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The Journal of Immunology, 2002, 168: 3550-3556.
Copyright © 2002 by The American Association of Immunologists

Tissue-Specific Mechanisms Control the Retention of IL-8 in Lungs and Skin1

Charles W. Frevert2,*, Richard B. Goodman*, Michael G. Kinsella{dagger}, Osamu Kajikawa*, Kimberly Ballman*, Ian Clark-Lewis{ddagger}, Amanda E. I. Proudfoot§, Timothy N. C. Wells§ and Thomas R. Martin*

* Medical Research Service and Division of Pulmonary/Critical Care Medicine, Department of Medicine, Seattle Department of Veterans Affairs Medical Center, Seattle, WA 98108; {dagger} Department of Vascular Biology, Hope Heart Institute, Seattle, WA 98105; {ddagger} Biomedical Research Center, University of British Columbia, Vancouver, British Columbia, Canada; and § Serono Pharmaceutical Research Institute, Geneva, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chemokines are a group of structurally related peptides that promote the directed migration of leukocytes in tissue. Mechanisms controlling the retention of chemokines in tissue are not well understood. In this study we present evidence that two different mechanisms control the persistence of the CXC chemokine, IL-8, in lungs and skin. 125I-labeled IL-8 was injected into the airspaces of the lungs and the dermis of the skin and the amount of 125I-labeled IL-8 that remained at specified times was measured by scintillation counting. The 125I-labeled IL-8 was cleared much more rapidly from skin than lungs, as only 2% of the 125I-labeled IL-8 remained in skin at 4 h whereas 50% of the 125I-labeled IL-8 remained in lungs at 4 h. Studies in neutropenic rabbits showed that neutrophils shortened the retention of 125I-labeled IL-8 in skin but not lungs. A monomeric form of IL-8, N-methyl-leucine 25 IL-8, was not retained as long in lungs as recombinant human IL-8, indicating that dimerization of IL-8 is a mechanism that increases the local concentration and prolongs the retention of 125I-labeled IL-8 in lungs. These observations show that the mechanisms that control the retention of IL-8 in tissue include neutrophil migration and dimerization, and that the importance of these varies in different tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chemokines are a structurally related group of cytokines that promote the migration of specific subsets of leukocytes (1, 2). IL-8, a CXC chemokine, is an effective polymorphonuclear neutrophil (PMN)3 chemotactic factor produced during acute inflammatory responses (3, 4). Whereas IL-8-mediated PMN recruitment is important to host defenses against microbial pathogens, animal models and clinical studies suggest that IL-8 plays a role in the pathogenesis of acute lung injury (5, 6, 7, 8, 9). Thus, determining the mechanisms that control the retention and the duration of the biological activity of IL-8 in tissue is of fundamental importance for understanding normal host defenses and pathological forms of inflammation.

A considerable literature has accumulated about the cellular production of chemokines and the interactions between chemokines and their high-affinity receptors (1, 2, 10). In contrast, little is known about how IL-8 and other chemokines function in tissue to promote directed migration of leukocytes. IL-8 was found to promote PMN migration 4–6 h after its instillation into the skin of rabbits, suggesting prolonged retention of IL-8 in skin (11). No data exist about the retention of IL-8 in lungs.

The three-dimensional structures of CXC and CC chemokines are highly conserved and contain two spatially separate binding domains, a high-affinity binding domain and a glycosaminoglycan (GAG)-binding domain (12, 13, 14, 15, 16, 17). The high-affinity binding domain of IL-8 is responsible for activation and chemotaxis of PMN, while the GAG-binding domain binds to the GAGs heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate in vitro (13, 15, 18, 19). The presence of a GAG binding domain on IL-8 has led several investigators to propose that, in tissue, chemokines are immobilized on the surface of endothelial cells and extracellular matrix through interactions with GAGs and that leukocytes migrate over fixed gradients (e.g., haptotactic gradients) rather than along soluble gradients (20, 21, 22, 23, 24, 25, 26, 27). Thus, the binding of IL-8 to GAGs in tissue is a potential mechanism that would prolong the retention of IL-8 by limiting diffusion.

Besemer et al. (28) found that PMN bind, internalize, and degrade IL-8 in vitro. PMN elastase decreases IL-8 chemotactic activity by proteolytic degradation of IL-8 into small fragments (29). The proteolytic degradation of IL-8 by PMN elastase was specific, as equimolar concentrations of serine proteases, urokinase, plasmin, thrombin, and cathepsin G did not result in a loss of IL-8 bioactivity. Therefore, proteolytic degradation of IL-8 by PMN is a potential mechanism that would decrease the retention and biological activity of IL-8 in tissue.

Structural differences in organs need to be considered when studying the retention of IL-8 in vivo. In lungs, IL-8 is produced by alveolar macrophages located in airspaces and must cross an epithelial and an endothelial cell barrier to establish tissue-bound or haptotactic gradients that can be sensed by migrating PMN (20). In contrast, in tissues such as skin, IL-8 is released into the dermis, a loose connective tissue underlying epithelial cells, and must move only across an endothelial cell barrier to promote PMN migration.

The goals of this study were to determine how long IL-8 persists in two different tissues, lungs, and skin, and to identify potential mechanisms that control the retention of IL-8 in these two locations. We demonstrate that IL-8 retention occurs in both organ systems, but that the retention time is significantly longer in the lungs as compared with skin. In the skin PMN contribute to a shorter retention time, whereas they do not affect the retention of IL-8 in lungs. We also show that dimerization of IL-8 is important in lungs but not skin and that this mechanism results in a 2-fold increase in the retention of IL-8 in lung tissue. Thus, mechanisms that are tissue specific regulate the amount of IL-8 in lungs and skin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of reagents

The 125I-labeled IL-8 (Amersham Pharmacia Biotech, Piscataway, NY), recombinant human (rh)IL-8 (PeproTech, Rocky Hill, NJ), and an obligate monomeric form of IL-8, N-methyl-leucine 25 (N-methyl-L25) IL-8 (provided by I. Clark-Lewis, University of British Columbia, Vancouver, Canada), were resuspended in sterile 0.9% NaCl with 0.1% pyrogen-free BSA (Irvine Scientific, Santa Ana, CA). 125I-labeled IL-8 was used at a concentration of 2 ng/ml (0.25 nM) when used alone or in combination with unlabeled IL-8. Unlabeled IL-8 and unlabeled N-methyl-L25 IL-8 were used at a concentration of 1000 ng/ml (125 nM). Vinblastine (Eli Lilly, Indianapolis, IN) was resuspended in sterile NaCl at 1 mg/ml. Colloidal carbon (Pelikan, Hanover, Germany) was added to the suspension of IL-8 at a final concentration of 1%. The IL-8 was mixed with 1% colloidal carbon (Pelikan) to aid in identifying the instilled areas in lungs at necropsy (30).

Animal protocols

The Animal Research Committee of the Veterans Affairs Puget Sound Health Care System approved all experiments. Female New Zealand White rabbits, specific pathogen-free, weighing 3–3.5 kg, were purchased from Western Oregon Rabbit Company (Philomath, OR) and were housed in the animal facility until the day of the experiment. Rabbits were anesthetized with a combination of i.v. ketamine (10 mg/kg) and xylazine (3 mg/kg). Anesthesia was maintained for the duration of the skin and lung studies with ketamine (50 mg/ml at 1.2 ml/h). Rabbits were allowed to breathe spontaneously for the duration of the study.

Preparation of neutropenic rabbits. To induce neutropenia, rabbits were treated with one dose of i.v. vinblastine (0.75 mg/kg), which causes leukopenia within 24 h, with the lowest point 72 h after the treatment (31). All studies with neutropenic rabbits were performed 72 h after the vinblastine treatment. Following treatment with vinblastine, rabbits were treated with enrofloxacin (5 mg/kg) to prevent the development of bacterial infections.

Intradermal and intratracheal instillation of 125I-labeled IL-8. Intradermal instillations of 125I-labeled IL-8 (100 µl of 0.25 nM) were performed on the back of normal and neutropenic rabbits at 0, 0.25, 0.5, 1, 2, 4, and 6 h before euthanasia. Instillations were performed using a 25-gauge needle. In later studies, intradermal instillations of 125I-labeled IL-8 (0.25 nM) and 125I-labeled IL-8 (0.25 nM) plus an excess of unlabeled rhIL-8 (125 nM) (100 µl total volume) were performed in neutropenic rabbits at 0, 0.25, 0.5, 1, 2, 3, and 4 h. Rabbits were anesthetized for the duration of the skin studies.

For intratracheal instillation, rabbits were anesthetized and placed in a right lateral recumbent position on a 20-degree incline with the head elevated. Then a 5-French catheter (AccuMark, Keene, NH) was advanced through the endotracheal tube, and a 0.5-ml solution of IL-8 was instilled in the right lung. First, the pulmonary retention of radiolabeled IL-8 was measured in normal rabbits using the same concentration of 125I-labeled IL-8 used in skin (0.25 nM). Next, the pulmonary retention of 0.25 nM 125I-labeled IL-8 was measured in neutropenic rabbits to determine whether PMN modify the retention of IL-8 in lungs. To test the effect of much greater PMN recruitment, a 500-fold excess of unlabeled IL-8 (125 nM) was combined with 125I-labeled IL-8 (0.25 nM) and the retention of the 125I-labeled IL-8 tracer was measured in normal and neutropenic rabbits at 4 h. This concentration of IL-8 was chosen based on two observations. First, the intratracheal instillation of IL-8 at 1000 ng/ml resulted in maximal PMN recruitment (1.91 x 107 ± 5.40 x 106) into the airspaces of the lungs of rabbits. Second, in patients with adult respiratory distress syndrome, Pugin et al. (9) and Miller et al. (8) have reported IL-8 concentrations as high 1000 ng/ml in pulmonary edema fluid, and this amount of IL-8 is consistent with reported values in bronchoalveolar lavage (BAL) fluid, allowing for 100-fold dilution of IL-8 by BAL (32). Finally, to determine whether the dimerization of IL-8 modifies the retention of radiolabeled IL-8 in lung tissue, an excess of unlabeled monomeric IL-8, N-methyl-L25 IL-8 (125 nM) (33), was mixed with 125I-labeled IL-8 (0.25 nM) and the retention of the 125I-labeled IL-8 (0.25 nM) was measured in neutropenic rabbits to eliminate contributions from migrating PMN. Rabbits were anesthetized for the duration of the lung studies using the same anesthetic protocol used for studies in skin.

Measurement of 125I-labeled IL-8 retained in skin and lungs. After the intradermal instillation of 125I-labeled IL-8, rabbits were euthanized and skin biopsies were taken from injection sites using an 8-mm punch biopsy (Miltex, Lake Success, NY). The amount of radioactivity in tissue was determined with a Packard 5000 series autogamma scintillation counter (Packard Instrument, Meriden, CT). Radioactivity is expressed either as the percentage of total 125I-labeled IL-8 that was instilled or as the amount of IL-8 (in nanograms) present in tissue calculated from the specific radioactivity of 125I-labeled IL-8. In studies where colloidal carbon was instilled in combination with 125I-labeled IL-8 in the skin, we found that the size of the instilled area marked by colloidal carbon did not change over time and that the biopsy removed the majority of the colloidal carbon. Therefore, the tissue biopsy provided a consistent sample of the instilled area at each time.

After the instillation of 125I-labeled IL-8 into the lungs, the rabbits were euthanized with pentobarbital (120 mg/kg) and exsanguinated by direct cardiac puncture. The trachea was isolated and cross-clamped and the trachea, lungs, and heart were removed en bloc. The trachea and lungs were dissected free from the heart and surrounding tissue and a plastic catheter was inserted into the middle portion of the trachea and secured with silk suture. Each lung was lavaged separately. First, the right mainstem bronchus was cross-clamped and the left lung was lavaged with five separate 15-ml aliquots of 0.9% NaCl containing 0.6 mM EDTA at 37°C. The left mainstem bronchus was then cross-clamped and the right lung was lavaged using the same protocol. The BAL fluid from each lung was spun at 200 x g to pellet cells, aliquots of the cell-free supernatant fluid were removed, and the radioactivity in the fluid was measured in a gamma scintillation counter. The cell pellet was resuspended in 5 ml 0.9% NaCl and a small aliquot (100 µl) was removed to perform cell count and differential in the high-dose group. The radioactivity of the cell pellet was measured in a gamma counter. Lung tissue was cut into small pieces (1 cm x 1 cm) and radioactivity in the right and left lung tissue was measured.

To calculate the amount of 125I-labeled IL-8 in the systemic circulation, radioactivity was measured in 1 ml of whole blood. The blood volume in the systemic circulation was estimated as 7.5% of total body weight to calculate the total radioactivity in blood. Radioactivity was expressed as the percentage of total 125I-labeled IL-8 that was instilled or as the amount of IL-8 present in tissue calculated from the specific activity of 125I-labeled IL-8.

Measurement of PMN migration in vitro and in vivo

Measurement of PMN chemotaxis in vitro. Rabbit PMN chemotaxis toward rhIL-8 was measured in vitro using a fluorescence-based microchemotaxis assay (34). Rabbit PMN were recovered from peripheral blood of healthy donor rabbits using a two-step density gradient (OptiPrep; Accurate Chemical and Scientific, Westbury, NY) and labeled with calcein AM (Molecular Probes, Eugene, OR). The bottom wells of the 96-well chemotaxis chamber were filled with IL-8 as well as positive and negative controls. A polycarbonate filter with a pore size of 8 µm was placed on the bottom chamber, calcein-labeled PMN were placed directly onto sites on the filter, and the chamber was incubated for 1 h (37°C and 5% CO2). PMN that did not migrate were removed from the top of the filter. The chemotaxis chamber was placed in a multiwell fluorescent plate reader (CytoFluor II; PerSeptive Biosystems, Framingham, MA) and the cells that migrated into the bottom chamber were measured using the calcein fluorescence signal (excitation, 485 nm; emission, 530 nm). PMN migration was expressed as the percentage of the total PMN that migrated in response to the different concentrations of IL-8 (total percentage).

Measurement of PMN migration into skin and lungs. Semiquantitative morphometry was used to measure PMN migration into skin using H&E tissue sections obtained from tissue biopsies. At the end of each study, tissue biopsies were placed in 10% formalin, embedded in paraffin, and stained with H&E using standard protocols. The number of PMN in 10 randomly selected high-power fields (hpf; x1000) were counted in each tissue biopsy by two independent individuals and reported as the number of PMN/10 hpf.

The number and identity of cells in BAL fluid was determined by counting cells on a hemacytometer at a magnification of x400. Cell size, nuclear detail, and the granularity of the cytoplasm were used to differentiate macrophages, lymphocytes, and PMN. The differential cell counts from the hemacytometer were confirmed by light microscopy by counting 200 cells on smears made from the BAL cell pellet stained with the Diff-Quik stain (American Scientific Products, McGraw Park, IL).

Data and statistical analysis

The tissue t1/2 in skin was calculated using the following equation: tissue t1/2 = 0.693/KE, where KE = ln (slope). When comparisons between two groups were made, statistical analysis was performed with the Mann-Whitney U test. A p value of <= 0.05 was considered significant. Comparisons between multiple groups were performed using ANOVA and secondary comparisons were performed with the Bonferroni test. Values are means ± SEM unless otherwise specified.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Retention of 125I-labeled IL-8 in the lungs and skin of normal rabbits

Our first approach was to compare the retention of human 125I-labeled IL-8 in the lungs and skin of normal rabbits. Initial experiments established that rabbit PMN recognize human IL-8 and migrate with a peak response to 10 ng/ml (i.e., 1.25 nM) rhIL-8 in vitro as previously reported (35). For all studies comparing retention of IL-8 in lungs and skin, the same experimental conditions were used and identical concentrations of radiolabeled IL-8 were injected into either the airspaces of the lungs or the dermis of the skin. The amount of 125I-labeled IL-8 that remained at specified times was determined by scintillation counting and expressed either as the percentage of instilled 125I-labeled IL-8 or as the absolute amount of IL-8 that remained using the specific activity of 125I-labeled IL-8 (measured in nanograms per site). Distinct differences were observed in the retention of radiolabeled IL-8 in skin and lungs, with a more rapid loss of 125I-labeled IL-8 from skin than from the lungs (Fig. 1Go). By 4 h, 125I-labeled IL-8 had been almost completely cleared from the skin sites (2% remaining), whereas 50% of the instilled 125I-labeled IL-8 remained in the lungs. In the lungs, there was a slow movement of IL-8 from the pulmonary airspaces toward the blood over time, as indicated by partitioning of 125I-labeled IL-8 in alveolar fluid (i.e., BAL fluid), lung tissue, and blood (Fig. 2Go). The amount of 125I-labeled IL-8 recovered in the alveolar fluid decreased with time, while 125I-labeled IL-8 in lung tissue remained constant and 125I-labeled IL-8 in blood increased steadily.



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  		FIGURE 1. Comparison of the retention of 125I-labeled IL-8 in lungs and skin at 0.25, 2, and 4 h after instillation. Data are the percentage of the instilled 125I-labeled IL-8. The amount of 125I-labeled IL-8 in skin is obtained from the tissue biopsy while the amount of 125I-labeled IL-8 in lungs is obtained from lung tissue and BAL fluid. Values are the mean ± SEM.

 


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  		FIGURE 2. 125I-labeled IL-8 in BAL fluid, lung tissue, and blood at 0.25, 2, and 4 h after the instillation of IL-8 into the airspaces of the lungs. The amount of 125I-labeled IL-8 recovered in the BAL fluid decreased with time, while 125I-labeled IL-8 in lung tissue remained constant and 125I-labeled IL-8 in blood increased steadily. The amount of 125I-labeled IL-8 in the lungs of animals was determined using the specific activity of 125I-labeled IL-8 and is expressed in nanograms per site. Values are the mean ± SEM (n = 2 for 0.25 h, n = 6 for 2 h, and n = 6 for 4 h).

 
To insure that the addition of colloidal carbon to 125I-labeled IL-8 did not affect the retention of IL-8 in lungs, additional rabbits were treated with 125I-labeled IL-8 (0.25 nM) alone (n = 4). In these animals there was no difference in the retention of 125I-labeled IL-8 in lungs as compared with animals in which colloidal carbon was used (51.34 ± 4.61 vs 50 ± 4.7%, respectively). Thus, the presence of colloidal carbon cannot explain the significant difference in the retention of 125I-labeled IL-8 in lungs and skin.

Retention of 125I-labeled IL-8 in the lungs and skin of normal and neutropenic rabbits

To determine the effect of PMN migration on the retention of IL-8 in tissue, we first compared the retention of 125I-labeled IL-8 in the skin of normal and neutropenic rabbits (Fig. 3Go). Neutropenia was induced by pretreatment with vinblastine and confirmed by measuring circulating leukocyte counts (normal = 2242 ± 713.85 PMN/mm3 and neutropenic = 255 ± 67.85 PMN/mm3). Direct comparisons of the amount of IL-8 that remained in skin of normal and neutropenic rabbits showed that significantly more 125I-labeled IL-8 remained in the skin of neutropenic rabbits at 2 and 4 h (Fig. 3Go, p < 0.005). The tissue t1/2 of IL-8 in skin of normal rabbits was 0.73 h, while the tissue t1/2 of IL-8 in skin of neutropenic rabbits was 0.94 h, an increase of 29%. Histologic assessment showed that, in normal rabbits, there was a significant recruitment of PMN into the skin (69 ± 16.09 PMN/hpf) in response to the intradermal instillation of 125I-labeled IL-8 (0.25 nM). In contrast, there were very few PMN in the skin of neutropenic rabbits following the intradermal instillation of the same dose of 125I-labeled IL-8 (0.33 ± 0.58 PMN/hpf). Thus, the absence of migrating PMN was associated with a significantly longer t1/2 of IL-8 in skin.



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  		FIGURE 3. The retention of IL-8 in the skin of normal and neutropenic rabbits at sequential times after its intradermal instillation. The retention of 125I-labeled IL-8 in the skin of neutropenic rabbits is significantly increased at 2 and 4 h after the intradermal instillation of IL-8 (*, p < 0.005). Values are the mean ± SEM (n = 6 at each time for normal rabbits and n = 5 for neutropenic rabbits).

 
In the next experiments, we determined the effect of PMN migration on the retention of 125I-labeled IL-8 in lungs using experimental methods similar to studies in skin. In contrast to skin, neutropenia did not prolong the retention of 125I-labeled IL-8 in lung tissue (Fig. 4Go). However, this dose of instilled IL-8 was associated with only modest PMN recruitment into the lungs, as reflected by histology. To provide a more definitive test of the effect of PMN migration, a 500-fold higher concentration of unlabeled IL-8 (125 nM) was instilled in combination with the 125I-labeled IL-8 (0.25 nM). This concentration of instilled IL-8 significantly enhanced PMN migration into the lungs of normal rabbits (1.58 ± 0.36 x 107 PMN in BAL fluid) as compared with neutropenic rabbits (8.75 ± 2.10 x 105 PMN in BAL fluid). However, even at the higher concentration of IL-8 there was no difference in the retention of 125I-labeled IL-8 in lung tissue of normal and neutropenic rabbits (normal = 28 ± 4.4% and neutropenic = 35 ± 5.7% of instilled 125I-labeled IL-8 retained at 4 h). Thus, in contrast to what was observed in the skin, neutropenia did not prolong the retention of 125I-labeled IL-8 in the lungs.



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  		FIGURE 4. Retention of 125I-labeled IL-8 in the lungs of normal and neutropenic rabbits 4 h after the administration of 125I-labeled IL-8 (0.25 nM). Neutropenia did not affect the retention of 125I-labeled IL-8 in the lungs. Values are the mean ± SEM (n = 6 for normal and n = 5 for the neutropenic rabbits).

 
The effect of unlabeled IL-8 (125 nM) on the retention of 125I-labeled IL-8 (0.25 nM) in the lungs and the skin

With the addition of excess unlabeled IL-8 with the 125I-labeled IL-8, we expected that increased PMN migration would shorten the t1/2 of the 125I-labeled IL-8 and/or that the excess unlabeled IL-8 would displace the 125I-labeled IL-8 from tissue binding sites and decrease the retention of radiolabeled IL-8 in the lungs. However, we were surprised to find that the addition of excess unlabeled IL-8 significantly increased the amount of 125I-labeled IL-8 that was recovered in lung tissue of neutropenic rabbits when compared with neutropenic rabbits treated with 125I-labeled IL-8 alone (p = 0.005) (Fig. 5GoA). This increase in the concentration of 125I-labeled IL-8 occurred despite a 500-fold dilution of the specific activity of the 125I-labeled IL-8 by the unlabeled IL-8. In neutropenic rabbits treated with 125I-labeled IL-8 alone, 15 ± 2.2% of the 125I-labeled IL-8 was recovered in lung tissue at 4 h. In contrast, in neutropenic rabbits treated with 125I-labeled IL-8 and an excess of unlabeled IL-8, 35 ± 4.7% of the 125I-labeled IL-8 remained in lung tissue at 4 h. This was over twice the amount of 125I-labeled IL-8 that remained in lung tissue of neutropenic rabbits treated with 125I-labeled IL-8 alone. Similar trends were seen for 125I-labeled IL-8 recovered from lung tissue of normal rabbits, but the pattern was more striking in the neutropenic rabbits, in which the effects of migrating PMN were minimized. Additional experiments were performed to determine whether the addition of excess unlabeled IL-8 modified the amount of 125I-labeled IL-8 in the skin of neutropenic rabbits (Fig. 5GoB). In contrast to the lungs, the addition of a 500-fold excess of unlabeled IL-8 did not increase the amount of 125I-labeled IL-8 in skin.



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  		FIGURE 5. Retention of 125I-labeled IL-8 in the lungs (A) and skin (B) of neutropenic rabbits after instillation of either 125I-labeled IL-8 (0.25 nM) alone or in combination with excess unlabeled IL-8 (125 nM). A, When neutropenic rabbits treated in the lungs with 125I-labeled IL-8 alone were compared with rabbits treated with an excess of unlabeled rhIL-8 combined with 125I-labeled IL-8, there was a significant increase in the amount of 125I-labeled IL-8 in the lung tissue (15 ± 2.2 and 35 ± 5.7%, respectively; *, p = 0.02) and decrease in the amount of 125I-labeled IL-8 in blood (9 ± 2.5 and 4 ± 0.4%, respectively; *, p = 0.05). B, In skin, no differences were observed when neutropenic rabbits treated with an excess of unlabeled rhIL-8 combined with 125I-labeled IL-8 were compared with rabbits receiving 125I-labeled IL-8 alone. Values are the means ± SEM (n = at least 4 rabbits per group).

 
IL-8 forms dimers in solution, and dimerization of IL-8 increases the amount of 125I-labeled IL-8 binding to immobilized heparin and endothelial cells in vitro (36). To determine whether dimerization was the mechanism responsible for the increased retention of 125I-labeled IL-8 in lung tissue, we instilled 125I-labeled IL-8 in combination with a 500-fold excess of either rhIL-8 or a modified form of IL-8, N-methyl-L25 IL-8, which does not form dimers (33, 36). When 125I-labeled IL-8 (0.25 nM) was instilled in combination with N-methyl-L25 IL-8 (125 nM), the recovery of the 125I-labeled IL-8 was not affected (Fig. 6Go), indicating that the monomeric form of IL-8 did not significantly increase the amount of 125I-labeled IL-8 retained in lung tissue. In contrast, treatment with an excess of rhIL-8 significantly increased the retention of 125I-labeled IL-8 in lung tissue when compared with 125I-labeled IL-8 combined with the monomeric form of IL-8 (Fig. 6Go; #, p = 0.015). Thus, rhIL-8, which forms dimers, increased the amount of 125I-labeled IL-8 in tissue, whereas a monomeric form of IL-8 did not increase the retention of 125I-labeled IL-8 in lung tissue.



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  		FIGURE 6. The amount of 125I-labeled IL-8 in the lungs of neutropenic rabbits 4 h after instillation of either 125I-labeled IL-8 (0.25 nM) alone or in combination with excess unlabeled IL-8 (125 nM) or unlabeled N-methyl-L25 IL-8 (125 nM). There is a significant increase in the amount of 125I-labeled IL-8 in the lung tissue of neutropenic rabbits treated with excess unlabeled IL-8 combined with 125I-labeled IL-8, when compared with rabbits treated with 125I-labeled IL-8 alone (*, p = 0.003). When unlabeled N-methyl-L25 IL-8, an obligate monomer, was instilled in combination with 125I-labeled IL-8, the retention did not differ from that seen in animals treated with 125I-labeled IL-8 alone. There was significantly less 125I-labeled IL-8 recovered from the lungs of rabbit treated with the cold monomeric form of IL-8 when compared with rabbits treated with cold rhIL-8, which is capable of dimerization (#, p = 0.015). Values are the means ± SEM (n = at least 4 rabbits per group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The goals of this study were to determine how long IL-8 persists in two locations, the lungs and the skin, and to identify mechanisms responsible for controlling the retention of IL-8 in tissue. We report three observations that provide new information about the clearance of IL-8 from tissue sites. First, the clearance of IL-8 differs dramatically in lungs and skin, with slow clearance from lungs and rapid clearance from skin. Second, migrating PMN shorten the t1/2 of IL-8 in skin but not lungs. Third, dimerization of IL-8 is an important mechanism that prolongs the retention of IL-8 in lungs, whereas in skin dimerization of IL-8 does not prolong the t1/2 of IL-8. Thus, the mechanisms that govern the fate of IL-8 in tissue depend on the site at which IL-8 is deposited.

The work of Besemer et al. (28) showing that PMN bind, internalize, and then degrade IL-8 in vitro and the work of Leavell et al. (29) showing that PMN elastase degrades IL-8 suggested that PMN migration through tissue would decrease the local concentration of 125I-labeled IL-8 in lungs and skin. As predicted, neutropenia significantly prolonged the retention of IL-8 in skin (Fig. 3Go). In contrast, neutropenia did not affect the retention of the same concentration of 125I-labeled IL-8 in lungs (Fig. 4Go). To test the effect of greater numbers of migrating PMN in the lungs, we instilled a 500-fold higher concentration of unlabeled IL-8 in combination with the 125I-labeled IL-8. This concentration of unlabeled IL-8 was chosen because it causes maximal recruitment of PMN into the lungs of rabbits and is in the range of concentrations detected in edema fluid and BAL fluid of patients with adult respiratory distress syndrome (8, 32, 37, 38). Despite a robust PMN response, PMN did not affect the retention of 125I-labeled IL-8 in lung tissue.

One possible explanation for the differential effect of PMN on the retention of 125I-labeled IL-8 in lungs and skin is the difference in PMN migration in these two tissues. When PMN migrate into lungs, they migrate from the pulmonary circulation into the airspaces (e.g., alveoli and airways). In contrast, in skin PMN migrate into the interstitial connective tissue and remain in this location, prolonging the time that PMN may be in contact with IL-8 and providing more time for internalization and degradation of 125I-labeled IL-8. A second possible explanation is that differences may exist in the effects of PMN migration on microvascular permeability in skin as compared with lungs (7, 39, 40). If the effect of IL-8-dependent PMN migration on microvascular permeability were greater in skin than lungs, this could result in a more rapid clearance and decreased retention of 125I-labeled IL-8 in skin.

Whereas IL-8 was cleared rapidly from skin, the retention of IL-8 in lung tissue was constant for the duration of the 4-h study (Fig. 2Go). The binding of IL-8 to GAG side chains on tissue proteoglycans is a potential mechanism for the stable retention of IL-8 in lung tissue. Proteoglycans are found on epithelial cells, in the extracellular matrix, and on endothelial cells in the lungs (41). IL-8 has been shown to bind to endothelial cells in skin and isolated GAGs such as heparin and heparan sulfate in vitro (15, 18, 20). The molecular interaction between IL-8 and GAG is specific and requires a GAG binding domain on IL-8 and complementary sequences on the GAG heparan sulfate (15, 42). The observation that IL-8 selectively binds to different GAG families with binding to heparin > heparan sulfate > dermatan sulfate > chondroitin sulfate is further evidence of specificity for this molecular interaction (19). In fact, specificity of protein-GAG interactions is even present within the same class of GAG, as was observed in a study in rats comparing the antithrombin-binding characteristics of heparan sulfate isolated from different tissues (43). In this study, it was shown that heparan sulfate isolated from skin bound antithrombin with higher affinity as compared with heparan sulfate isolated from lungs (43). Thus, differences in the composition of GAGs or the binding affinity of a specific subset of GAGs, such as heparan sulfate, for IL-8 in lungs vs skin may account for the differences observed in IL-8 retention between these two locations.

In the experiments designed to investigate whether PMN decreased the retention of IL-8 in lung tissue, we enhanced PMN migration by combining an excess of unlabeled IL-8 (125 nM) with 125I-labeled IL-8 (0.25 nM). Surprisingly, the addition ofexcess unlabeled IL-8 increased the retention of 125I-labeled IL-8 in lung tissue of neutropenic rabbits (Fig. 5GoA). The binding of IL-8 to its high-affinity receptors (i.e., CXCR-1, CXCR-2, and Duffy Ag/receptor for chemokines) occurs at a Kd of ~2 nM (12), so the addition of a large excess of unlabeled IL-8 (125 nM) should have reduced the binding of 125I-labeled IL-8 to high-affinity receptors and accelerated clearance from tissue. Thus, the binding of 125I-labeled IL-8 to high-affinity receptors cannot account for the enhanced retention of 125I-labeled IL-8 in lung tissue caused by the excess of unlabeled IL-8. In addition, the observation that excess unlabeled IL-8 caused a 2-fold increase in the retention of 125I-labeled IL-8 in neutropenic rabbits suggests that this increase did not depend on PMN migration. In contrast to the lungs, an excess of unlabeled IL-8 did not increase the retention of 125I-labeled IL-8 in the skin, suggesting that the mechanisms that control the retention of IL-8 in lung and skin differ (Fig. 5GoB).

We were surprised to find that adding an excess of cold IL-8 to the tracer 125I-labeled IL-8 actually increased the retention of radiolabeled IL-8 in tissue. A possible mechanism that could explain this observation is dimerization, a process whereby IL-8 binds to itself to form higher m.w. multimers (14, 44). Hoogewerf et al. (36) showed that dimerization of IL-8 on immobilized heparin and on endothelial cells in vitro resulted in a 2.5-fold increase in the amount of 125I-labeled IL-8 that bound. This increased binding of 125I-labeled IL-8 occurred despite a 1000-fold dilution of 125I-labeled IL-8 with unlabeled IL-8 and required two essential conditions, the dimerization of IL-8 and the presence of GAGs on endothelial cells. Our in vivo data are remarkably consistent with these in vitro findings, as we found that an excess of unlabeled IL-8 caused a 2-fold increase in the retention of 125I-labeled IL-8 in vivo. In contrast, an excess of unlabeled monomeric IL-8 did not significantly increase the amount of 125I-labeled IL-8 in lung tissue (Fig. 6Go). These data suggest that dimerization of IL-8 is required for the increased retention of 125I-labeled IL-8 in lung tissue. The role of GAGs in facilitating dimerization of IL-8 in lung tissue needs to be determined.

The effect of dimerization of IL-8 in lung tissue was more apparent in neutropenic animals than in normal animals. This suggests that PMN may have a modest effect on the clearance of IL-8 from lung tissue, in contrast to skin, where the effect of PMN on clearance of IL-8 is more dramatic and dimerization does not have a major role (Fig. 5Go). This supports the conclusion that the mechanisms that control the retention of IL-8 vary in different tissues depending on the specific characteristics of the local environment.

To our knowledge this is the first report that dimerization of a chemokine is significant in vivo, resulting in a 2-fold increase in the amount of IL-8 recovered in lungs but not skin at 4 h. Although Horcher et al. (45) reported that dimerization of IL-8 was not functionally important in the recruitment of PMN into the peritoneum of mice, we have recently found that dimerization is important in the recruitment of PMN into the lungs. In mice, the intratracheal instillation of rhIL-8 (125 nM) resulted in a significant increase in the pulmonary recruitment of PMN when compared with the monomeric form of rhIL-8 (125 nM) (16.4 ± 4 x 104 PMN vs 7.4 ± 0.8 x 104 PMN, respectively, at 4 h; p < 0.05 and n = 4; unpublished data). These findings in mice provide further support for the existence of tissue-specific mechanisms that control IL-8 function in vivo, as dimerization of IL-8 appears to be functionally important in the lungs but not in the peritoneum of mice. Further work will be required to determine the mechanisms whereby the dimerization of IL-8 increases the biological activity of IL-8 in lungs in vivo.

An additional question is whether 125I remains incorporated in 125I-labeled IL-8 in vivo. Initial attempts to isolate intact 125I-labeled IL-8 in blood and lungs using SDS-PAGE and autoradiography were unsuccessful because of the small amount of radioactivity used in the studies. Nevertheless, several lines of evidence suggest that most of the radioactivity reflects intact 125I-labeled IL-8. First, <0.2% of the instilled 125I was recovered in the thyroid, suggesting that the majority of the 125I is bound to IL-8 rather than free. Second, we immunoprecipitated radioactivity from RBCs using an anti-IL-8 Ab, suggesting that a majority of the 125I-labeled IL-8 in whole blood binds to the Duffy receptor on RBC (46). Third, the radioactivity recovered in blood and BAL fluid has a m.w. of >3000 based on studies in which plasma and BAL fluid were spun through 3000-m.w. cutoff filters. Finally, the t1/2 of radioactive vasoactive intestinal peptide (m.w. = 3450) following its instillation into the airspaces of the lungs was 19 min, suggesting that radiolabeled vasoactive intestinal peptide was rapidly degraded in lungs (47). In contrast, the tissue t1/2 of 125I-labeled IL-8 in the lungs is at least 4 h, suggesting that this molecule is not rapidly degraded (Fig. 5Go).

In conclusion, we have found that IL-8 is retained much longer in the lungs than skin, that PMN migration reduces retention of IL-8 in skin but not lungs, and that dimerization of IL-8 increases the retention of 125I-labeled IL-8 in lungs. These data suggest that mechanisms that control the retention of IL-8 are tissue specific. Understanding the mechanisms that control the retention of IL-8 in tissue is of fundamental importance for understanding the regulation of the biological activity of IL-8 in vivo.


   Acknowledgments
 
We thank Venus Wong, Charie Vathanaprida, Steve Mongovin, Tonja Hagen, Kristine Wynant, and Lena Strait for expert technical assistance.


   Footnotes
 
1 This work was supported in part by the Medical Research Service of the U.S. Department of Veterans Affairs, the American Heart Association of Washington (to C.W.F.), the Francis Families Foundation (to C.W.F.), and National Institutes of Health Grant AI29103. Back

2 Address correspondence and reprint requests to Dr. Charles W. Frevert, Division of Pulmonary/Critical Care Medicine, Department of Medicine, Seattle Department of Veterans Affairs Medical Center, 151L, 1660 South Columbian Way, Seattle, WA 98108. E-mail address: cfrevert{at}u.washington.edu Back

3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; BAL, bronchoalveolar lavage; GAG, glycosaminoglycan; rh, recombinant human; hpf, high-power field; N-methyl-L25, N-methyl-leucine 25. Back

Received for publication August 10, 2001. Accepted for publication January 25, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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