As the tissue microenvironment plays a critical role in regulating inflammation, it is increasingly clear that extracellular matrix degradation products are not only the result of inflammation, but also active participants in the perpetuation of the inflammatory process. LMW HA fragments are able to initiate innate immune responses via engagement of TLRs, further inducing an inflammatory response (2
). Opposing this inflammation, adenosine, which is released into the extracellular space during inflammation and tissue destruction, acts as a negative regulator of both inflammation and immune-mediated tissue destruction (12
). The anti-inflammatory properties of adenosine have been shown to be mediated by the A2aR (26
). In this article, we demonstrate for the first time a mechanism by which fragments of the extracellular matrix component HA can further augment inflammation by down-regulating the anti-inflammatory A2a receptor. This HA-mediated receptor inhibition is independent of TLRs, but is dependent on the HA receptor, CD44, and PKC. Furthermore, in an in vivo
model of lung inflammation, blocking LMW HA prevents the down-regulation of A2aR expression. We propose a novel mechanism by which the extracellular matrix, in the form of LMW HA fragments, plays an important role in modulating the magnitude and quality of an immune response via interaction with the adenosine A2aR.
Endogenous ligands, such as LMW HA fragments, released at the site of tissue injury, have the ability to activate the innate immune system and act as “danger signals” (2
). HMW HA is broken down into LMW species that promote inflammation by inducing the release of reactive oxygen species, cytokines, chemokines, and destructive enzymes, and facilitating the recruitment of CD4+
). Whereas HMW HA acts to maintain homeostasis and potentially down-regulate inflammation, the generation of LMW HA fragments may act as an endogenous danger signal, leading to the activation of both innate and acquired immunity (2
). The fact that lack of clearance of LMW HA leads to excess damage, whereas overexpression of HMW HA is protective in the noninfectious bleomycin-induced lung injury model, supports this hypothesis (20
). In addition, administration of LMW HA fragments directly into murine lungs has recently been shown to induce inflammation (28
). Normally, inflammation is self-limiting, and the biologically active LMW HA fragments are removed as healing occurs. However, in states of ongoing inflammation and fibrosis, such as sarcoidosis, chronic bronchitis, and idiopathic pulmonary fibrosis, there is ongoing tissue destruction and remodeling, leading to persistence of HA degradation products (29
). Similarly, during inflammation and tissue destruction, cells release adenosine, which acts to mitigate the inflammation via the A2aR (26
). In fact, we have previously demonstrated that engagement of the A2aR inhibits LMW HA–induced inflammatory genes and augments antifibrotic genes (18
). Now we demonstrate that HA fragments can circumvent the anti-inflammatory effects of adenosine by directly down-regulating A2aR expression on inflammatory cells. Thus, the extent and degree of inflammation is not only dictated by the nature and extent of tissue injury/insult, but also by the balance between tissue-derived adenosine, A2aR modulation of LMW HA–induced inflammatory genes, as well as by LMW HA inhibition of A2aR function.
It is known that HA binds to several receptors, such as CD44, TLRs, and receptor for HA-mediated motility (CD168) (2
). The CD44 family of cell surface glycoproteins consists of numerous isoform receptors with variable HA-binding characteristics that are cell and tissue dependent. Indeed, HMW HA–CD44 interactions have been implicated in leukocyte trafficking, tissue repair, and tumor metastasis (32
). However, it is not at all clear what role, if any, the HA-CD44 interaction play in LMW HA–induced inflammatory gene regulation. We have shown that macrophages from CD44-null mice respond similarly to WT macrophages upon stimulation with LMW HA in terms of inflammatory gene expression (2
). However, CD44 is important in the clearance of LMW HA by macrophages in vivo
, as evident in CD44-null mice that have increased bleomycin-induced lung injury due to the increased accumulation of LMW HA in the lung (27
). This increased lung injury was partially reversed by transplant of the CD44-null mice with WT bone marrow that were capable of clearing the HA (27
). The ability of LMW HA to induce inflammatory gene expression has been shown to be dependent on TLR-dependent signaling (20
). Recently, we have demonstrated that LMW HA binds specifically to TLR2 in macrophages and induces inflammatory gene expression via a MyD88-, IRAK-, TRAF6-, PKCζ-, and NF-κB–dependent pathway (2
). Importantly, the induction of inflammatory genes by LMW HA was independent of CD44 expression. Here, we demonstrate a dissociation between two important proinflammatory roles of HA fragments: the TLR-dependent HA fragment induction of inflammatory genes, and TLR-independent, CD44-dependent down-regulation of the anti-inflammatory A2aR.
The role of adenosine in lung inflammation is emerging as being both important and complex. One of the mechanisms whereby A2aR on macrophages are considered to exert an anti-inflammatory effect is through the up-regulation of IL-10 production, and our data suggest that A2aR-induced IL-10 would be decreased in the presence of LMW HA. Adenosine deaminase–null mice, which lack the enzyme that metabolizes adenosine, have massive accumulation of adenosine in their lungs (35
). In these mice, too much adenosine results in nonspecific engagement of all the adenosine receptors (both pro- and anti-inflammatory), resulting in excessive inflammation (35
). If adenosine deaminase is only partially knocked out, the mice accumulate adenosine slowly over time, and develop a progressive lung fibrosis with increased myofibroblasts and collagen deposition, presumably by overstimulation of the profibrotic A2bR (36
). In these models, it is unclear which of the adenosine receptors are the culprits, although adenosine deaminase/A1 double-knockout mice have increased inflammation (38
). In addition, IL-13 transgenic mice, which have increased A1, A2b, and A3, but not the anti-inflammatory A2aR, have recently been shown to have high levels of lung adenosine and increased lung inflammation (39
). These data are consistent with the previously described proinflammatory role of A2bR in adenosine-dependent lung inflammation via stimulation of fibroblast proliferation, matrixmetallo proteinase (MMP)-2 activity, and collagen deposition (40
). Interestingly, whereas A2b has been shown to have proinflammatory activity in airway epithelial cells, A2aR stimulation in lung epithelial cells has been shown to promote wound healing (42
). Furthermore, numerous studies have demonstrated the anti-inflammatory, antifibrotic role of A2aR stimulation in preventing toxin-induced hepatic fibrosis, allergen, or tobacco-induced lung inflammation, and acute lung injury after hemorrhagic shock (44
). Thus, these data clearly define a role for adenosine in modulating lung inflammation and fibrosis, and our data identify a role for augmentation of LMW HA–induced inflammation and fibrosis by down-regulating the A2aR function.
An understanding of the pathways involved in extracellular matrix–induced inflammation may lead to potential targets of pharmacologic intervention. We believe that our studies provide important preclinical data supporting the development of A2aR-specific agonists for the treatment of inflammation. Likewise, blocking PKCα or CD44 could be beneficial as ancillary approaches in preventing inflammation. In addition, given the ubiquitous nature of HA and adenosine, our studies could be generalized to many inflammatory diseases, such as arthritis, hepatitis, myocarditis, and atherosclerosis.