Expression and Function of the Receptors CXCR1 and CXCR2 in Sepsis C. James Cummings, Thomas R. Martin, Charles W. Frevert, Joanne M. Quan, Venus A. Wong, Steven M. Mongovin, This information is current as Tonja R. Hagen, Kenneth P. Steinberg and Richard B. of September 28, 2021. Goodman J Immunol 1999; 162:2341-2346; ; http://www.jimmunol.org/content/162/4/2341 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Expression and Function of the Chemokine Receptors CXCR1 and CXCR2 in Sepsis1

C. James Cummings, Thomas R. Martin, Charles W. Frevert, Joanne M. Quan, Venus A. Wong, Steven M. Mongovin, Tonja R. Hagen, Kenneth P. Steinberg, and Richard B. Goodman2

Neutrophils (polymorphonuclear ; PMN) and a redundant system of chemotactic () have been implicated in the pathogenesis of the acute respiratory distress syndrome in patients with sepsis. PMN express two cell surface receptors for the CXC chemokines, CXCR1 and CXCR2. We investigated the expression and function of these receptors in patients with severe sepsis. Compared with normal donors, CXCR2 surface expression was down-regulated by 50% on PMN from septic patients (p < 0.005), while CXCR1 expression persisted. In vitro migratory responses to the CXCR1 ligand, IL-8, were similar in PMN from septic patients and normal donors. By contrast, the migratory response to the CXCR2 ligands, epithelial cell-derived activator (ENA-78) and the growth-related oncogene proteins, was markedly suppressed in PMN from Downloaded from septic patients (p < 0.05). Ab specific for CXCR1 blocked in vitro migration of PMN from septic patients to IL-8 (p < 0.05), but not to FMLP. Thus, functionally significant down-regulation of CXCR2 occurs on PMN in septic patients. We conclude that in a complex milieu of multiple CXC chemokines, CXCR1 functions as the single dominant CXC in patients with sepsis. These observations offer a potential strategy for attenuating adverse inflammation in sepsis while preserving host defenses mediated by bacteria-derived peptides such as FMLP. The Journal of Immunology, 1999, 162: 2341–2346. http://www.jimmunol.org/ eutrophils (polymorphonuclear neutrophils; PMN)3 play In contrast to the multiple CXC chemokines, only two CXC a dual role in patients with sepsis. While they are critical chemokine receptors, CXCR1 and CXCR2 (also known as IL-8RA N to host defense against infection, PMN also contribute to and IL-8RB), have been shown to mediate the responses to CXC the undesirable sequelae of sepsis, including the multiple organ chemokines in PMN (12–16). The two receptors have different dysfunction syndrome and the acute respiratory distress syndrome ligand binding affinities. CXCR1 binds with high affinity to IL-8, (ARDS) (1, 2). Understanding the molecular mechanisms of PMN but binds with low affinity to ENA-78, neutrophil-activating pep- recruitment to sites of inflammation is central to designing the tide-2, and GRO-␣,-␤, and -␥, whereas, CXCR2 binds all these safest and most effective strategies to limit the untoward effects of

CXC chemokines with high affinity (17–19). Moreover, the surface by guest on September 28, 2021 acute inflammation while preserving host defenses against bacte- expression of the two CXCRs is regulated differently in vitro (20, rial infection. 21). On unstimulated PMN the two receptors are expressed in ap- The chemotactic cytokines (chemokines) are potent and specific proximately equal numbers, and in the presence of stimulating chemoattractants for inflammatory cells (3). The CXC chemokines ligand both receptors are rapidly internalized. However, CXCR1 is comprise several proteins that are regulated and produced by res- rapidly re-expressed (within minutes), whereas the re-expression ident cells in the lungs and other organs. These include IL-8, the of CXCR2 is considerably slower. Thus, the expected net effect of epithelial cell-derived neutrophil activator (ENA-78), and the stimulation of PMN with a CXC chemokine is down-regulation of ␣ ␤ ␥ GRO subfamily of proteins (GRO- , GRO- , and GRO- ). Their CXCR2. Even though IL-8 binds both CXCRs with high affinity, critical role in PMN migration has been shown in humans and in blockade of CXCR1 alone is sufficient to suppress the chemotactic animal models of disease (4–11). The redundancy of these host- activity of normal PMN toward IL-8 in vitro (22, 23). The rele- derived signals for PMN recruitment complicates the selection of vance of these receptor mechanisms and their effects on CXCR therapeutic targets to reduce inflammation. expression on circulating PMN in vivo in patients with sepsis is unknown, but the available data suggest that CXCR1 may be a rational target for therapeutic interventions, particularly in patients Medical Research Service, Seattle Veterans Affairs and Harborview Medical Centers, with elevated blood levels of CXC chemokines. and Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Washington School of Medicine, Seattle, WA 98195 Therefore, we investigated the expression and function of Received for publication August 18, 1998. Accepted for publication November CXCR1 and CXCR2 in patients with severe sepsis. We hypothe- 5, 1998. sized that in circulating PMN 1) CXCR2 would be down-regu- The costs of publication of this article were defrayed in part by the payment of page lated; 2) CXCR2 down-regulation would be associated with charges. This article must therefore be hereby marked advertisement in accordance blunted chemotactic responses to the CXC chemokines, which de- with 18 U.S.C. Section 1734 solely to indicate this fact. pend on CXCR2 for signal transduction (e.g., ENA-78 and 1 This work was supported in part by National Institutes of Health Grants HL51072, ␣ ␤ ␥ AI29103, HL30542, and GM37696; the American Heart Association; and the Medical GRO- ,- , and - ); and 3) blockade of CXCR1 alone would be Research Service of the Department of Veterans Affairs. sufficient to block chemotactic activity toward IL-8 despite the 2 Address correspondence and reprint requests to Dr. Richard B. Goodman, Veterans ability of IL-8 to engage either receptor. Further, we hypothesized Affairs Medical Center, 111B; 1660 S Columbian Way; Seattle, WA 98108. E-mail that CXCR1 blockade would not affect PMN migration toward address: [email protected] other relevant chemoattractants such as the bacterial-derived che- 3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophils; ARDS, acute respiratory distress syndrome; ENA-78, epithelial cell-derived neutrophil acti- moattractant, FMLP, or the chemotactically active complement vator; GRO, growth-related oncogene; ZAS, zymosan-activated human serum. fragment, C5a, both of which bind distinct receptors on PMN

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 2342 CXC CHEMOKINE RECEPTORS IN SEPSIS

(24–26). Herein, we present data supporting these hypotheses. Table I. Sources of sepsis and organ dysfunction in the study patientsa These data simplify our understanding of the molecular mecha- nisms for PMN recruitment in patients with sepsis and organ fail- n (%) ure, and offer a rationale for targeting CXCR1 to block chemokine- Sepsis source(s) mediated PMN migration while potentially preserving PMN Pneumonia 9 (64) recruitment to sites of bacterial infection. Burn 3 (21) Necrotizing Fasciitis 2 (14) Materials and Methods Endocarditis 1 (7) SBP 1 (7) Patient population Dysfunctional organ(s) Patients in the intensive care units of Harborview Medical Center (Seattle, Cardiovascular 13 (93) WA) were prospectively identified between December 1996 and April Respiratory 13 (93) 1997 as having severe sepsis and organ dysfunction according to the Amer- ALI 7 (50) ican College of Chest Physicians/Society for Critical Care Medicine Con- ARDS 6 (43) sensus Conference definitions (27). Criteria for organ dysfunction were CNS 8 (57) specifically defined. Patients had respiratory dysfunction if they met crite- Renal 5 (36) ria for either ARDS or acute lung injury according to the American/Euro- Hematologic 3 (21) pean Consensus Conference definitions (28). Cardiovascular dysfunction Hepatic 3 (21) was defined as systemic vascular resistance Ͻ800 dyne-s/cm2, systolic Metabolic 2 (14) blood pressure Ͻ90 mm Hg, or need for vasopressors. Hematologic dys- a Ͻ SBP, spontaneous bacterial peritonitis, ALI, acute lung injury, ARDS, acute function was defined as thrombocytopenia (platelets 80K) or dissemi- respiratory distress syndrome; CNS central nervous system. Downloaded from nated intravascular coagulation. Renal dysfunction was defined as urine output of Ͻ30 ml/h and an abrupt rise in creatinine. Hepatic dysfunction was defined as serum bilirubin Ͼ3.5 mg/dl. Metabolic dysfunction was defined as an unexplained anion gap Ͼ15 mEq/l or serum lactate human serum (ZAS; 10%; containing 1 ϫ 10Ϫ8 M C5a by RIA) and FMLP Ͼ2 mmol/l. Central nervous system dysfunction was defined as a Glascow (Calbiochem, San Diego, CA) served as positive controls, and PBS served coma scale value Ͻ12 unexplained by sedation. Patients were excluded if as a negative control. All samples were tested in triplicate. they were Ͻ18 yr of age, pregnant, neutropenic (Ͻ1000 white blood cells/ Inhibition of was determined in the presence or the absence ␮l), recently transfused (Ͼ3 U of blood within the preceding 24 h), known of an affinity-purified polyclonal rabbit IgG specific for CXCR1 that blocks http://www.jimmunol.org/ to have HIV infection, or entered into an interventional trial designed to binding of IL-8 to the CXCR1 receptor (22). PMN were incubated for 20 ameliorate the systemic inflammatory response before sample acquisition. min at room temperature in the presence of 50 ␮g/ml of blocking Ab or All subjects were enrolled within 96 h of the onset of severe sepsis and met nonimmune rabbit IgG (Jackson ImmunoResearch Laboratories). This con- the entry criteria at the time of enrollment. Eight normal nonsmoking vol- centration of anti-CXCR1 Ab is sufficient to block binding of 1 ϫ 10Ϫ8 M unteers served as controls. The study protocol was approved by the Uni- IL-8 to cloned CXCR1 by competitive radioligand binding (22). The che- versity of Washington human subjects committee, and informed consent motactic index represents the percentage of the total PMN migrating. It was was obtained from all subjects. calculated from the mean fluorescence of PMN migrating toward chemoat- tractant (IL-8, ZAS, or FMLP) minus the mean fluorescence of PMN mi- Neutrophil isolation grating to PBS divided by the mean fluorescence of chambers containing ϫ 6 Heparinized blood (40 ml) was obtained and transported immediately on 3 10 calcein-labeled PMN/ml (total cells available for migration) mul- by guest on September 28, 2021 ice to the laboratory. Plasma was obtained from an aliquot of heparinized tiplied by 100. All conditions were tested in triplicate, and the values were blood within 20 min of acquisition and was stored at Ϫ20°C for subsequent averaged. analysis of chemokine concentrations by immunoassay. The remainder of Plasma chemokine determinations the whole blood was layered onto a Ficoll-Hypaque density gradient (Mono-Poly Resolving Medium, ICN Biomedicals, Costa Mesa, CA), and Plasma concentrations of IL-8, GRO-␣, and ENA-78 were determined in neutrophils were isolated by centrifugation. RBC were eliminated by hy- 11 patients and 5 normal subjects by sandwich ELISA according to the potonic lysis and dextran sedimentation. Each subject’s neutrophil sample manufacturer’s protocol (R & D Systems, Minneapolis, MN). Specimens was divided, and half was used for determination of CXCR1 and CXCR2 with undetectable chemokine concentrations were assigned a value equal to expression by flow cytometry. The other half was used for measurement of the lower limit of detection to permit statistical analysis. Samples were chemotaxis in vitro. stored at Ϫ20°C until assayed, and each sample was assayed in duplicate. Flow cytometry Statistical analysis PMN were suspended at a concentration of 1 ϫ 106 cells/ml in ice-cold The expression of CXCR1 and CXCR2 on patient and normal PMN was HBSS containing 0.1% NaN3 and 0.1% BSA. To detect CXCR1, PMN compared using Student’s unpaired t test with unequal variance. Chemo- were incubated with 1 ␮g/ml of mouse IgG2b mAb that is specific for taxis dose-response curves were compared by ANOVA with a Bonferroni/ CXCR1 (22). Anti-protein C mouse IgG2b Ab (Sigma, St. Louis, MO) Dunn post-hoc analysis. Chemokine concentrations in plasma of patients served as a control for nonspecific binding. FITC-conjugated goat anti- and normal volunteers were compared using the Mann-Whitney U test for Ј mouse IgG F(ab )2 (Jackson ImmunoResearch Laboratories, West Grove, nonparametric data. Correlations between chemokine concentrations were PA) was used as a detecting Ab. To detect CXCR2, PMN were incubated determined using Spearman’s correlation. In all cases, p Ͻ 0.05 was ␮ Ј with 10 g/ml of an affinity-purified polyclonal rabbit IgG F(ab )2 that is accepted as significant. Ј specific for CXCR2 (22). Nonimmune rabbit IgG F(ab )2 was used as a Ј control, and FITC-conjugated goat anti-rabbit IgG F(ab )2 was used as the detecting Ab (both from Jackson ImmunoResearch Laboratories). Flow Results cytometry was performed using a FACScan instrument (Becton Dickinson, Study population Mountain View, CA) as previously described (22). We studied 14 patients and 8 normal subjects. The causes of sepsis Neutrophil chemotaxis and the number of dysfunctional organ systems are shown in Table I. The patients were critically ill, with APACHE II scores of PMN chemotaxis was measured as previously described (29). Briefly, Ϯ Ϯ PMN were labeled with 5 ␮M calcein-AM (Molecular Probes, Eugene, 25.4 10 (mean SD) and a 28-day mortality rate of 57%. OR) and diluted to a concentration of 3 ϫ 106 cells/ml in RPMI 1640 medium (Sigma). PMN chemotactic activity for IL-8 is maximal between Chemokine receptor expression 10–100 nM in this assay. PMN migration was detected by measuring the CXCR1 and CXCR2 receptors were identified by flow cytometry. fluorescence (Cytofluor II, PerSeptive Biosystems, Framingham, MA) of calcein-labeled PMN migrating through an 8.0-␮m pore size polyvinylpyr- Fig. 1 compares receptor expression on PMN from a normal vol- rolidone-free polycarbonate filter during a 90-min incubation in a 96-well unteer and a septic patient. The expression of CXCR1 was only chemotaxis chamber (NeuroProbe, Cabin John, MD). Zymosan-activated slightly reduced, whereas the expression of CXCR2 was markedly The Journal of Immunology 2343

FIGURE 1. Representative flow cytometry histograms for CXC recep- tor expression. PMN from a normal donor (dashed lines) and a septic patient (solid lines) were incubated with saturating concentrations of either mouse monoclonal anti-CXCR1 IgG (1 ␮g/ml; left panel), affinity-purified Ј ␮ polyclonal rabbit anti-CXCR2 F(ab )2 (10 g/ml; right panel), or the re- spective nonspecific control Abs (dotted lines). CXCR2 expression is sig- nificantly reduced in PMN from the septic patient compared with that in normal PMN, whereas CXCR1 expression is only minimally reduced. Downloaded from

reduced on the PMN from this septic patient. Similar changes were seen for each subject. The CXCR1 fluorescence intensity was nor- mal (i.e., Ϯ1 SD of the mean of CXCR1 fluorescence on PMN

from normal donors) in nine of 14 septic patients, was reduced in http://www.jimmunol.org/ four, and was increased in one. In contrast, CXCR2 fluorescence intensity was normal in only three of 14 septic patients and was significantly reduced (reduced by Ͼ1 SD from the mean of FIGURE 3. Chemotaxis dose response of PMN from septic and normal CXCR2 fluorescence on PMN from normal donors) in 11 of 14 subjects to five CXC chemokines. The migrations of PMN from normal septic patients. donors (filled circles; n ϭ 7) and septic patients (open circles; n ϭ 12) were The peak (mode) fluorescence intensity was averaged for the compared using serial dilutions of IL-8, GRO-␣, GRO-␤, GRO-␥, and eight normal donors and the 14 septic patients (Fig. 2). CXCR1 ENA-78. The data points represent the mean (ϮSEM) chemotactic index expression on PMN from septic patients was not significantly dif- for the indicated concentrations of each chemokine. Chemotaxis to IL-8 ferent from that on normal PMN. By contrast, CXCR2 expression was only slightly reduced in PMN from septic patients compared with that by guest on September 28, 2021 was significantly reduced ( p Ͻ 0.005) on PMN from septic pa- in PMN from normal subjects (A), whereas the responses to the chemo- tients. Similar differences were seen when the data were expressed kines that bind with high affinity to only CXCR2 were significantly reduced Ͻ ء as the fold increase over control fluorescence. Thus, in contrast to (B–E; , p 0.05).

the normal expression of CXCR1 receptor, CXCR2 receptor is significantly down-regulated on circulating PMN from septic pa- tients.

Receptor function The migration of PMN from 12 septic patients and seven normal donors toward five different CXC chemokines was measured (Fig. 3). The chemokine concentrations selected fell within a biologi- cally relevant range (8). All the CXC chemokines were effective chemoattractants for PMN from normal subjects (Fig. 3, A–E, filled circles). The migratory response to IL-8 was robust in PMN from normal donors and septic patients (Fig. 3A). In contrast, PMN from septic patients demonstrated significantly reduced chemotac- tic activity to GRO-␣, GRO-␤, GRO-␥, and ENA-78 (Fig. 3, B–E, open circles), the chemokines that bind with high affinity to only CXCR2. These data indicate that the down-regulation of CXCR2 FIGURE 2. CXC receptor expression on PMN from normal and septic on PMN from septic patients, seen by flow cytometry (Fig. 2), is patients. Peak (mode) fluorescence was recorded by flow cytometry on functionally significant. PMN from each subject labeled with Ab to CXCR1 (A), CXCR2 (B), or the appropriate nonspecific control Ab, and background (nonspecific) fluores- Functional effect of CXCR1-blocking Ab cence was subtracted. The average values (ϮSEM) for the group of eight normal volunteers (solid bars) and 14 septic patients (hatched bars) are PMN from 14 septic patients and eight normal donors were incu- ␮ p Ͻ 0.005) by bated in the presence of nonimmune rabbit IgG (50 g/ml) or ,ءء) shown. The average CXCR2 expression was reduced 50% on PMN from septic patients compared with that on normal PMN. affinity-purified rabbit anti-human CXCR1 polyclonal IgG (50 ␮g/ CXCR1 expression on PMN from septic patients was not significantly ml). This concentration of Ab is sufficient to block binding of Ϫ different from that on normal PMN. radiolabeled IL-8 (up to 1 ϫ 10 8 M) to recombinant CXCR1 2344 CXC CHEMOKINE RECEPTORS IN SEPSIS

FIGURE 5. Plasma chemokine concentrations. IL-8, GRO-␣, and FIGURE 4. Inhibition of PMN chemotaxis with blocking Ab to ENA-78 concentrations were measured by ELISA in plasma from normal CXCR1. PMN from normal donors (n ϭ 8) or septic patients (n ϭ 14) were donors (n ϭ 5; open circles) and septic patients (n ϭ 11; filled circles). incubated in the presence of a blocking polyclonal IgG specific for CXCR1 Bars indicate median values. IL-8 was undetectable in all normal speci- (anti-CXCR1; hatched bars) or control Ab (nonimmune IgG; solid bars), mens and is displayed as a value at the lower limit of detection. Statistical and migration was measured toward 1 ϫ 10Ϫ8 M concentrations of IL-8 comparisons are indicated. and FMLP and a 10% concentration of ZAS. Anti-CXCR1 significantly p Ͻ Downloaded from ,ءء) inhibited chemotaxis of both normal and septic PMN to IL-8 0.005), but the effect was more pronounced with septic PMN. As expected, p ϭ 0.05 Discussion ,ء) anti-CXCR1 had minimal effect on PMN migration to FMLP with normal PMN, nonsignificant with septic PMN) and ZAS (p ϭ NS). The major goal of this study was to investigate the effect of severe sepsis on the expression and function of the two CXC chemokine expressed on BHK cells, and the Ab does not recognize CXCR2 receptors on circulating PMN. We found that CXCR2 expression Ϫ8 (22). PMN chemotaxis to 1 ϫ 10 -M concentrations of IL-8, was reduced by 50% in septic patients, whereas CXCR1 expres- http://www.jimmunol.org/ Ϫ FMLP, or 10% ZAS (which contained 1 ϫ 10 8 M C5a) was sion was preserved. Similarly, we found that the chemotactic re- measured. Anti-CXCR1 significantly inhibited the migration of sponses to the CXC chemokines which bind with high affinity to normal and septic PMN to IL-8 ( p Ͻ 0.005), but the inhibitory only CXCR2 (GRO-␣,-␤, and -␥ and ENA-78) were markedly effect was more pronounced with septic PMN (Fig. 4). Anti- suppressed in PMN from septic patients, whereas the chemotactic CXCR1 inhibited chemotaxis of normal PMN by 42%, but inhib- response to IL-8, which binds with high affinity to either CXCR, ited chemotaxis of septic PMN by 73%. As expected anti-CXCR1 was preserved. Finally, specific blockade of CXCR1 had a more had little effect on chemotaxis to FMLP or ZAS. Although CXCR1 pronounced suppressive effect on the chemotactic function of Ab produced a slight reduction in chemotaxis of normal PMN to PMN from septic patients than on that of PMN from normal do- FMLP, which reached statistical significance in this series of ex- nors. Taken together, these observations indicate that CXCR2 is by guest on September 28, 2021 periments ( p ϭ 0.05), the magnitude of the effect was minimal functionally down-regulated in severe sepsis, leaving CXCR1 as (20% decrease), and the effect was not seen in previous experi- the dominant receptor for mediating the effects of the CXC che- ments with this or other concentrations of FMLP (22). There was mokines in PMN from these patients. no significant effect of the Ab on FMLP-induced migration of Previous reports (13, 21, 30) demonstrate that in normal PMN, PMN from septic patients. These data show that despite the septic CXC receptors are transiently internalized following in vitro stim- condition of these patients, their PMN had normal chemotactic ulation by IL-8. Subsequently, CXCR1 is rapidly re-expressed on responses to FMLP and C5a and only a slightly reduced response the cell surface, whereas CXCR2 is re-expressed at a considerably to IL-8. In PMN from septic patients, blockade of CXCR1 sub- slower rate (21). The primary rationale for this study was to in- stantially impaired the migratory response to IL-8 without affect- vestigate the relevance of these in vitro observations for patients ing migratory responses to FMLP or C5a. whose circulating PMN were stimulated in vivo by an active in- flammatory process. We prospectively defined a population of pa- Plasma chemokine levels tients with severe sepsis and organ dysfunction that we predicted Plasma concentrations of IL-8, GRO-␣, and ENA-78 were deter- would have elevated circulating chemokine concentrations. We mined by immunoassay in plasma from 11 septic patients and five found chemokine values that were elevated to an extent similar to normal donors (Fig. 5). The median concentration of IL-8 in those reported by others (31–34). Moreover, we found that plasma from septic patients was 0.157 ng/ml (19 pM), whereas CXCR2 was significantly down-regulated in these patients. In ad- levels were undetectable in all five normal subjects ( p Ͻ 0.005). dition, there were trends suggesting that higher plasma chemokine The median concentration of GRO-␣ was 0.170 ng/ml (20 pM) in concentrations are correlated with lower CXCR2 surface expres- plasma from septic patients compared with 0.030 ng/ml (4 pM) in sion. The plasma chemokine concentrations were considerably less that from normal subjects ( p ϭ 0.07). By contrast, the median than those required for receptor down-regulation in vitro (21), sug- concentration of ENA-78 was 0.140 ng/ml in plasma from septic gesting that CXCR2 may be modulated by very low chemokine patients, which was not significantly different from values in concentrations in vivo in patients with severe sepsis. plasma from normal subjects. Thus, the clinical selection criteria Other mechanisms may also contribute to the down-regulation of severe sepsis identified patients with elevated plasma concen- of CXCR2 in patients with severe sepsis. Cytokines such as TNF-␣ trations of IL-8 and GRO-␣, whereas plasma concentrations of have been detected in the plasma of septic patients (35) and can ENA-78 were normal in patients with sepsis. As predicted, there down-regulate CXCRs on PMN in vitro (36). TNF-␣ may also were inverse relationships of CXCR2 expression with plasma IL-8 induce proteolytic degradation of CXCR2 (37). Hypoxic condi- levels (r ϭϪ0.44) and with plasma GRO-␣ levels (r ϭϪ0.53); tions in vitro can affect PMN cell surface expression of CXCRs however, the number of patients was insufficient to reach statistical (38). Granulocyte CSF up-regulates the transcription and expres- significance ( p ϭ 0.17 and p ϭ 0.09, respectively). sion of both receptors, and LPS down-regulates each receptor by The Journal of Immunology 2345 decreasing transcription and reducing the half-lives of their mR- sepsis will reduce PMN migration to CXC chemokines, yet pre- NAs (36). In the complex milieu of sepsis, all these serve PMN responsiveness to bacterial products. mechanisms may contribute to CXCR expression on PMN. Our studies were designed to examine the net effect of these multiple Acknowledgments mechanisms of CXCR regulation on circulating PMN in vivo un- We thank Michelle Goodman, Doreen Anardi, Donna Davis, and Patsy der clinically relevant conditions. We found that CXCR2 expres- Treece for assistance with patient identification, and Ellen Caldwell for sion and function are down-regulated on circulating PMN from assistance with statistical analysis. We thank Drs. Donald C. Foster and patients with sepsis. Gary Rosenberg for their guidance in cloning the IL-8R and in isolating the Our observations are relevant to the mechanisms of PMN em- CXCR1 mAb. igration from the bloodstream in patients with severe sepsis and organ dysfunction. Although Soejima and colleagues (39) showed References in chronic stable lung disease that CXCR down-regulation can 1. Repine, J. E., and C. J. Beehler. 1991. Neutrophils and adult respiratory distress occur as a result of the process of migration, we have shown that syndrome: two interlocking perspectives in 1991. Am. Rev. Respir. Dis. 144:251. changes in receptor expression occur on circulating PMN even 2. Steinberg, K. P., J. A. Milberg, T. R. Martin, R. J. Maunder, B. A. Cockrill, and L. D. Hudson. 1994. Evolution of bronchoalveolar cell populations in the adult before they migrate into the tissues in critically ill patients with respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 150:113. systemic inflammation. 3. Luster, A. D. 1998. Chemokines: chemotactic cytokines that mediate inflamma- Multiple CXC chemokines are produced in the organ tissues of tion. N. Engl. J. Med. 338:436. 4. Kunkel, S. L., N. Lukacs, and R. M. Strieter. 1995. Chemokines and their role in septic patients. For example, patients with sepsis-related ARDS human disease. Agents Actions 46:11. have significantly increased concentrations of IL-8, ENA-78, and 5. Donnelly, S. C., R. M. Strieter, S. L. Kunkel, A. Walz, C. R. Robertson, Downloaded from GRO-␣ in their bronchoalveolar lavage fluids (8, 40). Interest- D. C. Carter, I. S. Grant, A. J. Pollok, and C. Haslett. 1993. Interleukin-8 and development of adult respiratory distress syndrome in at-risk patient groups. Lan- ingly, in patients with ARDS, the average concentrations of cet 341:643. ENA-78 and GRO␣ are higher than that of IL-8 (8, 40). A simi- 6. Jorens, P. G., J. Van Damme, W. De Backer, L. Bossaert, R. F. De Jongh, A. G. Herman, and M. Rampart. 1992. (IL-8) in the bronchoalveolar larly broad spectrum of CXC chemokines is produced by macro- lavage fluid from patients with the adult respiratory distress syndrome (ARDS) phages simply by stimulation with endotoxin in vitro (41). Another and patients at risk for ARDS. Cytokine 4:592. important CXC chemokine, granulocyte chemotactic peptide-2, 7. Miller, E. J., A. B. Cohen, S. Nagao, D. Griffith, R. J. Maunder, T. R. Martin, http://www.jimmunol.org/ J. P. Weiner-Kronish, M. Sticherling, E. Christophers, and M. A. Matthay. 1992. binds with high affinity to CXCR1 and is a potent chemoattractant Elevated levels of NAP-1/interleukin-8 are present in the airspaces of patients for PMN, although its characterization in clinical fluids is limited with the adult respiratory distress syndrome and are associated with increased (42–44). The host-derived signals for PMN recruitment to tissues mortality. Am. Rev. Respir. Dis. 146:427. 8. Goodman, R. B., R. M. Strieter, D. P. Martin, K. P. Steinberg, J. A. Milberg, appear to be highly redundant. R. J. Maunder, S. L. Kunkel, A. Walz, L. D. Hudson, and T. R. Martin. 1996. Despite the multiplicity of CXC chemokines, however, the data Inflammatory cytokines in patients with persistence of the acute respiratory dis- tress syndrome. Am. J. Respir. Crit. 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