Role of Hyaluronan in Inflammatory Effects on Human Articular Chondrocytes

Mary K Cowman,Claire Shortt,Jai Rathore,Yuhong Fu,Jemma Villavieja,Gyu Ik Jung,Thorsten Kirsch,Shivani Arora,Xiayun Huang,Tatini Rakshit

doi:10.1007/s10753-019-01043-9

Abstract

Hyaluronan (HA) fragments have been proposed to elicit defensive or pro-inflammatory responses in many cell types. For articular chondrocytes in an inflammatory environment, studies have failed to reach consensus on the endogenous production or effects of added HA fragments. The present study was undertaken to resolve this discrepancy. Cultured primary human articular chondrocytes were exposed to the inflammatory cytokine IL-1β, and then tested for changes in HA content/size in conditioned medium, and for the expression of genes important in HA binding/signaling or metabolism, and in other catabolic/anabolic responses. Changes in gene expression caused by enzymatic degradation of endogenous HA, or addition of exogenous HA fragments, were examined. IL-1β increased the mRNA levels for HA synthases HAS2/HAS3 and for the HA-binding proteins CD44 and TSG-6. mRNA levels for TLR4 and RHAMM were very low and were little affected by IL-1β. mRNA levels for catabolic markers were increased, while type II collagen (α1(II)) and aggrecan were decreased. HA concentration in the conditioned medium was increased, but the HA was not degraded. Treatment with recombinant hyaluronidase or addition of low endotoxin HA fragments did not elicit pro-inflammatory responses. Our findings showed that HA fragments were not produced by IL-1β-stimulated human articular chondrocytes in the absence of other sources of reactive oxygen or nitrogen species, and that exogenous HA fragments from oligosaccharides up to about 40 kDa in molecular mass were not pro-inflammatory agents for human articular chondrocytes, probably due to low expression of TLR4 and RHAMM in these cells.

Highlights

The effects of changes in the molecular mass (M) of hyaluronan (HA) in the pericellular environment during inflammatory processes are still incompletely understood
We first confirmed that treatment of our human articular chondrocyte cultures with IL-1β, one of the main catabolic and inflammatory cytokines in OA, resulted in the expected marked increase in messenger RNA (mRNA) levels of catabolic and inflammatory markers, including Cox-2, IL-6, iNOS, and MMP-13, and a decrease in expression of anabolic cartilage markers, including aggrecan and type II collagen (α1(II)) [4, 22, 28, 38, 39]
In agreement with previous reports [40, 41], we observed the mRNA levels for the hyaluronan synthases HAS2 and HAS3 to be increased by IL-1β

Summary

INTRODUCTION

The effects of changes in the molecular mass (M) of hyaluronan (HA) in the pericellular environment during inflammatory processes are still incompletely understood. Via the receptors CD44 and/or ICAM-1, high M HA reduces the catabolic effects of interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), and lipopolysaccharide (LPS) [4,5,6] It shields the cell from reactive oxygen and nitrogen species (ROS/RNS) generated under inflammatory conditions, by serving as a first target [7]. We hypothesized that quantification and control of the size and concentration of HA fragments, as well as characterization of the expression levels of HA binding proteins, receptors, and synthetic/degradative enzymes would aid in clarifying the potential signaling role of HA fragments in articular chondrocytes. Cultures of primary human articular chondrocytes were exposed to the inflammatory cytokine IL-1β, and tested for changes in HA content/size in conditioned medium, and for the expression of genes important in HA binding or metabolism, and in other catabolic/anabolic responses. The results show that HA fragments are not pro-inflammatory in cultured human articular chondrocytes that express low levels of the TLR4 and RHAMM receptors

MATERIALS AND METHODS

RESULTS

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