Skip to main content
PMC home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2002 Sep 18;137(3):315–322. doi: 10.1038/sj.bjp.0704872

Augmentation of allergic inflammation in prostanoid IP receptor deficient mice

Yoshimasa Takahashi 1, Shota Tokuoka 1, Taisei Masuda 1, Yousuke Hirano 1, Masafumi Nagao 1, Hiroyuki Tanaka 1, Naoki Inagaki 1, Shuh Narumiya 2, Hiroichi Nagai 1,*
PMCID: PMC1573495  PMID: 12237250

Abstract

  1. To evaluate the role of prostaglandin I2 (PGI2) in allergic inflammation, allergic responses in the airway, skin and T cells were studied in mice lacking the receptor for PGI2 (the prostanoid IP receptor) through gene disruption.

  2. Three inhalations of antigen caused an increase in plasma extravasation, leukocyte accumulation and cytokine (interleukin (IL)-4 and IL-5) production in the airway of sensitized mice. These airway inflammatory responses were significantly greater in IP receptor deficient mice than in wild-type mice.

  3. The vascular leakage caused by passive cutaneous anaphylaxis, substance P and 5-hydroxytryptamine was markedly increased in the skin of IP receptor deficient mice, compared with comparably treated wild-type mice.

  4. The inhalation of antigen in sensitized mice resulted in increased serum antigen specific IgE, total IgE and IgG levels. The magnitude of the elevations of each immunoglobulin level in IP receptor deficient mice is notably higher than that in wild-type mice. To elucidate the mechanism of an enhancement of immunoglobulin production, the activity of T cells in sensitized and non-sensitized mice was studied by means of the production of cytokines. The antigen-induced IL-4 production by spleen cells from sensitized IP receptor deficient mice was almost three times greater than that in wild-type mice. On the contrary, the anti-CD3 antibody-induced interferon-γ production by CD4+ T cells from non-sensitized IP receptor deficient mice was significantly lower than that in wild-type mice.

  5. The present data indicate that IP receptor deficiency reinforced an allergic airway and skin inflammation by augmentation of vascular permeability increase and the T helper 2 cell function. These findings suggest a regulatory role of PGI2 in allergic inflammation.

Keywords: Allergic response, IP receptor, prostaglandin I2, T cell function

Introduction

Prostaglandins (PGs) are cyclo-oxygenase metabolites of arachidonic acid and are generated in response to various stimuli to cells. They are locally acting autacoids with pleiotropic roles in physiologic and pathophysiologic processes (Arias, 2000; Campbell & Halushka, 1996; Imig, 2000; Seibert et al., 1999). Under physiological conditions, they mainly work in the gastrointestinal tract, the circulatory system and regenerative organs. Moreover, PGs play a role in a variety of pathophysiological situations including inflammation (Ghosh et al., 2000; Romay et al., 2000; Murata et al., 1997) and allergic response.

As for the role of PGs in allergic response, Gavett et al. (1999) reported that allergic lung responses were enhanced in cyclo-oxygenase-deficient mice. They demonstrated exaggeration of lung eosinophilia, IgE production and airway hyper-responsiveness in cyclo-oxygenase-1 and cyclo-oxygenase-2 deficient mice. Their findings indicated that the inhibition of PG production resulted in an enhancement of allergic responses.

Concerning the role of individual PGs, the production of each PG in allergic lesions and the effect of extrinsic PGs on allergic responses were investigated. The release of many PGs has been demonstrated after allergic reaction in several cells or tissues (Dahlen et al., 1983; Lahti et al., 1983; Meijer et al., 1996; Montuschi et al., 1999; Oosterhoff et al., 1995; Schulman et al., 1981; Wang et al., 1995; Woodward et al., 1995; Yamasaki et al., 1997). Among PGs, the role of PGD2 and PGE2 was thoroughly investigated. In previous studies, we have demonstrated a role for PGD2 in allergic asthmatic responses by studies in PGD2 receptor gene-disrupted mice (Matsuoka et al., 2000). More recent studies also indicate that PGD2 plays a role in T helper (Th) 2- and eosinophil-dependent inflammation (Hirai et al., 2001; Tanaka et al., 2000). In addition to PGD2, the role of PGE2 in allergic inflammation was also investigated (Gauvreau et al., 1999; Sestini et al., 1996; Szczeklik et al., 1996). Whereas PGE2 showed an inhibition of allergic mediator release and antigen-induced broncoconstriction, it potentiated the in vitro Th2 response, e.g. IgE production (Betz & Fox, 1991; Gold et al., 1994; Hilkens et al., 1995; Kalinski et al., 1997; Paliogianni & Boumpas, 1996; Parker et al., 1995; van der Pouw Kraan et al., 1995). These results suggest that PGE2 has an opposite pharmacological action in allergic inflammation.

In addition to the above two PGs, another PG, PGI2, could be involved in allergic reactions. PGI2 was produced by allergic reaction in human lung, human skin, guinea-pig lung and other tissues (Dahlen et al., 1983; Lahti et al., 1983; Schulman et al., 1981). PGI2 also showed an inhibition of allergic mediator release and eosinophil recruitment in human and experimental animals (Burka & Garland, 1977; Lellouch-Tubiana et al., 1988; Wu et al., 1998). These findings suggest the participation of PGI2 in allergic responses. Therefore, this study was conducted to clarify the role of PGI2 in allergic responses by measuring those responses in mice with disruption of the gene for the PGI2 receptor (IP receptor deficient mice).

Methods

Animals

Seven- to twelve-week-old male mice, with the gene for the IP receptor disrupted (129Sv×C57BL/6 background; backcross to C57BL/6 five times (N5)) weighing 21–30 g were obtained by the same method previously described (Matsuoka et al., 2000). Age- and generation-matched male wild-type animals were used as controls. The animals were housed in plastic cages in an air-conditioned room at 22±1°C with a relative humidity of 60±5%, fed a standard laboratory diet and given water ad libitum. Experiments were performed following the guidelines for the care and use of experimental animals set by the Japanese Association for Laboratory Animals Science in 1987.

Agents

Ovalbumin (OA; Seikagaku Kogyo, Tokyo, Japan), bovine serum albumin (BSA; Seikagaku Kogyo), acetylcholine chloride (Ach; Nacalai Tesque, Kyoto, Japan), Türk solution (Wako Pure Chemical Industries Ltd., Osaka, Japan), pentobarbitone sodium (Abbott Laboratories, Chicago, IL, U.S.A.), disodium ethylenediaminetetraacetic acid (EDTA-2Na; Nacalai Tesque), Diff-Quick solution (International Reagent Corp., Kobe, Japan) were purchased.

Sensitization and antigen challenge for airway inflammation

Mice were actively sensitized by intraperitoneal injections of 50 μg OA with 1 mg alum on days 0 and 12. Starting on day 22, they were exposed to aerosolized OA (10 mg ml−1 in 0.9% NaCl solution) for 30 min, three times every fourth day (days 22, 26 and 30) according to the method reported by Nagai et al. (1996). The aerosol (particle size; 2.0–6.0 μm3) was generated by a nebulizer (Ultrasonic nebulizer TN-701, Azwell, Osaka, Japan) driven by a vacuum pump. Mice were exposed to aerosolized antigen in a perspex cylinder chamber (diameter 5.5 cm, height 12 cm). Control wild-type animals were exposed to saline in a similar manner. Negative control animals were injected with saline and then exposed to saline.

Measurement of leukocyte number, albumin level and cytokine levels in bronchoalveolar lavage fluid (BALF)

The following experiments were carried out according to previously described methods (Nagai et al., 1996). The mice were sensitized and challenged with antigen by the methods described above. Eight hours after the last inhalation of antigen (30 days after the first immunization), the mice were killed with an intraperitoneal injection of sodium pentobarbitone (100 mg kg−1). The trachea was cannulated and the airway lumen was washed four times with 1 ml of calcium- and magnesium-free phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) and 0.05 mM EDTA-2Na (total volume; 3 ml, recovery>85%). Bronchoalveolar lavage fluid (BALF) from each animal was pooled in a plastic tube, cooled in ice, and centrifuged (150×g) at 4°C for 10 min. Cell pellets were resuspended in the same buffer (1 ml). BALF was stained with Türk solution and the number of nucleated cells was counted in a chamber. A differential count was made on a smear prepared with a cytocentrifuge (Cytospine II, Shandon, U.K.) and stained with Diff-Quick solution (based on standard morphologic criteria) on at least 300 cells (magnification×500). To measure the amount of albumin, interleukin (IL)-4 and IL-5, BALF was obtained by employing the same buffer described above (without BSA). Measurement was carried out using enzyme-linked immunosorbent assay (ELISA) (Funakoshi, Tokyo, Japan and Endogen Inc., Cambridge, MA, U.S.A.).

Histological study

To confirm the presence of airway inflammation, histopathological studies were carried out. The mice were killed 24 h after the third antigen inhalation, and the whole lungs were distended by instillation of 10% buffered formalin via the trachea, removed, and immersed in the same fixative with the trachea clamped for 24 h. The tissue was sliced and embedded in paraffin, and 6 μm sections were stained with haematoxylin and eosin for light microscopic examination.

Vascular leakage in the skin

Vascular leakage in the skin was caused by three different stimuli, passive cutaneous anaphylaxis (PCA), substance P and 5-hydroxytryptamine. PCA was carried out by the method as described in Inagaki et al. (1986). Ten microlitres of 30 fold diluted anti-dinitrofluorobenzene (DNFB) monoclonal IgE antibody was injected into both ears of C57BL/6 mice. After 48 h, PCA was elicited by an intravenous injection of 0.25 mg of dinitrophenylated bovine serum albumin dissolved in 0.5% Evans blue saline solution in a volume of 0.25 ml. Thirty minutes after the challenge, mice were sacrificed by cervical dislocation and both ears were removed. Both ears were dissolved with 0.7 ml of IN KOH solution at 37°C overnight and 9.3 ml of a mixture of 0.6 NH3PO4 solution and acetone (5 : 13) was added. After vigorous shaking, precipitates were filtered off and the amount of dye was measured colorimetrically at 620 nM. Female C57BL/6 mice received 20 μl of substance P or 5-hydroxytryptamine at a concentration of 10 nM into one ear. As a vehicle control, 20 μl of saline was injected into the other ear. Immediately after the injection of substance P, 5-hydroxytryptamine or saline, 0.25 ml of 0.5% Evans blue in saline was injected intravenously. Extraction and measurement of extravasated dye was carried out as described above for the PCA.

Measurement of immunoglobulins in serum

In order to understand the effect of IP receptor deficiency on immunoglobulin production, mice were immunized by intraperitoneal injection and given repeated inhalations of antigen as described previously in the airway inflammation. Blood was collected at the time before antigen-provocation, and each serum was obtained by centrifugation and stored at −80°C. Antigen-specific IgE, total IgE and total IgG levels in the mouse serum was measured using the ELISA method as described by us (Nagai et al., 1999).

In brief, to measure the specific IgE, flat-bottom 96-well microliter plates (Nunc Immuno-Plate I 96-F, Roskilde, Denmark) coated with anti-mouse IgE (Serotec, Oxford, U.K.) were used. One hundred microlitres of diluted sample sera were added to each well, followed by incubation at room temperature for 1 h. After washing with phosphate buffered saline containing 0.1% Tween 20 (T-PBS), 100 μl of diluted biotinylated-OA was added to each well and incubated at room temperature for 1 h. Then, after extensive washing with T-PBS, 100 mg of diluted peroxidase-conjugated streptavidin (Dakopatts a/s, Glostrup, Denmark) was added to each well. The enzymatic reaction was stopped by adding 100 μl of 2 M H2SO4 after incubation at room temperature for exactly 30 min. The optical densities of the enzymatic reactions were read using an automatic ELISA plate reader (Titertek Multiscan MCC/340; Lab Systems Oy, Helsinki, Finland) at 492 nm (reference 690 nm). To make a standard curve, sequentially diluted anti-OA IgE monoclonal antibody (mAb; donated by Dr M. Kiniwa, Taiho Pharmaceutical Co. Ltd., Saitama, Japan) was used.

To measure the total IgE and IgG, similar procedures were carried out by using peroxidase-labelled polyclonal anti-mouse IgE (Nordic Immunological Laboratories, Tilburg, The Netherlands) and goat anti-mouse IgG (Capple, Cooper Biomedical Inc., Malvern, PA, U.S.A.).

Cell preparation and cytokine production

To analyse the effect of IP receptor gene deficiency on T-cell function, cytokine production by T cells was examined. Spleen cells and splenic CD4+ T-lymphocytes were used for either antigen-stimulated or anti-CD3 stimulated cytokine production, respectively. The spleen was removed from IP receptor deficient and wild-type mice 12 days after the first immunization, and a dissociated cell suspension (sensitized spleen cells) was prepared and washed twice in RPMI 1640 medium with 5% foetal bovine serum (FBS, Intergen, NY, U.S.A.). The cells (1×106) were then suspended in 200 μl of RPMI 1640 medium supplemented with 10% FBS, 10 mM glutamine, 100 u ml−1 penicillin, 100 μg ml−1 streptomycin and 0.05 mM 2-mercaptoethanol, and were cultured in duplicate with or without OA (100 mg ml−1) in a 96-well round-bottom plate at 37°C for 48 h in humidified 5% CO2/95% air. After the culture, the culture supernatant was collected and centrifuged at 400×g at 4°C. The cell-free supernatants were stored at −80°C until the cytokine assay.

In a separate experiment, the spleen was removed from non-sensitized IP receptor deficient and wild-type mice, and a single-cell suspension was prepared and a T-lymphocyte rich fraction was obtained by centrifugation at 1000×g for 20 min at room temperature using Lympholyte-M (Cedarlane, Ontario, Canada), which was washed twice in PBS with 2 mM EDTA-2Na and 0.5% BSA. The T-lymphocyte rich fraction was treated with magnetic beads conjugated with anti-CD4 (L3T4) monoclonal antibody (Miltenyi Biotec, Bergisch Gladbach, Germany), and purified using a VarioMACS (Miltenyi Biotec) assembly fitted with a VS+ column (Miltenyi Biotec). The purified CD4+ T-lymphocytes were washed and counted. The cells (5×105) were then resuspended in 1 ml of RPMI 1640 medium as described above, and cultured in triplicate with anti-CD28 mAb (10 μg ml−1, 37.51, Pharmingen, CA, U.S.A.) in the 48-well plate precoated with anti-CD3 mAb (10 μg ml−1, 145-2C11, Pharmingen) at 37°C for 72 h. The cells were washed, counted, and then were cultured in RPMI 1640 medium supplemented with murine IL-2 (50 u ml−1, Genzyme/Tecne, CA, U.S.A.) for 3 days. The cells were then washed, counted, and resuspended in 1 ml of RPMI 1640 medium without IL-2, and re-stimulated with anti-CD3 mAb (10 μg ml−1) for 24 h. The culture supernatant was collected and centrifuged at 400×g at 4°C. The cell-free supernatants were stored at −80°C until the cytokine assay.

Statistical analysis

Values are represented as the mean±s.e.mean. Statistical significance between two groups was estimated using the two-tailed Student's t-test or the Mann–Whitney U-test.

Results

Airway inflammation in IP receptor deficient mice

In a previous study, we reported the time course of antigen-induced airway inflammation (Nagai et al., 1996). The number of leukocytes and the amount of cytokines in BALF increased significantly after the three inhalations of antigen. They reached a maximum level 8 h after the third antigen inhalation in sensitized mice. In this study, therefore, the BALF study was carried out 8 h after the last antigen inhalation. The number of total cells, eosinophils, neutrophils and lymphocytes in BALF were significantly increased in antigen-inhaled sensitized wild-type mice (Figure 1). Similar increases were observed in antigen challenged sensitized IP receptor deficient mice. The magnitude of increase in the number of leukocytes in IP receptor deficient mice was three or five times more than those in the wild-type mice (P<0.01). In the saline treated group, no significant change was observed in both wild-type and IP receptor deficient mice.

Figure 1.

Figure 1

Open in a new tab

Antigen-induced leukocyte accumulation in bronchoalveolar lavage fluid (BALF) 8 h after the last antigen challenge in 129×C57BL/6 (F5; wild-type) or IP receptor deficient (KO) mice. Results are presented as the means±s.e.mean of 4–8 animals. N.D.: not detected, OA: ovalbumin. *, **: P<0.05 and 0.01 (vs saline; Mann–Whitney U-test). ††: P<0.01 (vs wild-type; Mann–Whitney U-test).

In addition to the BALF study, histological studies on lung tissue were carried out. Marked infiltration of eosinophils and mononuclear cells were observed in the peribronchial and perivascular regions of the lung in both wild-type and IP receptor deficient mice (Figure 2). The degree of leukocyte infiltration in the area of inflammatory lesion around pulmonary arterioles and bronchioles was measured under microscopic examination. The number of leukocytes in five different areas of each group was counted. The magnitude of eosinophilic inflammation was more severe in IP receptor deficient mice in comparison with those of wild-type mice.

Figure 2.

Figure 2

Open in a new tab

Morphological features of antigen-induced airway inflammation obtained from wild-type or IP receptor deficient (KO) mice. The tissue was stained with H & E (×100). Representative histology showing profound infiltration of inflammatory cells around small bronchioles of OA challenged IP deficient mice. (A) Wild-type, saline treated, (B) Wild-type, OA treated, (C) KO, saline treated, (D) KO, OA treated.

Simultaneously, the levels of albumin, IL-4 and IL-5 in BALF were measured. The leakage of albumin into the BALF (representing increased vascular permeability) was increased 8 h after the antigen challenge in both groups of animals (Figure 3). The level of albumin in the BALF from IP receptor deficient mice was almost 3.2 fold more than that from wild-type mice, after antigen inhalation (P<0.05).

Figure 3.

Figure 3

Open in a new tab

The levels of albumin, IL-4 and IL-5 in bronchoalveolar lavage fluid (BALF) obtained 8 h after the last antigen challenge in wild-type or IP receptor deficient (KO) mice. Results are presented as the mean±s.e.mean of 4–8 animals. Albumin and cytokine levels were determined using ELISA. N.D.: not detected (<5 pg ml−1), OA: ovalbumin. *, **: P<0.05 and 0.01 (vs saline; Mann–Whitney U-test). †, ††: p<0.05 and 0.01 (vs wild-type; Student's t-test or Mann–Whitney U-test).

The levels of IL-4 and IL-5 in BALF were significantly increased in both wild-type and IP receptor deficient mice after the antigen challenge compared with those after saline challenge. However, after challenge with antigen, levels of IL-4 and IL-5 in IP receptor deficient mice were increased almost five times and seven times, respectively, higher than those in wild-type mice (P<0.05) (Figure 3).

Vascular leakage in the skin

In the skin of wild-type mice, the PCA reaction and substance P (10 nM per site) showed a tendency to increase the dye leakage (Figure 4), whereas 5-hydroxytryptamine (10 nM per site) caused a significant dye leakage in these mice. The amount of extravasated dye in IP receptor deficient mice caused by PCA (P<0.05), substance P (P<0.01) or 5-hydroxytryptamine (P<0.05) was significantly greater than that in wild-type mice treated equivalently.

Figure 4.

Figure 4

Open in a new tab

Dye leakage caused by PCA, substance P (10 nM per site) and 5-hydroxytryptamine (5HT; 10 nM per site) in wild-type mice and IP receptor deficient (KO) mice. Each value and bar represents the mean±s.e.mean of 4–6 mice. **P<0.01 (vs saline treated group; Student's t-test), †, ††: P<0.05 and 0.01 (vs wild-type; Mann–Whitney U-test).

Antigen-induced increases in serum immunoglobulin levels

The levels of serum OA-specific IgE, total IgE and total IgG were significantly elevated in both wild-type and IP receptor deficient mice after each immunization and antigen inhalation. Figure 5 shows the immunoglobulin levels in the serum obtained 9 days after the secondary sensitization by intraperitoneal injection of OA. There was no significant change in all immunoglobulin levels after the treatment with saline. The level of each immunoglobulin in IP receptor deficient mice was significantly higher than that of wild-type mice (P<0.05 or P<0.01). OA-specific serum IgE levels in saline-treated mice were below the detection limit (<10 ng ml−1).

Figure 5.

Figure 5

Open in a new tab

Antigen-induced increases in serum immunoglobulin levels in wild-type and IP receptor deficient (KO) mice. Sera were obtained 9 days after the secondary sensitization with OA. Results are presented as the mean±s.e.mean of 4–6 animals. Immunoglobulin levels were determined using ELISA. N.D.: not detected (<1 ng ml−1), OA: ovalbumin. *, **, ***: P<0.05, 0.01 and 0.001 (vs saline; Student's t-test or Mann–Whitney U-test). †, ††: P<0.05 and 0.01 (vs wild type; Student's t-test or Mann–Whitney U-test).

To elucidate the mechanism for an increased production of immunoglobulin, the activity of T cells in sensitized and non-sensitized mice was studied by the antigen- or anti-CD3 antibody-induced production of cytokines by spleen cells. The production of either IL-4 or interferon (IFN)-γ was increased after antigen challenge in sensitized wild-type mice as shown in Figure 6A. Production of both cytokines was higher in IP receptor deficient mice after the antigen-stimulation. The amount of IL-4 was almost 3 fold increased with statistical significance (P<0.01). IFN-γ was also increased about 3 fold, but without statistical significance. The production of both cytokines by anti-CD3 antibody by splenocytes from non-sensitized mice is shown in Figure 6B. The mean amount of IL-4 increased 2 fold in IP receptor deficient mice, but without statistical significance. In contrast, IFN-γ production was significantly lower by almost one half, in IP receptor deficient mice (P<0.01).

Figure 6.

Figure 6

Open in a new tab

Antigen-induced cytokine production by splenocytes obtained from sensitized IP receptor deficient (KO) mice or wild-type mice (A) and anti-CD3 antibody induced cytokine production by splenic CD4+ T cells from IP receptor deficient or wild-type mice (B). Each level represents the mean±s.e.mean of 3–5 mice. The sensitized spleen cells were incubated with antigen for 48 h and CD4+ non-sensitized T cells were incubated with anti-CD3 antibody for 24 h. Cytokine levels were measured by ELISA. N.D.: not detected (<5 pg ml−1), N.S.: not significantly different, OA: ovalbumin. *, **: P<0.05 and 0.01 (vs medium; Student's t-test or Mann–Whitney U-test). ††: P<0.01 (vs wild-type; Student t-test or Mann–Whitney U-test).

Discussion

In this study, we demonstrated an enhancement of allergic inflammation in the airway and skin of IP receptor deficient mice. In addition, IgE and IgG production were augmented in IP receptor deficient mice. These findings suggest that PGI2 plays an important suppressive role in allergic inflammation. In previous studies, Murata et al. (1997) reported a lowered response in carrageenin-induced inflammation in IP receptor deficient mice, implying a pathological role for PGI2 in non-allergic inflammation. However, the allergic responses measured in the present study were elevated in IP receptor deficient mice. These differences were caused by several different factors in allergic and non-allergic inflammatory responses, especially the participation of immunological processes.

Regarding the allergic airway inflammation, deficiency of cyclo-oxygenase resulted in the enhancement of airway eosinophilia and bronchial hyper-responsiveness (Gavett et al., 1999), suggesting that cyclo-oxygenase products play a role as a protective factor in allergic airway inflammation. We have shown diminished allergic asthmatic responses in mice, deficient in the DP receptor gene (Matsuoka et al., 2000). Therefore, we can conclude that PGD2 plays a role as a causative substance in allergic inflammation of airway. In addition to PGD2, other prostanoids, such as PGF2α and thromboxane A2, may play pathophysiological roles in allergic bronchoconstriction (Godard et al., 1981; Lahti et al., 1983; Montuschi et al., 1999; Schulman et al., 1981; Yamasaki et al., 1997). In contrast, PGE2 and PGI2 were reported to be suppressive mediators in allergic reactions. Therefore, the balance of the production of each PG during the allergic response is important in determining the severity of allergic airway inflammation.

Regarding the role of PGI2 in cutaneous vascular permeability, contrasting results have been reported. PGI2 and a PGI2 analogue inhibited the increase in vascular permeability caused by histamine, 5-hydroxytryptamine, leukotriene B4 and tumor necrosis factor (TNF)-α (Erlansson et al., 1989; Muller et al., 1987; Suzuki et al., 2001). In contrast, PGI2 participated in the increase of vascular permeability caused by bacterial lipopolysacharide, bradykinin and carrageenan (Moller & Grande, 1997; Okiji et al., 1989). Moreover, as described previously, Murata et al. (1997) reported the suppression of oedema caused by carrageenan in IP receptor deficient mice. These results suggest that the role of PGI2 in vascular permeability depends on the stimulus inducing the change in permeability. In addition, Jahr & Grande (1996) and Neppl et al. (1991) reported the action of PGI2 on capillary permeability was affected by the concentration of PGI2. As shown in the present results, IP receptor deficient mice showed an augmentation of vascular permeability caused by antigen challenge, substance P and 5- hydroxytryptamine. Considering together the previous and present findings, we would conclude that PGI2 plays a protective role in vascular permeability, at least in allergic conditions.

Concerning the effect of PGs on immune responses, little attention has been paid to the role of PGI2. The present studies employing IP receptor deficient mice indicate enhancement of immunoglobulin production and alteration of T-cell function after allergic reaction. The enhancement of T-cell function is demonstrated in antigen- or anti-CD3 antibody induced cytokine production by spleen cells from IP receptor deficient mice. The antigen-induced production of IL-4 by spleen cells from sensitized IP receptor deficient mice is significantly higher than that from wild-type mice. Moreover, IP receptor deficient mice showed a tendency to increase the production of IFN-γ compared with that from wild-type mice. These results were supported by the in vivo experiments, measuring antigen-induced cytokine production in BALF, and showing that amounts of IL-4 and IL-5 in IP receptor deficient mice were greater than that in wild-type mice. In contrast, the anti-CD3 antibody-induced cytokine production by splenocytes from non-sensitized mice resulted in a different pattern. Whereas the production of IL-4 was increased slightly, IFN-γ production was significantly decreased by the disruption of the IP receptor gene. This suggests that the functional activity of Th1 cells in IP receptor deficient mice was congenitally lower than wild-type mice. However, when the antigen was added to sensitized spleen cells, the Th2 cells are activated significantly. These data suggest the dominance of Th2 response in IP receptor deficient mice over the Th1 response. Since there are many findings to indicate the importance of the Th1 and Th2 balance in allergic reactions (Calder, 2000; Godard et al., 1981; Prahalad, 2000; Ray & Cohn, 2000), further experiments are necessary to clarify the role of PGI2 especially concerning the balance between Th1 and Th2 cells during immunological processes. In addition, the activities of other immunological cells including antigen presenting cells, B cells and others were not studied. The present data only indicate the alteration of T-cell activities in IP receptor deficient mice. Further experiments on the activities of immune competent cells, apart from T, cells in IP receptor deficient mice are necessary.

This study was conducted to investigate the role of PGI2 in allergic responses by generating these responses in IP receptor deficient mice. Since Murata et al. (1997) demonstrated an abrogation of the pharmacological actions of a PGI2 analogue in IP receptor deficient mice, it is possible to evaluate the role of PGI2 by using IP receptor deficient mice. However, there are some reports that high concentrations of PGI2 can activate other prostanoid receptor sub-types (Coleman et al., 1989; Dong et al., 1986). Further studies employing specific IP receptor agonists will be necessary to confirm the roles of PGI2 in allergic inflammation.

In summary, the findings of this study suggest an increased allergic response in the airways and skin of IP receptor deficient mice. The enhancement of this allergic response is closely related to an increase in capillary permeability in each tissue. Moreover, IP receptor deficient mice showed a higher immune response to antigen, in terms of the production of serum IgE and IgG and T cell reactivity, especially the Th2 response. These findings suggest a protective role for PGI2 in the allergic response.

Abbreviations

Ach

acetylcholine chloride

BALF

bronchoalveolar lavage fluid

BSA

bovine serum albumin

DNFB

dinitrofluorobenzene

EDTA

ethylenediaminetetraacetic acid

ELISA

enzyme-linked immunosorbent assay

FBS

foetal bovine serum

IFN

interferon

IL

interleukin

OA

ovalbumin

PBS

phosphate-buffered saline

PCA

passive cutaneous anaphylaxis

PG

prostaglandin

Th

T helper

TNF

tumour necrosis factor

References

  1. ARIAS F. Pharmacology of oxytocin and prostaglandins. Clin. Obstet. Gynecol. 2000;43:455–468. doi: 10.1097/00003081-200009000-00006. [DOI] [PubMed] [Google Scholar]
  2. BETZ M., FOX B.S. Prostaglandin E2 inhibits production of Th1 lymphokines but not of Th2 lymphokines. J. Immunol. 1991;146:108–113. [PubMed] [Google Scholar]
  3. BURKA J.F., GARLAND L.G. A possible modulatory role for prostacyclin (PGI2) INIgGa-induced release of slow-reacting substance of anaphylaxis in rats. Br. J. Pharmacol. 1977;61:697–699. doi: 10.1111/j.1476-5381.1977.tb07564.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. CALDER P.C. Dietary nucleic acids and Th1/Th2 balance: a clue to cow's milk allergy. Clin. Exp. Allergy. 2000;30:908–911. doi: 10.1046/j.1365-2222.2000.00880.x. [DOI] [PubMed] [Google Scholar]
  5. CAMPBELL W.B., HALUSHKA P.V. The Pharmacological Basis of Therapeutics 1996New York: McGraw-Hill; 601–616.eds. Hardman, J.G. & Limbird, L.E. pp [Google Scholar]
  6. COLEMAN R.A., KENNEDY I., HUMPHREY P.P.A., BUNCE K., LUMLEY P.Prostanoids and their receptors Comprehensive Medicinal Chemistry 1989Oxford: Pergamon Press; 643–714.eds. Hansch, C., Sammes, P.G., Taylor, J.B. & Emmett, J.C. pp [Google Scholar]
  7. DAHLEN S.E., HANSSON G., HEDQVIST P., BJORCK T., GRANSTROM E., DAHLEN B. Allergen challenge of lung tissue from asthmatics elicits bronchial contraction that correlates with the release of leukotrienes C4, D4, and E4. Proc. Natl. Acad. Sci. U.S.A. 1983;80:1712–1716. doi: 10.1073/pnas.80.6.1712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. DONG Y.J., JONES R.L., WILSON N.H. Prostaglandin E receptor subtypes in smooth muscle: agonist activities of stable prostacyclin analogues. Br. J. Pharmacol. 1986;87:97–107. doi: 10.1111/j.1476-5381.1986.tb10161.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. ERLANSSON M., SVENSJO E., BERGQVISTt D. Leukotriene B4-induced permeability increase in postcapillary venules and its inhibition by three different antiinflammatory drugs. Inflammation. 1989;13:693–705. doi: 10.1007/BF00914313. [DOI] [PubMed] [Google Scholar]
  10. GAUVREAU G.M., WATSON R.M., O'BYRNE P.M. Protective effects of inhaled PGE2 on allergen-induced airway responses and airway inflammation. Am. J. Respir. Crit. Care. Med. 1999;159:31–36. doi: 10.1164/ajrccm.159.1.9804030. [DOI] [PubMed] [Google Scholar]
  11. GAVETT S.H., MADISON S.L., CHULADA P.C., SCARBOROUGH P.E., QU W., BOYLE J.E., TIANO H.F., LEE C.A., LANGENBACH R., ROGGLI V.L., ZELDIN D.C. Allergic lung responses are increased in prostaglandin H synthase-deficient mice. J. Clin. Invest. 1999;104:721–732. doi: 10.1172/JCI6890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. GHOSH A.K., HIRASAWA N., NIKI H., OHUCHI K. Cyclooxygenase-2-mediated angiogenesis in carrageenin-induced granulation tissue in rats. J. Pharmacol. Exp. Ther. 2000;295:802–809. [PubMed] [Google Scholar]
  13. GODARD P., CHANTREUIL J., CLAUZEL A.M., CRASTES DE PAULET A., MICHEL F.B. Plasma concentrations of prostaglandins E2 and F2a in asthmatic patients. Respiration. 1981;42:43–51. doi: 10.1159/000194402. [DOI] [PubMed] [Google Scholar]
  14. GOLD K.N., WEYAND C.M., GORONZY J.J. Modulation of helper T cell function by prostaglandins. Arthritis Rheum. 1994;37:925–933. doi: 10.1002/art.1780370623. [DOI] [PubMed] [Google Scholar]
  15. HILKENS C.M., VERMEULEN H., VAN NEERVEN R.J., SNIJDEWINT F.G., WIERENGA E.A., KAPSENBERG M.L. Differential modulation of T helper type 1 (Th1) and T helper type 2 (Th2) cytokine secretion by prostaglandin E2 critically depends on interleukin-2. Eur. J. Immunol. 1995;25:59–63. doi: 10.1002/eji.1830250112. [DOI] [PubMed] [Google Scholar]
  16. HIRAI H., TANAKA K., YOSHIE O., OGAWA K., KENMOTSU K., TAKAMORI Y., ICHIMASA M., SUGAMURA K., NAKAMURA M., TAKANO S., NAGATA K. Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2. J. Exp. Med. 2001;193:255–261. doi: 10.1084/jem.193.2.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. IMIG J.D. Eicosanoid regulation of the renal vasculature. Am. J. Physiol. Renal Physiol. 2000;279:F965–F981. doi: 10.1152/ajprenal.2000.279.6.F965. [DOI] [PubMed] [Google Scholar]
  18. INAGAKI N., GOTO S., NAGAI H., KODA A. Homologous passive cutaneous anaphylaxis in various strains of mice. Int. Arch. Allergy Appl. Immun. 1986;81:58–62. doi: 10.1159/000234108. [DOI] [PubMed] [Google Scholar]
  19. JAHR J., GRANDE P.O. Prostacyclin counteracts the increase in capillary permeability induced by tumor necrosis factor-alpha. Intensive. Care. Med. 1996;22:1453–1460. doi: 10.1007/BF01709568. [DOI] [PubMed] [Google Scholar]
  20. KALINSKI P., HILKENS C.M., SNIJDERS A., SNIJDEWINT F.G., KAPSENBERG M.L. Dendritic cells, obtained from peripheral blood precursors in the presence of PGE2, promote Th2 responses. Adv. Exp. Med. Biol. 1997;417:363–367. doi: 10.1007/978-1-4757-9966-8_59. [DOI] [PubMed] [Google Scholar]
  21. LAHTI A., OIKARINEN A., VIINIKKA L., YLIKORKALA O., HANNUKSELA M. Prostaglandins in contact urticaria induced by benzoic acid. Acta. Derm. Venereol. 1983;63:425–427. [PubMed] [Google Scholar]
  22. LELLOUCH-TUBIANA A., LEFORT J., SIMON M.T., PFISTER A., VARGAFTIG B.B. Eosinophil recruitment into guinea pig lungs after PAF-acether and allergen administration. Modulation by prostacyclin, platelet depletion, and selective antagonists. Am. Rev. Respir. Dis. 1988;137:948–954. doi: 10.1164/ajrccm/137.4.948. [DOI] [PubMed] [Google Scholar]
  23. MATSUOKA T., HIRATA M., TANAKA H., TAKAHASHI Y., MURATA T., KABASHIMA K., SUGIMOTO Y., KOBAYASHI T., USHIKUBI F., AZE Y., EGUCHI N., URADE Y., YOSHIDA N., KIMURA K., MIZOGUCHI A., HONDA Y., NAGAI H., NARUMIYA S. Prostaglandin D2 as a mediator of allergic asthma. Science. 2000;287:2013–2017. doi: 10.1126/science.287.5460.2013. [DOI] [PubMed] [Google Scholar]
  24. MEIJER F., TAK C., van HAERINGEN N.J., KIJLSTRA A. Interaction between nitric oxide and prostaglandin synthesis in the acute phase of allergic conjunctivitis. Prostaglandins. 1996;52:431–446. doi: 10.1016/s0090-6980(96)00123-2. [DOI] [PubMed] [Google Scholar]
  25. MOLLER A.D., GRANDE P.O. Low-dose prostacyclin has potent capillary permeability-reducing effect in cat skeletal muscle in vivo. Am. J. Physiol. 1997;273:H200–H207. doi: 10.1152/ajpheart.1997.273.1.H200. [DOI] [PubMed] [Google Scholar]
  26. MOLLER A.D., GRANDE P.O. Low-dose prostacyclin is superior to terbutaline and aminophylline in reducing capillary permeability in cat skeletal muscle in vivo. Crit. Care. Med. 1999;27:130–136. doi: 10.1097/00003246-199901000-00040. [DOI] [PubMed] [Google Scholar]
  27. MONTUSCHI P., CURRO D., RAGAZZONI E., PREZIOSI P., CIABATTONI G. Anaphylaxis increases 8-iso-prostaglandin F2α release from guinea-pig lung in vitro. Eur. J. Pharmacol. 1999;365:59–64. doi: 10.1016/s0014-2999(98)00859-0. [DOI] [PubMed] [Google Scholar]
  28. MULLER B., SCHMIDTKE M., WITT W. Action of the stable prostacyclin analogue iloprost on microvascular tone and permeability in the hamster cheek pouch. Prostaglandins Leukot. Med. 1987;29:187–198. doi: 10.1016/0262-1746(87)90008-4. [DOI] [PubMed] [Google Scholar]
  29. MURATA T., USHIKUBI F., MATSUOKA T., HIRATA M., YAMASAKI A., SUGIMOTO Y., ICHIKAWA A., AZE Y., TANAKA T., YOSHIDA N., UENO A., OH-ISHI S., NARUMIYA S. Altered pain perception and inflammatory response in mice lacking prostacyclin receptor. Nature. 1997;388:678–682. doi: 10.1038/41780. [DOI] [PubMed] [Google Scholar]
  30. NAGAI H., UEDA Y., TANAKA H., HIRANO Y., NAKAMURA N., INAGAKI N., TAKATSU K., KAWADA K. Effect of overproduction of interkeukin 5 on dinitrofluorobenzene-induced allergic cutaneous responses in mice. J. Pharmacol. Exp. Ther. 1999;288:43–50. [PubMed] [Google Scholar]
  31. NAGAI H., YAMAGUCHI S., MAEDA Y., TANAKA H. Role of mast cells, eosinophils and IL-5 in the development of airway hyperresponsiveness in sensitized mice. Clin. Exp. Allergy. 1996;26:642–647. [PubMed] [Google Scholar]
  32. NEPPL H., NEUHOF H., AFFLERBACH F., LLACH PUIG NEPPL J., BERGHOFER A. Bradykinin-induced oedema formation proceeds from B2 receptor stimulation and is potentiated by concomitantly released prostaglandins. Acta. Physiol. Scand. 1991;142:141–147. doi: 10.1111/j.1748-1716.1991.tb09141.x. [DOI] [PubMed] [Google Scholar]
  33. OKIJI T., MORITA I., SUNADA I., MUROTA S. Involvement of arachidonic acid metabolites in increases in vascular permeability in experimental dental pulpal inflammation in the rat. Arch. Oral. Biol. 1989;34:523–528. doi: 10.1016/0003-9969(89)90090-3. [DOI] [PubMed] [Google Scholar]
  34. OOSTERHOFF Y., KAUFFMAN H.F., RUTGERS B., ZIJLSTRA F.J., KOETER G.H., POSTMA D.S. Inflammatory cell number and mediators in bronchoalveolar lavage fluid and peripheral blood in subjects with asthma with increased nocturnal airways narrowing. J. Allergy Clin. Immunol. 1995;96:219–229. doi: 10.1016/s0091-6749(95)70011-0. [DOI] [PubMed] [Google Scholar]
  35. PALIOGIANNI F., BOUMPAS D.T. Prostaglandin E2 inhibits the nuclear transcription of the human interleukin 2, but not the IL-4, gene in human T cells by targeting transcription factors AP-1 and NF-AT. Cell Immunol. 1996;171:95–101. doi: 10.1006/cimm.1996.0178. [DOI] [PubMed] [Google Scholar]
  36. PARKER C.W., HUBER M.G., GODT S.M. Modulation of IL-4 production in murine spleen cells by prostaglandins. Cell Immunol. 1995;160:278–285. doi: 10.1016/0008-8749(95)80039-l. [DOI] [PubMed] [Google Scholar]
  37. PRAHALAD S. Atopy, autoimmunity, and the T(H)1/T(H)2 balance. J. Pediatr. 2000;137:446–449. doi: 10.1067/mpd.2000.110300. [DOI] [PubMed] [Google Scholar]
  38. RAY A., COHN L. Altering the Th1/Th2 balance as a therapeutic strategy in asthmatic diseases. Curr. Opin. Investig. Drugs. 2000;1:442–448. [PubMed] [Google Scholar]
  39. ROMAY C., LEDON N., GONZALEZ R. Effects of phycocyanin extract on prostaglandin E2 levels in mouse ear inflammation test. Arzneimittelforschung. 2000;50:1106–1109. doi: 10.1055/s-0031-1300340. [DOI] [PubMed] [Google Scholar]
  40. SCHULMAN E.S., NEWBALL H.H., DEMERS L.M., FITZPATRICK F.A., ADKINSON N.F. Anaphylactic release of thromboxane A2, prostaglandin D2, and prostacyclin from human lung parenchyma. Am. Rev. Respir. Dis. 1981;124:402–406. doi: 10.1164/arrd.1981.124.4.402. [DOI] [PubMed] [Google Scholar]
  41. SEIBERT K., LEFKOWITH J., TRIPP C., ISAKSON P., NEEDLEMAN P. Cox-2 inhibitors- Is there cause for concern. Nature Med. 1999;5:621–622. doi: 10.1038/9465. [DOI] [PubMed] [Google Scholar]
  42. SESTINI P., ARMETTI L., GAMBARO G., PIERONI M.G., REFINI R.M., SALA A., VAGHI A., FOLCO G.C., BIANCO S., ROBUSCHI M. Inhaled PGE2 prevents aspirin-induced bronchoconstriction and urinary LTE4 excretion in aspirin-sensitive asthma. Am. J. Respir. Crit. Care. Med. 1996;153:572–575. doi: 10.1164/ajrccm.153.2.8564100. [DOI] [PubMed] [Google Scholar]
  43. SUZUKI N., KUDO K., SANO Y., ITO K. Can Mycobacterium tuberculosis infection prevent asthma and other allergic disorders. Int. Arch. Allergy. Immunol. 2001;124:113–116. doi: 10.1159/000053686. [DOI] [PubMed] [Google Scholar]
  44. SZCZEKLIK A., MASTALERZ L., NIZANKOWSKA E., CMIEL A. Protective and bronchodilator of prostaglandin E and salbutamol in aspirin-induced asthma. Am. J. Respir. Crit. Care. Med. 1996;153:567–571. doi: 10.1164/ajrccm.153.2.8564099. [DOI] [PubMed] [Google Scholar]
  45. TANAKA K., OGAWA K., SUGAMURA K., NAKAMURA M., TAKANO S., NAGATA K. Cutting edge: differential production of prostaglandin D2 by human helper T cell subsets. J. Immunol. 2000;164:2277–2280. doi: 10.4049/jimmunol.164.5.2277. [DOI] [PubMed] [Google Scholar]
  46. VAN DER POUW KRAAN T.C., BOEIJE L.C., SMEENK R.J., WIJDENES J., AARDEN L.A. Prostaglandin-E2 is a potent inhibitor of human interleukin 12 production. J. Exp. Med. 1995;181:775–779. doi: 10.1084/jem.181.2.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. WANG D., CLEMENT P., SMITZ J., DERDE M.P. Concentrations of chemical mediators in nasal secretions after nasal allergen challenges in atopic patients. Eur. Arch. Otorhinolaryngol. Suppl. 1995;1:S40–S43. doi: 10.1007/BF02484433. [DOI] [PubMed] [Google Scholar]
  48. WOODWARD D.F., NIEVES A.L., HAWLEY S.B., JOSEPH R., MERLINO G.F., SPADA C.S. The pruritogenic and inflammatory effects of prostanoids in the conjunctiva. J. Ocul. Pharmacol. Ther. 1995;11:339–347. doi: 10.1089/jop.1995.11.339. [DOI] [PubMed] [Google Scholar]
  49. WU C.Y., WANG K., MCDYER J.F., SEDER R.A. Prostaglandin E2 and dexamethasone inhibit IL-12 receptor expression and IL-12 responsiveness. J. Immunol. 1998;161:2723–2730. [PubMed] [Google Scholar]
  50. YAMASAKI M., MATSUMOTO T., FUKUDA S., NAKAYAMA T., NAGAYA H., ASHIDA Y. Involvement of thromboxane A2 and histamine in experimental allergic rhinitis of guinea pigs. J. Pharmacol. Exp. Ther. 1997;280:1471–1479. [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES