TSG-6 Potentiates the Antitissue Kallikrein Activity of Inter–α-inhibitor through Bikunin Release

Abstract

TSG-6 (the protein product of TNF-stimulated gene-6), an inflammation-associated protein, forms covalent complexes with heavy chains (HCs) from inter-␣-inhibitor and pre-␣-inhibitor and associates noncovalently with their common bikunin chain, potentiating the antiplasmin activity of this serine protease inhibitor. We show that TSG-6 and TSG-6·HC complexes are present in bronchoalveolar lavage fluid from patients with asthma and increase after allergen challenge. Immunodetection demonstrated elevated TSG-6 in the airway tissue and secretions of smokers. Experiments conducted in vitro with purified components revealed that bikunin·HC complexes (byproducts of TSG-6·HC formation) release bikunin. Immunoprecipitation revealed that bikunin accounts for a significant proportion of tissue kallikrein inhibition in bronchoalveolar lavage after allergen challenge but not in baseline conditions, confirming that bikunin in its free state, but not when associated with HCs, is a relevant protease inhibitor in airway secretions. In primary cultures of differentiated human airway epithelial and submucosal gland cells, TSG-6 is induced by TNF-␣ and IL-1␤, which suggests that these cells are responsible for TSG-6 release in vivo. Bikunin and HC3 (i.e., pre-␣-inhibitor) were also induced by TNF-␣ in primary cultures. Our results suggest that TSG-6 may play an important protective role in bronchial epithelium by increasing the antiprotease screen on the airway lumen.

Determining the Molecular Basis for the pH-dependent Interaction between the Link Module of Human TSG-6 and Hyaluronan

“…a salt concentration much higher than physiological). Here we have shown that the pH-dependent component of the TSG-6-HA interaction relies upon the change in ionization state of His 4 , and therefore it is to be expected that the pH dependence will not be observed in a salt concentration as high as 0.5 M NaCl because this would severely weaken all ionic interactions. We also note that a study using confocal-fluorescent recovery after photobleaching (FRAP), has also demonstrated that the pH dependence of the Link_TSG6 interaction with polymeric HA occurs in solution.…”

“…His 45 as well as His 4 ). As described under "Results," it seems likely that proteinprotein interactions occur between TSG-6 Link modules when the protein associates with HA chains containing multiple binding sites and that such interactions might involve His 4 . We have found previously that Link_TSG6 (and the full-length protein) can form stable complexes with polymeric HA that have enhanced binding to CD44 on leukocyte cell lines compared with free HA (12,38); these complexes, which may work through the induction of receptor clustering, are presumed to correspond to cross-linked hyaluronan fibers, which are formed when there is sufficient TSG-6 to saturate all possible protein-binding sites on the HA and when the HA is above a threshold concentration.…”

TSG-6 Protein Is Crucial for the Development of Pulmonary Hyaluronan Deposition, Eosinophilia, and Airway Hyperresponsiveness in a Murine Model of Asthma

“…Following segmental allergen challenge of asthmatic individuals, TSG-6 (in addition to the Th2 cytokines IL-3, IL-4, and IL-5) is one of 24 genes that are induced by 4-fold or more (36). Additionally, TSG-6 and TSG-6-HC complexes are also found in the BAL fluid of asthmatics after segmental allergen challenge (37). Nevertheless, how TSG-6 contributes to the pathogenesis of inflammatory responses in asthma has not been well defined.…”

“…Furthermore, because TSG-6 catalyzes the transesterification reactions that form HA-HC complexes (34,35,37), we speculated that the absence of endogenous TSG-6 would affect the formation of these HA-HC complexes. We used Streptomyces hyaluronidase to degrade HA and to dissociate HA-associated molecules, including HCs from pulmonary tissue obtained from WT and TSG-6 Ϫ/Ϫ animals undergoing OVA challenge.…”

“…The lack of this transesterification of HCs destabilizes the HA matrix of the cumulus celloocyte complexes and contributes to the infertility of female TSG-6 Ϫ/Ϫ mice (33)(34)(35). In asthmatics, both TSG-6 mRNA expression and TSG-6-HC complexes have been shown to increase after allergen challenge (36,37), suggesting that TSG-6 may play an important role in the asthmatic airway. Nevertheless, how TSG-6 contributes to the pathogenesis of inflammatory responses in asthma remains to be defined.…”