Pneumolysin Induces 12-Lipoxygenase–Dependent Neutrophil Migration During Streptococcus Pneumoniae Infection

Pneumolysin Induces 12-Lipoxygenase− Dependent Neutrophil Migration during Streptococcus pneumoniae Infection

This information is current as Walter Adams, Rudra Bhowmick, Elsa N. Bou Ghanem, of September 24, 2021. Kristin Wade, Mikhail Shchepetov, Jeffrey N. Weiser, Beth A. McCormick, Rodney K. Tweten and John M. Leong J Immunol published online 27 November 2019 http://www.jimmunol.org/content/early/2019/11/26/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 27, 2019, doi:10.4049/jimmunol.1800748 The Journal of Immunology

Pneumolysin Induces 12-Lipoxygenase–Dependent Neutrophil Migration during Streptococcus pneumoniae Infection

Walter Adams,*,† Rudra Bhowmick,*,1 Elsa N. Bou Ghanem,*,2 Kristin Wade,‡,3 Mikhail Shchepetov,x,4 Jeffrey N. Weiser,{ Beth A. McCormick,‖ Rodney K. Tweten,‡ and John M. Leong*

Streptococcus pneumoniae is a major cause of pneumonia, wherein infection of respiratory mucosa drives a robust influx of neutrophils. We have previously shown that S. pneumoniae infection of the respiratory epithelium induces the production of the 12-lipoxygenase (12-LOX)–dependent lipid inflammatory mediator hepoxilin A3, which promotes recruitment of neutrophils into the airways, tissue damage, and lethal septicemia. Pneumolysin (PLY), a member of the cholesterol-dependent cytolysin (CDC) family, is a major S. pneumoniae virulence factor that generates ∼25-nm diameter pores in eukaryotic membranes and promotes acute inflammation, tissue damage, and bacteremia. We show that a PLY-deficient S. pneumoniae mutant was impaired in Downloaded from triggering human neutrophil transepithelial migration in vitro. Ectopic production of PLY endowed the nonpathogenic Bacillus subtilis with the ability to trigger neutrophil recruitment across human-cultured monolayers. Purified PLY, several other CDC family members, and the a-toxin of Clostridium septicum, which generates pores with cross-sectional areas nearly 300 times smaller than CDCs, reproduced this robust neutrophil transmigration. PLY non–pore-forming point mutants that are trapped at various stages of pore assembly did not recruit neutrophils. PLY triggered neutrophil recruitment in a 12-LOX–dependent manner in vitro. Instillation of wild-type PLY but not inactive derivatives into the lungs of mice induced robust 12-LOX– http://www.jimmunol.org/ dependent neutrophil migration into the airways, although residual inflammation induced by PLY in 12-LOX–deficient mice indicates that 12-LOX–independent pathways also contribute to PLY-triggered pulmonary inflammation. These data indicate that PLY is an important factor in promoting hepoxilin A3–dependent neutrophil recruitment across pulmonary epithelium in a pore-dependent fashion. The Journal of Immunology, 2020, 204: 000–000.

he Gram-positive bacterium Streptococcus pneumoniae is A hallmark of a S. pneumoniae lung infection is a robust the leading cause of community-acquired pneumonia and proinflammatory host response characterized by a massive influx T also causes several other infections, including otitis me- of neutrophils (polymorphonuclear leukocytes [PMNs]) into the by guest on September 24, 2021 dia, bacteremia, and meningitis. Asymptomatic colonization alveoli. PMNs, which confront the invading S. pneumoniae with a by S. pneumoniae has been estimated to be as high as 95% in number of antibacterial effector mechanisms, are beneficial for the children and 40% in adults and is considered to be an important host during early stages of the infection (6). Indeed, murine in- prerequisite for invasive disease (1, 2). In the United States fection studies have found that decreased neutrophil recruitment alone, there are ∼900,000 cases of pneumococcal pneumonia leads to higher bacterial loads in the lungs by 12–24 hours after annually, with a mortality rate of 5–7%, making the disease both pulmonary challenge with S. pneumoniae (7, 8). However, a strong a significant health and financial burden (3, 4). According to the PMN response to control bacterial outgrowth during the first World Health Organization, S. pneumoniae pneumonia accounts phase of infection can also lead to epithelial barrier disrup- for ∼500,000 deaths in children under 5 years old in developing tion, pulmonary edema, and significant lung damage (9, 10), countries (5). and mice containing high numbers of pulmonary PMNs several

*Department of Molecular Biology and Microbiology, Tufts University, Boston, MA This work was supported by National Institutes of Health Award 5R37AI037657- 02111; †Department of Biological Sciences, San Jose State University, San Jose, CA 22 (to R.K.T.), by Award Number K12GM074869 from the National Institute of 95192; ‡Department of Microbiology and Immunology, University of Oklahoma General Medical Sciences (to W.A.), and American Lung Association Senior Re- Health Sciences Center, Oklahoma City, OK 73104; xDepartment of Microbiology, search Training Fellowship RT 194942 N (to R.B.). University of Pennsylvania, Philadelphia, PA 19104; {Department of Microbiology, ‖ Address correspondence and reprint requests to Prof. John M. Leong, Tufts New York University School of Medicine, New York, NY 10016; and Department of University School of Medicine, 136 Harrison Avenue, Boston, MA 02111. E-mail Microbiology and Physiological Systems, University of Massachusetts Medical address: [email protected] School, Worcester, MA 01655 The online version of this article contains supplemental material. 1Current address: School of Chemical Engineering, Oklahoma State University, Stillwater, OK. Abbreviations used in this article: AA, arachidonic acid; ABTS, 2,29-azinobis- 3-ethylbenzotiazoline-6-sulfonic acid; BALF, bronchoalveolar lavage ﬂuid; 2Current address: Department of Microbiology and Immunology, University at bcn, baicalein; CDC, cholesterol-dependent cytolysin; Ci-Di-Cy, cinnamyl- Buffalo School of Medicine, Buffalo, NY. 3,4-dihydroxy-a-cyanocinnamate; cPLA2a, cytosolic phospholipase A2a; H292, hu- 3 Current address: Manufacturing Sciences and Technology Department, Cytovance man pulmonary mucoepidermoid carcinoma-derived NCI-H292; HXA3, hepoxilin Biologics, Oklahoma City, OK. A3; ILY, intermedilysin; i.t., intratracheal(ly); 12-LOX, 12-lipoxygenase; MPO, myeloperoxidase; PFO, perfringolysin O; PI, propidium iodide; PLY, pneumo- 4Current address: Pathology Department, Children’s Hospital of Philadelphia, lysin; PLY , PLY cholesterol binding site toxoid; PLY , PLY early prepore Philadelphia, PA. CBS EP toxoid; PLYLP, PLY late prepore toxoid; PMN, polymorphonuclear leukocyte; ORCIDs: 0000-0001-7168-8090 (J.N.W.); 0000-0001-9293-9899 (R.K.T.); 0000- RD2, RedDot2; SLO, streptolysin O; WT, wild-type. 0003-0240-6402 (J.M.L.). Ó Received for publication May 29, 2018. Accepted for publication October 16, 2019. Copyright 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800748 2 PLY ELICITS 12-LIPOXYGENASE–DEPENDENT NEUTROPHIL MIGRATION days postinfection suffer bacteremia and succumb to infection dilutions on Tryptic Soy Agar plates supplemented with 5% sheep blood (11, 12). These findings suggest that for a favorable host out- agar (Northeast Laboratory Services, Winslow, ME). Bacillus subtilis come the timing and degree of PMN recruitment must be carefully strains were grown overnight at 37˚C in Luria Broth. The TIGR4 ply mutant (Dply) was a gift from A. Camilli, and D39 and 23F WT Dply orchestrated. mutants and revertant strains were a gift from J. Weiser. Strains D39 and S. pneumoniae encodes pneumolysin (PLY), a 53-kDa member 23F WT and Dply mutants have been previously described (30–34). The of a large family of cholesterol-dependent cytolysins (CDCs) that D39 revertant strain was generated by transformation of the D39Dply form ∼25-nm diameter pores in eukaryotic membranes (13). mutant with D39 chromosomal DNA and screening for hemolytic trans- formants. Insertion of WT ply at the original locus was confirmed by CDCs have been identified in over 40 bacterial species and include sequencing. intermedilysin (ILY) of S. intermedius, streptolysin O (SLO) of S. pyogenes, and perfringolysin O (PFO) of Clostridium perfringens Bacterial growth conditions (14). Pore formation by CDCs is a multistep process involving S. pneumoniae strains TIGR4 (serotype 4), D39 (serotype 2), E134 membrane binding, oligomerization, and membrane insertion, and (serotype 23F) were grown in Todd Hewitt Broth (BD Biosciences) several PLY toxoids that are defective at discrete steps have been supplemented with 0.5% yeast extract and used in late log phase. For characterized (15–17). CDCs damage diverse host cell types and mouse experiments and human pulmonary mucoepidermoid carcinoma- derived NCI-H292 (H292) membrane repair experiments, bacteria were contribute to disease in many infection models (18). Indeed, after stored at 280˚C in media with 25% (v/v) glycerol, thawed, and diluted in pulmonary challenge in mice, S. pneumoniae deficient for PLY PBS to appropriate concentrations as required. Bacterial titers in thawed exhibit decreased tissue damage and inflammation, lower bacterial stocks were confirmed by plating serial dilutions on blood agar. B. subtilis burden, and less bacteremia (12, 19, 20). In addition to directly strain 168 was grown overnight at 37˚C in Luria Broth. damaging host cells, PLY has a major influence on the host im- Growth and maintenance of epithelial cells Downloaded from mune response. Relative to infection by a PLY-deficient strain, H292 cells were grown on the underside of collagen-coated Transwell wild-type (WT) S. pneumoniae infection results in an earlier and filters (0.33 cm2; Corning Life Sciences) in RPMI 1640 medium greater influx of PMNs and in greater numbers, resulting in more (American Type Culture Collection) with 2 mM L-glutamine, 10% FBS, severe lung damage (19–21). PLY also activates complement, an and 100 U penicillin/streptomycin. activity that has been shown to contribute to cellular influx during Animals pulmonary infection (22, 23). http://www.jimmunol.org/ 2/2 PLY triggers an early step in movement of PMNs into airways C57BL/6J female mice and Alox12/15 knockout (Alox15 ) female mice [i.e., transmigration across the endothelial cell barrier (24)]. (B6.129S2ALOX15 tm1Fun/J) were obtained from The Jackson Labora- tory. Mice were housed in a specific pathogen–free facility at Tufts Uni- For example, a PLY-deficient S. pneumoniae mutant exhibits a 2- versity, and all procedures were performed in accordance with Institutional to 4-fold defect for inducing PMN migration across cultured en- Animal Care and Use Committee–approved protocols. dothelial monolayers, and purified PLY is capable of promoting Toxin purification PMN movement (24). Although the specific host signaling molecules underlying this process have not been identified, purified The expression and purification of recombinant toxins and toxin deriv- PLY activates phospholipase A in endothelial cells, with concomi- atives from Escherichia coli were carried out as previously described (35), with the following modifications. Growth of E. coli XL-1 Blue by guest on September 24, 2021 tant release of arachidonic acid (AA), suggesting that eicosanoid containing pRT20 or derivatives thereof was initiated by inoculating signaling molecules may be involved (25). 1 l of sterile Terrific Broth (36) with a 1:100 inoculum of an overnight The final step of PMN entry into airways during S. pneumoniae culture grown at room temperature with 100 mg/ml ampicillin. The 1-l infection is transmigration across the lung epithelium, a step that culture was incubated at 37˚C with shaking at 200 rpm. At OD600 0.5–0.6, expression of toxin was induced by the addition of isopropyl has been associated with disruption of the mucosal barrier func- b-D-thiogalactopyranoside (Thermo Fisher Scientific) to a final concen- tion and spread of S. pneumoniae into the bloodstream (11). In- tration of 1 mM. Ampicillin was also added to a final concentration of 100 terestingly, given that PLY activates phospholipase in cultured mg/ml. The induced culture was grown overnight at room temperature and endothelial cells (25), we previously showed that the final step in pelleted by centrifugation. Cell pellets were resuspended in a total of 30 ml PMN movement into the airways is promoted by epithelial of PBS. Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific) was added to prevent proteolytic degradation of the toxin. Cells were lysed by production of 12-lipoxygenase (12-LOX), which is required for two passages through a microfluidizer, and cell debris was removed by the synthesis of the potent eicosanoid chemoattractant hepoxilin centrifugation at 10,000 3 g for 30 min at 4˚C. The supernatant containing A3 (HXA3) (26). HXA3 has been implicated in both intestinal the polyhistidine-tagged toxin was loaded onto a Ni-NTA Agarose column and pulmonary inflammation induced during bacterial infection (QIAGEN). The column was then washed with a 20–120 mM gradient imidazole to remove additional contaminating proteins. Bound toxin was (26–29). Disruption of 12-LOX activity by chemical inhibition or then eluted (2 ml/min) with 10 ml of PBS containing 500 mM imidazole. genetic ablation dramatically reduces pulmonary inflammation, SDS-PAGE was performed on the collected fractions to confirm toxin bacteremia, and host morbidity in a murine infection model (11). purity. 10% (v/v) glycerol and 5 mM Tris(2-carboxyethyl)phosphine hy- In this study, we identify S. pneumoniae PLY as a bacterial drochloride was added to toxin-containing fractions, which were then 2 factor necessary and sufficient to induce 12-LOX–dependent PMN flash-frozen and stored at 80˚C until use. migration across epithelial monolayers. We found that the pore- Toxin activity assay forming activity of PLY is central to induction of inflammation Toxin aliquots were thawed on ice on and then spun at 14,000 rpm in a and that purified PLY triggered recruitment of PMNs into the microcentrifuge at 4˚C for 10 min to remove protein precipitate. Toxin murine airway in a manner dependent on both its pore-forming concentration was determined using Bradford reagent (Bio-Rad Labora- activity and host 12-LOX activity. tories) per the manufacturer’s instructions. H292 cells were placed in 100-ml volumes in a nonadherent 96-well plate. Toxin was incubated at various concentrations in triplicate with the H292 cells for 15 min at 37˚C. Materials and Methods Addition of 5% Triton X-100 and HBSS plus Ca/Mg were used as positive Bacterial strains and negative controls, respectively. The plate was then spun at 1200 rpm for 5 min, and cells were resuspended in 1 mg/ml propidium iodide (PI). Midexponential growth phase aliquots of S. pneumoniae TIGR4, D39, and Cells were filtered through 100-mM filters into 100 ml of PBS with 10% 23F strains (serotype 4) were grown in Todd Hewitt Broth (BD Biosci- serum, placed on ice, and kept in the dark until analysis by flow cytometry. ences) supplemented with 0.5% yeast extract in 5% CO2 and Oxyrase Cells were run through a FACSCalibur flow cytometer (BD Biosciences), (Oxyrase, Mansfield, OH) and were frozen in growth media with 20% (v/v) and a minimum of 5 3 104 events were analyzed per replicate. Collected glycerol. Bacterial titers in aliquots were confirmed by plating serial data were analyzed using FlowJo software (TreeStar) to determine the The Journal of Immunology 3 toxin concentration that resulted in 50% of the H292 cells becoming PI+. H292 membrane repair assay This concentration was defined as “1 suspension unit” of toxin activity. 3 5 m Transwell monolayers were then treated with various suspension units to A total of 2 10 H292 cells were placed in 200- l volumes per well in a determine the number of units that resulted in 50% of the polarized H292 nonadherent 96-well plate and incubated at 37˚C with CO2. One milligram cells on the Transwell becoming PI+. This concentration was defined as per milliliter of PI was added to each well, and the plate was protected “1 Transwell unit” of toxin activity, which we refer to as “1 U” of toxin from light. H292 cells were infected with WT TIGR4 or the TIGR4 ply- activity. For some experiments, ionomycin activity was determined iden- deficient derivative, and the plate was briefly spun at 1000 rpm for 3 min. tically to the method described above, except ionomycin, not toxin, was The plate was incubated at 37˚C with CO2 and protected from light for 1 h. incubated at various concentrations in triplicate with the H292 cells for The plate was then spun at 1200 rpm for 5 min, and cells were resuspended 15 min at 37˚C. in FACS buffer before RedDot2 (RD2) was added to designated wells, transferred to a round-bottom 96-well plate and spun at 1200 rpm for Preparation and assessment of polarized H292 monolayers 5 min. Cells were filtered through 100-mM filters into a final volume of 250 ml of FACS buffer. Cells were placed on ice and kept in the dark until Polarized H292 monolayers were prepared as previously described (37). analysis by flow cytometry. Cells were run through a FACSCalibur flow The transepithelial resistance of lung epithelial monolayers are typically cytometer (BD Biosciences) and a minimum of 2 3 104 events were an- very low (Ref. 38 and W. I. Adams, unpublished data]. Hence, to assess the alyzed per replicate. Collected data were analyzed using FlowJo software generation of intact, confluent H292 monolayers for a sampling of polar- (TreeStar). ized monolayers, we measured the ability of HRP added to the basolateral compartment to be detected in the apical chamber after 20 min, as pre- Leached PLY activity assay viously described (28, 39). In addition, some samples were analyzed by fluorescent microscopy to confirm confluent and intact monolayers. The apical surface of H292 cell monolayers on Transwell filters was treated To prepare samples for fluorescent microscopy, monolayers were per- with the indicated units of toxin or incubated with HBSS plus Ca/Mg at meabilized with 0.1% Triton X-100 in PBS plus 3% BSA and fixed in 4% 37˚C for 1 h. After toxin treatment, monolayers were washed and placed in paraformaldehyde. Monolayers were then stained with DAPI and phal- 24-well plates containing HBSS plus Ca/Mg for 2.5 h at 37˚C. Basolateral Downloaded from loidin (data not shown) and visualized on excised filters. Finally, defects in and apical supernatants were collected and added to a 96-well plate. A 3 Transwell filter integrity after collagen coating were detected by buffer total of 7.8 3 10 PMNs were added to wells containing basolateral or loss, and changes in pH of cell media were used to detect potential defects apical supernatant and incubated for 1.5 h at 37˚C. Addition of 0.05 U of in cell growth. Approximately 3% of H292 monolayers were excluded PLY (as defined by the toxin activity assay) and HBSS plus Ca/Mg were from our experiments. used as positive and negative controls, respectively. The plate was then spun at 1200 rpm for 5 min, and cells were resuspended in 1 mg/ml PI. PMN transepithelial migration assay m m

Cells were filtered through 100- M filters into 100 l of PBS with 10% http://www.jimmunol.org/ 3 7 serum, placed on ice, and kept in the dark until analysis by flow cytometry. The apical surface of inverted Transwells was infected with 1 10 Cells were run through a FACSCalibur flow cytometer (BD Biosciences) bacteria for 2.5 h, treated with the indicated units of toxin for 1 h, treated and a minimum of 1 3 104 events were analyzed per replicate. Collected with the indicated units of ionomycin for 1 h, or incubated with HBSS data were analyzed using FlowJo software (TreeStar) to measure the plus Ca/Mg at 37˚C. After toxin treatment, Transwells were washed percentage of PI+ cells. and placed in 24-well plates containing HBSS plus Ca/Mg. A total of 1 3 106 PMNs, isolated from whole blood obtained from healthy human Murine infection studies volunteers, as previously described (28, 40), were added to the basolateral chamber, and after 2.5 h, PMNs in the apical chamber were Mice were intratracheally (i.t.) instilled with PLY or the indicated PLY quantified by myeloperoxidase (MPO) assay, as described previously toxoids (in 50 ml of PBS). Control mice received PBS alone. Mice were (40). Briefly, monolayers and their underlying Transwell filters were sacrificed at 48 h, and H&E-stained lung sections were prepared. Sections by guest on September 24, 2021 removed from individual wells of 24-well plates, leaving the 24-well were examined by light microscopy (at original magnification 320). plate containing the apical buffer and migrated neutrophils for each Bronchoalveolar lavage fluid (BALF) was collected, and cells in the BALF sample and control well. Fifty microliters of 10% Triton X-100 was were quantitated by flow cytometry as described below. added to each well and gently rocked for 20 min at 4˚C. Fifty microliters of citrate buffer was added to each sample, and the 24-well plate was Flow cytometry gently rocked for 20 min at 4˚C. 2,29-azinobis-3-ethylbenzotiazoline- For flow cytometry studies, anti–Ly-6G-PE (clone 1A8), anti-CD11c–FITC 6-sulfonic acid (ABTS) solution was freshly prepared and 50 mlof (clone N418), and anti-F4-80-PE-Cy7 (clone BM8) were obtained from hydrogen peroxide was added to the ABTS solution. One hundred mi- BD Biosciences. Mice were euthanized at 48 h, and BALF was collected. croliters from each well was transferred to a 96-well plate, and 100 ml Cells present in the BALF were stained on ice for 30 min with appropriate of ABTS solution was added to each sample in the 96-well plate. mAbs and then washed. Cells were then run through a FACSCalibur flow The 96-well plate was incubated in the dark at room temperature for cytometer (BD Biosciences), and the fluorescence intensities of the stained 5–10 min until it was read on a microplate reader for absorbance at a cells were determined. Data were analyzed using FlowJo software wavelength of 405 nm. fMLF and HBSS plus Ca/Mg were used as (TreeStar) to determine the numbers of PMNs (Ly-6G+) in the BALF. positive and negative controls, respectively. To determine whether epithelial cells in the absence of PMNs were Presentation of data and statistical analyses capable of generating chemoattractants after treatment with PLY, Transwell filters were incubated in 24-well plates containing HBSS plus Because of intrinsic donor-to-donor variability of human PMNs and their Ca/Mg for an additional 2.5 h at 37˚C to collect potential chemo- transmigration, efficiency of transmigration was compared within in- attractants released by epithelial monolayers into the apical supernatant. dividual experiments but not between experiments. The conclusions Treated Transwell filters were removed, and naive Transwell filters were drawn were those found to be reproducible and statistically significant placed in the apical supernatant, with the remainder of the transmigration across independent experiments. Statistical analysis was carried out assay proceeding as described above to detect chemoattractant activity. using the program GraphPad Prism (GraphPad Software, San Diego, CA). All p values , 0.05 were considered significant. For all graphs, For select Transwell filters, we used fluorescence microscopy to visually 6 assess the effect of toxin treatment on the health of the H292 mono- the mean values SEM are shown. layers. To prepare samples for fluorescent microscopy, monolayers were permeabilized with 0.1% Triton X-100 in PBS plus 3% BSA and fixed in Results 4% paraformaldehyde. Monolayers were then stained with DAPI and PI PLY is required for maximal S. pneumoniae–induced + + and visualized on excised filters. The number of PI and DAPI cells transepithelial PMN migration were counted manually in a blinded manner and then the percentage of PI+ cells was calculated. We previously showed that apical infection of polarized H292 Treatment of monolayers with inhibitors lung epithelial cell monolayers cultured on Transwell filters with S. pneumoniae triggers the basolateral-to-apical transmigration a Cinnamyl-3,4-dihydroxy- -cyanocinnamate (Ci-Di-Cy) and baicalein of human PMNs, as assessed by MPO activity (see Materials and (bcn) were obtained from Enzo Life Sciences (Plymouth Meeting, PA). Cells were pretreated with Ci-Di-Cy (25 mM) (41) or bcn (0.5 mM) Methods) (26, 29, 42). We confirmed that S. pneumoniae strain (11, 27) for 3–5 h. To minimize potential direct effects of the inhibitors, TIGR4, a capsular serotype 4 clinical isolate, triggers 35-fold drugs were removed prior to addition of toxin to the Transwells. higher levels of transmigration compared with the buffer control, 4 PLY ELICITS 12-LIPOXYGENASE–DEPENDENT NEUTROPHIL MIGRATION although that is somewhat less than the transmigration trig- 2.5 U of PLY, which triggered permeabilization of 62% of cells gered by imposed gradients of the potent chemoattractant fMLF (Supplemental Fig. 1A–D), were sufficient to induce significant (Fig. 1A). An isogenic TIGR4 ply-deficient derivative recruited PMN migration when applied to H292 cell–polarized mono- 4-fold less PMNs (p , 0.0001), indicating that PLY contributes to layers (Fig. 2B). Five units induced maximal PMN transmigra- S. pneumoniae–induced transmigration across respiratory epithelial tion, whereas no transmigrationwasobservedat20Uormore monolayers (Fig. 1A). To determine whether PLY-induced trans- (Fig. 2B), which resulted in PI staining of 100% of H292 cells migration was a common property of S. pneumoniae,weapically (Supplemental Fig. 1A–D). Treatment of human embryonic infected polarized H292 monolayers with WT or ply-deficient de- kidney cells and neuroblastoma SH-SY5Y cells with the equiv- rivatives of the capsular serotype 2 S. pneumoniae strain D39 and alent of 20 U of PLY results in irreversible cell lysis (47), pro- theserotype23FS. pneumoniae strain E134 (32, 30). The WT viding an explanation for the lack of PMN transmigration at the versions of these strains elicited robust PMN transmigration, at least high doses observed in our study. 8-fold greater than buffer control (Fig. 1B, 1C). The degree of PMN The lack of PMN transmigration in response to levels of PLY that migration varied with each experiment, a finding common to human induced pore formation (i.e., PI+ staining) in 100% of cells raised PMN migration studies and thought to be related to donor variation. the question as to whether any PI+ H292 cells might be viable and Consistent with our findings with strain TIGR4, the ply-deficient actively produce chemoattractant. In fact, limited pore formation derivatives of both D39 and 23F recruited fewer PMNs relative to by PLY or the related CDC SLO triggers rapid membrane repair the WT parental strain (p , 0.05, p , 0.0001; Fig. 1B, 1C). Re- that prevents cell death (48, 49). At a PLY dose that induces version of these ply mutants by insertion of a WT ply at the original permeabilization of 70% of human embryonic kidney cells locus(43)resultedinrestorationofPMNmigrationtoatleastWT and neuroblastoma SH-SY5Y cells, all but ∼5% are capable of Downloaded from levels. These findings indicate that PLY is required for maximal PMN repairing their cytoplasmic membrane (47). To test whether lim- transepithelial migration upon apical infection by S. pneumoniae. ited PLY-mediated pore formation was also reversible, we mock infected H292s or infected them with either WT or PLY-deficient PLY is sufficient to recruit transepithelial PMN migration S. pneumoniae TIGR4 in the presence of PI 1 h. After washing to To determine whether PLY requires one or more S. pneumoniae– remove bacteria and PI, H292s were incubated with RD2, which

specific factors to promote PMN transepithelial migration in vitro, would stain host cells only if the plasma membrane remained http://www.jimmunol.org/ we infected polarized H292 monolayers either with strain AC4052 compromised (50). Using flow cytometry, we found that 40% of of the nonpathogenic soil bacterium B. subtilis or strain AC4050, H292 cells were permeabilized (“PI+”) by WT infection, a per- a derivative that ectopically expresses surface-localized PLY centage that was at least 14-fold higher (p , 0.01) than the per- (44, 45). B. subtilis strain AC4052 elicited low levels of PMN centage of permeabilized cells after mock infection or infection by migration, consistent with the notion that this bacterium encodes the ply-deficient strain (Supplemental Fig. 1E). Importantly, using some molecules [e.g., peptidoglycan (46)] that are capable of trig- RD2 staining, we found that 69% of cells had intact (PI+RD22) gering inflammation. In contrast, the B. subtilis strain expressing membranes, compared with 31% with permeabilized (PI+RD2+) PLY exhibited 3-fold more PMN migration relative to strain membranes (Supplemental Fig. 1F, RD22 or RD2+,respec- AC4050 (p , 0.0001), indicating that PLY plays a role in eliciting tively). Thus, the majority of H292s permeabilized in a PLY- by guest on September 24, 2021 the host immune response independent of S. pneumoniae–specific dependent manner retain sufficient viability to undergo rapid factors (Fig. 2A). membrane repair. To determine whether bacterial factors other than PLY are re- In our transmigration assays, although monolayers were washed quired to elicit PMN transmigration in vitro, we measured the PMN after apical treatment with PLY prior to the addition of PMNs to the response to purified PLY. We found that the pore-forming ac- basolateral chamber, PLY might diffuse into the basolateral tivity of an N-terminally His-tagged PLY varied, even when chamber and directly alter PMN function. Contrary to this hy- stored as aliquots at 280˚C (see Materials and Methods). Thus, pothesis, by PI staining of PMNs, no pore-forming activity was to expose monolayers to standardized levels of PLY activity, we detected in buffer collected from the basolateral chamber 2.5 h after defined the concentration of PLY as 1 U that resulted in per- apical treatment of H292 cell monolayers with 2.5 or 5 U of PLY meabilization (indicated by PI staining) of 50% of polarized (Supplemental Fig. 2A; see Materials and Methods). PLY that H292 cells (see Materials and Methods). We found that as few as binds to the H292 cell monolayers and then is released into the

FIGURE 1. PLY is required for PMN transepithelial migration during S. pneumoniae infection. (A–C) Apical sides of H292 monolayers on Transwells were infected with 1 3 107 CFU per Transwell of WT, ply-deficient, or complemented S. pneumoniae strains for 2.5 h. Postinfection, 1 3 106 human PMNs were added to the basolateral side. PMN transepithelial migration was quantified by MPO activity. fMLF and buffer alone were used as positive and negative controls, respectively. Shown for each panel is a representative of two to four independent experiments, in which each condition was tested in triplicate per experiment. Asterisk(s) indicate migration is significantly greater than the corresponding S. pneumoniae Δply mutant, as determined by one- way ANOVA with a post hoc Tukey test. *p , 0.05, ****p , 0.0001. The Journal of Immunology 5

After 2.5 h of incubation, this buffer also was found to contain no PLY when assayed for the ability to generate pores in PMNs (Supplemental Fig. 2B). We conclude that the PLY-induced transmigration in our Transwell system is due to its interaction with H292 cells rather than PMNs. To rigorously test whether PLY could elicit chemoattractant from epithelial cells in the complete absence of PMNs, we collected supernatant from PLY-treated H292 cells. This supernatant was found to be capable of eliciting neutrophil migration across naive monolayers and in a 12-LOX–dependent manner (Supplemental Fig. 3), demonstrating that PLY-epithelial interaction in the absence of neutrophils is sufﬁcient to drive initial basolateral-to- apical neutrophil migration. Diverse pore-forming agonists induce PMN migration The CDCs show substantial structural and functional conservation, so we investigated whether other CDCs could also induce PMN migration. We treated polarized H292 cell monolayers with 2.5 or

5 U of ILY, SLO, and PFO, with activity units determined as Downloaded from described above (see also Materials and Methods). ILY, SLO, and PFO triggered a dose-dependent increase in PMN migration that at 5 U reached at least 6-fold greater than buffer controls, respectively (p , 0.05; Fig. 3). Similar to our other PMN migration experiments, each toxin induced signiﬁcant PMN migration in a

dose-dependent manner, although we observed donor-to-donor http://www.jimmunol.org/ variability in maximal levels of PMN migration. To assess whether a non-CDC toxin that generates pores of a different nature could also elicit PMN migration, we tested a-toxin of C. septicum, which generates pores 1.3–1.6-nm in diameter, with cross-sectional areas some 300 times smaller than those generated by CDCs (51, 52). We found that 0.5 and 2.5 U of C. septicum a-toxin induced PMN migration at 14-fold and 30-fold higher levels than the buffer-treated control (p , 0.05) (Fig. 3). These results indicate that the ability to induce PMN by guest on September 24, 2021 trafficking is a property of diverse pore-forming agonists. PLY requires pore-forming activity to induce PMN migration Pore formation by PLYis the result of a multistep process (53). First, CDC monomers bind to cholesterol in the host cell membrane. Thirty to fifty monomers then oligomerize to form a large “ring,” termed an “early prepore complex,” on the cell surface (35, 54, 55). FIGURE 2. PLY is sufficient to induce PMN transepithelial migration in a dose-dependent manner. (A) Apical sides of H292 monolayers on The oligomer undergoes a conformational change to generate a Transwells were infected with 1 3 107 WT B. subtilis or B. subtilis “late prepore complex” which is stable to dissociation by SDS and expressing ply. Postinfection, transepithelial migration of 1 3 106 human heat. Finally, the late prepore complex refolds two a-helical bundles PMNs added to the basolateral side was quantified by MPO assay. fMLF from each monomer in the prepore complex into membrane- and buffer alone were used as positive and negative controls, respectively. spanning b-hairpins to form the large 25-nm b-barrel pore (56). Shown is a representative of two independent experiments, in which each To determine which step of pore formation is associated with condition was tested in triplicate per experiment. Asterisks indicate mi- triggering PMN migration, we tested whether PLY mutants de- gration is significantly greater than migration induced by the parental fective at different stages retain the ability to induce PMN mi- B. subtilis strain, as determined by one-way ANOVAwith a post hoc Tukey gration. The PLY mutant PLY cholesterol binding site toxoid test. ****p , 0.0001. (B) Apical sides of H292 monolayers were treat- (PLY ), containing a single amino acid substitution in the ed with indicated units of recombinant PLY. Transepithelial migration of CBS 1 3 106 PMNs added to the basolateral side postinfection was quantified. cholesterol binding sequence in domain 4, is deficient in mem- fMLF and buffer alone were used as positive and negative controls, re- brane association and pore formation (57). Treatment of epithelial spectively. Shown is one representative experiment of four independent monolayers with as much as 2.5 mMofPLYCBS, a concentration experiments, in which each condition was tested in triplicate per experi- that corresponds to 10 U of WT PLY, did not induce PMN mi- ment. Asterisks indicate migration is significantly greater than migration gration significantly over background levels (Fig. 4A), whereas as induced by buffer alone, as determined by one-way ANOVA with a post little as 2.5 U of WT PLY induced considerable PMN migration. hoc Dunnett test. **p , 0.005, ****p , 0.0001. PLY early prepore toxoid (PLYEP), which is locked at the early prepore stage because of single amino acid substitutions in do- apical medium might also interact with PMNs, potentially contrib- mains 2 and 3, and PLY late prepore toxoid (PLYLP), which is uting to transmigration by directly eliciting PMN chemoattractant locked at the late prepore stage because of a double substitution secretion. However, we attempted to capture this putative “leached” mutation in domain 3, demonstrated only 3 and 2% of WT PLY by apically treating H292 cell monolayers with 2.5 or 5 U PLY pore-forming activity, respectively. Neither toxoid triggered of PLY, washing the monolayers, and adding fresh apical buffer. PMN migration across epithelial monolayers at concentrations of 6 PLY ELICITS 12-LIPOXYGENASE–DEPENDENT NEUTROPHIL MIGRATION Downloaded from

FIGURE 3. Diverse pore-forming agonists induce PMN transmigration. The apical face of H292 monolayers were treated with the indicated units of ILY of S. intermedius, SLO of S. pyogenes, PFO of C. perfringens,or a-toxin of C. septicum. Transepithelial migration of 1 3 106 human PMNs added to the basolateral side was quantiﬁed by MPO assay. Buffer alone was used as negative control. Each panel depicts one representative of http://www.jimmunol.org/ between two and four independent experiments, in which each condition was tested in triplicate per experiment. Asterisks indicate migration is signiﬁcantly greater than migration induced by buffer alone, as determined by one-way ANOVAwith a post hoc Dunnett test. *p , 0.05, **p , 0.005, ***p , 0.0005, ****p , 0.0001.

0.75 or 1.5 mM, whereas equivalent molar concentrations of WT PLY, corresponding to 2.5 and 5 U, respectively, were sufficient to by guest on September 24, 2021 induce robust PMN migration (Fig. 4B). These results indicate that PLY-mediated pore formation is necessary for PLY-induced PMN migration. 12-LOX inhibitors impair PMN migration S. pneumoniae–induced PMN epithelial transmigration is dependent on 12-LOX, an enzyme that converts AA into 12(S)-HPETE and is required for the production of HXA3 (11, 26, 58, 59). As noted above, purified PLY has been previously shown to activate phospholipase A in endothelial cells, with concomitant release of AA (25). To determine whether neutrophil migration in response to PLY is dependent on the 12-LOX pathway, we tested the ability FIGURE 4. PLY requires pore-forming activity to induce PMN migra- of 12-LOX pharmacologic inhibitors Ci-Di-Cy or bcn to inhibit tion. (A) Apical sides of H292 monolayers were treated for 1 h with neutrophil transmigration in response to apically applied PLY. equimolar concentrations of functional PLYor a PLY cholesterol binding site B H292 cells treated with either 25 mM Ci-Di-Cy or 0.5 mM bcn mutant (PLYCBS). ( ) Apical sides of H292 monolayers were treated for 1 h with equimolar concentrations of PLY, a PLY mutant locked in an early (see Materials and Methods) exhibited significantly less PMN prepore conformation (PLYEP), or a PLY mutant locked in a late prepore migration (Fig. 5). We also investigated whether neutrophil mi- 6 conformation (PLYLP). A total of 1 3 10 human PMNs were added to the gration in response to other pore-forming toxins (e.g., ILY, PFO, basolateral side, and PMN transepithelial migration was quantified by MPO SLO, and a-toxin) is dependent on the 12-LOX pathway, using the assay. Buffer alone and fMLF were used as negative and positive controls, same pharmacologic inhibitors, but our results were inconsistent respectively. Shown is one representative experiment of two independent (data not shown); so definitive identification of the signaling experiments, in which each condition was tested in triplicate per experiment. pathway(s) activated by these other pore-forming toxins will re- Asterisks indicate migration induced by WT PLYis significantly greater than quire further study. Nevertheless, the observed neutrophil migra- equimolar concentrations of toxoid as determined by (A) Welch t test or B , , tion in response to PLY is dependent on the 12-LOX pathway, ( ) one-way ANOVA with a post hoc Tukey test. *p 0.05, **p 0.005, , supporting the hypothesis that PLY contributes to the neutrophil ***p 0.0005. transmigration induced by S. pneumoniae. is required for this phenotype, we instilled i.t. into mice WT PLY, PLY-mediated pore-forming activity triggers 12-LOX– or PLY mutants that cannot form pores or are severely deficient dependent pulmonary inflammation in PLY-induced mice in pore formation. As expected, when evaluated at 48 h post- PLY is sufficient to induce pulmonary inflammation upon i.t. in- instillation, buffer alone induced no pathologic condition, and stillation (60–62). To determine whether PLY pore-forming activity BALF contained less than 13 PMNs/ml (Fig. 6A), whereas WT The Journal of Immunology 7 Downloaded from

FIGURE 5. 12-LOX is required for PLY-induced PMN transmigration. H292 monolayers were incubated for 3 h with buffer alone, the 12-LOX inhibitor Ci-Di-Cy, or the 12-LOX inhibitor bcn. Following this incubation, apical sides of H292 monolayers were treated for 1 h with 2.5 U of PLY. A total of 1 3 106 human PMNs were added to the basolateral side, and PMN transepithelial migration was quantified by MPO assay. Buffer http://www.jimmunol.org/ alone and fMLF were used as negative and positive controls, respectively. Shown is a representative of six independent experiments, in which each condition was tested in triplicate per experiment. Treatment with inhibitors reached statistical significance in five out of six independent experiments. Asterisks indicate that PMN migration is significantly greater than migration induced by the negative control of buffer without PLY, 2.5 U PLY with Ci-Di-Cy, and 2.5 U PLY with bcn, as determined by one-way ANOVA with a post hoc Tukey test. ****p , 0.0001. by guest on September 24, 2021 PLY triggered considerable infiltration of PMNs into the airways and more than 900 PMNs/ml in the BALF (Fig. 6B, 6G, p , 0.05). PLYCBS,PLYEP,orPLYLP mutant toxoids did not trigger pulmonary inflammation when evaluated by histopathology, and there was no significant difference in PMN infiltration between these treatment conditions and buffer alone. In fact, the number of PMNs in BALF of these mice was at least 28-fold lower than the number FIGURE 6. PLY requires pore formation and the 12-LOX pathway to induce robust PMN migration in vivo. Female WT mice were challenged in BALF from mice instilled with WT PLY (Fig. 6C–E, 6G). 2/2 i.t. with PLY, PLYCBS,PLYEP,orPLYLP. Female Alox15 mice were As in vitro inhibition of 12-LOX activity diminishes PLY- challenged i.t. with PLY. Control mice received buffer alone. (A–F) Mice mediated PMN transmigration across polarized H292 cell mono- were sacrificed, and H&E-stained lung sections were prepared. Sections layers, we asked whether pulmonary inflammation also displays a were examined by light microscopy (at original magnification 320). similar dependence on 12-LOX. To address this question, we in- Histology images shown are representative data from one of three inde- 2 2 stilled purified PLY into the lungs of Alox15 / mice, which are pendent experiments (using three to five mice per condition). (G) BALF deficient for 12/15–LOX (26, 63, 64). Histopathological analysis samples were collected, and the number of PMNs was measured by flow revealed that PLY-mediated inflammation, like inflammation cytometry. The data presented are the average value for each treatment triggered by infection with S. pneumoniae (11), was dramatically condition across three independent experiments (using three to five mice diminished in 12-LOX–deficient mice, and the number of PMNs per condition). The “#” symbol indicates that PMN number in the BALF was significantly greater in WT mice challenged with PLY than in all other in BALF was 3-fold lower than in WT mice (Fig. 6F, 6G). These 2/2 groups except Alox15 mice challenged with PLY, as determined by findings support our model that PLY-mediated pore-forming ac- one-way ANOVA with a post hoc Tukey test. p = 0.1012. Alox152/2 mice tivity triggers activation of the HXA3 synthesis pathway, resulting challenged with PLY were not significantly different from WT mice that in robust pulmonary inflammation, a hallmark of pneumococcal received buffer alone, as determined by one-way ANOVA with a post hoc pneumonia. Tukey test. p = 0.7135, #p , 0.05.

Discussion PMN recruitment to the lung in response to a microbial intrusion is migration across endothelial monolayers (24). After traversing the a multistage process that is dictated by a combination of haptotactic basement membrane and lung interstitium, the final step in mi- and chemotactic gradients. In vitro modeling of the initial step, exit gration of PMNs to the site of infection is movement across the from the vasculature, indicates that WT S. pneumoniae elicits lung epithelium and into the alveoli (65). We previously dem- significantly more PMN recruitment than a PLY-deficient strain onstrated that S. pneumoniae infection of pulmonary epithe- (24) and that purified PLY alone is sufficient to induce PMN lial monolayers leads to PMN transmigration in vitro and that 8 PLY ELICITS 12-LIPOXYGENASE–DEPENDENT NEUTROPHIL MIGRATION blocking this step not only diminished pulmonary inflammation, also induced significant PMN migration (Supplemental Fig. 4). 2+ but abrogated high-level bacteremia and lethal outcome (11). This Furthermore, HXA3 itself induces Ca flux, first rapidly from earlier work also revealed that the S. pneumoniae polysaccharide intracellular stores, then slowly from the extracellular environ- capsule promoted PMN recruitment, but because capsule-deficient ment (76), potentially amplifying Ca2+-dependent signaling such strains still elicited PMN migration, we posited that other bacterial as that mediated by cPLA2a. products must also contribute to this process. In this study we PLY has been reported to recognize TLR4 (77), activation found that ply-deficient S. pneumoniae strains of multiple sero- of which has been found to induce cPLA2 activity, and TLR4- types were significantly impaired in eliciting PMN migration deficient mice exhibit lower levels of PMNs in the BALF fol- across polarized respiratory epithelial cell monolayers. Similarly, lowing PLY instillation (77–80). In addition, PLY has been ectopic expression of PLY by B. subtilis boosted the ability of this demonstrated to activate complement (22), raising the possibility nonpathogen to elicit PMN movement. (Note that B. subtilis alone that TLR4 binding or complement activation plays a central role was also able to induce low levels of PMN migration, suggesting in inducing PMN chemotaxis. However, the set of PLY toxoids that bacterial components that are also produced by B. subtilis, described in this manuscript harbor single or double amino acid such as peptidoglycan or lipoproteins, may trigger PMN traffick- substitutions that are (collectively) distributed throughout all four ing with less efficiency than PLY, consistent with our observation PLY domains (16, 17, 81, 82): PLYCBS (L460D) is altered in that high inoculums of ply-deficient S. pneumoniae induce PMN domain 4, whereas PLYLP (G25C-E159C) and PLYEP (K288C- migration; data not shown). In addition, we found that purified V303C) are altered in domains 2 and/or 3 (16, 17, 81, 82). PLY was sufficient to induce transepithelial PMN migration. This Given the diversity of the location of the lesions, defects in their activity of purified PLY is consistent with previous findings that interaction with TLR4 or complement that are common to all three Downloaded from instillation of PLY into mouse lungs causes acute lung injury, mutants would likely reflect global misfolding. Contrary to this including hyperpermeability, pulmonary edema, and PMN in- possibility, each of the mutants retains the native structure of WT filtration (60, 61, 66), that mice infected with ply-deficient PLY (78, 79, R.K. Tweten, unpublished data). For example, PLYLP S. pneumoniae exhibit reduced PMN recruitment (20), and that and PLYEP retain cholesterol binding activity, indicating that do- in vitro infection by ply-deficient S. pneumoniae induced a di- main 4, the cholesterol binding domain, is functional, and both

minished number of specific transcriptional immune responses in these toxoids retain full pore-forming activity when reduced http://www.jimmunol.org/ epithelial cells compared with WT TIGR4 infection (67). Thus, (78, 79, R.K. Tweten, unpublished data), indicating that the PLY plays a central role in eliciting an acute inflammatory re- structure of domains 1–3 has not been perturbed. For PLYCBS sponse, promoting movement of PMNs across both endothelial (L460D), which does not bind cholesterol (57), the sidechain and epithelial barriers in response to S. pneumoniae infection. affected does not significantly interact with other nearby amino Purified PLYactivates phospholipase A in endothelial cells, with acids. Rather, the major interactions of L460 are exclusively concomitant release of AA (25). We previously showed that via its backbone amide and carbonyl, which are not altered by S. pneumoniae infection of cultured pulmonary epithelial cells the mutation. Note, also, that although the putative comple- activates cytosolic phospholipase A2a (cPLA2a) and the synthesis ment activation site near N385 (83) is in domain 4, N385 lies by guest on September 24, 2021 pathway for the AA metabolite HXA3, a potent PMN chemo- at the extreme end of the domain away from L460 (16, 17). attractant (11, 68). Hence, we investigated whether the synthetic These considerations rule out the possibility that the lack of pathway for HXA3 is required for robust PMN migration in re- chemoattractant-inducing activity of the toxoids is due to an sponse to purified PLY. Two structurally unrelated pharmacolog- inability to bind TLR4 or activate complement. Instead, these ical inhibitors that block HXA3 synthesis, used at concentrations findings show that the inability to form pores by mutants thought to specifically inhibit 12-LOX (11, 27, 41), each signifi- inhibited at three different stages of the pore-forming mecha- cantly decreased PLY-induced PMN migration in vitro. In addi- nism (i.e., binding, early prepore oligomer assembly, and late tion, when we instilled purified PLY into a mouse genetically prepore oligomer assembly) prevents chemoattractant secretion ablated for HXA3 production, the numbers of PMNs present in the by epithelial cells. Therefore, PLY-mediated pore formation is airways were markedly reduced relative to WT mice. These necessary to stimulate the observed chemoattractant secretion findings support a model in which PLY, by activating phospholi- and subsequent influx of neutrophils, an assertion consistent pase activity, releases AA from the plasma membrane and stim- with our analysis of other pore-forming toxins. ulates the production of HXA3 to promote PMN influx. Although PLY-mediated pore formation is required to trigger PLY might activate phospholipase activity through its best PMN transmigration, the production and secretion of chemo- characterized activity, the formation of ∼25-nm pores in eukary- attractant is not simply a reflection of cell lysis, but instead, is an otic membranes (13, 60). In fact, the pore-forming activity of PLY active process. Exposure of H292 cells to 20 U or more of PLY, is required to activate phospholipase A in cultured endothelial which triggers pore formation in 100% of H292 cells, did not cells (25). In this study, we found that PLY derivatives deficient in induce PMN migration. In contrast, 2.5 U of PLY, which resulted in any of the several steps leading to pore formation, such as cho- membrane permeabilization (indicated by PI staining) of 62% of lesterol binding or conformational changes leading to pore in- H292 cells, triggered significant chemoattractant activity. Several sertion into the lipid bilayer, were unable to elicit significant studies have revealed that host cell repair mechanisms are quickly neutrophil migration in vitro or in the lung after i.t. instillation. initiated to prevent cell death after CDC-dependent membrane Three related CDCs, ILY, SLO, and PFO, as well as C. septicum permeabilization (47–49), and in this study, we found that the a-toxin, which generates pores with cross-sectional areas nearly majority of H292 cells that became permeabilized upon exposure 300-times smaller than the CDCs, all induced PMN transmigra- to PLY-producing S. pneumoniae strain TIGR4 were capable of tion in vitro. Given that cPLA2a is calcium-regulated (69, 70), an membrane repair, as indicated by exclusion of a subsequently attractive hypothesis is that PLY-induced influx of ions, which added (different) membrane impermeant dye. Finally, we found includes Ca2+, into host cells (71–75) leads to the activation of that chemical inhibitors of 12-LOX reduced PMN transmigra- cPLA2a or other phospholipases and the production of chemo- tion, indicating that an active metabolic pathway capable of attractant lipids such as HXA3. Indeed, we found that treatment of producing HXA3 is required for production and/or release of lung epithelial monolayers with the calcium ionophore ionomycin chemoattractant activity. The Journal of Immunology 9

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