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INFLAMMATION AND IMMUNOPHARMACOLOGY

In Vivo Activity of a Phospholipase C Inhibitor, 1-(6-((17-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), in Acute and Chronic Inflammatory Reactions

Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research and Development, L.L.C., Raritan, New Jersey

Received for publication September 26, 2003
Accepted December 22, 2003.

	Abstract

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To investigate the role of phospholipase C (PLC) in inflammatory processes, we tested 1-(6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122 [GenBank] ), a widely used PLC inhibitor, in several in vitro and in vivo assays. We first examined the effects of U73122 [GenBank] on human phospholipase C- (PLC-) isozymes and found that U73122 [GenBank] significantly inhibited recombinant human PLC-2, with an IC₅₀ of 6 µM. U73122 [GenBank] had little effect on PLC-1, PLC-3, or PLC-4. Consistent with its ability to inhibit PLC-2 enzymatic activity, U73122 [GenBank] reduced interleukin-8 and leukotriene B₄-induced Ca²⁺ flux and chemotaxis in human neutrophils in a concentration-dependent manner. In vivo, U73122 [GenBank] blocked carrageenan-induced hind paw edema in rats, carrageenan-induced macrophage and lymphocyte accumulation into subcutaneous chambers in dogs, lipopolysaccharide-induced macrophage, lymphocyte infiltration and prostaglandin E₂ production in a mouse peritonitis model, and 12-O-tetradecanoylphorbol-13-acetate-induced ear edema in mice. These results implicate PLC-dependent signaling pathways in the development of acute and chronic inflammatory responses in vivo.

To understand the role of PLC in the inflammatory responses in vivo, in the present study we investigated the effect of U73122 [GenBank] , a membrane-permeable aminosteroid PLC inhibitor, on both acute and chronic inflammation models in rats, dogs, or mice. U73122 [GenBank] was reported to selectively inhibit the PLC-dependent process in human platelets and neutrophils (Bleasdale et al., 1990; Smith et al., 1990; Thompson et al., 1991; Vickers, 1993; Wang, 1996; Jan et al., 1998), and therefore has subsequently proven useful in the evaluation of the role that PLC plays in cell activation. We have evaluated the effects of U73122 [GenBank] on carrageenan-induced hind paw edema in rats, carrageenan-induced macrophage accumulation in fluid exudates from subcutaneous chambers in dogs, LPS-induced macrophage infiltration into peritoneal lavage fluid of mice, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema in mice, and LPS-induced macrophage infiltration in peritoneal fluid of mice. In addition, we have examined the effect of U73122 [GenBank] on prostaglandin E₂ (PGE₂) production in LPS-induced peritonitis in mice. As part of our search for anti-inflammatory agents, we describe here for the first time the in vivo effect of U73122 [GenBank] on acute and chronic inflammation. The effects of U73122 [GenBank] on inflammatory parameters such as swelling and leukocyte infiltration, as well as specific PLC isozyme activities, are presented.

	Materials and Methods

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Animals. Male Sprague-Dawley rats, male Balb/C mice, and Swiss-Webster mice were purchased from ACE Animals (Boyertown, PA). Purpose bred dogs were purchased from Marshall Farms (North Rose, NY) and housed in an American Association for Accreditation of Laboratory Animal Care-accredited facility. All procedures conformed to the requirements of the Animal Welfare Act and were conducted according to the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee approved the studies. The animals were maintained on a 12-h light/dark cycle, the room temperature was set at 64°-84°F, and the humidity set at 30 to 70%. They were fed food and water ad libitum. The dogs were housed singly in 12-sq. ft. stainless steel cages and were fed Purina High Density Canine Diet once daily.

Chemicals and Reagents. The PLC inhibitor U73122 [GenBank] and arachidonic acid were obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). [³H]PIP₂ and scintillation precoated 96-well plates were purchased from PerkinElmer Life Sciences (Boston, MA). Ficoll-pague and dextran T-500 were purchased from Amersham Biosciences Inc. (Piscataway, NJ). Fluo-3 was obtained from Molecular Probes (Eugene, OR). IL-8 was purchased from R&D Systems (Minneapolis, MN). The cell culture media and serum were purchased from CellGro (Kansas City, MO). The PGE₂ enzyme immunoassay kit was obtained from Assay Design (Ann Arbor, MI). The plethysmograph was from Buxco Electronics (Sharon, CT). Ionomycin was from Calbiochem (La Jolla, CA). LTB₄, chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine, complement C5a, C3a, TPA, carrageenan, indomethacin, and the rest of the chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO).

PLC Assays. A scintillant, precoated 96-well microplate (PerkinElmer Life Sciences) was coated with 0.1 µCi of [³H]PIP₂ (PerkinElmer Life Sciences) in buffer, incubated at 4°C for at least 72 h, and then aspirated and washed three times with PBS (Ca²⁺-and Mg²⁺-free) before use. The amount of [³H]PIP₂ immobilized on the plates was determined by reading the plate in reaction buffer (50 mM Tris/HCl, pH 7.2, 1 mM EDTA, pH 7.3, 80 mM KCl, 10 mM LiCl, 0.04% deoxycholic acid sodium salt, and 3 mM CaCl₂) on a microplate scintillation and luminescence counter (PerkinElmer Life Sciences). The reaction was started with the addition of enzyme to wells in the presence or absence of U73122 [GenBank] with varying concentrations except the control wells. The wells were mixed and the plates were incubated at 37°C for 1.5 h. The plates were then read on a microplate scintillation and luminescence counter after termination of the reactions, and these counts were used as the postcounts for the assay. The percentage of inhibition of drugs was then calculated as a function of the amount of hydrolysis of the substrate.

Calcium Mobilization Assay in Human Neutrophils. Fresh human neutrophils were isolated from peripheral blood obtained from human volunteers and loaded with 4 µM Fluo-3 (Molecular Probes), a calcium-sensitive fluorescence dye, in Hanks' balanced salt solution (HBSS), pH 7.4, containing 20 mM HEPES, pH 7.5, 3.2 mM CaCl₂, 1% fetal bovine serum, 2.5 mM probenecid, and 0.04% Pluronic acid, in the dark at room temperature for 45 min with gentle rocking. The excess dye was removed by washing cells with HBSS after centrifugation. The cells were then resuspended in HBSS buffer and added to clear bottom black 96-well plates. U73122 [GenBank] (BIOMOL Research Laboratories) with indicated concentrations was added to the neutrophils and incubated at 37°C for 30 min. Agonists in appropriate concentration were prepared. Changes in intracellular free Ca²⁺ concentration were measured with a fluorometric imaging plate reader instrument immediately after the addition of agonist. The final concentration for IL-8 (R&D Systems) and LTB₄ (Sigma-Aldrich) was 10 nM.

IL-8 and LTB₄-Induced Chemotaxis Assay in Human Neutrophils. IL-8 or LTB₄ was prepared at a final concentration of 0.01 µg/ml in RPMI 1640 medium containing 0.5% bovine serum albumin. Using Costar 24-well Transwell filter plates, medium containing IL-8 or LTB₄ was added to the bottom chambers of the plate. To the top chamber of the Transwell plate, 1 x 10⁶ of freshly prepared human neutrophils was added. Next, testing compound or vehicle was added both to the cells and to the bottom chamber. The filters were loaded onto the bottom chambers of the plate, and the plate was incubated for 3 h at 37°C in a CO₂ incubator. The cells that have migrated into the bottom chambers of the plate were then counted using a hemocytometer.

Carrageenan-Induced Macrophage Accumulation in Fluid Exudate from Subcutaneous Chambers in Dogs. Carrageenan-induced inflammation assays in dogs were conducted as described previously (Kirchner et al., 1997). Groups of five beagle dogs of either sex, approximately 1 year of age and weighing 8 to 13 kg, were used in this study. After a postoperative recovery period of at least 1 month, a 1-ml sample of exudate from each dog was aspirated from within the chamber using a 1-ml syringe and 20-gauge 1-inch needle inserted through one of the perforations in the ball. The area was cleansed with 70% ethanol before each sample was collected. Immediately after obtaining the sample, U73122 [GenBank] (administered to render 2 µM final concentration in the chamber, based on 25-ml average chamber exudate) was injected into the chamber. Then, an inflammatory response was generated by injecting 1.5 ml of 0.33% carrageenan (Sigma-Aldrich) directly into the chamber. Fluid samples of 0.5 ml were obtained from within the chamber using a 21-gauge needle and 1-ml syringe. The skin area was treated with 70% ethanol before injections into and aspirations from the chamber. Exudate samples were obtained again at 1, 2, 3, and 4 days postcarrageenan administration and were analyzed for macrophage accumulation as an indicator of inflammatory activity using a Technicon H^*1E Hematology analyzer (Miles Technicon, Tarrytown, NY) to determine white blood cell count. The H^*1E is a laser light scattering flow system and colorimetric automated hematology analyzer that has four cytochemistry subsystem modules (RBC/PLT, Peroxidase, Baso, and Hemoglobin). The instrument performs a complete blood cell count with differential leukocyte cell count and morphology observations on whole blood. Due to the nature of the exudate samples, only the white blood cell value is used from the instrument. Differential cell counts were then performed manually. A cytospin centrifuge was used to spread the fluid out on a slide to make the cells easier to identify and the cells were then stained with Wright's stain. The percentage of cell types was determined after counting 100 cells under a microscope. Antibody and compound effects were compared with the chemotactic response of the control group. The exudate within the chambers was evacuated after the last sample was obtained and the dogs were allowed a recovery period of at least 4 weeks between carrageenan challenges. The dogs were used repeatedly and they served as their own controls when measuring treatment effects on the inflammatory responses elicited by various inflammatory provocations such as carrageenan, tumor necrosis factor-, IL-8, and monocyte chemoattractant protein-1 (Kirchner et al., 1997).

LPS-Induced Peritonitis and Macrophage Accumulation in Mice. Swiss-Webster mice (ACE Animals) were dosed intraperitoneally with test compound in sterile saline. Thirty minutes later, they were injected intraperitoneally with 1 ml of LPS (5 µg/ml) to induce peritonitis. The mice were dosed with U73122 [GenBank] 30 mg/kg i.p. on day 0, and once daily for a total of nine doses. The animals were then sacrificed in a CO₂ chamber on day 9 and the peritoneal cavities lavaged with 3 ml of Dulbecco's PBS to collect cells. Lavage samples were analyzed by differential cell counts using the same procedure as described in the canine subcutaneous chamber model. Percentage of inhibition was evaluated by comparison with the increase in macrophage count between the baseline control group and the positive control group.

TPA-Induced Ear Edema in Mice. TPA-induced ear edema was performed by the method reported previously by Rao et al. (1993). TPA (1.0 µg) dissolved in 20 µl of acetone was applied to the dorsal surface of the right ear of mice. U73122 [GenBank] was administered intravenously before the TPA administration. Six hours later, the animals were sacrificed and 7-mm biopsy punches were taken from each ear. The punches were weighed and the difference between treated and untreated ears determined. The percentage of inhibition was calculated by comparing the difference in ear weight of vehicle-treated with compound-treated mice.

Carrageenan-Induced Paw Edema in Rats. Male Sprague-Dawley rats (Ace Animals), weighing ca. 200 g, were fasted overnight. Then, 100 µl of a 0.5% carrageenan solution was injected into the subplantar tissue of one hind paw 1 h after drug or vehicle pretreatment. Paw volume displacement was measured using a mercury plethysmograph (Buxco Electronics) at 1, 3, and 5 h after induction of inflammation, and the edema was expressed as an increase in paw volume due to carrageenan injection. Indomethacin (10 mg/kg p.o.), a cyclooxygenase (COX) inhibitor, was used as a control.

In Vitro COX-2 Assay. Evaluation of specific COX-2 activity of the compound was performed using a whole cell assay with ECV-304 (human, endothelial, umbilical cord) cells (American Type Culture Collection, Rockville, MD) as described previously (Kirchner et al., 1997; Miralpeix et al., 1997). These cells were cultured in Media 199/10% bovine serum albumin (Cambrex Bio Science Walkersville, Inc., Walkersville, MD) at 37°C and 5% CO₂, and then trypsinized and plated at a density of 9 x 10⁴ cells/well of a 96-well plate before assay. Approximately 28 h later, 50 µg/ml phorbol 12-myristate 13-acetate (Sigma-Aldrich) and 2 µM ionomycin (Sigma-Aldrich) (final concentration) were added to each well. Cells were incubated in the presence of vehicle or drug for 18 h. PGE₂ production was determined via radioimmunoassay (Assay Design) after the addition of 30 µM arachidonic acid. The data are expressed as percentage of inhibition of products compared with vehicle treatment.

Statistical Analysis. The results of in vivo experiments were expressed as the mean ± S.E.M. Differences between means were tested for significance by Student's t test or (for multiple comparisons with the same control) by an analysis of variance followed by the Bonferroni's multiple comparison test (Prism; GraphPad Software Inc., San Diego, CA). P < 0.05 or lower was considered to be statistically significant using Student's t test.

	Results

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Effects of U73122 [GenBank] on Phospholipase C- Isozyme Activities. We conducted our PLC assays by using the exogenously immobilized specific substrate [³H]PIP₂ and human PLC- enzymes. U73343 [GenBank] , a close analog of U73122 [GenBank] , was used as a negative control. U73122 [GenBank] did not significantly inhibit human recombinant PLC-1 at concentrations 25 µM (Fig. 1A). However, U73122 [GenBank] markedly inhibited the ability of human recombinant PLC-2 to hydrolyze [³H]PIP₂. The half-maximal and maximal inhibitory effect of U73122 [GenBank] on PLC-2 was observed at 6 and 25 µM, respectively (Fig. 1B). U73122 [GenBank] moderately inhibited the hydrolysis of the substrate by PLC-3_, isolated from human brain, with 35% inhibition at 25 µM (Fig. 1C). Similarly, up to 25 µM, U73122 [GenBank] did not significantly inhibit the hydrolysis of [³H]PIP₂ by PLC-4 (Fig. 1D). The maximum inhibitory effect of U73122 [GenBank] on PLC-4, isolated from a human retina cell line, was 30% inhibition. The rank order of potency among the isozymes for the inhibitory effect of U73122 [GenBank] was PLC-2 > PLC-3 PLC-4 and PLC-1. Therefore, our results demonstrate that U73122 [GenBank] preferentially inhibits PLC-2 activity. U73343 [GenBank] had no effect on any of the four PLC- isozymes.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Effects of U73122 [GenBank] and its analog U73343 [GenBank] on the hydrolysis of [3H]PIP2 by PLC- isozymes. The PLC- isozyme protein was incubated with the vehicle or indicated concentrations of U73122 [GenBank] or U73343 [GenBank] at 37°C for 1.5 h as described under Materials and Methods. The reduction in [3H]PIP2 level before and after the enzymatic reaction was quantitatively represented as percentage of inhibition. The effect of U73122 [GenBank] and U73343 [GenBank] on PLC-1 (A), PLC-2 (B), PLC-3 (C), and PLC-4 activity (D) was presented. The results are representative of three independent experiments.

Effect of U73122 [GenBank] on IL-8- and LTB₄-Induced Ca²⁺ Flux in Human Neutrophils. U73122 [GenBank] was then tested in cellular functional assays. As shown in the Fig. 2, A and B, U73122 [GenBank] inhibited IL-8- and LTB₄-induced Ca²⁺ fluxes in human neutrophils in a dose-dependent manner, with an IC₅₀ of 6 µM in each case, consistent with its potency in the PLC-2 enzyme assay.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Effect of U73122 [GenBank] on IL-8- and LTB4-induced Ca2+ mobilization in human neutrophils. Fluo-3-loaded human neutrophils were pretreated with increasing concentrations of U73122 [GenBank] for 15 min followed by 100 nM IL-8 or LTB4. The changes in fluorescence representing the changes in Ca2+ concentration were measured by fluorometric imaging plate reader at 37°C. Data are percentage of inhibition of IL-8- or LTB4-induced Ca2+ fluorescence counts by U73122 [GenBank] . The inhibitory effect of U73122 [GenBank] on IL-8 (A) and LTB4 (B) are presented. The results shown are representative of three separate studies.

Effect of U73122 [GenBank] on IL-8- and LTB₄-Induced Human Neutrophil Migration. It has been reported that cells from PLC-2 knockout mice show decreased chemoattractant-induced Ca²⁺ fluxes, consistent with our results mentioned above, but have normal or supranormal chemotactic responses. Therefore, we next tested U73122 [GenBank] in IL-8- and LTB₄-induced chemotaxis assays. As shown in Fig. 3, A and B, IL-8 and LTB₄ were potent chemotactic factors for human neutrophils. Surprisingly, we found that U73122 [GenBank] markedly inhibited those chemotactic responses with IC₅₀ values of 5 µM, consistent with its effects in the enzymatic and Ca²⁺ mobilization assays described above.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Effect of U73122 [GenBank] on neutrophil migration in response to IL-8 and LTB4. Using Costar 24-well filter plates, medium containing IL-8 or LTB4 was added to the bottom chambers of the plate. Then 1 x 106 freshly prepared human neutrophils were added to the top chamber in the absence or presence of U73122 [GenBank] . The Transwell plate was incubated at 37°C for 3 h. The cells that have migrated into the bottom chambers of the plate were then counted using a hemacytometer. Data shown are percentage of inhibition of IL-8- (A) and LTB4 (B)-induced chemotaxis in human neutrophils by U73122 [GenBank] . The results shown are representative of five separate studies.

Effect of U73122 [GenBank] on Carrageenan-Induced Hind Paw Edema in Rats. We examined the effect of U73122 [GenBank] on the development of inflammation in vivo in rats. Indomethacin was used as a positive control. At a dose of 30 mg/kg i.p., U73122 [GenBank] significantly inhibited carrageenan-induced inflammation, and its effect was more complete than a 10 mg/kg p.o. dose of indomethacin (Fig. 4). U73122 [GenBank] inhibited swelling by 65 and 80% at 1 and 3 h postcarrageenan challenge, as shown in Fig. 4.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Anti-inflammatory effects of U73122 [GenBank] and indomethacin on carrageenan-induced hind paw edema in rats. Vehicle or indomethacin (10 mg/kg p.o.) suspended in 0.5% methylcellulose solution or U73122 [GenBank] (30 mg/kg i.p.) suspended in saline was administered as indicated in Fig. 4. Footpad edema was induced 1 h later by subplantar injection of carrageenan [0.5% (w/v) in saline]. Footpad volume was measured 3 h after treatment as described under Materials and Methods. Values are the mean ± S.E.M. (n = 6). The results are expressed the mean ± S.E.M. of six rats. **, P < 0.01 versus control using Student's t test.

Effect of U73122 [GenBank] on COX-2 Activity. To determine whether U73122 [GenBank] exhibits COX-2 inhibitory activity, we evaluated U73122 [GenBank] in our in vitro COX-2 assay using ECV-304 cells, as described previously (Kirchner et al., 1997; Miralpeix et al., 1997). U73122 [GenBank] showed only 10% inhibition at 10 µM, indicating that the anti-inflammatory effect of U73122 [GenBank] was not due to the inhibition of the COX-2 enzyme.

Effect of U73122 [GenBank] on Carrageenan-Induced Macrophage Accumulation in Dogs. We then evaluated U73122 [GenBank] for its anti-inflammatory effect on carrageenan-induced inflammation in s.c. chambers in dogs. We found that U73122 [GenBank] significantly inhibited the carrageenan-induced macrophage and lymphocyte infiltration into the exudates in s.c. chambers in dogs 24 h postchallenge. The maximum effect of U73122 [GenBank] (0.1 mg/ml) on the macrophage and lymphocyte influx was 65 and 74% inhibition, respectively (Fig. 5, A and B). In contrast, U73122 [GenBank] did not have a significant effect on the influx of neutrophils into the chambers (Fig. 5C).

View larger version (14K): [in this window] [in a new window]   Fig. 5. Effect of U73122 [GenBank] on carrageenan-induced inflammatory cell accumulation in fluid exudates from subcutaneous chambers in dogs. One milliliter of sample exudate from each dog was aspirated from within the chamber. Vehicle or U73122 [GenBank] suspended in 1.5 ml of sterile saline was injected into the chamber. Then an inflammatory response was generated by injecting 1.5 ml of 0.33% carrageenan directly into the chamber as described under Materials and Methods. Fluid samples of 0.5 ml were obtained from within the chamber 5 and 24 h postcarrageenan challenge and analyzed for macrophage (A), lymphocyte (B), and neutrophil (C) accumulation via a Technicon H*1E hematology analyzer. The open bars represent the control levels 5 and 24 h after treatment. The solid columns show the inhibitory effect of U73122 [GenBank] on the cell levels at 5 and 24 h. Each column represents the mean of levels from five dogs with S.E.M. *, P < 0.05 versus control using Student's t test.

Effect of U73122 [GenBank] on LPS-Induced Peritoneal Macrophage Accumulation in Mice. In our LPS-induced mouse peritonitis model, the numbers of neutrophils in the peritoneal cavity rapidly decrease after LPS injection, whereas the macrophages and lymphocytes gradually increase and peak at day 9. As shown in Fig. 6A, U73122 [GenBank] (30 mg/kg i.p.) totally inhibited the LPS-induced increase in macrophages. It also completely blocked the LPS-induced increase in lymphocytes (Fig. 6B); in fact, the lymphocyte count was below the background level in non-LPS-injected animals. However, U73122 [GenBank] did not inhibit the LPS-induced loss of neutrophils from the peritoneal cavity (Fig. 6C).

View larger version (18K): [in this window] [in a new window]   Fig. 6. U73122 inhibits LPS-induced peritoneal inflammatory cell accumulation in mice. Vehicle or U73122 [GenBank] (30 mg/kg) suspended in sterile saline was administered i.p. 30 min before LPS (5 µg/ml, 1 ml i.p.) challenge to induce peritonitis. The mice were dosed every day. On day 9, the peritoneal cavities were lavaged with 3 ml of PBS to collect cells. Lavage samples were analyzed for macrophage (A), lymphocyte (B), and neutrophil (C) accumulation. The open columns show the control number of cells. The striped column indicates the LPS-induced cell level. The solid column represents the effect of U73122 [GenBank] on LPS-induced cell accumulation. The data are means ± S.E.M. of six samples in each group. **, P < 0.01 and ***, P < 0.001 versus control using Student's t test.

Effect of U73122 [GenBank] on LPS-Induced PGE₂ Production in Peritoneal Lavage Fluid in Mice. In addition, we studied the effect of U73122 [GenBank] on LPS-induced PGE₂ production in the same experimental model. As shown in Fig. 7, U73122 [GenBank] inhibited LPS-induced PGE₂ production in the peritoneal cavity by 80%.

View larger version (23K): [in this window] [in a new window]   Fig. 7. U73122 inhibits LPS-induced PGE2 production in peritoneal lavage fluid. The effect of U73122 [GenBank] on LPS-induced PGE2 production was determined with the same lavage samples as described in Fig. 6. The PGE2 level was determined by using an enzyme immunoassay PGE2 assay kit. The open columns represent the control level of PGE2. The striped columns show the LPS-induced PGE2 production level. The solid column indicates the inhibitory effect of U73122 [GenBank] . The data are means ± S.E.M. of six samples in each group. ***, P < 0.001 versus control using Student's t test.

Effect of U73122 [GenBank] on TPA-Induced Mouse Ear Edema. We also investigated the effect of U73122 [GenBank] on TPA-induced ear edema in mice. Administration of U73122 [GenBank] at 1, 3, and 10 mg/kg i.v. effectively suppressed TPA-induced ear edema in a dose-dependent manner (Fig. 8).

View larger version (24K): [in this window] [in a new window]   Fig. 8. Effect of U73122 [GenBank] on TPA-induced ear edema in mice. Vehicle, control compound or indicated concentrations of U73122 [GenBank] was administered 1 h before TPA challenge. Twenty microliters of TPA (1 µg) was topically applied onto the dorsal surface of the right mouse ear. Ear edema was measured 6 h after challenge by using a 7-mm biopsy punch. The open column represents the TPA-induced ear edema. The striped bars show the effect of tepoxalin on edema. The solid columns indicate the dose-dependent inhibitory effect of U73122 [GenBank] on TPA-induced mouse ear edema. The results are means ± S.E.M. of six samples. *, P < 0.05 versus control using Student's t test

Effect of U73122 [GenBank] on Glucocorticoid and Other Steroid Receptor Binding. Because U73122 [GenBank] is an aminosteroid, it is important to rule out the possibility that its anti-inflammatory effects were mediated by binding to one or more of the steroid receptors. We then tested the binding of U73122 [GenBank] to several steroid receptors, including glucocorticoid receptor, estrogen receptor (ER), ER, and androgen receptor (AR). As shown in Table 1, U73122 [GenBank] did not bind to the human glucocorticoid receptor at 10 µM. In contrast, the IC₅₀ value of dexamethasone was 26 nM. Similarly, U73122 [GenBank] had little or no binding affinity for ER, ER, and AR, respectively, at 10 µM, whereas the appropriate control compounds had expected IC₅₀ values in the low nanomolar range in each case.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Activation of phospholipase C is a central component of the signal transduction in numerous cells, including leukocytes from human peripheral blood (Wu et al., 2000). However, the role of PLC in the inflammatory process in vivo is still largely unknown.

In this study, we demonstrated that a PLC inhibitor, U73122 [GenBank] , inhibited recombinant human PLC-2, with an IC₅₀ value of 6 µM. U73122 [GenBank] had little effect on PLC-1, PLC-3, and PLC-4, implying that U73122 [GenBank] preferentially inhibits PLC-2 over PLC-1, PLC-3, and PLC-4. Consistent with previous reports (Bleasdale et al., 1990; Smith et al., 1990), U73343 [GenBank] had no effect on any of the four PLC- isozymes. In addition, we observed in our studies that U73122 [GenBank] blocked IL-8-, LTB₄-induced Ca²⁺ mobilization and chemotaxis in human neutrophils.

Of particular interest, in vivo, we found, for the first time, that a PLC inhibitor, U73122 [GenBank] , markedly inhibited LPS-induced leukocyte, macrophage, and lymphocyte infiltration into lavage fluid of LPS-induced peritonitis in mice. In addition, U73122 [GenBank] inhibited LPS-induced PGE₂ production in lavage fluid of LPS-induced mouse peritonitis. The inhibition of peritoneal macrophage infiltration by U73122 [GenBank] correlated with the inhibition of the PGE₂ level in the lavage fluid of mouse peritonitis, indicating the anti-inflammatory effect of U71322 [GenBank] in vivo on the inflammatory models. In addition, it seems that U73122 [GenBank] could inhibit LPS-induced leukocyte and macrophage infiltration, but not the basal peritoneal leukocyte and macrophage in mice.

Furthermore, in our studies, U73122 [GenBank] inhibited carrageenan-induced macrophage and lymphocyte influx into subcutaneous chambers in dogs. In this model, carrageenan administration produces a significant influx of leukocytes into the exudates, which peaked at 24 h, and was maintained over a period of three or more days (Kirchner et al., 1997).

Next, we observed that U73122 [GenBank] significantly inhibited carrageenan-induced hind paw edema in rats and TPA-induced ear edema in mice, suggesting that U73122 [GenBank] was efficacious in vivo in inhibiting the inflammatory process.

Macrophages produce a variety of inflammatory mediators during inflammation (Laskin and Pendino, 1995; Guha and Mackman, 2001). One of the mediators is PGE₂ (Smith et al., 1996, 2000). Clinically, an increase in PGE₂ is associated with numerous pathophysiological conditions during inflammation (O'Neil and Ford-Hutchinson, 1993; Lockhart and McNicol, 1996; Barrios-Rodiles et al., 1999; Gilroy et al., 1999; Simon, 1999; Bandeira-Melo et al., 2000; Ianaro et al., 2001; Lawrence et al., 2002; Zhou et al., 2002). An acute inflammation is significantly suppressed by COX-2 inhibitors and corticosteroids. With respect to this, we evaluated U73122 [GenBank] in our COX-2 assay in vitro, using ECV-304 cells. Our data show that U73122 [GenBank] showed only 10% inhibition of PGE₂ production at 10 µM, indicating that the anti-inflammatory effect of U73122 [GenBank] was not due to the inhibition of COX-2. Thus, our hypothesis is that the anti-inflammatory effect of U73122 [GenBank] through the PLC pathway might contribute to the mechanisms of controlling PGE₂ synthesis in regulating inflammatory reactions.

We also demonstrated that U73122 [GenBank] did not bind to the human glucocorticoid receptor at 10 µM. Similarly, no significant binding of U73122 [GenBank] to estrogen-, estrogen-, and androgen receptor was observed at 10 µM. These results indicate that the anti-inflammatory activity of U73122 [GenBank] is not through glucocorticoid regulation of inflammatory signaling, but may be indeed through the PLC and PKC signaling pathways.

Cotransfection experiments in COS-7 and human embryonic kidney 293 cells suggest that PLC-2 may function downstream of chemoattractant receptors. Transfection of receptors for complement C5a and fMet-Leu-Phe, interleukin-8, and monocyte chemoattractant protein-1 demonstrated that each of the receptors activates PLC-2 through the pertussis toxin-sensitive G proteins. It was thought that PLC-2 may be a primary signaling pathway in neutrophils, because the PLC activity elicited through chemoattractant receptors also seems to function through the G_i-mediated release of subunits (Jiang et al., 1997). The role of PLC-2 in chemoattractant-mediated responses was studied in mice lacking PLC-2. These studies revealed that PLC-2 deficiency blocked chemoattractant-induced Ca²⁺ release, superoxide production, Mac-1 up-regulation in neutrophils and regulation of protein kinases, but not chemotaxis (Jiang et al., 1997; Li et al., 2000). To date, there are no literature reports about the role of a PLC in the inflammatory process.

Our findings in the present study suggest that PLC-dependent signaling pathways are implicated in the development of acute and chronic inflammatory responses in vivo. Alternatively spliced forms of PLC-2 have been reported in hematopoietic cells (Mao et al., 2000). They differ in the carboxyl-terminal sequence implicated in interaction of PLC- enzymes with G_q, particulate association, and nuclear localization. The PLC-2 splice variants may be regulated differentially with distinct roles in signal transduction. The insight into the mechanism of the involvement of PLC pathways in the inflammatory reactions is currently under investigation.

In conclusion, our data provide direct evidence, for the first time, that PLC pathways may play an important role in leukocyte function at the cellular level and in animal model in vivo.

	Acknowledgements

 
We thank Dr. T. K. Harden for providing human phospholipase C- enzymes. We also thank Dr. David Ritchie for scientific suggestions and discussion, Lynn Varacallo for leukocyte analysis of lavage fluid samples, and Dr. George Allan for the evaluation of U73122 [GenBank] in the steroid receptor binding assays.

	Footnotes

 
DOI: 10.1124/jpet.103.060574.

ABBREVIATIONS: PLC, phospholipase C; PIP₂, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; LTB₄, leukotriene B₄; IL-8, interleukin-8; U73122 [GenBank] , 1-(6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; U73343 [GenBank] , 1-(6-((17-3-methoxylestra-1,3,5(10)-trien-17yl)amino)hexyl)-2,5-pyrrolidine-dione; LPS, lipopolysaccharide; TPA, 12-O-tetradecanoylphorbol-13-acetate; PGE₂, prostaglandin E₂; PBS, phosphate-buffered saline; HBSS, Hanks' balanced salt solution; COX, cyclooxygenase; ER, estrogen receptor; AR, androgen receptor.

Address correspondence to: Dr. Cuifen Hou, Johnson and Johnson Pharmaceutical Research and Development, L.L.C., Drug Discovery, 1000 Route 202, Rm B-110, P.O. Box 300, Raritan, NJ 08869. E-mail: chou{at}prdus.jnj.com

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