Hyaluronidase expression and activity is regulated by pro-inflammatory cytokines in human airway epithelial cells

Abstract

Hyaluronan (HA) is present at the apical surface of airway epithelium as a high-molecular-weight polymer. Since HA depolymerization initiates a cascade of events that results in kinin generation and growth factor processing, in the present work we used primary cultures of human bronchial epithelial (HBE) cells grown at the air-liquid interface (ALI) to assess hyaluronidase (Hyal) activity by HA zymography, gene expression by quantitative real-time PCR, and localization by confocal microscopy. Because TNF-alpha and IL-1beta induce Hyals in other cells, we tested their effects on Hyals expression and activity. We found that Hyal-like activity is present in the apical and basolateral secretions from HBE cells where Hyals 1, 2, and 3 are expressed, and that IL-1beta acts synergistically with TNF-alpha to increase gene expression and activity. Confocal microscopy showed that Hyals 1, 2, and 3 were localized intracellularly, while Hyal2 was also expressed at the apical pole associated with the plasma membrane, and in a soluble form on the apical secretions. Tissue sections from normal individuals and from individuals with asthma showed a Hyal distribution pattern similar to that observed on nontreated HBE cells or exposed to cytokines, respectively. In addition, increased expression and activity were observed in tracheal sections and in bronchoalveolar lavage (BAL) obtained from subjects with asthma when compared with normal lung donors and healthy volunteers. Our observations indicate that Hyal 1, 2, and 3 are expressed in airway epithelium and may operate in a coordinated fashion to depolymerize HA during inflammation associated with up-regulation of TNF-alpha and IL-1beta, such as allergen-induced asthmatic responses.

Figures

Figure 1.

Hyaluronidases 1, 2, and 3 are expressed in human bronchial epithelial (HBE) cells grown at the air–liquid interface. (A) Hyals on HBE cells were assessed by HA zymography for Hyal-like activity in: cell lysates (CL), apical washes (A), and basolateral media (BL). Control is culture media not exposed to cell (Ctrl). (B) QPCR of Hyal1, Hyal2, and Hyal3 by Taq-Man as described in Materials and Methods. Figure depicts results obtained from one lung, representative of the results observed in cultures obtained from three lung donors.

Figure 2.

Hyaluronidases 1, 2, and 3 are localized intracellularly. Hyal 2 it is also present at the apical membrane and in a soluble form at the mucus layer in HBE cells. Cultures were labeled with (A) Hyal1- (red) and Hyal2- (green), or (B) Hyal1- (red) and Hyal3- (green) specific antibodies as described in Materials and Methods. (C) Control cultures were incubated with mouse and rabbit non-immune serum. Nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI). Images were obtained by confocal microscopy (magnification: ×63). Above each panel the corresponding Z-axis reconstruction images are included.

Figure 3.

Tissue distribution of Hyals in human airways is similar to the one observed in HBE cultures. Confocal microscopy (magnification: ×63) of human tracheal sections immuno-labeled with rabbit anti-Hyal1 (C and D), mouse anti-Hyal2 (E and F) or mouse anti-Hyal3 (G and H), control nonimmune (A and B). Specific staining was visualized using Alexa 488–conjugated secondary antibodies. Bottom: same as top images merged with differential interference contrast for better visualization of the ciliary border. White arrows show the epithelial surface localization of Hyal2. Nuclei were visualized with DAPI.

Figure 4.

Pro-inflammatory cytokines induce Hyals gene expression in HBE cells. Hyaluronidases gene expression was assessed by QPCR after basolateral exposure to media alone (controls), TNF-α, IL-1β, or both (TNF-α + IL-1β). Results were normalized to GAPDH and expressed as fold changes with respect to media control. Bars represent mean ± SEM obtained from three different lung donors. *P < 0.05 compared with controls.

Figure 5.

Hyaluronidase activity is induced by pro-inflammatory cytokines in HBE cells. Top: Hyal activity was determined by HA zymography in cell lysates (25 μg of proteins were loaded per well) after exposure to PBS (controls), TNF-α, IL-1β, or both (T+I). Bottom: Relative activity expressed as fold changes above media control.

Figure 6.

Hyaluronidase activity is elevated in the BAL of individuals with asthma and is further increased after segmental allergen challenge: HA-zymogram was used to assess Hyal activity on BAL samples obtained from healthy subjects and from subjects with asthma 24 hours after segmental allergen challenge (allergen). Saline instillation (Saline) on the contralateral lung was used as a control. A total of 50 μg (total proteins) from each sample were loaded per well.

Figure 7.

Hyaluronidases 1, 2, and 3 immunoreactivity is increased in airway tissue sections obtained from asthmatic lung donors: Human tracheal sections from subjects without (A, B, F, and G) and from subjects with asthma (C–E and H–J) were double-labeled with (A–E) anti-Hyal1 (green) and anti-Hyal2 (red) or (F–J) anti-Hyal1 (green) and anti-Hyal3 (red). Specific staining was visualized using Alexa 488–conjugated (Hyal1, green) and Alexa 555–conjugated (Hyals 2 and 3, red) secondary antibodies. Tissue samples obtained from three different individuals with asthma depicting denuded (C, H), intact (D, I), goblet cells (E, J) tissues were chosen. Images were merged with differential interference contrast to better visualize the epithelial morphology. Green arrows indicate infiltrated leukocyte expressing Hyals, yellow arrows indicate ciliated epithelial shedding, and white arrows show goblet cells. Nuclei were visualized with DAPI.