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Originally published online as doi:10.1189/jlb.0805458 on October 4, 2005

Published online before print October 4, 2005
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(Journal of Leukocyte Biology. 2005;78:1265-1272.)
© 2005 by Society for Leukocyte Biology

The combined CXCR1/CXCR2 antagonist CXCL8(3–74)K11R/G31P blocks neutrophil infiltration, pyrexia, and pulmonary vascular pathology in endotoxemic animals

John R. Gordon*,1, Fang Li{dagger}, Xiaobei Zhang*, Wenjun Wang*, Xixing Zhao* and Aarti Nayyar*

* Immunology Research Group, University of Saskatchewan, Saskatoon, Canada; and
{dagger} Department of Immunology, Dalian Medical University, Peoples Republic of China

1 Correspondence: Department of Veterinary Microbiology, 52 Campus Dr., Saskatoon, Saskatchewan, Canada S7N 5B4. E-mail: john.gordon{at}usask.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CXC chemokine receptor 2 (CXCR2) antagonism alone can reduce neutrophil infiltration of some inflammatory sites, but the CXCR1 and CXCR2 critically regulate neutrophil responses to Glu-Leu-Arg-CXC chemokines. Herein, we assessed a combined CXCR1/CXCR2 antagonist, CXC chemokine ligand 8(3–74) [CXCL8(3–74)]K11R/G31P, for its ability to blunt neutrophil-influx and ancillary pathology in severe endotoxemia. Guinea pigs challenged via the airways with Escherichia coli lipopolysaccharide (LPS; 5 µg/kg) were given CXCL8(3–74)K11R/G31P (subcutaneously) before or after the onset of symptoms. The airways of the LPS-challenged animals contained high levels of endogenous pyrogens interleukin (IL)-1 and tumor necrosis factor (TNF) at 2–4 h, and the animals developed pyrexia, which peaked at {approx}6 h; strong pulmonary, neutrophilic inflammation; and marked pleural hemorrhagic consolidation, as assessed at {approx}15 h. CXCL8(3–74)K11R/G31P treatment before LPS challenge reduced lung pleural hemorrhagic consolidation and airway neutrophilia by >90% and essentially abrogated the IL-1, TNF, and fever responses. When given 3 or 6 h after LPS, CXCL8(3–74)K11R/G31P reduced pulmonary neutrophilia by up to 85% and pleural hemorrhagic consolidation by 50–85%. The 3-h treatment reduced the 6- to 24-h fever response to background. Delays of 6 or 9 h in beginning treatment had significant effects on the fever decay curve, but only the 6-h treatment had a significant effect on the 24-h fever. These results indicate that combined CXCR1/CXCR2 antagonism can have significant therapeutic effects on pulmonary inflammation and hemorrhage, as well as pyrexia in endotoxemic animals.

Key Words: inflammation • IL-8 • chemokine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophilic inflammatory responses are induced by many stimuli that trigger the innate immune system, including challenge with bacteria or their secretion products [e.g., lipopolysaccharide (LPS) endotoxins], as well as events of nonmicrobial origin (e.g., ischemia/reperfusion injury) [1 ]. Numerous mediators are expressed by multiple cellular sources (e.g., epithelial and endothelial cells, macrophages) in these lesions, including Glu-Leu-Arg (ELR)-CXC chemokines [e.g., CXC chemokine ligand 8 (CXCL8)/interleukin (IL)-8], arachidonic acid metabolites, and complement split products [2 3 4 ]. Many reports document that the ELR-CXC chemokines are critical to the pathogenesis of such inflammatory lesions so that their neutralization significantly reduces pathology or mortality [5 6 7 8 9 ]. Indeed, in many clinical settings, the levels of CXCL8, for example, correlate better with morbidity or mortality than do other inflammatory markers [10 , 11 ].

The ELR-CXC chemokines are ligands of the CXC chemokine receptor 1 (CXCR1) or CXCR2, and as such, importantly regulate neutrophil recruitment and activation. The CXCR1 is the receptor for CXCL8 and CXCL6 (granulocyte chemoattractant protein-2), and the CXCR2 binds these as well as all other ELR-CXC chemokines; CXCL8 binds with high affinity to both receptors, while the others are lower affinity ligands [12 13 14 ]. Independent signaling via the CXCR1 or CXCR2 triggers neutrophil chemotactic and degranulation responses, and simultaneous signaling through both receptors leads to additive responses. This redundancy in these particular functions clearly suggests, conceptually at least, that to fully control CXCL8 triggering of neutrophil responses, the CXCR1 and CXCR2 would need to be controlled. In accord with this concept, it has been shown that the CXCR2 blockade by itself, for example, does not optimally antagonize neutrophil elastase release [15 ]. The importance of this issue to neutrophil therapeutics is confirmed by the reports that elastase release plays an important role in exacerbating neutrophilic pathology in multiple conditions [16 , 17 ]. Above and beyond such redundancy of function, these receptors also differ qualitatively in their responses to ligand signaling. For example, the CXCR1, but not CXCR2, activates reduced nicotinamide adenine dinucleotide phosphate oxidase and thereby regulates neutrophil superoxide production [18 ], which has been implicated as pathologically important in ischemia-reperfusion injury [19 , 20 ], myeloproliferative pathology [21 ], and sepsis [22 ], among other conditions. CXCR2 antagonism alone has been shown to partially (40–50%) block synovial neutrophilia following local LPS or antigen challenge in an arthritis model [23 ], and this raises the issue of whether simultaneous CXCR1 and CXCR2 antagonism in this model would have led to still greater therapeutic effects.

We have recently developed a broad-spectrum ELR-CXC chemokine antagonist, CXCL8(3–74)K11R/G31P [24 ]. In vitro, it effectively blocks neutrophil responses to CXCR1- and CXCR2-specific agonists, and ablates neutrophil responses to all mediators present in clinical bacterial pneumonia lesions [24 , 25 ]. We hypothesized that the ability of CXCL8(3–74)K11R/G31P to antagonize the CXCR1 and CXCR2 should provide it with a strong therapeutic potential in vivo in neutrophilic inflammation. Thus, herein, we tested its efficacy in blocking severe endotoxemia-induced pulmonary inflammation, including neutrophil influx, local inflammatory mediator expression, pyrexia, and vascular damage. Our results indicate that CXCL8(3–74)K11R/G31P is highly effective in blocking multiple aspects of neutrophilic inflammation in airway endotoxemia, whether given at the time of endotoxin challenge or after the onset of symptoms.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and supplies
The following reagents were purchased commercially: endotoxin (Escherichia coli LPS, serotype 0127B8, Sigma Chemical Co., Mississauga, Canada); a Diff-Quick staining kit (American Scientific Products, McGaw Park, IL); human CXCL5/epithelial neutrophil-activating peptide-78 and CXCL8/IL-8 (R&D Systems Inc., Minneapolis, MN); Dulbecco’s modified Eagle’s medium (DMEM) and Hanks’ balanced salt solution (HBSS; Gibco, Grand Island, NY); horseradish peroxidase-streptavidin (Vector Labs, Burlingame, CA); 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) enzyme substrate (Kirkegaard and Perry Labs, Gaithersburg, MD); and bovine serum albumin and lymphocyte separation medium (ICN Pharmaceuticals, Aurora, IL). CXCL8(3–74)K11R/G31P was generated and characterized as noted [24 , 25 ]. Female Hartley guinea pigs (7 weeks old) were purchased from Charles River Laboratories (Charles River, MA); all experiments were carried out according to the guidelines established by the Canada Council on Animal Care and were approved by our institutional animal ethics review panel.

Animal procedures
The animals were held gently in {approx}45° dorsal recumbency for intranasal intubation and challenged with 0–100 µg/kg endotoxin in 200 µl saline. In preliminary experiments, we found no differences in the lung neutrophil responses to LPS delivered in this manner versus that delivered by direct transtracheal injection (data not shown). At the time of challenge and at various times thereafter, the rectal temperatures of the animals were determined. In addition, at 0, 3, 6, or 9 h postchallenge, the animals were given 0–250 µg/kg CXCL8(3–74)K11R/G31P or an equivalent volume [1 ml, subcutaneously (s.c.)] of saline, and at 15 h, they were killed with isofluorane. The respiratory tree of each animal was removed from each animal on euthanasia, gently washed, and blotted, and then, the dorsal and ventral aspects of each were photographed. The photographic images were scored for the proportion of the surface area that was grossly hemorrhagic using a computer-driven image analyzer. Bronchoalveolar lavage (BAL) was performed on the resected respiratory trees as noted previously [26 ] using 5 ml vol HBSS. In preliminary experiments, we determined that maximal neutrophil responses occurred at 15 h postchallenge (data not shown). All samples from each animal were assayed independently and examined immediately or stored at –80°C prior to assay.

Neutrophil isolation, bronchoalveolar cells, and fluids
Red blood cells were sedimented from heparin-anticoagulated blood by dextran sedimentation using 70 kD molecular weight dextran, then, the leukocytes were fractionated on lymphocyte separation medium, and residual red cells in the granulocyte fraction were lysed hypotonically. Blood smears and cytocentrifuge slides of airway cells obtained by BAL were stained with Wright’s solution, and differential counts were performed on 200 cells per sample [26 , 27 ].

Neutrophil chemotaxis
Microchemotaxis assays were run in duplicate-modified Boyden microchemotaxis chambers using polyvinyl pyrrolidone-free 5 µm pore polycarbonate filters, and the results were tabulated as noted [25 , 27 ]. The chemoattractants included recombinant human CXCL1, CXCL5, or CXCL8 or BAL fluids from the lungs of the treated guinea pigs (diluted 1:1–1:40), and the antagonists comprised mouse anti-human CXCL8 or CXCL8(3–74)K11R/G31P. In some assays, we preincubated the samples with the antibodies (5 µg/ml) for 60 min on ice [28 ]. For assays with recombinant CXCL8(3–74)K11R/G31P, the inhibitor was mixed directly with the samples immediately prior to testing.

Lung histopathology
The cranial right lung lobe of each animal was fixed for 3 h in acid-alcohol formaldehyde and routinely processed to 6 µm paraffin sections. For routine histopathology, the tissue sections were stained with Giemsa solution and examined in a blinded manner at 400x magnification [29 ]. The tissue neutrophils were enumerated (numbers of cells/40x field) by an individual blinded to the sample identity.

Cytokine bioassays
IL-1 and IL-6 were detected in BAL fluids using LM-1 and 7TD1 cell proliferation assays, respectively, as noted [30 ], and tumor necrosis factor (TNF) was detected using a L929 cell cytotoxicity assay, also as noted [31 ]. In each case, recombinant human cytokine standard curves were used.

IL-1 assay
IL-1-starved LM-1 cells were plated at 4 x 105 cells/ml in Click’s medium containing 10% fetal calf serum (FCS), L-glutamine, and 1% penicillin/streptomycin/fungizone, together with varying volumes of BAL fluids (1–100 µl/well) and control or IL-1-specific antibodies. After 5 days, the proliferative responses were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The sensitivity of this assay was {approx}10 pg/ml IL-1.

IL-6 assay
For this assay, 7TD1 cells were plated at 1.25 x 104 cells/well in RPMI 10 containing 10% FCS, L-glutamine, and 1% penicillin/streptomycin/fungizone, together with varying volumes of BAL fluids (1–25 µl/well) and control or IL-6-specific antibodies. MTT uptake was used to assess cell proliferation as above. The sensitivity of this assay was {approx}25 pg/ml IL-6.

TNF assay
The details of the L929 cell cytotoxicity assay for TNF have been reported previously. Briefly, varying volumes of BAL fluids were incubated overnight with subconfluent monolayers of L929 cells in DMEM supplemented with 2.5 µg/ml actinomycin D and control or TNF-specific antibodies. The cytotoxicity in each well was determined by MTT uptake, as noted above, with the activity interpolated from a standard curve of recombinant cytokine. The sensitivity of this assay was {approx}10 pg/ml recombinant cytokine.

Statistical analyses
Multigroup data were analyzed by ANOVA and post-hoc Fisher-protected least significant difference testing, and two-group comparisons were made using the Student’s t-test (two-tailed). Linear regression analysis was used to compare the impact on body temperature of delayed antagonist treatments versus saline treatment, using the software program Statview 4.1 (Abacus Concepts Inc., Berkley, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CXCL8(3–74)K11R/G31P blocks the chemotactic responses of guinea pig neutrophils to CXCR1 and CXCR2 ligands
As guinea pig ELR-CXC chemokines are not commercially available, we used human chemokines for our in vitro studies. We confirmed that human CXCL5 and CXCL8, ligands of the CXCR1 and/or CXCR2, were chemotactic for guinea pig neutrophils (Fig. 1 ) and examined the impact of treating guinea pigs with CXCL8(3–74)K11R/G31P on the responses of their neutrophils to these agonists. Cells from saline-injected guinea pigs were responsive to the human chemokines, and CXCL8 and CXCL5 induced optimal chemotactic responses at 10 and 100 ng/ml, respectively. Neutrophils from guinea pigs treated with 250 µg/kg CXCL8(3–74)K11R/G31P animals were essentially unresponsive to CXCL5 or CXCL8 in vitro, and those from guinea pigs given 100 µg/kg antagonist retained some (low-level) responsiveness (data not shown). These experiments confirmed that guinea pig neutrophils do respond to CXCR1 and CXCR2 ligands and that CXCL8(3–74)K11R/G31P can block their responses to these agonists.



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  		Figure 1. CXCL8(3–74)K11R/G31P blocks chemotactic responses of guinea pig neutrophils to human CXCR1 and CXCR2 ligands. Neutrophils were purified from the peripheral blood of healthy guinea pigs and used in modified Boyden chamber microchemotaxis assays against a series of concentrations of recombinant human CXCL8 or CXCL5 (open bars). We also added bovine CXCL8(3–74)K11R/G31P (G31P; 10 ng/ml) to the lower chambers of some samples (solid bars) along with the chemokine to assess its abilities to inhibit chemokine-driven neutrophil responses. Each sample was assessed in duplicate wells in at least three different experiments, and the responses were assessed by counting the numbers of cells within nine 40x objective microscope fields of the stained chemotaxis membranes. The results presented are from one representative experiment and are expressed as the mean numbers of cells per 40x field ± SEM. For each sample, except the CXCL5-negative control, the CXCL8(3–74)K11R/G31P treatment reduced the neutrophil response significantly (P≤0.01).

 
CXCL8(3–74)K11R/G31P can protect against acute neutrophilic inflammation in airway endotoxemia
Before assessing the effects of CXCL8(3–74)K11R/G31P on airway endotoxemia, we first optimized the endotoxin challenge dose in our model, instilling 0, 5, 25, or 100 µg/kg E. coli endotoxin into the airways of each guinea pig. At 15 h after challenge, we killed the animals and found that the 5 or 25 µg/kg doses of LPS induced a significant peripheral blood, pulmonary, and airway neutrophilia, and these responses were reduced substantially in the 100-µg/kg challenge-dose animals (Fig. 2 ). Conversely, the levels of grossly observable pleural hemorrhage increased in direct accord with the LPS dose, rather than being attenuated at the highest dose (data not shown). Based on these results, we chose 5 µg/kg LPS as our standard challenge dose. As early as 3 h after challenge with 5 µg/kg LPS, the animals developed mild pyrexia (LPS, 100.2±0.3°C; saline, 98.2±0.1°C), and their airways contained significant amounts of endogenous pyrogens IL-1 and TNF but not neutrophil chemotactic activities or neutrophils themselves at this time (data not shown).



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  		Figure 2. Endotoxin dose-response curve for endotoxemia-induced airway, lung parenchyma, and peripheral blood neutrophil responses. E. coli endotoxin (LPS; serotype 0127B8) or saline alone (0 dose) was instilled into the airways of conscious guinea pigs by intranasal intubation, and 15 h later, the animals were killed. The airway neutrophil responses were assessed from differential cell counts of bronchoalveolar (BAL) cell cytocentrifuge preparations, the tissue neutrophilia from Giemsa-stained paraffin sections of the lung tissues, and the peripheral blood responses from Wright’s solution-stained blood smears. The 5 and 25 µg/kg doses of LPS induced strong pulmonary neutrophil responses and moderate peripheral blood neutrophilia (**, P≤0.01, vs. saline control values), and the 100 µg/kg dose strongly suppressed the neutrophil responses ({dagger}{dagger}, P≤0.01, vs. the lower LPS dose values) but not overall gross pathology (data not shown).

 
At 15 h postchallenge, the endotoxemic animals were suffering from marked pulmonary inflammatory responses, including hemorrhagic consolidation, across the majority of pleural surfaces of the lungs and infiltration of the airways and lung parenchyma with neutrophils (Fig. 3 ). CXCL8(3–74)K11R/G31P treatments (100 or 250 µg/kg, s.c.), just prior to LPS challenge, markedly blunted the development of this endotoxemic pulmonary response. The antagonist treatments significantly reduced the development of pleural hemorrhagic consolidation, such that the pleura of the LPS-infused animals given 250 µg/kg CXCL8(3–74)K11R/G31P were {approx}85% less hemorrhagic than those of the saline-treated animals (P≤0.001), although the low-dose antagonist treatments did not significantly affect this response (P>0.05; Fig. 3 ). The airway neutrophilia of the animals treated with 100 or 250 µg/kg CXCL8(3–74)K11R/G31P was reduced by 73.9 ± 6 or 86.3 ± 7.5%, respectively, relative to saline-treated animals (P≤0.01 vs. saline-treated, LPS-challenged animals). These treatments also reduced the numbers of neutrophils infiltrating the pulmonary tissues, although to a lesser extent at the lower antagonist dose (Fig. 3) , and reduced the circulating neutrophil counts by {approx}65% and 82%, respectively (P≤0.01 and 0.005), relative to the saline-treated, LPS-challenged animals.



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  		Figure 3. CXCL8(3–74)K11R/G31P can block pleural hemorrhagic consolidation and neutrophil infiltration in endotoxic animals. Endotoxemia was induced in guinea pigs by airway instillation of 5 µg/kg LPS, as in Fig. 2 . Some groups of animals were given CXCL8(3–74)K11R/G31P (G31P; 100 or 250 µg/kg, s.c) immediately prior to LPS instillation, and then, the animals were killed at 15 h postchallenge. (A) The entire cardiopulmonary tree of each animal was removed and photographed using a ventral approach (upper panel), then subjected to BAL, and processed to paraffin sections for histopathology (lower panel). In the gross pictures, the heart can be seen protruding out from between the cranial left and right lung lobes, and the medial and caudal lobes are visible in the lower half of each photograph. The lungs of the saline-challenged animals (saline control) were normal in gross and histological appearance, and those of the LPS-challenged, saline-treated animals (LPS control) were grossly hemorrhagic and histologically inflamed. Neutrophils were the primary population infiltrating the airways (inset, LPS control histology). The lungs of the LPS-challenged animals which were treated with 100 or 250 µg/kg G31P were markedly less hemorrhagic and inflamed than the saline-treated animals. (B) Differential counts were performed on the BAL cells from each group of animals and the proportions of neutrophils enumerated (Airway PMN), while the tissue neutrophilia was assessed by direct counting of Giemsa-stained lung tissue sections (Tissue PMN). In addition, the pleural surface hemorrhagic responses were quantified by computerized photo image analysis of the resected respiratory tree from each animal, and the data were expressed as the mean (±SEM) proportion of the total pleural surface which appeared grossly hemorrhagic. Both doses of CXCL8(3–74)K11R/G31P significantly reduced the 15-h airway and tissue neutrophil responses, but only the 250-µg/kg dose significantly reduced the hemorrhagic response. The results presented are from one experiment representative of two such titrations performed. {dagger}{dagger}, P ≤ 0.01, versus the saline-treated, LPS-challenged animal values. WBC, White blood cells.

 
CXCL8(3–74)K11R/G31P treatments prevent pyrexia and endogenous pyrogen expression in the lungs of endotoxemic animals
To determine whether this CXCL8(3–74)K11R/G31P-dependent blockade of neutrophilic inflammation might have tangential effects, we also assessed its impact on the fever responses in our animals. The saline-treated, LPS-challenged animals developed pyrexia which was detectable at 3 h, continued to develop until at least 6 h, and then waned slowly over the next 18 h (Fig. 4A ). Conversely, treatment with CXCL8(3–74)K11R/G31P at the time of LPS challenge reduced this response to background (P>0.05 vs. saline-challenged animals at each time-point), suggesting that the neutrophil responses could be critical to the development of the fever response. The apparent increase in mean temperature in this group at 3 h was not statistically significant relative to the 0-h time-point (P>0.05). As the fever response in endotoxemia is strongly related to the expression of endogenous pyrogens, we also examined the impact of our antagonist treatments on the early expression of the pyrogens IL-1 and TNF in the airways, as well as the inflammation-associated cytokine IL-6 (Fig. 4B) . The CXCL8(3–74)K11R/G31P treatments reduced the expression of each of these, such that the airway IL-1 responses were lowered by {approx}78 ± 3%, the IL-6 responses by {approx}91 ± 2%, and the TNF responses by 90 ± 5% (P<0.01 relative to the saline-treated, LPS-challenged animals).



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  		Figure 4. CXCL8(3–74)K11R/G31P treatment of endotoxic guinea pigs can modulate pyrexia and endogenous pyrogen expression. Airway endotoxemia was induced as in Figure 3 , although a negative control group of animals was given saline alone. (A) Animals in the endotoxemia groups were treated with saline (LPS-saline) or 250 µg/kg CXCL8(3–74)K11R/G31P [LPS-G31P (0 h)] immediately prior to LPS challenge, and then, the rectal temperature of all animals was determined at the time of LPS challenge (0 h) and 3, 6, 9, and 24 h later. There were no statistically significant increases in body temperature over the 0-h time-point in the animals given saline alone (P≥0.05), and the saline-treated endotoxemic animals developed a marked fever response, which was reduced to background levels at all times by the antagonist treatments (P≤0.01 vs. LPS-saline; P>0.05 vs. negative control). (B). In a separate experiment, at 4 h after LPS challenge groups, animals were killed, and their airways were subjected to BAL. The levels of IL-1, IL-6, and TNF in the BAL fluids were assessed by bioassay, using LM-1, 7TD9, and L929 cells, respectively, as noted in Materials and Methods. The BAL fluids of the saline-treated endotoxemic animals contained substantial levels of each mediator, and the samples from the antagonist-treated endotoxemic animals contained only low levels of each (P≤0.01 vs. saline-treated LPS animals). In other experiments, we did not detect any of these cytokines in the airways or lung tissues of endotoxemic animals at 15 h (data not shown). The data are presented as the mean (±SEM) cytokine values and in each case, are representative of two experiments performed.

 
Moderate delays in CXCL8(3–74)K11R/G31P treatment can reverse recently established pyrexia and neutrophilic inflammation
In clinical settings, patients would not be identified as endotoxemic until some time after the onset of symptoms. Thus, in our next set of experiments, we assessed the impact of delaying treatment with CXCL8(3–74)K11R/G31P for varying periods of time after endotoxin challenge. We treated the animals with CXCL8(3–74)K11R/G31P at 0, 3, 6, or 9 h after endotoxin challenge and then assessed their responses as above (Fig. 5 ). Administration of the antagonist at 3 h postchallenge, when low-grade pyrexia was apparent, actually reversed the developing fever response such that by 9 h, the animal’s temperatures had normalized (P<0.05 vs. negative control animals). When assessed by linear regression analysis, there were significant differences in the fever decay curves (i.e., between the 6- and 24-h time-points) for the saline-treated, LPS-challenged animals and those treated at 6 (F[1, 13]=8.47; P≤0.01) or 9 (F[1, 13]=15.95; P≤0.01) h postchallenge (Fig. 5A) . Notwithstanding, there were no differences in the mean 24-h temperatures of the untreated and 9 h treatment groups (P>0.05), although 24 h temperatures of the untreated and 6 h treatment group animals were different (P≤0.05). Delay in ELR-CXC chemokine antagonism until at or after the time of onset of pyrexia also affected the airway inflammatory responses (Fig. 5B) . When given at 0, 3, or 6 h postchallenge, CXCL8(3–74)K11R/G31P reduced the total numbers of airway cells by 77.2, 72.0, or 58%, respectively (P≤0.01 vs. the saline-treated endotoxemic animals), and the airway neutrophilia by 80, 65.5, or 72.7%, respectively (P≤0.01, 0.05, or 0.01, respectively, vs. the saline-treated, endotoxemic animals).



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  		Figure 5. Delayed treatment of airway endotoxemia with CXCL8(3–74)K11R/G31P also affects fever and inflammatory responses. Guinea pigs were given LPS or saline as in Figure 4 . (A) The temperatures of all animals were assessed as in Figure 4A , but the endotoxemic animals were given saline alone at time 0 (LPS-sal; crossed boxes) or 250 µg/kg CXCL8(3–74)K11R/G31P (s.c) at 3 h ({square}), 6 h ({diamondsuit}), or 9 h ({circ}) after LPS challenge. The horizontal, solid line at 97.1°F represents, for reference, the mean temperature (±SEM; dotted lines) of negative control animals. The temperatures of the animals treated at 3 h post-LPS challenge were reduced dramatically at 6, 9, and 24 h (P≤0.01 vs. the 3-h time-point temperature). When assessed by linear regression analysis, the 6- and 9-h post-LPS treatments had significant effects on pyrexia (P≤0.01 vs. saline-treated endotoxemic animals). The 24-h temperatures of animals in the 6-h treatment and LPS-saline groups were significantly different (P≤0.05), although there was no statistically significant difference in the 24-h temperatures of the animals in the 9-h and LPS-saline groups (P>0.05). The data are presented as the mean rectal temperatures (±SEM) and are from one representative experiment of three performed. (B) In a separate experiment, the animals were killed at 15 h, their airway cells were recovered by BAL, and the total numbers of BAL fluid cells per animal (CELL NO.) as well as the proportions of neutrophils in each sample (% PMN) were determined. LPS challenge induced a significant influx of cells, especially neutrophils, into the airway. Treatment of the LPS-challenged animals before or up to 6 h postchallenge significantly reduced both of these responses. * or **, P ≤ 0.05 or 0.01, respectively, versus the saline-treated, LPS-challenged animals. These data are from one experiment representative of two performed.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We had previously generated CXCL8(3–74)K11R as a high-affinity CXCR1 and CXR2 agonist [32 ] and used rationale design and site-directed mutagenesis approaches with this construct to generate a number of potential ELR-CXC chemokine antagonists [24 ]. We identified CXCL8(3–74)K11R/G31P, among several candidates, as a high-affinity ligand of the CXCR1 and CXCR2; its ability to antagonize neutrophil responses to CXCL1, CXCL2, and CXCL8 further confirmed this specificity [24 ]. We also previously reported that CXCL8(3–74)K11R/G31P can block the in vitro responses of neutrophils to the chemoattractants found in wash fluids from the lungs of animals with pneumonic pasteurellosis [25 ] and that one treatment with CXCL8(3–74)K11R/G31P could block cutaneous neutrophil responses to serial intradermal endotoxin challenges for 2–3 days in calves [25 ]. Herein, we significantly extended our knowledge of the anti-inflammatory abilities of this combined CXCR1/CXCR2 antagonist, showing that when used prophylactically, it can dramatically reduce pulmonary and neutrophilic inflammation, as well as its attendant hemorrhagic consolidation and pyrexia in airway endotoxemia. We also found that this antagonist had significant therapeutic effects when used at the time of, or shortly after, onset of symptoms (i.e., pyrexia) in endotoxemic animals.

This report represents what we believe to be the first addressing the efficiency with which a high-affinity, combined CXCR1/CXCR2 antagonist can affect a disease complex as pathologically diverse as airway endotoxemia. Others have used CXCR2-specific antagonists [33 34 35 ] or substantially lower affinity antagonists based on CXCL8/IL-8 or other ELR-CXC chemokines [15 , 36 37 38 ], and these have had beneficial effects to varying degrees in a number of systems. As noted above, a low molecular weight, nonpeptide, CXCR2-specific antagonist has been shown to reduce by {approx}50% the numbers of synovial neutrophils following endotoxin or antigen infusion in an arthritis model, and these effects were also mirrored by similar reductions in knee swelling or even greater reductions in local CXCL8 or TNF levels [23 ]. A similar nonpeptide CXCR2 antagonist reduced transient neutrophil margination during intravenous CXCL8 infusion into rabbits [33 ], and an amino-truncated growth-related oncogene-{alpha} (CXCL1) analog was demonstrated to effectively reduce neutrophil infiltration of endotoxin-challenged skin air pouches in mice or neutrophil responses to peritoneal chemokine challenge in these animals [35 ]. These data demonstrate conclusively that CXCR2-specific antagonists can have beneficial effects, sometimes striking ones in neutrophilic inflammation. The most striking effects were in mice, which are considered not to express a CXCR1, and the CXCR2 antagonists were not quite as effective in rabbits (which express a CXCR1 and CXCR2). This suggests that perhaps both receptors play important roles in inflammatory responses in CXCR1/CXCR2-replete species. We found that CXCL8(3–74)K11R/G31P effectively blocked neutrophilic inflammation and its downstream effects in endotoxemic animals, which would suggest that simultaneous CXCR1/CXCR2 antagonism may indeed be highly desirable.

The fever response to endotoxin challenge is dependent on endogenous pyrogens (e.g., IL-1, TNF) expressed by endotoxin-stimulated cells [39 , 40 ] but likely also on other mediators (e.g., prostaglandins) expressed under the influence of these cytokines [40 ]. IL-1, IL-6, and TNF were expressed at high levels in our endotoxemic animals at 3–4 h after challenge, but these responses had waned to background by 15 h. Our ELR-CXC chemokine antagonist pretreatments essentially ablated the 4-h IL-1 and TNF responses, as well as the 3- to 24-h fever response in the endotoxemic animals. A 3-h delay in CXCL8(3–74)K11R/G31P treatment fully reversed the developing fever response, and a 6- or 9-h delay only minimally modulated pyrexia. This suggests that the mediators that fall under the influence of the CXCR1 or CXCR2, directly or indirectly, and that trigger the fever response in airway endotoxemia are only expressed early on (i.e., before 6–9 h) after endotoxin induction. Conversely, delay of treatment to 6 h more than 70% reduced the 15-h airway neutrophil response, indicating that full realization of the effector mechanisms required to induce strong neutrophil responses is not required for successful implementation and support of significant fever responses. Thus, it is possible that pyrexia itself may not be a highly accurate indicator of local pathology.

Neutrophils can express IL-1 [41 ] and TNF [42 ], and indeed, the levels at which neutrophils secrete these pyrogens can increase in proportion to their relative numbers [42 ], suggesting that these cells may use a synergistic response system under inflammatory conditions. Such synergies have been reported for IL-1 and TNF, for example, in the induction of CXCL8/IL-8 expression by monocytes [43 ]. Others have reported that neutrophil depletion down-regulates epithelial IL-1 expression under inflammatory conditions (e.g., ref. [44 ]). In preliminary experiments, we have found that coincubation of purified human neutrophils with monolayers of bronchial epithelial (A549) cells leads to high-level release of IL-1 and CXCL8 (J. R. Gordon and A. Dean Befus, unpublished). We postulate that the activated neutrophils in our untreated, endotoxemic animals would exacerbate local responses through the mediators they release (e.g., elastase, reactive oxygen intermediates) and that reciprocally, TNF, IL-1, and CXCL8 secreted locally could lead to additional neutrophil recruitment [45 ] and inflammatory mediator release [42 , 46 47 48 49 ]. Although we have not documented the molecular mechanisms mediating the therapeutic efficacy of CXCL8(3–74)K11R/G31P in this model, it seems feasible that its reversal of the neutrophil response would have the effect of short-circuiting this feed-forward cascade.

Received August 16, 2005; accepted August 17, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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