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Chemotactic Migration Triggers IL-8 Generation in Neutrophilic Leukocytes¹

Rafat A. Siddiqui^*,, Luke P. Akard^*, J. G. N. Garcia, Yi Cui^* and Denis English^2,*,§

^* Experimental Cell Research Program, Methodist Research Institute, and Department of Biology, Indiana University/Purdue University, Indianapolis, IN 46201; and Departments of Medicine and ^§ Allied Health Sciences, Indiana University School of Medicine, Indianapolis, IN 46202

	Abstract

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Neutrophils recovered from inflammatory exudates possess increased levels of IL-8, but exposure of neutrophils to chemoattractants results in only a modest stimulation of IL-8 generation. This study was undertaken to explore the hypothesis that IL-8 generation in these cells is dependent upon the process of migration. Neutrophils synthesized up to 30 times as much IL-8 during migration in response to a gradient of diverse chemoattractants than they did when stimulated directly by the attractants in the absence of a gradient. This IL-8 response was dependent on migration since it was not observed in cells exposed to concentration gradients of chemoattractants under conditions that prevented cell movement. While actinomycin-D (1 µg/ml) had little influence on the generation of IL-8 during chemotaxis, the protein synthesis inhibitor cycloheximide (10 µg/ml) markedly blunted the accumulation of cell-associated IL-8, suggesting that new protein synthesis from preexisting mRNA was responsible for the effect. Consistent with this interpretation, migrating cells incorporated over 10 times as much [³H]leucine into IL-8 as did nonmotile neutrophils exposed to chemoattractants. A substantial portion of the IL-8 generated during chemotaxis was released upon subsequent metabolic stimulation. Thus, the IL-8 synthesized during chemotaxis is uniquely positioned to exert a regulatory influence on inflammatory processes governed by neutrophilic leukocytes responding to inflammatory and infectious stimuli.

	Introduction

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Neutrophils activated by various metabolic agonists, including opsonized zymosan, PMA, LPS, granulocyte-macrophage-CSF, and TNF-, synthesize and release several cytokines including TNF, IL-1, IL-6, IL-8, and IL-12 (1, 2, 3, 4, 5, 6). These factors are important regulators of the inflammatory response. In particular, IL-8 is a potent chemoattractant that promotes the mobilization of neutrophils into sites of infection and inflammation. Recent studies have demonstrated that, in some instances, protein synthesis rather than release of stored material accounts for the enhanced secretion of IL-8 by activated neutrophils (7). Activated neutrophils potentially modulate IL-8 synthesis at either the level of mRNA transcription or translation. Northern blot analysis revealed increased expression of IL-8 mRNA upon stimulation of neutrophils with certain cytokines (8, 9), LPS (9, 10), culture supernatants (11), and selectin ligands (12). Transcriptional regulation of IL-8 has also been shown in several other systems, including epithelial cells infected with Helicobacter pylori (13) or Pseudomonas aeroginosa (14) and monocytes responding to cytokines (15). In addition to transcription, IL-8 generation during inflammation may also be regulated at the level of mRNA translation. Kuhns and Gallin found that IL-8 production in ionophore-stimulated neutrophils was partially suppressed by cycloheximide, a protein synthesis inhibitor (7). Under similar conditions, the RNA synthesis inhibitor actinomycin-D had little effect, suggesting that the synthesis of IL-8 was under translational rather than transcriptional control.

After migration to sites of infection and inflammation, neutrophils putatively release enhanced amounts of IL-8 and other cytokines in response to metabolic stimulation. At these sites, cytokines secreted by chemotactic neutrophils exert potent effects on many aspects of the inflammatory response (16, 17, 18). However, the ability of chemotactically responsive cells to release IL-8 upon further stimulation has not, to our knowledge, been directly tested. In preliminary experiments, we observed that neutrophils recovered after chemotactic migration not only displayed a remarkably enhanced ability to release IL-8 upon metabolic stimulation but also possessed markedly elevated levels of IL-8 in comparison with nonmotile cells that were exposed directly to chemoattractants, suggesting that IL-8 synthesis may have occurred during chemotactic migration. The present investigation was undertaken to explore this possibility. The results indicate that chemotactic migration induces IL-8 synthesis by activating a cellular process that results in enhanced translation of mRNA present in resting cells.

	Materials and Methods

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Human IL-8, ELISA IL-8 assay kits, and monoclonal anti-IL-8 Abs were obtained from R&D systems (Minneapolis, MN). Platelet-activating factor (PAF)³ was obtained from Calbiochem (San Diego, CA). L-[4,5-³H]leucine (140 Ci/mmol) was from New England Nuclear (Boston, MA). Tissue solubilizer NSC-II was from Amersham Canada (London, Ontario, Canada). Precast gels and reagents for SDS gel electrophoresis were from Bio-Rad (Hercules, CA). Endothelial cell culture reagents were from Cell Systems (Kirkland, WA). Protein A/G plus beads were from Oncogene Science (Cambridge, MA). Dulbecco’s minimal essential media (DMEM) was from Grand Island Biological (Grand Island, MI). Zymosan, FMLP, recombinant C5a, PMA, casein, DMSO, HBSS, cytochalasin-D, actinomycin-D, and cycloheximide were obtained from Sigma (St. Louis, MO). Cycloheximide was dissolved in distilled water at a concentration of 1 mg/ml. Actinomycin-D was dissolved in ethanol at a concentration of 100 µg/ml. Cytochalasin-D and PMA were dissolved in DMSO at a concentration of 1 mg/ml. Chemoattractant solutions were prepared as described below.

Isolation of neutrophils

Neutrophils were isolated from the blood of healthy donors using Ficoll-Hypaque density gradient centrifugation as previously described (19). After erythrocyte sedimentation at room temperature, buffy coats were layered on a 3-ml cushion of Ficoll-Hypaque and centrifuged at 800 x g for 20 min. The pellets were washed once with HBSS, and contaminating erythrocytes were removed by isotonic ammonium chloride lysis. Neutrophils were washed and suspended at a final concentration of 1.0 x 10⁷/ml in PBS unless otherwise indicated.

Preparation of chemoattractants

Zymosan-activated serum (ZAS) was prepared by incubating 15 mg of zymosan in 1 ml of fresh serum for 1 h at 37°C as described (20). After incubation, the reaction was heat inactivated at 56°C for 30 min, and the supernatant was collected after centrifugation at 500 x g for 15 min at 4°C. The activated serum was stored at -70°C until use. Casein (5 mg/ml) was dissolved in HBSS heated at 56°C for 10 min. The solution was then cooled at 4°C and centrifuged to remove undissolved particles. The solution was stored at -20°C until used. PAF was dissolved in ethanol at a final concentration of 2 mM and stored at -20°C. Stock solutions of FMLP (10 µM) were prepared in DMSO and stored at -70°C. Stock solutions of recombinant C5a (10 µM) were prepared in HBSS containing 1% BSA and stored at -70°C until use.

Endothelial cell culture

Bovine pulmonary aortic endothelial cells were cultured as described previously (21). In brief, bovine pulmonary aortic endothelial cells were grown in DMEM containing 100 µM nonessential amino acids, 2 µM glutamine, 20% colostrum-free bovine serum, 20 µg/ml penicillin, 20 µg/ml streptomycin, 0.05% µg/ml fungisone, 0.2 ml of sodium heparin/100 ml, and 2 µg/ml endothelial cell growth supplement under a humidified atmosphere of 95% air and 5% CO₂ in gelatin-coated flasks. Between their 16th and 22nd passage, the cells were trypsinized and dislodged. Single cell suspensions were then prepared in DMEM. These suspensions were seeded onto collagen-treated 3-µm pore size polycarbonate filters at the base of inserts of 24-mm Transwell chambers (Costar, Cambridge, MA) at an average density of 10⁴ cells/well, as previously described (21). The inserts were returned to wells containing 0.5 ml of endothelial cell growth media and cultured until confluent growth was attained (3–5 days), at which time the inserts were rinsed and used for chemotaxis assays as described below.

Chemotaxis assay

Chemotaxis was performed using 24-mm Transwell chambers (Costar), wherein the chemoattractant was separated from cells within replaceable inserts by either an untreated or endothelialized 3-µm pore size polycarbonate membrane. To expose neutrophils to chemoattractant gradients under conditions that physically limited migration, a 0.4-µm pore size filter, through which no migration was observed, was used. Preliminary experiments revealed that the smaller pore size did not markedly restrict the diffusion of macromolecules from lower to upper compartments of chemotaxis chambers, as assessed with blue dextran (Fig. 1). While the pore size of these filters is not small enough to impede the transfer of even large molecules, the pore density of the 0.4-µm filters is markedly greater than that of the 3.0-µm filters, as assessed microscopically. Thus, cells in the upper compartments of chambers with either 3.0- or 0.4-µm filters were exposed to a continuously increasing concentration gradient of chemoattractant at approximately the same rate. In experiments designed to examine neutrophil responses during transendothelial migration, an endothelial monolayer was grown on the filters as described above. Chemoattractants were deposited in lower compartments in a final volume of 1.5 ml and prewarmed to 37°C. After warming, 1 x 10⁷ neutrophils in 1.0 ml of HBSS were aliquoted into the detachable inserts, which were placed over the chemoattractant solutions. Loaded chambers were incubated for 90 min at 37°C in a humidified atmosphere of 5% CO₂ in air. At the end of incubation, the cells that migrated into the bottom chambers were dislodged by gentle scraping, harvested, and enumerated electronically. In some experiments, cells and media from upper chambers were recovered after incubation and processed for IL-8 analysis. Suspensions of recovered cells were cooled on melting ice and pelleted by centrifugation at 500 x g for 5 min. Supernatants were recovered and saved for assay of IL-8 levels. Washed pellets were lysed with 0.2% Triton X-100 on ice for 10 min and centrifuged, and the supernatants were stored at -70°C for further analysis.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Kinetics of diffusion of macromolecules from lower to upper compartments of chambers with 3.0- and 0.4-µm pore size filters. Lower compartments of 12-mm diameter Transwell chambers were loaded with 0.9 ml of blue dextran (m.w. 5000, 20 mg/ml, in HBSS; Sigma), inserts were positioned, and upper compartments were filled with 0.2 ml of HBSS. At various times thereafter, upper compartments were aspirated, and the blue dextran content within them was measured by adsorption at 595 nm to assess the rate of diffusion of macromolecules from lower to upper compartments. The results of one experiment that was repeated twice are shown. Each data point reflects OD values assessed from an individual chamber. The OD of the blue dextran solution introduced into lower compartments was 7.2.

Quantitation of IL-8 was performed using a commercially available ELISA kit (R&D Systems) that employs IL-8 Ab-coated 96-well plastic microtiter plates. After incubation with recovered extracts or IL-8 standards, the wells were washed three times and then incubated with a peroxidase-conjugated anti-IL-8 Ab to ligate IL-8 bound to the fixed Ab. After washing, a peroxidase substrate (tetramethyl benzidine) and hydrogen peroxide were added, and oxidation of the substrate was monitored spectrophotometrically. This method was capable of detecting IL-8 levels as low as 100 pg/ml (10 pg/10⁶ cells), and linear responses were detected between 1 ng and 10 µg IL-8/ml. Where indicated, cells were preincubated for 10 min at 37°C with 10 µg/ml cytochalasin-D, 1 µg/ml actinomycin-D, or 10 µg/ml cycloheximide before chemotactic stimulation.

Measurement of IL-8 synthesis

Neutrophils were labeled with L-[4,5-³H]leucine (100 µCi/ml) for 3 h at 37°C in a humidified 5% CO₂ and 95% air in HBSS containing 10% dialyzed FCS. After incubation, cells were washed extensively with warm (37°C) HBSS and used in chemotaxis chambers as described above. After migration, cells were recovered and lysed with 0.2% Triton X-100. Lysates from 10⁶ cells were spiked with 5 µg of nonlabeled IL-8 to facilitate identification and recovery of labeled IL-8 after immunoprecipitation and gel electrophoresis. Extracts (1 ml) were incubated with 5–10 µg monoclonal anti-IL-8 overnight at 4°C and subsequently precipitated with 30 µl of protein A/G Plus beads for 2 h at room temperature. Beads were centrifuged and washed three times with RIPA buffer (20 mm HEPES, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, pH 7.4), and the resulting pellets were boiled with 20 µl of 2x sample buffer for 5 min. Supernatants obtained after boiling were then separated on a 5–20% gradient polyacrylamide gel. After staining with silver nitrate, IL-8 bands were identified, excised, and solubilized in NSC-II. Solubilized samples were then mixed with 10 ml of scintillation fluid, and the samples were assayed for radioactivity in a liquid scintillation counter.

	Results

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Effect of chemoattractants on IL-8 synthesis

Several chemoattractants including ZAS, PAF, FMLP, C5a, and casein were characterized for their ability to induce IL-8 synthesis in neutrophils. With the exception of PAF, each of these chemoattractants induced a potent chemotactic response resulting in the migration of 44–53% of upper chamber cells into the lower compartments. The response induced by PAF, which attracted 24% of upper chamber cells, was lower than that induced by other chemoattractants but still markedly greater than that observed in chambers without chemoattractant, wherein less than 1% of the cells deposited in the upper chambers were recovered in lower compartments (Table I).

Neutrophils recovered after migration to each of the chemoattractants were found to possess far greater levels of IL-8 than levels observed in cells exposed directly to chemoattractants. ZAS and casein were again the most potent attractants in this respect; levels of IL-8 in cells responding to these attractants were over 100 times greater than levels found in unstimulated cells and 10- to 30-fold higher than levels found in cells stimulated with chemoattractants in the absence of a gradient. Cells migrating in response to these chemoattractants released a small portion of this increased IL-8. Cells that migrated in response to other chemoattractants (PAF, FMLP, C5a) also possessed levels of IL-8 that were markedly greater than those of cells that were directly stimulated with these chemoattractants but did not secrete detectable levels of IL-8 into the extracellular medium during chemotaxis. Cells retrieved from upper chambers after migration did not possess similarly increased levels of IL-8 (not shown).

Involvement of migration in the IL-8 response

The results of the experiments described above indicate that exposure of neutrophils to gradients of chemoattractants potentiates IL-8 synthesis to an extent that is not observed in cells exposed to attractants in the absence of a gradient. While exposure of cells to chemoattractant gradients may directly induce IL-8 synthesis, the response may be secondary to processes activated during migration induced by these gradients. Experiments were designed to explore the actual role of chemotactic migration in induction of the IL-8 response. In the first approach, we employed a system to expose cells to a gradient of chemoattractant under conditions that prevented migration by limiting the pore size of the membrane within chemotaxis chambers to 0.4 µm (see Fig. 1). As shown in Table II, cells exposed to chemoattractant gradients produced higher levels of IL-8 than did cells directly exposed to chemoattractants, whether or not migration was allowed to proceed. However, cells recovered after migration displayed markedly higher levels of IL-8 than did cells exposed to chemoattractant gradients under conditions in which migration was prevented.

The studies described above demonstrate that chemotactic neutrophils possess enhanced quantities of IL-8 as a result of processes activated during migration. However, the origin of this IL-8 is not clear. To investigate the origin of chemotaxis-induced IL-8, we examined IL-8 levels of cells induced to migrate in the presence of the protein and RNA synthesis inhibitors cycloheximide and actinomycin-D, respectively. Actinomycin-D (1 µg/ml) had little effect on IL-8 increases associated with chemotactic migration (Fig. 2). In contrast, cycloheximide (10 µg/ml) markedly attenuated increases in both cell-associated and released IL-8 during chemotactic migration. These observations are consistent with the proposal that increases in IL-8 levels observed in migrating neutrophils result from increased protein synthesis directed by preexisting mRNA.

View larger version (42K): [in this window] [in a new window]   FIGURE 2. Effects of cycloheximide and actinomycin-D on the chemoattractant-induced IL-8 response. Neutrophils were incubated with either cycloheximide (10 µg/ml) or actinomycin-D (1 µg/ml) or vehicle before introduction into upper compartments of chemotaxis chambers containing 10% ZAS in lower compartments. Chemotaxis was allowed to proceed for 90 min, and responsive cells were recovered from lower chambers. Concentrations of IL-8 were measured by ELISA as described in the legend of Table I. Results are the mean ± SD of three experiments.

To demonstrate the de novo synthesis of IL-8 during chemotactic migration, we employed neutrophils preincubated with L-[4,5-³H)]leucine as described under Materials and Methods. The results shown in Fig. 3 demonstrate that chemotactic migration resulted in over a 10-fold increase in [³H]leucine incorporation into IL-8 as compared with cells treated with chemoattractant directly. Furthermore, when cells were treated with cycloheximide (10 µg/ml), the incorporation of L-[4,5-³H)]leucine into IL-8 during migration was reduced by 40%.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. IL-8 synthesis during chemotactic migration. Neutrophils were labeled with L-[4,5-3H]leucine (100 µCi/ml) for 3 h at 37°C in a humidified 5% C02 and 95% air incubator in HBSS containing 10% dialyzed FCS. After incubation, cells were washed extensively with warmed (37°C) HBSS. Washed cells were exposed directly to chemoattractant (chemoattractant-treated cells) or recovered after chemotaxis induced by 10% ZAS (chemotactic cells) as described in Table I. Where indicated, chemotaxis was conducted in the presence of cycloheximide (10 µg/ml). Recovered cells were lysed with 0.2% Triton X-100 and immunoprecipitated with monoclonal anti IL-8 Ab, and the immunoprecipitates were separated on a 5–20% gradient polyacrylamide gel. The bands corresponding to authentic IL-8 were localized, excised, and assayed for radioactivity. Results reflect mean and SD of three determinations.

To evaluate chemotaxis-dependent IL-8 synthesis in a physiologically relevant system, we examined cells after migration through endothelial monolayers. The results demonstrated that migration of neutrophils through endothelial monolayer results in markedly potentiated increases in cell-associated IL-8. In these experiments, neutrophils that migrated through endothelial monolayers were found to possess 6989 ± 1250 pg of IL-8/10⁶ cells. During migration, these cells released 2050 ± 125 pg of IL-8/10⁶ cells into the surrounding medium. Exposure of neutrophils to an endothelial cell monolayer in the absence of chemoattractants did not result in enhanced levels of neutrophil IL-8. The amount of IL-8 released by endothelial cells cultured on inserts and exposed to 10% ZAS in either the absence or the presence of neutrophils (1.0 x 10⁷ cells in 1.0 ml) was below the limits of detection of the assay used. Thus, the elevated IL-8 levels observed in neutrophils migrating through the endothelial monolayer could not be attributed to uptake of IL-8 released from stimulated endothelial cells.

Release of IL-8 by metabolic stimulation of chemotactic neutrophils

Finally, we examined the ability of neutrophils to release the IL-8 that was generated during chemotaxis in response to metabolic stimulation. Cells were recovered from the lower compartment of chemotaxis chambers after migration induced by 10% ZAS, washed, and exposed to 100 nM PMA for 30 min at 37°C. Results were compared with those obtained with neutrophils from the same preparation that were not subject to chemotactic migration or chemoattractant exposure. Upon exposure to PMA, chemotactic cells released 3132 ± 156 pg IL-8/10⁶ cells while control cells released 31.7 ± 1.6 pg IL-8/10⁶ cells. Since the amount of IL-8 released after chemotaxis is much greater than the total amount of IL-8 observed in cells exposed to chemoattractants directly (see Table I), the IL-8 generated during chemotaxis accounts for the increased ability of chemotactic cells to release the cytokine upon metabolic stimulation.

	Discussion

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Neutrophil IL-8 generation is a key component of the inflammatory response (16, 17). Released from migrating cells, IL-8 is a potent autokine that possesses the ability to attract other leukocytes and potentiate cellular function. The IL-8 that is retained by the neutrophil presumably exerts its effects later, after the cell has reached its target. In addition, IL-8 released by damaged neutrophils may exert important effects at the site of inflammatory reactivity, possibly acting in concert with lipids and other protein mediators confined to membrane fragments of disrupted cells. How the neutrophil is prompted to generate IL-8 along its path to the inflammatory sites was the focus of the present investigation.

IL-8 synthesis by metabolically stimulated neutrophils has been well characterized (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 17, 18, 23, 24), and phagocytosing cells release enhanced levels of the cytokine (25, 26). Since cells recovered from inflammatory exudates possess elevated levels of IL-8 (7), we examined the IL-8 response of chemotactic neutrophils. Not surprisingly, we observed that neutrophils recovered from lower compartments after chemotactic migration displayed a remarkably enhanced capacity to release IL-8 upon metabolic stimulation. Chemotactic cells released far greater levels of IL-8 than resting cells possessed. In addition, the extent of IL-8 release by these cells could not be accounted for by direct chemoattractant-stimulated IL-8 synthesis. These considerations led us to explore the possibility that IL-8 was generated in neutrophils during chemotaxis as a result of cellular processes dependent upon or activated by chemotactic migration. The experiments presented in this report demonstrate that this clearly is the case; chemotaxis per se is an effective and potent stimulus of neutrophil IL-8 generation. Neutrophils migrating in response to a gradient of chemoattractant consistently displayed increased levels of cell-associated IL-8. While some of this IL-8 was released into the surrounding media, the majority was retained by the cells. A number of different chemoattractants that are known to activate widely divergent signaling pathways (27) induced a similar IL-8 response, suggesting that the response depended more on the functional consequence of stimulation than on the manner by which that response was triggered. As noted above, cells exposed directly to chemoattractants in the absence of a gradient displayed a markedly lower IL-8 response than that observed in migrating cells. In addition, physical limitation of migration markedly limited the IL-8 response of cells exposed to concentration gradients of chemoattractants. Finally, no response was observed in cells exposed to gradients of chemoattractants in the presence of the inhibitor of actin polymerization, cytochalasin-D, used at levels that effectively prevented migration. While cytochalasin-D may have effects on cells that potentially influence their reactivity independently of the disruption of actin polymerization, it did not inhibit IL-8 generation in cells exposed to a single concentration of chemoattractant (Table III). Thus, IL-8-treated cells retain the machinery necessary to generated IL-8 in response to chemoattractant stimulation. Therefore, it certainly appears that migration itself rather than a biochemical process activated by either chemoattractants or concentration gradients of chemoattractants (as discussed in Refs. 28 and 29) was necessary for optimal expression of the response documented in this report. Neutrophils arriving at sites of infection and inflammation may be expected to possess enhanced levels of IL-8 not as a result of enhanced metabolic reactivity after migration but rather as a result of cellular process activated by migration.

Although cellular migration apparently played a major role in inducing IL-8 synthesis, we cannot entirely discount the possibility that migration facilitated a response that was triggered by a more traditional mechanism. Many cells, including neutrophils, are encumbered by an "adaptation" response that continuously resets the magnitude of responses to certain stimuli, including chemoattractants (see Ref. 28 and references therein). In some noteworthy instances, the rate at which the cell resets is such that no response is initiated by small rates of stimulus increase; at higher rates of stimulus increase, the cell will respond maximally. Our data (Fig. 1) demonstrate that nonmigrating neutrophils in upper compartments of chemotaxis chambers are exposed to a linear increase in chemoattractant levels. It is possible that such a linear increase is not sufficient to trigger an IL-8 response. However, when the cells are migrating through the filter toward the chemoattractant, the rate of increase of attractant to which they are exposed would increase appreciably, probably in an exponential manner. Such an exponential rate of increase may be sufficient to trigger the maximal IL-8 response. If this is the explanation for the results observed, it should be possible to initiate with chemoattractants a maximal response in static cells by ramping the stimulus concentration exponentially. Experiments are underway to determine whether this interesting hypothesis provides the basis for our dramatic results.

While heterogeneity in neutrophil responses to chemoattractants may have influenced our results, we do not believe neutrophil subpopulations can explain the dramatic increase in IL-8 levels observed in cells that have migrated. Indeed, there are well-documented precedents for heterogeneous responses of neutrophil subpopulations to chemoattractants and other metabolic stimuli (30, 31, 32, 33). Subpopulation heterogeneity results in differences in a diverse array of responses, including Ca²⁺ mobilization, surface Ag expression, phagocytosis, and surface electric charge. There may be differences in migratory ability as well. Thus, it is conceivable that cells selected on the basis of migration are enriched in a subpopulation that displays a very brisk IL-8 response to chemoattractants, independent of cell migration. However, it should be noted that, under the conditions used, a high percentage of upper chamber cells migrated in response to each chemoattractant. Such a large subpopulation cannot account for the discrepant IL-8 values between chemoattractant-stimulated and chemotactic cells, since this population would markedly elevate levels in the initial cell preparations as well. Small subpopulations of cells that migrate and exhibit exceptionally elevated responses to chemoattractants independent of migration may play a role in the responses observed, but it is difficult to envision how such an effect explains the dramatic results shown in this study. However, many factors may influence the final tally of average IL-8 levels within such a complex population of cells. It may be necessary to evaluate IL-8 levels within individual cells to finally resolve the influence of cell migration on IL-8 generation in neutrophilic leukocytes.

The nature of the processes activated during migration that lead to enhanced IL-8 levels remains to be defined, but our results provide some important insights regarding the mechanisms involved. Thus, while the protein synthesis inhibitor cycloheximide markedly attenuated the response, the RNA synthesis inhibitor actinomycin-D had little effect. In addition, it is clear that de novo synthesis of IL-8 contributed to the response, since radiolabeled amino acids were incorporated into the parent molecule during migration. Therefore, chemotaxis apparently potentiates the translation of preexisting mRNA. Previous studies have demonstrated regulation of neutrophil IL-8 synthesis at both the levels of RNA transcription and translation. For example, Kuhns and Gallin observed inhibition of ionophore-stimulated IL-8 by cycloheximide but not actinomycin-D (7), similar to the effects found herein. In contrast, using Northern blot analyses, Cassatella et al. demonstrated increased levels of IL-8 mRNA in neutrophils stimulated with LPS (10). In addition, Hachicha et al. recently reported increased levels of IL-8 mRNA in neutrophils phagocytizing zymosan and certain microbial pathogens (18). The failure of actinomycin-D to inhibit responses in the present study cannot be taken as absolute evidence for the conclusion that transcription was not involved, since we did not directly validate the effect of the inhibitor on RNA synthesis. Thus, while actinomycin-D at levels known to interrupt mRNA synthesis in other systems had little influence on the IL-8 response reported in the present study, we cannot entirely exclude the possibility that increased expression of IL-8 mRNA may have contributed to this response.

IL-8 is a chemotactic cytokine that possesses the ability to attract cells to sites of inflammation and infection. IL-8 released by stimulated neutrophils is thought to exert important autocrine effects, amplifying the inflammatory response by recruiting additional cells to the affected area (34). The present study reveals an additional facet of this system of inflammatory regulation: the potentiation of IL-8 synthesis by chemotactic migration. How the IL-8 associated with neutrophils that arrive at inflammatory targets exerts its effect is unclear. However, cell-associated IL-8 would be released from cells that are either metabolically stimulated or disrupted after chemotaxis. In addition, cells with enhanced levels of IL-8 may possess heightened functional reactivity as a result of endogenous cytokine or in response to low but continuous levels of released cytokine. Finally, intracellular IL-8 potentially serves a paracrine-signaling function when intact but apoptotic neutrophils are ingested by macrophages.

	Footnotes

 
¹ This work was supported by project 3 of National Institutes of Health Grant 1 PO1 HL 58064-01, by a grant from the Indiana affiliate of the American Heart Association, and by a grant from the Phi Beta Psi Sorority.

² Address correspondence and reprint requests to Dr. Denis English, Experimental Cell Research Program, The Methodist Research Institute, 1701 North Senate, Indianapolis, IN 46201.

³ Abbreviations used in this paper: PAF, platelet-activating factor; DMEM, Dulbecco’s minimal essential media; ZAS, zymosan-activated serum.

Received for publication April 27, 1998. Accepted for publication September 30, 1998.

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