Pulmonary Inflammation Neutrophil Migration Differs During Acute ...

CD18 Dependency of Transendothelial Neutrophil Migration Differs During Acute Pulmonary Inflammation1 A. Jill Mackarel,2 Kenneth J. Russell, Clodagh M. Ryan, Shirley J. Hislip, Jacqueline C. Rendall, Muiris X. FitzGerald, and Clare M. O’Connor Neutrophil extravasation during inflammation can occur either by a mechanism that requires the neutrophil integrin complex, CD18, or by an alternative CD18-independent route. Which of the two pathways is used has been shown to depend on the site and nature of the inflammatory insult. More recent evidence suggests that selection may also depend on whether inflammation is chronic or acute, but why this is the case remains unknown. Using an in vitro model that supports both migratory mechanisms, we examined the CD18 dependency of migration of neutrophils isolated from patients with either chronic or acute pulmonary infection. Chronic neutrophils were found to behave like normal neutrophils by migrating to IL-8 and leukotriene B4 using the CD18-independent pathway, but to the bacterial product, FMLP, using the CD18-dependent route. In contrast, migration of acute neutrophils to all of these stimuli was CD18 dependent. Normal neutrophils could be manipulated to resemble acute neutrophils by exposing them to FMLP before migration, which resulted in a “switch” from the CD18-independent to -dependent mechanism during migration to IL-8 or leukotriene B4. Although treatment of normal neutrophils with FMLP caused selective down-regulation of the IL-8 receptor, CXCR2, and acute neutrophils were found to have less CXCR2 than normal, a functional relationship between decreased CXCR2 and selection of CD18-dependent migration was not demonstrated. Results indicate that selection of the CD18-dependent or -independent migration mechanism can be controlled by the neutrophil and suggest that the altered CD18 requirements of acute neutrophils may be due to priming in the circulation during acute infection. The Journal of Immunology, 2001, 167: 2839 –2846.

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uring infection, neutrophils migrate out of the bloodstream and move to the source of infection where their function is to eliminate contaminating pathogens. This extravasation process involves a sequence of events initiated by the firm adhesion of the neutrophil to the endothelial lining of the blood vessel wall. If sufficiently activated, the neutrophil leaves the bloodstream by traversing the endothelium and is directed along a concentration gradient of chemoattractant to the inflammatory locus. Extravasation of neutrophils during inflammation has been demonstrated to either involve the ␤2 integrin (or CD18) complex on the neutrophil surface (CD18-dependent migration) or to occur by an alternative as yet uncharacterized pathway that does not involve CD18 (CD18-independent migration). Neutrophil migration out of postcapillary venules during systemic inflammation occurs predominantly by the CD18-dependent pathway (reviewed in Ref. 1). The CD18-independent route for neutrophil migration was first described in animal models of pulmonary inflammation and was initially thought to be specific to the lung (reviewed in Ref. 2). However, the kidney (3), liver (4), heart (5), and peritoneum (6) have also been demonstrated to support both CD18-dependent and CD18-independent migration.

Department of Medicine and Therapeutics, University College Dublin, Dublin, Ireland Received for publication October 30, 2000. Accepted for publication June 20, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Health Research Board of Ireland and the Irish Lung Foundation. 2

Address correspondence and reprint requests to Dr. A. Jill Mackarel, Lung Fibrosis Unit, Department of Medicine and Therapeutics, Woodview, University College Dublin, Belfield, Dublin 4, Ireland. E-mail address: [email protected] Copyright © 2001 by The American Association of Immunologists

To date, the adhesion molecules or interactions that mediate CD18-independent migration have not been identified, but studies with animal and in vitro models of pulmonary inflammation have been successful in uncovering some general characteristics of this alternative migratory route. The stimulus responsible for initiating inflammation appears to be an important determinant in whether CD18-dependent or -independent migration occurs, with some stimuli eliciting CD18-dependent migration while others cause predominantly CD18-independent migration or a mixture of the two (2). For example, i.v. infusion of function-blocking Abs specific for CD18 has been demonstrated to prevent neutrophils migrating into the lungs of rabbits during PMA-induced pneumoniae but did not block migration stimulated by Streptococcus pneumoniae, Staphylococcus aureus, or hydrochloric acid and only partially inhibited migration in response to Escherichia coli endotoxin or C5a (7, 8). It is suggested that this stimulus selectivity may not be a direct effect of the initial insult itself, but may be controlled by the inflammatory mediators produced in response to the original stimulus (2, 9). In support of this, we have recently demonstrated that CD18-independent transendothelial migration of neutrophils can be stimulated in vitro by the host-derived chemoattractants IL-8 and leukotriene B4 (LTB4)3 (10). In the same model, the bacterial-derived chemoattractant, FMLP, activated neutrophils to migrate using the CD18-dependent pathway. Further studies using animal models of inflammation have revealed that the CD18 dependency of migration in response to a stimulus can change depending on whether the insult is acute or chronic. Using a rabbit model of Pseudomonas aeruginosa-induced pneumoniae, Kumasaka and coworkers demonstrated that 3

Abbreviations used in this paper: LTB4, leukotriene B4; CF, cystic fibrosis; HPAEC, human pulmonary artery endothelial cell; cHBSS, HBSS containing Ca2⫹ and Mg2⫹; MPO, myeloperoxidase; RFI, relative fluorescence index; COPD, chronic obstructive pulmonary disease; FEV1, forced expiratory volume at 1 s. 0022-1767/01/$02.00

2840

NEUTROPHIL MIGRATION IN CHRONIC AND ACUTE INFLAMMATION

neutrophil influx during acute inflammation was dependent on CD18, but when recurrent pneumoniae was induced at the same site as the original infection, migration occurred by the CD18independent pathway (11). In another study examining the CD18 dependency of neutrophil migration stimulated by i.p. instillation of protease peptone or live E. coli bacteria, early (4 h) migration was found to be CD18 dependent whereas migration after 24 h occurred by the CD18-independent route (6). These studies suggest that the same stimulus is capable of inducing both CD18dependent and CD18-independent migration, depending on the timing and duration of exposure. Arising from this, the aim of the present study was to assess the CD18 dependency of the migration of neutrophils isolated directly from patients with acute and chronic inflammation. In addition, as a recent study (12) reported that circulating neutrophils from acutely infected patients have an altered surface expression of the two receptors for IL-8 (CXCR1 and CXCR2), we examined whether the CD18 dependency of neutrophil migration to IL-8 was related to changes in IL-8 surface receptor levels.

Materials and Methods

fected with S. aureus. Sputum from the third CF patient grew S. aureus and not P. aeruginosa. No pathogens were cultured from sputum samples of the non-CF patients. Peripheral blood counts revealed a mean white cell count for this chronic inflammation study population of 10.5 ⫾ 1.6 ⫻ 109/L blood, which is at the upper end of the normal range (3.5–11.0 ⫻ 109/L blood). The control population for these studies consisted of seven age- and sex-matched, normal healthy volunteers. For quantification of the chemokine receptors, CXCR1 and CXCR2, on circulating neutrophils, blood was collected from 12 patients with acute pulmonary infection: 6 were CF patients (4 male, 2 female; age range 20 –32 years, median age 28 years; percentage predicted FEV1 range 23– 42%, median 32%) and 6 were non-CF patients (3 male, 3 female; age range 69 – 80 years, median 72 years; percentage predicted FEV1 range 26 – 82%, median 33%). Blood was also collected from 12 patients with chronic pulmonary inflammation: 8 of these were CF patients (three male, five female; age range 17–25 years, median age 21 years; percentage predicted FEV1 range 30 – 89%, median 66%) and 5 were patients with non-CF bronchiectasis (2 male, 3 female; age range 54 –73 years, median age 68 years; percentage predicted FEV1 range 30 –92%, median 69%). When blood was collected from each patient, a sample of blood was simultaneously collected from an age- and sex-matched normal, healthy volunteer and processed for the quantification of CXCR1 and CXCR2 in parallel with the patient sample. This reference normal control group consisted of 25 healthy volunteers (12 male, 13 female; age range 21– 82 years, median age 35 years).

Materials

Cell culture

FMLP, LTB4, human placental type IV collagen, and ABTS were purchased from Sigma (Dorset, U.K.). Human rIL-8 was obtained from R&D Systems (Oxon, U.K.). Functional-blocking mAb specifically recognizing the ␤2 integrin subunit, CD18 (clone L130), and an isotype-matched IgG1 control mAb were purchased from Becton Dickinson (Oxford, U.K.). FITC-conjugated anti-CXCR1 (clone 5A12), PE-conjugated anti-CXCR2 (clone 6C6), and the corresponding FITC- and PE-labeled isotype-matched IgG control Abs were also obtained from Becton Dickinson, as were the function-blocking purified anti-human CXCR1 (clone 5A12) and CXCR2 (clone 6C6) mAbs and the corresponding isotype-matched Ig controls.

Human pulmonary artery endothelial cells (HPAECs) were grown in 100% humidity and 5% CO2 at 37°C in endothelial growth medium supplemented with 5% FCS, epidermal growth factor (10 ng/ml), hydrocortisone (1 ␮g/ml), bovine brain extract containing heparin (10 ␮g/ml), gentamicin (50 ng/ml), and amphotericin-B (50 ng/ml) (Clonetics, San Diego, CA). When ⬃80% confluent, cells were harvested, resuspended in fresh endothelial growth medium, and seeded as previously described (13) at a density of 1.5 ⫻ 105 cells in 200 ␮l onto Transwell polycarbonate membrane filters (6.5 mm diameter, 3.0 ␮m pore size; Corning Costar, Cambridge, MA) that had been coated with human type IV collagen. The Transwell filter inserts were suspended in 24-well culture plates so that the filter separated the upper and lower compartments. Then 600 ␮l culture medium was placed in the lower compartment and the cells were cultured for 4 days. Scanning and transmission electron microscopy confirmed monolayer confluence and integrity (13). All experiments were conducted on HPAECs between passage 6 and 9, and, following completion of experiments, the cells were confirmed to be free of Mycoplasma infection. Chromosome analysis also confirmed that cells remained diploid.

Study populations Circulating neutrophils were isolated from patients with acute pulmonary infection, patients with chronic pulmonary infection (inflammation), and normal controls. Patients with acute infection comprised patients with acute pneumoniae and cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD) patients with acute infective exacerbation. Those with chronic inflammation comprised patients with stable COPD, CF patients in a stable state, and patients with non-CF bronchiectasis. Blood was also collected from a control population that consisted of age- and sexmatched healthy volunteers. In all studies, neutrophils isolated from CF and non-CF patients were compared to ensure that the observations noted were not specific to CF. All subjects gave their informal consent, and ethical permission for the study was obtained from the Ethics Committee in St. Vincent’s Hospital (Dublin, Ireland). For transmigration assays examining the involvement of CD18, blood samples were collected from a total of eight patients with acute pulmonary infection: five CF patients (four female, one male, age range 20 –23 years, median age 20 years; forced expiratory volume at 1 s (FEV1) of 35%, ranging from 30 to 52%) and three non-CF patients with acute pulmonary inflammation (age range 19 – 83 years, median age 62 years; no pulmonary function data available). Sputum cultures revealed that all of the CF patients were infected with P. aeruginosa while two patients were also infected with S. aureus. The mean white cell count for the acute CF patients was 8.3 ⫾ 0.8 ⫻ 109/L blood. The non-CF patient group comprised a COPD patient hospitalized for an acute exacerbation, a patient recovering from a mild stroke who developed a chest infection, and a young male with no previous medical history who was admitted to hospital through casualty with pneumonia. These patients had elevated peripheral blood white cell counts at 20.2 ⫾ 4.6 ⫻ 109/L blood, and they presented with increased temperature, a productive cough, and chest x-rays demonstrating the presence of infiltrates. The mean white cell count for the combined CF and non-CF acute patient group was 14.3 ⫾ 3.4 ⫻ 109/L blood. The chronic inflammation study population consisted of a total of six patients in a stable state: three CF patients (two female, one male, age range 20 –23 years, median age 22 years; percentage predicted FEV1 range of 54 –77%, median 58%) and three non-CF patients with COPD (age range 62–79 years, median 66 years; percentage predicted FEV1 range 25–79%, median 64%). Of the CF patients, sputum cultures revealed that two of the three subjects were infected with P. aeruginosa, and one of these subjects was also in-

Isolation of neutrophils For transmigration assays, human peripheral venous blood was collected by venipuncture into sterile Vacutainers (BD Biosciences, Franklin Lakes, NJ) containing 0.105 M sodium citrate as an anticoagulant and allowed to cool to room temperature for 10 –15 min. Neutrophils were isolated using density gradient centrifugation on Polymorphprep (Nycomed Pharma, Oslo, Norway) at 450 ⫻ g for 35 min at 20°C. Contaminating erythrocytes were removed by hypotonic lysis, and the isolated neutrophils were resuspended in HBSS without Ca2⫹ or Mg2⫹ for cell number and viability to be assessed before being resuspended in HBSS containing Ca2⫹ and Mg2⫹ (cHBSS) at a concentration of 1 ⫻ 107 neutrophils/ml. Neutrophils isolated in this way were 97% pure and ⬎95% viable.

Transmigration assay After 4 days in culture, HPAECs on the Transwell filter inserts were transferred to a fresh 24-well tissue culture plate, and neutrophil transmigration was monitored as previously reported (13). In brief, culture medium was carefully removed from all filter inserts, and, following gentle rinsing of the cells with cHBSS prewarmed to 37°C, medium was replaced with cHBSS in the upper compartment of the Transwell system and either chemoattractant in cHBSS or cHBSS alone was added to the lower compartments. After a 45-min preincubation period, the migration assay was initiated by the addition of 1 ⫻ 106 neutrophils to all upper compartments. The plates were incubated at 37°C in 100% humidity and 5% CO2 for 3 h. After the 3-h incubation time, the plate was placed on ice, and nonadherent neutrophils (upper compartment) and migrated neutrophils (lower compartment) were collected by gentle washing with cHBSS followed by centrifugation at 300 ⫻ g for 10 min. The neutrophils were then lysed by suspension in cHBSS containing 0.25% (w/v) Brij-35. The HPAEC monolayer with associated adherent neutrophils was removed by carefully cutting the filter membrane out of the insert and lysed by addition of

The Journal of Immunology cHBSS containing 0.25% Brij-35. For each experiment, a range of neutrophil concentrations were prepared in cHBSS and incubated for 3 h at 37°C, after which time the neutrophils were collected by centrifuging at 300 ⫻ g for 10 min before being resuspended in cHBSS containing 0.25% Brij-35 and lysed concurrent with the migration assay samples. All neutrophil lysates were assayed for myeloperoxidase (MPO) activity using an adaptation (13) of the method of Madara et al. (14). A standard curve of number of neutrophils vs MPO activity was constructed, and the number of nonadhered, adherent, and migrated neutrophils was quantified by extrapolation of the MPO activity present in upper, monolayer, and lower compartments, respectively. A linear relationship between number of neutrophils and MPO activity was obtained in the range of 0.05–1.0 ⫻ 106 neutrophils/ml. Incubation of neutrophils for the duration of the transmigration assay with either FMLP, IL-8, or LTB4 at the concentrations used to stimulate migration did not cause any release of MPO.

Assessment of the role of CD18 in neutrophil transmigration of HPAEC

2841 tween two groups were assessed using either Student’s unpaired t test or the Mann-Whitney U test for nonparametric data. In all cases, statistical significance was considered at p ⬍ 0.05.

Results Migration of neutrophils from patients with chronic or acute pulmonary infection Neutrophils isolated from patients with chronic or acute pulmonary infection could be stimulated to migrate across monolayers of HPAECs in response to a chemotactic gradient of IL-8, LTB4, or FMLP (Fig. 1A). In the absence of chemoattractant, no neutrophil migration was detected. For both chronic and acute neutrophils, a similar amount of migration was stimulated by all three chemoattractants with ⬃50% of total neutrophils migrating after the 3-h

The involvement of CD18 in neutrophil migration across HPAECs was analyzed by preincubating neutrophils for 15 min at 37°C with either antiCD18 or the corresponding isotype-matched IgG1 control mAb (a saturating concentration of 1 ␮g of purified Ig/5 ⫻ 105 neutrophils was used) before adding them to the HPAEC monolayers. Migration of untreated neutrophils, and of neutrophils treated with Ab, was analyzed in response to FMLP (10 nM), IL-8 (10 nM), and LTB4 (0.1 ␮M) by the method detailed in Transmigration assay above. When the effect of preexposure to FMLP on the CD18 dependency of neutrophil migration was being examined, FMLP (10 nM) was included in the 15 min preincubation of neutrophils with either anti-CD18 mAb or the isotype-matched Ig control. After this incubation period, neutrophils were added directly to HPAEC monolayers that had been preincubated for 45 min with either IL-8 (10 nM) or LTB4 (0.1 ␮M) as described in Transmigration assay.

Quantification of CXCR1 and CXCR2 on neutrophils FITC- and PE-conjugated Abs were used to fluorescently dual-label the chemokine receptors, CXCR1 and CXCR2, respectively, on the surface of neutrophils. Isotype-matched Ig labeled with either FITC or PE was used to assess nonspecific binding. Human peripheral venous blood was collected by venipuncture into sterile Vacutainers containing 0.105 M sodium citrate as an anticoagulant, and a sample of whole blood was immediately fixed with an equal volume of 0.4% formaldehyde in PBS for 10 min on ice. Following lysis of erythrocytes, remaining cells were washed and resuspended in ice-cold PBS. Then 100-␮l aliquots of cell suspension containing 1 ⫻ 105 neutrophils were incubated with a saturating amount (0.4 – 1.0 ␮g) of both FITC-labeled anti-CXCR1 Ab and PE-labeled anti-CXCR2 Ab for 60 min in the dark at 4°C. After the incubation, the cells were washed twice in PBS containing 2% (v/v) FCS before being resuspended in 1% (w/v) paraformaldehyde in PBS. Two-color flow cytometry was used to simultaneously quantify fluorescence from labeled CXCR1 and CXCR2 using a FACScan flow cytometer (Becton Dickinson). Fluorescence from each receptor was expressed as the relative fluorescence index (RFI), where RFI was calculated as the ratio of the mean fluorescence intensity of the specific vs nonspecific FITC or PE fluorescence intensity for each sample. Isotype-matched FITC- and PE-labeled Ig was used to assess nonspecific binding. When the effect of exposure to FMLP, IL-8, and LTB4 on CXCR1 and CXCR2 levels was being assessed, neutrophils from whole blood were incubated alone or in the presence of each chemoattractant at the concentration used in the migration assay for either 15 min or 3 h at 37°C. After the incubation period, neutrophil CXCR1 and CXCR2 levels were quantified as described above.

Assessment of the role of CXCR1 and CXCR2 in IL-8-stimulated neutrophil transmigration of HPAEC The involvement of CXCR1 and CXCR2 in the migration of neutrophils across HPAEC in response to IL-8 was examined by preincubating neutrophils for 15 min at 37°C with function-blocking Abs specific for CXCR1 or CXCR2, either alone or in combination, or with the corresponding isotype-matched IgG control mAb (1 ␮g of purified Ig/106 neutrophils) before adding them to the HPAEC monolayers. The Abs were present for the duration of the migration assay. Migration of untreated neutrophils, and of neutrophils treated with Ab, was analyzed in response to IL-8 (10 nM) by the method detailed in Transmigration assay.

Statistical analysis Results are summarized as means ⫾ SEM. Multiple comparisons were performed using ANOVA with a Bonferroni post-test. Comparisons be-

FIGURE 1. Transendothelial migration (A) and adhesion (B) of neutrophils from patients with chronic or acute pulmonary infection, compared with normal. Neutrophils were isolated from normal healthy subjects (n ⫽ 7) and from patients with either chronic (n ⫽ 8) or acute (n ⫽ 6) pulmonary infection and were allowed to migrate across HPAEC in response to IL-8 (10 nM), LTB4 (0.1 ␮M), or FMLP (10 ␮M) as described in Materials and Methods. The percentage of adherent and migrated neutrophils was determined by comparison of MPO in neutrophils recovered from the HPAEC monolayers and the lower compartment of the Transwell system, respectively, with the total MPO content of neutrophils added to the system. Neutrophils that had not adhered to or migrated across the HPAEC monolayers were recovered from the upper compartment of the Transwell system and accounted for the remaining percentage of total neutrophils added to the system. Results are means ⫾ SEM (*, p ⬍ 0.05; **, p ⬍ 0.01). PMN, polymorphonuclear neutrophils.

2842


incubation time. However, when this migration was compared with that of neutrophils from normal healthy subjects, it was found to be significantly lower when IL-8 or LTB4 was the migratory stimulus (Fig. 1A). In response to IL-8, 71.6 ⫾ 4.5% of normal neutrophils migrated, whereas migration of chronic and acute neutrophils was significantly less ( p ⬍ 0.01) at 53.2 ⫾ 3.4 and 45.2 ⫾ 1.2%, respectively. For LTB4, a similar ⬃30% decrease in total migration ( p ⬍ 0.01) was observed with chronic and acute neutrophils compared with control neutrophils: 52.6 ⫾ 3.2% of chronic neutrophils and 52.2 ⫾ 3.5% of acute neutrophils migrated to LTB4 in contrast to 78.9 ⫾ 4.8% migration of normal neutrophils. The decrease in migration observed with chronic and acute neutrophils was not as significant when FMLP was the migratory stimulus, with 51.2 ⫾ 1.9% and 50.4 ⫾ 4.7% of chronic and acute neutrophils migrating to FMLP, respectively, in contrast to 62.3 ⫾ 3.5% migration observed with normal neutrophils. The decreased migration observed with chronic and acute neutrophils was not due to decreased ability to adhere to HPAEC. The percentage of total chronic and acute neutrophils associated with the HPAEC monolayers was greater than that observed with normal neutrophils, indicating that the decrease in migration observed was the result of blunted ability to migrate but not to adhere (Fig. 1B). Assessment of the CD18 dependency of neutrophil migration from patients with chronic or acute pulmonary infection When migration was stimulated by either IL-8 or LTB4, chronic and acute neutrophils differed in the effect the presence of a function-blocking Ab to CD18 had on migration (Fig. 2). Chronic neutrophils, similar to control neutrophils, migrated to IL-8 and LTB4 in a predominantly CD18-independent manner. Migration of chronic neutrophils to IL-8 (Fig. 2A) in the presence of anti-CD18 Ab (46.6 ⫾ 2.7%) was not significantly less than migration in the presence of the isotype-matched control Ab (49.3 ⫾ 2.7%). Similarly, migration of chronic neutrophils to LTB4 (Fig. 2B) was not affected by the anti-CD18 Ab: 45.5 ⫾ 2.3% of neutrophils migrated in the presence of anti-CD18 Ab compared with 48.8 ⫾ 1.9% of neutrophils treated with isotype-matched control Ab. By comparison, neutrophils from patients with acute pulmonary infection migrated to IL-8 and LTB4 in a predominantly CD18dependent manner (Fig. 2, A and B). Compared with migration in the presence of the isotype-matched control Ab, the presence of the anti-CD18 Ab decreased migration of acute neutrophils to IL-8 (Fig. 2A) by ⬃70% (from 44.3 ⫾ 3.3% to 12.7 ⫾ 1.4%, p ⬍ 0.01) and to LTB4 (Fig. 2B) by ⬃66% (from 45.8 ⫾ 3.6% to 15.5 ⫾ 2.9%, p ⬍ 0.01). In contrast to IL-8 and LTB4, the bacterial-derived chemoattractant, FMLP, stimulated all neutrophils to migrate using the CD18dependent mechanism (Fig. 2C). Greater than 70% of migration of control, chronic, and acute neutrophils was blocked by the presence of the anti-CD18 Ab: migration of neutrophils from chronically infected patients was decreased from 46.1 ⫾ 1.1% (isotypematched control) to 13.6 ⫾ 2.9% ( p ⬍ 0.01), while migration of neutrophils from acutely infected patients was decreased from 46.8 ⫾ 3.8% to 10.1 ⫾ 1.6% ( p ⬍ 0.01). Effect of preincubating normal neutrophils with FMLP on the CD18 dependency of migration to IL-8 and LTB4 To determine whether exposure to bacterial products such as FMLP in the bloodstream during acute infection might account for the switch to CD18-dependent migration observed with neutrophils from patients with acute pulmonary infection, the CD18 dependency of migration of normal neutrophils to IL-8 and LTB4 was examined after the neutrophils had been incubated with and

FIGURE 2. Effect of anti-CD18 Ab on migration of neutrophils from normal, healthy subjects (n ⫽ 8) and from patients with chronic (n ⫽ 6) or acute (n ⫽ 6) pulmonary infection. Neutrophils were incubated in the presence of antibody to CD18 or with isotype-matched IgG1 for 15 min at 37°C and then allowed to migrate across HPAECs in response to 0.1 ␮M IL-8 (A), LTB4 (B), or 10 nM FMLP (C) as described in Materials and Methods. The percentage of migrated neutrophils was determined by comparison of MPO in neutrophils recovered from the lower compartment of the Transwell system with the total MPO content of neutrophils added to the system. Results are means ⫾ SEM (**, p ⬍ 0.01). PMN, polymorphonuclear neutrophils; aCD18 mAb, anti-CD18 mAb.

The Journal of Immunology without FMLP. As was observed in our previous study (10), normal neutrophils migrated to IL-8 and LTB4 in a predominantly CD18-independent manner. Preexposure to FMLP did not alter the total amount of migration that occurred over the 3-h assay period but caused a significant shift in the CD18 dependency of migration, with the CD18-dependent route taking over as the predominant migratory route (Fig. 3). Preexposure to FMLP decreased the percentage of CD18-independent migration to IL-8 from 68.5 ⫾ 10.9% to 37.7 ⫾ 5.5% ( p ⬍ 0.05) so that the predominant migratory route was CD18 dependent. Likewise, the percentage of migration in response to LTB4 that was CD18 independent was decreased from 73.8 ⫾ 5.6 to 40.3 ⫾ 5.4% ( p ⬍ 0.05) by preexposure to FMLP (Fig. 3). To determine whether this ability to cause neutrophils to “switch” to using the CD18-dependent pathway was specific for FMLP, the effect of pretreating neutrophils with TNF-␣ on the CD18 dependency of migration was examined. Preexposure to TNF-␣ (1 ng/ml), in a similar manner to FMLP, decreased the percentage of CD18-independent migration to IL-8 from 70.0 ⫾ 4.4% to 42.4 ⫾ 8.4% ( p ⬍ 0.05; n ⫽ 4 independent experiments). Examination of the relationship between the CD18 dependency of migration and expression of IL-8 receptors By causing a switch from CD18-independent to -dependent migration, stimulation with FMLP caused normal neutrophils to behave like neutrophils from patients with acute pulmonary infection when migrating to IL-8 and LTB4. Because FMLP treatment of normal neutrophils has been shown by others to result in selective down-regulation of CXCR2 (15) and a similar down-regulation of this IL-8 receptor has been observed (12) on neutrophils from patients with acute infection (sepsis/adult respiratory distress syndrome), we decided to examine whether there was a functional link

FIGURE 3. Effect of preexposure to FMLP on the CD18 dependency of migration of normal neutrophils to IL-8 and LTB4. Neutrophils were incubated with (hatched bars) and without (cross-hatched bars) FMLP (10 nM) in the presence of Ab to CD18 or with isotype-matched IgG1 for 15 min at 37°C and then allowed to migrated across HPAECs in response to either IL-8 (0.1 ␮M) or LTB4 as described in Materials and Methods. The percentage of CD18-dependent migration represents the amount of migration that was blocked by treatment with CD18 Ab compared with migration of neutrophils treated with the isotype-matched control Ig. Migration was quantified by comparison of MPO in neutrophils recovered from the lower compartment of the Transwell system with the total MPO content of neutrophils added to the system. Results are means ⫾ SEM for four independent experiments (*, p ⬍ 0.05).

2843 between decreased CXCR2 (or CXCR1 dominance) and selection of the CD18-dependent pathway. To address this hypothesis, we first examined the effect of a 15-min exposure to FMLP on the surface expression of the IL-8 receptors on normal neutrophils. Quantification of CXCR1 and CXCR2 using dual-label flow cytometry revealed that, while levels of CXCR1 remained unchanged, levels of CXCR2 on FMLP-treated neutrophils were decreased by ⬃60% from 8.9 ⫾ 0.6 RFI units (CXCR2 level on untreated neutrophils after the 15-min incubation) to 3.7 ⫾ 0.9 RFI units ( p ⬍ 0.05). Having thus confirmed that preexposure of normal neutrophils to FMLP in our system did cause selective downregulation in CXCR2, to examine the possibility that there may be a functional relationship between CXCR1 dominance and CD18-dependent migration, levels of CXCR1 and CXCR2 were quantified on neutrophils from patients with either chronic or acute pulmonary infection and compared with levels on age- and sexmatched normal, healthy controls. If there was a functional link between decreased CXCR2 levels and selection of the CD18dependent migratory pathway, then CXCR2 levels on chronic neutrophils should not be less than normal because chronic neutrophils resembled normal neutrophils by migrating to IL-8 using the CD18-independent pathway. As expected (12, 16), normal neutrophils had approximately equal amounts of CXCR1 and CXCR2 (Fig. 4). In contrast, both chronic and acute neutrophils were found to have less CXCR2 than CXCR1 on their surfaces (Fig. 4). Compared with normal neutrophils, the amount of surface CXCR1 was not altered on chronic or acute neutrophils (Table I). However when CXCR2 levels were compared, chronic and acute neutrophils

FIGURE 4. Surface CXCR1 and CXCR2 levels on peripheral blood neutrophils taken from a representative normal, healthy subject or a patient with either chronic or acute pulmonary infection. CXCR1 and CXCR2 were simultaneously labeled on blood neutrophils by incubating the neutrophils at 4°C for 60 min with FITC-labeled Ab against CXCR1 and PE-labeled Ab against CXCR2 (0.4 –1 ␮g Ab/1 ⫻ 105 neutrophils) as described in Materials and Methods. After two washings in PBS containing 2% (v/v) FBS, the neutrophils were resuspended in 1% (w/v) paraformaldehyde. Cell surface fluorescence was quantified using a FACScan flow cytometer. Data are presented as overlaid distribution histograms of surface fluorescence intensity from labeled CXCR1 (dashed black line) and CXCR2 (solid black line) on neutrophils from a representative donor. Isotype-matched FITC- and PE-labeled Ig was used to assess nonspecific binding for the Ab specific for CXCR1 (dashed gray line) and CXCR2 (solid gray line), respectively. FL1-H, fluorescence.

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Table I. Levels of CXCR1 and CXCR2 on neutrophils from normal, healthy control subjects and on neutrophils from patients with either chronic or acute pulmonary infectiona Surface Levels of IL-8 Receptors (relative fluorescence index) Neutrophil Type

Control Chronic Acute

CXCR1

CXCR2

11.18 ⫾ 0.71 12.13 ⫾ 0.85 10.62 ⫾ 1.02

13.87 ⫾ 1.22 7.98 ⫾ 0.65* 7.49 ⫾ 1.13*

a CXCR1 and CXCR2 were simultaneously labeled on blood neutrophils by incubating the neutrophils at 4°C for 60 min with FITC-labeled Ab against CXCR1 and PE-labeled Ab against CXCR2 (0.4 –1 ␮g Ab/1 ⫻ 105 neutrophils) as described in Materials and Methods. After washing twice in PBS containing 2% (v/v) FBS, the neutrophils were resuspended in 1% (w/v) paraformaldehyde. Cell-surface fluorescence was quantified using a FACScan flow cytometer and expressed as the ratio of the mean fluorescence intensity of the specific versus nonspecific FITC or PE fluorescence intensity for each sample. Results are mean ⫾ SEM for 23 normal, healthy control subjects, 12 patients with chronic pulmonary infection, and 12 patients with acute pulmonary infection. ⴱ, p ⬍ 0.01 for the difference in CXCR2 levels on neutrophils from chronic and acute patients compared to CXCR2 levels on neutrophils from controls.

were found to have 43% and 46% less CXCR2 than normal ( p ⬍ 0.01), respectively. Therefore, decreased expression of CXCR2 does not appear to be directly linked to the switch from CD18-independent to -dependent migration observed with neutrophils from patients with acute pulmonary infection during migration to IL-8. Additional studies with functional-blocking Abs specific for the IL-8 receptors revealed that CXCR1 was the receptor mediating IL-8-stimulated CD18-independent migration of normal neutrophils. Compared with the isotype-matched control Ig, the antiCXCR1 Ab decreased migration of normal neutrophils to IL-8 by 54.1 ⫾ 6.9%. Blocking CXCR2 had no effect on migration or on the CD18 dependency of migration (Table II). A combination of anti-CXCR1 and anti-CXCR2 Abs had a slight additive effect but the functionally active receptor mediating CD18-independent migration of normal neutrophils to IL-8 appeared to be CXCR1. Blocking this receptor did not alter the CD18 dependency of migration, in that including a function-blocking Ab against CD18 in addition to the anti-CXCR1 Ab did not affect migration (Table II). Interestingly, although a 15-min exposure to FMLP at 37°C was found to cause a significant decrease in CXCR2 levels on neutrophils, this selective down-regulation of CXCR2 was not specific for FMLP, with exposure to IL-8 or LTB4 causing a similar decrease. Compared with CXCR2 levels on untreated neutrophils incubated under the same conditions (9.0 ⫾ 0.6 RFI units), FMLP Table II. Effect of function-blocking Abs against the IL-8 receptors, CXCR1 and CXCR2, on the migration of normal neutrophils to IL-8a

Function-Blocking Ab

CXCR1 CXCR1 ⫹ CD18 CXCR2 CXCR2 ⫹ CD18 CXCR1 ⫹ CXCR2

Neutrophil Migration to IL-8 (% of IgG control)

46.9 ⫾ 6.9 51.0 ⫾ 6.1 104.6 ⫾ 13.9 98.9 ⫾ 11.4 29.8 ⫾ 6.2

a The involvement of CXCR1 and CXCR2 in the migration of neutrophils across HPAEC in response to IL-8 was examined by preincubating neutrophils (isolated from normal, healthy subjects) for 15 min at 37°C with function blocking Abs specific for CXCR1 or CXCR2, either alone or in combination, or with the corresponding isotype-matched IgG control mAb before adding them to the HPAEC monolayers. When the CD18-dependency of migration was being examined, function-blocking Ab specific for CD18 or the corresponding isotype-match control Ab was included in the neutrophil pre-incubation. The Abs were present for the duration of the migration assay. Migration of untreated neutrophils and of neutrophils treated with Ab was analysed in response to IL-8 (10 nM) as detailed in Materials and Methods.

treatment decreased CXCR2 to 3.8 ⫾ 0.9 RFI units ( p ⬍ 0.05), while IL-8 and LTB4 decreased CXCR2 to 2.9 ⫾ 1.1 ( p ⬍ 0.01) and 4.0 ⫾ 1.2 ( p ⬍ 0.05) RFI units, respectively. A similar pattern was observed when neutrophils were exposed to the different chemoattractants for 3 h (i.e., the time period of the transmigration assay) with CXCR2 being decreased from 7.8 ⫾ 1.0 RFI units on untreated neutrophils to 3.7 ⫾ 0.8 ( p ⬍ 0.01), 2.3 ⫾ 0.2 ( p ⬍ 0.001) and 5.6 ⫾ 0.6 (NS) on FMLP-, IL-8-, and LTB4-treated neutrophils, respectively.

Discussion In this study, the involvement of CD18 was examined during transendothelial migration of neutrophils from patients with either chronic or acute pulmonary infection in an in vitro model that supports both CD18-dependent and -independent migration. We report that chronic neutrophils behave in a similar manner to previous observations with normal neutrophils (10) in migrating to IL-8 and LTB4 in a CD18-independent manner. In contrast, acute neutrophils differ by migrating to these stimuli using the CD18dependent pathway. Results indicate that during acute infection, a switch in the CD18 dependency of the migratory route used by neutrophils occurs, with the CD18-dependent pathway predominating. Such association of chronic inflammation with the CD18independent pathway and of acute inflammation with the CD18dependent migratory pathway reflects previous reports from animal studies that examined CD18 involvement during neutrophil migration in acute and chronic inflammation. In rabbits, blocking CD18 prevented neutrophil migration during acute pneumoniae induced by airway instillation of P. aeruginosa, but did not block migration during recurrent inflammation when P. aeruginosa was reinstilled at the same site 7 days later (11). Similarly, in a mouse model of inflammation, early (4 h) migration of neutrophils stimulated by i.p. instillation of either protease peptone or live E. coli bacteria was demonstrated to be CD18 dependent, whereas late (24 h) migration was CD18 independent (6). Such observations of differences in the migratory pathway used by neutrophils during acute and chronic inflammation led to suggestions that the duration of the inflammatory insult is an important factor in determining the migratory pathway taken by the neutrophil during inflammation. Results of the present study offer explanation for these differences by demonstrating that circulating neutrophils are inherently altered during acute but not chronic infection, possibly as a result of activation/priming in the bloodstream. Neutrophils in the circulation during sepsis have been shown to display characteristics that suggest they are activated or primed. They are less deformable than normal (17), have increased surface expression of CD11/CD18, reduced levels of L-selectin, and display increased oxidative capacity (reviewed in Refs. 18 and 19). It is interesting that, although neutrophils from patients with chronic infection displayed blunted chemotactic responses in a similar manner to neutrophils from patients with acute infection (Fig. 1), they behaved like normal neutrophils in the CD18 dependency of migration. The fact that preexposure to FMLP or TNF-␣ caused normal neutrophils to switch to using the CD18-dependent route when migrating to IL-8 and thereby resemble acute neutrophils, suggests that activation or priming of neutrophils in the circulation during acute infection may account for the differences in the CD18 dependency of migration observed in this study. Such priming by elevated levels of inflammatory mediators in the bloodstream during acute infection/inflammation would explain our observations that, although “acute” neutrophils were obtained from patients with very varied clinical circumstances, a very uniform and reproducible alteration of the migration behavior of their peripheral blood neutrophils was observed.

The Journal of Immunology These findings provide valuable insight into what determines selection of the CD18-dependent or -independent migratory pathway, a topic that has been the center of recent discussion (20). Animal (reviewed in Ref. 2) and in vitro (10, 21) models of inflammation have demonstrated that the initial inflammatory stimulus is a major factor in determining the CD18 dependency of migration, and results of the present study are supportive of this. Chronic and normal neutrophils migrated to the host-derived stimuli, IL-8 and LTB4, using the CD18-independent pathway, whereas when the chemotactic stimulus was FMLP, migration occurred by the CD18-dependent route. The ability of normal and chronic neutrophils to switch between the two pathways depending on what inflammatory stimulus is activating them to migrate, the observation that neutrophils isolated from patients with acute infection differed in migrating to all stimuli using the CD18-dependent pathway, and the fact that normal neutrophils could be manipulated to switch from using the CD18-independent to the -dependent pathway and mimic acute neutrophils all confirm that the neutrophil alone can control the migratory pathway selected. Simultaneous to our finding that acute neutrophils differ from chronic and normal neutrophils in the way in which they migrate to IL-8, Cummings and coworkers reported that the balance of the two surface receptors for IL-8, CXCR1 and CXCR2, was altered on neutrophils from patients with acute infection (12). Unlike normal neutrophils that have approximately equal amounts of CXCR1 and CXCR2, neutrophils from patients with acute inflammation/ infection were found to have decreased levels of CXCR2 (12). When the functionality of this receptor “imbalance” was examined, it was found that CXCR1 was the functionally dominant receptor on these acute neutrophils. The IL-8 receptors (22, 23), along with the two FMLP (24) and LTB4 (25) receptors, are members of a family of chemoattractant receptors that are heptahelical G protein-coupled transmembrane proteins. Although it was originally believed that a common signaling pathway mediated the activation of neutrophils by all chemoattractants, it is now recognized that a diverse range of intracellular signaling pathways are coupled to the activation of chemoattractant receptors on neutrophils (26). Therefore, it is feasible to suggest that the signaling pathways that result in CD18-dependent migration could be different to those that culminate in CD18-independent migration, or that binding of IL-8, for example, to one of its high-affinity receptors stimulates CD18-dependent migration whereas binding via the other specific receptor results in CD18-independent migration. Coupling Cummings and colleagues’ findings that neutrophils from patients with sepsis have decreased expression and function of the CXCR2 receptor for IL-8 with our observations that neutrophils from patients with acute infection differed from normal in the way they migrated to IL-8, we extended our study to examine whether there may be a functional link between CXCR1 dominance (CXCR2 down-regulation) and selection of the CD18-dependent migratory route. Our working hypothesis was that decreased expression of CXCR2 predisposes acute neutrophils to migrate in response to IL-8 activation in a CD18-dependent manner, whereas a normal balance of the two receptors enables neutrophils to use the CD18-independent migratory route in response to IL-8, as observed with neutrophils from normal, healthy subjects (10). We report here that, in contradiction to our hypothesis, there does not seem to be a direct functional link between decreased CXCR2 expression and “selection” of the CD18-dependent migratory route. We demonstrate that CXCR1, not CXCR2, mediates CD18-independent migration of normal neutrophils and that neutrophils from patients with chronic pulmonary inflammation, similar to neutrophils from patients with acute infection, have

2845 decreased expression of CXCR2 yet migrate to IL-8 in a CD18independent manner. Although the finding that CXCR2 levels on chronic neutrophils were similar to acute neutrophils disproved our original hypothesis that there may be a functional link between CXCR1 dominance and CD18-dependent migration, the observation that chronic neutrophils had decreased surface expression of CXCR2 is an important and novel finding. Both CXCR1 and CXCR2 are high-affinity receptors for IL-8 and can mediate IL-8-stimulated increases in intracellular calcium concentrations, release of granule contents, and chemotaxis (reviewed in Ref. 27). However, IL-8-induced activation of the respiratory burst and release of reactive oxygen species by neutrophils is exclusively mediated by CXCR1 (28, 29). Dominance of CXCR1 as a result of selective down-regulation of CXCR2 has been associated with enhanced reactive oxygen production by neutrophils and has been suggested to explain the increased oxidative capacity observed with circulating neutrophils from patients with sepsis (29). To date, decreased expression and function of CXCR2 has only been reported with neutrophils from patients with acute inflammation, i.e., patients with sepsis (12) or trauma (30). We demonstrate here that decreased CXCR2 expression is not specific to acute inflammation but that neutrophils from clinically stable patients with chronic inflammation display a similar phenotype. In conclusion, the results of the present study demonstrate that neutrophils from patients with acute but not chronic infection differ to neutrophils from normal, healthy subjects in the selection of the migratory route they use during migration to IL-8 and LTB4 in vitro. Neutrophils appear to be primed during acute infection to use the CD18-dependent pathway during migration, an effect that could be mimicked in vitro by treating normal neutrophils with FMLP or TNF-␣. These results indicate that selection of the migratory pathway can be controlled solely by the neutrophil.

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