Bacterial lipopolysaccharide induces endothelial ... - The FASEB Journal

Bacterial lipopolysaccharide induces endothelial cells to synthesize a degranulating factor for neutrophils EDWARD A. GILL,*,1,2 TADA-ATSU IMAIZUMI,*,1,3 HOLLY CARVETH,*,† MATTHEW K. TOPHAM,*,†,‡ E. BART TARBET,* THOMAS M. McINTYRE,*,†,§ STEPHEN M. PRESCOTT,†,‡,H AND GUY A. ZIMMERMAN*,†,4 *Nora Eccles Harrison Cardiovascular Research and Training Institute, ‡Eccles Program in Human Molecular Biology and Genetics, and †Departments of Medicine, HBiochemistry, and §Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84112–5000, USA

Enzymes and other factors secreted by degranulating neutrophils (polymorphonuclear leukocytes, PMNs) mediate endothelial injury, thrombosis, and vascular remodeling. In bacteremia and sepsis syndrome and their consequent complications (including acute respiratory distress syndrome and systemic ischemia-reperfusion resulting from septic shock), neutrophil degranulation is an important mechanism of injury. In related studies, we found that human endothelial cells regulate neutrophil degranulation and that inflammatory cytokines induce synthesis of degranulating factors by human endothelial cells. Here we show that lipopolysaccharides (LPS) from gram-negative bacteria were the most potent agonists for release of degranulating activity by endothelial cells when compared to several cytokines and stimulatory factors. LPS also induced the release of degranulating signals for PMNs from a human endothelial cell line, EA.hy 926. Interleukin 8 (IL-8) is synthesized by endothelial and EA.hy 926 cells in response to LPS and induces neutrophil degranulation. However, complementary strategies using receptor desensitization, translation of messenger RNA by Xenopus laevis oocytes, and purification and analysis of factors from conditioned supernatants demonstrated that degranulating factors distinct from IL8 are generated in response to LPS. The characteristics of a partially purified degranulating factor isolated from conditioned supernatants distinguished it from known chemokines and other factors that induce PMN degranulation and are generated by endothelial cells in response to LPS. Thus, cultured human endothelial cells and endothelial cell lines synthesize several unique signaling molecules that can trigger neutrophil granular secretion. If produced in vivo in response to LPS or other pathologic agonists, these degranulating signals may activate PMNs in combination or in sequence, initiating or propagating vascular damage.—Gill, E. A., Imaizumi, T.-a., Carveth, H., Topham, M. K., Tarbet, E. B., McIntyre, T. M., Prescott, S. M., Zimmerman, G. A. Bacterial lipo-

ABSTRACT

polysaccharide induces endothelial cells to synthesize a degranulating factor for neutrophils. FASEB J. 12, 673–684 (1998) Key Words: PMN · polymorphonuclear leukocytes · PMN degranulation · lactoferrin

HUMAN ENDOTHELIAL cells respond to stimulation by expressing adhesion and signaling molecules for polymorphonuclear leukocytes (PMNs; neutrophils),5 monocytes, and other leukocytes (1, 2). Coordinate and regulated expression of adhesion and signaling factors by endothelial cells is a key feature in the orchestration of inflammatory responses, promoting precise targeting and activation of leukocyte subclasses in a temporally dependent pattern (2–4). In contrast, dysregulated adhesion or signaling of leukocytes by endothelial cells may be an important mechanism of inflammatory injury (1, 2, 5). The sub1 Edward Gill and Tada-atsu Imaizumi contributed equally to the work described in this paper. 2 Current address: Department of Medicine, University of Colorado Health Sciences Center, Box B130, 4200 E. Ninth Ave., Denver, CO 80262, USA. 3 Current address: Department of Pathologic Physiology, Institute of Neurological Diseases, Hirosaki University School of Medicine, Hirosaki, Japan. 4 Correspondence: University of Utah, CVRTI, 95 S. 2000 E. Back, Salt Lake City, UT 84112–5000, USA. 5 Abbreviations: LPS, lipopolysaccharides; PMNs, polymorphonuclear leukocytes; IL, interleukin; TNF-a, tumor necrosis factor; HBSS/A, Hank’s balanced salt solution containing human serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; r(h)GM-CSF, recombinant human granulocytemacrophage colony stimulating factor; fMLP, n-formyl-methionyl-leucyl-phenylalanine; BSA, bovine serum albumin; OPD, o-phenylenediamine; SEAP, secreted form of human placental alkaline phosphatase; DEPC-H2O, diethyl pyrocarbonatetreated water; ALP, alkaline phosphatase; PNPP, p-nitrophenylphosphate; OD, optical density; LF, lactoferrin; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; PBS-A, 1% BSA in PBS; PBS-T, 0.05% Tween-20 in PBS; FN, fibronectin; IFN, interferon; KPO4, potassium phosphate; Ig, immunoglobulin; PMA, phorbol myristate acetate; r(h)IL-8, recombinant human interleukin 8.

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types of leukocytes that accumulate and are active in defensive or injurious inflammatory conditions are, in part, temporally regulated. In acute inflammation, the PMN is a key effector cell that is tethered and activated by adhesion and signaling factors (3, 6). Signaling molecules for PMNs that are generated by stimulated endothelial cells include lipids (3), cytokines and chemokines (3, 6–8), and modulating factors such as nitric oxide (9). Human endothelial cells produce platelet-activating factor, a phospholipid that activates PMNs (2, 3), and interleukin-1 (IL-1), interleukin-6, and granulocyte-macrophage colony stimulating factor (GM-CSF), which are cytokines (10). Human endothelial cells also produce chemokines of the C-X-C and C-C classes (3, 6–8, 11). These polypeptide signaling factors are thought to be particularly important in the transfer of information between endothelial cells and leukocytes (8). The pattern of chemokines produced by human endothelium varies with the agonist, the time of stimulation, and other factors (12). IL-8, earlier called NAF and also given other designations, is the most intensely studied endothelial chemokine (3–11, 13, 14): it is a member of the C-X-C family and an important mediator of interactions with PMNs. There is evidence that proinflammatory peptides other than IL-8 may be synthesized by stimulated endothelium (13–15), although their identities and their individual and combinatorial actions on PMNs are largely unknown. We found recently that a second C-X-C chemokine, ENA-78, is synthesized by stimulated human endothelial cells and that it induces functional alterations in PMNs in combination with IL-8 (16). Endothelial activation occurs in bacteremia, the sepsis syndrome, and septic shock (1). Synthesis of signaling factors by endothelial cells in response to microbial toxins then leads to activation of PMNs, resulting in the generation of oxygen radicals and extracellular liberation of their granular contents. Release of enzymes and other constituents by degranulating neutrophils may be especially important in subsequent vascular injury and can cause damage to endothelial cells independent of the injury induced by oxygen radicals (17–21). Lipopolysaccharide (LPS; endotoxin), a major toxin of gram-negative bacteria, can initiate an injurious cycle of endothelial stimulation and consequent signaling and activation of locally sequestered PMNs (1). In addition to its direct effects on endothelial cells, LPS also ‘primes’ PMNs for enhanced degranulation (19) and can directly induce degranulation under some conditions (22). In companion studies, we found that human endothelial cells regulate PMN degranulation and that the cytokines IL-1 and tumor necrosis factor (TNFa) induce cultured human endothelial cells to synthesize and release degranulating factors for PMNs (23). Expression of signaling factors, including those 674

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that trigger degranulation of neutrophils, is important in physiologic responses of the endothelium (8, 23, 24), but it may also be a mechanism of vascular injury if induced in an uncontrolled fashion or by a pathologic agonist (1). Here we show that LPS was the most potent stimulus for generation of degranulation signals by endothelial cells when compared to cytokines under the conditions of our experiments. We also demonstrate that degranulating factors distinct from IL-8 are generated when human endothelial cells and a human endothelial cell line, EA.hy 926 (25), are stimulated with LPS and we report initial characterization of one of the degranulating factors.

MATERIALS AND METHODS Reagents Hank’s balanced salt solution (HBSS), Dulbecco’s modified Eagle’s medium (DMEM), and penicillin/streptomycin were from Whittaker M.A. Bioproducts (Walkersville, Md.). Fetal calf serum was from HyClone Laboratories, Inc. (Logan, Utah). Human serum albumin was from Miles Laboratories, Inc. (Elkhart, Ind.). Recombinant human interleukin 8 [r(h)IL-8, endothelial cell type, 77 amino acids], recombinant human granulocyte-macrophage colony stimulating factor [r(h)GM-CSF], r(h)IL-6, murine monoclonal anti-human GM-CSF antibody, and polyclonal rabbit anti-human GM-CSF antibody were from Genzyme (Cambridge, Mass.). Polyclonal goat anti-human IL-8 antibody was from R&D Systems (Minneapolis, Minn.). Anti-rabbit immunoglobulin (Ig), peroxidase-linked, species-specific Fab*2 fragment (from donkey) was from Amersham (Arlington Heights, Ill.). Human lactoferrin (LF) standard was from J. T. Baker, Inc. (Phillipsburg, N.J.). LPS from Escherichia coli serotype 0III:B4, gelatin, ethyl-3aminobenzoate, collagen type II, n-formyl-methionyl-leucylphenylalanine (fMLP), rabbit anti-human LF antibody, peroxidase-conjugated avidin, bovine serum albumin (BSA), o-phenylenediamine (OPD), L-homoarginine, CNBr-activated Sepharose 6MB, guanidinium thiocyanate, and N-laurylsarcosine were from Sigma (St. Louis, Mo.). Oligo(dT)-cellulose spun columns were from Pharmacia (Gaithersburg, Md.). HindIII and cap structure analog m7-GpppG were from Stratagene (La Jolla, Calif.); SP6 RNA polymerase was from Promega (Madison, Wis.). Twelve-well tissue culture plates and EIA microtiter plates were from Costar (Cambridge, Mass.). The plasmid vector pGEM-4Z containing a cDNA encoding the secreted form of human placental alkaline phosphatase (SEAP) was a kind gift from Dr. J. Kaplan (Salt Lake City, University of Utah). Cell culture EA.hy 926 cells were a generous gift from Dr. C.-J.S. Edgell (University of North Carolina). EA.hy 926 cells were cultured in T-75 flasks in DMEM (high glucose) supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and other additives as described (25) at 377C in a humidified atmosphere of 95% air and 5% CO2. For generation of samples for purification of degranulating factors, EA.hy 926 cells were grown on Cytodex beads (Pharmacia). The cells adherent to beads were maintained at a density of 6.5 1 106 cells/ml in spinner flasks (Bellco). LPS (5 mg/ml) was added to the culture medium and the cells were incubated for 1 h with constant stirring. At the end of the incubation

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period, the medium was separated from the beads and they were washed once with HBSS. Fresh HBSS containing 0.5 mg/ ml human serum albumin (HBSS/A) was added, the cells were incubated for an additional 4 h, and the supernatant was collected. Preliminary experiments demonstrated that EA.hy 926 cells on beads could be used repetitively to generate conditioned supernatants if enough time was allowed between stimulation periods. Conditioned supernatants were frozen until volumes sufficient for analysis were in hand. They were then pooled and fractionated by chromatography as described below. Human umbilical vein endothelial cells were cultured as described (26). Cytosol from endothelial cells was prepared according to a published method (27).

Levels of alkaline phosphatase (ALP) in the medium of oocytes were assayed by monitoring the rate of hydrolysis of pnitrophenylphosphate (PNPP) essentially as described (29). An aliquot (50 ml) of the medium from each oocyte or standard p-nitrophenol was transferred to a well of a 96-well plate containing 50 ml/well of assay buffer (1 M diethanolamine, pH 9.8, 0.5 mM MgCl2, 0.02 mM, ZnSO4, and 20 mM L-homoarginine). The plate was warmed to 377C for 10 min, and 50 ml/well of warmed 50 mM PNPP was added to each well. After the incubation for 60 min, the optical density (OD) at 405 nm was recorded with a microplate reader (Molecular Devices, Menlo Park, Calif.).

Preparation of mRNAs

Assays of PMN degranulation

Messenger RNA was prepared for heterologous expression of degranulating factors in Xenopus oocytes (see below). Confluent EA.hy 926 cells were stimulated with 5 mg/ml LPS and the cells were incubated for 4 h at 377C. After the LPS-containing medium was removed, the cells were washed twice with HBSS. Control monolayers were treated with buffer alone. RNA was extracted from the EA.hy 926 cells with guanidinium thiocyanate and N-larylsarcosine by a modification of conventional methods (28). The poly(A)/mRNA was purified by using oligo(dT)-cellulose spun columns. mRNA of SEAP was produced from the plasmid pGEM-4Z containing cDNA encoding SEAP (29). The plasmid vector was amplified, purified, and linearized by digestion with HindIII. The linearized plasmid was transcribed with SP6 RNA polymerase in the presence of the cap structure analog m7-GpppG. RNA concentration was quantitated by its absorbency at 260 nm. Translation of mRNAs in Xenopus oocytes Injection of mRNA into oocytes and translation of factors (30) were performed as previously described (28), with minor modifications. Adult female Xenopus laevis frogs were anesthetized by immersion in 0.3% ethyl-3-aminobenzoate. Ovarian follicles were surgically removed through a small incision in the abdomen and immediately placed in a Ca2/-free OR2 solution (82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1 mM Na2HPO4, 5 mM HEPES, pH 7.4). The follicles were separated into small clumps of approximately 10 to 30 large oocytes. The oocytes were then treated with 2 mg/ml collagenase for 2 h at room temperature in the Ca2/-free OR2 solution with mild agitation. The denuded oocytes were washed thoroughly and then stored overnight at 197C in modified Barth’s solution (MBSH: 88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 2.4 mM NaHCO3, 10 mM HEPES, pH 7.4). Xenopus oocytes were injected with poly(A)/mRNA from LPS-stimulated or control, unstimulated EA.hy 926 cells. Control oocytes were injected with diethyl pyrocarbonate-treated water (DEPC-H2O) or with the cRNA for SEAP (see above). The poly(A)/mRNA from EA.hy 926 cells or SEAP cRNA was redissolved in DEPC-H2O at a concentration of 1 mg/ml or 0.1 mg/ml, respectively. Healthy-appearing stage V–VI oocytes were injected with either 50 nl of DEPC-H2O containing RNA or DEPC-H2O alone. The injected oocytes were individually transferred to wells of 96-well flat-bottom culture plates and maintained in 100 ml of MBSHII solution (MBSH supplemented with 1 mg/ml BSA, 1 mg/ml Ficoll, 100 U/ml penicillin, 100 mg/ml streptomycin) at 197C. The culture medium was changed daily for up to 6 days and then assayed for degranulating activity.

Alkaline phosphatase assay

We used the release of LF, a specific granule constituent, from PMNs as a marker of degranulation (31). Human blood was obtained from healthy volunteers after informed consent. PMNs were isolated and suspended at a final concentration of 5.5 1 106 cells/ml in HBSS/A. The wells of Costar culture plates were coated with 0.2% gelatin at 47C overnight. A PMN suspension (450 ml) was layered over the gelatin matrix and 50 ml of oocyte medium, r(h)IL-8, r(h)GM-CSF, r(H)IL-6, or fMLP solution was added. After incubation for 90 min at 377C (5% CO2), the PMNs were removed and centrifuged to pellet the cells (12,000 g, for 3 min). The supernatants were collected and the concentration of LF was assayed by enzymelinked immunosorbent assay (ELISA). In some experiments, PMNs were incubated with agonists for 120 instead 90 min. Using 96-well EIA microtiter plates, 100 ml/well of 10 mg/ ml rabbit anti-human LF polyclonal antibody in coating buffer (0.16 M Na2CO3, 0.35 M NaHCO3, pH 9.6, 10 mg/ml) was immobilized by incubating at 47C for 12 h. The plate was then washed six times with 0.05% Tween-20 in phosphate-buffered saline (PBS, pH 7.4) (PBS-T). Immediately before use, PMN supernatants were diluted with 1% BSA in PBS (PBS-A) to appropriate concentrations for the assay (usually 50-fold). The samples and LF standards (100 ml each) were added to the plate and incubated at 377C for 90 min. The plate was washed six times with PBS-T, and then 100 ml of horseradish peroxidase-conjugated rabbit anti-human LF IgG (24 mg/ml in PBSA) was added and incubated at 377C for 60 min. The plate was again washed six times with PBS-T and 100 ml of the peroxidase substrate (0.4 mg/ml OPD, 0.01% Hili) in citric acid buffer (103 mM NaHPO4, 49 mM citric acid, pH 5.0) was added. After incubation at room temperature for 5–10 min to allow development, 50 ml/well of 1 N H2SO4 was added to stop the reaction; the OD at 490 nm was then determined. We used an arbitrary unit of degranulation in certain experiments to compare degranulating activities in conditioned supernatants generated under various conditions—in fractions of partially purified degranulating factors, for example. A unit of degranulating activity was defined as that which induced the release of 1000 ng/ml LF when PMNs were assayed as detailed above. In some experiments, elastase release from PMNs was measured as a marker of primary granule secretion, as described previously (31). Immunoprecipitation of LF releasing activity Goat anti-human IL-8, rabbit anti-human GM-CSF, or goat anti-human fibronectin (FN) polyclonal antibodies were coupled to CNBr-activated Sepharose 6MB, as described (16). Approximately 500 mg of each antibody was coupled to 750 ml swollen gel. We incubated 150 ml of oocyte medium, r(h)IL-8 (final concentration 2 1 1008 M), r(h)GM-CSF (20 ng/ml),

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or fMLP (final concentration 1007 M) with an equal volume of antibody-coupled Sepharose 6MB saturated with 50 mM Tris-HCl (pH 7.5) at 377C for 2 h with rocking. After incubation, the Sepharose gel was removed by centrifugation and the LF releasing activity in the supernatant was assayed as above.

starting material. The column was perfused at a rate of 1 ml/ min and fractions were collected and assayed for degranulating activity.

ELISAs for IL-8 and GM-CSF

RESULTS

These assays were performed by using methods that have already been described (16, 31). Microtiter plates were coated with 100 ml/well of goat anti-human IL-8 polyclonal antibody (5 mg/ml) or mouse anti-human GM-CSF monoclonal antibody (2 mg/ml) at 47C for 12 h, and the plates were washed six times with PBS-T. The samples or standards (100 ml/well) were added and incubated at 377C for 90 min. The plates were washed six times with PBS-T. Then 100 ml/well of second antibody in PBS-A (5 mg/ml biotin-labeled goat polyclonal antihuman IL-8 antibody or 2 mg/ml biotin-labeled rabbit polyclonal anti-human GM-CSF polyclonal antibody) was added and incubated at 377C for 60 min. The plates were washed again six times, and 100 ml/well of horseradish peroxidase-conjugated avidin (1.25 mg/ml in PBS-A) or horseradish peroxidase-conjugated donkey anti-rabbit Ig (1/500 diluted in PBS-A) was added. The plates were incubated at 377C for another 60 min, washed six times, peroxidase substrate was added, stopped, and the OD at 490 nm was determined as shown above.

LPS is a potent agonist for the generation of degranulating activity by primary human endothelial cells and the EA.hy 926 endothelial cell line

Purification of degranulating factors from supernatants of EA.hy 926 cells stimulated with LPS

We showed previously that IL-1 and TNF-a induce cultured human endothelial cells to release degranulating factors for human neutrophils (23). To identify conditions that would be optimal for further characterization of these signaling factors for granular secretion, we compared different agonists known to stimulate synthetic responses in human endothelial cells for their ability to stimulate release of degranulating activity. Treatment of endothelial cell monolayers with LPS induced this response (Fig. 1 and data not shown). LPS was considerably more potent than IL-1 or TNF-a and generally was equivalent to phorbol myristate acetate (PMA), a powerful en-

Modifications of general strategies and methods for protein purification (32) were used as outlined in the following sections. DEAE Sepharose CL-6B Supernatant (1–2 l) from LPS stimulated EA.hy 926 cells were collected and fractionated on an 800 ml DEAE Sepharose CLB6 column equilibrated with 20 mM potassium phosphate (KPO4) buffer (pH 7.2). The supernatant was diluted 1:2 with this buffer, loaded onto the column, and washed with 1.5 column volumes of KO4 buffer. A 1.6 l linear gradient from 0– 400 mM KCl, followed by an 800 ml wash with 400 mM KCl, was used to elute bound protein in 9.5 ml fractions. Neutrophil degranulating activity was measured in these fractions as described above. Activity peaks of interest were concentrated over a YM-30 filter to a volume of 10 ml and used to perform the next purification step, which employed dye chromatography. Reactive green 19 Concentrated buffer (10 ml) from the DEAE chromatography step was used. A 60 ml reactive green 19 column was equilibrated with 20 mM KPO4 buffer (pH 7.2). Samples of 3 ml were collected and assayed for activity. The peaks of activity obtained from this step were concentrated over YM-10 filters to a volume of 2 ml for Superdex chromatography. Superdex 16/60 (sizing) A Superdex 16/60 column (Pharmacia) was equilibrated with 20 mM KPO4 buffer containing 170 mM NaCl and 16.2 mM cholate (pH 8.0). Buffer (1 ml) containing the activity obtained from the reactive green 19 column was used as the 676

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Figure 1. LPS induces the release of degranulating factors for PMNs from cultured human endothelial cells. Cultured human umbilical vein endothelial cells were incubated in medium alone or with LPS (20 mg/ml), IL-1 (50 U/ml), TNF-a (100 U/ml), or PMA (1006 M) for 2 h a 377C in 5% CO2, 95% air. The medium was removed, the monolayers were washed with HBSS, and the cells were covered with fresh HBSS/A. After a 2 h incubation at 377C in 5% CO2, 95% air, the HBSS/ A was removed and assayed for PMN degranulating activity, using lactoferrin release as the index marker (see Methods). The protocols for the times of incubation of the endothelial cells with the agonists and collection of degranulating activity were based on earlier experiments with human umbilical vein endothelial cells stimulated with IL-1 (23). The figure depicts individual assays from one experiment and is representative of three experiments. The data are expressed in arbitrary units of degranulation, which were devised to facilitate comparison of the magnitude of degranulation in certain experiments and to compare yields in subsequent strategies aimed at purifying degranulating factors (see Methods).

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dothelial agonist, although in some experiments the degranulating activity released from endothelial cells treated with LPS was greater than that secreted in response to PMA (Fig. 1). Many other cytokines and agonists reported to activate endothelial cells did not induce release of degranulating activity, including IL2, interferon gamma (IFN-g), platelet-derived growth factor, neurokinin, and platelet-activating factor. Pretreatment with IFN-g did not alter the release of degranulating activity when endothelial cells were subsequently stimulated with LPS, although this cytokine modulates certain endothelial responses to LPS (10, 33). The degranulating activity present in supernatants collected from LPS-stimulated endothelial cells triggered release of substantial amounts of LF from PMNs. For example, the supernatants collected in the experiment shown in Fig. 2 at 1, 3, and 4 h after stimulation induced secretion of 43%, 55%, and 53%, respectively, of the total LF in a lysate of unstimulated PMNs from the same suspension. However, the fraction of total LF secreted in response to degranulating activity generated by LPS-stimulated endothelial cells varied considerably from experiment to experiment and was influenced by the specific conditions used; therefore,

Figure 2. Degranulating factors are rapidly synthesized and secreted by LPS-stimulated human endothelial cells. HUVEC monolayers were incubated with LPS for the times shown as described in Fig. 1. They were then washed, covered with HBSS/A, and incubated for an additional 2 h. At the end of the incubation periods, the supernatants were collected, the cell monolayers were lysed, and cytosol was prepared. Supernatants and lysates from each time point were assayed for degranulating activity. The total degranulating activity (supernatant / cellular lysate) at 1, 2, and 4 h of incubation was 6, 7, and 6.2 units, respectively. The degranulating activity in endothelial cells treated with control buffer for 4 h in the place of LPS was 0.28 units. See Methods and Fig. 1 for a definition of the arbitrary unit of degranulation.

the magnitude of granular secretion is expressed in concentrations of LF or in arbitrary units of degranulation (see Fig. 1 and Methods), depending on the experimental protocol. Generation of degranulating activity depended on the concentration of LPS (1–20 mg/ml) and the duration of its incubation with endothelial cells before collection of conditioned buffer for assay. Degranulating activity was generated by 1 h after stimulation with LPS and was variably present as long as 24 h after stimulation. Most activity was released into the supernatant buffer, although 5– 20% of total activity remained associated with cell lysates depending on the time of incubation (Fig. 2). LPS from a variety of different bacteria induced release of degranulating activity. Direct measurements of LPS concentrations in conditioned supernatants revealed that between 1% and 10% of the starting amount used to stimulate the endothelial cells was carried over, depending on the experiment. However, the degranulating activity was not due to carryover of LPS itself, as shown in experiments in which polymyxin B was added to neutralize residual LPS (34) in incubations of conditioned supernatants with PMNs. In a second strategy, direct addition of LPS to PMN preparations in the concentrations equivalent to those that may have been carried over from stimulated endothelial cells did not induce degranulation. Finally, pretreatment of endothelial monolayers with the transcriptional blocking agent actinomycin D partially inhibited release of degranulating activity, indicating that a response of the endothelial cells to LPS was required. The EA.hy 926 cell line, produced by fusion of human umbilical vein endothelial cells with human A549 carcinoma cells, has many phenotypic features of primary endothelial cells (25, 35–38). We found that treatment of monolayers of EA.hy 926 cells with LPS also induced release of degranulating activity. The characteristics were similar to those of primary HUVEC monolayers, including the concentrations of LPS required and the time course of release of degranulating activity. As with HUVEC, LPS was more potent as an agonist for generation of degranulating activity than was IL-1 or TNF-a. This was demonstrated in three experiments, including one in which EA.hy 926 cells and primary HUVEC monolayers were compared in parallel. The two cell types were different in that TNF-a was a poor agonist for EA.hy 926 cells but was equivalent to IL-1 in experiments with HUVEC. The degranulating activity released by LPS-stimulated EA.hy 926 cells partitioned into the aqueous phase of an organic/water biphasic system and was degraded by treatment with trypsin and other proteases. These results indicated that the degranulating activity includes one or more proteins or polypeptides.



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Desensitization of receptors for IL-8 on neutrophils does not block degranulation in response to LPSinduced factors We found that EA.hy 926 cells stimulated with LPS express mRNA for IL-8 when assayed by Northern analysis and secrete IL-8 into the incubation medium (not shown). Because it can induce neutrophil degranulation under the conditions of our experiments (23), we used homologous desensitization (39, 40) to determine whether this chemokine accounted for the degranulating activity and whether engagement of its receptors on the PMN is required for the response. Treatment of PMNs with IL-8 causes internalization of the majority of copies of CXCR1 and CXCR2, the receptors that recognize it, within minutes (reviewed in ref 40). Under appropriate conditions, this desensitizes the leukocytes to subsequent stimulation by a second application of IL-8. We pretreated PMNs with concentrations of recombinant IL8 that, in preliminary experiments, were determined to desensitize them to a second stimulation with IL8 but did not prevent degranulation in response to fMLP, indicating that the desensitization was specific and homologous (not shown). We then incubated neutrophils treated in this fashion with supernatants from LPS-stimulated EA.hy 926 cells and found that degranulation was minimally inhibited (Fig. 3). De-

Figure 3. Supernatants from LPS-stimulated EA.hy 926 cells contain a degranulating factor that is not blocked by desensitizing PMN IL-8 receptors. PMNs were pretreated with buffer or recombinant IL-8 (2.511008 M) for 15 min at 377C. They were then pelleted by centrifugation and resuspended in supernatant from EA.hy 926 cell monolayers treated with LPS as in Fig. 1, HBSS/A containing recombinant IL-8 (1007 M), or control buffer, as indicated below the bars. After 2 h incubation, the amount of lactoferrin released was measured by ELISA. The filled bars indicate the values from PMNs pretreated with buffer, and the hatched bars indicate those from PMNs pretreated with IL-8. In a second experiment, there was a similar pattern with supernatants from LPS-stimulated HUVEC monolayers. 678

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sensitization of PMNs under these conditions also failed to block the degranulating response of PMNs when they were treated with supernatants from LPSstimulated HUVEC. These findings indicated that a degranulating factor other than IL-8 is released by LPS-stimulated EA.hy 926 cells. They also indicate that this factor is not ENA-78 (16) or one of the Gro homologues (12, 41), since these C-X-C chemokines are recognized by the low-affinity receptor for IL-8 on neutrophils (CXCR2; IL-8RB) (42–44). Xenopus oocytes injected with poly(A)/mRNA from LPS-stimulated EA.hy 926 cells secrete degranulating activity We used the X. laevis oocyte expression system to further define the contributions of individual factors to the degranulating activity produced by EA.hy 926 cells in response to LPS. LF release from PMNs was again used as the reporting assay for degranulation. Oocytes were injected with poly(A)/mRNA isolated from LPS-stimulated EA.hy 926 cells, and the medium bathing the oocytes was collected daily thereafter. We found degranulating activity in these samples, with peak activity present 3 days after injection (Fig. 4, upper panel). In general, the activity released by oocytes was lower than that released by LPS-stimulated EA.hy 926 cells. In some experiments we coinjected oocytes with the cRNA for an independent secreted marker, secretory alkaline phosphatase (29), and found that the profile of its release paralleled that of degranulating activity (Fig. 4, lower panel). In contrast, there were background levels of both activities in media collected from oocytes injected with DEPC-H2O, the vehicle for RNA when it was introduced into the oocytes (Fig. 4). We found that 25–45% of oocytes injected with poly(A)/mRNA from LPS-stimulated EA.hy 926 cells secreted degranulating activity (LF released by treated PMNs ú 150– 200 ng/ml), whereas only 10% or fewer oocytes secreted activity when injected with poly(A)/mRNA from control cells that had been treated with buffer instead of LPS. Together, these results demonstrate that degranulating factors are translated from mRNA in LPS-stimulated EA.hy 926 cells; the results exclude the release of degranulating activity by oocytes as a nonspecific response to the physical process of injection or to injecting control or irrelevant RNAs, and show that this system could be used for additional characterization of degranulating factors. The degranulating activity released by injected oocytes was sensitive to protease digestion (not shown), as was the activity released from LPS-stimulated EA.hy 926 cells and HUVEC (see above). Immunologic analysis of degranulating activity released by injected oocytes We assayed the conditioned oocyte medium and found that IL-8 was present (see below), again similar

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CSF (20 ng/ml) assayed under similar conditions. Neither antibody reduced the degranulating activity of fMLP (1007 M) when added exogenously in parallel incubations, indicating their specificity. We then examined the effect of these antibodies on the degranulating activity secreted by oocytes injected with mRNA from LPS-stimulated EA.hy 926 cells and found only a 26% reduction by immunoprecipitation with anti-IL-8 (Fig. 5). Incubation of aliquots of the same samples with anti-GM-CSF reduced the activity by a mean of 8%. These findings supported the conclusion that IL-8 alone does not account for the degranulating activity, as indicated by desensitization experiments (above), and indicated that factors other than GM-CSF are present. To further document the conclusion that IL-8 alone did not account for the secreted degranulating activity, we performed the desensitization experiments described above and found that pretreatment of neutrophils with IL-8 did not desensitize them to degranulating factors present in conditioned oocyte medium injected with mRNA from LPS-stimulated monolayers (not shown). In parallel, we measured the concentrations of IL-8 and GM-CSF secreted into the medium by oocytes injected with RNA from LPS-stimulated EA.hy 926 cells and found that the amounts present could not account for the degranulating activity when compared to the concentration-response relationships of recombinant IL-8 (Fig 6) and GM-CSF (not shown). Figure 4. Xenopus laevis oocytes secrete degranulating factors for PMNs when injected with mRNA from LPS-stimulated EA.hy 926 cells. Oocytes were microinjected with 50 nl of solution containing 50 ng poly(A)/mRNA from LPS-stimulated EA.hy 926 cells, 5 ng cRNA for SEAP, or DEPC-H2O. The oocytes were then cultured at 197C for 6 days. The medium was removed every 24 h; LF releasing activity (upper panel) or ALP activity (lower panel) was assayed as an index of degranulating factor release and secretion of a newly translated protein, respectively (see text). The values are the means of 31 oocytes injected with cRNA for SEAP or 16 oocytes injected with DEPC-H2O (lower panel) and the means of 17 oocytes injected with poly(A)/mRNA from LPS-stimulated EA.hy 926 cells or 5 oocytes injected with DEPC-H2O (upper panel).

to our observations with endothelial and EA.hy 926 cells. We then determined whether the degranulating activity in the conditioned medium from microinjected oocytes was immunoprecipitated by an antibody against IL-8 coupled to Sepharose beads. As a control, we used an antibody against GM-CSF, which is also synthesized by endothelial cells in response to LPS (T. Imaizumi et al., unpublished observations). In preliminary experiments, anti-human IL-8 precipitated 73% of the degranulating activity when exogenous r(h)IL-8 (211008 M) was added to the control oocyte medium. Anti-human GM-CSF antibody precipitated 67% of the activity of r(h)GM-

A degranulating factor distinct from IL-8 can be physically separated from conditioned supernatants from LPS-stimulated EA.hy 926 cells We collected large volumes of supernatant (Ç1–2 l) from EA.hy 926 cells that were grown on Cytodex beads and stimulated with LPS, and used these samples for partial purification of degranulating factors. Fractionation of supernatants from LPS-stimulated cells by ion exchange chromatography yielded three major peaks of degranulating activity (Fig. 7, upper panel). This pattern was seen in multiple experiments using the same or similar chromatographic conditions. Peak III caused potent degranulation as measured by release of LF (Fig. 7, upper panel) and elastase (not shown), approaching that induced by direct treatment of PMNs with PMA in parallel incubations, and contained background levels of IL-8 and GM-CSF by ELISA. This fraction was therefore used for further purification. We found that dye chromatography using a reactive green 19 column yielded an approximate 50-fold increase in specific activity, although this was variable from run to run; the eluted material was again free of IL-8 and GMCSF by ELISA. Material isolated by sequential ion exchange and green 19 chromatography was then subjected to gel filtration chromatography (Fig. 7,



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Figure 5. Degranulating factors in addition to IL-8 are secreted by oocytes injected with mRNA from LPS-stimulated EA.hy 926 cells. The medium from oocytes injected with poly(A)/mRNA from LPS-stimulated EA.hy 926 cells was collected, pooled from 2 to 4 days after the injection, and the LF releasing activity was assayed. The pooled conditioned oocyte medium, r(h)IL-8 (211008 M), r(h)GM-CSF (20 ng/ml), or fMLP (1007 M; control agonist) diluted in fresh oocyte medium was incubated at 377C for 2 h with antibodies against human FN (control), human IL-8, or human GM-CSF, which were coupled to Sepharose 6MB. After incubation, the antigen–antibody–Sepharose complexes were removed by centrifugation and the lactoferrin releasing activity remaining in the supernatant was determined. In parallel, recombinant human IL-8 (211008 M), recombinant human GM-CSF (20 ng/ml), or fMLP (1007 M) in HBSS/A was incubated with the same panel of antibodies, and neutralization of their PMN degranulating activities was determined. The mean values { SD from three experiments are shown.

lower panel). The molecular mass of the major peak of activity, migrating in and around fraction 26, was estimated to be 30–40 kDa. Analysis of a portion of this peak by sodium dodecyl sulfate gradient gel

yielded a single major band corresponding to this molecular mass as well as several minor bands. Additional samples prepared by sequential ion exchange, dye, and gel filtration chromatography were used for further biologic characterization of the degranulating factor. The purified material induced adhesiveness of PMNs in an assay that measures inside-out signaling of b2 integrins and also induced shedding of L-selectin and superoxide production (data not shown). Buffering of intracellular Ca2/ by treatment of the PMNs with BAPTA (1,2 bis(o-aminophenoxy)ethane-N,N,N*N*-tetraacetate) blocked degranulation in response to the purified factor. Thus, the degranulating factor induces other activation responses of PMNs and requires an increase in intracellular Ca2/ for its action, a mechanism known to be required for degranulation in response to a number of other neutrophil agonists (45).

DISCUSSION Figure 6. The concentration of IL-8 present in conditioned medium from injected oocytes is insufficient to account for the degranulating activity. The medium from 40 oocytes injected with poly(A)/mRNA from LPS-stimulated EA.hy 926 cells was collected from 2–4 days after injection. The concentration of IL-8 secreted into the medium by each individual oocyte was measured by ELISA in an aliquot of the conditioned medium and the mean IL-8 concentration was determined. The PMN degranulating activity was also measured in an aliquot of the medium from each oocyte and the mean degranulating activity was determined. Purified PMNs were incubated with various concentrations of recombinant IL-8, as shown on the abscissa. By comparing the mean degranulating activity in the oocyte media (dashed line indicated by the open arrow) with the mean concentration of IL-8 in these samples (indicated by the filled arrow) and the IL-8 concentration– response curve (filled circles connected by solid line), it can be seen that insufficient IL-8 is present in the conditioned oocyte medium to account for the activity. 680

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We show here that potent signaling factors that induce neutrophil degranulation are produced when primary endothelial cells and the EA.hy 926 endothelial cell line are stimulated with LPS. Although IL8 is thought to be the principal polypeptide signaling molecule for PMNs produced by stimulated endothelium (4, 6–8, 11, 13, 14), we found that additional factors that induce degranulation are synthesized. This conclusion is based on complementary experimental strategies using receptor desensitization, a X. laevis translation system, and protein separation. With the latter approach, we provide initial characterization of a new degranulating factor. Although we originally evaluated LPS as an agonist for production of degranulating factors by endothelial cells in order to identify optimal conditions for purification of

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Figure 7. Physical separation of a degranulating factor synthesized by LPS-stimulated EA.hy 926 cells. Upper panel: EA.hy 926 cells were grown on Cytodex beads in spinner flasks and stimulated with LPS. The conditioned supernatants were collected, pooled, and fractionated by DEAE chromatography as described in Methods. Lower panel: The third peak of activity (peak III, upper panel) was subjected to sequential dye chromatography on green 19 and gel filtration chromatography on Superdex 16/60. The peak of activity migrating in and around fraction 25 was further subjected to biologic characterization as described in the text.

these signaling molecules, its ability to induce this response has important implications for the pathogenesis of vascular injury. In experiments using both primary umbilical vein endothelial cells and EA.hy 926 cells as sources of signaling factors, desensitization of neutrophil receptors for IL-8 either had no effect or only partially inhibited the response of the leukocytes to degranulating activity in conditioned supernatants from LPS-stimulated cells. This result indicates that degranulating factors are present that bind to receptors different from CXC R1 (the IL-8 type A receptor), which recognizes IL-8, and CXC R2 (the IL-8 type B receptor), which recognizes IL-8, ENA-78, and members of the Gro family of C-X-C chemokines (40, 42– 44). Desensitization strategies have been used before to identify and characterize signaling factors that act by binding to specific receptors on PMNs and other target cells (46, 47). The results of our desensitization experiments are supported by studies that used the X. laevis oocyte translation system and demonstrated that degranulating factors immunologically distinct from IL-8 are synthesized when mRNA from LPSstimulated EA.hy 926 cells are microinjected. Earlier we had found that antibodies against IL-8 failed to neutralize the degranulating activity released by IL1-stimulated endothelial cells, indicating the presence of one or more additional signaling molecules (23). The desensitization studies (Fig. 3) and experiments using the oocyte system (Figs. 4–6) shown here complement and extend these earlier experi-

ments and support the conclusion that stimulated endothelial cells produce degranulating factors different from IL-8. In an additional approach, we found that a degranulating factor was physically separated from IL-8 when supernatants from LPS-stimulated EA.hy 926 cells were subjected to fractionation by using sequential chromatographic procedures (Fig. 7). The estimated molecular mass, 30–40 kDa, is considerably larger than that of IL-8, ENA-78, and Gro (42), supporting the results from desensitization experiments that indicated that these chemokines do not account for the degranulating activity (see foregoing text). The degranulating factor we purified is also larger than neutrophil-activating peptides previously reported to be released by LPS-stimulated human endothelial cells (48). Together, our experiments indicate that a molecule previously unknown to signal neutrophil degranulation is synthesized by LPS-stimulated endothelial cells and cell lines. IL-8independent signaling of PMN transmigration has been reported (13–15, 33), although the alternative factors were not identified and it is unknown whether they induce degranulation. In experiments not shown here, we surveyed a variety of factors that have putative neutrophil-activating properties and are synthesized by stimulated human endothelial cells, including platelet-derived growth factor, IL-6, and others (10, 49), and found that they did not individually reproduce the degranulating activity. Thus, the degranulating factor released under the conditions of our experiments may be a novel signaling mole-



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cule for neutrophils. Our initial characterization studies indicate that an increase in intracellular calcium is required when the degranulating factor triggers exocytosis by neutrophils and that it induces other activation-dependent functional responses in the leukocytes (see Results section), consistent with features of other signaling molecules that stimulate granular secretion (45). The first signaling factor for PMNs to be identified as a product of stimulated endothelial cells, plateletactivating factor (2, 3), is a phospholipid and is clearly different from the degranulating factor we describe here (Fig. 7). Although weak as an agonist for secretion of primary and specific granular contents (50), PAF can prime PMNs for enhanced degranulation in a juxtacrine fashion when coexpressed with P-selectin at the surfaces of endothelial cells (51). In addition, PAF induces translocation of secretory vesicles to the neutrophil surface (50). Limited degranulation involving this PMN organelle may be involved in transmigration (14). ENA-78, which is synthesized by endothelial cells stimulated by inflammatory cytokines or LPS (16), is a weak direct degranulating factor (T. Imaizumi, unpublished experiments), but may also prime PMNs for this response (44). As shown in our companion studies (23), IL-8 is a direct degranulating signal for neutrophils and contributes to the total degranulating activity released by cytokine-stimulated endothelial cells, although to a lesser degree than other factors when this is examined at specific times. Thus, a complex system of signaling molecules that influence PMN degranulation can be expressed by endothelial cells when they are stimulated or injured. The factor we purified here has potent direct degranulating actions (Fig. 7) and must be added to this list. One or more of these signaling factors may be critical effectors of leukocyte degranulation and consequent vascular damage if its synthesis is induced by LPS in bacteremia or sepsis or by other pathologic agonists (see below). These degranulating factors may also act together in an overlapping or synergistic fashion or in sequence. The pattern of synthesis of the factors that directly or indirectly influence degranulation is not stereotyped but varies with the endothelial agonist (23, 31; T. Imaizumi et al., unpublished observations). Neutrophil degranulation may be important in host defense against LPS and bacteria that synthesize this toxin. Bacterial permeability enhancing factor, which binds and inactivates LPS (34), is a constituent of PMN granules and is released during the process of degranulation (52). A fraction of the intracellular acetyloxyacylhydrolase that hydrolyzes LPS is also released during PMN degranulation (53). LF, the index marker of neutrophil degranulation used in our study, is reported to bind LPS and inhibit its ability to prime neutrophils (54). Also, activation of PMNs by signaling factors that induce other effector re682

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sponses in addition to degranulation may lead to the internalization and intracellular degradation of LPS (53). Thus, release of degranulating factors for neutrophils by endothelial cells may in some cases be a protective mechanism that reduces the effective local concentration of LPS at infected sites. However, synthesis of neutrophil degranulating factors by endothelial cells in response to LPS is unlikely to be uniformly protective under all conditions. There is abundant evidence that PMN degranulation is a mechanism of vascular injury when it is uncontrolled. Neutrophil granular proteases cause detachment of human endothelial cells from monolayer culture, potentially accounting for an oxygen radical-independent mechanism of vascular injury (17, 18). PMNs primed with LPS and stimulated with signaling factors cause injury to cultured human microvascular endothelial cells. The injury is mediated in large part by the secretion of elastase (19), a mechanism that may account for LPS-induced vascular permeability in vivo (55). Exocytosis of other granular constituents may also cause cellular injury and increased permeability of microvascular structures (21, 56, 57). PMN granular proteases are also synergistic with oxygen radicals in the killing of endothelial cells by PMNs (20). This synergistic interaction between neutrophil granular proteases and oxygen radicals may be a central feature of severe inflammatory tissue damage (58). Elastase and other granular proteases cleave proteoglycans from the endothelial cell plasma membrane in the absence of cytolysis, leading potentially to altered regulation of coagulant mechanisms at the vascular surface and thrombosis (59). Thus, neutrophil degranulating factors expressed by endothelial cells in response to LPS or other pathologic agonists (31) may be critical effectors of vascular injury. These in vitro observations are relevant to syndromes of vascular injury in humans. Release of granular enzymes by stimulated neutrophils appears to be a key pathogenetic mechanism of alveolar capillary injury in the acute respiratory distress syndrome, which is frequently caused by endotoxemia and gram-negative bacterial shock (60–62). In addition, the magnitude of PMN degranulation has been correlated with the development of multiple organ failure and with patient outcome after trauma and infection (63). The degranulating factor we describe here can be released into solution (Fig. 2) and can therefore potentially act at sites distant from the original focus of infection or endothelial injury, which may account in part for the disseminated vascular injury that is a feature of multiple organ failure under these conditions. Neutrophil degranulation triggered by dysregulated expression of endothelial signaling factors may also be a feature of severe vascular injury induced by microbial toxins other than LPS (1) and by toxins from organisms besides bacteria

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that cause tissue destruction in humans (31). Identification of the specific signaling factors involved and characterization of the individual and combinational biologic actions of the degranulating factor described here as well as other factors with this property are critical to understanding the pathogenesis of these injury states and may identify new strategies for therapeutic intervention.

15.

The authors thank Cora-Jean Edgell, Jerry Kaplan, and Yoshigi Yamada for contributing cells, reagents, and/or technical advice and for helpful discussions. We also thank the nurses and staff of the Labor and Delivery Service of LDS Hospital for help in collecting umbilical vein samples; Donnie Benson, Sue Cowley, and Margaret Vogel for cell culture and neutrophil isolation; and Leona Montoya and Michelle Bills for preparation of the manuscript. This work was supported by the Nora Eccles Treadwell Foundation and a Special Center of Research in Acute Respiratory Distress Syndrome (P50 HL50153 sponsored by the National Institutes of Health).

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Received for publication September 25, 1997. Revised for publication January 19, 1998.

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