In the present study, we demonstrate increased synthesis and expression of the promigratory molecule RHAMM in macrophages accumulating in areas of the lung injured by intratracheal instillation of bleomycin. Elevated expression of RHAMM occurred in association with increased macrophage motility observed after injury. In addition, using antibody inhibition, we show that cell surface RHAMM is not only necessary for this increased motility, but also contributes to lung macrophage accumulation after injury. Taken together with our previous studies demonstrating the ability of HA-binding peptide to decrease inflammation and fibrosis after bleomycin injury (17
), the results of the current study suggest that RHAMM:HA interactions play a critical role in the recruitment of inflammatory cells after lung injury by bleomycin.
HA exerts direct effects on cells and on the extracellular matrix that may be relevant to its role in wound repair. Thus, the proinflammatory cytokines TNF-α and IL-1β have been shown to induce cell surface expression of HA (41
), which in turn promotes adhesion of leukocytes to the endothelium (41
). Further, HA has been shown to stimulate the migration and proliferation of smooth muscle cells (29
), fibroblasts (27
), immune cells (15
), and endothelial cells (43
). HA also activates monocytes into macrophages (45
), and increases cytokine gene expression by macrophages (25
) and fibroblasts (46
). Increased accumulation of HA is associated with inflammation after acute injury to several organ systems including the lung (reviewed in Refs. 10 and 31). However, recent studies have suggested that HA promotes inflammation. Thus, HA-binding peptide specifically blocks macrophage motility in vitro
and, when systemically administered to rats during bleomycin injury, results in decreased alveolar macrophage motility and accumulation with subsequently reduced collagen content in the lung (17
). In rats, subcutaneous injection of HA-binding peptide before mechanical ventilation blocks neutrophil influx in the lung (47
). Importance of HA to inflammation and the likely universal nature of this response was confirmed by Mummert and colleagues (16
). In a model of contact hypersensitivity, this group demonstrated that systemic, local, or topical administration of a different HA-binding peptide to reactive hapten-sensitized mice blocked skin-directed homing of inflammatory leukocytes. Together, these studies suggest that the interaction of HA with its receptors precedes and promotes the inflammatory response to injury.
Several other authors have also demonstrated increased cell surface RHAMM expression in leukocytes (48
). Further, we have previously shown that inflammatory cell chemotaxis and random migration is dependent on RHAMM (15
). Interestingly, RHAMM expression is unaltered in CD44 null mice that exhibit increased inflammation after collagen-induced arthritis and anti-RHAMM antibody blocks the inflammatory cell influx in the absence of CD44 (51
). Thus, RHAMM:HA interactions may be responsible for leukocyte transmigration through the endothelium and for chemotaxis within injured areas of the lung. However, other reports have noted an absence of cell surface RHAMM after injury. Teder and colleagues (52
) proposed two mechanisms for the increased accumulation of HA after bleomycin injury in rats. First, they showed that fibroblasts exposed to BAL from injured animals increase their production of HA, a response that was largely abrogated by blocking antibodies to TGF-β1 (52
). Further, alveolar macrophages obtained 5 d after bleomycin injury bound less [3
H]-hyaluronan than those from saline-treated controls, suggesting lower HA receptor expression in macrophages after injury (53
). This lower expression of HA receptors was also thought to contribute to elevated HA, since less HA would be internalized by these cells. Indeed, in their study, no RHAMM could be detected on the surface of these cells. Further, Weiss and coworkers (9
) examined the HA-binding properties of activated monocytes. Immunostaining of skin samples from allergic contact dermatitis found little HA on vascular endothelial cells or activated lymphocytes and no RHAMM expression in monocytes infiltrating sites of cutaneous inflammation. In vitro
activation of peripheral blood monocytes by tissue culture, plastic adhesion, and treatment with LPS and IFN-γ did increase HA binding, but did not upregulate RHAMM expression. Instead, FACS analysis showed that activated monocytes expressed increased cell surface CD44, coinciding with increased CD44 mRNA and protein (9
The lack of increased HA receptor expression in macrophages after bleomycin injury as reported by Teder and colleagues (53
) is contradictory to other reports and the data presented in the current study. For instance, the expression of the standard form of CD44 (CD44 s) is increased in alveolar macrophages and in the interstitium of the lung in areas of thickened alveolar wall, while the expression of CD44v6, thought to be the epithelial form, is decreased in type II pneumocytes (54
). Further, CD44, presumably acting thorough its ability to bind HA, has been implicated in novel rolling and adhesion pathways leading to leukocyte adhesion to activated endothelium (41
). Further, the recruitment of lymphocytes to the lung after antigenic challenge to sensitized mice could be blocked using an anti-CD44 antibody (55
Thus, there are conflicting data with respect to increased expression of CD44 and RHAMM after lung injury. It is unclear why these differences in observations exist. However, it is relevant to this discrepancy that we have noticed that the increased motility observed in macrophages isolated from bleomycin-injured animals is rapidly lost after 2 h following culture (data not shown). Since Teder and colleagues (53
) examined macrophages after 24 h in culture, the decreased surface expression of HA receptors observed could be the result of downregulated cell surface expression of HA receptors.
Upregulation of growth factors such as TNF and TGF-β after bleomycin-induced lung injury coincides with increased HA production (59
). In lungs injured by bleomycin, the increased expression of HA receptors in macrophages coincides with the temporal increase in TGF-β1 expression observed in the same cells after similar injury (8
). This similarity in expression suggests in vivo
regulation of HA receptors in macrophages by TGF-β1. In fibroblasts, the expression of RHAMM has been shown to be regulated by TGF-β1 by a mechanism that involves increased message stability (61
), and the RHAMM:HA interaction is required for TGF-β1–stimulated increases in cell locomotion (62
). TGF-β1 may thus be responsible for an autocrine system in which production of this growth factor results in upregulation of HA and its receptors in macrophages and a paracrine effect on fibroblasts to produce HA and increased collagen deposition, thereby influencing both inflammation and fibrosis after injury. Our data, however, showing a decrease in macrophage accumulation with HA-binding peptide treatment (17
), and with anti-RHAMM antibody treatment (this study) suggests that RHAMM: HA interactions are, at least in part, responsible for the inflammatory response to acute lung injury.
In summary, the results of this study suggest that HA and RHAMM are critical components of the inflammatory and fibrotic processes resulting from acute lung injury and that these molecules represent novel targets to limit the adverse consequences of lung injury in humans.