Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

The interaction between endothelial cells and polymorphonuclear leukocytes (PMNs) is a central process in acute inflammation, both physiologic and pathologic (1). Specific molecular events govern this interaction. When appropriately stimulated, human endothelial cells express tethering (adhesion) and signaling factors that are recognized by counterligands and receptors on PMNs (1). Cytokines, including interleukin (IL)-1 and tumor necrosis factor  (TNF), are among the proinflammatory agonists that trigger these responses. As an index example, endothelial cells that are stimulated with IL-1 or TNF, or with bacterial lipopolysaccharide (LPS), respond by transcribing and synthesizing a tethering factor, E-selectin (2). It is transiently expressed for several hours on the plasma membranes of endothelial cells of postcapillary venules and other vessels, where it mediates rolling and adhesion of PMNs and other leukocytes by binding to one or more counterligands on the leukocyte surfaces (1). Other adhesion molecules besides E-selectin are expressed by stimulated endothelial cells under different conditions, providing a mechanism for differential targeting of particular classes of leukocytes (1, 3, 4).

The signaling factors expressed by human endothelial cells are of diverse classes (1) and are recognized by a number of different receptors on the PMN plasma membrane (5, 6). The first signaling molecule for PMNs to be identified as a product of stimulated endothelial cells, platelet-activating factor (7), is a biologically-active phospholipid (7, 10). More recently, it was shown that stimulated human endothelial cells synthesize members of the chemokine family (1, 11, 12). The prototype member of this group, IL-8, is a C-X-C chemokine (6, 11) that is synthesized by endothelium in response to IL-1, TNF, and LPS and is locally released (14). IL-8 binds to serpentine G-protein-linked receptors on PMNs (5) and activates them, leading to complex changes in their adhesive function (15, 16) and mediating transmigration across endothelial monolayers (17, 18). IL-8 associates with glycosaminoglycans on the endothelial plasma membranes via heparin-binding sequences (19). When associated with the endothelial cell surface, IL-8 may then activate PMNs in a juxtacrine fashion (1, 20). This mode of signaling may be particularly important when it is coexpressed with E-selectin (1, 20). Coexpression of tethering and signaling factors is an important mechanism for spatially regulating leukocyte activation in acute inflammatory responses (1, 20, 21). Coordinate action of signaling and tethering factors by endothelial cells regulates the phenotype and biologic functions of PMNs and other leukocytes during and after their transmigration, in addition to targeting them to particular sites (1, 22, 23). As with tethering molecules, signaling factors of different specificities are synthesized by endothelial cells depending on the agonist and time of stimulation, providing a molecular system for localized activation of specific classes of leukocytes as they are recruited (1, 14, 23).

In some instances, two or more signaling molecules with similar biologic properties are produced by endothelial cells at a particular time. They may be of the same or different classes (18; M. Topham and colleagues, manuscript in preparation). The significance of coincident generation of leukocyte signaling factors with similar biologic properties by endothelial cells is not yet established, although in some cases it has been postulated that each factor plays a particular role in transmigration of PMNs and in other complex responses of these cells (18). In other types of cell-cell interactions, multiple factors generated by one cell may deliver combinatorial signals that induce unique functional responses in the second (or "target") cell or may amplify the magnitude of a particular response; also, sequential and overlapping expression of factors with similar biologic properties can be a mechanism to sustain intercellular interactions over time (26). Thus, specific patterns of expression of signaling factors by endothelial cells may be a critical determinant of the activation responses of PMNs and other leukocytes and of their sustained actions at inflammatory sites, including the inflamed or injured lung (26). Here we report that human endothelial cells synthesize and secrete epithelial neutrophil activating peptide-78 (ENA-78) when stimulated with IL-1 and other agonists. ENA-78 is a recently identified C-X-C chemokine (27) that was previously thought to be expressed exclusively by epithelial cells (5, 13, 27, 28). ENA-78 has biologic activity similar to that of IL-8 and the two factors are coexpressed by stimulated endothelial cells. However, the synthesis and release of ENA-78 by stimulated endothelial cells varies from that of IL-8 in a fashion that indicates differential regulation.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Reagents

Hank's balanced salt solution (HBSS) and medium 199 (M199) were from Whittaker M.A. Bioproducts (Walkersville, MD). Human serum albumin (25%) was from Miles Laboratories, Inc. (Elkhart, IN). Recombinant human IL-1 and recombinant human IL-8 (77-amino-acid form) were from Genzyme Corp. (Cambridge, MA), and recombinant TNF was from Genentech (South San Francisco, CA). Recombinant human ENA-78 and polyclonal goat antibodies against human ENA-78 and IL-8 were from R&D Systems (Minneapolis, MN). See below for specificities of these antibodies. A polyclonal goat antihuman fibrinogen antibody was from Cappel (West Chester, PA). Peroxidase-conjugated avidin, bovine serum albumin, o-phenylenediamine (OPD), N-hydroxysuccimide biotin, dimethylformamide, cyanogenbromide (CNBr)-activated Sepharose 6MB, guanidinium thiocyanate, and N-laurylsalcosine were from Sigma (St. Louis, MO). Primer oligo(dT)₁₅ and M-Mulv reverse transcriptase were from Gibco-BRL (Gaithersburg, MD). All oligonucleotide primers for polymerase chain reaction (PCR) were synthesized by the University of Utah DNA/peptide user facility.

Cell Culture

Human umbilical vein endothelial cells (HUVEC) were cultured in 6-well plates in M199 supplemented with 10% human serum at 37°C in a humidified atmosphere of 95% air and 5% CO₂, as described (29). Except where specified, only primary monolayers that were tightly confluent were used for these experiments. We found that endothelial monolayers cultured under these conditions contain 1% or fewer contaminating leukocytes (30). First-passage endothelial monolayers were used in certain experiments, as indicated, and were cultured as described (29, 30).

Incubation of Endothelial Cells for Measurement of ENA-78 and IL-8 Secretion

HUVEC were incubated in complete culture medium alone, or with IL-1 or other agonists, for the indicated times at 37°C in 95% air/5% CO₂. After the incubation, the medium was removed and the cells were washed twice with HBSS. After an additional 1-h incubation in 0.5 ml of HBSS containing 0.5% human serum albumin (HBSS-HSA), the "conditioned" incubation buffer was collected for assay of the concentrations of ENA-78 and IL-8, and for measurements of proadhesive biologic activity (see below).

ELISAs for ENA-78 and IL-8

The concentrations of ENA-78 and IL-8 were measured using "sandwich" enzyme-linked immunosorbent assays (ELISAs) as described or with minor modification of methods published by others (31, 32). The capture antibodies were goat polyclonal anti-ENA-78 or anti-IL-8, and the second antibodies were biotinylated goat polyclonal anti-ENA-78 or anti-IL-8. Avidin-conjugated horseradish peroxidase was used for detection, and OPD was used as the indicator substrate. Recombinant human (r[h]) ENA-78 or r(h) IL-8 was used to generate standard curves. The detection limits were 80 to 200 pg/ml for ENA-78 and 20 to 100 pg/ml for IL-8.

The antibodies against ENA-78 and IL-8 are specific and do not recognize other chemokines or cytokines, according to the manufacturer. We confirmed this by demonstrating that neither ENA-78 nor IL-8 crossreacted in the ELISA for the other chemokine when tested at concentrations as high as 10⁴ pg/ml (not shown). In addition, neither antibody recognized recombinant growth-related oncogene (GRO)- or neutrophil activating protein (NAP)-2 in concentrations from 10² to 5 × 10⁴ pg/ml. GRO- and NAP-2 share significant sequence homology with ENA-78 (27).

Preparation of RNAs and Reverse Transcriptase-mediated Polymerase Chain Reaction (RT-PCR)

Total RNA was isolated from the cells with guanidinium thiocyanate by standard techniques (33). Single-strand cDNA for the PCR template was synthesized from 1 µg of total RNA using M-Mulv reverse transcriptase and primer oligo(dT)₁₅ under the conditions indicated by the manufacturer. Primers were designed from the cDNA sequences for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (34), ENA-78 (35), and IL-8 (36). The primers were: GAPDH-F, 5'-CCACCCATGGCAAATTCCATGGCA-3'; GAPDH-R, 5'-TCTAGACGGCAGGTCAGGTCCACC-3'; ENA-78-F, 5'-GCCCGTGTCCCCGGTCCTTCGAG-3'; ENA-78-R, 5'-CTGGATCAAGAC-AAATTTCCTTC-3'; IL-8-F, 5'-ATGAGTCTAAAGAACTTCGA-3'; and IL-8-R, 5'-TGAATTCTCAGCCCTCTTCAA-3'.

The reaction conditions were 1 × (94°C, 5 min); 30 × (94°C, 1 min 15 s; 55°C, 2 min; 72°C, 2 min); and 1 × (72°C, 10 min). The products were analyzed on 1.5% agarose gel containing ethidium bromide. Controls for precision of the reaction (33) and negative controls (no template) were included in each assay.

Bioassay for PMN Adherence

The adherence of PMNs to a gelatin matrix, which depends on inside-out signaling of ₂ integrins (CD11/CD18 integrins), was assayed by a modification of previously described methods (15, 37, 38). Briefly, peripheral blood was obtained from healthy volunteers after informed consent. PMNs were isolated by density centrifugation and resuspended at a final concentration of 3.5 × 10⁵/ml in HBSS-HSA. A total of 225 µl of PMN solution was layered onto gelatin-coated 15-mm wells, and 25 µl of media from HUVEC or other agonists (r[h] ENA-78, r[h] IL-8, HBSS-HSA as a negative control, or fMLP as a positive control) were added. After 5 min at 37°C, nonadherent PMNs were removed. The wells were washed twice with HBSS, and the adherent PMNs were fixed with 2.5% glutaraldehyde in HBSS. The adherent PMNs were counted by microscopy using a video imaging system.

Immunoprecipitation of Biologic Activity Released by Stimulated Endothelial Cells

The contributions of ENA-78 and IL-8 to the proadhesive activity for PMNs released into medium incubated with HUVEC treated with IL-1 were examined by immunoprecipitation with anti-ENA-78 or anti-IL-8 antibodies. Goat antihuman ENA-78, anti-IL-8, or anti-fibrinogen (control) polyclonal antibodies were coupled to CNBr- activated Sepharose 6MB under the conditions specified by Pharmacia. Approximately 800 µg of each antibody were coupled to 1.5 ml of swollen sepharose gel. We then incubated 400 µl of media collected from HUVEC stimulated with IL-1 (50 U/ml for 21 h) with 0.5 ml of antibody-coupled Sepharose 6MB, saturated with 10 mM Tris-HCl (pH 7.5), at 38°C for 2 h with rocking. After the incubation, the sepharose gel was removed by centrifugation, and the signaling activity for PMNs recovered in the supernatant was examined by assay of PMN adhesion to gelating matrices (see above).

Immunocytochemical Localization of ENA-78 and IL-8

We examined adult human lung tissue obtained at autopsy and vascular tissue obtained at surgery. The lung tissue was collected from 3 patients whose death was attributed to the acute respiratory distress syndrome (ARDS), 1 patient with bacterial pneumonia, and 3 patients who died of nonpulmonary causes (ruptured thoracic aortic aneurysm, glioblastoma multiforme of the brain, and traumatic head injury). Blood was passively drained from the lung samples prior to fixation. The vascular tissue was taken from the edges of aortic resections from patients with aneurysmic or occlusive disease (3 each) of the abdominal aorta. The protocols for collecting the tissue were approved by the Institutional Review Board at the University of Utah.

The tissues were fixed in 10% buffered neutral formalin or HistoChoice MB (Amresco Inc., Solon, OH) for 2 h at room temperature and stored in 70% ethanol until embedment in paraffin. Tissue (4-5 µm) were collected on PLUS slides (VWR, Inc., Media, PA) for immunohistochemistry. Briefly, the deparaffinized sections were treated with 3% H₂O₂ in methanol for 10 min at 37°C to remove endogenous peroxidase. The sections were washed with phosphate-buffered saline, blocked with normal goat serum, and then incubated with the primary antibodies (polyclonal goat-antihuman; R&D Systems, Minneapolis, MN) at 4°C overnight. Optimal dilutions were 1:250 for ENA-78 and 1:200 for IL-8. Staining controls included polyclonal goat-antihuman von Willebrand factor (vWF) (1:5,000 dilution), omission of the primary antibody, and omission of the secondary antibody (biotinylated rabbit antigoat IgG). Antigen detection was done by the avidin-biotin-horseradish peroxidase method (ABC Elite kit; Vector Laboratories, Burlingame, CA). We used Gill's #3 hematoxylin to counterstrain the sections. Photography was done using a Zeiss Axioplan microscope.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Stimulated Human Endothelial Cells Express and Secrete ENA-78 as well as IL-8

The concentrations of ENA-78 and IL-8 in buffer collected from endothelial cells incubated in the absence of an agonist was at or below the threshold of detection in our assays. In contrast, HUVEC stimulated with IL-1 secreted both ENA-78 and IL-8 into the incubation medium (Figures 1 and 2). Both chemokines were released by passed HUVEC that were stimulated with IL-1 (not shown), as well as by primary monolayers (Figures 1 and 2). This experiment was done to exclude trace numbers of contaminating mononuclear leukocytes as the source of the two mediators.

View larger version (16K): [in this window] [in a new window]   Figure 1.   IL-1 induces the secretion of both ENA-78 (A) and IL-8 (B) in a concentration-dependent manner. HUVEC were stimulated with various concentrations of IL-1 for 21 h. The media were removed, the cells were washed twice with HBSS, and the monolayers were covered with 0.5 ml HBSS-HSA. After an additional 1-h incubation the HBSS-HSA was collected and the concentrations of ENA-78 and IL-8 were determined by ELISAs. The points in the figure represent the mean of duplicate determinations in one experiment. The results were similar in a second experiment.

View larger version (17K): [in this window] [in a new window]   Figure 2.   ENA-78 (A) and IL-8 (B) are secreted from stimulated HUVEC with different time courses. HUVEC were stimulated with 50 U/ml IL-1 for various times. After the medium was removed the cells were washed, a new volume of HBSS-HSA was added, the cells were incubated for 1 h, and the concentrations of ENA-78 and IL-8 were determined as in Figure 1. The points in the figures indicate the mean of duplicate determinations.

The secretion of ENA-78 and IL-8 depended on the concentration of IL-1 used to stimulate endothelial monolayers in vitro (Figure 1). However, while both chemokines were induced by stimulation, the levels of ENA-78 in the incubation medium were less than 15% of those of IL-8 measured in parallel. Also, the two chemokines were secreted with different time courses (Figure 2). Release of IL-8 into the medium was measurable by 1 h after stimulation of the endothelial cells and reached a maximal level at 8 h (Figure 2, lower panel). In contrast, secretion of ENA-78 was not detected until 4 to 8 h after stimulation and increased thereafter (Figure 2, upper panel).

In addition to IL-1, TNF and LPS triggered secretion of ENA-78 from cultured human endothelial cells. Secretion of IL-8 was also induced by these agonists, as previously reported (14). The secretion of both of the chemokines in response to TNF and LPS depended on the concentration of the agonist and the time of incubation (not shown). The rank order of potency of the agonists for both ENA-78 and IL-8 secretion was IL-1 > LPS > TNF when concentrations of the agonists that induced maximal secretion were compared. Notably, however, TNF was a weak stimulus for ENA-78 release, causing an approximate 2-fold increase in ENA-78 over the background levels measured in buffer incubated with unstimulated endothelial cells (Figure 3 and data not shown). In 4 experiments the pattern was as shown in Figure 3, while in a fifth experiment TNF did not cause secretion of ENA-78 over baseline even though IL-1 and LPS induced its release. In contrast, TNF was nearly as potent as an agonist for IL-8 secretion from cultured endothelial cells as were IL-1 and LPS in each experiment (Figure 3 and data not shown).

View larger version (25K): [in this window] [in a new window]   Figure 3.   LPS and TNF induce secretion of ENA-78 and IL-8 by human endothelial cells. HUVEC were stimulated with 10 µg/ ml LPS, 1,000 U/ml TNF, or 50 U/ml IL-1 for 21 h. After the media were removed, the cells were washed and HBSS-HSA was incubated over the stimulated monolayers for an additional 1 h. The conditioned HBSS-HSA was removed and the concentrations of ENA-78 (upper panel) and IL-8 (lower panel) were determined by ELISA. The figure represents the mean ± SD of three experiments.

Human Endothelial Cells in Inflamed Tissue Express ENA-78

We then examined vessels in inflamed tissues by immunohistochemistry to determine whether ENA-78 is present in endothelial cells. Endothelial cells in vessels in the lungs of patients who died with ARDS were stained by the antibody to ENA-78 (Figure 4). The positively stained cells were identified as endothelial cells by anti-vWF staining in serial sections (Figure 4). In some areas there were extravascular infiltrates of neutrophils around vessels lined by endothelial cells that reacted with the anti-ENA-78 antibody (not shown). Vessels with endothelium that stained positively for ENA-78 were also surrounded by PMNs in areas of lung from a subject with bacterial pneumonia. Omission of either the primary or secondary antibody eliminated immunostaining for both ENA-78 and vWF (Figure 4). In additional experiments, a species-matched polyclonal antibody against human insulin used in the first step did not stain cells that were stained positively by the anti-ENA-78 antibody. Only rare endothelial cells were positive for ENA-78 in lung sections from patients who died without clinical pulmonary disease, whereas endothelial cells of arteries, veins, and microvessels in the same tissue sections were positive for vWF. In sections from lungs of patients who died with ARDS, hyperplastic alveolar type II cells, alveolar macrophages, PMNs, and, in some areas, vascular smooth muscle were positive for ENA-78 (Figure 4). In contrast, only occasional alveolar type II cells and macrophages in lung sections from patients who died from nonpulmonary causes were positive.

View larger version (134K): [in this window] [in a new window]   Figure 4.   ENA-78 is present in endothelial cells in inflamed human lung. Panel a shows brown immunoperoxidase staining of the endothelium (E; arrow) and vascular smooth muscle cells (M) in the wall of a pulmonary vessel (PV). Some of the alveolar macrophages (AM) in the airspace also are immunostained. Panel b demonstrates endothelial cell (E) immunoperoxidase staining (arrow) for the von (Figure 4 legend continued) Willebrand factor that is similar to the endothelial cell staining pattern for ENA-78 (panel a). Alveolar capillary endothelial cells (C) are also stained. Panel c illustrates brown immunostaining for ENA-78 (arrow) in hyperplastic alveolar type II epithelial cells (Ep). Inflammatory cells, notably neutrophils (N), in the airspace are immunostained for ENA-78. Panel d shows a staining control in which the primary antibody was omitted. There is no immunostaining of the hyperplastic alveolar type II epithelial cells (Ep) in a tissue section cut adjacent to the section shown in panel c. Nuclei are counterstained with Gill's #3 hematoxylin. All four panels are the same magnification.

We found that endothelium of postcapillary venules and other microvessels in the adventitia of abdominal aortic segments adjacent to aneurysmal dilatations or occlusive lesions also stained positively for ENA-78 (Figure 5). Regions of the adventia in arteries involved by aneurysmal dilatations, or containing atheromatous plaques, are inflamed (39, 40) and are sites at which chemokines and other mediators may be generated. Thus, our findings indicate that ENA-78 is not uniquely expressed by lung endothelium (Figure 4). In the aortic tissue samples that we studied, the regions of the adventitial microvessels that were positive for ENA-78 also stained positively for von Willebrand factor in serial sections (data not shown). This feature, and the location of the ENA-78-positive cells on the luminal surfaces of venules and other microvessels (Figure 5), confirmed their identity as endothelial cells. In addition to endothelial cells, macrophages (but not aggregates of lymphocytes) in the areas of adventitial inflammation were positive for ENA-78 by immunostaining. Omission of either the primary or the secondary antibody eliminated the immunostaining of endothelial cells and macrophages in these sections (Figure 5 and data not shown). In some areas, venular smooth muscle was also positive for ENA-78 (Figure 5).

View larger version (102K): [in this window] [in a new window]   Figure 5.   ENA-78 is present in venular endothelium in inflamed aortic adventitia. Panels a and b show two venules stained by an immunoperoxidase-conjugated antibody against ENA-78 (MATERIALS AND METHODS). The endothelium (E) is stained brown, demonstrating expression of ENA-78 (arrows). Vascular smooth muscle cells (M) also are immunostained. Nuclei are counterstained with Gill's #3 hematoxylin.

mRNAs for ENA-78 and IL-8 are Differentially Expressed by Stimulated Human Endothelial Cells in Culture

In order to further explore the differences in secretion of chemokines by stimulated endothelium, we examined the expression of mRNAs for ENA-78 and IL-8 by RT-PCR. PCR products for both chemokines were amplified from stimulated primary (Figure 6) and passed (not shown) HUVEC monolayers. As with secretion of immunoreactive proteins (Figures 1 and 2), the levels of mRNA for ENA-78 and IL-8 depended on the concentration of IL-1 (Figure 6A) and the times of incubation (Figure 6B). The mRNA for ENA-78 was detected at 1 h, but at a very low level. The signal then gradually increased over the next 19 h. In contrast, mRNA for IL-8 was clearly present at 1 h and reached maximal levels at 4 to 8 h when measured in the same cells. These patterns are generally similar to those for protein secretion (Figure 2) and suggest that the temporal difference in release of the two chemokines is due, in part, to transcriptional regulation. Interestingly, however, transcripts for ENA-78 were present prior to significant protein secretion, which was not detectable until 4 h or later (compare Figures 2 and 6). Also, the RT-PCR bands for ENA-78 were more intense than those for IL-8 at times of maximal expression (Figure 6B), and more intense in response to maximal stimulation with IL-1 (Figure 6A). This contrasts with the relative amounts of the immunoreactive patterns secreted into the medium (Figures 1 and 2, and data not shown), and suggests that post-transcriptional mechanisms also regulate the synthesis and/or secretion of ENA-78. RT-PCR products for ENA-78 and IL-8 were also detected in endothelial cells stimulated with LPS or TNF (Figure 7).

View larger version (55K): [in this window] [in a new window]   View larger version (49K): [in this window] [in a new window]   Figure 6.   IL-1 induces the expression of mRNAs for ENA-78 and IL-8 in a concentration-dependent manner (upper panel ) and with different time courses (lower panel ). HUVEC were stimulated with IL-1 as described in MATERIALS AND METHODS and Figures 1 and 2, and RNA was extracted. The RNA was reverse transcribed and the cDNAs for ENA-78, IL-8, and GAPDH were amplified by PCR (30 cycles). The sizes of the PCR products for ENA-78, IL-8, and GAPDH were 255, 219, and 598 bases, respectively.

View larger version (45K): [in this window] [in a new window]   Figure 7.   TNF and LPS induce expression of mRNAs for ENA-78 and IL-8 in human endothelial cells when measured by RT-PCR. HUVEC were treated with control buffer ("unstimulated") or were stimulated with LPS, TNF, or IL-1 as in Figure 3, and the expression of mRNAs for ENA-78 and IL-8 was examined by RT-PCR as in Figure 5.

ENA-78 Signals PMN Adhesiveness

IL-8 is known to induce activation-dependent alterations in PMN adhesiveness (1, 12, 15, 16). To determine whether ENA-78 has this property, we first examined the activity of the recombinant chemokine in an assay that detects neutrophil adhesion via their ₂ integrins (37, 38). PMN adhesiveness was induced by r(h) ENA-78 (Figure 8A). Consistent with earlier observations (15; reviewed in 11 and 12), IL-8 also induced PMN adhesiveness (Figure 8B). The potencies of the two recombinant chemokines were similar, although ENA-78 appeared slightly more potent than IL-8 at lower concentrations and slightly less potent at higher concentrations (Figures 8A and 8B).

View larger version (21K): [in this window] [in a new window]   Figure 8.   PMN adhesiveness is induced by r(h) ENA-78, r(h) IL-8, and by factors secreted by HUVEC stimulated with IL-1. PMNs were incubated with agonist or control buffer on a gelatin matrix at 37°C for 5 min, and the adherent cells were counted as described in MATERIALS AND METHODS. The closed circles and closed triangles indicate the response to r(h) ENA-78 (panel A), and to r(h) IL-8 (panel B), respectively. The open squares in each panel indicate the adhesive activity in HBSS-HSA incubated with unstimulated endothelial monolayers, and the open circles indicate the adhesive activity in buffer incubated with HUVEC monolayers stimulated with IL-1 (50 U/ml, 21 h). The open squares and open circles are positioned on the figures so that they indicate the concentrations of ENA-78 (panel A) and IL-8 (panel B) measured in the corresponding samples. The data indicate the means of duplicate determinations for each point.

ENA-78 and IL-8 Contribute to the Proadhesive Activity for PMNs that Is Released by IL-1-stimulated Endothelial Cells

We next examined the agonist effects of ENA-78 and IL-8 released from stimulated endothelial cells, using assays of proadhesive activity. The supernatant medium from unstimulated HUVEC did not contain activity that induced PMN adhesion, consistent with our earlier observations (8). In contrast, the supernatant medium from endothelial monolayers that had been stimulated with IL-1 contained proadhesive activity (open circles, Figure 8). ENA-78 was present and, based on the ability of the recombinant chemokine to induce inside-out signaling of ₂ integrins (see above), could potentially be the factor that induces adhesion. However, the concentration of ENA-78 measured in the buffer incubated with stimulated endothelial cells was clearly lower than the concentration of recombinant ENA-78 required to induce the same magnitude of PMN adhesiveness. This can be seen by examining the open circles in Figure 8A, which indicate the magnitude of PMN adhesiveness induced by the conditioned buffer on the ordinate, and comparing them with the concentrations of ENA-78 measured in the same samples of incubation buffer by ELISA (abscissa) and with the concentration-response curve for recombinant ENA-78 (closed circles). Similarly, the concentration of IL-8 in buffer incubated with stimulated endothelial cells was lower than the concentration of recombinant IL-8 that would be required to induce the same degree of PMN adhesiveness, although the discrepancy was not as great as with ENA-78 (Figure 8B). These results suggested that neither IL-8 nor ENA-78 alone accounted for the proadhesive activity released by IL-1-stimulated endothelial cells. To directly explore the contributions of each factor to the secreted bioactivity, we performed immunoprecipitations to selectively remove the chemokines. This approach was taken because the antibodies that we used are not neutralizing under the conditions of these experiments, but they can be used to deplete the chemokines from the conditioned buffer. Anti-ENA-78 and anti-IL-8 antibodies removed 32% and 81% of the proadhesive activity, respectively, when compared with a control antibody (Figure 9). Neither the anti-ENA-78 nor the anti-IL-8 antibody blocked the proadhesive activity of fMLP when it was exogenously added to buffer and then used to activate PMNs in parallel control incubations (not shown). These experiments indicate that ENA-78 and IL-8 secreted by stimulated endothelial cells act cooperatively to induce PMN adhesiveness. We do not yet know their relative potencies in biologic fluids or at endothelial surfaces (19), or whether ENA-78 inhibits PMN adhesion under some conditions, as has been reported for IL-8 (16).

View larger version (21K): [in this window] [in a new window]   Figure 9.   The activity for PMN adherence was immunoprecipitated with antibodies against ENA-78 or IL-8. The medium from HUVEC stimulated with 50 U/ml IL-1 at 37°C for 21 h was incubated with sepharose gel coupled with antibodies against human fibrinogen (control), human ENA-78 (anti-ENA-78), or human IL-8 (anti-IL-8), and recovered as described in MATERIALS AND METHODS. HBSS-HSA incubated in the absence of endothelial cells ("buffer"), incubated with unstimulated HUVEC ("control EC"), and incubated with IL-1-stimulated HUVEC were removed and subjected to immunoprecipitation with antibodies as indicated, the PMNs were added on a gelatin matrix, and the proadhesive activity for PMN was assayed as described in MATERIALS AND METHODS. The figure represents the mean ± SD of triplicate determinations. In a second experiment, anti-ENA-78 and anti-IL-8 removed 47% and 85% of the proadhesive activity, respectively.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

ENA-78 and IL-8 are members of the C-X-C chemokine superfamily, which also includes NAP-2, the growth-related oncogene products (GRO-, GRO-, Gro-), and others (5, 6, 12, 13). Several human cells are known to produce more than one chemokine of this class, but the relation of the synthesis of these factors to one another in a given cell type, their regulation, and their individual and combined contributions to specific intercellular signaling events are largely unknown. Here we show that ENA-78 is synthesized and secreted when cultured human endothelial cells are stimulated with inflammatory cytokines or LPS, as is IL-8. We also found that ENA-78 and IL-8 each contribute to the proadhesive activity for neutrophils that is released by stimulated endothelial cells. Thus, ENA-78 is a signaling molecule that can account for IL-8-independent mechanisms of PMN activation by endothelial cells (see below). We demonstrated the presence of ENA-78 in endothelial cells in inflamed human lung and vascular tissue (Figures 4 and 5, and micrographs not shown), providing clinical and in vivo correlates to our observation that cultured endothelial cells express this factor. It was previously mentioned that ENA-78 is present in endothelial cells in synovial samples from patients with arthritis, although the observation was not documented (41). A single experiment indicating that cultured human endothelial cells produce ENA-78 in response to IL-1 was reported earlier (32), although there were no controls to exclude its synthesis by contaminating mononuclear cells and there was no further examination of the response. We characterized the expression of ENA-78 by human endothelial cells and found that ENA-78 and IL-8 are concurrently expressed. However, the amount of ENA-78 secreted and the time course of its expression and secretion varied from these features of the production of IL-8, indicating that the two factors are differentially regulated. In addition, the secretion of ENA-78 and IL-8 were different depending on the agonist used to stimulate the endothelial cells.

The gene encoding human ENA-78 has been mapped to chromosome 4q13-q21, which is the locus of other members of the C-X-C class (35, 42). ENA-78 shares significant amino acid sequence homology with other C-X-C chemokines, including NAP-2 (53%), GRO- (52%), and IL-8 (22%), and has the ability to induce chemotaxis of PMNs (27), which is a common biological feature of these C-X-C chemokines (5, 11). ENA-78 activates PMNs through a receptor that also recognizes IL-8, the type B IL-8 receptor (CXCR2; 5), based on studies of desensitization and radiolabeled ligand binding (13, 27, 43). ENA-78 was first identified in the conditioned medium of the cultured pulmonary type II epithelial carcinoma cell line A549 (27), and it is generally thought to be exclusively a product of epithelial cells (6, 13, 28, 44). We found that it is present in hyperplastic alveolar epithelial cells in sections from patients with acute lung injury (Figure 4). This is consistent with the observations using the A549 cell line (27) but for the first time, to our knowledge, demonstrates that ENA-78 is present in in situ epithelium of the inflamed human lung.

ENA-78 was subsequently reported to be produced by isolated cells other than the A549 line in preliminary studies (32). Our observation that ENA-78 is secreted by cultured endothelial monolayers stimulated with IL-1 is consistent with a previous experiment in which endothelial cells were stimulated with IL-1 in vitro (32) and with experiments in A549 cells (27) and human embryonic kidney 293 cells transfected with the gene for ENA-78 (35), where IL-1 was found to drive its expression. In the A549 and 293 cell lines, TNF was also an agonist for ENA-78 expression, equivalent in potency to IL-1, when PMN chemotactic activity or the activity of a luciferase reporter construct was used as an assay (27, 35). In contrast, we found that TNF was much less potent than was IL-1 as a stimulus for ENA-78 secretion by primary human endothelial cells, although it was similar in potency to IL-1 when IL-8 secretion was measured. Taken together, our observations using human endothelial cells and the previously published reports describing studies with cell lines (27, 35) indicate that the expression of ENA-78 is different in cells of different origin and in primary cells versus transfected cells. This is also true of IL-8 (6).

When we compared the handling of ENA-78 and IL-8 by stimulated human endothelial cells, an unexpected feature was that the time course of secretion of ENA-78 was different from that of IL-8 in monolayers stimulated with IL-1 (Figure 2). This varies from the situation in the A549 cell line, where production of the two chemokines was reported to occur concomitantly (35). In stimulated endothelial cells the expression of MRNA for IL-8 generally paralleled secretion of the protein, whereas mRNA for ENA-78 was present considerably earlier than release of detectable levels of the factor into the incubation medium (Figures 2 and 6). The latter finding suggests that translational steps and/or post-translational events are points of regulation of expression of ENA-78 at the protein level. Transcriptional regulation also appears to be important, since little or no mRNA could be amplified from endothelial cells under resting conditions (Figure 6).

Based on analysis of reporter constructs and other experimental strategies, both IL-8 and ENA-78 require nuclear factor B (NF-B) translocation to the nucleus for initiating transcription, since transcription is abrogated when the B sites in the 5'-untranslated regions of each gene are altered (35, 45, 46). IL-8 requires another factor, enhancer binding protein-like factor (45, 46; reviewed in 6), whereas ENA-78 does not (35). Differential action of specific patterns of trans-activating factors (47), which may be particularly important in the regulation of NF-B-dependent genes (48), may allow different patterns of expression of ENA-78 and IL-8 in particular cell types under varying conditions. Of note, our preliminary studies of inflamed human lung and vascular tissue using immunocytochemical analysis indicate that under some conditions ENA-78 is present in stimulated endothelial cells when IL-8 is not (K. H. Albertine, unpublished observations). Similarly, in cultured human endothelial cells treated with certain agonists ENA-78 is produced whereas IL-8 is not (49).

We asked if the concomitant production of ENA-78 and IL-8 by endothelial cells stimulated with IL-1 leads to biologic activity with a contribution from each chemokine. We found that an activity that induces PMN adhesiveness is released into buffer incubated with stimulated endothelial cells for 21 h (Figures 8 and 9), a time of secretion of both factors (Figures 1 and 2). We then determined the contributions of ENA-78 and IL-8 to this activity. Both r(h) ENA-78 and r(h) IL-8 caused PMNs to adhere to gelatin matrices (Figure 8). This response is dependent on activation of ₂ integrins on the PMN surface (37, 38). Although this property has not been reported previously, the ability of ENA-78 to induce PMN adhesiveness by inside-out signaling of ₂ integrins is expected from its ability to trigger an intracellular Ca²⁺ transient in PMNs and to stimulate their chemotaxis (13, 27). ENA-78 also induces quantitative upregulation of ₂ integrins on the PMN plasma membrane (43). We then compared the concentrations of ENA-78 and IL-8 in the samples of conditioned buffer from stimulated endothelial cells to the concentration- response relationships for PMN adhesion induced by recombinant chemokines (Figure 8). This analysis suggested that both factors contribute to the proadhesive activity in an additive fashion. Results of immunoprecipitation experiments supported this conclusion: antibodies against ENA-78 or IL-8 precipitated ~ 30% and ~ 80% of the activity that induces PMN adhesiveness in the conditioned buffer, respectively (Figure 9). This suggests that one biologic consequence of coexpression of ENA-78 and IL-8 is to amplify PMN adhesive responses and related activation events, including transmigration.

Several laboratories have previously shown that PMN adhesion to endothelial cells stimulated with IL-1 for 18 to 24 h is primarily dependent on activation of the leukocyte ₂ integrins (50), but the factor or factors generated by the endothelial cells that mediate this signaling have not been clearly defined. At earlier time points (4-8 h) engagement of E-selectin is a dominant mechanism that acts together with ₂ integrins to tether PMNs to the endothelial surface (1, 2, 4), whereas at the later time points (18- 24 h) the expression of E-selectin has waned (1, 2) but factors that induce PMN activation, and consequent ₂ integrin-dependent adhesiveness, are still present (50). The data in Figures 8 and 9 indicate that ENA-78 contributes to this endothelial-leukocyte signaling, in concert with IL-8. An antibody against ENA-78 reduced PMN sequestration in vivo in a model of acute lung injury (54), consistent with the ability of ENA-78 to induce PMN adhesiveness (Figures 8 and 9). In a second animal model, an antibody against ENA-78 reduced neutrophil accumulation in vessel walls in a model of deep vein thrombosis (55). Because ENA-78 triggers PMN adhesiveness (Figures 8 and 9) and chemotaxis (27), it may account for the IL-8-independent mechanism of neutrophil migration across endothelial monolayers stimulated with cytokines (17, 56). ENA-78, like other C-X-C chemokines, may have additional functions at the vascular wall as well. This is suggested by the fact that in some tissue sections that we studied there was clear expression of ENA-78 in endothelial cells without associated neutrophil influx (Figure 5, and data not shown).

Recently it was reported that the levels of ENA-78 in synovial fluid and peripheral blood are increased in rheumatoid arthritis (41). ENA-78 and IL-8 each accounted for approximately 40% of the chemotactic activity for PMNs in synovial fluid, indicating that the two chemokines may contribute parallel signals when present together in an inflammatory milieu in human tissue. ENA-78 and IL-8 are also present in bronchoalveolar lavage samples from subjects with ARDS, and correlate positively with the concentrations of PMNs in these fluids (57). Our observations indicate that endothelial cells can generate both factors in inflammatory lesions. Characterization of the regulation of the two chemokines in human endothelial cells will provide important information relevant to their actions in inflammation, and insights into how the chemokines influence the complex biologic responses of target cells that recognize them. It will also provide a basis for determining whether these chemokines become dysregulated in pathologic inflammation of the lung and other organs, an issue of importance because impaired regulation of signaling factors may be a fundamental mechanism of disease (20, 30, 31).