin reductase (EC1.3.1.24) to convert to bilirubin-IXalpha , the terminal product of the HO-mediated heme degradation. In mammals, two forms have been identified. HO-1 is inducible by various stimuli such as cytokines, heavy metals, hormones, endotoxin, oxidants, and protoheme IX, the substrate for HO by itself (1), whereas HO-2 appears to be constitutive. When tissues are preexposed to the HO-1 inducers, the resulting damages and/or inflammatory responses are markedly attenuated in a variety of models, such as carrageenin-induced pleuritis (40), endotoxin shock (25), lethal ischemic lung injury (1, 6, 23), and transplantation of cardiatic allografts. Previous studies (28) showed the preventive effects of HO-1 gene transfer on posttransplantation graft injury. Several mechanisms through which the HO-1 induction attenuates the inflammatory responses have been proposed in relation to biological actions of the reaction products of HO. Despite its direct action to augment oxidative stress through catalysis of the Fenton reaction, free reduced iron can form a complex with iron-responsive proteins that facilitates stabilization of ferritin mRNA and thereby upregulates this iron-chelating protein (4). On the other hand, CO has the ability to attenuate microvascular disturbances (9, 32, 39) or to ameliorate thrombogenesis through suppression of a plasminogen activator inhibitor (6). Finally, biliverdin and bilirubin are thought to serve as potent radical-scavenging substances that ameliorate oxidative stress and thereby reduce inflammatory responses (12, 30).

Despite a growing body of evidence for protective actions of the HO products, it has not fully been examined what types of cells could be involved in executing such HO-1-dependent antioxidative and anti-inflammatory mechanisms. Previous studies revealed the protective roles of HO-1 in functional changes or damages in microvascular endothelial cells, a key device that gates the delivery of circulating leukocytes into the interstitial space. Gene transfection of HO-1 or pretreatment with the enzyme induction in endothelial cells is known to protect the cells from oxidative stress (7, 42). Other important cellular components that regulate the leukocyte recruitment are those producing chemotactic factors in the extravascular space. Among these cells, mast cells have been shown to serve as a primary detector sensing tissue injury and proinflammatory stimuli and can release varied inflammatory mediators such as histamine and platelet-activating factor on their stimulus-induced degranulation and thereby stimulate leukocyte-endothelial cell interactions in vivo (14, 41). The current study examined whether induction or gene transfection of HO-1 in mast cells could alter their sensitivity of agonist-induced degranulation. The results provided evidence that mast cells can sense local stressor insults to induce HO-1 and thereby reduce their sensitivity to degranulation stimuli.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Analyses of HO-1 expression. This study protocol was approved by the Animal Care and Utilization Committee of Keio University School of Medicine. Male Wistar rats weighing 300-330 g were fed ad libitum with water and laboratory chow until the start of the experiments. All reagents were purchased from Sigma (St. Louis, MO), unless stated otherwise. Rats were anesthetized with ether and given an intraperitoneal injection of hemin, a potent inducer of HO-1, at desired doses. Mesenteric tissues were collected for Western blot analysis of the HO-1 induction from 12-h hemin-treated and untreated rats anesthetized with an intramuscular injection of 50 mg/kg pentobarbital sodium. Animals treated with hemin and vehicle were designated as the hemin-treated and control rats, respectively. Bilirubin-IXalpha , an end product of the HO-mediated heme degradation, was measured in plasma and peritoneal lavage samples using enzyme-linked immunosorbent assay (41). To sample peritoneal lavage, the anesthetized rats were treated with an intraperitoneal injection of physiological saline at 15 ml/kg. Fluorescence immunohistochemistry with the use of an anti-rat mast cell rabbit antibody (Ab) (Leinco Technologies) and anti-rat HO-1 monoclonal Ab (MoAb) GTS-1 was performed in mesenteric tissues sampled from the hemin-treated and control rats. The immunoreactivities of GTS-1 were visualized with a secondary anti-mouse IgG antibody labeled with fluorescein isothiocyanate. Localization of the anti-rat mast cell serum was visualized with a secondary anti-rabbit IgG tagged with phycoerythrin. Whole mount preparations of mesenteric tissues were fixed with acetone and followed by treatment with Ab. The stained tissues were epi-illuminated at 488 nm using a laser confocal microscopy equipped with an intensified charge-coupled device camera and a computer-assisted image processor (model C5810, Hamamatsu Photonics; Hamamatsu City, Japan). We also examined histochemically effects of topical application of compound 48/80 on the density of degranulated mast cells in the mesenteric tissue using the toluidine blue staining method (35). Briefly, mesenteric tissues of the anesthetized rats that underwent the 12-h treatment with hemin or with its vehicle were gently exposed and superfused with Krebs-Henseleit buffer containing compound 48/80 at a final concentration of 0.5 µg/ml for 20 min. The rate of superfusion was controlled at 1.0 ml/min, unless stated otherwise. The buffer containing 0.1% toluidine blue was then superfused for 10 min, followed by a 2-min rinse with phosphate-buffered saline. After excess administration of pentobarbital sodium, the tissue was excised, air-dried, and fixed with acetone for morphometrical analysis of the cell degranulation (33). Under microscopic observation, the cells were characterized by typical metachromasia representing cytosolic staining in purple, bright, and round-shaped nuclei, and the absence of granules scattered from the cell body were regarded as undegranulated cells, whereas other cells were considered degranulated. The percent values of degranulated cells versus total cell numbers observed in the individual microscopic fields were calculated in 10 different fields in a single mesenterig tissue. In some experiments, zinc protoporphyrin (ZnPP) IX (Aldrich), an HO inhibitor, was injected intraperitoneally at a dose of 5 µmol/kg twice, at 6 h and 1 h, before exteriorization of the mesenteric tissues. The effects of superfusion of the mesentery with bilirubin or with CO, terminal products of HO-mediated heme degradation, on the mast cell degranulation were also examined (12). As shown in RESULTS, the bilirubin concentrations in the peritoneal space of the hemin-pretreated rats could be estimated to be ~5 µM, assuming that the volume of the peritoneal fluid was not >1.0 ml. On the basis of these results, effects of supplementation with bilirubin were examined by adding the reagent to the perfusate between 2.5 and 10 µM. When CO was applied, the CO-saturated Krebs-Ringer buffer was stored in a gastight syringe and injected into the superfusion circuit with the use of an apparatus pump (Harvard). The flow rate of the CO-containing buffer was carefully controlled so that the final concentration of CO judged by myoglobin-assisted spectrophotometry became ~10 µM (41). The perfusion buffer containing bilirubin was kept under light-excluding conditions to minimize spontaneous degradation of the reagent and was superfused from 5 min before the start of the application of compound 48/80 until the end of experiments.

We also examined expression of HO-1 in peritoneal cells, including mast cells in vitro. To this end, the peritoneal lavage samples containing mast cells were double-stained with anti-rat HO-1 MoAb GTS-1 and the anti-mast cell Ab and examined with two-color fluorescence-associated cell scanning system (Becton-Dickinson; Tokyo, Japan) (9).

Analysis of venular leukocyte adhesion in vivo. Rats pretreated with or without hemin were used to examine effects of compound 48/80 on leukocyte adhesion in mesenteric postcapillary venules using intravital videomicroscopy (33, 34). The exteriorized mesentery was superfused with Krebs-Henseleit buffer saturated with carbogen at a rate of 1.0 ml/min. After the 20-min stabilization period, compound 48/80 and/or other interventions such as ZnPP were added to the perfusate. The erythrocyte velocity (Vr) at the centerline of venules was measured continuously by a temporal correlation velocimeter (IPM; San Diego, CA) (33, 34). The mean rolling velocity of leukocytes versus Vr (Vw/Vr) was determined to examine alterations in adhesion energy between venular endothelium and rolling leukocytes (34, 36). The densities of the adherent cells were expressed as the number per 100-µm length of a venular segment (12). At the end of experiments, H2O2 was superfused on the mesentery at 500 µM; this oxidant is known to induce venular leukocyte adhesion, which is inhibitable by coperfusion of bilirubin or by the HO-1 induction (12).

Isolation of connective tissue mast cells and degranulation assay. Connective tissue mast cells (CTMCs) were isolated by peritoneal lavage from rats, as described previously (21). CTMCs were resuspended at a concentration of 1 × 106 cells/ml in the Hanks buffer (Nissui; Tokyo, Japan). The buffer (100 µl) that contained compound 48/80, a stimulator of mast cell degranulation, was incubated with the cell suspension in the presence or absence of desired concentrations of bilirubin, biliverdin, and CO for 10 min at 37°C (26, 32). Separately, we examined whether the cell degranulation elicited by the calcium ionophore A-23187 or by anti-IgE serum could be attenuated by bilirubin. In these experiments, either A-23187 or anti-IgE serum was added in the cell suspension simultaneously with bilirubin.

Secretagogue activation of mast cells was examined by measuring the release of the enzyme beta -hexosaminidase (beta -hex), as described elsewhere (17). The release of this enzyme serves as an index of secretagogue activation of the cells and occurs in parallel with that of histamine (17, 27). Results were shown as percentages of beta -hex released into the supernatant versus total amounts of the enzyme in the cells (17).

HO-1 gene transfection and degranulation assay for rat mastocytoma RBL2H3 cells. Effects of the HO-1 gene transfer on degranulation sensitivity were examined using the rat mastocytoma cell line RBL2H3. The rat HO-1 cDNA expression plasmid, pEFneo-rHO-1, was transfected into RBL2H3 cells by electroporation (9, 13). Among cell clones surviving against a screening with geneticin (GIBCO-BRL; Gaithersburg, MD) at 1 mg/ml, several stable transformants that express the rat HO-1 protein were established. RBL2H3 is known to exhibit degranulation in response to calcium ionophore but not to compound 48/80. Thus, in the following experiments, A-23187 was used as a stimulus that caused degranulation. The parent cells and those transfected with rHO-1 cDNA were cultured in minimum essential medium supplemented with 10% fetal calf serum and were plated onto 24-well culture dishes at 2.5 × 104 cells/ml for 72 h. The number of cells determined after the 72-h incubation period did not differ significantly among groups (data not shown). The cells incubated in the Hanks' buffer were stimulated with varied concentrations of A-23187.

To check whether the gene transfection of HO-1 could actually elicit overproduction of bilirubin, differences in contents of the heme-degrading product between the mock and HO-1 transfectants were examined by fluorescence immunocytochemistry using anti-bilirubin-IXalpha MoAb 24G7 (24). Cells were fixed with 2.5% paraformaldehyde and treated with saponin for membrane permeablization. The MoAb 24G7 was applied as the primary antibody and its immunoreactivities were visualized by fluorescein isothiocyanate-labeled anti-mouse IgG. Control experiments were carried out in the presence of nonspecific mouse IgG instead of the primary Ab. The aforementioned laser confocal microscope for fluorescence immunohistochemistry of the mesentery was used to semiquantify the bilirubin-associated fluorescence intensities. Microfluorographs collected through the microscope were processed digitally with an eight-bit image analyzer (NIH Image version 1.62 for Power Macintosh G4) (16). To compare the fluorescence intensities with those of known concentrations of bilirubin, parent RBL2H3 cells in culture were treated with bilirubin at 5 or 20 µM for 3 min, and their medium was replaced with the bilirubin-free buffer. As shown in RESULTS, cultured RBL2H3 cells were characterized by their round-shaped cell bodies with dendritic processes. The cell images were thus scanned at two different optical planes along the y-axis; cell soma, which was distal from the bottom of culture dishes, and dendritic structure, which was spread over the dish surface. Gray levels in dendritic processes were determined using optical windows (2 × 2 µm2) in >10 cells from 2-3 separate experiments. At least 10 different portions in each cell were analyzed for such measurements.

Statistical evaluation. Differences in mean values among groups were determined statistically by one-way analysis of variance with Fisher's multiple-comparison test. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mast cells are capable of sensing hemin and exhibit HO-1 induction. Figure 1 illustrates induction of HO-1 and resultant alterations in bilirubin generation in mesenteric tissues of rats undergoing the 12-h hemin treatment. Western blot analysis using the anti-HO-1 MoAb GTS-1 in Fig. 1A showed that the mesenteric tissues untreated with hemin or treated with 10 µmol/kg did not exhibit any detectable levels of the HO-1 protein expression, whereas those undergoing the intraperitoneal injection at doses of 40 µmol/kg displayed a marked increase in the protein expression. On the basis of these data, we chose the dose at 40 µmol/kg and 12 h as the time interval suitable for the pretreatment protocol, which guaranteed a reproducible HO-1 expression. As reported previously (12), hemin-induced HO-1 protein expression became increased time dependent and reached a maximum level at 12 h (data not shown). On the other hand, the HO-1 induction was not evident in tissues treated with vehicle. The hemin-elicited HO-1 induction coincided with marked alterations in amounts of bilirubin in circulation as well as in the peritoneal cavity. As shown in Fig. 1B, the bilirubin concentrations peaked at 12 h in both plasma and the peritoneal lavage samples and decreased backward to the basal levels at 24 h. When the rats were pretreated with intraperitoneal injections of ZnPP (two arrows on x-axis in Fig. 1B; 6 and 1 h before the animals were euthanized), the hemin-induced elevation of the bilirubin generation was attenuated almost completely. These results showed that the protocol for administration of ZnPP used in this study was sufficient enough to suppress the inducible HO reaction in vivo.


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  		Fig. 1.   Induction of heme oxygenase-1 (HO-1) protein and overproduction of bilirubin-IXalpha (BR-IXalpha ) in plasma and peritoneal compartments by treatment with hemin. A: Western blotting analysis of HO-1 as a function of doses of hemin. mm, Molecular marker at 31 kappa Da. B: alterations in plasma and peritoneal contents of BR-IXalpha in the hemin-treated rats. Open and closed circles indicate data measured in plasma and peritoneal lavage samples, respectively. Open and closed squares indicate data measured in zinc protoporphyrin (ZnPP)-treated rats. Large arrow refers to hemin treatment. Small arrows on the x-axis refer to intraperitoneal injections of ZnPP. Data are means ± SE of 4-6 separate experiments. * P < 0.05 compared with the data measured at 0 min. dagger  P < 0.05 compared with the data in the ZnPP-untreated rats. Note that peritoneal BR-IXalpha concentrations indicate those in the 5-ml peritoneal lavage samples.

Figure 2 illustrates HO-1 immunohistochemistry of the mesenteric tissue undergoing the 12-h hemin treatment. As seen in Fig. 2A, the hemin-untreated control exhibited only small amounts of the HO-1-associated immunoreactivities in the parenchyma. On the 12-h hemin treatment, the HO-1 expression became evident in both microvascular walls and cells distributing in the interstitial space (Fig. 2C), being in good agreement with our previous study (12). We thus examined whether mast cells are responsible for the HO-1 expression. As shown in Fig. 2D, the HO-1-associated green fluorescent activities were superimposed on mast cell associated phycoerythrin immunoreactivities and the cells were stained in yellow as a result, suggesting that mast cells constitute a cellular component for the HO-1 induction in the interstitial space.


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  		Fig. 2.   Representative photos of the HO-1 induction in the hemin-treated mesentery. A: HO-1 staining in the hemin-untreated control mesentery. B: double immunohistochemical staining with anti-rat HO-1 monoclonal antibody (MoAb) GTS-1 (green) and anti-mast cell serum (orange) in the hemin-untreated control. C: HO-1 staining in the hemin-treated mesentery. D: double immunohistochemical staining with anti-rat HO-1 MoAb GTS-1 (green) and anti-mast cell serum (orange) in the hemin-treated mesentery. Note the superimposition of the both immunoreactivities as indicated by yellow staining, illustrating the HO-1 induction in mast cells. Bar represents 50 µm.

With the use of fluorescence-activated cell sorter (FACS) analyses, we attempted to confirm the HO-1 expression in mast cells collected from peritoneal lavage samples in the hemin-treated rats. As seen in the dot-plotted analyses in Fig. 3, the peritoneal cells collected from the hemin-untreated control rats illustrated that ~10% of the total cells was identified as anti-mast cell Ab-positive cells (arrow in Fig. 3A). The HO-1 expression in these cells was little in the control group, if any. On the other hand, in the 12-h hemin-treated group, the cells displaying positive immunoreactivities to the anti-mast cell Ab exhibited a marked expression of the HO-1 protein, as indicated by the asterisk in Fig. 3A. At the same time, a subpopulation of cells that displayed negative immunoreactivities to the anti-mast cell Ab also increased the HO-1 expression on the hemin exposure. Histograms showing the expression of HO-1 protein in mast cells were established in the two groups by gating the cells displaying positive immunoreactivities with the anti-mast cell Ab: as seen in Fig. 3B, mean intensities of the HO-1-associated fluorescence of the mast cells became ~10-fold greater in the hemin-treated group than in the controls. These results indicated that mast cells constitute a cellular population responsible for the HO-1 induction in the peritoneal interstitium of hemin-treated rats.


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  		Fig. 3.   Demonstration of the HO-1 induction in connective tissue mast cells (CTMCs) in peritoneal lavage samples collected from the hemin-treated rats using fluorescence-activated cell sorter (FACS) analyses. A: dot-plot analyses of immunoreactivities to the anti-mast cell Ab (Anti-MC Ab) and the anti-rat HO-1 MoAb GTS-1. Top, mouse IgG was used as a negative control. Bottom, 12-h hemin treatment (H12) markedly induces HO-1 in anti-MC Ab-positive cells (asterisk), whereas the same cells in vehicle-treated controls (arrow) did not show such changes. B: histogram analyses of the HO-1 expression in the anti-MC Ab-positive cells between the two groups.

Downregulation of mast cell-dependent leukocyte adhesion by the hemin pretreatment. Figure 4 illustrates compound 48/80-induced alterations in mast cell degranulation (Fig. 4A) and venular leukocyte adhesion in postcapillary venules of the mesentery (Fig. 4B), and effects of pretreatment with hemin on these indexes. As seen in the closed bar, in the hemin-untreated control, the superfusion of 0.5 µg/ml of compound 48/80 evolved a marked stimulation of mast cell degranulation. The compound 48/80-elicited mast cell degranulation was accompanied by an increase in the density of venular leukocyte adhesion. These changes induced by the mast cell activator were attenuated significantly in the 12-h hemin-pretreated rats. To examine whether preventive actions of the hemin pretreatment is attributable to an increase in the enzyme activity of HO-1, we tested effects of pretreatment with ZnPP, a HO inhibitor: as seen, the ZnPP treatment significantly cancelled out the preventive effects of hemin on the compound 48/80-elicited mast cell degranulation and leukocyte adhesion, suggesting that the HO enzyme activity is necessary for mechanisms through which the hemin pretreatment attenuates the compound 48/80-induced responses. On the other hand, the mesentery undergoing the 24-h hemin treatment exhibited the cell degranulation and venular leukocyte adhesion to extents comparable with the controls.


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  		Fig. 4.   Effects of the HO-1 induction by hemin on compound 48/80 (C48/80)-elicited mast cell degranulation and venular leukocyte adhesion in vivo. A and B: relative population of degranulated mast cells and the density of adherent leukocytes in venules, respectively. Data are means ± SD of 7-9 separate experiments in each group. * P < 0.05 compared with the unstimulated controls. dagger  P < 0.05 compared with the data collected from the C48/80-stimulated preparations. #P < 0.05 compared with the data collected from C48/80-stimulated rats undergoing the 12-h hemin treatment.

We then examined whether direct treatment of the mesenteric tissue with products of the HO reaction could ameliorate the compound 48/80-induced mast cell degranulation and venular leukocyte adhesion. Interestingly, superfusion of bilirubin at a final concentration of 10 µM attenuated the compound 48/80-induced mast cell degranulation almost completely. The bilirubin superfusion also abolished the increase in the venular leukocyte adhesion elicited by compound 48/80, suggesting that bilirubin can mimic the inhibitory effects of the hemin pretreatment on the compound 48/80-induced changes. We also examined the effects of superfusion of CO at a final concentration of 10 µM, but without any detectable inhibition of mast cell degranulation or leukocyte adhesion. These results suggest that decreases in the compound 48/80-elicited mast cell degranulation and leukocyte adhesion in the HO-1-inducing mesentery are ascribable to biological actions of bilirubin rather than to those of CO.

Topical ZnPP application did not restore hemin-induced reduction of mast cell degranulation. Aforementioned data collectively suggest that an elevation of local concentrations of bilirubin plays an important role in HO-1-dependent amelioration of mast cell degranulation and venular leukocyte adhesion. We thus examined whether the HO-1-dependent attenuation of the microvascular changes could be cancelled by the local superfusion of the HO inhibitor ZnPP. As seen in the closed circles and squares of Fig. 5, mesenteric venules undergoing the hemin pretreatment did not exhibit any notable changes in rolling and adhesion of leukocytes on superfusion with compound 48/80. Such a paucity of the adhesive responses was reproducible when the stimulus was replaced by 500 µM H2O2, being in good agreement with our previous results (12), indicating that microvessels undergoing the hemin exposure acquire tolerance to the cell adhesion equally between these proadhesive reagents. On the other hand, under the topical ZnPP superfusion, the adhesive responses exhibited great differences between the two reagents; as shown by closed circles and squares, the superfusion of compound 48/80 did not stimulate rolling and adhesion of leukocytes. On replacement of the stimulus with H2O2, both rolling and adhesion became evident significantly. When the mesentery was superfused with 500 µM H2O2 from the beginning of experiments, we observed similar magnitudes of rolling and adhesion. Under these circumstances, the H2O2-elicited changes were attenuated by the 12-h hemin treatment and were restored by supplementation with the local superfusion with ZnPP (data not shown), being in agreement with our previous observation (12) that the topical ZnPP superfusion restores H2O2-induced adhesion in the hemin-pretreated mesentery. These results suggest that HO-1-mediated downregulation of the compound 48/80-elicited leukocyte adhesion involves different mechanisms from that elicited by the H2O2 exposure.


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  		Fig. 5.   Hemin-induced suppression of C48/80- or H2O2-elicited venular leukocyte rolling and adhesion and its recovery by topical superfusion of ZnPP (ZnPPtop). Hatched (0-20 min) and shaded (20-40 min) intervals indicate those for application of 0.5 µg/ml C48/80 and 500 µM H2O2, respectively. All data in this figure were collected from rats undergoing the 12-h hemin treatment. Open or closed circles indicate alterations in the relative rolling velocity (Vw/Vr) in the presence and absence of the ZnPP superfusion at 0.5 µM, respectively. Open or closed squares illustrate alterations in the density of leukocyte adhesion in the presence or absence of the ZnPP superfusion. Data are means ± SD of 7-9 separate experiments in each group. * P < 0.05 compared with data collected from the ZnPP-untreated controls.

Effects of exogenous bilirubin on stimulus-induced degranulation of CTMCs. Considering that H2O2-induced venular leukocyte adhesion is unlikely to be accompanied by notable mast cell degranulation (12), the results shown in Fig. 5 led us to hypothesize that the HO-1 induction could stabilize mast cells through the actions of the reaction products at least in part, and thereby downregulate compound 48/80-induced venular leukocyte adhesion. To test this hypothesis, effects of the heme-degrading end products such as biliverdin, bilirubin, and CO on stimulus-dependent mast cell degranulation were examined in vitro using primary cultured CTMCs. As seen in Fig. 6A, compound 48/80 at 1 µg/ml induced a significant release of beta -hex, which reached ~40% of the total beta -hex amounts in the cells. The compound 48/80-induced degranulation was attenuated dose dependently by bilirubin (note open circles). Such dose-dependent stabilizing actions of bilirubin on the cell degranulation were observed when the stimulus was replaced by the anti-IgE serum or by A-23187. The inhibitory effects of bilirubin on the mast cell degranulation were observed in the presence of albumin, a physiological carrier of bilirubin in circulation (Fig. 6B). The compound 48/80-induced degranulation was also attenuated by application of biliverdin. We also examined effects of CO, another product of the HO reaction, but this substance did not attenuate the compound 48/80-induced degranulation, being in good agreement with our current data shown in vivo in Fig. 4.


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  		Fig. 6.   Inhibitory effects of BR on stimulus-induced CTMCs degranulation. A: dose-dependent inhibition of the stimulus-dependent release of beta -hexosaminidase (beta -hex). C48/80, 1 µg/ml. Anti-IgE serum, 1:100 dilution. Calcium ionophore A-23187, 1 µM. B: effects of BR, albumin-bound bilirubin (BR+Alb), biliverdin (BV), and carbon monoxide (CO) on C48/80-induced mast cell degranulation. Concentrations of BR, BV, and CO were 30 µM. Data indicate means ± SD of 4-10 separate experiments in each group.

Stabilization of RBL2H3 degranulation by bilirubin or by HO-1 gene transfection. Because mast cells constitute a major cellular component expressing HO-1 in the hemin-treated rats, it is not unreasonable to hypothesize that overexpression of the enzyme per se could render the cells less sensitive to degranulation stimuli. To address this hypothesis, effects of the HO-1 gene transfer on stimulus-dependent degranulation of mast cells were examined in RBL2H3 cells. As shown by Western blot analysis using anti-rat HO-1 MoAb GTS-1 (Fig. 7A), two cell lines used for the current experiments displayed a marked increase in the baseline expression of HO-1 protein. We also checked the HO-1 protein expression by FACS using the same MoAb and confirmed a single-peak population of the cells that abundantly expressed the protein, whereas the mock transfectant did not exhibit any significant elevation of the protein expression compared with the parent cells (Fig. 7B). The actual HO activities of the RBL2H3-pEFneo-rHO-1 cells were approximately sixfold greater than those in the parent cells or in the mock transfectant cells (Fig. 7C).


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  		Fig. 7.   Characterization of the rat HO-1 cDNA transfected RBL2H3 cells and alterations in their sensitivity of stimulus-elicited degranulation. A: Western blot analysis of expression of HO-1 protein. P, parent cells; Mo, mock transfectant. H1A and H1B denote two different cell lines of wild-type HO-1 transfectants. B: characterization of the HO-1 protein expression in different cell lines by FACS analyses. C: differences in the HO activities among the cell lines established in the current study. D: differences in the beta -hex release among the cell lines treated with A-23187 at 1 µM. Open and closed bars denote data collected from unstimulated and stimulated RBL2H3 cells, respectively. Data are means ± SD of 5 separate experiments in each group. * P < 0.05 compared with the unstimulated control data. dagger  P < 0.05 compared with the data collected from cells untreated with ZnPP. E: dose-dependent effects of bilirubin on A-23187-induced degranulation of RBL2H3 cells. Open and closed circles denote data collected from the cells stimulated with 50 nM and 1 µM A-23187, respectively. Data are expressed as the percent drop of the beta -hex release versus the control enzyme release measured in the absence of bilirubin, indicating means ± SD of 3-4 separate experiments. * P < 0.05 compared with the control values measured in the absence of bilirubin in the same group.

We examined differences in the release of beta -hex on stimulation with A-23187 among established cell lines. Figure 7D summarizes differences in the degranulation sensitivity among these cells. A-23187 induced a marked release of beta -hex in parent and mock-transfected cells. On the other hand, two different cell lines of the HO-1 transfectants (H1A and H1B) exhibited ~60% reduction of the beta -hex release on the same stimulus. The reduction of the enzyme release in the HO-1 transfectant was restored partly but significantly by 5 µM ZnPP, an HO inhibitor. We also studied whether parent RBL2H3 cells could decrease their degranulation sensitivity with application of bilirubin. When the cells were stimulated with 1 µM A-23187, bilirubin in the range of 1-10 µM did not significantly attenuate their degranulation judged by the beta -hex release. On the other hand, under milder stimulation with the same reagent at 50 nM, the beta -hex release was attenuated dose dependently with bilirubin at the same range of concentrations (Fig. 7E). At 10 µM, its inhibitory effect was significant but limited to ~20%.

The inhibitory action of bilirubin on the cell degranulation tempted us to examine if the cells transfected with the HO-1 gene actually generate sufficient amounts of bilirubin in culture. As seen in Fig. 8, the HO-1-transfected RBL2H3 cells displayed notable immunoreactivities to bilirubin-IXalpha compared with the mock-transfectant control. Such an elevation was evident not only in their dendritic processes (Fig. 8, A and B) but also in the cell bodies (Fig. 8, C and D). On the other hand, the mock-transfectant cells undergoing a 3-min exposure to 20 µM bilirubin in culture displayed notable immunoreacvtivities comparable to those observed in the HO-1 transfectant (Fig. 8F). Differences in bilirubin-IXalpha -associated immunoreactivities were then analyzed semiquantitatively among different groups of the cells. Under the given optical conditions, 8-bit gray level intensities in the mock- and HO-1-transfected cells were 39.8 ± 4.8 and 108.5 ± 33.3 (means ± SE of 20 and 10 cells, P < 0.05), respectively. On the other hand, those of the mock transfectants undergoing 3-min exposure to exogenous bilirubin at 5 µM and 20 µM were 62.9 ± 22.3 and 102.9 ± 32.0 (means ± SE of 12 and 11 cells; P < 0.05 in the both group vs. the control). These results suggest that RBL2H3 cells transfected with the HO-1 gene have the ability to produce enough amounts of bilirubin comparable to explain the stabilizing effect of exogenously applied one.


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  		Fig. 8.   Laser confocal microfluorographs showing an increase in BR-IXalpha -associated immunoreactivities in RBL2H3 cells transfected with rat HO-1 cDNA. A and B: dendritic processes of the mock transfected and HO-1cDNA-transfected cells, respectively. C and D: cell bodies captured at the different focusing plane in the same fields of A (mock transfectants) and B (HO-1 transfectants), respectively. E: mock transfectants labeled with anti-mouse IgG as a primary antibody. F: mock transfectants undergoing a 3-min exposure to bilirubin in culture. Bar = 30 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The current study provided evidence that mast cells constitute an important cellular apparatus, which can sense proinflammatory stressors such as free heme molecules by inducing HO-1, rendering themselves less sensitive to degranulation-eliciting stimuli. The inhibitory effects of the HO-1 induction on the cell degranulation and resultant downregulation of venular leukocyte adhesion are likely to be ascribable to biological actions of the HO-derived products such as biliverdin and/or bilirubin but not to those of CO, and thus shed light on an inhibitory role of these heme-degrading pigments in mast cell-mediated inflammatory responses. Furthermore, bilirubin by itself has the potent inhibitory action on the stimulus-dependent degranulation of the cells with its micromolar concentrations, which could occur under in vivo situations, even when the cells do not upregulate expression of the HO-1 protein. Most importantly, the mast cell-desensitizing action of bilirubin occurs independently of the choice of stimuli. There are several distinct mechanisms for stimulus-elicited calcium mobilization and a resultant degranulation of the cells. First, endogenous stimuli, such as substance P and bradykinin, are known to elicit degranulation primarily through G protein-dependent pathways. In the current study, compound 48/80 was used as the representative reagent mimicking effects of these mediators (8). On the other hand, anti-IgE antibody can cross-link surface-bound IgE molecules and activate the Fcepsilon receptor to elicit intracellular calcium mobilization (29). Finally, A-23187 serves as a stimulus that can directly increase intracellular calcium concentrations to switch on the degranulation. The observation that bilirubin ubiquitously blocks all of these degranulation responses led us to hypothesize that this heme-degrading product could exert its action on final common processes of these pathways. Although the exact mechanisms responsible for the inhibitory action are unknown, the current results suggest that bilirubin or the HO-1 induction can block degranulation of mast cells no matter which pathways for degranulation the stimulus turns on in tissues.

Under physiological conditions, plasma concentrations of bilirubin range ~5-20 µM in humans. When the concentration becomes >300 µM, bilirubin is thought to exert its cytotoxic action, including neural dysfunction in neonates and organ dysfunction, presumably as a consequence of the interference with energy metabolism in mitochondria and protein synthesis in the target cells (30, 31). On the other hand, with its physiological concentrations, beneficial biological actions of bilirubin have long been suggested because the generation of bilirubin through an energetically expensive reaction of biliverdin reductase was introduced in mammals during evolutional processes. Recent studies (2) in vitro have also suggested that neuronal cells can phosphorylate HO-2, a constitutive HO isozyme, through protein kinase C and thereby upregulate bilirubin to be utilized as a neuroprotective molecule. However, the anti-inflammatory roles of bilirubin have not fully been demonstrated yet. We have shown that pretreatment with the HO-1 induction in microvascular endothelial cells suppresses oxidant-elicited translocation of endothelial P-selectin and thereby downregulate venular leukocyte adhesion elicited by H2O2 (12). Mechanisms through which the HO-1 induction attenuates venular adhesion of leukocytes appear to involve amelioration of endothelial oxidative insults by bilirubin. On the other hand, their adhesion elicited by local superfusion of histamine, a nonoxidant proadhesive reagent, which stimulates P-selectin translocation (15), was not altered markedly by the HO-1 induction, suggesting that the bilirubin effect in this particular model results sorely from its anti-oxidative actions on endothelial cells (12). In this context, the current study sheds light on a novel anti-inflammatory effect of bilirubin as an endogenous stabilizer of mast cells.

It has been suggested that mast cells could serve as a primary detector mechanism for tissue infection or invasion of proinflammatory reagents in tissues in that they have the ability to release chemical mediators required for triggering leukocyte recruitment to the appropriate site at risk (7). Such mast cell-derived mediators stimulating tissue leukocyte accumulation involve histamine and platelet activating factor (33, 35). In this regard, the current findings showing that mast cells utilize either the induction of HO-1 or bilirubin to be desensitized and to attenuate mast cell-mediated leukocyte adhesion shed light on pathophysiological implications in regulation of local inflammatory responses. Namely, once mast cells are exposed to greater amounts of bilirubin than those in the ordinary conditions, the cells may reduce their ability to elicit leukocyte adhesion at the jeopardized sites where proinflammatory reagents are invaded. In other words, as a result of stress responses, the HO-1 induction and/or bilirubin overproduction could suppress the mast cell-dependent defense mechanism against bacterial invasion while contributing to attenuation of excessive leukocyte recruitment at the local inflammed regions. Such circumstances involve varied disease conditions causing hyperbilirubinemia, such as spontaneous bacterial peritonitis in decompensatory liver cirrhosis or sepsis associated with postoperative liver dysfunction or neonatal hyperbilirubinemia (19, 22). Previous clinical analyses (20, 37, 38) indicated that hyperbilirubinemia is one of the best predictors of aggravation of bacterial infection under aforementioned disease conditions. Although it is not known whether the stabilization of mast cells plays a role in downregulation of leukocyte recruitment in varied disease models, studies (10, 12) from our laboratory and other laboratories shed light on the putative mechanisms through which bilirubin compromises the host-defense capacity (10, 12).

In the current study, iron protoheme IX was used as a tool to induce HO-1 in mast cells in vivo. However, considering the diversity of inducers that elicit a transcriptional upregulation of this enzyme, it is not unreasonable to hypothesize that mast cells could reduce their sensitivity of degranulation through the HO-1 induction under varied inflammatory diseases causing increases in the enzyme inducers. Cytokines such as interferon-gamma , interleukin-1 and -6, hypoxia, and pro-oxidant reagents such as nitric oxide (NO) are involved in such HO-1-inducing reagents (1, 5, 11, 43). Among these stimuli, the role of interferon-gamma has recently attracted interest in mechanisms for modulation of mast cell degranulation (3). Although this cytokine can stimulate inducible NO synthase in the accessory cells surrounding mast cells and desensitize the cells through NO-dependent manners, the mechanisms through which NO reduces sensitivity to degranulation-stimulating substances are still unknown (3). It has also been shown that continuous exposure of cells to NO evokes an induction of HO-1 and subsequent acceleration of heme degradation and bilirubin generation (16). Thus the current observation raised a possibility that bilirubin-dependent suppression of mast cell degranulation could serve as a putative negative feedback mechanism against inflammatory responses induced by cytokines. Whether the HO-1 induction in mast cells and a resultant downregulation of leukocyte adhesion could be involved in the mechanism for tolerance against endotoxin or shock conditions deserves further study given evidence for contribution of biological actions of the HO products. Attempts to elucidate the whole picture of HO-1-mediated and mast cell-dependent mechanisms for amelioration of inflammation are currently underway in this laboratory.


    ACKNOWLEDGEMENTS

The authors thank Kenjiro Matsuno and Makoto Naito for immunohistochemistry expertise.


    FOOTNOTES

This work was supported by grants from Keio University School of Medicine and from Keio Medical Fund, by Grant-in-Aid for Creative Scientific Research, Japan Society for the Promotion of Science Grant 13GS0015, and in part by Research on Advanced Medical Technology in Health Sciences Research Grants from Ministry of Health and Welfare.

Address for reprint requests and other correspondence: M. Suematsu, Dept. of Biochemistry and Integrative Medical Biology, School of Medicine, Keio Univ., 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan (E-mail: msuem{at}sc.itc.keio.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

April 11, 2002;10.1152/ajpheart.00740.2001

Received 17 August 2001; accepted in final form 1 April 2002.


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Am J Physiol Heart Circ Physiol 283(3):H861-H870
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