The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ballinger, M. N.
Right arrow Articles by Moore, B. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ballinger, M. N.
Right arrow Articles by Moore, B. B.
The Journal of Immunology, 2006, 177: 5499-5508.
Copyright © 2006 by The American Association of Immunologists, Inc.

Critical Role of Prostaglandin E2 Overproduction in Impaired Pulmonary Host Response following Bone Marrow Transplantation1

Megan N. Ballinger*,{dagger}, David M. Aronoff{ddagger}, Tracy R. McMillan*, Kenneth R. Cooke§, Krystyna Olkiewicz§, Galen B. Toews*, Marc Peters-Golden* and Bethany B. Moore2,*

* Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, {dagger} Graduate Program in Immunology, {ddagger} Division of Infectious Diseases, and § Bone and Marrow Transplantation Program, University of Michigan, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The success of bone marrow transplantation (BMT) as a therapy for malignant and inherited disorders is limited by infectious complications. We previously demonstrated syngeneic BMT mice are more susceptible to Pseudomonas aeruginosa pneumonia due to defects in the ability of donor-derived alveolar macrophages (AMs), but not polymorphonuclear leukocytes (PMNs), to phagocytose bacteria. We now demonstrate that both donor-derived AMs and PMNs display bacterial killing defects post-BMT. PGE2 is a lipid mediator with potent immunosuppressive effects against antimicrobial functions. We hypothesize that enhanced PGE2 production post-BMT impairs host defense. We demonstrate that lung homogenates from BMT mice contain 2.8-fold more PGE2 than control mice, and alveolar epithelial cells (2.7-fold), AMs (125-fold), and PMNs (10-fold) from BMT animals all overproduce PGE2. AMs also produce increased prostacyclin (PGI2) post-BMT. Interestingly, the E prostanoid (EP) receptors EP2 and EP4 are elevated on donor-derived phagocytes post-BMT. Blocking PGE2 synthesis with indomethacin overcame the phagocytic and killing defects of BMT AMs and the killing defects of BMT PMNs in vitro. The effect of indomethacin on AM phagocytosis could be mimicked by an EP2 antagonist, AH-6809, and exogenous addition of PGE2 reversed the beneficial effects of indomethacin in vitro. Importantly, in vivo treatment with indomethacin reduced PGE2 levels in lung homogenates and restored in vivo bacterial clearance from the lung and blood in BMT mice. Genetic reduction of cyclooxygenase-2 in BMT mice also had similar effects. These data clearly demonstrate that overproduction of PGE2 post-BMT is a critical factor determining impaired host defense against pathogens.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Bone marrow transplantation (BMT)3 is a common therapeutic option for various malignant and inherited disorders. However, the overall success of BMT is diminished by a myriad of serious complications including the following: graft failure (1), graft-vs-host disease (2), multiorgan complications (3), opportunistic/nosocomial infections (4), and sepsis (5). The lung is a particular target organ for complications following BMT. Infectious and noninfectious pulmonary complications occur in up to 60% of hemopoietic stem cell transplant recipients and nearly one-third of these patients require admission to the intensive care unit (4). A variety of infectious agents cause pulmonary complications including viruses, bacteria, and fungi (reviewed in Refs. 4, 5, 6, 7). As such, pneumonia is the leading infectious cause of death after transplantation (4). Although the risk for such infections is greatest during the pre-engraftment period of neutropenia, vulnerability to pneumonia continues well after successful homing and engraftment of stem cells in the host. In the setting of allogeneic transplants, antirejection medicines certainly contribute to the state of immunosuppression; however, immunodeficiency persists even in recipients of autologous transplants in the absence of pharmacological immunosuppression (8). Clinical studies document that BMT patients exhibit long-term immune dysfunction for months or even years posttransplantation (8).

The lung has the largest interface with the external environment of any internal organ, and therefore has generated an elaborate host defense system to protect it. Resident alveolar macrophages (AMs) and recruited polymorphonuclear leukocytes (PMNs) are key cells responsible for the innate immune response in the lung (9, 10). Our laboratory has previously developed a murine model of bacterial pneumonia post-BMT (11). We showed that 3 wk postsyngeneic transplantation, BMT mice were unable to clear a Pseudomonas aeruginosa infection. Although susceptibility to infection was not explained by decreased circulatory or lung levels of leukocytes, it was associated with defective phagocytosis by the AMs and an inability of these AMs to produce activating cytokines such as TNF-{alpha} (11). Interestingly, the phagocytic ability of PMNs post-BMT appeared intact (11). However, the fundamental etiology of the innate immune dysregulation and impaired antimicrobial defense in this model remains unknown.

In the present study, we tested the hypothesis that altered production and/or signaling of eicosanoids contribute to immunosuppression post-BMT. Eicosanoids are arachidonic acid (AA)-derived lipid mediators synthesized and secreted by virtually all cell types and are capable of eliciting a wide spectrum of biological responses (reviewed in (12)). Through either cyclooxygenase (COX) or 5-lipoxygenase (5-LO) enzymatic pathways, AA is converted to either PGs or leukotrienes (LTs), respectively. LTs serve as important mediators of innate immunity (13), as evidenced by their ability to augment bacterial phagocytosis and killing by AMs (14, 15, 16) and PMNs (17, 18, 19), induce TNF-{alpha} production by AMs (20) and PMNs (21), and recruit PMNs to the site of infection (22). By contrast, PGE2 is an important negative regulator of host innate immunity, with the ability to inhibit leukocyte chemotaxis (23), AM phagocytosis and killing (24, 25), PMN host defense functions (26), reactive oxygen intermediate production (27), AA release (28), LT synthesis (29), and generation of multiple proinflammatory cytokines (30, 31, 32). PGE2 also enhances the production of the immunosuppressive cytokine IL-10 (33).

PGE2 exerts its effects via binding to G protein-coupled seven-transmembrane spanning E prostanoid (EP) receptors (34). There are four discrete EP receptors, each coupled to distinct G proteins and intracellular signaling pathways. The inhibitory effects of PGE2 on AMs are largely mediated via increases in cAMP. Although both the EP2 and EP4 receptors can raise cAMP levels (34), we have shown that, in AMs, EP2 is the predominant inhibitory receptor for PGE2 (25).

Previous work has shown that BMT patients have increased levels of circulating PGE2 in their plasma for weeks to months posttransplant (35). However, no studies have examined the production of PGE2 within the lung post-BMT or its consequences for antimicrobial defense. We hypothesized that the overproduction of PGE2 post-BMT could contribute to the impaired pulmonary host defense functions of resident or recruited phagocytes in response to a Gram-negative infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

C57BL/6 or Ptgs2tm1Jed (PG-deficient COX-2+/–) (36) and their controls, B6;129S7, mice were obtained from The Jackson Laboratory. In some cases, B6Ly5.2 mice, purchased from the Fredrick Cancer Research Facility (Fredrick, MD), were used as BMT donors for irradiated B6Ly5.1 (The Jackson Laboratory) recipients so that donor vs host leukocytes could be distinguished by staining for the CD45.1 and CD45.2 alleles using Abs commercially available from BD Pharmingen. Mice were housed under specific pathogen-free conditions and were monitored daily by veterinary staff. All mice were euthanized by CO2 asphyxiation. The University of Michigan Committee on the Use and Care of Animals approved these experiments.

Bone marrow transplantation

The protocol for syngeneic BMT has been previously described (11, 37, 38). Briefly, recipient mice received 13 Gy of total body irradiation (137Cs source) delivered in two fractions separated by 3 h. A cell mixture of 5 x 106 bone marrow (BM) cells and 1 x 106 purified splenic T cells was resuspended in Leibovitz’s L-15 medium (Invitrogen Life Technologies) and transplanted into syngeneic recipients via tail vein infusion (total volume, 0.25 ml). All experiments with BMT mice were performed 3–6 wk post-BMT and no differences were observed within this time span. By performing transplants using B6Ly5.2 mice as donors into control C57BL/6 (Ly5.1) mice and staining for CD45.1 (Ly 5.2) and CD45.2 (Ly5.1) alleles, we have previously shown that, at 1 wk post-BMT, >99% of PMNs are donor derived and, at 3 wk post-BMT, 89.5% of the AMs are donor derived (11).

Harvesting lavage fluid

Bronchoalveolar lavage fluid (BALF) was obtained by lavaging the alveolar space with PBS containing 5 mM EDTA using a previously described method (11). Bronchoalveolar lavage was performed by instilling 1 ml of lavage buffer, followed by gentle suction with an ~0.8-ml volume return. This was repeated three times for a total return of ~2.5 ml of BALF.

Peritoneal lavage fluid (PLF) was obtained by lavaging the peritoneal cavity with PBS. Briefly, the skin over the peritoneal cavity (but not the peritoneal lining), was removed and 7 ml of PBS was carefully infused into the peritoneal cavity via an 18-gauge needle. The abdomen was massaged, followed by a gentle suction with a ~6-ml volume return. This was repeated twice for a total return of ~12 ml of PLF.

Alveolar epithelial cell (AEC) purification

Type II AECs were isolated using dispase and DNase digestion of lower lungs as previously described (39, 40). Bone marrow-derived cells were removed via anti-CD32 and anti-CD45 magnetic depletion. Mesenchymal cells were removed by overnight adherence in a petri dish. The nonadherent cells, after this initial plating, were plated in 96-well plates coated with fibronectin. Purity was >94% as determined by staining with cytokeratin and vimentin. Supernatants from the AECs were harvested 72 h after fibronectin adherence.

Harvesting AMs and lung PMNs

Resident AMs from mice were obtained via ex vivo lung lavage, using a previously described protocol (41). Briefly, these cells were collected in lavage fluid consisting of complete medium (DMEM, 1% penicillin-streptomycin, 1% L-glutamine, 10% FCS, 0.1% Fungizone) and 5 mM EDTA. The cells were enumerated by counting on a hemocytometer before plating. Cells were allowed to adhere to tissue culture plates for 1 h then were washed to remove nonadherent cells, resulting in >95% pure AM culture as determined by modified Wright-Giemsa stain.

Lung PMNs were elicited as previously described (9). Briefly, lung PMNs were obtained by bronchoalveolar lavage 18 h after intratracheal (i.t.) injection with 25 µg of LPS derived from P. aeruginosa (Sigma-Aldrich). At this time point, the percentage of PMNs in the lavage ranged from 87 to 93% as determined by differential staining. PMNs were collected by centrifugation, washed two times, and allowed to adhere for 30 min in serum-free medium (DMEM, 0.1% BSA, 1% penicillin-streptomycin, 1% L-glutamine, 0.1% amphotericin B), and then immediately used for analysis.

To account for possible differences in the adherence of leukocytes from normal or BMT mice to tissue culture plates, which could potentially confound experimental results, the relative numbers of viable, adherent cells were determined using a commercial assay for intracellular lactate dehydrogenase (Roche Diagnostic) as previously described (9). Results from comparative studies (described below) were normalized for differences in cell adherence.

Eicosanoid measurements in supernatants of isolated cells and lavage fluid

AMs from control or BMT mice were cultured for 24 h with and without LPS (10 µg/ml) or for 1 h with Ca2+ ionophore A23187 (5 µM). Likewise, PMNs from control or BMT mice were cultured overnight with and without LPS or for 1 h with Ca2+ ionophore. After stimulation, cell-free supernatants were collected and analyzed by specific enzyme immunoassay (EIA) kits for PGE2, 6-keto PGF1{alpha} (a metabolite of PGI2), leukotriene B4 (LTB4), or total cysteinyl leukotrienes (cys-LTs: LTC4, LTD4, and LTE4) (Cayman Chemical) according to the manufacturer’s instructions.

In vitro phagocytosis assays

AMs were harvested as described above and the ability of the AMs from control and BMT mice to phagocytose via opsonin-dependent or -independent pathways was examined. The ability of AMs to phagocytose unopsonized bacteria was examined using FITC-labeled Escherichia coli (Vybrant phagocytosis assay; Molecular Probes), and modified slightly from a previously described method (9, 11, 25). Briefly, AMs were plated on a half-area black 96-well plate and were incubated overnight at 37°C. The next day, cells were pretreated as follows with the COX inhibitor indomethacin (5 µM, 30 min), the inhibitor of internalization cytochalasin D (5 µg/ml, 30 min), the EP2 antagonist, AH-6809 (50 µM, 30 min), the cys-LT, LTD4 (10 nM, 10 min), and/or PGE2 (1 µM, 10 min). After the appropriate pretreatment, 50 µl of FITC-labeled E. coli was added to each well (which corresponds to a ratio of bacteria to phagocytes of 60:1). After 2 h at 37°C, phagocytosis was quantified as previously described (25).

The phagocytosis of opsonized particles was examined by using IgG-opsonized SRBC and was modified slightly from a previously described protocol (25, 42). Briefly, AMs were cultured overnight in a 96-well plate. SRBCs (ICN Pharmaceutical) were opsonized with a subaggutinating concentration of polyclonal, rabbit anti-SRBC IgG (Cappel Organon Teknika) as previously described (25, 43). AMs were then washed and pretreated as described above. Following the pretreatment interval, opsonized or unopsonized SRBCs were added at a ratio of 50:1 (SRBCs to AMs) and cultures were incubated for 90 min at 37°C. Wells were then washed to remove uningested erythrocytes and lysed with 0.3% SDS in PBS. Lastly, o-phenylenediamine dihydrochloride solution (25, 42) was added to each well as a chromogen. Following a 30- to 90-min incubation, the absorbance (A450) was evaluated with an automated reader (VERSAmax; Molecular Devices). The number of SRBCs per well was derived from A450 data using a standard curve. The phagocytic index (number of ingested SRBCs) represented the number of SRBCs in an experimental well (ingested plus adherent SRBCs) minus the mean number of SRBCs in the cytochalasin D-treated wells (adherent SRBCs) and was expressed as a percentage of the control.

Tetrazolium dye reduction assay of bacterial killing

The ability of AMs and PMNs from control and BMT mice to kill P. aeruginosa was quantified using a tetrazolium dye reduction assay, as described elsewhere (16, 44). Briefly, AMs or PMNs from control and BMT mice were aliquoted into duplicate 96-well plates: one experimental (37°C) plate and one control (4°C). Cells from both plates were infected with opsonized (4% mouse-derived anti-P. aeruginosa immune serum (15)) or unopsonized P. aeruginosa (2 x 108 CFU/ml; multiplicity of infection, 50:1) for 30 min at 37°C. Briefly, the cells on the experimental plate were washed, and then incubated at 37°C for 90 min, while the cells on the control plate were washed, and then lysed with tryptic soy broth and 0.5% saponin (Sigma-Aldrich) and placed at 4°C. After 90 min, the cells from the experimental plate were lysed with tryptic soy broth and 0.5% saponin. Both plates were then incubated at 37°C for 2.5 h. MTT (5 mg/ml; Sigma-Aldrich) was added to each plate and incubated for 30 min. Solubilization solution was added to dissolve fomazan salts and the absorbance was read at 595 nm. Results were expressed as percentage of survival of ingested bacteria, where the survival of ingested bacteria = (A595 control plate/A595 experimental plate) x 100%.

Semiquantitative real-time PCR

Semiquantitative RT-PCR was performed on an ABI Prism 7000 thermocycler (Applied Biosystems). Gene-specific primers and probes were designed using Primer Express software (PerkinElmer/PE Applied Biosystems). The sequences for all primers and probes used can be found in Table I. Briefly, the reaction mixture contained 250 ng of RNA, 12.5 µl of TaqMan 2x Universal PCR Master Mix, 0.625 µl of 40x MultiScribe and RNase Inhibitor Mix (Applied Biosystem; Roche), 250 nM FAM probe, and forward and reverse primers at 300 nM in a final volume of 25 µl. For each experiment, samples from mice (n = 2–3) were run in triplicate. The average cycle threshold (CT) was determined for each mouse from a given experiment. Relative gene expression (using the formula 2–{Delta}{Delta}CT) was calculated using the comparative CT method (45), which assesses the difference in gene expression between the gene of interest and an internal standard gene (beta-actin) for each sample to generate the {Delta}{Delta}CT. The average of the control sample was set to 1 for each experiment, and the relative gene expression for each experimental sample was compared with that.


View this table:
[in this window]
[in a new window]
  		Table I. Primers and probes for semiquantitative real-time PCR

 
Intratracheal infection with P. aeruginosa

P. aeruginosa PAO1 stock was grown as previously described (11) in tryptic soy broth (Difco) for 18 h at 37°C. Bacterial concentration was determined spectrophotometrically (A600) compared with a predetermined standard curve. Mice were anesthetized and the trachea was exposed in sterile fashion. The appropriate dose of P. aeruginosa inoculum (or saline control) was administered i.t. To determine the effects of inhibiting PG synthesis during an infection, mice received an i.p. injection of 1.2 mg/kg of indomethacin (46) (Sigma-Aldrich) at the same time as the i.t. infection. Inoculum dose was confirmed by CFUs as described below.

Quantification of bacterial burden in lung and blood

Mice were euthanized by CO2 asphyxiation 24 h after P. aeruginosa inoculum with and without indomethacin treatment. The thoracic cavity was exposed and blood was collected by puncture of the right ventricle using syringes prelubricated with heparin. Lungs were suspended in 1 ml of normal saline and homogenized. Then, each specimen (lung and blood) was plated on blood agar plates using serial 10-fold dilutions and CFU/milliliter of blood or CFU/whole lung were determined 24 h later.

Statistical analysis

Statistical significance was analyzed using Prism 3.0 statistical program (GraphPad Software). Comparisons between two experimental groups were performed with Student’s t test. Comparisons among three or more experimental groups were performed with ANOVA and a post hoc Bonferroni’s test to determine significance. A value of p < 0.05 was considered significant


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
BMT mice exhibit elevated levels of PGE2 in vivo

PGE2 has been shown to be a negative regulator of host defense (23, 24, 25, 27, 28, 29, 30, 31, 32, 33). To determine whether BMT mice were characterized by elevated levels of PGE2 in vivo, we measured the amount of PGE2 in the BALF and the lung homogenates of control and BMT mice. BMT mice contained elevated levels of PGE2 in the lung when compared with control mice as determined by both approaches (Fig. 1). PGE2 overproduction post-BMT was likely a systemic phenomenon because increased levels of PGE2 were also noted in PLF from BMT mice (3.89 ± 0.2 ng/ml) vs control mice (1.25 ± 0.2 ng/ml) (n = 5, p < 0.01).


Figure 1
View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 1. BMT mice produce excess PGE2 in the lung microenvironment. Mice were given a syngeneic BMT and harvested 3 wk posttransplant. A, BALF was collected from both BMT and control mice at 3 wk post-BMT (p = 0.04, n = 4). B, Lung homogenates were obtained from control and BMT mice (p = 0.05, n = 4) at 3 wk post-BMT. BALF and lung homogenates were analyzed for PGE2 levels by specific EIA.

 
Lung cells from BMT mice demonstrate dysregulated eicosanoid synthesis in vitro compared with cells from control mice

AECs represent a major cellular source of PGE2 in the lung. To determine whether AECs might contribute to enhanced PGE2 production in the lung, we purified AECs from control and BMT mice and determined the amount of PGE2 produced constitutively. Purified AECs from BMT mice produced 7.64 ± 0.8 ng/ml PGE2, whereas AECs from control mice produced only 2.83 ± 0.2 ng/ml (n = 5; p < 0.001).

We next characterized the production of eicosanoids by resident AMs and recruited PMNs from control and BMT mice. AMs from BMT mice produced increased PGE2 at baseline and when maximally stimulated (Fig. 2A) and decreased cys-LTs with ionophore stimulation (B) compared with control AMs. The synthesis of LTB4 by control and BMT AMs was very low and was not significantly different (data not shown). BMT PMNs also produced increased amounts of PGE2 in vitro compared with control PMNs (Fig. 2C). Interestingly, PMNs from BMT mice produced increased cys-LT (Fig. 2D) and LTB4 (E) with Ca2+ ionophore stimulation compared with PMNs from control mice. Consistent with these observations, we noted that the mRNA for the PG synthetic enzyme, COX-2, was elevated 5.2-fold in AMs and 4.7-fold in PMNs post-BMT (data not shown). Similarly, 5-LO’s helper protein, 5-LO activating protein (FLAP), but not 5-LO mRNA, was decreased 2-fold in BMT AMs (data not shown). To determine whether other prostanoids were elevated, we measured production of 6-keto PGF1{alpha} as a marker of PGI2 synthesis. LPS-stimulated AMs from BMT mice produced 947.8 ± 281 pg/ml 6-keto PGF1{alpha} compared with 239.5 ± 30.6 pg/ml produced by control AMs (n = 3, p < 0.05). In LPS-stimulated PMN cultures, however, levels of 6-keto PGF1{alpha} were not elevated in BMT mice (data not shown). Thus, PGI2 and PGE2 production are both elevated post-BMT.


Figure 2
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 2. AMs and PMNs from BMT mice produce a dysregulated eicosanoid profile compared with control cells. Resident AMs were harvested from control and BMT mice by ex vivo lung lavage. PMNs from control and BMT mice were elicited 18 h after i.t. injection with 25 µg of P. aeruginosa-derived LPS. AMs and PMNs from control and BMT mice were enumerated and 0.5 x 106 AMs or PMNs per milliliter were cultured overnight in the presence or absence of LPS (A and C) or were cultured in the presence or absence of Ca2+ ionophore (A23187) for 1 h before supernatant harvest (B, D, and E). Supernatants were analyzed by specific EIA for PGE2, cys-LT, or LTB4 (*, p < 0.05; **, p < 0.01; ***, p < 0.001 with respect to control treatment; data are representative of three experiments with cells pooled from four mice each).

 
AMs and PMNs from BMT mice have altered PGE2 receptor expression

We next determined the expression levels of the inhibitory cAMP-coupled EP2 and EP4 receptors in AMs (Fig. 3A) and PMNs (B) from BMT and control mice. The profile of EP receptors on phagocytes (both AMs and PMNs) is EP2>EP1>EP4>EP3 when assessed by mRNA levels (data not shown). Semiquantitative real-time PCR analysis indicated that EP4 expression is decreased, but EP2 expression is 3.6-fold greater in BMT vs control AMs (Fig. 3A). Interestingly, in PMNs, EP2 was elevated 2.2-fold, whereas EP4 was increased 4.7-fold. The expression of cys-LT receptor 1 and 2 was similar in control and BMT phagocytes (data not shown). Thus, AMs and PMNs from BMT mice are characterized not only by increased PGE2 synthesis (Fig. 2) but also by altered EP2 and EP4 receptor expression. In particular, both cell types manifest increased expression of the primarily inhibitory receptor, EP2.


Figure 3
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 3. AMs and PMNs from BMT mice display elevated levels of EP2 and EP4 compared with control cells. Resident AMs and elicited PMNs were purified via lavage from control and BMT mice as described in Materials and Methods. RNA was prepared and analyzed by semiquantitative real-time PCR for the expression of EP2 and EP4 on AMs (A) and PMNs (B). For each sample, the expression level of the gene of interest was normalized to beta-actin. The average of the normalized control sample was set to 1 for each gene, and the relative expression of that gene in the BMT samples was measured by the {Delta}{Delta}CT method. The dotted line represents the expression of each gene in the control samples normalized to 1 (*, p < 0.05; **, p < 0.01; ***, p < 0.001 with respect to control cells; n = 5).

 
Impaired phagocytosis in BMT AMs is improved by augmenting LTs or inhibiting PGs

We previously demonstrated that BMT AMs display defective phagocytosis of unopsonized bacteria (11). We have confirmed these results (Fig. 4A) and extended them to demonstrate that BMT AMs are similarly defective in FcR-mediated phagocytosis of particles opsonized by IgG (B). Because activated BMT AMs were defective in cys-LT production, we tested the phagocytic ability of AMs in the presence of an exogenously added cys-LT. The addition of LTD4 to BMT AMs increased the phagocytosis of both unopsonized and opsonized targets by BMT AMs. This increase in phagocytosis was significant with respect to untreated BMT samples. Addition of LTD4 to control AMs also increased phagocytosis.


Figure 4
View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 4. Defective phagocytosis by BMT AMs was rescued in vitro with the addition of exogenous LTs or by inhibiting PG production. AMs were harvested from control or BMT mice and cultured at 2 x 105 cells/well. Pharmacologically, the eicosanoid balance was altered in the AMs by the presence or absence of 5 µM indomethacin, which blocks PG production, or the presence or absence of 10 nM LTD4. Non-Fc-mediated phagocytosis (A) or Fc-mediated phagocytosis (B) was assessed as described in Materials and Methods. AMs were next harvested from mice that had received BM from COX-2+/– deficient mice. AMs from COX-2+/– BMT mice that were unable to up-regulate the production of PGs, displayed improved phagocytosis compared with cells from a WT BMT (C) (*, p < 0.05; **, p < 0.01; ***, p < 0.001 with respect to untreated control AMs; n = 5).

 
In view of the excess PGE2 produced by BMT AMs (Fig. 2A), we were interested in whether pharmacologic or genetic blockade of PG synthesis would restore the phagocytic ability of BMT AMs. Indomethacin restored non-Fc-mediated phagocytosis by BMT AMs to untreated control levels (Fig. 4A), and augmented Fc-mediated phagocytosis to levels that were significantly greater than cells from untreated control mice (Fig. 4B). Similarly, pretreatment of control AMs with indomethacin was able to significantly enhance the phagocytic ability of these cells as well. Additionally, COX-2+/– mice were used as BM donors into wild-type (WT) recipients and the phagocytic ability of AMs from these chimeric mice were examined 3 wk post-BMT. As expected, we observed a defect in the ability of WT into WT BMT AMs to phagocytose opsonized targets compared with control AMs; however, the phagocytic ability of AMs from COX-2+/– into WT BMT chimeras was significantly improved (Fig. 4C).

PGE2 signaling through the EP2 receptor inhibits AM phagocytosis

In previous experiments, indomethacin was able to restore the phagocytic defect observed in AMs post-BMT (Fig. 4). We next sought to verify that the beneficial effects of indomethacin were mediated via blockade of PGE2 production and signaling via EP2. AMs were harvested from control and BMT mice and were treated with indomethacin and/or PGE2. Indomethacin was able to augment the phagocytic ability of both control and BMT AMs; however, the addition of 1 µM PGE2 completely abrogated the phagocytic ability of both groups (Fig. 5A). We next sought to determine whether the inhibitory signaling of PGE2 was mediated via EP2. We were unable to use EP2–/– mice to study this due to a compensatory up-regulation of EP4 on both AMs and PMNs in EP2–/– mice (data not shown). Thus, to avoid this confounding variable, we chose a pharmacologic approach to limit EP2 signaling through the use of a specific EP2 antagonist, AH-6809. The treatment of BMT AMs with AH-6809 resulted in increased phagocytic ability of these cells comparable to that of control AMs treated with vehicle alone (Fig. 5B). Collectively, these data indicate that indomethacin treatment restores phagocytosis primarily by interfering with synthesis of PGE2 and its signaling via EP2.


Figure 5
View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 5. Impaired phagocytosis in BMT AMs is related to PGE2 production, and EP2 signaling AMs were harvested from control and BMT mice and cultured at 2 x 105 cells/well. AMs were treated either in the presence or absence of 5 µM indomethacin for 30 min and/or 1 µM PGE2 for 10 min (A) or with a specific EP2 antagonist, AH-6809 (50 µM, 30 min) (B). Phagocytosis of unopsonized bacteria was assayed as previously described (*, p < 0.05; **, p < 0.01; ***, p < 0.001 with respect to untreated control AMs; n = 4).

 
AMs and PMNs from BMT mice exhibit defective killing of P. aeruginosa in vitro

To test whether AMs and PMNs from BMT mice were deficient in their bactericidal activity, we examined killing of ingested unopsonized (Fig. 6, A and C) and opsonized (B and D) P. aeruginosa in vitro. BMT AMs were equally deficient in their ability to kill both unopsonized (Fig. 6A) and opsonized (B) P. aeruginosa in vitro. Despite the intact phagocytic capacity of BMT PMNs (11), these cells were unable to kill either unopsonized (Fig. 6C) or opsonized (D) P. aeruginosa in vitro as well as control untreated PMNs. In all BMT cells, the addition of 5 µM indomethacin decreased the amount of surviving intracellular bacteria, which reflects increased killing, in BMT AMs and PMNs (Fig. 6). Surprisingly, there was no increase in the bactericidal killing of control AMs or PMNs with indomethacin treatment.


Figure 6
View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 6. Both AMs and PMNs post-BMT are defective in their ability to kill ingested P. aeruginosa in vitro compared with control cells. Resident AMs and elicited lung PMNs were harvested and cultured in duplicate plates at 2 x 105 cells/well in the presence or absence of 5 µM indomethacin for 30 min. Unopsonized (A and C) or opsonized (B and D) P. aeruginosa was added to the wells, and the cells were allowed to ingest the bacteria. The amount of bacteria surviving at the end of the assay was assessed as described in Materials and Methods. Note that an increase in the percentage of bacteria surviving correlates with impaired phagocyte killing (*, p < 0.05; ***, p < 0.001, with respect to untreated control cells; n = 8).

 
Inhibition of PGs in vivo normalizes eicosanoid synthesis and pulmonary clearance of P. aeruginosa infection in BMT mice

We next sought to determine whether blockade of PG synthesis by a COX inhibitor altered the susceptibility of BMT mice to P. aeruginosa in vivo. BMT mice were given an i.p. injection of either vehicle or 1.2 mg/kg of indomethacin at the same time that they were given an i.t. inoculum of P. aeruginosa (3.3 x 106 CFU/ml; 6.5 log10 scale). Twenty-four hours later, whole lungs were removed and homogenized to examine eicosanoid levels after indomethacin treatment. PGE2 levels were significantly higher in infected BMT mice than control animals. We also identified a significant reduction in the cys-LT levels of infected BMT mice compared with controls. The lung homogenates from BMT mice treated with indomethacin exhibited decreased amounts of PGE2 (Fig. 7A) and increased cys-LT (B) compared with vehicle-treated BMT mice, and were no longer statistically different from control mice. Twenty-four hours after i.t. infection, whole lung and blood were collected from the mice to measure bacterial burden and dissemination. As expected, BMT mice were more susceptible than control mice to an i.t. infection of P. aeruginosa, as shown by increased bacterial burden in the lung (Fig. 7C) and increased dissemination into the blood of BMT mice (D). Administration of indomethacin restored the innate pulmonary clearance mechanisms of the BMT mice, as evidenced by reductions in both local lung bacterial burden and bacterial dissemination to the blood. Similarly, when PG-deficient COX-2+/– into WT BMT mice were examined for in vivo host defense, we noted a trend of ~20% improvement in lung CFU when compared with WT into WT BMT mice (data not shown). These results demonstrate that reductions in donor leukocyte-derived PGE2 alone can help to restore the innate immune defects seen in vivo post-BMT. As predicted, the improvement in BMT mice treated with indomethacin is greater than that seen in the COX-2+/– into WT BMT mice because both donor and host cells, as well as both COX-1 and COX-2 enzymes, are targeted by the indomethacin treatment.


Figure 7
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 7. Indomethacin treatment restores host defense capabilities of BMT mice in vivo. Control or BMT mice were injected i.t. with P. aeruginosa (3.3 x 106 CFU/ml; 6.5 log10 scale). At the same time as the P. aeruginosa infection, one cohort of BMT mice was also treated with 1.2 mg/kg indomethacin while the other was treated with vehicle control (0.01% DMSO in PBS). Twenty-four hours later, the animals were euthanized, and lungs and blood were collected for eicosanoid measurements and CFU analysis. Treatment with indomethacin decreased the PGE2 production (A) and increased the cys-LT production (B) in the lungs of mice post-BMT. In addition, there was decreased bacterial burden in the lungs (C) and decreased bacterial dissemination into the blood (D) with indomethacin treatment. Thus, indomethacin treatment was effective at restoring the clearance ability of BMT mice to levels equivalent to or better than control mice. Data are from one experiment, representative of two (*, p < 0.05; ***, p < 0.001, with respect to control mice; n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our results in a murine model provide mechanistic insight into the enhanced susceptibility to infection that is noted post-BMT in patients despite hemopoietic reconstitution. We demonstrate that 1) PGE2 synthesis is increased in both donor-derived leukocytes and resident host cell types following BMT, 2) EP2/EP4 receptors are elevated on donor-derived phagocytes post-BMT, 3) the increased PGE2 production and signaling in phagocytes post-BMT is critical to impaired phagocytosis and killing of Gram-negative bacteria, and 4) pharmacologic and genetic strategies to block PG production lead to improved host defense post-BMT in vivo (summarized in Table II). These results are relevant to human BMT, because elevated levels of systemic PGE2 have been noted previously in patients undergoing stem cell transplants (35). However, neither the biological significance of this finding nor its applicability to the lung had previously been determined.


View this table:
[in this window]
[in a new window]
  		Table II. Summary of differences in lung phagocytes pre- and post-BMT

 
Our data clearly demonstrate that eicosanoid dysregulations occur post-BMT. We have shown that PGE2 levels are elevated in the alveolar spaces (Fig. 1) post-BMT. This increase represents elevated PGE2 production both by donor-derived AMs (Fig. 2A) and PMNs (C), and also by resident lung cell types such as AECs. Our conditioning regimen employs total body irradiation, and thus it is possible that resident lung cells up-regulate PGE2 production as a consequence of exposure to ionizing radiation (47). However, the mechanisms responsible for elevated PGE2 production in the donor leukocytes are less clear. Of note, PGI2 production is also elevated in AMs post-BMT, suggesting that other prostanoids may be similarly dysregulated. Although both AMs and PMNs display increased COX-2 mRNA post-BMT, the ultimate regulation of eicosanoid synthesis may be cell-type specific. The eicosanoid profile of AMs post-BMT is characterized by both elevated PGE2 (Fig. 2A) and PGI2 and decreased cys-LT production (B). Consistent with these observations, FLAP expression is diminished by one-half in AMs post-BMT. In contrast, PMNs post-BMT display increased generation of PGE2 and LTs (Fig. 2, C–E). The fact that AMs from PG-deficient COX-2+/– BMT mice showed improved host defense when compared with AMs from control BMT mice suggests that leukocyte-derived PG production is a critical regulator of host defense post-BMT. Further evidence in support of this hypothesis is that the restoration of AM phagocytosis by indomethacin is abrogated by the addition of PGE2 in vitro (Fig. 5A).

Although there were alterations in both LTs and PGs post-BMT that might exert an immunosuppressive effect, the host defense defects are more likely attributable to the global increase in PGE2 synthesis. In support of this hypothesis, 1) although LT production was decreased only in AMs, PGE2 synthesis was increased in both AMs and PMNs post-BMT; 2) PGE2 levels in lung homogenates postinfection in BMT mice were 700-fold greater than the corresponding levels of cys-LTs (Fig. 7); 3) expression of the inhibitory PGE2 signaling receptors (EP2 and/or EP4) was elevated on AMs and PMNs post-BMT compared with cells from control mice, whereas expression of LT receptors was not different on AMs or PMNs post-BMT; 4) an EP2 receptor antagonist, AH-6809, augmented BMT AM phagocytosis in vitro; and 5) exogenous addition of PGE2 ameliorated the beneficial effects of indomethacin on AM phagocytosis. Additionally, our results demonstrate that PGE2 is produced at much greater levels than PGI2 by AMs post-BMT. Collectively, these results strongly implicate the PGE2-EP2 axis as the major inhibitor of innate immune function post-BMT. However, the fact that indomethacin is slightly more potent in vitro than AH-6809 suggests that additional PG products (such as PGI2), additional receptors (such as EP4), or indomethacin-induced increases in LT production may play some role as well.

Elevated PGE2 production in AMs was associated with both impaired phagocytosis (Fig. 4) and bactericidal activity (Fig. 6), whereas in PMNs, elevated PGE2 levels were only associated with impaired bacterial killing (Fig. 6). We previously demonstrated that PMN phagocytosis post-BMT was normal (11). Thus, this discrepancy may reflect differential PGE2 signaling in these two cell types. EP2 expression was elevated on both AMs and PMNs post-BMT, whereas EP4 expression was only elevated on PMNs post-BMT (Fig. 3). Although both EP2 and EP4 are Gs-coupled receptors known to signal by increasing cAMP levels, the intracellular coupling of these receptor signaling pathways may be cell type specific. Alternatively, we have shown that PGE2-mediated inhibition of phagocytosis in AMs via EP2 involves activation of the guanine exchange protein activated by adenyl cyclase (EPAC-1), whereas bactericidal killing in AMs is mediated through activation of both EPAC and protein kinase A (24, 25). Thus, a second formal possibility for differential effects of PGE2 on phagocyte function post-BMT may involve differential expression or activation of EPAC and protein kinase A. Studies to differentiate between these possibilities are currently underway. Finally, the PGI2 receptor, IP, is also a Gs-coupled receptor that signals via elevations in cAMP. It is possible that elevated levels of PGI2 post-BMT or IP receptor signaling may differentially affect AM and PMN functions.

Our data demonstrate that indomethacin treatment can restore or enhance both phagocytic and killing function of AMs and PMNs from BMT mice. Interestingly, however, indomethacin can augment phagocytosis by control AMs as well. This result verifies earlier work demonstrating that endogenous production of PGE2 by AMs can negatively influence phagocytosis (25). This may reflect the ability of indomethacin to not only inhibit PG production, but to stimulate LT production. Surprisingly, indomethacin has no apparent effect on bacterial killing in control phagocytes (Fig. 6), which is in agreement with earlier work as well (48). The reasons for this discrepancy between phagocytosis and killing are unclear to us, and may simply reflect the fact that the phagocytosis assays are more sensitive than the bacterial killing assays.

Eicosanoid imbalances have been noted previously in a variety of situations associated with increased susceptibility to pneumonia. For example, impaired LT synthesis has been noted in HIV infection, cigarette smoking, diabetes mellitus, malnutrition, and neonates (13). Moreover, increased COX-2 expression and PGE2 production has been noted in cancer, aging, and stem cell transplant recipients (35, 49, 50, 51). Importantly, PG blockade has been shown to improve host defense in models of sepsis (52, 53) and pneumonia (54, 55). In the clinical setting, Klingemann et al. (56) demonstrated a role for PGE2 in the suppression of lymphocyte function observed in human stem cell transplant recipients, which was reversible in vitro by pharmacological COX inhibition. It was also shown that the direct application of PGE2 to the oral mucosa of patients after transplantation increased the risk of reactivating latent herpes simplex virus infections (57). We have now clearly demonstrated that both donor and host cells contribute to increased PGE2 post-BMT and that pharmacologic and genetic blockade of PG production post-BMT improves outcomes in response to infections.

Our findings have two important implications. First, from a therapeutic standpoint, COX inhibitors might be used in patients post-BMT to ameliorate their immune dysfunction. In this regard, oral administration of the omega-3 fatty acid eicosapentaenoic acid to BMT patients inhibited leukocyte PG production and improved their survival (58). Second, our results demonstrate a need for caution when using BMT to generate chimeric mice for a variety of experimental purposes. Given the wide range of effects that eicosanoids can have on virtually every biological system, investigators should be aware that systemic eicosanoid abnormalities associated with this procedure may confound many experimental results.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by National Institutes of Health Grants HL071586 (to B.B.M.), P50HL56402 (to B.B.M., G.B.T., and M.P.-G.), HL078727 (to D.M.A.), and RG8909N (to D.M.A.), and a Career Investigator Award (to B.B.M.) from the American Lung Association of Michigan. Back

2 Address correspondence and reprint requests to Dr. Bethany B. Moore, Internal Medicine/Pulmonary and Critical Care Medicine, 4062 Biomedical Science Research Building, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200. E-mail address: Bmoore{at}umich.edu Back

3 Abbreviations used in this paper: BMT, bone marrow transplantation; BM, bone marrow; AM, alveolar macrophage; PMN, polymorphonuclear leukocyte; AA, arachidonic acid; COX, cyclooxygenase; 5-LO, 5-lipoxygenase; FLAP, 5-LO activating protein; LT, leukotriene; EP, E prostanoid; BALF, bronchoalveolar lavage fluid; PLF, peritoneal lavage fluid; AEC, alveolar epithelial cell; EIA, enzyme immunoassay; WT, wild type; i.t., intratracheal. Back

Received for publication April 28, 2006. Accepted for publication August 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Jacquet, E. G., C. L. Schanie, I. Fugier-Vivier, S. S. Willer, S. T. Ildstad. 2003. Facilitating cells as a venue to establish mixed chimerism and tolerance. Pediatr. Transplant. 7: 348-357. [Medline]
  2. Reddy, P., J. L. Ferrara. 2003. Immunobiology of acute graft-versus-host disease. Blood Rev. 17: 187-194. [Medline]
  3. Leiper, A. D.. 2002. Non-endocrine late complications of bone marrow transplantation in childhood: part I. Br. J. Haematol. 118: 3-22. [Medline]
  4. Kotloff, R. M., V. N. Ahya, S. W. Crawford. 2004. Pulmonary complications of solid organ and hematopoietic stem cell transplantation. Am. J. Respir. Crit. Care Med. 170: 22-48. [Abstract/Free Full Text]
  5. Collin, B. A., H. L. Leather, J. R. Wingard, R. Ramphal. 2001. Evolution, incidence, and susceptibility of bacterial bloodstream isolates from 519 bone marrow transplant patients. Clin. Infect. Dis. 33: 947-953. [Medline]
  6. Krowka, M. J., E. C. Rosenow, III, H. C. Hoagland. 1985. Pulmonary complications of bone marrow transplantation. Chest 87: 237-246. [Medline]
  7. Winston, D. J.. 1993. Prophylaxis and treatment of infection in the bone marrow transplant recipient. Curr. Clin. Top. Infect. Dis. 13: 293-321. [Medline]
  8. Auletta, J. J., H. M. Lazarus. 2005. Immune restoration following hematopoietic stem cell transplantation: an evolving target. Bone Marrow Transplant. 35: 835-857. [Medline]
  9. Ballinger, M. N., R. Paine, III, C. H. Serezani, D. M. Aronoff, E. S. Choi, T. J. Standiford, G. B. Toews, B. B. Moore. 2006. Role of GM-CSF during Gram-negative lung infection with Pseudomonas aeruginosa. Am. J. Respir. Cell Mol. Biol. 34: 766-774. [Abstract/Free Full Text]
  10. Delclaux, C., E. Azoulay. 2003. Inflammatory response to infectious pulmonary injury. Eur. Respir. J. Suppl. 42: 10s-14s.
  11. Ojielo, C. I., K. Cooke, P. Mancuso, T. J. Standiford, K. M. Olkiewicz, S. Clouthier, L. Corrion, M. N. Ballinger, G. B. Toews, R. Paine, III, B. B. Moore. 2003. Defective phagocytosis and clearance of Pseudomonas aeruginosa in the lung following bone marrow transplantation. J. Immunol. 171: 4416-4424. [Abstract/Free Full Text]
  12. Funk, C. D.. 2001. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 294: 1871-1875. [Abstract/Free Full Text]
  13. Peters-Golden, M., C. Canetti, P. Mancuso, M. J. Coffey. 2005. Leukotrienes: underappreciated mediators of innate immune responses. J. Immunol. 174: 589-594. [Abstract/Free Full Text]
  14. Bailie, M. B., T. J. Standiford, L. L. Laichalk, M. J. Coffey, R. Strieter, M. Peters-Golden. 1996. Leukotriene-deficient mice manifest enhanced lethality from Klebsiella pneumonia in association with decreased alveolar macrophage phagocytic and bactericidal activities. J. Immunol. 157: 5221-5224. [Abstract]
  15. Mancuso, P., T. J. Standiford, T. Marshall, M. Peters-Golden. 1998. 5-Lipoxygenase reaction products modulate alveolar macrophage phagocytosis of Klebsiella pneumoniae. Infect. Immun. 66: 5140-5146. [Abstract/Free Full Text]
  16. Serezani, C. H., D. M. Aronoff, S. Jancar, P. Mancuso, M. Peters-Golden. 2005. Leukotrienes enhance the bactericidal activity of alveolar macrophages against Klebsiella pneumoniae through the activation of NADPH oxidase. Blood 106: 1067-1075. [Abstract/Free Full Text]
  17. Mancuso, P., P. Nana-Sinkam, M. Peters-Golden. 2001. Leukotriene B4 augments neutrophil phagocytosis of Klebsiella pneumoniae. Infect. Immun. 69: 2011-2016. [Abstract/Free Full Text]
  18. Coffey, M. J., S. M. Phare, M. Peters-Golden. 2004. Role of leukotrienes in killing of Mycobacterium bovis by neutrophils. Prostaglandins Leukot. Essent. Fatty Acids 71: 185-190. [Medline]
  19. Coffey, M. J., S. M. Phare, S. George, M. Peters-Golden, P. H. Kazanjian. 1998. Granulocyte colony-stimulating factor administration to HIV-infected subjects augments reduced leukotriene synthesis and anticryptococcal activity in neutrophils. J. Clin. Invest. 102: 663-670. [Medline]
  20. Menard, G., E. Y. Bissonnette. 2000. Priming of alveolar macrophages by leukotriene D4: potentiation of inflammation. Am. J. Respir. Cell Mol. Biol. 23: 572-577. [Abstract/Free Full Text]
  21. Sayers, T. J., T. A. Wiltrout, C. A. Bull, A. C. Denn, III, A. M. Pilaro, B. Lokesh. 1988. Effect of cytokines on polymorphonuclear neutrophil infiltration in the mouse: prostaglandin- and leukotriene-independent induction of infiltration by IL-1 and tumor necrosis factor. J. Immunol. 141: 1670-1677. [Abstract]
  22. Tarlowe, M. H., K. B. Kannan, K. Itagaki, J. M. Adams, D. H. Livingston, C. J. Hauser. 2003. Inflammatory chemoreceptor cross-talk suppresses leukotriene B4 receptor 1-mediated neutrophil calcium mobilization and chemotaxis after trauma. J. Immunol. 171: 2066-2073. [Abstract/Free Full Text]
  23. Armstrong, R. A.. 1995. Investigation of the inhibitory effects of PGE2 and selective EP agonists on chemotaxis of human neutrophils. Br. J. Pharmacol. 116: 2903-2908. [Medline]
  24. Aronoff, D. M., C. Canetti, C. H. Serezani, M. Luo, M. Peters-Golden. 2005. Cutting edge: macrophage inhibition by cyclic AMP (cAMP): differential roles of protein kinase A and exchange protein directly activated by cAMP-1. J. Immunol. 174: 595-599. [Abstract/Free Full Text]
  25. Aronoff, D. M., C. Canetti, M. Peters-Golden. 2004. Prostaglandin E2 inhibits alveolar macrophage phagocytosis through an E-prostanoid 2 receptor-mediated increase in intracellular cyclic AMP. J. Immunol. 173: 559-565. [Abstract/Free Full Text]
  26. He, L. K., L. H. Liu, E. Hahn, R. L. Gamelli. 2001. The expression of cyclooxygenase and the production of prostaglandin E2 in neutrophils after burn injury and infection. J. Burn Care Rehabil. 22: 58-64. [Medline]
  27. McLeish, K. R., G. T. Stelzer, J. H. Wallace. 1987. Regulation of oxygen radical release from murine peritoneal macrophages by pharmacologic doses of PGE2. Free Radic. Biol. Med. 3: 15-20. [Medline]
  28. Elliott, G. R., A. P. Lauwen, I. L. Bonta. 1989. Prostaglandin E2 inhibits and indomethacin and aspirin enhance, A23187-stimulated leukotriene B4 synthesis by rat peritoneal macrophages. Br. J. Pharmacol. 96: 265-270.
  29. Christman, B. W., J. W. Christman, R. Dworski, I. A. Blair, C. Prakash. 1993. Prostaglandin E2 limits arachidonic acid availability and inhibits leukotriene B4 synthesis in rat alveolar macrophages by a nonphospholipase A2 mechanism. J. Immunol. 151: 2096-2104. [Abstract]
  30. Caggiano, A. O., R. P. Kraig. 1999. Prostaglandin E receptor subtypes in cultured rat microglia and their role in reducing lipopolysaccharide-induced interleukin-1beta production. J. Neurochem. 72: 565-575. [Medline]
  31. Kunkel, S. L., M. Spengler, M. A. May, R. Spengler, J. Larrick, D. Remick. 1988. Prostaglandin E2 regulates macrophage-derived tumor necrosis factor gene expression. J. Biol. Chem. 263: 5380-5384. [Abstract/Free Full Text]
  32. Iwasaki, K., K. Noguchi, H. Endo, H. Kondo, I. Ishikawa. 2003. Prostaglandin E2 downregulates interleukin-12 production through EP4 receptors in human monocytes stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans and interferon-{gamma}. Oral Microbiol. Immunol. 18: 150-155. [Medline]
  33. Harizi, H., M. Juzan, V. Pitard, J. F. Moreau, N. Gualde. 2002. Cyclooxygenase-2-issued prostaglandin E2 enhances the production of endogenous IL-10, which down-regulates dendritic cell functions. J. Immunol. 168: 2255-2263. [Abstract/Free Full Text]
  34. Narumiya, S., Y. Sugimoto, F. Ushikubi. 1999. Prostanoid receptors: structures, properties, and functions. Physiol. Rev. 79: 1193-1226. [Abstract/Free Full Text]
  35. Cayeux, S. J., P. C. Beverley, R. Schulz, B. Dorken. 1993. Elevated plasma prostaglandin E2 levels found in 14 patients undergoing autologous bone marrow or stem cell transplantation. Bone Marrow Transplant. 12: 603-608. [Medline]
  36. Hodges, R. J., R. G. Jenkins, C. P. Wheeler-Jones, D. M. Copeman, S. E. Bottoms, G. J. Bellingan, C. B. Nanthakumar, G. J. Laurent, S. L. Hart, M. L. Foster, R. J. McAnulty. 2004. Severity of lung injury in cyclooxygenase-2-deficient mice is dependent on reduced prostaglandin E2 production. Am. J. Pathol. 165: 1663-1676. [Abstract/Free Full Text]
  37. Cooke, K. R., W. Krenger, G. Hill, T. R. Martin, L. Kobzik, J. Brewer, R. Simmons, J. M. Crawford, M. R. van den Brink, J. L. Ferrara. 1998. Host reactive donor T cells are associated with lung injury after experimental allogeneic bone marrow transplantation. Blood 92: 2571-2580. [Abstract/Free Full Text]
  38. Down, J. D., P. Mauch, M. Warhol, S. Neben, J. L. Ferrara. 1992. The effect of donor T lymphocytes and total-body irradiation on hemopoietic engraftment and pulmonary toxicity following experimental allogeneic bone marrow transplantation. Transplantation 54: 802-808. [Medline]
  39. Charbeneau, R. P., P. J. Christensen, C. J. Chrisman, R. Paine, III, G. B. Toews, M. Peters-Golden, B. B. Moore. 2003. Impaired synthesis of prostaglandin E2 by lung fibroblasts and alveolar epithelial cells from GM-CSF–/– mice: implications for fibroproliferation. Am. J. Physiol. 284: L1103-L1111.
  40. Corti, M., A. R. Brody, J. H. Harrison. 1996. Isolation and primary culture of murine alveolar type II cells. Am. J. Respir. Cell Mol. Biol. 14: 309-315. [Abstract]
  41. Peters-Golden, M., P. Thebert. 1987. Inhibition by methylprednisolone of zymosan-induced leukotriene synthesis in alveolar macrophages. Am. Rev. Respir. Dis. 135: 1020-1026. [Medline]
  42. Smacchia, C., P. Rebulla, F. Drago, F. Morelati, M. Pappalettera, G. Sirchia. 1997. A micro colorimetric assay using cryopreserved monocytes to evaluate antibody-mediated red cell-monocyte interaction. Haematologica 82: 526-531. [Abstract/Free Full Text]
  43. Araki, N., M. T. Johnson, J. A. Swanson. 1996. A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. J. Cell Biol. 135: 1249-1260. [Abstract/Free Full Text]
  44. Peck, R.. 1985. A one-plate assay for macrophage bactericidal activity. J. Immunol. Methods 82: 131-140. [Medline]
  45. Fink, L., W. Seeger, L. Ermert, J. Hanze, U. Stahl, F. Grimminger, W. Kummer, R. M. Bohle. 1998. Real-time quantitative RT-PCR after laser-assisted cell picking. Nat. Med. 4: 1329-1333. [Medline]
  46. Moore, B. B., M. J. Coffey, P. Christensen, S. Sitterding, R. Ngan, C. A. Wilke, R. McDonald, S. M. Phare, M. Peters-Golden, R. Paine, III, G. B. Toews. 2000. GM-CSF regulates bleomycin-induced pulmonary fibrosis via a prostaglandin-dependent mechanism. J. Immunol. 165: 4032-4039. [Abstract/Free Full Text]
  47. Tessner, T. G., F. Muhale, S. Schloemann, S. M. Cohn, A. R. Morrison, W. F. Stenson. 2004. Ionizing radiation up-regulates cyclooxygenase-2 in I407 cells through p38 mitogen-activated protein kinase. Carcinogenesis 25: 37-45. [Abstract/Free Full Text]
  48. Laegreid, W., H. Liggitt, R. Silflow, J. Evermann, S. Taylor, R. Leid. 1989. Reversal of virus-induced alveolar macrophage bactericidal dysfunction by cyclooxygenase inhibition in vitro. J. Leukocyte Biol. 45: 293-300. [Abstract]
  49. Seo, J. Y., E. K. Kim, S. H. Lee, K. C. Park, K. H. Kim, H. C. Eun, J. H. Chung. 2003. Enhanced expression of cylooxygenase-2 by UV in aged human skin in vivo. Mech. Ageing Dev. 124: 903-910. [Medline]
  50. Davies, G., L. A. Martin, N. Sacks, M. Dowsett. 2002. Cyclooxygenase-2 (COX-2), aromatase and breast cancer: a possible role for COX-2 inhibitors in breast cancer chemoprevention. Ann. Oncol. 13: 669-678. [Abstract/Free Full Text]
  51. Gately, S., W. W. Li. 2004. Multiple roles of COX-2 in tumor angiogenesis: a target for antiangiogenic therapy. Semin. Oncol. 31: 2-11. [Medline]
  52. Reddy, R. C., G. H. Chen, K. Tateda, W. C. Tsai, S. M. Phare, P. Mancuso, M. Peters-Golden, T. J. Standiford. 2001. Selective inhibition of COX-2 improves early survival in murine endotoxemia but not in bacterial peritonitis. Am. J. Physiol. 281: L537-L543.
  53. Strong, V. E., P. J. Mackrell, E. M. Concannon, H. A. Naama, P. A. Schaefer, G. W. Shaftan, P. P. Stapleton, J. M. Daly. 2000. Blocking prostaglandin E2 after trauma attenuates pro-inflammatory cytokines and improves survival. Shock 14: 374-379. [Medline]
  54. Fontan, P. A., C. R. Amura, D. O. Sordelli. 1993. The effect of a cyclooxygenase inhibitor on the in vivo polymorphonuclear leukocyte migration to Pseudomonas aeruginosa peptide chemotactins. Agents Actions 40: 106-109. [Medline]
  55. Sordelli, D. O., M. C. Cerquetti, P. A. Fontan, R. P. Meiss. 1989. Piroxicam treatment protects mice from lethal pulmonary challenge with Pseudomonas aeruginosa. J. Infect. Dis. 159: 232-238. [Medline]
  56. Klingemann, H. G., M. S. Tsoi, R. Storb. 1986. Inhibition of prostaglandin E2 restores defective lymphocyte proliferation and cell-mediated lympholysis in recipients after allogeneic marrow grafting. Blood 68: 102-107. [Abstract/Free Full Text]
  57. Labar, B., M. Mrsic, Z. Pavletic, V. Bogdanic, D. Nemet, I. Aurer, I. Radman, N. Filipovic-Grcic, D. Sertic, S. Kalenic, et al 1993. Prostaglandin E2 for prophylaxis of oral mucositis following BMT. Bone Marrow Transplant. 11: 379-382. [Medline]
  58. Takatsuka, H., Y. Takemoto, N. Iwata, A. Suehiro, T. Hamano, T. Okamoto, A. Kanamaru, E. Kakishita. 2001. Oral eicosapentaenoic acid for complications of bone marrow transplantation. Bone Marrow Transplant. 28: 769-774. [Medline]



This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
C. H. Serezani, M. N. Ballinger, D. M. Aronoff, and M. Peters-Golden
Cyclic AMP: Master Regulator of Innate Immune Cell Function
Am. J. Respir. Cell Mol. Biol., August 1, 2008; 39(2): 127 - 132.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
M. N. Ballinger, L. L. N. Hubbard, T. R. McMillan, G. B. Toews, M. Peters-Golden, R. Paine III, and B. B. Moore
Paradoxical role of alveolar macrophage-derived granulocyte-macrophage colony-stimulating factor in pulmonary host defense post-bone marrow transplantation
Am J Physiol Lung Cell Mol Physiol, July 1, 2008; 295(1): L114 - L122.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. M. Aronoff, Y. Hao, J. Chung, N. Coleman, C. Lewis, C. M. Peres, C. H. Serezani, G.-H. Chen, N. Flamand, T. G. Brock, et al.
Misoprostol Impairs Female Reproductive Tract Innate Immunity against Clostridium sordellii
J. Immunol., June 15, 2008; 180(12): 8222 - 8230.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
T. R. McMillan, B. B. Moore, J. B. Weinberg, K. M. Vannella, W. B. Fields, P. J. Christensen, L. F. van Dyk, and G. B. Toews
Exacerbation of Established Pulmonary Fibrosis in a Murine Model by Gammaherpesvirus
Am. J. Respir. Crit. Care Med., April 1, 2008; 177(7): 771 - 780.
[Abstract] [Full Text] [PDF]

Home page