Hyaluronan as an Immune Regulator in Human Diseases

A. Hyaluronan synthase 1 (HAS1)

Human HAS1 gene is located at 19q13.3-q13.4, whereas mouse Has1 is located at Chromosome 17 (396). Human HAS1 protein shows significant homology with the hasA gene product of Streptococcus pyogenes, a glycosaminoglycan synthetase (DG42) from Xenopus laevis, and a murine HA synthase (395). HAS1 protein is a bona fide synthase since it alone is able to synthesize HA and different amino acid residues on the cytoplasmic central loop domain are involved in transferring N-acetyl-D-glucosamine and D-glucuronic acid residues, respectively (488).

HA accumulation and dysregulated expression of HA synthases have been demonstrated in many diseases in both animal studies and in humans. HA deposition is prominent in MRL-Fas^(lpr) mice with renal disease and could be mediated by local synthesis through HAS1 and HAS2. The enhanced synthesis of HA could be promoted by proinflammatory cytokines in vivo (96). Has1 mRNA was expressed predominantly in bone marrow mesenchymal progenitor cells derived from multiple myeloma patients when compared to mesenchymal progenitor cells from normal individuals (43). Bone marrow mesenchymal progenitor cells from myeloma synthesize more HA than those from healthy donors, suggesting that myeloma mesenchymal progenitor cells could be an important component of the myeloma pathophysiology in vivo by their increased expression of extracellular matrix components relevant to plasma cell growth and survival (43). Accumulation of HA is a hallmark of rheumatoid arthritis. In human fibroblast-like synoviocytes, HAS1 expression can be enhanced by TGF-β transcription. The TGF-β-induced transcription is mediated by a p38 MEK dependent, not JNK, pathway. TGF-β treatment leads to an increase in synthase activity and in HA production (402).

B. Hyaluronan synthase 2 (HAS2)

Human hyaluronan synthase 2 is located on chromosome 8, whereas mouse Has2 is on chromosome 15 (396). In an in vitro transfection assay, Kimata and associates demonstrated that all three isoforms of HA synthase exhibited a de novo, massive formation of a HA matrix in nontransformed rat 3Y1 embryonic fibroblast cells (157). Hyaluronan and its matrix can modulate contact inhibition of cell growth and migration (157). Inhibition of a phosphatidylinositol 3-kinase pathway resulted in reacquisition of the normal phenotype of HAS2 transfectants, suggesting that the intracellular phosphatidylinositol 3-kinase signaling regulates diminution of contact inhibition induced by formation of the massive HA matrix (157).

Targeted deletion of HAS2 has been a major development in the field, providing in vivo evidence of the functions of HA (45). There are major abnormalities in heart and blood vessel development, resulting in an embryonic lethal phenotype (44, 45). HAS2-deficient embryos at embryonic day 9.5 completely lack endocardial cushions (44, 45), consistent with early observations that high content of HA is present in normal human heart valves (435). These defects resemble the features in naturally occurring versican knockouts (276). These studies suggest that HA-ECM interactions play a significant role in cardiac development. Elucidating the role for HAS2 in animal models has been difficult due to the embryonic lethal phenotype in the HAS2-deficient mice.

HAS2 also plays a role in limb development. Overexpression of Has2 in the mesoderm of the chick limb bud in vivo results in the formation of shortened and severely malformed limbs that lack one or more skeletal elements. Skeletal elements in limbs overexpressing Has2 are reduced in length, exhibit abnormal morphology, and are positioned inappropriately (236). In vitro, sustained HA production in micromass cultures of limb mesenchymal cells inhibits formation of precartilage condensations and subsequent chondrogenesis, indicating that proper regulation of HA is essential for formation of the precartilage condensations that trigger cartilage differentiation (236). Conditional inactivation of HAS2 showed a role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb (258).

Recently, transcription factors Sp1 and Sp3 have been identified as principal mediators of HAS2 constitutive transcription. Sp1 and Sp3 bind to three sites immediately upstream of the HAS2 transcription initiation site and that mutation of the consensus recognition sequences within these sites ablated their transcriptional response. In contrast, NF-Y, CCAAT, and NF-κB binding proteins may not be involved in HAS2 transcription (278). A recent study showed that transgenic expression of Tbx2, a central intermediary of Bmp-Smad signaling, induced Has2 and TGF-β2 expression, facilitating endocardial cushions formation (382).

C. Hyaluronan synthase 3 (HAS3)

The human HAS3 gene is localized to chromosome 16q22.1 and the mouse Has3 to chromosome 8 (396). The HAS3 protein is transmembrane protein. Using green fluorescent protein to tag HA synthases in keratinocytes, HAS2 and HAS3 were found to travel through endoplasmic reticulum, Golgi, plasma membrane, and endocytic vesicles, and a distinct enrichment of plasma membrane HAS was observed in cell protrusions (351). The trafficking of HAS was correlated with its activity, since inhibition of HA synthesis by substrate UDP-glucuronic acid starvation using 4-methyl-umbelliferone prevented HAS access to the plasma membrane (351). Similarly, in cells transfected with green fluorescent protein-tagged Has3, the dorsal surface was decorated by up to 150 slender, 3 to 20-μm-long microvillus-like plasma membrane protrusions, which consisted of filamentous actin, CD44, and lipid raft microdomains, in addition to Has3 (212). Enzymatic activity of HAS was required for the growth of the microvilli. The microvilli induced by HAS3 gradually withered with the introduction of an inhibitor of HA synthesis and rapidly retracted by hyaluronidase digestion, whereas they were independent of HA receptors (212). Keratinocyte growth factor activates keratinocyte migration and stimulates wound healing. At same time, keratinocyte growth factor stimulates epidermal keratinocytes to accumulate intermediate-sized HA in the culture medium and within keratinocytes and leads to a rapid increase of Has2 and Has3 mRNA (188), suggesting that Has2 and Has3 are the targets of keratinocyte growth factor in keratinocytes. Enhanced HA synthesis acts an effector for the migratory response of keratinocytes in wound healing, whereas it may delay keratinocyte terminal differentiation (188).

A. CD44

CD44 is the major cell-surface HA binding protein (10). CD44 is a widely researched molecule and it has been shown to have multiple roles in a variety of biomedical fields. CD44 is a polymorphic type I transmembrane glycoprotein whose diversity is determined by differential splicing of at least 10 variable exons encoding a segment of the extracellular domain and by cell-type-specific glycosylation (232). It is widely expressed in almost every cell type in the human and in the mouse. It is also an abundant protein on many cell types. It is believed to be one of the most abundant cell surface proteins on macrophages. Although glycosaminoglycan side chains associated with some CD44 isoforms can bind a subset of heparin-binding growth factors, cytokines, and ECM proteins such as fibronectin, most of the functions ascribed to CD44 thus far can be attributed to its ability to bind and internalize HA (381). N-glycosylation regulates CD44 structure by altering both the affinity and avidity of CD44-HA binding (422). Most cells express the standard isoform, which is an 85-kDa protein that undergoes posttranslational modification (232). Most cells including stromal cells such as fibroblasts and smooth muscle cells, epithelial cells, and immune cells all express CD44 (381).

HA-CD44 interactions play an important role in development, inflammation, T cell recruitment and activation, and tumor growth and metastasis (232). CD44 cytoplasmic domain can be phosphorylated when ligand binds to transduce signaling. CD44 is also required for BMP-7 signaling. The cytoplasmic domain of CD44 is required for BMP-7 induced Smad1 nuclear translocation (325). Smad1 was found to interact with the cytoplasmic domain of CD44 demonstrated with coimmunoprecipitation. The integrity of extracellular HA-cell interactions is required for BMP-7-mediated Smad1 phosphorylation, nuclear translocation of Smad1 or Smad4, and SBE4-luciferase reporter activation, suggesting a functional link between the BMP signaling cascade and CD44 (325).

When CD44 phosphorylation mutants were transfected into a murine fibroblast line expressing low levels of endogenous CD44, CD44 phosphorylation mutants were as efficient as wild type CD44 in mediating cell adhesion but were unable to support HA-dependent fibroblast migration, demonstrating a control mechanism specific for CD44-mediated cell motility (323). Work with transformed cells indicates that these cells use the cell surface matrix receptor CD44 for migration and invasion (139). Lung tissue from patients who died from acute alveolar fibrosis after lung injury reveals CD44-expressing mesenchymal cells throughout newly formed fibrotic tissue. CD44 was found uniformly over the cell surface and was found densely labeling filopodia and lamellipodia. By blocking the function of CD44 with monoclonal antibodies, fibroblast invasion into a fibrin matrix was inhibited (408). Fibroblast CD44 functions as an adhesion receptor for provisional matrix proteins and is capable of mediating fibroblast migration and invasion of the wound provisional matrix resulting in the formation of fibrotic tissue (408). Hyaluronan inhibits PDGF-BB-induced activation of PDGF receptor and cell motility, while blockage of the binding of HA to CD44 restored PDGF-receptor activation and motility, indicating that CD44 mediates the inhibiting effect on PDGF receptor (234). PDGF receptor and CD44 form a complex and the inhibitory effect of HA is neutralized by inhibition of tyrosine phosphatases, suggesting that HA-activated CD44 modulate PDGF receptor signaling by recruiting tyrosine phosphatase to the receptor (234).

The role of CD44 was investigated by genetic targeting in vivo. Transgenic mice expressing an antisense CD44 cDNA under the control of keratin-5 promoter had defective keratinocyte proliferation indicating the role of CD44 in the regulation of keratinocyte proliferation in response to extracellular stimuli (192). Given the importance of CD44 as demonstrated in many biological and pathological conditions, it was very surprising that the genetic disruption of CD44 in the mouse did not show much abnormality (371). Our laboratory has demonstrated that CD44 is required for the resolution of pulmonary inflammation (421). CD44-deficient mice succumb to unremitting inflammation following bleomycin-induced noninfectious lung injury, characterized by impaired clearance of apoptotic neutrophils, persistent accumulation of HA fragments at the site of tissue injury, and impaired activation of TGF-β1 (421). The phenotype was partially reversed by reconstitution with CD44⁺ cells, thus demonstrating a critical role for this receptor on leukocytes in resolving lung inflammation. CD44 deficiency results in enhanced inflammation in E. coli but not S. pneumoniae-induced pneumonia, suggesting a role for CD44 in limiting the inflammatory response to E. coli (454). We and others demonstrated that inflammation is more aggravated in CD44-knockout mice than in wild type mice (237, 294). We further found that the induction of the negative regulators of TLR signaling IL-1R-associated kinase-M (202), Toll-interacting protein, and A20 by intratracheal lipopolysaccharide in vivo and in macrophages in vitro was significantly reduced in CD44^−/− mice, suggesting that CD44 plays a role in preventing exaggerated inflammatory responses to LPS by promoting the expression of negative regulators of TLR4 signaling (237).

In addition, recent studies suggested that CD44 expression was strongly induced in the infarcted myocardium and was localized on infiltrating leukocytes, wound myofibroblasts, and vascular cells. Although CD44 deficient mice showed enhanced inflammation following myocardial infarction, CD44 deficient infarcts showed decreased fibroblast infiltration, reduced collagen deposition, and diminished proliferative activity (156). Isolated primary CD44 deficient cardiac fibroblasts had reduced proliferation upon stimulation with serum and decreased collagen synthesis in response to TGF-β in comparison to wild type fibroblasts, suggesting that CD44-mediated interactions are critically involved in infarct healing (156). After tissue injury, fibroblast migration from the peri-wound collagenous stroma into the fibrin-laden wound is critical for granulation tissue formation and subsequent healing. Fibroblast transmigration or invasive migration from a collagen matrix into a fibrin matrix required the presence of fibronectin, several integrins, CD44 (59), and syndecan-4 (238). A recent study suggested that CD44 expression is not a general requirement for cell migration and gradient sensing; rather, it elicits a ligand-specific response (441). Expression of CD44 alone is not sufficient to drive chemotaxis towards HA, as NIH-3T3 fibroblasts were unable to respond to a HA gradient even when transfected with high levels of human CD44 (441). For NIH-3T3 cells to bind exogenous HA, it was necessary to both increase the level of receptor expression and remove a HA pericellular matrix (441). CD44 is required for myoblast migration and differentiation, since primary myoblasts from CD44-deficient mice displayed attenuated differentiation and subsequent myotube formation at early times in a differentiation-inducing in vitro environment (287). Chemotaxis of CD44 deficient myoblasts toward hepatocyte growth factor and basic fibroblast growth factor was totally abrogated (287). Very recently, it was demonstrated that CD44-deficient fibroblasts have fewer stress fibers, and focal adhesion complexes (1). Migration of CD44-deficient fibroblasts was increased in velocity, but was directionless (1).

B. RHAMM

RHAMM (for receptor for hyaluronan-mediated motility expressed protein, also called CD168) binds to biotinylated HA (129, 482). RHAMM is a functional receptor in many cell types including endothelial cells (240, 365). It is believed to be an HA receptor involved in tumor cell locomotion (129). Transfection experiments in fibroblasts suggest that RHAMM plays a role in Ras-dependent oncogenesis (122), but this role has been challenged by others (145), reflecting the complexity of the interactions between HA and HABPs in biological and pathological conditions. Nevertheless, RHAMM-HA interactions do play an important role in tissue injury and repair (491). Upregulation of RHAMM expression in bovine aortic smooth muscle cells was readily detected in an in vitro injury model (366). While HA stimulated the random locomotion of bovine aortic smooth muscle cells, an antibody to RHAMM that blocks HA binding with this receptor abolished smooth muscle cell migration following injury (366), suggesting that RHAMM is necessary for the migration of smooth muscle cells during wound repair.

Increased expression of RHAMM in macrophages after bleomycin injury in rats has been reported with a function blocking anti-RHAMM antibody (491). HA-stimulated macrophage chemotaxis was also inhibited by anti-RHAMM antibody (491). Daily administration of anti-RHAMM antibody to injured animals resulted in a 40% decrease in macrophage accumulation and lung architecture were improved with anti-RHAMM antibody treatment (491).

Inflammation is more aggravated in CD44-knockout mice than in wild type mice (294). Compensation for the loss of the CD44 gene occurs not because of enhanced expression of the redundant RHAMM gene, but rather because the loss of CD44 allows increased accumulation of the HA substrate, with which both CD44 and RHAMM engage, thus enabling augmented signaling through RHAMM (294). RHAMM knockout mice are viable and show no significant defect, suggesting that RHAMM is not an absolute requirement for normal development (431). RHAMM is expressed at high levels in aggressive fibromatosis. When crossed with mice that harbor a targeted mutation in the tumor suppressor APC gene predisposing animals to tumors including aggressive fibromatosis tumor, RHAMM deficiency significantly decreased the number of aggressive fibromatosis tumors formed (431). RHAMM promotes fibroblast proliferation under conditions of low density (431). RHAMM-deficient fibroblasts fail to repair wounds in an in vitro wound healing assay (430). ERK1,2 activation and fibroblast migration/differentiation is also defective during the repair of RHAMM-deficient excisional skin wounds and results in aberrant granulation tissue in vivo. These results identify RHAMM as an essential regulator of CD44-ERK1,2 fibroblast motogenic signaling required for wound repair (430).

C. TSG-6/TNFIP6

Tumor necrosis factor-stimulated gene-6 (TSG-6), also called TNFα-induced protein 6 (TNFIP6) contains Link module and binds HA (204, 228, 322). The solution structure of the Link module from human TNFIP6 was determined and found to consist of two alpha helices and two antiparallel beta sheets arranged around a large hydrophobic core. This defines the consensus fold for the Link module family, which includes CD44, cartilage link protein, and aggrecan (204). The interaction of TNFIP6 with HA is pH-dependent (32). TNFIP6 expression is upregulated in many cell types in response to a variety of proinflammatory mediators and growth factors. TNFIP6 is induced by IL-1 and TNFα (228, 474), and LPS (474). This protein is detected in several inflammatory disease states such as rheumatoid arthritis and in the context of inflammation-like processes is often associated with extracellular matrix remodeling. TNFIP6 is a potent inhibitor of neutrophil migration in an in vivo model of acute inflammation (473).

The recombinant Link module from human TNFIP6 has an inhibitory effect on neutrophil influx into zymosan A-stimulated murine air pouches, equivalent to that of full-length protein, resulting in a significant reduction in the concentrations of various inflammatory mediators in air pouch exudates (110). TNFIP6 is expressed by cultured vascular smooth muscle cells in response to serum and growth factor IL-1 but not to TNF or IL-6 (484). TNFIP6 is upregulated in proliferating vascular smooth muscle in the rat neointima after injury (484). TNFIP6-overexpressing cells grew faster than control vascular smooth muscle cells (484). TNFIP6 forms both covalent and non-covalent complexes with inter-α-inhibitor and potentiates its anti-plasmin activity (273).

Knockout of Tnfip6 in mice with a BALB/c background did not change the onset of proteoglycan-induced arthritis, but progression and severity were significantly greater in Tnfip6-deficient mice when compared to wild type BALB/c mice (409). An early and more extensive infiltration of the synovium with neutrophil leukocytes was the most prominent histopathologic feature of proteoglycan-induced arthritis in Tnfip6-deficient mice (409). Thus, TNFIP6 is a multifunctional anti-inflammatory protein that is produced at the site of inflammation and can be retained by the HA-rich extracellular matrix. A major effect of TNFIP6 is the inhibition of the extravasation of PMN cells, predominantly neutrophils, into the site of inflammation, most likely via a CD44/HA/TNFIP6-mediated blocking mechanism (409). TNFIP6 is a down-regulated gene during osteoblastic differentiation. TNFIP6 inhibits osteoblastic differentiation of human mesenchymal stem cells induced by osteogenic differentiation medium and BMP-2 (437). TNFIP6 is specifically expressed by expanding cumulus cell-oocyte complexes (103). Cumulus cell-oocyte complexes fail to expand in Tnfip6-deficient female mice because of the inability of the cumulus cells to assemble their HA-rich extracellular matrix, demonstrating that TNFIP6 is a key catalyst in the formation of the cumulus extracellular matrix and indispensable for female fertility (104).

D. Brevican

Brevican (also call BEHAB, for brain enriched hyaluronan binding) is a brain-specific proteoglycan in perineuronal nets. Brevican occurs as secreted and cell-surface, glycosylphosphatidylinositol-anchored, isoforms (166). Brevican binds not only to HA (167, 378) but also to chondroitin sulfate (480). Brevican increases the invasiveness of glioma cells in vivo and has been suggested to play a role in central nervous system fiber tract development (108, 167). Brevican deficient mice are viable and fertile and have a normal life span (41). Extracellular matrix perineuronal nets formed but appeared to be less prominent in mutant than in wild type mice. Brevican-deficient mice showed significant deficits in the maintenance of hippocampal long-term potentiation, but no significant deficits in learning and memory (41). Expression of brevican was upregulated in response to a stab wound to the adult rat brain (166).

E. Neurocan

Neurocan is a nervous tissue-specific chondroitin sulfate proteoglycan of the aggrecan family which has been shown to interact with neural cell adhesion molecules (348) and tenascin (347). Neurocan binds to HA and chondroitin sulfate in the brain (75). Through its interactions with neural cell adhesion and extracellular matrix molecules, neurocan is a potent inhibitor of neuronal and glial adhesion and neurite outgrowth (249). Neurocan-deficient mice are viable and fertile and have no obvious deficits in reproduction and general performance (499). Brain anatomy, morphology, and ultrastructure are similar to those of wild type mice, indicating that neurocan has a redundant function in the development of the brain (499). The distribution and function of neurocan and HA are associated with the remodeling process of brain (346). Hyaluronan is organized into fiber-like structures along migratory pathways in the developing mouse cerebellum. Thus, HA-rich fibers are concentrated at sites where specific neural precursor cell types migrate, and the anisotropic orientation of these fibers suggests that they may support guided neural migration during brain development (18). Tissue injuries such as brain injury (12, 266) and spinal cord injury (181) induced neurocan expression. Proteoglycan decorin suppresses neurocan and brevican expression and inflammation, and promotes axon growth across adult rat spinal cord injuries (68).

F. HABP1/C1QBP

HABP1 (for hyaluronan-binding protein 1, also called C1QBP, for complement component 1, q subcomponent binding protein) was first purified from the liver (67) and the brain (66). cDNA cloning confirmed that HABP1 is identical to complement component 1, q subcomponent binding protein (C1QBP) (111), and to pre-mRNA splicing factor SF2 (147, 208). It is about 64 to 68 kDa and binds to HA (66, 67) as well as mannosylated albumin (213). In addition to its mitochondrial location, HABP1 was also localized in the Golgi and completely dispersed throughout the cell during mitosis. This distinctive distribution pattern of HABP1 during mitosis resembles its ligand HA (377).

HABP1 exists as a highly acidic, noncovalently associated trimer in equilibrium with a small fraction of a covalently linked dimer of trimers (172). Structural studies demonstrated that HABP1 exhibits structural plasticity, which is influenced by the ionic environment under in vitro conditions near physiological pH (173). HA interacts only with compact HABP1, while C1q and mannosylated albumin can bind to loosely held oligomeric HABP1 as well. Thus, structural changes in HABP1 mediated by changes in the ionic environment are responsible for recognizing different ligands (173). The more stable conformation of HABP1 was attained at higher ionic strength or at acidic pH showed maximum affinity toward HA (171). Therefore, cellular HA-HABP1 interaction can be regulated by pH and ionic strength. Endogenous HABP1 can be phosphorylated, and was coimmunoprecipitated with activated extracellular signal-regulated kinases (246). HABP1 is a substrate for extracellular signal-regulated kinases and an integral part of the mitogen-activated protein kinase cascade (246). In addition to HA, HABP1/C1QBP also binds to the globular heads of complement subcomponent C1q molecules and inhibit C1 activation (111).

HABP1 overexpressing cells showed extensive vacuolation and reduced growth rate, underwent apoptosis in normal rat skin fibroblasts (187, 267). Overexpression of HABP1 was seen during Leishmania donovani infection. HABP1 binds with 2 proteins of promastigotes as well as amastigotes of L. donovani, suggesting a possible role for HABP in adhesion during the interaction of promastigotes and macrophages (344). It is unknown whether HA is required in this interaction. HABP1 has been demonstrated to play a role in spermatogenic differentiation. Stage-specific expression of HABP1 precursor during spermatogenesis in the rat (24). The sperm surface HABP1 level was correlated with the degree of sperm motility, an important criterion for fertilization (112). The appearance of HABP1 proprotein in the pachytene spermatocytes and the round spermatids during the initial stages of postnatal testis development suggests that this expression may be crucial for spermatogenesis (427).

G. HARE

HARE (for hyaluronan receptor for endocytosis, also called stabilin-2 and FEEL-2) was identified in the abundant expression of 175- and approximately 300-kDa HARE species from sinusoidal endothelial cells of the liver, lymph node, and spleen (497). The expression of HA receptors Stab2 and LYVE1 depended on the tissue and developmental stage of liver endothelial cells in mice (307). The human gene that encodes HARE is on chromosome 12 (495). The HARE protein features multiple B(X₇)B HA-binding motifs, several fasciclin-like adhesion domains, and 18–20 epidermal-growth-factor domains (329). HARE protein binds to HA (130, 262), dermatan sulfate, and chondroitin sulfates A, C, D, and E, but not chondroitin, heparin, heparan sulfate, or keratan sulfate (131, 459).

While HARE/Stab-2 mRNA is confined to the liver and spleen, HARE/Stab-2 protein expression can be detected in the endothelial sinuses of the liver, lymph nodes, spleen, and bone marrow, and in specialized structures of the eye, heart, brain, and kidney in mice (93). Cloning and functional expression of the rat 175-kDa HARE demonstrated that the recombinant HARE is an authentic endocytic receptor for HA and was expressed on the cell surface (498). HARE was substantially colocalized with clathrin, but not with internalized HA that was delivered to lysosomes (498). The liver contains two distinct endothelial cell types: vascular and sinusoidal. The anti-HARE monoclonal antibodies showed diffuse strong staining of nonneoplastic liver sinusoidal endothelium. No staining of nonsinusoidal endothelium or the endothelial lining of the hemangiomas was seen with anti-HARE (82). Antibodies to HARE are not only markers for endothelial cells and the sinusoidal cells of rat liver, spleen, and lymph nodes, but also proved a useful tool in studying the functions of HARE and the physiological significance of HA clearance (458, 497). HARE mediates systemic clearance of glycosaminoglycans from the circulatory and lymphatic systems via coated pit-mediated uptake. A majority of each HARE isoform was intracellular, within the endocytic system, suggesting transient surface residency typical of an active endocytic recycling receptor (130).

H. LYVE1

LYVE1 (for lymphatic vessel endothelial hyaluronan receptor 1, also called CRSBP-1 for cell surface retention sequence binding protein-1) was cloned as a lymph-specific HA receptor on the lymph vessel wall in human (19) and in mice (337). It is a type I integral membrane glycoprotein, homologue of CD44, contains a link module, and binds both soluble and immobilized HA (19). Expression of mouse LYVE1 remains restricted to the lymphatics in homozygous knockout mice lacking a functional gene for CD44 (337). However, LYVE1 is also present in normal hepatic blood sinusoidal endothelial cells in mice and humans (282). LYVE1 expression was also found on the endothelial cells of the lymphatic sinus and in reticular cells in the lymph nodes. (476). LYVE1 is expressed in DC-SIGN⁺ macrophages within the chorionic villi in the rapidly differentiating placenta (33). TNFa promotes LYVE1 internalization from cell surface to lysosomes leading to its degradation. However, the internalization of LYVE1 is independent of HA (179). LYVE1 has being widely used as a lymphatic vessel specific marker (4, 163, 280, 479). It also offers a prognostic parameter for head and neck squamous cell carcinomas, since intratumoral LYVE1⁺ lymphatic vessels were clearly associated with a higher risk for local relapse as well as with poor disease-specific prognosis (259). LYVE1 is expressed in tissue macrophages. In murine tumor models and excisional wound healing, LYVE1 expression occurred in a subset of CD11b⁺, F4/80⁺ tissue macrophages (370). DC-SIGN⁺CD163⁺ macrophages have been shown to express LYVE-1 (33). LYVE1 is also implicated in the trafficking of cells within lymphatic vessels and lymph nodes (162).

LYVE1 plays a role in the transport of HA from tissue to lymph by uptaking HA via lymphatic endothelial cells (163, 337). The occurrence of LYVE1-expressing lymphatic compartments and the alteration of chemokine CCL21 expression in the lymphatics may be involved in defective thymocyte differentiation and migration, and play a significant role in insulitic and diabetic processes (174). Despite the implied importance of this molecule, LYVE1-deficient mice are grossly normal (105, 155). One study did not observe obvious alterations in lymphatic vessel ultrastructure or function or any apparent changes in secondary lymphoid tissue structure or cellularity in LYVE1-deficient mice (105). HA homeostasis is unperturbed in LYVE-deficient mice (105). This suggests that LYVE1 is not obligatory for normal lymphatic development and function and that either compensatory receptors exist or LYVE1 has a more specific role than previously envisioned (105). However, a second study identified morphological and functional alterations of lymphatic capillary vessels in certain tissues, marked by constitutively increased interstitial-lymphatic flow and lack of typical irregularly shaped lumens in LYVE1-deficient mice (155). LYVE1 is not required for either entry or migration of dendritic cells through the afferent lymphatics, and leukocyte populations are unaltered in LYVE1-deficient mice (105), in line with the finding that lymphatic adhesion/transmigration is largely mediated by ICAM-1 and VCAM-1 rather than LYVE1 (178).

B. PMN

Both polarization and directed migration of neutrophils are dependent on the expression of CD44 and its interaction with HA, which could modulate neutrophil migration into inflamed tissues (7). TNFIP6 inhibits polymorphonuclear cell efflux and neutrophil invasion into the inflammatory site (409). A recent study suggested that CD44 does not appear to affect subsequent migration within inflamed tissues, although CD44 mediates neutrophil adhesion and emigration (195). CD44 deficiency leads to enhanced neutrophil migration and lung injury in Escherichia coli pneumonia in mice CD44 deficiency results in enhanced inflammation in E. coli but not S. pneumoniae-induced pneumonia, suggesting a role for CD44 in limiting the inflammatory response to E. coli (454). This study is consistent with our recent observation that inflammation is more aggravated in CD44-knockout mice than in wild type mice (237). Endothelial CD44 rather than neutrophil CD44 mediates neutrophil migration (356). Ligation of CD44 on neutrophils with anti-CD44 and HA induced IL-6 gene transcription and IL-6 protein secretion. Interferon γ-induced IL-6 production is dependent on CD44 cross-linking (373). The cross-linking of specific epitopes of the CD44 molecule can rapidly induce neutrophil apoptosis in vitro and inhibit neutrophil-dependent renal injury in vivo (412). Thus physiological ligands of the CD44 molecule may play an important role in eliminating neutrophils from sites of inflammation, including inflammatory kidney disease (412).

C. Macrophages

Ligation of human macrophage surface CD44 by bivalent monoclonal antibodies rapidly and profoundly augments the capacity of macrophages to phagocytose apoptotic neutrophils in vitro (450). CD44-deficient mice succumb to unremitting inflammation following noninfectious lung injury, characterized by impaired clearance of apoptotic neutrophils, persistent accumulation of HA fragments at the site of tissue injury, and impaired activation of transforming growth factor-β1 (421). This phenotype was partially reversed by reconstitution with CD44⁺ cells, thus demonstrating a critical role for this receptor in resolving lung inflammation (421). CD44 regulates phagocytosis of apoptotic neutrophil granulocytes, but not apoptotic lymphocytes, by human macrophages (132).

There are several reports demonstrating that HA fragments can influence dendritic cell maturation. Goldstein and colleagues showed that 135-kDa fragments of HA induce dendritic cell maturation and initiate alloimmunity (426). Activation of dendritic cells with HA enhances their ability to stimulate allogeneic and antigen-specific T cells markedly (81).

Langerhans cells are skin-specific members of the dendritic cell family and crucial for the initiation of cutaneous immune responses. Systemic, local, or topical administration of HA blocking peptide prevented hapten-induced Langerhans cell migration from the epidermis and inhibited the hapten-induced maturation of Langerhans cells in vivo (283). Small HA fragments of tetra- and hexasaccharide size increase dendritic cell production of the cytokines IL-1β, TNFα, and IL-12 as well as their allostimulatory capacity (425). These small HA fragments induce dendritic cell maturation independently of CD44 or RHAMM and are dependent upon TLR4 (423).

D. Mast cells

Mast cell granules are a rich source of HA, and this may account for the striking concurrence of HA accumulation with a mastocytotic condition in many tissues undergoing pathologic changes (84). Cultured human mast cells adhere to HA-coated surfaces (102). Human mast cells expressing standard form of CD44, but not the v5, v6, v7, and v8 variants, appears to mediate the attachment of these cells to HA (102). However, CD44 was not found to be consistently elevated in serum obtained from patients with mastocytosis or individuals experiencing anaphylaxis (102).

The number of mast cells was higher in patients with sarcoidosis than in controls (86). Sarcoidosis patients had significantly increased bronchoalveolar lavage (BAL) fluid concentrations of HA, fibronectin, and type III procollagen peptide, as well as albumin and lymphocytes, compared to controls (86). Mast cell activation and stress play a role in the pathogenesis of interstitial cystitis (363). Intravesical sodium hyaluronate has been used to treat interstitial cystitis due to its possible replenishment of bladder glycosaminoglycans (186). HA inhibited mast cell activation and the secretion of proinflammatory mediators, providing a mechanism of HA as a therapeutic option for interstitial cystitis (36).

B. Endothelial cells

Oligosaccharides of HA enhance the production of collagens by endothelial cells (354). Early-response gene signaling is induced by angiogenic oligosaccharides of HA in endothelial cells, while is inhibited by high molecular weight HA (74). HA fragments, but not high molecular weight HA, induce iNOS in liver, having the greatest effects on endothelial and Kupffer cells. Thus, HA fragments may be an important stimulus for NO production in various forms of liver disease, particularly as a cofactor with inflammatory cytokines (353).

Local cytokine production within inflamed vascular beds may enhance surface HA expression on endothelial cells, thereby creating local sites receptive to the CD44/HA interaction and thus extravasation of inflammatory cells (277). IL 15 induces endothelial HA expression in vitro and promotes activated T cell extravasation through a CD44-dependent pathway in vivo (91). Endogenous components of the extracellular matrix HA can stimulate endothelia to trigger recognition of injury in the initial stages of the wound defense and repair response (417). HA-induced proliferation of endothelial cells is CD44 receptor mediated and accompanied by early-response-gene activation (389). HARE/stabilin-2 is found on sinusoidal endothelial cells of the liver, lymph node, and spleen (496). HARE mediates the systemic clearance of HA from the circulatory and lymphatic systems, through a clathrin-dependent, coated pit-mediated uptake (497).

Hyaluronan fragments stimulate endothelial cell differentiation. Hyaluronan dodecasaccharides induce capillary endothelial cell sprouting by the binding to CD44 and by the induction of CXCL1/GRO1 (411). HA regulates vascular endothelial cell barrier function through CD44v10 isoform interaction with S1P receptors, S1P receptor transactivation, and RhoA/Rac1 signaling to the EC cytoskeleton (386). CD44 is an important regulator of HGF/c-Met-mediated in vitro and in vivo barrier enhancement, a process with essential involvement of Tiam1, Rac1, dynamin 2, and cortactin (387).

C. Fibroblasts

Fibroblasts are the main cell type that release HA (164, 242, 397, 429). Fibroblasts synthasize all three isoforms of HA synthases and release HA upon tissue injury (17, 255) and the stimulation of inflammatory factors such as IL-1β and TNFα (472). On the other hand, HA fragments stimulate fibroblasts to release cytokines and to regulate inflammatory responses (242, 255), and facilitate TGF-β-mediated fibroblast proliferation (270). Furthermore, upon myofibroblastic differentiation, more HA appeared in the conditioned medium and became associated with the cells (170). Moreover, HA modulates TGF-β dependent myofibroblast differentiation (456, 457).

Hyaluronan-binding proteins regulate fibroblast functions through their interactions with HA. For example, acute lung injury fibroblast migration and invasion of a fibrin matrix is mediated by CD44 (408). HA modulates contact inhibition of cell growth and migration (157). RHAMM deficient fibroblasts are defective in CD44-mediated ERK1/2 motogenic signaling, leading to defective skin wound repair, demonstrating a role of RHAMM in CD44 signaling and in fibroblast migration (430). A recent study suggested that HA-activated CD44 modulates PDGF receptor signaling by recruiting tyrosine phosphatases to the receptor and influences cell motility (234).

Consistent with the role of CD44 in mediating fibroblast invasion and subsequent tissue fibrosis, immunohistochemical analysis of lung tissue from patients who died from acute alveolar fibrosis after lung injury reveals CD44-expressing mesenchymal cells throughout newly formed fibrotic tissue (408). Anti-CD44 antibody blocked fibroblast migration on the provisional matrix proteins fibronectin, fibrinogen, and HA (408). Therefore, fibroblast CD44 functions as an adhesion receptor for provisional matrix proteins and is capable of mediating fibroblast migration and invasion of the wound provisional matrix resulting in the formation of fibrotic tissue (408). A recent study reported that high molecular weight HA increased Snail2 leading to fibroblast invasion (60).

B. HA and hematopoietic stem cells

The fate of hematopoietic stem cells (HSC) is determined by microenvironmental niches. Hyaluronan is part of the extracellular environment in bone marrow. HA not only provides a physical scaffold or support within the marrow to facilitate localization and retention of HSCs to the stem cell niche but moreover, through ligation with its counter-receptors is able to directly affect the cellular functions of HSCs (133).

HA is required for in vitro hematopoiesis, since the removal of HA with hyaluronidase in long-term bone marrow cultures results in reduced production of both progenitor and mature cells (193). HA enhanced hematopoietic activity, committed progenitors, and the total number of mature bone marrow cells (256). HA synthesized by primitive hemopoietic cells participates in their lodgment at the endosteum following transplantation (301). Therefore, HA is required for the regulation of the hematopoiesis-supportive function of bone marrow accessory cells and participates in hematopoietic niche assembly (256). In vivo exposure to cigarette smoke and in vitro treatment of long-term bone marrow cultures with nicotine, a major constituent of cigarette smoke, result in inhibition of hematopoiesis (193, 194, 256).

CXCL12/SDF-1 is a major chemokine in promoting HSC homing, migration, and proliferation (219, 269, 372). HA oligosaccharides enhance CXCL12-dependent chemotactic effect on peripheral blood hematopoietic CD34⁺ cells (367). HA-CD44 interaction is essential for homing into the bone marrow and spleen of non-obese diabetic/severe combined immunodeficient mice and engraftment by human HSCs (15). Hematopoietic progenitor cells migrating on HA toward a gradient of CXCL12 acquired spread and polarized morphology with CD44 concentrating at the pseudopodia at the leading edge. These morphologic alterations were not observed when the progenitors were first exposed to monoclonal anti-CD44 antibodies, demonstrating a crosstalk between CD44 and CXCR4 signaling (15).

Although RHAMM and CD44 are expressed by the mobilized blood hematopoietic progenitor cells, function blocking monoclonal antibodies identified RHAMM as a major HA binding receptor, with a less consistent participation by CD44. The G-CSF-associated alterations in RHAMM distribution and the RHAMM-dependent motility of hematopoietic progenitor cells suggest a potential role for HA and RHAMM in the trafficking of hematopoietic progenitor cells and the possible use of HA as a mobilizing agent in vivo (328).

B. Asthma

HA appears in low concentrations in BAL fluid from healthy individuals, while increased amounts have been reported in lavage fluid from patients with allergic asthma (358, 391). Furthermore, HA levels in BAL fluids were significantly increased in patients with persistent asthma, in comparison to patients with intermittent asthma (449), were correlated with the pulmonary function of the patients (39).

One of the reasons for increased HA levels in asthma is an increase in TGF-β levels which promote airway smooth muscle cells to secret glycosaminoglycans such as HA (30). IL-1β and TNFα were the most potent stimulators of HA synthesis and when combined, caused synergistic increases in HA accumulation in human lung fibroblasts. Fluticasone inhibited IL-1β and TNFα induced HA synthesis, and attenuated IL-1β and TNFα stimulated HA synthase-2 mRNA (472). On the other hand, low molecular weight HA increased GM-CSF, TGF-β and intercellular adhesion molecule-1 expression by eosinophils, leading to eosinophil survival. HA-eosinophil interaction may contribute to the regulation of airway inflammation and airway remodeling (313).

Fibroblasts from subjects with the most hyperresponsive airways in asthma produced much more total proteoglycans such as HA, perlecan, and versican than cells from subjects with less hyperresponsive or normal responsive airways (467). Hyaluronan, secreted from submucosal gland cells, plays a role in mucosal host defense by retaining lactoperoxidase and possibly other substances important for first line host defense at the apical surface ‘ready for use’ and protected from ciliary clearance (359).

Aerosol HA administration significantly reduces the bronchial hyper-reactivity to muscular exercise in asthmatics (326). Such an effect could be attributed to the correction of the pathological remodeling, one of the main features of asthma: a correction which could be attributed to the unique physicochemical properties of this major component of the loose connective amorphous matrix of the airways, which is undoubtedly involved in the remodeling process (326). Others found that inhaled HA does not significantly protect against exercise-induced bronchoconstriction in a randomized double-blinded placebo-controlled crossover study, suggesting that HA at the testing dose is not effective as a prophylaxis for exercise-induced bronchoconstriction in patients with asthma (214).

C. COPD/Pulmonary Emphysema

Young COPD patients with higher concentrations of HA and fibronectin in BAL had higher inflammatory cells in BAL, and lower in pulmonary function measurements, than the COPD patients with lower HA levels (393). HA levels were significantly higher in the sputum from patients with COPD than in those from controls. The COPD population appeared to consist of two subpopulations with either high or moderate HA levels. The subgroup of patients with high HA levels had lower FEV₁ than the moderate HA group (393). In addition, neutrophil influx and levels of IL-8, and the soluble TNF receptors were significantly higher in patients with high HA levels than in those with moderate HA levels and controls, indicating a relationship between HA levels, local inflammation and severity of disease, and suggesting enhanced degradation of HA in the lungs of patients with COPD (80). Hyaluronan, chondroitin 4-sulfate, and chondroitin 6-sulfate levels decreased significantly in animals exposed to ozone for 20 months when compared with those in control animals (338). Cigarette smoke exposure leads to enhanced deposition of mostly low molecular weight HA in alveolar and bronchial walls by altering the expression of HA modulating enzymes (40). However it is uncertain if the changes of these glycosaminoglycans contribute the pathogenesis of emphysema.

Aerosolized low molecular weight HA following endotoxin administration significantly increased lung inflammation, whereas pretreatment with HA had the opposite effect (288). Aerosolized HA may be an effective means of preventing pulmonary emphysema and perhaps other lung diseases that involve elastic fiber injury (48). Although clinical trials involving nebulized HA are not expected to yield a measurable treatment effect for at least several years, it is proposed that the special ability of this polysaccharide to retain water may increase the elasticity of lung elastic fibers, producing a relatively rapid improvement in pulmonary mechanics (49). Compared to untreated/smoked controls, aerosolized HA-treated animals showed statistically significant reductions in mean linear intercept and elastic fiber breakdown products in BAL fluid. The aerosolized HA showed preferential binding to elastic fibers, suggesting that it may protect them from injury (47). The effect of HA on inflammation appears to be related to its molecular size, with larger polysaccharide chains having anti-inflammatory activity and smaller ones having proinflammatory properties. The breakdown of inhaled HA into smaller fragments could possibly induce an inflammatory reaction in the lung that counteracts any beneficial effect. Consequently, the proposed therapeutic use of HA will require development of treatment strategies aimed at minimizing its proinflammatory activity (46).

B. Lupus Nephritis

Increased serum concentration of HA was noted in dermatomyositis patients (211). HA expression was increased in the mesangium, and in the periglomerular and tubular distribution in kidney biopsies of patients with lupus nephritis (490). Lupus nephritis patients showed increased levels of circulating HA, especially during active disease, which correlated with anti-DNA antibody titers (490). Fibroblasts derived from active lesions of nephrogenic fibrosing dermopathy synthesize elevated levels of HA when compared with normal controls (83). Given that HA plays a pivotal role during inflammatory responses, influences cellular behavior and assists in the recruitment of lymphocytes to sites of injury, it is likely that HA contributes to the pathogenesis of lupus nephritis (490).

The authors wish to thank the members of the Noble laboratory for their hard work in the areas of hyaluronan, innate immune and lung injury. This work has been supported by NIH grants HL060539, {"type":"entrez-nucleotide","attrs":{"text":"AI052201","term_id":"3308192","term_text":"AI052201"}}AI052201 and {"type":"entrez-nucleotide","attrs":{"text":"HL084917","term_id":"1051655325","term_text":"HL084917"}}HL084917.

GRANTS

This work was supported by the National Institutes of Health grants R01-HL 060539, R01 AI052201, and RO1 HL 077291.