Endothelial Activation and Increased Heparan Sulfate Expression in Cystic Fibrosis
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美国呼吸和危急护理医学 2005年第10期
Respiratory Cell and Molecular Biology Division, Southampton University School of Medicine, Southampton, United Kingdom
Department of Allergy, Immunology Respiratory Medicine, Alfred Hospital, Prahran, Victoria, Australia
ABSTRACT
Rationale: Pulmonary disease in cystic fibrosis (CF) is characterized by an exaggerated interleukin (IL)-8eCdriven, neutrophilic, inflammatory response to infection. Binding of IL-8 to heparan sulfate (HS)eCcontaining proteoglycans (HSPG) facilitates binding of the chemokine to its specific receptor, stabilizes and prolongs IL-8 activity, and protects it from proteolysis. We hypothesized that increased expression of HSPG contributes to the sustained inflammatory response in CF bronchial tissue.
Objectives: Our objectives were to analyze the distribution and abundance of IL-8 and HS, in intact and cleaved forms, in bronchial tissue from adult patients with CF or chronic obstructive pulmonary disease (COPD) and a control group without inflammatory airway disease.
Methods: Immunostaining and quantitative image analysis were applied to ethanol-fixed and paraffin-embedded tissue obtained at transplant in patients with CF or COPD, or postmortem in the control group.
Measurements and Main Results: Quantitative immunohistochemical analysis demonstrated significant disease-related differences. Intact HS was significantly more abundant in epithelial and endothelial basement membranes in CF than in COPD or the control group. Conversely, cleaved HS was significantly more abundant in COPD than the other groups. More IL-8eCpositive blood vessels were observed in CF and COPD compared with the control group, whereas more extensive IL-8 expression in the epithelium was observed in CF compared with COPD.
Conclusions: Sustained neutrophil recruitment in the CF airway may therefore be related not only to increased IL-8 expression but also to the increased stability and prolonged activity and retention of IL-8 when it is bound to HSPG in bronchial tissue.
Key Words: chemokine inflammation neutrophil
Pulmonary disease in patients with cystic fibrosis (CF) is dominated by an exaggerated interleukin (IL)-8eCdriven neutrophilic inflammatory response to infection of the airways (1, 2). A deficiency in IL-10 may contribute to the higher levels of IL-8 expressed by pulmonary macrophages and bronchial epithelial cells in patients with CF compared with healthy control subjects (3, 4). Endotoxin is a potent stimulus for macrophage cytokine production (2), whereas neutrophil elastase (5) and adherence of Pseudomonas aeruginosa (6) are stimuli for IL-8 expression by CF bronchial epithelial cells. For circulating neutrophils to detect this chemoattractant signal, it appears that IL-8 must establish a concentration gradient by diffusing from the epithelium, across the tissue matrix to the endothelial barrier. Supporting this notion, a recent study has demonstrated that radiolabeled IL-8 instilled into the airways moved slowly from the airspaces toward the vascular compartment, partitioning between the bronchoalveolar lavage fluid, tissue, and blood (7).
Neutrophil migration across the endothelium occurs in a multistep process that is mediated via the interaction of inflammatory cells with endothelial cell adhesion molecules and immobilized chemoattractants (8). It was originally proposed by Rot (9, 10) that neutrophils respond to a gradient of IL-8 bound to endothelial cell surfaces. In vitro studies have indicated that glycosaminoglycan side chains of proteoglycans act as IL-8 binding sites on human endothelial cells (11). Specifically, the heparan sulfate (HS) side chains associated with syndecan-1 bind IL-8 and are required for successful transendothelial migration of normal human neutrophils (12). Much less is known regarding the distribution of IL-8 in whole tissue. In rabbit lungs, IL-8 is bound to both HS and chondroitin sulfate, strengthening the argument that chemokine gradients are tissue-bound and not soluble (13). Binding to HS induces structural stabilization of IL-8, prolonging its biological activity (14), increases the local concentration of chemokine, and facilitates binding to its receptor (11, 15). Binding to the glycosaminoglycans may also protect IL-8 from elastase-mediated degradation (16) and prolong its half-life in the inflamed lung (7).
Higher levels of soluble IL-8 in airway samples, sputum, or bronchoalveolar lavage fluid than those in the circulation suggest that the recruitment of neutrophils into the airways of patients with CF is directed by concentration gradients of IL-8 (17). We now hypothesize that increased expression of both IL-8 and HS-containing proteoglycans (HSPG) may contribute to sustained neutrophil recruitment in CF. We used an immunohistochemical approach, with quantitative image analysis, to compare the distribution and abundance of IL-8, HS, and, because HS is susceptible to cleavage by activated neutrophils (18), cleaved forms of HS within bronchial tissue of patients with CF, patients with chronic obstructive pulmonary disease (COPD), which is also characterized by IL-8eCmediated neutrophilic inflammation (19), and control subjects. Some of the results of these studies have been previously reported in the form of an abstract (20).
METHODS
Tissue Samples
Airway tissue samples were obtained from patients with CF (n = 8; four men; age, 23.4 ± 2.8 years) and COPD (n = 8; five men; age, 49.5 ± 4.1 years) at lung transplantation and from patients without CF or COPD (n = 7; four men; age, 51.4 ± 7.1 years) at postmortem. Two of the control subjects had primary pulmonary hypertension; the remainder were normal at postmortem.
Materials
Monoclonal antibodies detecting intact (F58-10E4) and cleaved (F69-3G10) forms of HS were obtained from AMS Biotechnology, Abingdon, United Kingdom. Antibody F58-10E4 reacts with epitopes containing N-sulfated glucosamine residues on intact HS side chains of proteoglycans (21). We previously demonstrated that the binding of IL-8 to HS does not interfere with recognition of HS by this antibody (12). Antibody F69-3G10 reacts with epitopes exposed by heparitinase digestion of HSPG to reveal residual HS stubs attached to the core proteins (21). Mouse monoclonal antihuman IL-8 antibody was obtained from Bender Medsystems (Vienna, Austria), and biotinylated antimouse Fab fragments from Dako (Ely, UK). Streptavidin-biotin blocking kit was obtained from Vector Laboratories (Peterborough, UK). Streptavidin biotineChorseradish peroxidase was obtained from Dako. Aminoethyl carbazole peroxidase substrate was obtained from Menarini Diagnostics (Wokingham, UK). Crystal mount was obtained from Biomedia (Poole, UK), and routine chemicals were obtained from Merck (Lutterworth, UK).
Tissue Preparation and Immunohistochemistry
Tissue was fixed in ethanol and processed into paraffin blocks. Sections of 4 e were cut and stained immunohistochemically using the streptavidin biotineCperoxidase technique and the monoclonal antibodies described above, at the following dilutions: 1 e/ml F58-10E4, 3 e/ml F69-3G10, and 3 e/ml antieCIL-8. Isotype controls were used to demonstrate the specificity of staining.
Image Analysis
The percentage of epithelial expression of cleaved HSPG and IL-8 was measured with the assistance of computerized image analysis based on red/blue/green color composition as previously described (22). The number of blood vessels immunoreactive for IL-8 was determined as a percentage of total vessels number, given by staining of a sequential section for EN4, a marker for endothelium. The intensity of staining for IL-8 in the vascular basement membrane and intact HSPG in both the epithelial and vascular basement membranes was assessed on a gray level scale of 0 to 255, using computerized image analysis.
Statistical Analysis
Results were subject to statistical analysis using the Mann-Whitney test and Spearman rank correlation analysis using SPSS 11 software (SPSS, Woking, UK).
RESULTS
Intact HS was detected in association with the epithelial and endothelial basement membranes in all subject groups (Figures 1A, 1D, and 1G). The relative intensity of HS staining on epithelial basement membranes was significantly higher in CF than in either normal (p = 0.014) or COPD (p = 0.002) tissue samples (Figure 2). Intact HS was also more abundant on the endothelial basement membranes in CF tissue compared with normal (p = 0.06) and COPD (p = 0.002) tissue (Figure 3). The abundance of intact HS on endothelial and epithelial basement membranes was highly significantly correlated in the COPD group (r = 0.862, p = 0.006, n = 8), but not in the other groups.
The distribution of the cleaved and intact forms of HS differed. In all tissues, the cleaved form of HS was evident not only in the basement membranes but also within endothelial and epithelial cells (Figures 1B, 1E, and 1H). In CF tissues, it was clear that inflammatory cells were also strongly positive for cleaved HS. On high-power magnification, these cells had the morphologic appearance of polymorphonuclear neutrophils. Analysis of the epithelial staining for cleaved HS showed that this was most abundant in COPD (Figure 4), and significantly (p = 0.013) higher than in control samples.
In all groups, IL-8 was distributed throughout the bronchial tissue, and was most abundant in the epithelium and endothelium, both cell-associated, within the cells and on the cell surface, and in the basement membranes (Figures 1C, 1F, and 1I). Inflammatory cells, including neutrophils, were also IL-8eCpositive in CF tissues (Figure 1I). Assessing the area of epithelium staining positively for IL-8 indicates the extent of epithelial activation. Epithelial IL-8 staining was evident even in the non-CF/non-COPD tissue samples, and this tended to be higher in the CF group (Figure 5). Epithelial staining was significantly more extensive in the CF than the COPD group (Figure 5). The most significant differences in IL-8 staining were in the greater number of IL-8eCpositive blood vessels that were counted in CF and COPD tissue (Figure 6). The intensity of IL-8 staining on the blood vessels was significantly higher in CF than the control group, but not different than the COPD group (Figure 7).
DISCUSSION
IL-8 is an important driver of neutrophilic inflammation, which we have previously shown to correlate with disease severity in CF (17). To our knowledge, there have been no previous reports of the distribution of HSPG and IL-8 in bronchial tissue from patients with CF or COPD. In summary, quantitative immunohistochemical analysis of IL-8 and HS, identified as intact and cleaved forms, has demonstrated significant disease-related differences. Intact HS was significantly more abundant in epithelial and endothelial basement membranes in CF than COPD or the control group. Conversely, cleaved HS was significantly more abundant in COPD than the other groups. An increased number of IL-8eCpositive blood vessels was observed in CF and COPD compared with the control group. Epithelial IL-8eCpositive staining was more extensive in CF compared with COPD, but neither was significantly greater than the control group.
IL-8eCdependent neutrophilic inflammation is characteristic of both CF (1) and of COPD (19), where it is particularly associated with infective exacerbation. However, neutrophil numbers in bronchoalveolar lavage fluid of young adult patients with CF are estimated to be at least two orders of magnitude higher than in patients with chronic bronchitis, even during an infective exacerbation of symptoms (23, 24). The more extensive expression of IL-8 at epithelial sites in CF compared with COPD most likely reflects the greater magnitude of the inflammatory response in CF compared with COPD. However, the extent of epithelial IL-8 expression was not significantly greater in either patient group compared with the control group. Previous reports of increased expression of IL-8 by airway epithelial cells in CF appear to depend on the cell line and model used, with primary cells producing more IL-8 in response to P. aeruginosa, but not IFN-, tumor necrosis factor , IL-1, or Haemophilus influenza (25). Other studies have indicated that the presence of mutated CFTR had no effect, directly or indirectly through IL-8 expression, on transepithelial migration of neutrophils (26). Conversely, the absence of IL-10, an inhibitor of cytokine synthesis, is believed to contribute to the increased response in CF (3, 4), and a prolonged inflammatory response to acute Pseudomonas challenge has been shown in IL-10 knockout mice (27). More recently, a deficiency in the collectins, surfactant protein-A, and surfactant protein-D, normally expressed by epithelial cells lining the respiratory tract, was proposed to increase the inflammatory response in CF (28).
The endothelium is the primary portal for inflammatory cell recruitment. Neutrophils in the circulation emigrate into bronchial tissue in response to signals from adhesion molecules and chemoattractants presented by the lumenal surface of postcapillary venular endothelial cells (8). We demonstrated an increased number of IL-8eCpositive blood vessels in the CF and COPD groups compared with the control group. However, in addition to increased expression, factors that increase the bioactivity, stability, and half-life of IL-8 at this site in bronchial tissue may also be involved. HS side chains on HSPG were shown to bind and induce dimerization of IL-8 on endothelial cells in culture, increasing the local concentration of the chemokine and influencing its interaction with its specific receptor (11, 15). Recent animal studies have indicated that a tissue-specific mechanism for dimerization of IL-8 in tissue is responsible for increased retention of IL-8 in the lungs compared with the skin and is important for the recruitment of neutrophils into the lungs in vivo (7). Previous studies suggest that this specificity may be determined by a unique HS sequence that promotes binding and dimerization of IL-8 (29). However, the distribution of HSPG in relation to IL-8 distribution in bronchial tissue in CF and COPD has not previously been investigated.
We previously demonstrated IL-8 binding to endothelial HS (12); therefore, their colocalization at these sites implies that IL-8 is bound to HSPG. However, this remains an assumption, and other potential IL-8 binding sites on the endothelium have been identified. Specific, high-affinity IL-8 receptors are expressed on endothelial cells (30), and it is also possible that IL-8 is bound to the Duffy antigen on endothelial cells of postcapillary venules (31). The functions of the high-affinity IL-8 receptor on endothelial cells remain an open question: it may be involved in autocrine signaling and/or clearance of IL-8 by internalization after the inflammatory response. In situ binding assays indicated, however, that specific receptors did not appear to contribute to IL-8 binding on endothelial cells (32). The Duffy antigen binds tissue-derived IL-8 and directs transcytosis of the chemokine to the lumenal surface of the endothelium (31). However, the role of the Duffy antigen in neutrophil migration is controversial, with in vivo studies in mice indicating both an anti- and a proinflammatory role, but that this may be redundant (33, 34). It was also proposed that the Duffy antigen may have an antiinflammatory role and HS a proinflammatory role (31). Because IL-8 bound to Duffy antigen on red blood cells does not activate neutrophils, other molecules, such as glycosaminoglycans, may be more important in chemokine presentation. However, it has yet to be established whether chemokines bound to HS can bind to specific receptors or whether they must first dissociate from HS to interact with their signaling receptors.
Our finding that the HS side chains of HSPG are increased in bronchial tissue in CF, but not in COPD, is novel. Increased expression of chondroitin sulfateeCcontaining proteoglycans (35) and increased sulfation of glycosaminoglycans (36) in the liver and pancreas, but not lung or nasal tissue (36), in CF has previously been reported. In sputum samples, chondroitin sulfate appeared to be associated with severe tracheobronchial infection in CF (37), and chondroitin sulfate was the most abundant proteoglycan released by neutrophil elastase and cathepsin GeCstimulated airway gland serous cells in culture (38). The demonstration of increased expression of HSPG in the inflamed airways of patients with CF, compared with a group of patients with COPD and those with noninflamed airways, may have important consequences for the progression of CF lung disease. The increase in HSPG at the bronchial epithelium may promote increased adherence of P. aeruginosa (39), whereas an increase in HS expression on endothelial structures is suggested to increase the local concentration of IL-8 and is further evidence for endothelial activation in CF (40). Of the HSPG expressed by the endothelium, only 1 to 5% is estimated to have anticoagulant functions (41); the remainder represents an array of specific HS structures, bound to perlecan, syndecan, and glypican core proteins, for which the function and regulation of expression is poorly understood. However, the 10E4 antibody we used in our study is believed to trace the mass of HS expressed on individual proteoglycans and at individual sites (21).
There is no evidence to suggest that the observed increase in HS expression is related to defective CFTR function. However, it may represent a secondary phenomenon, reflecting the proinflammatory cytokine profile of the inflamed CF airway. Although the factors that enhance expression of HS in the airways are not well described, IL-1 and IL-8 stimulate renal epithelial cell HS expression (42, 43), and IFN- and tumor necrosis factor enhance the expression of 10E4-positive HS epitopes on endothelial cells (44). HS is the most common cell surface glycosaminoglycan, and the syndecan family of proteoglycans is the major source of HS on all cell types (45). Therefore, it may be relevant that antimicrobial peptides induce endothelial syndecans (46) and hypoxia induces epithelial expression of both proteoglycans and IL-8 (47). Because there was no difference between noninflammatory control and COPD samples, it is possible that the inflammatory cytokine profile in COPD does not support increased HS expression, or that there is increased catabolism of proteoglycans, which was indicated by the increased staining for cleaved HS chains, or that the significant difference in the ages of the patients with CF compared with the other groups was a factor, because the abundance of lung glycosaminoglycans decreases with age (48). However, inflammation is associated with both increased synthesis and shedding of HSPG (49), and the observed increased abundance of HS in CF obviously reflects the balance of these processes.
Little is known about how IL-8 functions in tissue to direct neutrophil recruitment from the circulation, but increased expression of both IL-8 and HS colocalized on endothelial cells is likely to enhance the inflammatory response to a given stimulus. IL-8 is only weakly bound to HS, with a dissociation constant of 0.4 to 2.6 e, whereas binding to the high-affinity receptor has a dissociation constant of 2 to 10 nM. However, the glycosaminoglycans provide a vast excess of low-affinity binding sites in lung tissue, and these determine the location where IL-8 binds in the lungs (13). The HS binding sites for IL-8 may be associated with either syndecan-1 (12) or syndecan-2 (50) on the endothelium. Our findings may offer an explanation for the observation (52) that, at high levels of infection, IL-8 production reaches a maximum that is similar in subjects with CF and control subjects, whereas neutrophil recruitment due to factors other than higher levels of endotoxin (2) continues to be enhanced in CF. Increased expression of HS at the endothelium in CF, compared with COPD, may indicate a unique feature of CF that contributes to increased stability, activation, and presentation of IL-8 to target cells, and the chronicity of the neutrophilic inflammatory response in CF.
Although HS-bound gradients of chemokines enriched in the endothelial basement membrane are likely to attract cells across the endothelium and into the tissue, the gradient is effectively the wrong way round on the epithelium, with chemokines enriched in the basement membrane retaining cells in the tissue and inhibiting their advancement into the airway. At the endothelial level, immobilized chemokines stimulate leukocyte migration, whereas at the epithelium, chemokines apparently need to be mobilized by proteolytic degradation of the binding proteoglycans for successful transepithelial migration (51).
In addition, our demonstration of abundant IL-8 at endothelial and epithelial sites indicates that neutrophils must move away from high concentrations of IL-8 at perivascular sites to migrate through bronchial tissue toward the airway. The in vitro studies of Foxman and colleagues (53, 54) provide evidence that this happens when neutrophil receptors are saturated and desensitized to one chemoattractant—in this case, endothelial IL-8—and the cells subsequently respond to a distant, different, and dominant chemoattractant. We speculate therefore that, at least in the first instance, this is not epithelial IL-8 but may be the dominant bacterial formyl-peptide chemoattractants or C5a, previously shown to be a major mediator of neutrophil chemotaxis in the airway fluids of patients with CF (55). In this way, it is suggested that neutrophils prioritize signals from their end targets, bacteria or immune complexes, over more general recruitment signals from host cells, such as IL-8 or LTB4 (53).
Lung proteoglycans are susceptible to cleavage by tumor necrosis factor eCactivated neutrophils (56). Neutrophil-derived serine proteases, notably elastase (57), cleave the proteoglycan core protein to release intact HS side chains. Conversely, neutrophil heparanase cleaves the HS side chain (18), and proteases enhance this process. In balance with increased expression of HS, we sought evidence for cleaved forms of HSPG in which the HS side chains had been degraded by heparanases, acting in concert with heparitinase(s), which generate the antigenic epitope recognized by the 3G10 antibody (21). The extent of HS present in a cleaved form at the epithelium was significantly increased in the COPD group compared with the control group. Neutrophil-mediated degradation of HSPG is reported confined to the core proteins in bronchiectasis (56), but protease-mediated shedding of intact HS would not be reflected as an increase in 3G10 staining. It appears, therefore, that HS side chains may be relatively stable in the inflamed CF airway. One explanation might be that extensive binding of IL-8 protects the HS chain from cleavage (29). Alternatively, because heparanase release from neutrophils and HS degradation was not demonstrated to be a feature of neutrophil migration across basement membranes (58), the increase in degraded HS in COPD might relate to heparanase released by platelets or T cells during recruitment (59). Because heparanase-generated disaccharides inhibit T-cell migration and activation, including secretion of tumor necrosis factor and IFN- (60), cytokines implicated in the stimulated expression of HS (44), feedback inhibition of T-celleCmediated immune responses may be in play. Disaccharides were not measured in this study, but future studies comparing levels of disaccharides in airway tissues and secretions in CF and COPD may provide further insight into mechanisms that regulate the inflammatory process.
HSPG are highly variable structures, which interact with a vast range of cytokines and growth factors via specific sequence domains that have a defined topologic distribution (61) and are likely to contribute to the regional control of immunity. Thus, increased expression of HS is likely to affect the distribution and function of many growth factors and cytokines in CF, and this is the subject of ongoing studies. The contribution of CFTR to the regulation of HS expression at epithelial, or endothelial (62), sites is unknown. It is also not known if expression of HS is increased in young patients with CF, including those with airway inflammation but no evidence of infection (63, 64), and this remains to be established. However, our current study suggests that factors that increase HS expression in bronchial tissue may be useful therapeutic targets to reduce IL-8eCdriven inflammatory responses in CF.
Acknowledgments
The authors thank the Heart-Lung Transplantation Service of the Alfred Hospital for their assistance with this study.
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Department of Allergy, Immunology Respiratory Medicine, Alfred Hospital, Prahran, Victoria, Australia
ABSTRACT
Rationale: Pulmonary disease in cystic fibrosis (CF) is characterized by an exaggerated interleukin (IL)-8eCdriven, neutrophilic, inflammatory response to infection. Binding of IL-8 to heparan sulfate (HS)eCcontaining proteoglycans (HSPG) facilitates binding of the chemokine to its specific receptor, stabilizes and prolongs IL-8 activity, and protects it from proteolysis. We hypothesized that increased expression of HSPG contributes to the sustained inflammatory response in CF bronchial tissue.
Objectives: Our objectives were to analyze the distribution and abundance of IL-8 and HS, in intact and cleaved forms, in bronchial tissue from adult patients with CF or chronic obstructive pulmonary disease (COPD) and a control group without inflammatory airway disease.
Methods: Immunostaining and quantitative image analysis were applied to ethanol-fixed and paraffin-embedded tissue obtained at transplant in patients with CF or COPD, or postmortem in the control group.
Measurements and Main Results: Quantitative immunohistochemical analysis demonstrated significant disease-related differences. Intact HS was significantly more abundant in epithelial and endothelial basement membranes in CF than in COPD or the control group. Conversely, cleaved HS was significantly more abundant in COPD than the other groups. More IL-8eCpositive blood vessels were observed in CF and COPD compared with the control group, whereas more extensive IL-8 expression in the epithelium was observed in CF compared with COPD.
Conclusions: Sustained neutrophil recruitment in the CF airway may therefore be related not only to increased IL-8 expression but also to the increased stability and prolonged activity and retention of IL-8 when it is bound to HSPG in bronchial tissue.
Key Words: chemokine inflammation neutrophil
Pulmonary disease in patients with cystic fibrosis (CF) is dominated by an exaggerated interleukin (IL)-8eCdriven neutrophilic inflammatory response to infection of the airways (1, 2). A deficiency in IL-10 may contribute to the higher levels of IL-8 expressed by pulmonary macrophages and bronchial epithelial cells in patients with CF compared with healthy control subjects (3, 4). Endotoxin is a potent stimulus for macrophage cytokine production (2), whereas neutrophil elastase (5) and adherence of Pseudomonas aeruginosa (6) are stimuli for IL-8 expression by CF bronchial epithelial cells. For circulating neutrophils to detect this chemoattractant signal, it appears that IL-8 must establish a concentration gradient by diffusing from the epithelium, across the tissue matrix to the endothelial barrier. Supporting this notion, a recent study has demonstrated that radiolabeled IL-8 instilled into the airways moved slowly from the airspaces toward the vascular compartment, partitioning between the bronchoalveolar lavage fluid, tissue, and blood (7).
Neutrophil migration across the endothelium occurs in a multistep process that is mediated via the interaction of inflammatory cells with endothelial cell adhesion molecules and immobilized chemoattractants (8). It was originally proposed by Rot (9, 10) that neutrophils respond to a gradient of IL-8 bound to endothelial cell surfaces. In vitro studies have indicated that glycosaminoglycan side chains of proteoglycans act as IL-8 binding sites on human endothelial cells (11). Specifically, the heparan sulfate (HS) side chains associated with syndecan-1 bind IL-8 and are required for successful transendothelial migration of normal human neutrophils (12). Much less is known regarding the distribution of IL-8 in whole tissue. In rabbit lungs, IL-8 is bound to both HS and chondroitin sulfate, strengthening the argument that chemokine gradients are tissue-bound and not soluble (13). Binding to HS induces structural stabilization of IL-8, prolonging its biological activity (14), increases the local concentration of chemokine, and facilitates binding to its receptor (11, 15). Binding to the glycosaminoglycans may also protect IL-8 from elastase-mediated degradation (16) and prolong its half-life in the inflamed lung (7).
Higher levels of soluble IL-8 in airway samples, sputum, or bronchoalveolar lavage fluid than those in the circulation suggest that the recruitment of neutrophils into the airways of patients with CF is directed by concentration gradients of IL-8 (17). We now hypothesize that increased expression of both IL-8 and HS-containing proteoglycans (HSPG) may contribute to sustained neutrophil recruitment in CF. We used an immunohistochemical approach, with quantitative image analysis, to compare the distribution and abundance of IL-8, HS, and, because HS is susceptible to cleavage by activated neutrophils (18), cleaved forms of HS within bronchial tissue of patients with CF, patients with chronic obstructive pulmonary disease (COPD), which is also characterized by IL-8eCmediated neutrophilic inflammation (19), and control subjects. Some of the results of these studies have been previously reported in the form of an abstract (20).
METHODS
Tissue Samples
Airway tissue samples were obtained from patients with CF (n = 8; four men; age, 23.4 ± 2.8 years) and COPD (n = 8; five men; age, 49.5 ± 4.1 years) at lung transplantation and from patients without CF or COPD (n = 7; four men; age, 51.4 ± 7.1 years) at postmortem. Two of the control subjects had primary pulmonary hypertension; the remainder were normal at postmortem.
Materials
Monoclonal antibodies detecting intact (F58-10E4) and cleaved (F69-3G10) forms of HS were obtained from AMS Biotechnology, Abingdon, United Kingdom. Antibody F58-10E4 reacts with epitopes containing N-sulfated glucosamine residues on intact HS side chains of proteoglycans (21). We previously demonstrated that the binding of IL-8 to HS does not interfere with recognition of HS by this antibody (12). Antibody F69-3G10 reacts with epitopes exposed by heparitinase digestion of HSPG to reveal residual HS stubs attached to the core proteins (21). Mouse monoclonal antihuman IL-8 antibody was obtained from Bender Medsystems (Vienna, Austria), and biotinylated antimouse Fab fragments from Dako (Ely, UK). Streptavidin-biotin blocking kit was obtained from Vector Laboratories (Peterborough, UK). Streptavidin biotineChorseradish peroxidase was obtained from Dako. Aminoethyl carbazole peroxidase substrate was obtained from Menarini Diagnostics (Wokingham, UK). Crystal mount was obtained from Biomedia (Poole, UK), and routine chemicals were obtained from Merck (Lutterworth, UK).
Tissue Preparation and Immunohistochemistry
Tissue was fixed in ethanol and processed into paraffin blocks. Sections of 4 e were cut and stained immunohistochemically using the streptavidin biotineCperoxidase technique and the monoclonal antibodies described above, at the following dilutions: 1 e/ml F58-10E4, 3 e/ml F69-3G10, and 3 e/ml antieCIL-8. Isotype controls were used to demonstrate the specificity of staining.
Image Analysis
The percentage of epithelial expression of cleaved HSPG and IL-8 was measured with the assistance of computerized image analysis based on red/blue/green color composition as previously described (22). The number of blood vessels immunoreactive for IL-8 was determined as a percentage of total vessels number, given by staining of a sequential section for EN4, a marker for endothelium. The intensity of staining for IL-8 in the vascular basement membrane and intact HSPG in both the epithelial and vascular basement membranes was assessed on a gray level scale of 0 to 255, using computerized image analysis.
Statistical Analysis
Results were subject to statistical analysis using the Mann-Whitney test and Spearman rank correlation analysis using SPSS 11 software (SPSS, Woking, UK).
RESULTS
Intact HS was detected in association with the epithelial and endothelial basement membranes in all subject groups (Figures 1A, 1D, and 1G). The relative intensity of HS staining on epithelial basement membranes was significantly higher in CF than in either normal (p = 0.014) or COPD (p = 0.002) tissue samples (Figure 2). Intact HS was also more abundant on the endothelial basement membranes in CF tissue compared with normal (p = 0.06) and COPD (p = 0.002) tissue (Figure 3). The abundance of intact HS on endothelial and epithelial basement membranes was highly significantly correlated in the COPD group (r = 0.862, p = 0.006, n = 8), but not in the other groups.
The distribution of the cleaved and intact forms of HS differed. In all tissues, the cleaved form of HS was evident not only in the basement membranes but also within endothelial and epithelial cells (Figures 1B, 1E, and 1H). In CF tissues, it was clear that inflammatory cells were also strongly positive for cleaved HS. On high-power magnification, these cells had the morphologic appearance of polymorphonuclear neutrophils. Analysis of the epithelial staining for cleaved HS showed that this was most abundant in COPD (Figure 4), and significantly (p = 0.013) higher than in control samples.
In all groups, IL-8 was distributed throughout the bronchial tissue, and was most abundant in the epithelium and endothelium, both cell-associated, within the cells and on the cell surface, and in the basement membranes (Figures 1C, 1F, and 1I). Inflammatory cells, including neutrophils, were also IL-8eCpositive in CF tissues (Figure 1I). Assessing the area of epithelium staining positively for IL-8 indicates the extent of epithelial activation. Epithelial IL-8 staining was evident even in the non-CF/non-COPD tissue samples, and this tended to be higher in the CF group (Figure 5). Epithelial staining was significantly more extensive in the CF than the COPD group (Figure 5). The most significant differences in IL-8 staining were in the greater number of IL-8eCpositive blood vessels that were counted in CF and COPD tissue (Figure 6). The intensity of IL-8 staining on the blood vessels was significantly higher in CF than the control group, but not different than the COPD group (Figure 7).
DISCUSSION
IL-8 is an important driver of neutrophilic inflammation, which we have previously shown to correlate with disease severity in CF (17). To our knowledge, there have been no previous reports of the distribution of HSPG and IL-8 in bronchial tissue from patients with CF or COPD. In summary, quantitative immunohistochemical analysis of IL-8 and HS, identified as intact and cleaved forms, has demonstrated significant disease-related differences. Intact HS was significantly more abundant in epithelial and endothelial basement membranes in CF than COPD or the control group. Conversely, cleaved HS was significantly more abundant in COPD than the other groups. An increased number of IL-8eCpositive blood vessels was observed in CF and COPD compared with the control group. Epithelial IL-8eCpositive staining was more extensive in CF compared with COPD, but neither was significantly greater than the control group.
IL-8eCdependent neutrophilic inflammation is characteristic of both CF (1) and of COPD (19), where it is particularly associated with infective exacerbation. However, neutrophil numbers in bronchoalveolar lavage fluid of young adult patients with CF are estimated to be at least two orders of magnitude higher than in patients with chronic bronchitis, even during an infective exacerbation of symptoms (23, 24). The more extensive expression of IL-8 at epithelial sites in CF compared with COPD most likely reflects the greater magnitude of the inflammatory response in CF compared with COPD. However, the extent of epithelial IL-8 expression was not significantly greater in either patient group compared with the control group. Previous reports of increased expression of IL-8 by airway epithelial cells in CF appear to depend on the cell line and model used, with primary cells producing more IL-8 in response to P. aeruginosa, but not IFN-, tumor necrosis factor , IL-1, or Haemophilus influenza (25). Other studies have indicated that the presence of mutated CFTR had no effect, directly or indirectly through IL-8 expression, on transepithelial migration of neutrophils (26). Conversely, the absence of IL-10, an inhibitor of cytokine synthesis, is believed to contribute to the increased response in CF (3, 4), and a prolonged inflammatory response to acute Pseudomonas challenge has been shown in IL-10 knockout mice (27). More recently, a deficiency in the collectins, surfactant protein-A, and surfactant protein-D, normally expressed by epithelial cells lining the respiratory tract, was proposed to increase the inflammatory response in CF (28).
The endothelium is the primary portal for inflammatory cell recruitment. Neutrophils in the circulation emigrate into bronchial tissue in response to signals from adhesion molecules and chemoattractants presented by the lumenal surface of postcapillary venular endothelial cells (8). We demonstrated an increased number of IL-8eCpositive blood vessels in the CF and COPD groups compared with the control group. However, in addition to increased expression, factors that increase the bioactivity, stability, and half-life of IL-8 at this site in bronchial tissue may also be involved. HS side chains on HSPG were shown to bind and induce dimerization of IL-8 on endothelial cells in culture, increasing the local concentration of the chemokine and influencing its interaction with its specific receptor (11, 15). Recent animal studies have indicated that a tissue-specific mechanism for dimerization of IL-8 in tissue is responsible for increased retention of IL-8 in the lungs compared with the skin and is important for the recruitment of neutrophils into the lungs in vivo (7). Previous studies suggest that this specificity may be determined by a unique HS sequence that promotes binding and dimerization of IL-8 (29). However, the distribution of HSPG in relation to IL-8 distribution in bronchial tissue in CF and COPD has not previously been investigated.
We previously demonstrated IL-8 binding to endothelial HS (12); therefore, their colocalization at these sites implies that IL-8 is bound to HSPG. However, this remains an assumption, and other potential IL-8 binding sites on the endothelium have been identified. Specific, high-affinity IL-8 receptors are expressed on endothelial cells (30), and it is also possible that IL-8 is bound to the Duffy antigen on endothelial cells of postcapillary venules (31). The functions of the high-affinity IL-8 receptor on endothelial cells remain an open question: it may be involved in autocrine signaling and/or clearance of IL-8 by internalization after the inflammatory response. In situ binding assays indicated, however, that specific receptors did not appear to contribute to IL-8 binding on endothelial cells (32). The Duffy antigen binds tissue-derived IL-8 and directs transcytosis of the chemokine to the lumenal surface of the endothelium (31). However, the role of the Duffy antigen in neutrophil migration is controversial, with in vivo studies in mice indicating both an anti- and a proinflammatory role, but that this may be redundant (33, 34). It was also proposed that the Duffy antigen may have an antiinflammatory role and HS a proinflammatory role (31). Because IL-8 bound to Duffy antigen on red blood cells does not activate neutrophils, other molecules, such as glycosaminoglycans, may be more important in chemokine presentation. However, it has yet to be established whether chemokines bound to HS can bind to specific receptors or whether they must first dissociate from HS to interact with their signaling receptors.
Our finding that the HS side chains of HSPG are increased in bronchial tissue in CF, but not in COPD, is novel. Increased expression of chondroitin sulfateeCcontaining proteoglycans (35) and increased sulfation of glycosaminoglycans (36) in the liver and pancreas, but not lung or nasal tissue (36), in CF has previously been reported. In sputum samples, chondroitin sulfate appeared to be associated with severe tracheobronchial infection in CF (37), and chondroitin sulfate was the most abundant proteoglycan released by neutrophil elastase and cathepsin GeCstimulated airway gland serous cells in culture (38). The demonstration of increased expression of HSPG in the inflamed airways of patients with CF, compared with a group of patients with COPD and those with noninflamed airways, may have important consequences for the progression of CF lung disease. The increase in HSPG at the bronchial epithelium may promote increased adherence of P. aeruginosa (39), whereas an increase in HS expression on endothelial structures is suggested to increase the local concentration of IL-8 and is further evidence for endothelial activation in CF (40). Of the HSPG expressed by the endothelium, only 1 to 5% is estimated to have anticoagulant functions (41); the remainder represents an array of specific HS structures, bound to perlecan, syndecan, and glypican core proteins, for which the function and regulation of expression is poorly understood. However, the 10E4 antibody we used in our study is believed to trace the mass of HS expressed on individual proteoglycans and at individual sites (21).
There is no evidence to suggest that the observed increase in HS expression is related to defective CFTR function. However, it may represent a secondary phenomenon, reflecting the proinflammatory cytokine profile of the inflamed CF airway. Although the factors that enhance expression of HS in the airways are not well described, IL-1 and IL-8 stimulate renal epithelial cell HS expression (42, 43), and IFN- and tumor necrosis factor enhance the expression of 10E4-positive HS epitopes on endothelial cells (44). HS is the most common cell surface glycosaminoglycan, and the syndecan family of proteoglycans is the major source of HS on all cell types (45). Therefore, it may be relevant that antimicrobial peptides induce endothelial syndecans (46) and hypoxia induces epithelial expression of both proteoglycans and IL-8 (47). Because there was no difference between noninflammatory control and COPD samples, it is possible that the inflammatory cytokine profile in COPD does not support increased HS expression, or that there is increased catabolism of proteoglycans, which was indicated by the increased staining for cleaved HS chains, or that the significant difference in the ages of the patients with CF compared with the other groups was a factor, because the abundance of lung glycosaminoglycans decreases with age (48). However, inflammation is associated with both increased synthesis and shedding of HSPG (49), and the observed increased abundance of HS in CF obviously reflects the balance of these processes.
Little is known about how IL-8 functions in tissue to direct neutrophil recruitment from the circulation, but increased expression of both IL-8 and HS colocalized on endothelial cells is likely to enhance the inflammatory response to a given stimulus. IL-8 is only weakly bound to HS, with a dissociation constant of 0.4 to 2.6 e, whereas binding to the high-affinity receptor has a dissociation constant of 2 to 10 nM. However, the glycosaminoglycans provide a vast excess of low-affinity binding sites in lung tissue, and these determine the location where IL-8 binds in the lungs (13). The HS binding sites for IL-8 may be associated with either syndecan-1 (12) or syndecan-2 (50) on the endothelium. Our findings may offer an explanation for the observation (52) that, at high levels of infection, IL-8 production reaches a maximum that is similar in subjects with CF and control subjects, whereas neutrophil recruitment due to factors other than higher levels of endotoxin (2) continues to be enhanced in CF. Increased expression of HS at the endothelium in CF, compared with COPD, may indicate a unique feature of CF that contributes to increased stability, activation, and presentation of IL-8 to target cells, and the chronicity of the neutrophilic inflammatory response in CF.
Although HS-bound gradients of chemokines enriched in the endothelial basement membrane are likely to attract cells across the endothelium and into the tissue, the gradient is effectively the wrong way round on the epithelium, with chemokines enriched in the basement membrane retaining cells in the tissue and inhibiting their advancement into the airway. At the endothelial level, immobilized chemokines stimulate leukocyte migration, whereas at the epithelium, chemokines apparently need to be mobilized by proteolytic degradation of the binding proteoglycans for successful transepithelial migration (51).
In addition, our demonstration of abundant IL-8 at endothelial and epithelial sites indicates that neutrophils must move away from high concentrations of IL-8 at perivascular sites to migrate through bronchial tissue toward the airway. The in vitro studies of Foxman and colleagues (53, 54) provide evidence that this happens when neutrophil receptors are saturated and desensitized to one chemoattractant—in this case, endothelial IL-8—and the cells subsequently respond to a distant, different, and dominant chemoattractant. We speculate therefore that, at least in the first instance, this is not epithelial IL-8 but may be the dominant bacterial formyl-peptide chemoattractants or C5a, previously shown to be a major mediator of neutrophil chemotaxis in the airway fluids of patients with CF (55). In this way, it is suggested that neutrophils prioritize signals from their end targets, bacteria or immune complexes, over more general recruitment signals from host cells, such as IL-8 or LTB4 (53).
Lung proteoglycans are susceptible to cleavage by tumor necrosis factor eCactivated neutrophils (56). Neutrophil-derived serine proteases, notably elastase (57), cleave the proteoglycan core protein to release intact HS side chains. Conversely, neutrophil heparanase cleaves the HS side chain (18), and proteases enhance this process. In balance with increased expression of HS, we sought evidence for cleaved forms of HSPG in which the HS side chains had been degraded by heparanases, acting in concert with heparitinase(s), which generate the antigenic epitope recognized by the 3G10 antibody (21). The extent of HS present in a cleaved form at the epithelium was significantly increased in the COPD group compared with the control group. Neutrophil-mediated degradation of HSPG is reported confined to the core proteins in bronchiectasis (56), but protease-mediated shedding of intact HS would not be reflected as an increase in 3G10 staining. It appears, therefore, that HS side chains may be relatively stable in the inflamed CF airway. One explanation might be that extensive binding of IL-8 protects the HS chain from cleavage (29). Alternatively, because heparanase release from neutrophils and HS degradation was not demonstrated to be a feature of neutrophil migration across basement membranes (58), the increase in degraded HS in COPD might relate to heparanase released by platelets or T cells during recruitment (59). Because heparanase-generated disaccharides inhibit T-cell migration and activation, including secretion of tumor necrosis factor and IFN- (60), cytokines implicated in the stimulated expression of HS (44), feedback inhibition of T-celleCmediated immune responses may be in play. Disaccharides were not measured in this study, but future studies comparing levels of disaccharides in airway tissues and secretions in CF and COPD may provide further insight into mechanisms that regulate the inflammatory process.
HSPG are highly variable structures, which interact with a vast range of cytokines and growth factors via specific sequence domains that have a defined topologic distribution (61) and are likely to contribute to the regional control of immunity. Thus, increased expression of HS is likely to affect the distribution and function of many growth factors and cytokines in CF, and this is the subject of ongoing studies. The contribution of CFTR to the regulation of HS expression at epithelial, or endothelial (62), sites is unknown. It is also not known if expression of HS is increased in young patients with CF, including those with airway inflammation but no evidence of infection (63, 64), and this remains to be established. However, our current study suggests that factors that increase HS expression in bronchial tissue may be useful therapeutic targets to reduce IL-8eCdriven inflammatory responses in CF.
Acknowledgments
The authors thank the Heart-Lung Transplantation Service of the Alfred Hospital for their assistance with this study.
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