Endotoxin-free buffers and reagents were scrupulously employed for all experiments. Saccharomyces cerevisiae derived cell wall β-glucans, the calcineurin disrupting agents TEMPO (2,2,6,6-Tetramethyl-1-piperidinyloxy, free radical, 2,2,6,6-Tetramethylpiperidine 1-oxyl) and cyclosporin B were purchased from Sigma Chemical Co, (St. Louis, MO). The calcium chelator BAPTA/AM (1,2-bis-(o-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester) was obtained from Alexis Biochemical. The glucosylceramide synthase inhibitor PDMP (D-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol•HCl) was purchased from Matreya, LLC (Pleasant Gap, PA), LPS from Escherichia coli 026:B6, EGTA, PD 98059, SB 202190, SB 202474, JNK inhibitor II and other general reagents were from Calbiochem (Gibbstown, NJ), unless otherwise specified. Pneumocystis carinii was derived originally from the American Type Culture Collection stock (Manassas, VA) and has been passaged though our immunosuppressed rat colony 25. All antibodies employed in these studies were purchased from Cell Signaling Technologies (Danvers, MA). The human airway epithelial cell line, 1HAEo^- cells, were generously provided by Dr. Dieter Gruenert (University of California, San Francisco) 26. The cells were routinely cultured in Modified Eagle's medium containing 10% fetal bovine serum and 2 mM L-glutamine, penicillin 10,000 units/liter, and streptomycin 1 mg/liter.

The NF-κB-dependent firefly luciferase reporter expression vector (κB-luc) was a kind gift of Dr. Carlos Paya (Mayo Clinic, Rochester, MN)27. The IL-8, IL-8 mutated in AP-1, and NF-κB sites promoter-luciferase reporter plasmids were gifts from Dr. Marc Hershenson (University of Michigan)28. The pRL-TK expression vector, which provides constitutive expression of Renilla luciferase, was purchased from Promega (Madison, Wisconsin).

The Mayo Institutional Animal Care and Usage Committee approved all animal experimentation. A β-glucan-rich cell wall fraction from P. carinii was prepared as we previously described 1118. Pneumocystis pneumonia was induced in dexamethasone-treated immunosuppressed Lewis rats (Harlan, Inc., Indianapolis, IN) 25. Pneumocystis organisms were isolated from lungs of heavily infected animals by homogenization and filtration through 10-μm filters. The organisms were autoclaved (120°C, 20 min) and disrupted by ultrasonication (200 W for 3 min, six times), and the glucans were isolated by NaOH digestion and lipid extraction as previously detailed 1118. As we prior reported, the final product contained predominantly carbohydrate (95.7%) and released 82% of its content as D-glucose following hydrolysis 11. Extensive measures were employed to ensure that the fractions were free of endotoxin. Prior to use in culture, the Pneumocystis cell wall fractions were washed with 0.1% SDS and then vigorously washed with distilled physiological saline to remove the detergent. The final preparation was assayed for endotoxin with the Limulus amebocyte lysate assay method and found to consistently contain < 0.125 units of endotoxin 11.

IL-8 was measured in the supernatants of cultivated 1HAEo^- cells by ELISA (BD OptEIA™, BD biosciences, San Diego, CA). Cells were cultured to ~70% confluence in a 96-well plates. Prior to activation with PCBG, the cells were weaned from serum for 18 hours. For some experiments, the cells were preincubated with various calcium disrupting agents or MAPKs inhibitors for one hour prior to stimulation. Supernatant was collected after 8 hour of stimulation with PCBG unless otherwise indicated and stored at -70°C. All experiments were performed in duplicate and repeated on a minimum of at least three occasions.

Cell viability was confirmed using the XTT Cell Proliferation Kit II (Roche Molecular Biochemicals, Mannheim, Germany). This assay measures the conversion of sodium-3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) to a formazan dye through electron coupling in metabolically active mitochondria using the coupling reagent N-methyldibenzopyrazine methyl sulfate. Only metabolically active cells are capable of mediating this reaction, which is detected by absorbance of the dye at 450-500 nm. Greater than 80% survival was considered acceptable cellular viability in all the experiments.

To measure intracellular Ca²⁺ fluxes, cells were plated in 8 well borosilicate coverglass chambers and were incubated with 5 μM Fura-2AM (acetoxy-methyl-2-[5-[bis[(acetoxymethoxy-oxomethyl)methyl]amino]-4-[2-[2-[bis[(acetoxymethoxy-oxo methyl)methyl]amino]-5-methyl-phenoxy]-ethoxy]benzofuran-2-yl]oxazole-5-carboxylate, a calcium imaging dye that binds to free Ca²⁺ in HBSS (Hanks balanced salt solution with 2.25 mM CaCl₂, 0.8 mM MgSO₄ and 12 mM glucose; pH 7.4) for 60 minutes at room temperature. Cells were then washed twice with fresh HBSS and subsequently maintained in HBSS. Cells were continuously perfused during the acquisition of Ca²⁺ measurements. Fluorescence excitation, image acquisition, and Ca²⁺ data analyses were controlled using a dedicated video fluorescence imaging system (Metafluor; Universal Imaging Corporation). Cells were imaged using an inverted Nikon Diaphot microscope equipped with a Nikon Fluor X20 objective lens. Fura 2-loaded cells were alternately excited at 340 and 380 nm using a Lambda 10-2 filter changer (Sutter Instrument Company). Fluorescence emissions were collected separately for each wavelength using a 510 nm barrier filter. Images were acquired using a Micromax 12 bit camera system (Princeton Instruments) approximately every 0.75 seconds. Intracellular Ca²⁺ concentrations were calculated from the ratio of intensities at 340 nm and 380 nm, by extrapolation from a calibration curve as previously described 29. For a positive control of intracellular calcium release, cells were stimulated in parallel with PAR-2 Peptide (Anaspec, San Jose, Ca (Protease activated receptor - 2)) at a final concentration of 100 μM.

To obtain total cellular proteins, cells were washed with cold phosphate-buffered saline (PBS) twice and lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 15 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA) freshly supplemented with 2 μM phenylmethylsulfonyl fluoride [PMSF], 10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 10 mM NaF and 300 μM Na orthovanadate. Cell lysates were centrifuged at 12,000 × g for 1 min at 4°C. The resultant supernatant contained total cellular protein. Protein concentrations in the clarified supernatants were determined using the Bio-Rad (Hercules, Calif.) protein assay. For Western immunoblotting, equal amounts of total cellular proteins were separated by 10% SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bedford, Mass.). Immunoblotting was performed with specific antibodies and visualized using the ECL enhanced chemiluminescence Western blotting detection kit (Amersham, Buckinghamshire, England). Densitometry analysis of the Immunoblots was performed using the computer program ImageJ 1.42d, National Institutes of Health, USA. The data was expressed as fold increase of the ratio between the protein of interest and the loading control.

Cells were seeded in 24-well plates. Lipofectamine Plus (Invitrogen) was used to transfect DNA plasmids into the 1HAEo^- cells according to the manufacturer's protocol. Following trasfection, the 1HAEo-cells were cultured for an additional 12 to 18 hours. Next, the cells were stimulated for eight hours with PCBG (100 μg/ml). One hour prior to stimulation, the cells were pretreated with PD98059 (16 μM), SB202190 (30 μM), JNK inhibitor II (10 μM) or BAPTA (1.2 μM). Following stimulation, the cells were washed twice in cold PBS and lysed with 50-100 μl of lysis buffer (Promega dual-luciferase reporter assay system). Firefly and Renilla luciferase activities from 10 μl of cell extracts were assayed with the Promega dual-luciferase reporter assay system reagents and a Berthold Lumat following the manufacturer's protocol. The κB-luc and IL-8 luc activities were normalized for Renilla expression. All transfection experiments were performed in duplicate.

Cells were cultured as previously described, and incubated with PDMP to reduce glycosphingolipid concentration, or media alone, for 72 hours prior to PCBG stimulation. Phosphorylation of p44/42 was analyzed from total cell lysates by immunoblotting and IL-8 was measured by ELISA in the culture supernatant. To exclude toxicity to the airway epithelial cells induced by PDMP, XTT viability assays were performed under identical conditions. Greater than 80% viability was considered as acceptable cellular viability for all experimental conditions.

All data are shown as the means ± SEM, unless otherwise stated. Data were assessed for significance using the Student t test or ANOVA with relevant posttests where appropriate. Statistical differences were considered to be significant if p was < 0.05. Statistical analysis was performed using GraphPad Prism version 5 (GraphPad Software, La Jolla, CA).

Since patients with severe Pneumocystis pneumonia exhibit an intense neutrophil infiltration in their lungs, we postulated that airway epithelial cells might participate in IL-8 secretion and subsequent recruitment of inflammatory cells in response to infection 53031. Our prior studies have been performed in rat primary alveolar epithelial cells 17. However, such primary cell cultures are of rodent origin and, as primary cultures, have limited ability to evaluate signaling pathways and promoter mechanisms. Therefore, in this investigation we utilized the 1HAEo-human airway epithelial cell line. Accordingly, we first determined whether IL-8 was secreted by 1HAEo-airway epithelial cells challenged with either PCBG or S. cerevisiae derived β-glucans. The 1HAEo^- cells were exposed to the fungal β-glucan preparations, or LPS, and IL-8 release was measured after 14 hours of challenge. P. carinii and to a lesser degree Saccharomyces derived β-glucans induced IL-8 secretion in a dose-dependent manner compared with both unstimulated and LPS challenged cells (Figure 1). Significantly, the absence of response of these cells to LPS excluded the possibility that endotoxin contamination of the β-glucan preparation was responsible for the observed inflammatory responses.

Since various microbial ligands are able to initiate intracellular calcium fluxes during cell stimulation, we next investigated whether PCBG challenge of airway epithelial cells triggered intracellular calcium release 3132. Consistent with this, we observed that PCBG-treated cells release intracellular calcium within a few seconds of stimulation (Figure 2A). As a positive control, a potent PAR-2 agonist peptide was tested in parallel. The peak wave of calcium release in PCBG treated cells appeared to be somewhat slower and maybe more prolonged than in PAR-2 treated cells. We believe that this is explained by the differences in formulation between the two compounds. While PAR-2 is a soluble reagent, and likely acts quicker on the cells, PCBG is a particulate agonist with slower action time.

Next, we sought to evaluate the importance of calcium release in IL-8 secretion of PCBG stimulation of 1HAEo^- epithelial cells. Accordingly, cells were pretreated with various calcium-signaling disrupting agents prior to PCBG stimulation and IL-8 release was determined in the culture supernatants, after 8 hours of stimulation (Figure 2B and 2C). Cells pretreated with EGTA, an extracellular calcium chelator 33, did not demonstrate any decrease in IL-8 secretion. In contrast, epithelial cells preincubated with the intracellular chelator BAPTA/AM 34, the calcineurin disrupting agents TEMPO, or cyclosporin A 35 each demonstrated significant decrease in IL-8 production (Figure 2B and 2C). Together, these data indicate that optimal secretion of IL-8 by airway epithelial cells stimulated with PCBG requires intra-cellular, rather than extra-cellular, calcium mobilization.

A variety of transcription factors including NF-κB and AP-1 binding sites have been identified within the IL-8 promoter 36373839404142. These transcription factors bind the promoter as dimers, and various combinations of AP-1 and NF-κB have been shown to be important for optimal activation of the IL-8 promoter, particularly in epithelial cells 43. Therefore, to further investigate the importance of NF-κB and AP-1, in IL-8 production induced by β-glucans, we measured IL-8 activation in 1HAEo-cells transiently transfected with the IL-8 luciferase reporter construct or with an IL-8 luciferase reporter construct that had targeted mutations in the NF-κB or AP-1 binding sites (Figure 3). PCBG failed to activate IL-8 transcription in cells transfected with either the mutant NF-κB or mutant AP-1 constructs, whereas IL-8 transcription was activated normally in cells transfected with the wild-type IL-8 promoter construct. From these observations, we can imply that both transcription factors are necessary for optimal activation of IL-8 transcription by airway epithelial cells following stimulation with PCBG.

Since MAPKs has been implicated in IL-8 secretion by airway epithelial cells, we next investigated whether MAPK activation was necessary for β-glucan stimulation of airway epithelial cells to release IL-8 314445. To accomplish this, 1HAEo^- cells were preincubated with PD98059, a specific pharmacological inhibitor of ERK, prior to stimulation with PCBG. Cells pre-treated with PD98059 exhibited a dose-dependent decrease in IL-8 production in response to the PCBG compared with untreated cells (Figure 4A). To further understand the kinetics of MAPK/ERK activation phosphorylation of ERK was determined by western immunoblotting after stimulation of the cells for different periods of time as indicated in Figure 4B. Phosphorylation of ERK p44/42 was detected within five minutes of stimulation, and remained slightly elevated as long as two hours after the initial challenge (Figure 4B and 4C). In addition, the calcineurin-disrupting agent TEMPO impaired ERK phosphorylation (Figure 4D and 4E).

Next, we evaluated whether p38, an independent major MAPKs pathway, participated in β-glucan mediated IL-8 secretion from airway epithelial cells in response to PCBG (Figure 5). The specific pharmacological inhibitor of p38, SB202190, was administered prior to and throughout PCBG stimulations of 1HAEo^- cells. Notably, SB202190 treated cells demonstrated significant reduction of IL-8 secretion in a dose-dependent manner, indicating the participation of p38 in the release of IL-8 (Figure 5A). In addition, we further investigated the kinetics of p38 activation following PCBG stimulation. Phosphorylation of p38 was detected as early as 15 minutes following stimulation, and reached its peak after 30 minutes. Following one hour of PCBG stimulation, phosphorylation of p38 had returned to baseline levels (Figure 5B and 5C). These data verify differential kinetics of these two MAPK signaling pathways, with the activation of p38 being substantially slower than the phosphorylation of ERK p44/42.

Finally, we investigated whether another important member of the MAPK signaling family, JNK, was also involved in IL-8 secretion by airway epithelial cells following challenge with PCBG (Figure 6). The JNK inhibitor II, a pharmacological antagonist of JNK was used prior to and through stimulation of 1HAEo-cells over PCBC for eight hours 46. Interestingly, we did not detect any inhibition of IL-8 secretion in PCBG stimulated cells in the presence of the JNK-II inhibitor. To verify that the inhibitor was functionally active, we further analyzed phosphorylation of JNK in PCBG stimulated cells in the presence of JNK inhibitor II in comparison to cells that were stimulated with PCBG in the absence of the inhibitor, verifying that JNK phosphorylation was indeed greatly reduced (data not shown). Nevertheless, IL-8 secretion was not impacted by this inhibitor, indicating that the participation of ERK and p38 MAPK in airway epithelial cells stimulated with PCBG is specifically restricted to those pathways, and that JNK does not participate in this cytokine response.

We have previously shown that MIP-2 neutrophil chemokine induced by PCBG in rodent primary lung epithelial cells is mediated by NF-κB activation (10). We next sought to determine whether MAPK activation following β-glucan stimulation of human 1HAEo^- cells resulted in downstream NF-κB dependant activation (Figure 7). To test this, we evaluated whether PCBG induced ERK and p38 signaling resulted in NF-κB promoter dependent activation in 1HAEo^- cells that were transiently transfected with an NF-κB-dependent luciferase reporter plasmid. Prior to PCBG stimulation, the 1HAEo^- cells were incubated with either; the PD98059, SB202190, or the JNK inhibitor II. Notably, pre-incubation of the cells with either PD98059 or SB202190 significantly reduced NF-κB dependent transcriptional activity in PCBG stimulated cells. However, the addition of JNK inhibitor II again had no effect on transcriptional activity related to NF-κB. These data suggest that PCBG mediated MAPKs activation results in downstream NF-κB-dependent transcriptional activation in target airway epithelial cells.

Previous data from our laboratory indicate that PCBG requires the glycosphingolipid lactosylceramide to induce MIP-2 release in murine epithelial cells 1747. We, therefore, sought to determine whether IL-8 secretion by PCBG in these human airway cells was also dependent on the presence of glycosphingolipids. To accomplish this, we evaluated IL-8 secretion in PCBG stimulated cells in the presence of PDMP, a potent glycosphingolipid synthesis inhibitor. Serum free media cultivated cells were treated with PDMP for 3 days prior to stimulation with PCBG. IL-8 release from β-glucan stimulated airway epithelial cells treated with the glycosphingolipid inhibitor was significantly decreased compared to non-treated cells (Figure 8A). We further investigated the effect of PDMP on ERK phosphorylation. Cells were cultured with media alone or in the presence of PDMP prior to activation with PCBG. Total cell lysates were analyzed for phospho-p44/42 by immunoblotting (Figure 8B and 8C). The phosphorylation of ERK p44/42 was reduced to baseline in cells treated with PDMP compared with non-treated cells. Taken together, these data strongly support our findings that glycosphingolipids are important for PCBG mediated ERK activation and subsequent IL-8 secretion by airway epithelial cells in response to PCBG.