Small hyaluronan oligosaccharides induce inflammation by engaging both toll-like-4 and CD44 receptors in human chondrocytes

Abstract

Small degradation fragments of hyaluronan (HA) may stimulate an inflammatory response in a variety of tissues at the injury site. HA oligosaccharides are endogenous ligands for the cluster determinant 44 (CD44) receptor as well as for toll-like receptor 4 (TLR-4). Previous data have shown that HA fragments may induce pro-inflammatory cytokine expression by interacting with both the CD44 receptor and TLR-4. CD44 and TLR-4 stimulation activates different inflammatory pathways that culminate with the activation of the transcriptional nuclear factor kappaB (NF-κB) which is responsible for the expression of inflammation mediators such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1 beta (IL-1β). The aim of this study was to investigate the inflammatory effects of very small HA oligosaccharides on both TLR-4 and CD44 involvement in normal human articular chondrocytes.

Adding HA fragments to chondrocyte cultures up-regulated CD44 and TLR-4 expression, activated NF-κB translocation and increased the pro-inflammatory cytokines TNF-α, IL-6 and IL-1β.

The addition of a specific CD44 blocking antibody reduced CD44 and all inflammatory cytokine expression as well as protein production. However, cytokine expression remained significantly higher than in untreated chondrocytes. TLR-4 expression was not affected. The treatment with TLR-4 blocking antibody decreased TLR-4 and inflammatory cytokine expression, although cytokine expression was significantly higher than in control cells. CD44 expression was unaffected. The addition of both CD44 and TLR-4 blocking antibodies significantly reduced CD44, TLR-4 and inflammatory cytokine expression.

Introduction

Toll-like receptors are a class of proteins that play a key role in the innate immune system. They recognize structurally conserved molecules derived from microbes. Once these microbes cross physical barriers such as the skin or intestinal tract mucosa, they are recognized by TLRs which activates the innate immune response [1]. Although each TLR recognizes specific ligands, they may trigger other different molecules. Recently, the glycosaminoglycan (GAG) hyaluronan (HA) has also been identified as an inducer of toll-like receptor 4 (TLR-4) activation, capable of causing the release of pro-inflammatory cytokines [2], [3].

ECM is a complex mixture of proteins, glycoproteins, proteoglycans (PGs) and GAGs. ECM plays several biological roles, in particular mechanical strength and also provides signals affecting cell adhesion, shape, migration, proliferation, survival and differentiation [4], [5]. ECM presents many domains that become active after proteolytic or glycolytic cleavage. These active ECM fragments are called matrikines and they may play different roles, in particular they may act as potent inflammatory mediators including collagen type one and four, elastin, fibronectin, laminins, entactin/nidogen, thrombospondin, HA [5]. Among these, HA is the most widely studied because of its varying activity depending on the different state of aggregation. HA may exist both as a soluble polymer as well as in complexes with non-covalently linked proteins called HA-binding proteins or hyaladherins. HA exists as a high molecular weight polymer (10⁶ Da) under physiological conditions. However, under inflammatory conditions HA has been shown to have greater polydispersity in size, with a preponderance of low molecular weight forms, especially after tissue injury [6]. HA functions are determined in part by the size of the molecule, in addition to the structure and its interaction with HA-binding proteins.

The cluster determinant 44 (CD44) is the best-known receptor of HA [7]. CD44 is a transmembrane glycoprotein that is widely distributed on leukocytes and several other cell types. CD44 stimulation with HA plays a role in various physiological functions, e.g. cell adhesion, cell–substrate interaction, and lymphocyte recruitment, as well as in pathological processes such as inflammation and metastasis [8]. Previous studies have reported that the separate stimulation of TLR-4 and CD44 receptors may prime pro-inflammatory intermediates through nuclear factor kappaB (NF-κB) activation [9], [10].

It has also been reported that fragments of HA or HA at low molecular weight are able to interact with TLR-4 and CD44 receptors, thereby stimulating inflammation or increasing the inflammatory mechanism previously induced by other agents in different cell types [11], [12], [13], [14], [15], [16]. Hence, the generation of lower molecular weight HA in pathologies may act as an endogenous danger signal, leading to the activation of both innate and acquired immunity.

As, in two separated experiments, we previously reported that low molecular weight HA (50 kDa) significantly activated NF-κB-induced transcription of pro-inflammatory mediators [15], [16] by stimulating TLR-4 or CD44 receptors, the aim of the present study was to evaluate whether the inflammation stimulated by low molecular weight HA or other small HA fragments could be the result of the activation of both TLR-4 and CD44 receptors. For this purpose we treated normal human chondrocytes with a specific HA oligosaccharide containing only six monosaccharides (6-mer) since previous reports [17], [18] had shown that HA at this size was able to stimulate inflammation by interacting with both receptors TLR-4 and CD44. We also evaluated the degree of inflammation by using specific selective blocking antibodies.