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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 464-472, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0084OC

Binding of Interleukin-8 to Heparan Sulfate and Chondroitin Sulfate in Lung Tissue

Charles W. Frevert, Michael G. Kinsella, Charie Vathanaprida, Richard B. Goodman, Denis G. Baskin, Amanda Proudfoot, Timothy N. C. Wells, Thomas N. Wight and Thomas R. Martin

Medical Research Service, VA Puget Sound Medical Center, Seattle; Division of Pulmonary and Critical and Care Medicine, Division of Metabolism Endocrinology and Nutrition, and Department of Medicine, University of Washington School of Medicine, Seattle; Department of Vascular Biology, Hope Heart Institute, Seattle, Washington; and Serono Pharmaceutical Research Institute, Geneva, Switzerland

Address correspondence to: Charles W. Frevert, D.V.M., Sc.D., Seattle VAMC Pulmonary Research Group, Seattle VA Medical Center, 151L, 1660 S. Columbian Way, Seattle, WA 98108. E-mail: cfrevert{at}u.washington.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interleukin (IL)-8, a member of the CXC chemokine family, is a potent neutrophil chemotactic factor. Mechanisms that regulate the activity of chemokines in tissue are not clear. The goal of this study was to determine whether IL-8–glycosaminoglycan interactions are responsible for the binding of IL-8 in lung tissue. Experiments were performed with a quantitative tissue-binding assay to measure the amount of 125I–IL-8 binding and an in situ tissue-binding assay to characterize the location of IL-8 binding in lung tissue. Confocal microscopy demonstrated IL-8 binding to specific anatomic locations such as cell surfaces and extracellular matrix that were enriched with heparan sulfate and chondroitin sulfate. Removal of heparan sulfate or chondroitin sulfate from lung tissue significantly decreased the binding of 125I–IL-8. Two forms of IL-8 with single amino acid mutations in the glycosaminoglycan-binding domain showed decreased binding. In addition, studies with normal and monomeric IL-8 showed that dimerization increased the binding of 125I–IL-8 in lung tissue. These findings suggest that IL-8–glycosaminoglycan interactions determine the location where IL-8 binds in lung tissue and provides a site for the dimerization of IL-8, which increases the local concentration of IL-8 in the lungs.

Abbreviations: interleukin, IL • dissociation constant, Kd • monocyte chemotactic protein, MCP • phosphate-buffered saline, PBS • polymorphonuclear neutrophils, PMN • recombinant human IL-8, rhIL-8


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chemokines, a group of structurally related chemotactic cytokines, are important in the activation and directed migration of leukocytes in tissue (1, 2). Interleukin (IL)-8, a member of the CXC chemokine family, is a potent neutrophil chemotactic factor and a critical component of early host defenses in the lungs. Whereas IL-8 plays an important role in pulmonary host defenses (3), animal models and clinical studies suggest that IL-8 also plays a role in the pathogenesis of acute lung injury (4–7). Therefore, identifying the mechanisms that control the duration of IL-8 biological activity in lung tissue are of fundamental importance for understanding normal pulmonary host defenses and pathological forms of inflammation such as the Acute Respiratory Distress Syndrome.

Although a considerable literature exists about the regulation of IL-8 production and the recognition of IL-8 by polymorphonuclear neutrophils (PMN), the mechanisms that regulate the duration of the biological activity of IL-8 in tissue are not well understood. IL-8 has three binding domains: a high-affinity binding domain, which mediates the binding to specific receptors on PMN; the glycosaminoglycan-binding domain; and the dimer interface, where IL-8 molcules bind to each other to form dimers and possibly tetramers (8–10). The high-affinity binding domain (dissociation constant [Kd] = 2–10 nM) is located in the N-terminus of IL-8, and is made up of the amino acids E4, L5, and R6, which are essential for the binding of IL-8 to CXCR-1 and CXCR-2 (11, 12). The interaction of IL-8 with these high-affinity receptors mediates neutrophil chemotaxis and activation (8).

In contrast to the high-affinity binding domain, there are two sites on IL-8 that mediate low-affinity binding, the glycosaminoglycan-binding domain (Kd = 0.4–2.6 µM) and the dimer interface (Kd = 4–18 µM) (13–15). The dimer interface is a region where IL-8 molcules bind to each other to form higher molecule weight multimers (9, 14–17). To determine the biological significance of dimerization, an obligate monomeric form of IL-8 has been synthesized, N-methyl-Leucine25 IL-8 (17). Studies using N-methyl-L25 IL-8 and wild-type IL-8 show that the two forms of IL-8 bind equally to high-affinity receptors and the glycosaminoglycan heparin (9, 17). The obligate monomeric form of IL-8 promotes neutrophil activation and chemotaxis in vitro and in vivo, leaving open the question as to the biological role of IL-8 dimerization (17, 18). In studies performed with N-methyl-L25 IL-8, Hoogewerf and coworkers (9) showed that dimerization is a mechanism that increases the local concentration of IL-8 on the surface of endothelial cells in vitro. Using the same monomeric form of IL-8, we showed that dimerization of IL-8 is the mechanism responsible for an unexpected increase in the amount of 125I–IL-8 binding to lung tissue in vivo (19).

The second low-affinity binding domain of IL-8 is the glycosaminoglycan-binding domain. IL-8 selectively binds to different glycosaminoglycan-families, with binding to heparin > heparan sulfate > dermatan sulfate > chondroitin sulfate (13). Binding to glycosaminoglycans is mediated by a group of basic amino acids located primarily in the C-terminal helix; however, residues in the proximal loop are also involved in this binding (10, 20). The interaction of the C-terminal {alpha}-helix of IL-8 with glycosaminoglycans enhances neutrophil migration in vitro (20), and the C-terminal helix of IL-8 is required for presentation of IL-8 to the luminal surface of endothelial cells in skin and for neutrophil emigration (21). Binding of IL-8 to glycosaminoglycans on endothelial cells facilitates the dimerization of IL-8 in vitro (9). The interaction of IL-8 with glycosaminoglycans has been proposed as a mechanism whereby tissue-bound or haptotactic gradients are formed to direct neutrophil migration through tissue (22–25).

There are four classes of glycosaminoglycans, including heparin/heparan sulfate, chondroitin sulfate/dermatan sulfate, keratan sulfate, and hyaluronan (26). All four classes of glycosaminoglycans are found in normal lungs, and except for hyaluronan all are present as side chains on the core proteins of complex macromolecules termed proteoglycans. The predominant glycosaminoglycan in normal lungs is heparan sulfate (40%) followed by chondroitin sulfate/dermatan sulfate (31%), hyaluronan (14%), and heparin (5%) (27). The sulfation pattern of glycosaminoglycans has important biological consequences, as sulfated disaccharides on heparan sulfate form the binding site for IL-8 (28). In the lungs, proteoglycans are found on cell surfaces (e.g., syndecan), in the extracellular matrix (e.g., decorin, perlecan and versican), and also in intracellular locations (e.g., serglycin) (29).

The goal of this study was to determine whether the interaction between IL-8 and glycosaminoglycans is responsible for the low-affinity binding of IL-8 in lung tissue. Removal of heparan sulfate or chondroitin sulfate from lung tissue significantly decreased the binding of 125I–IL-8, showing that IL-8 binds to these two glycosaminoglycans in lung tissue. Confocal microscopy demonstrated IL-8 binding to specific anatomic locations enriched with heparan sulfate and chondroitin sulfate. The use of mutant forms of IL-8 showed that the glycosaminoglycan-binding domain of IL-8 was responsible for this binding. In addition, dimerization increased the binding of 125I–IL-8 in lung tissue. In conclusion, the results suggest that the binding of IL-8 to glycosaminoglycans determines the location of IL-8 binding in lungs and provides a site for the dimerization of IL-8, which increases the amount of IL-8 binding in the lungs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and Antibodies
The 125I–IL-8 (Amersham, Piscataway, NY), recombinant human IL-8 (rhIL-8; Peprotech, Rocky Hill, NJ), R6A–IL-8, R68A–IL-8, K20A–IL-8, and N-methyl-L25 IL-8 (a kind gift of Dr. Ian Clark-Lewis, University of British Columbia, Vancouver, BC, Canada) were resuspended in sterile 0.9% NaCl with 0.1% pyrogen-free bovine serum albumin (Irvine Scientific, Santa Ana, CA). Low molecular weight heparin and chondroitin sulfate A were purchased from Sigma (St. Louis, MO). The heparinases I and II were purchased from Sigma, and the chondroitin ABC lyase was purchased from ICN (Aurora, OH). Polyclonal goat anti-human IL-8 was purchased from R&D Systems (Minneapolis, MN). The mouse monoclonal antibody, HepSS1, which recognizes heparan sulfate glycosaminoglycans, was purchased from Seikagaku (Tokyo, Japan) (30), and the mouse monoclonal antibody, CS-56, which recognizes chondroitin sulfate glycosaminoglycans, was purchased from Sigma (31). For bright field microscopy, the diaminobenzidine substrate was purchased from Sigma, and the 1% methyl green was purchased from Stephens Scientific (Kalamazoo, MI). For confocal microscopy, the TSA-Biotin kit was purchased from NEN (Boston, MA), the Strepavidin-Alexa 568 and the ToPro-3 were purchased from Molecular Probes (Eugene, OR).

Quantitative Tissue-Binding Studies
To measure the amount of IL-8 that binds in lung tissue, a quantitative tissue-binding assay that was previously described was modified (32). Briefly, lung tissue was embedded in O.C.T. compound (Miles, Inc., Elkhart, IN) and flash-frozen in liquid nitrogen. Tissue was then sectioned into 20-µm slices using a Jung Frigocut 2800E cryostat (Leica Microsystems, Heidelberg, Germany) and mounted on glass slides that were stored at –80°C. On the day of the experiment, slides were thawed at room temperature, then placed on a 35°C warming tray for 10 min. The tissue sections were incubated in blocking buffer (3% rabbit serum and 5% nonfat milk powder in phosphate-buffered saline [PBS] with protease inhibitors), rinsed briefly in PBS, and then incubated with 125I–IL-8 [1 x 10-10 M] in combination with serial dilutions of recombinant human IL-8 for 30 min at room temperature. To remove unbound 125I–IL-8, the sections were washed once in PBS, then once in cold H2O. Slides were air-dried and positioned on a Phosphor Imaging Screen (Bio-Rad, Hercules, CA) overnight. The amount of bound 125I–IL-8 was determined with a phospho imaging system and the Molecular Analyst program (Bio-Rad) on a Macintosh computer (Apple, Cupertino, CA).

Bacterial Lyase Treatment
To remove specific classes of glycosaminoglycans for the quantitative tissue-binding studies, cryostat sections of normal lung tissue were pretreated with bacterial lyases before incubation with soluble rhIL-8 (Peprotech) and 125I–IL-8 (Amersham, Piscataway, NY). Heparinase I and II (Sigma) were used at 20 U/ml in heparinase buffer (100 mM Tris-HCl, 5 mM calcium acetate, pH 7.04). Chondroitin ABC lyase was used at 1 U/ml in chondroitinase buffer (100 mM Tris buffer, 15 mM sodium acetate, pH 8.0). The tissues were incubated with the lyases for 4 h at 37°C, then washed with PBS and incubated with 125I–IL-8 [1 x 10-10 M] in combination with rhIL-8 [1 x 10-6] M for 30 min at room temperature. The bound 125I–IL-8 was detected as described above.

IN SITU TISSUE-BINDING STUDIES
Recombinant human IL-8 binding in paraffin-embedded tissue was visualized with an in situ tissue-binding assay that has been previously described (33, 34). To preserve morphologic details and allow for better visualization of the locations where IL-8 binds, fixed tissue was used in this assay. Preliminary studies showed that the binding of rhIL-8 to frozen and fixed tissue was similar. To prepare tissue for the in situ tissue-binding assay, normal rabbit lung tissue was removed en bloc and fixed with 4% paraformaldehyde via the trachea. The tissue was immersed in 4% paraformaldehyde overnight at room temperature, embedded in paraffin then cut into 4-µm-thick sections. On the day of the experiment, slide-mounted tissue sections were deparaffinized, rinsed twice with PBS for 5 min, and then digested for 10 min in 0.05% pronase to expose heparan sulfate epitopes. They were then rinsed twice with PBS for 5 min and blocked in PBS containing 3% normal rabbit serum (Vector, Burlingame, CA) and 5% nonfat milk protein for 60 min at room temperature. Next, recombinant human IL-8 or modified forms of IL-8 with single amino acid mutations (i.e., R6A–IL-8, R68A–IL-8, and K20A–IL-8; 1.0 µM) were incubated with the lung sections for 6 h at 4°C. Unbound IL-8 was washed from the tissue, and the bound IL-8 was visualized by immunohistochemistry using the Vector "Elite" ABC-HP kit (Burlingame, CA). Briefly, 5 µg/ml of a polyclonal goat anti-human IL-8 (R&D Systems) was incubated with the tissue sections for 4 h in a moist chamber at 4°C. Then the sections were rinsed twice with PBS and incubated with biotinylated rabbit anti-goat IgG antibody for 2 h at 4°C. The tissue sections were rinsed twice with PBS, incubated with 0.3% H2O2 in methanol for 30 min to block endogenous peroxidases, rinsed twice with PBS, and then incubated with ABC-HP in a moist chamber for 60 min at 21°C. After two rinses with PBS, they were incubated in a moist chamber with diaminobenzidine substrate (Sigma) for 1 – 15 min. in the dark at 21°C. The slides were counterstained with 1% methyl green for 5 min. (Stephens Scientific). Preliminary studies verified that the anti–rhIL-8 antibody recognized rhIL-8, R6A–IL-8, R68A–IL-8, and K20A–IL-8 equally in a direct enzyme-linked immunosorbent assay (data not shown). In several experiments, rhIL-8 was preincubated with a number of different concentrations of heparin (1 x 10-6 to 1 x 10-3 M) or chondroitin sulfate (1 x 10-3 M) before incubation with normal lung tissue.

To visualize the spatial distribution of rhIL-8 binding in lung tissue, the in situ tissue-binding assay was adapted for confocal microscopy. IL-8 tissue binding was performed as described above with minor modifications: the addition of 3% rabbit IgG to the blocking buffer and the use of reagents provided with the TSA-Biotin Kit (NEN). After the horseradish peroxidase incubation step, the tissue sections were rinsed in PBS, incubated with biotinyl tyramide for 8.5 min at room temperature, rinsed twice with PBS, and then incubated with Strepavidin-Alexa 568 (Molecular Probes) at 1:200 and To-Pro-3 (Molecular Probes) at 1 µM in PBS for 1 h in the dark. The slides were rinsed with PBS and mounted with Vectashield mounting media (Vector). The IL-8 in tissue was visualized using a Leica TCS-SP Confocal Microscope using an upright Leica DMR Microscope and Leica Confocal Software (Leica).

Immunohistochemistry for Heparan Sulfate and Chondroitin Sulfate
Immunohistochemistry for heparan sulfate and chondroitin sulfate was performed with paraffin-embedded tissue. On the day of the experiment, slide-mounted tissue sections were deparaffinized, rinsed twice with PBS for 5 min, and then digested for 10 min in 0.05% pronase (EM Science, Gibbstown, NJ) to expose heparan sulfate epitopes. In preliminary studies, digestion of lung tissue with pronase was required to visualize heparan sulfate. This confirms a previous report that the epitope for heparan sulfate is insensitive to pronase digestion (35). When performing immunohistochemistry for chondroitin sulfate, no tissue digestion was performed. Preliminary studies showed that the monoclonal antibody, CS-56, did not detect chondroitin sulfate in pronase digested lung tissue, confirming an earlier report that pronase removes the glycosaminoglycan chondroitin sulfated from tissue (31). The tissue sections were then incubated overnight with one of the following antibodies: (i) mouse monoclonal antibody, HepSS1 (IgM), which recognizes heparan sulfate glycosaminoglycans (30); or (ii) mouse monoclonal antibody, CS-56 (IgM), which recognizes chondroitin sulfate glycosaminoglycans (31). This was followed by incubation with a biotinylated anti-murine IgM antibody (Vector Laboratories) for 2 h at 4°C. To detect the location of the antibody: antigen complex, the tissue samples were processed as previously described for bright field and confocal microscopy.

Statistical Analysis
Comparisons between multiple groups were performed using one-way ANOVA. 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
 
The Role of Heparan Sulfate and Chondroitin Sulfate for the Binding of IL-8 in Lung Tissue
To test the hypothesis that glycosaminoglycans bind IL-8 in lung tissue, heparinase I/II was used to digest heparin and heparan sulfate, and chondroitinase ABC was used to digest chondroitin sulfate and dermatan sulfate in lung tissue sections. The quantitative tissue-binding assay was performed with a trace amount of 125I–IL-8 (1 x 10-10 M) in the presence of cold IL-8 (1 x 10-6 M), which is a concentration of IL-8 that is close to the Kd of IL-8 binding to glycosaminoglycans in vitro (13). Lung tissue treated with either heparinase or chondroitinase bound significantly less 125I–IL-8, indicating that glycosaminoglycans are required for the binding of IL-8 in lung tissue (Figure 1).



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  		Figure 1. Effect of removal of the glycosaminoglycans heparan sulfate and chondroitin sulfate on low-affinity binding of 125I–IL-8 in lung tissue. Lung tissue was treated with heparinase I/II (I/II) and Chondroitinase ABC (ABC) before incubation of tissue with a trace amount of 125I–IL-8 (1 x 10-10 M) in combination with an excess of unlabeled IL-8 (1 x 10-6 M). The binding of 125I–IL-8 in lung tissue was measured with a PhosphorImager screen and is reported as a percentage of the binding of 125I–IL-8 alone. The values are the mean ± SEM (n = 4, *p < 0.02).

 
Effect of Dimerization on the Amount of 125I–IL-8 Bound to Lung Tissue
To determine if dimerization increases the amount of 125I–IL-8 that binds to normal lung tissue ex vivo, competition-binding studies were performed using a trace amount of 125I–IL-8 (1 x 10-10 M) and increasing concentrations of either unlabeled rhIL-8 or unlabeled N-methyl-L25-IL-8 (1 x 10-8 M to 1 x 10-5 M). When increasing amounts of rhIL-8 were added to the trace amount of 125I–IL-8, the amount of 125I–IL-8 bound to lung tissue significantly increased (Figure 2). In contrast, when increasing amounts of the monomeric form of IL-8 were added, no increase in the binding of 125I–IL-8 was observed.



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  		Figure 2. Competition binding study showing the effect of increasing amounts of unlabeled rhIL-8 (closed circles) and of unlabeled monomeric form of IL-8 (open squares) (1 x 10-8 to 1 x 10-5 M) on the binding of a trace amount of 125I–IL-8 (1 x 10-10 M) in lung tissue in vitro. The binding of 125I–IL-8 in lung tissue was measured with a PhosphorImager screen and is reported as a percentage of the binding of 125I–IL-8 alone. Values are the mean ± SEM (n = 4, *p = 0.0005 and {phi}p = 0.0001) with one-factor ANOVA.

 
In Situ Tissue-Binding of IL-8 in Lung Tissue
To compare the binding of mutant forms of recombinant human IL-8 to lung tissue, all studies were performed with rhIL-8. Recombinant rabbit IL-8 produced in our laboratory (36) had the same binding characteristics as rhIL-8 (not shown). rhIL-8 bound diffusely to alveolar septa and alveolar macrophages in normal rabbit lung tissue (Figure 3A).



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  		Figure 3. Evaluation of IL-8 binding in lung tissue using the in situ binding assay. (A) Binding of rhIL-8 (brown) to lung tissue. (B, C) Binding of rhIL-8 combined with either heparin (1 x 10-3 M) or chondroitin sulfate (1 x 10-3 M) before incubation with lung tissue. (D) Binding of the positive control, R6A–IL-8, a mutant from of IL-8 that binds to glycosaminoglycans but not to high affinity receptors. (E, F) Binding of K20A– and R68A–IL-8 to lung tissue, two mutant forms of rhIL-8 that have decreased binding to glycosaminoglycans but normal binding to high-affinity receptors. Magnification: x400.

 
The Role of the Glycosaminoglycan-Binding Domain for the Binding of IL-8 in Lung Tissue
To determine whether the glycosaminoglycan-binding domain is required for the binding of IL-8 to lung tissue, increasing amounts of heparin (1 x 10-6 to 1 x 10-3 M) were combined with rhIL-8 before incubation with normal lung tissue. These studies showed that heparin at a concentration of 1 x 10-3 M (1,000 µg/ml) completely blocked the binding of rhIL-8 to lung tissue (Figures 3A and 3B). Chondroitin sulfate (1 x 10-3 M) also decreased the binding of IL-8 to lung tissue, but the inhibition was less than that seen with heparin (Figure 3C). Next, three forms of mutant IL-8 with different single amino acid mutations, R6A–IL-8, K20A–IL-8, and R68A–IL-8, were used in the in situ binding assay (Figures 3D, 3E, and 3F). The binding of rhIL-8 and R6A-IL-8 was similar, showing that R6A-IL-8 binds normally to lung tissue, although it does not bind to high-affinity receptors (10), (Figures 3A and 3D). In contrast, minimal binding was observed with R68A–IL-8 and K20A–IL-8, which have single amino acid mutations in the glycosaminoglycan-binding domain (Figures 3E and 3F). Thus, the glycosaminoglycan-binding domain of IL-8 is required for the binding of IL-8 to lung tissue ex vivo.

Location of IL-8 Binding in Lung Tissue
Using bright field microscopy, rhIL-8 appeared to bind diffusely to alveolar septa and alveolar macrophages in normal rabbit lung tissue (Figures 3A and 4A). In a large pulmonary vessel, rhIL-8 bound to endothelial cells and perivascular space, but not to the smooth muscle layer (Figure 4A). Adaptation of the in situ tissue-binding assay to confocal microscopy confirmed that rhIL-8 binds to distinct anatomic locations in lung tissue, including endothelial cells, perivascular space, alveolar septa, and alveolar macrophages (Figure 4B). At higher magnifications, rhIL-8 was identified on the surface of alveolar macrophages and airway epithelial cells (Figure 4C). No positive staining was observed in the negative control section (Figure 4D), which was an adjacent section incubated without rhIL-8, and subjected to the same detection steps as for sections incubated with rhIL-8.



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  		Figure 4. Comparison of bright field and confocal microscopy for localization of rhIL-8 binding in lung tissue. (A) Bright field image showing the binding of rhIL-8 (brown) to alveolar macrophages (arrow), endothelial cells (small arrowhead) and perivascular space (large arrowheads) around a pulmonary artery. (B, C, D) Distribution of rhIL-8 binding in lung tissue imaged with confocal microscopy. (B) rhIL-8 (red) binds to alveolar septa, alveolar macrophages (white arrow), endothelial cells (small arrowhead) and perivascular space (large arrowhead) but not the media of the vessel wall. (C) rhIL-8 (red) binds to the cell surface of airway epithelial cells (yellow arrow) and an alveolar macrophage (white arrow). (D) The negative control is a serial tissue section incubated with PBS instead of rhIL-8. Nuclei are stained with methylene green in the bright field images (A) and ToPro-3 (blue) in the confocal images (B, C, D). Tissue autofluorescence (green) was used to improve the image quality of the confocal images.

 
Immunolocalization of Heparan Sulfate, Chondroitin Sulfate, and IL-8 Binding in Lung Tissue
To determine the location of heparan sulfate and chondroitin sulfate in normal lung tissue, immunohistochemistry was performed with antibodies to these two classes of glycosaminoglycans (Figures 5A–5D). Bright field images of lung sections labeled with an anti–heparan sulfate antibody show diffuse positive staining in macrophages and alveolar septa (Figure 5A). Confocal microscopy more precisely localizes heparan sulfate to the cell surface of alveolar macrophages, and to specific regions of the alveolar septa (Figure 5C). Positive immunostaining for chondroitin sulfate (brown) showed a patchy distribution throughout the alveolar septum (Figure 5B), and this distribution was confirmed by confocal microscopy (Figure 5D).



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  		Figure 5. Bright field and confocal images illustrating the immunolocalization of heparan sulfate (A, C) and chondroitin sulfate (B, D) in tissue with comparison to the distribution of rhIL-8 binding in serial tissue sections using the in situ binding assay and confocal microscopy (E, F). (A, C) Immunolocalization of heparan sulfate in lung tissue using bright field microscopy and confocal microscopy respectively. (A) Diffuse staining of heparan sulfate (brown) was observed in the alveolar septa and alveolar macrophages (arrow) in images obtained with bright field microscopy. (C) Positive staining for heparin sulfate (red) was observed in specific locations on the alveolar septa and on the cell surface of alveolar macrophages (arrow) when tissue sections where imaged with the confocal microscope. (B, D) Immunolocalization of chondroitin sulfate with bright field microscopy showed a patchy distribution throughout the alveolar septum (B, brown), this distribution was confirmed by confocal microscopy (D, red). (E) Binding of rhIL-8 (red) using the in situ binding assay and lung tissue treated with pronase. (F) Binding of rhIL-8 (red) using the in situ binding assay and lung tissue not treated with pronase. Nuclei are stained with methylene green in the bright field images (A, B) and ToPro-3 (blue) in the confocal images (C–F). Tissue autofluorescence (green) was used to improve the image quality.

 
Next, we performed the in situ binding assay with rhIL-8 using tissue prepared as described for immunohistochemistry for heparan sulfate and chondroitin sulfate. When tissue was treated with pronase, which is required for the visualization of heparan sulfate, rhIL-8 binds to regions rich in heparan sulfate (Figures 5C and 5E). In contrast, using the method that allows for visualization of chondroitin sulfate, rhIL-8 binds to regions rich in chondroitin sulfate (Figures 5D and 5F). Thus, the presence of glycosaminoglycans heparan sulfate or chondroitin sulfate colocalizes to the location of IL-8 binding in lung tissue ex vivo.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The primary goal of this study was to determine whether IL-8–glycosaminoglycan interactions are responsible for the binding of IL-8 in intact lung tissue. First, the removal of heparan sulfate and chondroitin sulfate from lung tissue decreased the binding 125I–IL-8, suggesting that the interaction between IL-8 and glycosaminoglycans is required for the low-affinity binding of IL-8 in lung tissue. In studies performed to determine the spatial distribution of IL-8 binding in lung tissue, it was shown that IL-8 binds to specific anatomic locations, enriched with heparan sulfate and chondroitin sulfate. The use of mutant forms of IL-8 showed that the glycosaminoglycan-binding domain of IL-8 was responsible for this binding. In addition, the dimerization of IL-8 increased the amount of 125I–IL-8 that bound to lung tissue. These results suggest that interactions between IL-8 and glycosaminoglycans determine the location where IL-8 binds in lung tissue and provides a site for the dimerization of IL-8, which increases the amount of IL-8 that binds in lungs.

To determine the amount of IL-8 that binds in lung tissue, studies were performed using the quantitative tissue-binding assay. Removal of heparan sulfate and chondroitin sulfate from lung tissue decreased the binding of 125I–IL-8 (Figure 1), suggesting that glycosaminoglycans provide low-affinity binding sites for IL-8 in lung tissue. In studies where both heparan sulfate and chondroitin sulfate were removed from lung tissue, binding of 125I–IL-8 still occurred (data not shown). This is not surprising, as there are many negatively charged molecules in tissue (e.g., fibronectin and hyaluronic acid) that could bind to IL-8, albeit with lower affinity than glycosaminoglycans. Once the glycosaminoglycans are removed, IL-8 may bind to other negatively charged structures, making it difficult to completely inhibit IL-8 binding in lung tissue. Nevertheless, the data show that the interactions between IL-8 and heparan sulfate or chondroitin sulfate provide an important mechanism for the binding of IL-8 in lung tissue (Figure 1).

Consistent with our in vivo findings (19), dimerization of IL-8 increased the binding of 125I–IL-8 in lung tissue (Figure 2). Hoogewerf and colleagues found that glycosaminoglycans on endothelial cells were required for the dimerization of IL-8 in vitro (9). Thus, our finding that IL-8 binds to glycosaminoglycans in lung tissue suggests that this interaction provides a site for the dimerization of IL-8 to occur in lungs.

The increased binding of 125I–IL-8 in the presence of excess cold IL-8 differs from what normally occurs with receptor ligand interactions (Figure 2). One explanation for this is that glycosaminoglycans in lung tissue provide a large number of low-affinity binding sites. The parenchymal content of glycosaminoglycans in normal rabbit lungs is estimated to be 0.4% of the dry tissue weight, suggesting that the glycosaminoglycan concentration in lung tissue is {bsim} 4 mg/gm (37). The average molecular weight of glycosaminoglycan sidechains is in the range of 20–40,000 kD (26). This suggests that the concentration of glycosaminoglycan side chains in lung tissue would be 7.5 x 10-4 M. Although there are potentially multiple IL-8–binding sites on each glycosaminoglycan chain, the presence of one binding site for IL-8 on a glycosaminoglycan side chain results in an excess number of IL-8–binding sites in lung tissue. Dimerization of IL-8 increases the amount of IL-8 that binds to glycosaminoglycan side chains. Thus, there appears to be a vast excess of low-affinity binding sites that would not be saturated at the concentrations of IL-8 used for these studies.

To identify whether interactions between IL-8 and glycosaminoglycans determine the spatial relationship of IL-8 binding in lung tissue, an in situ tissue-binding assay was used. In initial studies, an excess of heparin or chondroitin sulfate were added to the suspension of rhIL-8 to determine whether these two glycosaminoglycans would reduce the interaction of the glycosaminoglycan-binding domain of IL-8 with heparan sulfate and chondroitin sulfate in lung tissue. The concentration of heparin used was determined by dose titration studies (data not shown) and chondroitin sulfate was used at equimolar concentrations to heparin. Addition of heparin and chondroitin sulfate to the solution of rhIL-8 decreased the staining of IL-8 in lung tissue (Figures 3B and 3C). The addition of heparin to the suspension of rhIL-8 appeared to block the binding of rhIL-8 in lung tissue to a greater extent than the addition of chondroitin sulfate, which is consistent with the work of Kuschert and coworkers showing that IL-8 binds to heparin > heparan sulfate > chondroitin sulfate (13). To specifically test the role of the glycosaminoglycan-binding domain in the binding of IL-8 in lung tissue, studies were performed using mutant forms of IL-8 with single amino acid mutations. The two forms of rhIL-8 with mutations in the glycosaminoglycan-binding domain (i.e., R68A– and K20A–IL-8) showed almost no binding in lung tissue, adding further support that the glycosaminoglycan-binding domain is required for the binding of IL-8 in lung tissue (Figure 3).

Confocal microscopy showed that IL-8 binds to specific anatomic locations in lung tissue and that this binding is localized to regions rich in heparan sulfate and chondroitin sulfate (Figures 4– 5). These findings suggest that the binding of IL-8 in lung tissue is determined by two factors, the glycosaminoglycan-binding domain on IL-8, and the presence of heparan sulfate and chondroitin sulfate in tissue. Heparan sulfate and chondroitin sulfate proteoglycans are found on the cell surfaces of alveolar macrophages, endothelial cells and epithelial cells, as well as in the extracellular matrix of the lungs (29), suggesting that the presence of these glycosaminoglycans determines the location where IL-8 binds in lungs (Figures 4 and 5). The binding of IL-8 to endothelial cell surfaces was first proposed by Rot and colleagues (23, 24) as a mechanism to establish haptotactic gradients in tissue. The spatial separation of the glycosaminoglycan-binding domain from the receptor binding domain suggests that IL-8 is able to bind heparan sulfate and chondroitin sulfate in tissue and still activate neutrophils (10).

The development of haptotactic chemokine gradients has been studied in vitro (38–40). In tissue it has been more difficult to show that haptotactic-gradients play a role in the directional-migration of neutrophils. The results of the present study strengthen the argument that in lungs, chemokine gradients are tissue-bound rather than soluble. We propose a hypothetical model whereby stable tissue-bound chemotactic gradients develop in the lungs through a specific interaction with glycosaminoglycan side chains of proteoglycans located on cell surfaces and in extracellular matrix. The interaction between IL-8 and glycosaminoglycans provides a large number of low-affinity binding sites for IL-8 in lung tissue. An important characteristic of the interaction between IL-8 and glycosaminoglycans is that the low-affinity binding allows IL-8 to develop stable tissue-bound chemokine gradients. Potential binding sites for IL-8 in lung tissue include proteoglycans such as syndecan 1–4 on the surface of epithelial and endothelial cells and perlecan and chondroitin sulfate proteoglycans in the extracellular matrix (29). Once IL-8 has bound to glycosaminoglycans, the dimerization or multimerization of IL-8 provides a second mechanism to increase the local concentration of IL-8 in lung tissue (19). A feature common to all chemokines is the ability to bind to glycosaminoglycans and form dimers, suggesting that this proposed model will not be limited to IL-8 (1, 41).

The development of tissue-bound gradients may not be the only way that IL-8-glycosaminoglycan interactions modify IL-8 function in vivo. The binding of IL-8 to glycosaminoglycans could also focus the inflammatory response to specific areas and prolong the retention of IL-8 in lung tissue. We have shown that IL-8 has a prolonged tissue half-life in the lungs with over 50% of the instilled 125I–IL-8 remaining at 4 h (19). Binding to glycosaminoglycans may also protect IL-8 from proteolytic cleavage, as basic fibroblast growth factor bound to heparan sulfate was protected from proteolytic degradation (42, 43).

The present study and the hypothetical model are based on normal lung tissue. Significant changes occur in the composition of glycosaminoglycans in inflamed lungs (44–49), and these may modify chemokine–glycosaminoglycan interactions. A consistent finding 24–48 h after the administration of lipopolysaccharide is an increase in the expression of chondroitin sulfate glycosaminoglycans in the lungs (44, 46). Increased expression of versican, a chondroitin sulfate proteoglycan, occurs in patients with adult respiratory distress syndrome, idiopathic pulmonary fibrosis, and bronchiolitis obliterans organizing pneumonia (47, 48). Versican has been shown to bind chemokines, suggesting that this proteoglycan may influence the inflammatory process in the lungs (50). Interestingly, versican binds to monocyte chemoattractant protein-1 (MCP-1) but does not bind to either IL-8 or GRO-{alpha} (50). This suggests that the increased expression of versican in inflamed lung tissue would favor the binding of the CC-chemokine, MCP-1, but not the CXC-chemokines, IL-8 or GRO-{alpha}. Therefore, changes in the expression of proteoglycans may influence the progression of inflammatory lung disease by favoring the retention of specific subsets of chemokines. In studies performed with human umbilical vein endothelial cells, the different strengths of the chemokine–glycosaminoglycan interactions in vitro were found to be RANTES > MCP-1 > IL-8 > macrophage inflammatory protein-1{alpha} (13). This suggests that the amount of IL-8 binding in lung tissue could be influenced by the presence of other chemokines that may compete for low-affinity tissue binding sites. Future studies will be required to determine whether changes in the composition of glycosaminoglycans or the presence of other chemokine in inflamed lungs influence the retention of IL-8 in lung tissue.

In conclusion, we show that IL-8 binds to specific anatomic locations in the lungs, and that this binding requires heparan sulfate and chondroitin sulfate in lung tissue and the glycosaminoglycan-binding domain on the IL-8 molecule. In addition, studies with normal and monomeric IL-8 showed that dimerization increased the binding of 125I–IL-8 in lung tissue. Thus, the binding of IL-8 to glycosaminoglycans determines the location where IL-8 binds in the lungs and provides a site for the dimerization of IL-8, which increases the local concentration of IL-8 in the lungs. These studies suggest that the low-affinity binding of IL-8 in lung tissue controls the location and the duration of IL-8 biological activity and may be of fundamental importance in the directed migration of neutrophils in lungs.


   Acknowledgments
 
This study was supported in part by grants from the Medical Research Service of the U.S. Department of Veterans' Affairs, American Heart Association of Washington (C.W.F.) and Francis Families Foundation (C.W.F.).

Received in original form June 11, 2002

Received in final form September 18, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rollins, B. J. 1997. Chemokines. Blood 90:909–928.[Free Full Text]
  2. Zlotnik, A., and O. Yoshie. 2000. Chemokines: a new classification system and their role in immunity. Immunity 12:121–127.[CrossRef][Medline]
  3. Abul, H., A. Abul, I. Khan, T. C. Mathew, A. Ayed, and E. Al-Athary. 2001. Levels of IL-8 and myeloperoxidase in the lungs of pneumonia patients. Mol. Cell Biochem. 217:107–112.[CrossRef][Medline]
  4. Miller, E. J., A. B. Cohen, S. Nagao, D. Griffith, R. J. Maunder, T. R. Martin, J. P. Weiner-Kronish, M. Sticherling, E. Christophers, and M. A. Matthay. 1992. Elevated levels of NAP-1/interleukin-8 are present in the airspaces of patients with the adult respiratory distress syndrome and are associated with increased mortality. Am. Rev. Respir. Dis. 146:427–432.[Medline]
  5. Baughman, R. P., K. L. Gunther, M. C. Rashkin, D. A. Keeton, and E. N. Pattishall. 1996. Changes in the inflammatory response of the lung during acute respiratory distress syndrome: prognostic indicators. Am. J. Respir. Crit. Care Med. 154:76–81.[Abstract]
  6. Miller, E. J., A. B. Cohen, and M. A. Matthay. 1996. Increased interleukin-8 concentrations in the pulmonary edema fluid of patients with acute respiratory distress syndrome from sepsis. Crit. Care Med. 24:1448–1454.[CrossRef][Medline]
  7. Goodman, R. B., R. M. Strieter, D. P. Martin, K. P. Steinberg, J. A. Milberg, R. J. Maunder, S. L. Kunkel, A. Walz, L. D. Hudson, and T. R. Martin. 1996. Inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 154:602–611.[Abstract]
  8. Lee, J., R. Horuk, G. C. Rice, G. L. Bennett, T. Camerato, and W. I. Wood. 1992. Characterization of two high affinity human interleukin-8 receptors. J. Biol. Chem. 267:16283–16287.[Abstract/Free Full Text]
  9. Hoogewerf, A. J., G. S. Kuschert, A. E. Proudfoot, F. Borlat, I. Clark-Lewis, C. A. Power, and T. N. Wells. 1997. Glycosaminoglycans mediate cell surface oligomerization of chemokines. Biochemistry 36:13570–13578.[CrossRef][Medline]
  10. Kuschert, G. S., A. J. Hoogewerf, A. E. Proudfoot, C. W. Chung, R. M. Cooke, R. E. Hubbard, T. N. Wells, and P. N. Sanderson. 1998. Identification of a glycosaminoglycan binding surface on human interleukin-8. Biochemistry 37:11193–11201.[CrossRef][Medline]
  11. Hebert, C. A., R. V. Vitangcol, and J. B. Baker. 1991. Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding. J. Biol. Chem. 266:18989–18994.[Abstract/Free Full Text]
  12. Clark-Lewis, I., C. Schumacher, M. Baggiolini, and B. Moser. 1991. Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs: critical role of NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis, exocytosis, and receptor binding activities. J. Biol. Chem. 266:23128–23134.[Abstract/Free Full Text]
  13. Kuschert, G. S., F. Coulin, C. A. Power, A. E. Proudfoot, R. E. Hubbard, A. J. Hoogewerf, and T. N. Wells. 1999. Glycosaminoglycans interact selectively with chemokines and modulate receptor binding and cellular responses. Biochemistry 38:12959–12968.[CrossRef][Medline]
  14. Burrows, S. D., M. L. Doyle, K. P. Murphy, S. G. Franklin, J. R. White, I. Brooks, D. E. McNulty, M. O. Scott, J. R. Knutson, D. Porter, P. R. Young, and P. Hensley. 1994. Determination of the monomer-dimer equilibrium of interleukin-8 reveals it is a monomer at physiological concentrations. Biochemistry 33:12741–12745.[CrossRef][Medline]
  15. Lowman, H. B., W. J. Fairbrother, P. H. Slagle, R. Kabakoff, J. Liu, S. Shire, and C. A. Hebert. 1997. Monomeric variants of IL-8: effects of side chain substitutions and solution conditions upon dimer formation. Protein Sci. 6:598–608.[Abstract]
  16. Paolini, J. F., D. Willard, T. Consler, M. Luther, and M. S. Krangel. 1994. The chemokines IL-8, monocyte chemoattractant protein-1, and I-309 are monomers at physiologically relevant concentrations. J. Immunol. 153:2704–2717.[Abstract]
  17. Rajarathnam, K., B. D. Sykes, C. M. Kay, B. Dewald, T. Geiser, M. Baggiolini, and I. Clark-Lewis. 1994. Neutrophil activation by monomeric interleukin-8. Science 264:90–92.[Abstract/Free Full Text]
  18. Horcher, M., A. Rot, H. Aschauer, and J. Besemer. 1998. IL-8 derivatives with a reduced potential to form homodimers are fully active in vitro and in vivo. Cytokine 10:1–12.[CrossRef][Medline]
  19. Frevert, C. W., R. B. Goodman, M. G. Kinsella, O. Kajikawa, K. Ballman, I. Clark-Lewis, A. E. Proudfoot, T. N. Wells, and T. R. Martin. 2002. Tissue-specific mechanisms control the retention of IL-8 in lungs and skin. J. Immunol. 168:3550–3556.[Abstract/Free Full Text]
  20. Webb, L. M., M. U. Ehrengruber, L. I. Clark, M. Baggiolini, and A. Rot. 1993. Binding to heparan sulfate or heparin enhances neutrophil responses to interleukin 8. Proc. Natl. Acad. Sci. USA 90:7158–7162.[Abstract/Free Full Text]
  21. Middleton, J., S. Neil, J. Wintle, L.-I. Clark, H. Moore, C. Lam, M. Auer, E. Hub, and A. Rot. 1997. Transcytosis and surface presentation of IL-8 by venular endothelial cells. Cell 91:385–395.[CrossRef][Medline]
  22. Rot, A. 1992. Binding of neutrophil attractant/activation protein-1 (interleukin 8) to resident dermal cells. Cytokine 4:347–352.[CrossRef][Medline]
  23. Rot, A. 1992. Endothelial cell binding of NAP-1/IL-8: role in neutrophil emigration. Immunol. Today 13:291–294.[CrossRef][Medline]
  24. Rot, A. 1993. Neutrophil attractant/activation protein-1 (interleukin-8) induces in vitro neutrophil migration by haptotactic mechanism. Eur. J. Immunol. 23:303–306.[Medline]
  25. Rot, A., E. Hub, J. Middleton, F. Pons, C. Rabeck, K. Thierer, J. Wintle, B. Wolff, M. Zsak, and P. Cukor. 1996. Some aspects of IL-8 pathophysiology III: chemokine interaction with endothelial cells. J. Leukoc. Biol. 59:39–44.[Abstract]
  26. Wight, T. N., D. K. Heinegard, and V. C. Hascall. 1991. Proteoglycans: structure and function. In E. Hay, editor. Cell Biology of the Extracellular Matrix, 2nd ed. Plenum Press, New York. 45–77.
  27. Sampson, P. M., R. B. Boyd, G. G. Pietra, and A. P. Fishman. 1984. Glycosaminoglycan biosynthesis in the isolated perfused rat lung. J. Appl. Physiol. 57:1648–1654.[Abstract/Free Full Text]
  28. Spillmann, D., D. Witt, and U. Lindahl. 1998. Defining the interleukin-8-binding domain of heparan sulfate. J. Biol. Chem. 273:15487–15493.[Abstract/Free Full Text]
  29. Roberts, C. R., T. N. Wight, and V. C. Hascall. 1997. Proteoglycans. In R. Crystal, J. West, E. Weibel, and P. Barnes, editors. The Lung: Scientific Foundations, 2nd ed. Lippinocott-Raven Press, Philadelphia. 757–767.
  30. Kure, S., and O. Yoshie. 1986. A syngeneic monoclonal antibody to murine Meth-A sarcoma (HepSS-1) recognizes heparan sulfate glycosaminoglycan (HS-GAG): cell density and transformation dependent alteration in cell surface HS-GAG defined by HepSS-1. J. Immunol. 137:3900–3908.[Abstract]
  31. Avnur, Z., and B. Geiger. 1984. Immunocytochemical localization of native chondroitin-sulfate in tissues and cultured cells using specific monoclonal antibody. Cell 38:811–822.[CrossRef][Medline]
  32. Baskin, D. G. 1993. Quantitative analysis of radioligand binding autoradiographs. In J. Wharton and J. Polak, editors. Receptor Autoradiography: Principles and Practice. Oxford University Press, New York. 107–130.
  33. Snow, A. D., R. Bramson, H. Mar, T. N. Wight, and R. Kisilevsky. 1991. A temporal and ultrastructural relationship between heparan sulfate proteoglycans and AA amyloid in experimental amyloidosis. J. Histochem. Cytochem. 39:1321–1330.[Abstract]
  34. Allen, B. L., M. S. Filla, and A. C. Rapraeger. 2001. Role of heparan sulfate as a tissue-specific regulator of FGF-4 and FGF receptor recognition. J. Cell Biol. 155:845–858.[Abstract/Free Full Text]
  35. van Kuppevelt, T. H., F. P. Cremers, J. G. Domen, and C. M. Kuyper. 1984. Staining of proteoglycans in mouse lung alveoli: II. Characterization of the Cuprolinic blue-positive, anionic sites. Histochem. J. 16:671–686.[CrossRef][Medline]
  36. Kajikawa, O., R. B. Goodman, M. C. Johnson, K. Konishi, and T. R. Martin. 1996. Sensitive and specific immunoassays to detect rabbit IL-8 and MCP-1 cytokines that mediate leukocyte recruitment to the lungs. J. Immunol. Meth. 197:19–29.[CrossRef][Medline]
  37. Horwitz, A. L., and R. C. Crystal. 1975. Content and synthesis of glycosaminoglycans in the developing lung. J. Clin. Invest. 56:1312–1318.
  38. Tanaka, Y., D. Adams, S. Hubscher, H. Hirano, U. Siebenlist, and S. Shaw. 1993. T-cell adhesion induced by proteoglycan-immobilized cytokine MIP-1ß. Nature 361:79–82.[CrossRef][Medline]
  39. McCormick, B. A., P. M. Hofman, J. Kim, D. K. Carnes, S. I. Miller, and J. L. Madara. 1995. Surface attachment of Salmonella typhimurium to intestinal epithelia imprints the subepithelial matrix with gradients chemotactic for neutrophils. J. Cell Biol. 131:1599–1608.[Abstract/Free Full Text]
  40. Netelenbos, T., S. Zuijderduijn, J. Van Den Born, F. L. Kessler, S. Zweegman, P. C. Huijgens, and A. M. Drager. 2002. Proteoglycans guide SDF-1-induced migration of hematopoietic progenitor cells. J. Leukoc. Biol. 72:353–362.[Abstract/Free Full Text]
  41. Clark-Lewis, I., K. S. Kim, K. Rajarathnam, J. H. Gong, B. Dewald, B. Moser, M. Baggiolini, and B. D. Sykes. 1995. Structure-activity relationships of chemokines. J. Leukoc. Biol. 57:703–711.[Abstract]
  42. Gospodarowicz, D., and J. Cheng. 1986. Heparin protects basic and acidic FGF from inactivation. J. Cell. Physiol. 128:475–484.[CrossRef][Medline]
  43. Saksela, O., D. Moscatelli, A. Sommer, and D. B. Rifkin. 1988. Endothelial cell-derive heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation. J. Cell Biol. 107:743–751.[Abstract/Free Full Text]
  44. Blackwood, R. A., J. O. Cantor, J. Moret, I. Mandl, and G. M. Turino. 1983. Glycosaminoglycan synthesis in endotoxin-induced lung injury. Proc. Soc. Exp. Biol. Med. 174:343–349.[Abstract]
  45. Karlinsky, J. B., and R. H. Goldstein. 1989. Regulation of sulfated glycosaminoglycan production by prostaglandin E2 in cultured lung fibroblasts. J. Lab. Clin. Med. 114:176–184.[Medline]
  46. Karlinsky, J. B., P. J. Bucay, D. E. Ciccolella, and M. P. Crowley. 1991. Effects of intratracheal endotoxin administration on hamster lung glycosaminoglycans. Am. J. Physiol. 261:L148–L155.[Abstract/Free Full Text]
  47. Bensadoun, E. S., A. K. Burke, J. C. Hogg, and C. R. Roberts. 1996. Proteoglycan deposition in pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 154:1819–1828.[Abstract]
  48. Bensadoun, E. S., A. K. Burke, J. C. Hogg, and C. R. Roberts. 1997. Proteoglycans in granulomatous lung diseases. Eur. Respir. J. 10:2731–2737.[Abstract]
  49. Venkatesan, N., T. Ebihara, P. J. Roughley, and M. S. Ludwig. 2000. Alterations in large and small proteoglycans in bleomycin-induced pulmonary fibrosis in rats. Am. J. Respir. Crit. Care Med. 161:2066–2073.[Abstract/Free Full Text]
  50. Hirose, J., H. Kawashima, O. Yoshie, K. Tashiro, and M. Miyasaka. 2001. Versican interacts with chemokines and modulates cellular responses. J. Biol. Chem. 276:5228–5234.[Abstract/Free Full Text]



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