Deficiency in the serum-derived hyaluronan-associated protein-hyaluronan complex enhances airway hyperresponsiveness in a murine model of asthma

Abstract

Background: Serum-derived hyaluronan (HA)-associated proteins (SHAPs), the heavy chains of inter-α-trypsin inhibitor, covalently bind to HA to form the SHAP-HA complex. The SHAP-HA complex is involved in the pathophysiology of inflammatory diseases, including rheumatoid arthritis. We investigated whether this complex is also involved in airway allergy.

Methods: SHAP-HA-deficient (bikunin knockout, KO) mice and wild-type (WT) mice were immunized twice by intraperitoneal injection of ovalbumin (OVA) and exposed to aerosol OVA for 30 min each day for 2 weeks. Twenty-four hours after the final OVA challenge, airway responsiveness to inhaled methacholine (MCh) was measured, and analysis of bronchoalveolar lavage fluid (BALF) and lung histological studies were done.

Results: Compared to WT mice, KO mice showed higher airway hyperresponsiveness to inhaled MCh and higher late-phase responses to OVA whereas the early-phase responses were similar. Cell differentials of BALF showed an increased number of macrophages and neutrophils in KO mice. Furthermore, decreased concentrations of soluble tumor necrosis factor receptor-1 (sTNFR1) were found in BALF from KO mice whereas the levels of Th1 and Th2 cytokines were not different from WT mice. Immunochemical study of the lung tissues revealed stronger staining of sTNFR1 in KO than in WT mice.

Conclusions: Our results suggest that in this murine asthma model, the SHAP-HA complex has an inhibitory role in the development of airway hyperresponsiveness and allergic airway inflammation which may be attributed, at least in part, to negative feedback mechanisms exerted by sTNFR1, the shedding of which from the cell surface might also be promoted by the SHAP-HA complex.

Fig. 1

Time schedules of OVA sensitization and airway challenge for asthma models.

Fig. 2

Airway responsiveness to inhaled MCh. a Airway responsiveness before OVA challenge. b Airway responsiveness after exposure to inhaled OVA for 2 weeks. * p < 0.05, ** p < 0.01.

Fig. 3

EPR and LPR to OVA provocation. Saline-treated WT mice served as controls. a Following OVA inhalation for 30 min, no significant difference was found between EPRs of KO and WT mice (NS). b Following OVA challenge, the KO mice showed a significantly higher LPR than the WT mice. ** p < 0.01, *** p < 0.001.

Fig. 4

Measurement of OVA-specific serum IgE (a) and IgG1 (b). No significant difference was found in the serum levels of OVA-specific IgE between the KO and WT mice (NS). However, a higher (*** p < 0.001) serum level of OVA-specific IgG1 was found in the KO mice compared with WT mice. Saline-treated WT mice served as controls.

Fig. 5

Total cell count (a) and differential cell count (b) in BALF. a The total cell number was not significantly different between KO and WT mice (NS). Very few cells were observed in the saline-treated control mice. b The numbers of alveolar macrophages (AM) and neutrophils (Neu) were significantly larger in KO mice than in WT mice (* p < 0.05, Mann-Whitney U test). No significant difference was found in the numbers of lymphocytes (Ly) and eosinophils (Eo).

Fig. 6

Measurement of HA (a) and SHAP-HA (b) in BALF by sandwich ELISA. a The BALF of OVA-treated WT mice contained more HA than that of OVA-treated KO mice, and the BALF of both WT and KO mice contained more HA than that of saline-treated controls. b Larger amounts of SHAP-HA were found in the BALF from OVA-treated WT mice than from saline-treated WT mice. The BALF from KO mice did not contain any SHAP-HA. * p < 0.05, *** p < 0.001.

Fig. 7

Determination of cytokines in BALF. a The cytokines in BALF were tested by a cytokine antibody array and analyzed by Multi Gauge version 3.0 for quantification. The BALFs were collected and pooled from mice in the same treatment groups, i.e. 6 animals in the control group, 7 animals in the WT group and 7 animals in the KO group. The levels of sTNFR-1 and IL-12p40 were lower in KO mice than in WT mice. b TGF-β1 concentration in BALF. No significant difference was found between KO and WT mice (NS), but both showed statistically significantly (p < 0.05) higher TGF-β1 concentrations than controls. Immunostaining was done twice and the determination was done in triplicate.

Fig. 8

Histological (a) and immunochemical (b) staining of lung specimens of OVA-treated WT and KO mice and of saline-treated WT control mice. a Representative sections stained with HE, PAS, Giemsa and EVG. The patterns suggest airway inflammation, goblet cell hyperplasia and airway fibrosis in OVA-treated lungs. Magnification: ×20 for all photographs. b Representative staining for HA, CD44, SHAP-HA complex and sTNFR1. Strong HA staining may be observed in the lungs of OVA-treated mice compared with the lung of a saline-treated WT mouse. In the staining for CD44, the lungs of OVA-treated WT and KO mice show similar staining levels; the lung of a saline-treated WT control shows weaker staining. Obvious SHAP-HA complex staining may be observed around the airways of an OVA-treated WT mouse, but there is no significant staining around those of a saline-treated WT mouse. There is no staining in the KO mouse lung. In the immunostaining for sTNFR1, the specimens were observed using a fluorescence microscope. sTNFR1 appears to be upregulated in an OVA-treated mouse, and the stain is stronger in the specimen from a KO mouse than in that from a WT mouse. Magnification: ×20 for all photographs.