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Division of Trauma, Department of Surgery, New Jersey Medical School, Newark, New Jersey
Department of Surgery, University of Rochester School of Medicine and Dentistry, Rochester, New York
ABSTRACT
Neutrophil hyperactivity contributes to organ failure, whereas hypofunction permits sepsis. The chemokine receptors CXCR1 and CXCR2 are central to polymorphonuclear neutrophil (PMN) function. We prospectively assessed CXCR function and expression in PMNs from trauma patients at high risk for pneumonia and their matched volunteer controls. CXCR2-specific calcium flux and chemotaxis were desensitized by injury, returning toward normal after 1 week. CXCR1 responses were relatively maintained. These defects appeared to be caused by preferential suppression of CXCR2 surface expression. To evaluate potential mechanisms of in vivo chemokine receptor regulation further we studied cross-desensitization of chemokine receptors in normal PMNs. Susceptibility to desensitization was in the order CXCR2 > CXCR1 > formyl peptide or C5a receptors. Trauma desensitizes CXC receptors, with CXCR2 being especially vulnerable. Desensitization is most marked immediately postinjury, generally resolving by Day 7. High-affinity chemoattractant receptors responsible for PMN chemotaxis from bloodstream to tissue appear to be regulated by injury. Receptors for end-target chemoattractants regulate CXCR1 and CXCR2 but resist suppression themselves and respond normally after injury. CXCR2 desensitization occurs before pneumonia, which developed in 44% of these patients. Suppression of high-affinity PMN receptors, like CXCR2, may predispose to pneumonia after trauma or other inflammatory conditions that lead to systemic inflammatory response syndrome.
Key Words: chemokines G-proteineCcoupled receptors injury pneumonia polymorphonuclear neutrophils
Polymorphonuclear neutrophils (PMNs) play a central role in the innate immune response. PMNs are recruited to sites of bacterial inoculation by a variety of G-proteineCcoupled (GPC) chemoattractants, including the CXC chemokines (1eC5). Conversely, pathologic PMN activation is associated with the systemic inflammatory response syndrome, acute lung injury, acute respiratory distress syndrome, and multisystem organ failure (6eC8). Chemokines as well as other GPC chemoattractants are produced in abundance at sterile injury sites (9, 10). Such distant inflammation may decrease PMN recruitment to the lung (11), but the mechanisms underlying this effect are unknown. Clinical outcomes after major trauma have been related to plasma levels of the CXC chemokine interleukin 8 (IL-8)/CXCL8 (12eC14). IL-8 binds to both CXC chemokine receptor 1 (CXCR1) and receptor 2 (CXCR2). Most CXC chemokines, however, only bind to the promiscuous CXCR2 (12, 13, 15eC17). Growth-related oncogene (GRO-; CXCL1) is an important example of this group, participating in both pulmonary responses to infection and in the pathogenesis of acute respiratory distress syndrome. Furthermore, in many systems, CXCR2 is the dominant receptor recruiting PMNs to the lung (15, 16, 18). GRO- is present in greater concentration in the lung than IL-8 in pneumonia and acute respiratory distress syndrome (3), and also binds CXCR2 with a higher affinity than does IL-8. Thus, under clinically relevant plasma conditions, chemokines active at CXCR2 may be present in concentrations that activate PMNs, whereas IL-8 may be inactive (3, 19). Conversely, though, CXCR2 is relatively labile and readily undergoes homologous desensitization after PMN exposure to IL-8 (19, 20).
Thus, interactions between CXC receptors as well as with other GPC receptors are critically important in regulating pulmonary PMN traffic both in clinical acute lung injury as well as pulmonary infection. CXCR2 expression is downregulated in clinical sepsis (21), and trauma places the lung at high risk for sepsis. Yet it is unclear whether CXCR2 is simply suppressed by sepsis or whether primary loss of CXCR2 might also be a risk factor for the development of pulmonary infections. Our preliminary studies suggested that loss of CXCR2 function might be associated with the subsequent development of pneumonia after trauma (22). Pneumonia is the most significant cause of septic morbidity and mortality in this population. We therefore decided to investigate the relationships between CXC receptor expression and infective events, prospectively studying the effects of major clinical trauma on CXC receptor function and expression, as well as their temporal relationship to the onset of pneumonia.
METHODS
All studies were performed in compliance with the institutional review board of New Jersey Medical School. Informed consent for blood sampling was obtained from the patient or his or her next of kin, as well as from volunteer donors.
Trauma Patients
Neutrophil samples were obtained prospectively from 32 major trauma patients admitted to the New Jersey State Trauma Center. Criteria for study required either (1) blunt truncal trauma with an injury severity score (ISS) of 25 or greater or (2) penetrating trauma with ISS 15 and requiring six or more units of packed red blood cells in the first 12 hours, or with a base deficit of seven units or more. These factors confer high risk for both organ failure and mortality (23, 24). Patients expected to die rapidly of head injuries or of uncontrollable bleeding were excluded. The mean injury severity score of the final study group was 27.4 (95% confidence interval 23.5eC31.1). Twenty-six patients were men; six were women. Patients ranged in age from 18 to 68 years, with a mean age of 35.1 years (95% confidence interval 30.3eC39.9). Twenty-five patients had a blunt mechanism of injury; seven suffered penetrating trauma. The mean length of intensive care unit stay was 11.1 days (95% confidence interval 7.0eC15.2). All patients survived. Samples were obtained on the day of admission (mean 14 ± 1 [SE] hours after injury) on Day 3 and on Day 7.
Volunteers
Age- (± 5 years), sex-, and ethnicity-matched healthy volunteer control subjects were identified and enlisted for each of the patients with trauma prospectively studied (n = 32). PMNs from the matched volunteers were isolated and studied contemporaneously with and identically to the patient samples.
Neutrophil Isolation
Our methods are described in more detail elsewhere (25). Heparinized whole blood samples (25 U/ml) were obtained via indwelling catheters or direct venipuncture and centrifuged at 150 x g for 10 minutes. The buffy coat and red blood cells were then layered onto Polymorphoprep centrifugation media (Robbins Scientific Corp., Sunnyvale, CA) and centrifuged at 300 x g for 30 minutes. The supernatants and mononuclear cell layers were discarded. The PMN layer was removed and mixed with an equal volume of 0.45% NaCl solution to restore osmolarity. After resting for 5 minutes, the cells were washed with RPMI solution and centrifuged for 10 minutes at 150 x g. PMN pellets were then resuspended in 2 ml of N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES) buffer solution (140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 10 mmol/L glucose, 20 mmol/L HEPES, and 0.1% fatty acideCfree bovine serum albumin, pH 7.4). PMNs were counted and assessed for purity using flow cytometry. These methods yield PMNs of 96 to 99% purity and greater than 98% viability by Trypan blue exclusion.
Calcium Dye Loading
Calcium concentration was adjusted to 1 mmol/L (mM) with CaCl2, and the cells were then incubated with 2 e/ml fura-2-acetoxymethyl ester (Molecular Probes, Eugene, OR) for 30 minutes in the dark at 37°C. PMNs were then divided into aliquots (2 x 106 PMN) and placed on ice in the dark. Immediately before study, cells were centrifuged for 5 seconds at 4,500 rpm in a programmable microcentrifuge, and the supernatants were removed. The cells were then resuspended in 200 e of HEPES buffer, with or without 1 mM CaCl2, and finally injected into cuvettes containing 2.8 ml of the same buffer for study.
Spectrofluorometry
[Ca2+]i was determined at 37°C with constant stirring by measuring fura-2-acetoxymethyl ester fluorescence at 505 nm using 340/380-nm dual wavelength excitation in a Fluoromax-2 spectrofluorometer (Jobin-SPEX, Edison, NJ) using our adaptations of the methods of Grynkiewicz and coworkers (26) and Hauser and colleagues (27). Calibration was achieved by permeabilizing neutrophils at the end of each experiment with 100 e digitonin and measuring the fura fluorescence in 1 mM Ca2+ solution (Rmax) and then adding 15 mM ethyleneglycol-bis-N-N'-tetraacetic acid for a zero-calcium solution (Rmin). The fluorescence of an aliquot treated with 100 e digitonin and 2 mM MnCl2 was subtracted from total fluorescence. The order of study of PMN isolates was alternated to avoid bias related to duration of dye loading or time of cell study.
Chemokine Receptor Responses of Clinical PMNs
PMN [Ca2+]i responses to chemokines change rapidly (within 1 minute) as a function of receptor expression (19, 20, 28). After study of basal [Ca2+]i for 30 seconds, volunteer or patient PMNs were stimulated with GRO- (1.25 nM) or IL-8 (1.25 nM), concentrations commonly found in the plasma (29) and bronchoalveolar lavage fluids (30) of severely injured patients. Clinical PMN aliquots were also stimulated by n-formyl met-leu-phe (fMLP; 10 nM). PMN [Ca2+]i responses to all these agonists were assessed as the peak transient [Ca2+]i change in nM/L. All transient results reported are measured as change from baseline. IL-8 binds both CXCR1 and CXCR2, but prior studies showed that total PMN [Ca2+]i responses to IL-8 are functionally CXCR1-dependent and unaffected by specific blockade of CXCR2 (19).
CXC Receptor Expression after Trauma In Vivo
To further understand the responses of trauma PMNs to chemokines, the expression of CXCR1 and CXCR2 was studied directly by flow cytometry in a second cohort of 10 matched trauma patients (ISS 29 ± 3) chosen in an identical fashion to the original group. All patients were transfused: 42% were acidotic and 31% were hypotensive on admission. Immediately after PMN isolation, cells were incubated with fluorescein isothiocyanateeCconjugated mouse antieChuman-CXCR1 antibody, phyoerythrin (PE)-conjugated mouse antieChuman-CXCR2 antibody (R&D Systems, Minneapolis, MN) or appropriate isotype controls. Samples were transferred to 5-ml polypropylene tubes, diluted with Hanks' balanced salt solution and placed on ice before analysis by flow cytometry. Samples were analyzed on an Epics XL flow cytometer (Beckman Coulter, Fullerton, CA) using CellQuest software (BD Biosciences, Franklin Lakes, NJ). A total of 15,000 events were counted per sample. Low-fluorescence debris was gated out of the analysis.
Chemotaxis Assays
The chemotaxis of PMNs from trauma patients (n = 9) and from volunteer subjects (n = 5) in response to GRO- was determined using a biomatrix-coated transwell system. Polycarbonate membrane inserts with 3-e pores (Corning, Inc., Corning, NY) were coated with biomatrix (Biomedical Technologies, Inc., Stoughton, MA) to create modified Boyden chambers. This gel approximates the composition of basement membranes. PMNs were isolated and suspended in HEPES buffer with 1 mM Ca2+ and loaded in 3 e/ml calcein-AM (Molecular Probes, Eugene, OR) for 30 minutes at 37°C in the dark. The PMN concentration was adjusted to 107 cells/ml, and 2 x 106 cells were set aside for the standard curve. For each condition, two transwells were set up with calcein-loaded PMN placed in the upper chambers. In one group of transwells, the lower chambers contained GRO- at various concentrations, whereas in the other, the lower chamber contained only buffer. These buffer-only "blank" transwells were used to determine the random migration (chemokinesis) of that aliquot of PMNs under those conditions. Blank results were subtracted from the results with chemoattractant to assess specific chemotaxis.
After each upper chamber was loaded with 106 PMNs/well, the system was incubated for 90 minutes at 37°C in the dark. Inserts were removed (without scraping) and migrated PMNs in the lower chamber were resuspended by simple pipetting. PMN aliquots from each lower chamber were then transferred, in duplicate, to a 96-well plate. Reserved PMNs were diluted in descending concentrations of PMN and used as a standard curve. The plate was read using an FL500 microplate fluorescence reader (Bio-Tek Instruments, Winooski, VT) at an excitation of 410/25 and an emission of 530/40. Fluorescent intensities were converted directly to the number of cells using the standard curve. The number of PMNs in each "blank" was subtracted from the total number of neutrophils migrating under experiment conditions to assess specific PMN chemotaxis. The final result was expressed as the percentage of PMNs undergoing chemotaxis in 90 minutes.
In Vitro Receptor Desensitization Studies
Heterologous desensitization of chemokine receptors by the chemoattractant GPC agonists fMLP and C5a was studied in volunteer PMNs (three studies per condition). To isolate CXCR1 function, PMNs were exposed to IL-8 in the presence of monoclonal antibodies specific for the n-terminal docking site of CXCR2 (courtesy of Dr. J. Bussiere; Genentec, South San Francisco, CA). This process is required because IL-8 can signal at CXCR2 if CXCR1 is not available. PMN calcium mobilization was studied in calcium-replete medium after stimulation by IL-8 at 1.25 nM. As noted, this approximates the conditions found in the plasma (29, 31) and alveolar fluids (30) of high-risk patients with trauma. IL-8 was either preceded or followed by fMLP or C5a. The concentrations of formylated peptides and C5a present at clinical sites are unknown; these agonists were therefore used at doses giving half-maximal peak PMN calcium mobilization. CXCR2 desensitization was studied by evaluating [Ca2+]i responses to GRO- at 1.25 nM before and after fMLP or C5a. As with IL-8, this concentration is found in clinical samples (29). CXCR1 blockade was not needed for these studies because GRO- is highly specific for CXCR2. We have previously reported the ability of CXCR1 to desensitize CXCR2 (27).
Onset of Pneumonia
Pneumonia is the most common infection in multiple trauma patients, and its development is believed to be related to immune failure (22, 32, 33). We therefore assessed the timing of onset of pneumonia in the prospectively studied patient cohort. Patients with clinical suspicion of pneumonitis (e.g., fever, increasing/foul sputum, worsening gas transport, deteriorating X rays) were evaluated for pneumonia by protocol using quantitative cultures obtained via bronchoalveolar lavage and accepting a count of more than 105 pathogens/ml as indicative of pneumonia (33). In two patients where bronchoalveolar lavage was relatively contraindicated, pneumonia was diagnosed by consensus criteria based on clinical presentation and sputum culture (34). Causative organisms were identified by review of the patients' clinical microbiology studies. The number and types of pneumonia were then stratified according to the timelines used for the PMN receptor function assays.
Statistical Analysis
All study data were assessed for statistical significance using one-way analysis of variance (ANOVA) with Tukey's post hoc test and unpaired t tests where appropriate. All data are reported as mean ± SEM. Statistical significance was accepted at p 0.05.
RESULTS
PMN CXCR2 Responses in Trauma Patients
[Ca2+]i mobilization by GRO-/CXCR2 was profoundly diminished immediately after injury (Figure 1) and returned progressively toward normal values over the week after trauma (p < 0.001, ANOVA). Responses were significantly diminished on Days 1 and 3 after injury when compared with volunteer control subjects (30 ± 5 and 46 ± 7 vs. 77 ± 8 nM; n = 32 for all groups, p < 0.05 by ANOVA/Tukey's test). By Day 7, responses to GRO- had recovered and were indistinguishable from control values (Figure 2A).
PMN CXCR1 Responses in Trauma Patients
Mean volunteer PMN [Ca2+]i responses to IL-8 were 142 ± 10 nM. In matched trauma patients (n = 27), IL-8 responses were depressed on Day 1 and then rebounded (p < 0.05, ANOVA). PMN responses on Day 1 were significantly less than control (97 ± 10 nM; p < 0.05, Tukey's test), becoming normalized by Day 3 (Figure 2B).
PMN Responses to fMLP in Trauma Patients
Peak volunteer PMN (Ca2+)i responses (n = 30) to 10 nM fMLP were 181 ± 12 nM. Trauma patients manifested essentially normal responses to fMLP at all time points studied (see Figure E1 in the online supplement). The minor decrease in [Ca2+]i response noted on Day 1 (to 150 ± 11 nM) did not achieve statistical significance.
PMN Chemotaxis toward GRO- in Trauma Patients
PMN chemotaxis to GRO- was assessed in a consecutive subgroup of the overall patients (n = 9) who were studied Day 3 after injury. This subset was representative of the group as a whole with respect to demographics, injuries, and injury severity scores. The responses of PMNs from normal volunteers studied simultaneously were used for comparisons. Studies were performed using GRO- concentrations from 10 to 250 ng/ml (1.25eC31 nM). Trauma patients showed significantly suppressed chemotaxis (p < 0.05) at all doses studied (Figure 3).
CXCR1 and CXCR2 Expression after Trauma In Vivo
Flow cytometric studies revealed that CXCR2 expression was significantly (p = 0.004) suppressed immediately after trauma. In contrast, surface expression of CXCR1 was not affected by injury (Figure 4). Suppression of CXCR2 was immediate, lasted throughout the week, and did not resolve until the second week after injury (Figure 5).
Cross-Desensitization of Chemokine Receptors
It has been shown that chemokines act in combination to recruit PMNs from the circulation to sites of infection (35eC37). We therefore sought to understand in more detail the regulation of CXC receptors by both chemokines and other important chemoattractants (38, 39) entering the circulation after trauma. CXCR2 stimulation (Figure 6) had no effect on C5a (Figure 6A) or fMLP (Figure 6C) signals. Conversely, prior exposure to either C5a (Figure 6C) or fMLP (Figure 6D) completely desensitized CXCR2. Thus, fMLP and C5a receptors are "dominant" or "higher ranking" than CXCR2. The effects of fMLP and C5a on CXCR1 (Figure 7) were similar to their effects on CXCR2. CXCR1 had no effect on fMLP and C5a signals, whereas fMLP and C5a desensitized CXCR1. CXCR1 stimulation was found to desensitize CXCR2 (Figure 8B). There was no tendency for CXCR2 stimulation to desensitize CXCR1. On the contrary, as we and others have previously noted, prior stimulation of CXCR2 enhances responses to IL-8 (19).
Onset of Pneumonia in the Study Population
Fourteen patients (44%) developed pneumonia. The peak incidence of pneumonia was found in the period between the Day 3 and Day 7 assays (Figure 9). Thus, the loss of CXC receptor function preceded the onset of pneumonia, which is typically the first infective complication seen in these patients. Most of the pneumonias found during the early period of PMN suppression were caused by gram-positive organisms and mixed flora. Pneumonias caused by gram-negative organisms predominated after the first week, when receptor function had returned to basal levels.
DISCUSSION
The CXC chemokines are important effectors of PMN recruitment and activation in the lung. In humans, CXC chemokines act through two receptors: CXCR1 binds predominantly IL-8, whereas CXCR2 is promiscuous, binding IL-8 as well as multiple related ELR+ CXC chemokines. Although the relationships between human CXC receptor systems and those in animal models are not precisely determined (40, 41), in many models, CXCR2 and the GRO series of CXC ligands are required for PMN recruitment to the lungs (42eC45), to wounds (46, 47), and to the peritoneal cavity (10). CXCR2, however, also undergoes desensitization by the CXCR1/CXCR2 agonist IL-8 (Figure 8) (19, 20). Other "end-target chemoattractants," such as complement fragments and formylated peptides, are mobilized immediately at sites of injury or by bacterial inoculation (38, 39). Because IL-8 as well as all of these other agents are released in response to injury (31, 48, 49), we postulated that the inflammatory milieu thus produced might alter subsequent PMN responses to chemokines. Because chemokines are required for PMN recruitment to infective sites, we further hypothesized that concurrent suppression of CXC receptors might occur after trauma, impacting PMN chemotactic function. Last, although CXCR2 is desensitized by sepsis (21), we postulated that CXCR2 receptor desensitization might occur in circulating PMNs before infection after injury, and in fact predispose to infection.
The present data confirm that both CXCR2 and CXCR1 are transiently desensitized after clinical injury. The suppression of PMN responses to GRO- mostly appears to reflect early suppression of CXCR2 surface expression. Surface expression of CXCR1, however, is not downregulated by injury. Thus, the early suppression of PMN calcium flux responses to IL-8 seen here may reflect either a loss of contribution by CXCR2 to IL-8 signaling or the development of a postreceptor regulation of CXCR1 function. In both cases, a return to normal function appears to occur over the first week after trauma (Figures 2 and 5). We further confirmed that the desensitization of chemokine receptors is functionally significant and that clinical PMN samples display suppressed chemotaxis toward GRO- (Figure 3) during this period. Last, distinct from CXCR2 suppression by infection per se, the suppression of CXC receptors by trauma clearly precedes the onset of pneumonia (Figure 9). It should be noted that PMN responses to IL-8 can be enhanced immediately after trauma in patients with early acute respiratory distress syndrome, as we (50) and others (51) have demonstrated. The current study therefore suggests that, although PMN chemotaxis to IL-8/CXCR1 is probably a key event in early, hyperacute respiratory distress syndrome, suppression of PMN responses to GRO/CXCR2 occurs over the first week after injury and appears closely related to the failure of subsequent antimicrobial surveillance.
Major trauma patients typically have elevated serum IL-8 levels. Thus, the present findings may, in part, reflect desensitization of CXCR2 by IL-8 in vivo (52, 53). In vitro, CXCR1 is more difficult to desensitize than CXCR2, requiring approximately 10-fold higher IL-8 levels. Moreover, when desensitization occurs, CXCR1 is recycled to the cell surface more rapidly than CXCR2 (20). Neutrophil responses after injury appear to act in a parallel fashion: CXCR1 suppression is less pronounced than CXCR2 suppression and resolves sooner. This relationship between CXCR2 and CXCR1 appears analogous to the relationship between leukotriene receptors BLT1 and BLT2, where the higher affinity receptors are relatively labile and repressed in inflammatory states, whereas the lower affinity member of the pair becomes activated (54). Similarly, the release of "classical" chemoattractants after tissue injury (49) may contribute to the desensitization of both chemokine and leukotriene receptors.
Thus, the pattern of differential receptor desensitization in vivo parallels the biology of the CXC receptors in vitro, and the expression of PMN chemokine receptors in trauma is probably inversely reflective of the plasma concentrations of the multiple inflammatory agonists that circulate after injury. A clear hierarchy is seen both in vitro and in vivo in trauma: CXCR2 undergoes suppression readily and for a protracted period. CXCR1 is more difficult to suppress. Responses to classical GPC agonists, like fMLP, persist at near-control levels. These findings suggest that differential desensitization of labile, high-affinity receptors like CXCR2 may be much more critical to PMN dysfunction after injury than desensitization of receptors for the classical GPC chemoattractants. On the other hand, others have noted that chemotactic deficits to such classical chemoattractants may also exist (55). We have also noted such effects in preliminary studies of PMN studied in the week after injury (data not shown). Thus, postreceptor defects in chemotaxis may exist across a broad array of receptors at this time. Nonetheless, early loss of high-affinity, easily downregulated receptors, such as those for LTB4 (56) and GRO-, may be an important generalized phenomenon in early susceptibility to sepsis.
Proof that clinical CXC receptor desensitization by inflammatory mediators directly predisposes to sepsis will require larger study populations. In this study population, however, chemokine receptor loss and decreased chemotaxis to GRO- clearly preceded infection and thus cannot be attributed to sepsis. CXCR2 is likely to be crucial for PMN recruitment from the bloodstream, where chemokines are present at picomolar levels (13, 16, 20) to sites of bacterial invasion. CXCR1 also plays a role in chemotaxis (16) but may be more important in PMN chemotaxis and activation (57) close to tissue sites. Where receptors have been desensitized, even adherent PMN will not transmigrate into tissues (58). Combinatorial chemotaxis has been proposed as a key mechanism of PMN recruitment, and to require differential receptor downregulation (58, 59). It appears therefore that suppression of chemotactic receptor function (as seen in trauma) may act both as a cause and effect of sepsis (Figure 10), thus contributing to the susceptibility of patients who are injured and septic to secondary infection.
Because early trauma is a model of sterile inflammation, the findings here suggest that some degree of PMN chemotactic desensitization may be common to disease processes that cause systemic inflammatory response syndrome. The degree and duration of desensitization probably varies both with the severity of systemic inflammatory response syndrome as well as the biology of the specific receptors. The current data as well as our other studies (54) suggest that premature downregulation of labile GPC receptors, such as CXCR2 and BLT1, may impair chemotactic delivery of PMN to sites of bacterial inoculation while still potentially allowing nonspecific PMN attack on "bystander" tissues. PMN activation by GPC agonists concurrent with the desensitization of their GPC receptors is an attractive explanation for the paradoxic association of hyperinflammation with sepsis that is commonly seen after systemic inflammatory response syndrome.
This manuscript has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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