Successful repair after tissue injury and inflammation requires resolution of the inflammatory response and removal of extracellular matrix breakdown products. We have examined whether the cell-surface adhesion molecule and hyaluronan receptor CD44 plays a role in resolving lung inflammation. CD44-deficient mice succumb to unremitting inflammation following noninfectious lung injury, characterized by impaired clearance of apoptotic neutrophils, persistent accumulation of hyaluronan fragments at the site of tissue injury, and impaired activation of transforming growth factor-beta1. This phenotype was partially reversed by reconstitution with CD44+ cells, thus demonstrating a critical role for this receptor in resolving lung inflammation.
CD44 is a major cell-surface receptor for hyaluronic acid (HA), a glycosaminoglycan component of extracellular matrix. HA-CD44 interactions have been implicated in leukocyte extravasation into an inflammatory site. This study examined the role of CD44 in acute inflammatory responses during pneumonias induced by Escherichia coli and Streptococcus pneumoniae using CD44-deficient mice. In E. coli-induced pneumonia, neutrophil accumulation in the lungs and edema formation was increased by 84% and 88%, respectively, in CD44-deficient mice compared to wild-type mice. In contrast, no difference was observed between these genotypes in S. pneumoniaeinduced pneumonia, and the HA content in the lungs decreased after instillation of S. pneumoniae, but not E. coli, in both genotypes. Studies to determine the mechanisms for this enhanced response showed that: 1) neutrophil apoptosis was not different between these two genotypes in either type of pneumonia; 2) CD44 deficiency resulted in enhanced mRNA expression of several inflammatory genes; and 3) CD44-deficient neutrophils migrated through Matrigel in response to chemoattractants faster and in greater numbers than wild-type neutrophils in vitro and this increase was in part dependent on HA content in the Matrigel. These data demonstrate that CD44 deficiency results in enhanced inflammation in E. coli but not S. pneumoniae-induced pneumonia, suggesting a previously unrecognized role for CD44 in limiting the inflammatory response to E. coli. Neutrophil accumulation at inflammatory sites is an important feature of acute inflammatory responses. During pulmonary inflammation, circulating neutrophils become sequestered within pulmonary capillaries, a process that does not require rolling. Neutrophils then migrate across the endothelium and through the pulmonary interstitium, which contains fibroblasts and extracellular matrix proteins such as collagens and proteoglycans.1-4 Finally, neutrophils migrate between alveolar epithelial cells, often between a type II and a type I pneumocyte, into the airspace. 1,5 CD44 is a type I transmembrane glycoprotein that is expressed by most cell types, including leukocytes, and is the major cell surface receptor for hyaluronan (HA). HA is a nonsulfated glycosaminoglycan component of the extracellular matrix and plays a major role in maintenance of tissue integrity.6 -8 CD44 has 10 different splice variants and neutrophils express the standard CD44 isoform, CD44s.9 CD44 may modulate immunological and inflammatory responses through at least two mechanisms. First, CD44 may play important roles in modulating leukocyte extravasation. Interaction between CD44 and HA is implicated in lymphocyte rolling and extravasation, and optimal binding of these two molecules is modulated by proinflammatory cytokines such as tumor necrosis factor-␣.10 -14 The role of CD44 in mediating neutrophil emigration during acute inflammatory responses is less well understood. Second, CD44 is capable of inducing signal transduction pathways and cell activation. Ligation of C...
Hyaluronan, a linear polysaccharide, is accumulated in lung interstitium during different pathological conditions, causing interstitial edema and thereby impaired lung function. We investigated the mechanism of local hyaluronan turnover during the early phase of bleomycin-induced fibrotic lung injury in rats. The binding of [3H]hyaluronan to alveolar macrophages (AM) established from bleomycin-treated rats 1 and 5 days after induction of injury was decreased 8- and 15-fold, respectively, compared with that of AM from saline-treated control counterparts, but at day 14 returned almost to the normal level. Data was confirmed by quantitative cytochemistry, using fluorescein-labeled hyaluronan. Analysis of the expression of CD44, a receptor for hyaluronan, by Western blotting revealed a 30% increase of CD44 molecules expressed on AM from bleomycin-treated rats at day 5 compared with control rats. In particular a lower molecular mass form of CD44 appeared. No expression of the receptor for hyaluronan-mediated motility (RHAMM) could be detected. The internalization and degradation of [3H]hyaluronan by AM, obtained from bleomycin-treated rats at days 1, 5, and 14, were decreased about 65%, 35%, and 30%, respectively, compared with AM from the control rats. The AM lysosomal hyaluronidase activity did not differ significantly between bleomycin-treated and control rats. Our results indicate that a decreased hyaluronan binding capacity of AM may account for the impairment of internalization and thereby degradation of excessive hyaluronan during the early phase of fibrotic lung injury.
Since the first identification of a vasoconstrictor activity present in endothelial cell supernatants (1) and the subsequent isolation of endothelin (ET)-1 in 1988 (2), there has been tremendous interest in the biology of endothelins. In addition to ET-1, further studies have demonstrated the existence of two other endothelins, ET-2 and ET-3, which differ from ET-1 with two and six amino acid residues, respectively (3). The family of endothelins has been an object of intensive research for scientists from many disciplines. The majority of research has focused on the cardiovascular system (for review see References 4 and 5), but there has also been a significant accumulation of data indicating the mediator role of endothelins in a variety of lung disorders (for further review see References 6 and 7). However, despite intense investigation and the identification of ET-1 expression under a number of pathologic conditions related to the lungs, such as pulmonary vascular disease, asthma, and pulmonary fibrosis, a direct link between ET-1 activity and a disease state has been elusive. This Perspective will focus on aspects of endothelin biology that pertain to the pulmonary system. Endothelins are synthesized from precursors known as preproendothelins (ppET), comprising 212 amino acid residues. The large precursors undergo an intermediate cleavage by endopeptidases to form the 38-amino-acid biologically inactive proendothelins, also called big endothelins. Endothelin-converting enzymes are membrane-bound metalloendopeptidases that further cleave proendothelins. The biologically active endothelins are 21-amino-acid peptides, with two disulfide bridges joining cysteins in positions 1-15 and 3-11 in the N-terminal half, and a cluster of hydrophobic amino acid residues at the C-terminal end of the structure. The structure of the N-terminal domain determines the affinity to the receptor, while the C-terminal domain contains the binding site of the peptide to the receptor (for review see Reference 8).Most tissues contain more ET-1 than ET-2 or ET-3, with the highest levels of ET-1 found in lung. ET-1 is secreted by endothelial cells (2, 9), epithelial cells (10, 11), alveolar macrophages (12, 13), polymorphonuclear leukocytes (14), and fibroblasts (15). Release of endothelins is (
The molecular mechanisms behind the accumulation of hyaluronan during bleomycin-induced lung injury in rats were investigated. The stimulatory effects of bronchoalveolar lavage fluid (BALF) and alveolar macrophage (AM)-conditioned media on hyaluronan synthesis in normal rat lung fibroblast cultures were studied as well as the hyaluronan binding activity on AM. BALF obtained on days 1 and 5 after bleomycin instillation exhibited hyaluronan stimulatory activity similar to that of 10% fetal serum; the activity returned to control values on day 14 after bleomycin treatment. Conditioned media from cultures of AM obtained from bleomycin-treated rats exhibited stimulatory effects higher than that of media from AM of control rats and equal to or higher than that of 10% fetal calf serum. The stimulatory activity in BALF was significantly inhibited by neutralizing antibodies against transforming growth factor-beta; the activity in AM-conditioned media was only partially affected. Neutralizing antibodies against platelet-derived growth factor-BB or -AA had no such inhibiting effect. Interestingly, AM from bleomycin-treated rats exhibited low hyaluronan binding activity. [3H]Hyaluronan binding by AM on days 1 and 5 after bleomycin administration was about 2-fold and 4-fold lower, respectively, compared with that by AM derived from saline-treated rats. This decrease was normalized 14 days after bleomycin treatment. In conclusion, our results indicate that factors with high potential to stimulate hyaluronan synthesis in rat lung fibroblasts are accumulated in BALF from bleomycin-treated rats and that AM are likely to be one source of such stimulatory factors.(ABSTRACT TRUNCATED AT 250 WORDS)
The composition and permeability of the pericellular coat surrounding normal human mesothelial (NHM) cells have been studied in vitro. NHM cells were grown in the presence of 3H-glucosamine and the amount of label recovered in hyaluronan and chondroitin sulfate was determined after selective enzymatic digestion of the polysaccharides in medium, pericellular, and intracellular pools. For comparison a similar analysis was carried out on mesothelioma cells (Mero-14). Of the labeled polysaccharides in the medium and pericellular pools of NHM cells about 80-90% could be ascribed to hyaluronan and only 3-5% to chondroitin sulfate. In contrast, Mero-14 synthesized only minute amounts of hyaluronan whereas chondroitin sulfate corresponded to 61% of the total glycosaminoglycans in the culture. The results exclude a structure of the pericellular layer of NHM cells similar to the hyaluronan-proteoglycan aggregates found in cartilage. The permeability of the pericellular layer was tested by the exclusion of polystyrene microspheres and bacteria of diameter 0.1-3.0 microns, as well as erythrocytes of diameter 7 microns. While the erythrocytes were excluded the smaller particles penetrated the coat. By adding 0.5 mg/ml of aggregating cartilage proteoglycan to the medium particles of 0.3 microns or larger were also excluded. Thus exogenous proteoglycans can reinforce the structure of the pericellular layer.
Cell lines established from human malignant mesotheliomas, but not from normal mesothelial cells, have been shown possess hyaluronan receptors, and to secrete factors that stimulate hyaluronan production by fibroblasts and normal mesothelial cells. In the present study we investigated the generality of this observation, namely the presence of hyaluro nan receptors and factors which induce stimulation of hyaluronan synthesis in primary mesothelioma and mesothelial cell cultures. Functionally active hyaluronan‐binding sites on the surface of malignant mesothelioma cells in primary cultures, established from pleural effusions of 3 different patients, were demonstrated using 3H‐hyaluronan. Primary cultures of normal mesothelial cells from non‐mesothelioma effusions did not exhibit any binding ability. Pleural fluids from mesothelioma patients both stimulated hyaluronan synthesis and promoted proliferation of normal mesothelial cells to a larger extent than non‐mesothelioma fluids. The hyaluronan‐stimulatory activity was only slightly neutralized by antibodies against PDGF‐BB TGF‐8; antibodies against bFGF had no effect. Although the concentration of hyaluronan was much higher in pleural fluids from mesothelioma than from non‐mesothelioma patients, molecular weight was almost the same. The hyaluronan‐binding capacity of early‐passage mesothelioma cells derived from pleural effusions can be an additional marker, in combination with other diagnostic tools, to distinguish between mesothelioma and mesothelial cells.
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