Roles of GAGs on neutrophil migration into the lungs
GAGs bind to various cytokines and chemokines, and sulfation of GAGs provides sites to which chemokines bind24-26) . Chemokines are a family of chemotactic cytokines which promote leukocyte migration into the tissues. Several studies have reported that chemokine-GAG interaction is critical for recruitment of leukocytes into the peritoneum and lungs20,27-29) .
Neutrophil migration into the lungs is involved in the pathogenesis of several lung diseases, particularly in lung infection and acute respiratory distress syndrome30,31) . IL-8/CXCL8 is a potent neutrophil chemokine which is produced by alveolar macrophages in the lungs during acute bacterial pneumonia and acute respiratory distress syndrome30-32) . All chemokines have a GAG-binding domain. The binding of chemokines to GAGs plays critical roles in leukocyte recruitment into tissues by facilitating both the formation of tissue-bound chemokine gradients and the presentation of chemokines to leukocytes in tissues25,33,34) .
The GAG-binding domain of CXCL8 includes basic residues located in the proximal loop (K20) and C-terminal α-helix (R60, K64, K67, and R68)35) (Figure 2). In the lungs, CXCL8 binds to heparin sulfate and chondroitin sulfate GAGs, and these interactions promote the dimerization of CXCL8, resulting in an increase in the amount of CXCL8 bound in lung tissues20,24) . Although these results suggest that the interaction of CXCL8 and GAGs plays a critical role in neutrophil migration in the lung and results of in vitro experiments support its possible role36-38) , the precise role of this interaction had not been clarified in vivo. Our research group uncovered the role of CXCL8 in neutrophil migration in the lung by conducting an in vivo experiment using two mutant forms of CXCL8 (R68A-CXCL8 and K64A/K67A/R68A-CXCL8), which do not bind to GAGs39) . When intratracheally instilled into the lungs of mice, the CXCL8 mutants recruited more neutrophils into the lungs and appeared more rapidly in systemic circulation than recombinant CXCL8 (Figure 3a). In addition, the CXCL8 mutants appeared in plasma at significantly higher concentrations (Figure 3b) and diffused more rapidly across the ECM in vitro. Furthermore, when we instilled another mutant CXCL8 (I64K-CXCL8), which has more binding activity to GAGs, into the lungs of mice, it recruited fewer neutrophils than recombinant CXCL8 (unpublished data). These results show that GAGs control the spatiotemporal formation of chemokine gradients and neutrophil migration in the lungs.
Fig. 2. Glycosaminoglycan-binding domain on CXCL8 dimer. CXCL8 has three binding domains: a high-affinity binding domain, which mediates binding to specific receptors on polymorphonuclear neutrophils; the glycosaminoglycan-binding domain (K20, R60, K64, K67, R68); and the dimer interface (R6), where CXCL8 molecules bind to each other to form dimers. Blue and red show positively and negatively charged regions, respectively.
Fig. 3. Neutrophil migration in response to rCXCL8 and CXCL8 mutants. a) The CXCL8 mutants (R68A-CXCL8 and K64A/K67A/R68A-CXCL8: TM-CXCL8) recruited more neutrophils into the lungs than recombinant CXCL8 (rCXCL8). b) The CXCL8 mutants appeared more rapidly in plasma after intratracheal instillation than rCXCL8.
*:p < 0.05 vs rCXCL8. #:p < 0.05 vs control.
Syndecan
Syndecan is one of the transmembrane HSPGs and consists of four isoforms. Syndecan-1, -2, and -3 are specifically expressed on the surface of epithelial cells or plasma cells, fibroblasts or endothelium, and nerve cells, respectively. On the other hand, syndecan-4 is expressed on a variety of cells12,40-43) . Heparan sulfate is the most abundant GAG in healthy lungs13) , and several types of proteoglycans exist in the lung as components of ECMs6) . Heparan sulfate GAG side chains of syndecans bind to various proteins such as cytokines, chemokines, and growth factors, and mediate biological activities of these proteins19,20,44,45) . However, the role of HSPGs in the lung had not been clarified in detail.
To clarify the role of HSPGs in acute lung inflammation, we evaluated mRNA expression of HSPGs using an LPS-induced murine lung inflammation model. After LPS instillation, syndecan-4 mRNA was rapidly and selectively upregulated among the HSPGs studied46) (Figure 4a). Therefore, we focused on syndecan-4 for our further studies. In the LPS-induced lung inflammation model, more neutrophils were found in bronchoalveolar lavage fluid (BALF) in syndecan-4 deficient mice compared to wild-type mice46) (Figure 4b, c). Moreover, in a model of lung inflammation induced by S. pneumonia, the survival rate of the syndecan-4 deficient mice was significantly lower than wild-type mice (Figure 5), while their total neutrophil counts in BALF, bacterial counts in blood, and plasma levels of inflammatory cytokines were significantly higher47) . In addition, pretreatment of recombinant syndecan-4 significantly inhibited LPS-induced CXCL8 upregulation in BEAS-2B bronchial epithelial cells46) . These results indicate that syndecan-4 can inhibit acute inflammation in the lungs. Furthermore, we evaluated the role of syndecan-4 in lung fibrosis48) . In a bleomycin-induced lung fibrosis model, the histopathological lung fibrosis score and collagen content in lung tissues were significantly higher in syndecan-4 deficient mice compared to wild-type mice at 21 days after intratracheal bleomycin instillation. In in vitro experiments using lung fibroblasts, TGF-β-induced Smad3 activation as well as collagen and α-smooth muscle actin upregulation were significantly inhibited by co-incubation of recombinant syndecan-4. These results show that syndecan-4 is involved in the pathogenesis of lung fibrosis.
To further explore the role of syndecan-4 in lung diseases, we analyzed the serum levels of syndecan-4 in patients with acute pneumonia and idiopathic interstitial pneumonia (IIP)47,49) . Although syndecans exist on cell surfaces, cell surface syndecans can be cleaved by several inflammatory factors such as matrix metalloproteinase (MMP)-7 and -9, or a disintegrin and metalloproteinase 1744,50-54) . In patients with acute pneumonia, serum syndecan-4 levels were significantly higher than those in healthy volunteers and were correlated negatively with the pneumonia severity score. Moreover, among patients who improved with short-term antibiotic therapy, serum syndecan-4 levels on admission were higher than in those who did not improve and gradually increased during the antibiotic therapy47) . IIP, including idiopathic pulmonary fibrosis (IPF), is a chronic, progressive, and intractable fibrosing lung disease55-57) . During its clinical course, acute respiratory failure (also referred to as acute exacerbation) may occur, and this is reported to be the most common cause of death (40%) in Japanese IPF patients58) . We found that serum syndecan-4 levels were significantly lower in patients with acute exacerbation of IIP than in those in the clinically stable phase, and the prognosis after acute exacerbation onset was significantly worse in the patients with higher baseline serum syndecan-4 levels than in those with lower baseline levels49) . Furthermore, the clinical significance of serum syndecan-4 levels in preterm infants with chronic lung disease was recently demonstrated59) . These results show that serum syndecan-4 is a clinically significant biomarker in lung diseases.
Fig. 4. Role of syndecan-4 in lipopolysaccharide-induced lung inflammation. a) Changes in mRNA for the heparan sulfate proteoglycans after intratracheal instillation of lipopolysaccharide (LPS) into wild-type mice. Among heparan sulfate proteoglycans, syndecan-4 mRNA was rapidly and selectively up-regulated. *: p < 0.05 vs syndecan-4-PBS. b, c) Intratracheal instillation of LPS induced more neutrophil recruitment into the lungs in syndecan-4 deficient mice (Sdc4 KO) than wild-type mice (WT). *: p < 0.05 vs WT.
Fig. 5. Survival of wild-type and syndecan-4 deficient mice after intranasal instillation of S. pneumoniae. The survival rate of syndecan-4 deficient (Sdc4 KO) mice was significantly worse after intranasal instillation of S. pneumoniae (5.0×106 CFU) than wild-type mice (WT).
Versican and hyaluronan
Versican, a large chondroitin sulfate proteoglycan belonging to the aggrecan family, has a molecular weight >1,000 kDa in its four isoforms, V0-V3, produced by alternative splicing60) . Versican alters the pericellular environment by binding to various mediators via the GAG domain. In addition, versican modifies the bioactivities of ECM proteins, such as hyaluronan, and plays important roles in cell morphology, adhesion, proliferation and migration61,62) . Hyaluronan, a non-sulfated GAG, is a major constituent of the ECM, and growing evidence shows its important roles in inflammation, injury, and repair in the lung63,64) .
In an LPS-induced murine lung inflammation model, we demonstrated that a rapid increase in mRNA expression of versican and hyaluronan synthase was associated with increased immunohistochemical staining for versican and hyaluronan. In addition, in vitro studies showed that LPS caused a rapid increase in versican mRNA, proteins, and hyaluronan synthase in M1 macrophages, but not in M2 macrophages65) . These results show important roles of versican and hyaluronan in the innate immune response to gram-negative lung infection.
In patients with IIP, significantly higher levels of serum hyaluronan were found compared to healthy volunteers, and positive correlations of hyaluronan levels in BALF with the percentage of inflammatory cells and the amount of CXCL8 were shown. In addition, patients with acute exacerbation had significantly higher serum hyaluronan levels compared with those in the stable phase, and patients with the highest serum hyaluronan had the worst 60-day outcomes66) . These results show that hyaluronan is involved in the pathogenesis of IIP, and serum hyaluronan is a possible biomarker in patients with IIP.
Proteases such as MMPs are involved in the pathogenesis of lung fibrosis, and MMP-1 and MMP-7 levels in blood, in particular, are reported to be prognostic biomarkers for IPF67,68) . In addition, it is reported that the serum levels of ECM products degraded by MMPs increased and were related to disease activity in IPF69) . In the PROFILE study, increased serum levels of proteoglycans degraded by MMPs were reported to be associated with disease activity in patients with IPF70) . We analyzed the serum levels of ECM degradation products in IIP patients, and found that type IV and VI collagen degradation products were significantly higher, while elastin and versican degradation products were lower during acute exacerbation than during the stable phase of IIP. Furthermore, lower levels of versican degradation products during acute exacerbation were associated with an increased risk of mortality71) .
Decorin
Decorin is a small, leucine-rich proteoglycan with one chondroitin/dermatan sulfate GAG side chain72) . Decorin binds to collagen and plays important roles in collagen fibril formation and fibrous spacing73-75) . It is reported that decorin-deficient mice have a phenotype of abnormal collagen fibril morphology and skin fragility73,76) . In addition to its role in collagen fibrogenesis, decorin also plays important roles in angiogenesis, innate immunity, inflammation, fibrosis, wound healing, tumor growth and autophagy73,74,77,78) .
In IPF, decorin is reportedly expressed in fibrotic collagen-deposited lesions and fibroblastic foci79) . We analyzed the serum decorin levels in IIP patients in the stable phase and at the time of acute exacerbation, and found that serum decorin levels at the time of acute exacerbation were significantly lower compared with those in the stable phase or in healthy volunteers. In addition, serum decorin levels in clinically stable IIP patients were significantly lower than those in healthy subjects. Moreover, those with serum decorin levels lower than the median, especially the patients with acute exacerbation of IPF, had significantly higher survival rates compared to those with higher-than-median serum decorin levels80).
