Evaluation of basement membrane degradation during TNF-α-induced increase in epithelial permeability

Abstract

We evaluated whether tumor necrosis factor (TNF)-alpha induces an increase in permeability of an alveolar epithelial monolayer via gelatinase secretion and basement membrane degradation. Gelatinase secretion and epithelial permeability to radiolabeled albumin under unstimulated and TNF-alpha-stimulated conditions of an A549 human epithelial cell line were evaluated in vitro. TNF-alpha induced both upregulation of a 92-kDa gelatinolytic activity (pro form in cell supernatant and activated form in extracellular matrix) and an increase in the epithelial permeability coefficient compared with the unstimulated condition (control: 1.34 +/- 0.04 x 10(-6) cm/s; 1 microg/ml TNF-alpha: 1.47 +/- 0.05 x 10(-6) cm/s, P < 0.05). The permeability increase in the TNF-alpha-stimulated condition involved both paracellular permeability, with gap formation visualized by actin cytoskeleton staining, and basement membrane permeability, with an increase in the basement membrane permeability coefficient (determined after cell removal; control: 2.58 +/- 0.07 x 10(-6) cm/s; 1 microg/ml TNF-alpha: 2.82 +/- 0.02.10(-6) x cm/s, P < 0.05). Because addition of gelatinase inhibitors [tissue inhibitor of metalloproteinase (TIMP)-1 or BB-3103] to cell supernatants failed to inhibit the permeability increase, the gelatinase-inhibitor balance in the cellular microenvironment was further evaluated by cell culture on a radiolabeled collagen matrix. In the unstimulated condition, spontaneous collagenolytic activity inhibited by addition to the matrix of 1 microg/ml TIMP-1 or 10(-6) M BB-3103 was found. TNF-alpha failed to increase this collagenolytic activity because it was associated with dose-dependent upregulation of TIMP-1 secretion by alveolar epithelial cells. In conclusion, induction by TNF-alpha of upregulation of both the 92-kDa gelatinase and its inhibitor TIMP-1 results in maintenance of the gelatinase-inhibitor balance, indicating that basement membrane degradation does not mediate the TNF-alpha-induced increase in alveolar epithelial monolayer permeability.

Neutrophil Proteinases in Hydrochloric Acid- and Endotoxin-Induced Acute Lung Injury: Evaluation of Interstitial Protease Activity by in situ Zymography

“…The absence of permeability changes in this model are in agreement with our recent findings demonstrating that gelatinase B does not contribute to the TNF-␣-induced increase of epithelial permeability in vitro (Lacherade et al, 2001). On the other hand, the absence of inhibition of neutrophil influx evidenced despite TIMP-1 overexpression could seem in disagreement with our previous findings, because we demonstrated that gelatinase B participates in neutrophil migration in vitro .…”

“…In accordance with the findings of Hayashi et al (1996), immunolocalization of gelatinase A showed a diffuse pattern of expression in lung tissue that was fully consistent with the in situ zymography data. These findings, together with those obtained in vitro with A549 alveolar epithelial cells previously described (Lacherade et al, 2001), demonstrate that basal gelatinase A expression is probably responsible for the physiologic turnover of the basement membrane and explain the diffuse gelatinolytic activities evidenced using in situ zymography.…”

“…This result is in keeping with the fact that bronchial epithelial cells and endothelial cells constitutively express gelatinase(s) involved in basement membrane turnover (Partridge et al, 1993;Yao et al, 1996). Furthermore we have recently shown, using in vitro experiments, that type 2 alveolar epithelial cells at confluence, which have been shown to constitutively secrete a gelatinase A (d'Ortho et al, 1997), were responsible for spontaneous matrix degradation (Lacherade et al, 2001). In accordance with the findings of Hayashi et al (1996), immunolocalization of gelatinase A showed a diffuse pattern of expression in lung tissue that was fully consistent with the in situ zymography data.…”

“…At least two mechanisms could theoretically lead to an increase in antiproteinase activity: increased endothelial permeability may have allowed serum inhibitors, namely TIMP and ␣2-macroglobulin, to gain access to the interstitial gelatinases and/or local inhibitor synthesis may have been up-regulated by proinflammatory cytokines. In vitro studies have demonstrated that leukocyte elastase inhibitors are up-regulated in alveolar type 2 cells by inflammatory cytokines (Sallenave et al, 1994), and in a recent study we demonstrated that TNF-␣ does not augment the gelatinase activity secreted by alveolar type 2 cells because up-regulation of both gelatinase B and TIMP-1 occurs concomitantly (Lacherade et al, 2001). Piedboeuf and colleagues (1994) reported that increased TIMP-1 expression occurred within 1 hour of oleic acid administration in foci of inflammation; recently, Gipson et al (1999) demonstrated a rapid increase of both TIMP-2 (0.5 to 4 hours) and secretory leukocyte proteinase inhibitor (1 to 8 hours) mRNAs in IgG immune complex-induced lung injury.…”

“…Since TNF-␣ has been shown to increase alveolar capillary wall permeability independent of neutrophils, both in vivo and in vitro (Horvath et al, 1988;Lacherade et al, 2001), its concentration in lung tissue was assessed. Despite the increased edema in HClinduced injury, no significant increase in TNF-␣ concentration was demonstrated; however, this result does not rule out the fact that this cytokine could have been involved, as recently demonstrated by Davidson et al (1999).…”