Interleukin-1β and tumor necrosis factor-α have opposite effects on fibroblasts and epithelial cells during basement membrane formation

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are typical proinflammatory cytokines that influence various cellular functions, including metabolism of the extracellular matrix. We examined the roles of IL-1beta and TNF-alpha in basement membrane formation in an in vitro model of alveolar epithelial tissue composed of alveolar epithelial cells and pulmonary fibroblasts. Formation of the basement membrane by immortalized rat alveolar type II epithelial (SV40-T2) cells, which ordinarily do not form a continuous basement membrane, was dose-dependently upregulated in the presence of 2 ng/ml IL-1beta or 5 ng/ml TNF-alpha. IL-1beta or TNF-alpha alone induced increased secretion of type IV collagen, laminin-1, and nidogen-1/entactin, all of which contributed to this upregulation. In contrast, while SV40-T2 cells cultured with a fibroblasts-embedded type I collagen gel were able to form a continuous basement membrane, they failed to form a continuous basement membrane in the presence of IL-1beta or TNF-alpha. Fibroblasts treated with IL-1beta or TNF-alpha secreted matrix metalloproteinase (MMP)-9 and MMP-2, and these MMPs inhibited basement membrane formation and degraded the basement membrane architecture. Neither IL-1beta- nor TNF-alpha-treated SV40-T2 cells increased the secretion of MMP-9 and MMP-2. These results suggest that IL-1beta participates in basement membrane formation in two ways. One is the induction of MMP-2 and MMP-9 secretion by fibroblasts, which inhibits basement membrane formation, and the other is induction of basement membrane component secretion from alveolar epithelial cells to enhance basement membrane formation.     

Mechanisms of TNF-alpha stimulation of amiloride-sensitive sodium transport across alveolar epithelium

Because tumor necrosis factor (TNF)-alpha can upregulate alveolar fluid clearance (AFC) in pneumonia or septic peritonitis, the mechanisms responsible for the TNF-alpha-mediated increase in epithelial fluid transport were studied. In rats, 5 microg of TNF-alpha in the alveolar instillate increased AFC by 67%. This increase was inhibited by amiloride but not by propranolol. We also tested a triple-mutant TNF-alpha that is deficient in the lectinlike tip portion of the molecule responsible for its membrane conductance effect; the mutant also has decreased binding affinity to both TNF-alpha receptors. The triple-mutant TNF-alpha did not increase AFC. Perfusion of human A549 cells, patched in the whole cell mode, with TNF-alpha (120 ng/ml) resulted in a sustained increase in Na(+) currents from 82 +/- 9 to 549 +/- 146 pA (P < 0.005; n = 6). The TNF-alpha-elicited Na(+) current was inhibited by amiloride, and there was no change when A549 cells were perfused with the triple-mutant TNF-alpha or after preincubation with blocking antibodies to the two TNF-alpha receptors before perfusion with TNF-alpha. In conclusion, although TNF- alpha can initiate acute inflammation and edema formation in the lung, TNF-alpha can also increase AFC by an amiloride-sensitive, cAMP-independent mechanism that enhances the resolution of alveolar edema in pathological conditions by either binding to its receptors or activating Na(+) channels by means of its lectinlike domain.     

Monocyte migration through the alveolar epithelial barrier: adhesion molecule mechanisms and impact of chemokines

Alveolar monocyte influx requires adherence and transmigration through the vascular endothelium, extracellular matrix, and alveolar epithelium. For investigating the monocyte migratory process across the epithelial barrier, we employed both the A549 cell line and isolated human alveolar epithelial cells. Under baseline conditions, spontaneous bidirectional transepithelial monocyte migration was noted, which was dose-dependently increased in the presence of the monocyte chemoattractant protein-1. TNF-alpha stimulation of the alveolar epithelium provoked the polarized apical secretion of monocyte chemoattractant protein-1 and RANTES and up-regulation of ICAM-1 and VCAM-1 expression, accompanied by markedly enhanced transepithelial monocyte traffic in the basal-to-apical direction. Multiple adhesive interactions were noted to contribute to the enhanced monocyte traffic across the TNF-alpha-stimulated alveolar epithelium: these included the beta 2 integrins CD11a, CD11b, CD11c/CD18, the beta 1 integrins very late Ag (VLA)-4, -5, and -6, and the integrin-associated protein CD47 on monocytes, as well as ICAM-1, VCAM-1, CD47, and matrix components on the epithelial side. In contrast, spontaneous monocyte migration through unstimulated epithelium depended predominantly on CD11b/CD18 and CD47, with some additional contribution of VLA-4, -5, and -6. In summary, unlike transendothelial monocyte traffic, for which beta 1 and beta 2 integrins are alternative mechanisms, monocyte migration across the alveolar epithelium largely depends on CD11b/CD18 and CD47 but required the additional engagement of the beta 1 integrins for optimal migration. In response to inflammatory challenge, the alveolar epithelium orchestrates enhanced monocyte traffic to the apical side by polarized chemokine secretion and up-regulation of ICAM-1 and VCAM-1.     

Caco-2 cell permeability and stability of two d-glucopyranuronamide conjugates of thyrotropin-releasing hormone

Caco-2 cell permeability and stability assays were used as an in vitro model to study the intestinal epithelial transport and stability of two analogues of thyrotropin-releasing hormone (TRH; Pyr-His-Pro-NH2). Peptide 1 (Pyr-His-Pro-D-glucopyranuronamide) was more permeable across the Caco-2 cell monolayer compared with the permeability of the parent TRH peptide (Papp=5.10+/-1.89x10(-6) cm/s c.f. Papp=0.147+/-0.0474x10(-6) cm/s respectively). The permeability of peptide 1 was improved threefold by attaching a 2-aminooctanoic acid moiety to the N-terminus to form peptide 2 (2-aminooctanoic acid-Gln-His-Pro-D-glucopyranuronamide) (Papp=16.3+/-2.47x10(-6) cm/s). The half-life for both peptide 1 and peptide 2 was approximately 20 min in a homogenate of Caco-2 cells compared with the half-life of TRH which is approximately 3 min. It was concluded that the permeability of peptides 1 and 2 was enhanced because of their increased stability, while the higher permeability of peptide 2 compared with peptide 1 may be attributed to its increased lipophilicity which results in enhanced passive diffusion.     

Basement membrane collagen type IV expression by human mesenchymal stem cells during adipogenic differentiation

During adipogenic differentiation human mesenchymal stem cells (hMSC) produce collagen type IV. In immunofluorescence staining differentiating hMSCs started to express collagen type IV when Oil Red O-positive fat droplets appeared intracellularly. Quantitative real time-polymerase chain reaction confirmed progressive increase of collagen type IV α1 and α2 mRNA levels over time, 18.6- and 12.2-fold by day 28, respectively, whereas the copy numbers of α3-α6 mRNAs remained rather stable and low. Type IV collagen was in confocal laser scanning microscopy seen around adipocytes, where also laminins and nidogen were found, suggesting pericellular deposition of all key components of the fully developed basement membrane. Immunofluorescence staining of matrix metalloproteinase-2 (MMP-2, 72 kD type IV collagenase, gelatinase A) and MMP-9 (92 kD type IV collagenase, gelatinase B) disclosed only faint staining of MSCs, but MMP-9 was strongly induced during adipogenesis, whereas MSC supernatants disclosed in zymography pro-MMP-2 and faint pro-MMP-9 bands, which increased over time, with partial conversion of pro-MMP-2 to its active 62 kD form. Differentiation was associated with increasing membrane type 1-MMP/MMP-14 and tissue inhibitor of metalloproteinase-2 (TIMP-2) staining, which may enable participation of type IV collagenases in basement membrane remodelling via ternary MT1-MMP/TIMP-2/MMP-2 or -9 complexes, focalizing the fully active enzyme to the cell surface. MMP-9, which increased more in immunofluorescence staining, was perhaps preferentially bound to cell surface and/or remodelling adipocyte basement membrane. These results suggest that upon MSC-adipocyte differentiation collagen type IV synthesis and remodelling become necessary when intracellular accumulation of fat necessitates a dynamically supporting and instructive, partly denatured adipogenic pericellular type IV collagen scaffold.     

Evaluation of extracellular matrix proteins and tissue inhibitor of matrix metalloproteinases-2 on bovine inner cell mass outgrowth in vitro

Effects of extracellular matrix proteins and tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) on bovine inner cell mass (ICM) outgrowth and proteinase production in vitro were determined. Inner cell masses were isolated immunosurgically from day 7 embryos (day 0 = onset of estrus) and cultured for 96 h. In experiment 1, cellular outgrowth and gelatinase production were evaluated for ICM cultured on collagen IV, fibronectin, or laminin. More (P < 0.05) ICM generated cellular outgrowth on fibronectin (71%). compared with collagen IV (0%) or laminin (15%). Inner cell mass and outgrowth areas were greatest (P < 0.05) on fibronectin after 96 h of culture, compared with laminin. Although the incidence of cellular outgrowth on laminin was limited, numbers of cells in outgrowths supported by laminin were similar (P > 0.10) to fibronectin except at 72 h of culture, where more (P < 0.05) cells were in laminin than in fibronectin outgrowths. Gelatinase activity was not detected in conditioned medium. In experiment 2, cellular outgrowth and plasminogen activator production by ICM cultured on fibronectin in medium containing 0 or 10 microg/ml TIMP-2 were evaluated. Inner cell mass and outgrowth areas, and numbers of cells in outgrowths were greater (P < 0.05) in 10 compared with 0 microg/ml TIMP-2 at 96 h of culture. Mean plasminogen activator activity in conditioned medium from ICM cultured in 10 microg/ml TIMP-2 was greater (P < 0.05) compared with 0 microg/ml TIMP-2 (16.2 +/- 4.8 versus 6.7 +/- 1.4 x 10(-3) IU/ml, respectively). These results demonstrate that cellular outgrowth from bovine ICM is supported by fibronectin and is stimulated by TIMP-2.     

Human T lymphocytes synthesize the 92 kDa type IV collagenase (gelatinase B)

In order for T cells to exit the circulatory system, traverse the endothelial basement membrane, and arrive in target tissues, these cells must attach to and degrade basement membrane proteins. 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to stimulate lymphoid cell adhesion to basement membrane components. We have used TPA to study the ability of human lymphoid cells to secrete type IV collagenases, enzymes capable of degrading basement membrane proteins. Here, we found that human primary T cells and H-9 lymphoid cells synthesize the 92 kDa type IV collagenase (gelatinase B) and TPA stimulates the synthesis and secretion of this protease. Peak TPA-stimulated gelatinase B secretion and mRNA accumulation were observed 9 hours after TPA treatment, while the peak adhesion to type IV collagen was observed only 3 hours after TPA treatment. The protein kinase C inhibitor, H-7, inhibited TPA-stimulated gelatinase B secretion. Both the primary T cells and H-9 lymphoid cells also expressed the mRNA for the tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that TPA - stimulated lymphoid cells adhere to type IV collagen and subsequently synthesize and secrete gelatinase B and TIMP-1. We conclude that lymphoid cell extravasation may involve cellular employment of adhesion mechanisms prior to degradation of the matrix, which is similar to the process of extravasation used by metastatic cells. © 1993 Wiley-Liss, Inc.     

TIMP-1, -2, -3, and -4 in idiopathic pulmonary fibrosis. A prevailing nondegradative lung microenvironment?

Fibroblast proliferation and extracellular matrix accumulation characterize idiopathic pulmonary fibrosis (IPF). We evaluated the presence of tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, and -4; collagenase-1, -2, and -3; gelatinases A and B; and membrane type 1 matrix metalloproteinase (MMP) in 12 IPF and 6 control lungs. TIMP-1 was found in interstitial macrophages and TIMP-2 in fibroblast foci. TIMP-3 revealed an intense staining mainly decorating the elastic lamina in vessels. TIMP-4 was expressed in IPF lungs by epithelial and plasma cells. TIMP-2 colocalized with Ki67 in fibroblasts, whereas TIMP-3 colocalized with p27 in inflammatory and epithelial cells. Collagenase-1 was localized in macrophages and alveolar epithelial cells, collagenase-2 was localized in a few neutrophils, and collagenase-3 was not detected. MMP-9 was found in neutrophils and subepithelial myofibroblasts. Myofibroblast expression of MMP-9 was corroborated in vitro by RT-PCR. MMP-2 was noticed in myofibroblasts, some of them close to areas of basement membrane disruption, and membrane type 1 MMP was noticed in interstitial macrophages. These findings suggest that in IPF there is higher expression of TIMPs compared with collagenases, supporting the hypothesis that a nondegrading fibrillar collagen microenvironment is prevailing.     

Characterization of Sertoli cells cultured in the bicameral chamber system: relationship between formation of permeability barriers and polarized secretion of transferrin

Sertoli cells from immature rats (18 days old) were cultured on Millipore filters impregnated with reconstituted basement membrane in bicameral chambers. Three types of cultures were obtained: 1) confluent monolayer cultures that formed a permeability barrier (impermeable), 2) confluent monolayer cultures that did not form a permeability barrier (permeable), and 3) subconfluent cultures (permeable). The relationships among fluid equilibrium, electrical resistance, and [3H]inulin transport between the apical and basal reservoirs of the chambers were examined. An impermeable confluent monolayer is defined when the cells of the Sertoli cell epithelial sheet are able to prevent hydrodynamic equilibration of fluid levels between the apical and basal reservoirs of a bicameral chamber. That is, a permeability barrier is present between the two sides of the chamber when fluid levels (volumes) do not change. In the impermeable confluent Sertoli cell monolayers, 7.5 +/- 0.6% of added [3H]inulin diffused across the monolayer during a 6-h collection period versus 13.7 +/- 0.5% in permeable cultures. Conversely, the electrical resistance was higher in the impermeable monolayers (41-71 ohm.cm2) than in the permeable layers (less than 33 ohm.cm2). A reciprocal linear relationship (Y = -4.68(X) + 91.50, r = 0.808) exists between inulin flux and electrical resistance, and this relationship is a function of cell density. Transferrin (Tf) was one of a few proteins detected in the basal medium of bicameral chambers, whereas most de novo synthesized proteins were secreted into the apical reservoir of the chamber. No significant differences in the total amount of Tf secreted by impermeable or permeable monolayers of Sertoli cells were observed. However, the Sertoli cell secretion ratios (apical/basal) of Tf during a 15-20-h collection period were 2.03 and 1.57 for impermeable monolayers plated at 2.4 x 10(6) and 3.6 x 10(6) cells/well, respectively, but less than 1.0 in permeable layers of cells. When fewer than 2 x 10(6) Sertoli cells were plated, the apical/basal polarity of Tf secretion declined to below 1 in a 24-h culture period, even though those chambers contained impermeable monolayers (recognized by the lack of hydrodynamic equilibrium). These results indicate that polarized secretion by Sertoli cells is dependent on (1) plating density and (2) formation of an impermeable epithelial sheet.     

Reconstituting the barrier properties of a water-tight epithelial membrane by design of leaflet-specific liposomes

To define aspects of lipid composition and bilayer asymmetry critical to barrier function, we examined the permeabilities of liposomes that model individual leaflets of the apical membrane of a barrier epithelium, Madin-Darby canine kidney type 1 cells. Using published lipid compositions we prepared exofacial liposomes containing phosphatidylcholine, sphingomyelin, glycosphingolipids, and cholesterol; and cytoplasmic liposomes containing phosphatidylethanolamine, phosphatidylserine, and cholesterol. The osmotic permeability of cytoplasmic liposomes to water (P(f)), solutes, and NH(3) was 18-90-fold higher than for the exofacial liposomes (P(f(ex)) = 2.4 +/- 0.4 x 10(-4) cm/s, P(f(cy)) = 4.4 +/- 0.3 x 10(-3) cm/s; P(glycerol(ex)) = 2.5 +/- 0.3 x 10(-8) cm/s, P(glycerol(cy)) = 2.2 +/- 0.02 x 10(-6) cm/s; P(NH3(ex)) = 0. 13 +/- 0.4 x 10(-4) cm/s, P(NH3(cy)) = 7.9 +/- 1.0 x 10(-3) cm/s). By contrast, the apparent proton permeability of exofacial liposomes was 4-fold higher than cytoplasmic liposomes (P(H+(ex)) = 1.1 +/- 0. 1 x 10(-2) cm/s, P(H+(cy)) = 2.7 +/- 0.6 x 10(-3) cm/s). By adding single leaflet permeabilities, we calculated a theoretical P(f) for a Madin-Darby canine kidney apical membrane of 4.6 x 10(-4) cm/s, which compares favorably with experimentally determined values. In exofacial liposomes lacking glycosphingolipids or sphingomyelin, permeabilities were 2-7-fold higher, indicating that both species play a role in barrier function. Removal of cholesterol resulted in 40-280-fold increases in permeability. We conclude: 1) that we have reconstituted the biophysical properties of a barrier membrane, 2) that the barrier resides in the exofacial leaflet, 3) that both sphingomyelin and glycosphingolipids play a role in reducing membrane permeability but that there is an absolute requirement for cholesterol to mediate this effect, 4) that these results further validate the hypothesis that each leaflet offers an independent resistance to permeation, and 5) that proton permeation was enhanced by sphingolipid/cholesterol interactions.     

Vasopressin-enhanced urea transport by rat inner medullary collecting duct cells in culture

The distal inner medullary collecting duct (IMCD) is critical in the urinary concentrating process, in part because it is the site of vasopressin (AVP)-regulated permeability to urea. The purpose of these experiments was to develop a cell culture model of the IMCD on permeable structure and to characterize the responsiveness to AVP. Rat IMCD cells were grown to confluence on collagen-coated Millipore filters glued onto plastic rings. To assess the time required to achieve confluence, the transepithelial resistance was measured periodically and was found to be stable after 2 weeks, at a maximal value of 595 +/- 22 omega cm2. In separate monolayers the effect of AVP on inulin and urea permeability was determined. While inulin permeability was unchanged after AVP, urea permeability increased from 6.0 +/- 0.4 to peak values of 16.0 +/- 3.8 (10 nM), 23.1 +/- 3.9 (1 microM) and 28.1 +/- 4.9 (10 microM) x 10(-6) cm s-1 (n = 24). In 10 other monolayers, after the addition of 1 mM 8-Br-cAMP, urea permeability increased from 5.1 +/- 0.3 to 8.1 +/- 1.6 x 10(-6) cm s-1 and, after 8-Br-cAMP + 3-isobutyl-1-methylxanthine, to 12.2 +/- 0.7 x 10(-6) cm s-1. We conclude that rat IMCD cells grown in culture exhibit the characteristics of a 'tight' epithelium. Inulin and urea permeability are not different in the absence of AVP, consistent with high resistance junctional complexes. Furthermore, IMCD cells retain the capacity for AVP-regulated urea permeability, a characteristic feature of this nephron segment in vivo.     

Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells.

Monolayers of a well differentiated human intestinal epithelial cell line, Caco-2, were used as a model to study passive drug absorption across the intestinal epithelium. Absorption rate constants (expressed as apparent permeability coefficients) were determined for 20 drugs and peptides with different structural properties. The permeability coefficients ranged from approximately 5 x 10(-8) to 5 x 10(-5) cm/s. A good correlation was obtained between data on oral absorption in humans and the results in the Caco-2 model. Drugs that are completely absorbed in humans had permeability coefficients greater than 1 x 10(-6) cm/s. Drugs that are absorbed to greater than 1% but less than 100% had permeability coefficients of 0.1-1.0 x 10(-6) cm/s while drugs and peptides that are absorbed to less than 1% had permeability coefficients of less than or equal to 1 x 10(-7) cm/s. The results indicate that Caco-2 monolayers can be used as a model for studies on intestinal drug absorption.     

Ontogeny of rabbit proximal tubule urea permeability

Urea transport in the proximal tubule is passive and is dependent on the epithelial permeability. The present study examined the maturation of urea permeability (P(urea)) in in vitro perfused proximal convoluted tubules (PCT) and basolateral membrane vesicles (BLMV) from rabbit renal cortex. Urea transport was lower in neonatal than adult PCT at both 37 and 25 degrees C. The PCT P(urea) was also lower in the neonates than the adults (37 degrees C: 45.4 +/- 10.8 vs. 88.5 +/- 15.2 x 10(-6) cm/s, P < 0.05; 25 degrees C: 28.5 +/- 6.9 vs. 55.3 +/- 10.4 x 10(-6) cm/s; P < 0.05). The activation energy for PCT P(urea) was not different between the neonatal and adult groups. BLMV P(urea) was determined by measuring vesicle shrinkage, due to efflux of urea, using a stop-flow instrument. Neonatal BLMV P(urea) was not different from adult BLMV P(urea) at 37 degrees C [1.14 +/- 0.05 x 10(-6) vs. 1.25 +/- 0.05 x 10(-6) cm/s; P = not significant (NS)] or 25 degrees C (0.94 +/- 0.06 vs. 1.05 +/- 0.10 x 10(-6) cm/s; P = NS). There was no effect of 250 microM phloretin, an inhibitor of the urea transporter, on P(urea) in either adult or neonatal BLMV. The activation energy for urea diffusion was also identical in the neonatal and adult BLMV. These findings in the BLMV are in contrast to the brush-border membrane vesicles (BBMV) where we have previously demonstrated that urea transport is lower in the neonate than the adult. Urea transport is lower in the neonatal proximal tubule than the adult. This is due to a lower rate of apical membrane urea transport, whereas basolateral urea transport is the same in neonates and adults. The lower P(urea) in neonatal proximal tubules may play a role in overall urea excretion and in developing and maintaining a high medullary urea concentration and thus in the ability to concentrate the urine during renal maturation.     

Surfactant replacement attenuates the increase in alveolar permeability in hyperoxia

Rabbits exposed to hyperoxia develop surfactant deficiency, abnormal lung mechanics, and increased permeability to solute. We investigated whether replenishment of depleted alveolar surfactant by the intratracheal instillation of calf lung surfactant extract (CLSE) would mitigate the increase in alveolar permeability to solute. Twenty-eight rabbits were exposed to 100% O2 for 72 h and received intratracheal instillations of 125 mg CLSE (approximately 170 mumol dipalmitoyl phosphatidylcholine) at 24 and 48 h. The interlobar and intralobar distribution of CLSE was quantified by adding [14C]dipalmitoyl phosphatidylcholine liposes into the instillate and measuring the levels of activity in lung tissue. CLSE was nonuniformly distributed in the different lung lobes, the right lower lobe receiving more CLSE than the rest. Alveolar epithelial permeability to solute was assessed by instilling 10 ml isotonic saline, which contained a trace amount of [57Co]cyanocobalamin, in the right lower lobe and measuring the disappearance of the tracer from the alveolar saline and its appearance in the arterial blood during a 1-h period. CLSE treatment was associated with significantly increased 72-h survival in hyperoxia compared with saline-treated controls (number of survivors: 16/17 vs. 5/11, P less than 0.01). CLSE treatment significantly reduced the rate constant for the movement of cyanocobalamin out of the alveolar space (24 +/- 5 vs. 42 +/- 6 min-1 x 10(-3), P less than 0.01) and tracer appearance in the blood at the end of the study (7 +/- 1 vs. 34 +/- 13%, P less than 0.01) when compared with values in saline controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulmonary interstitial pressure and tissue matrix structure in acute hypoxia

Pulmonary interstitial pressure was measured via micropuncture in anesthetized rabbits in normoxia and after breathing 12% O(2). In normoxia [arterial PO(2) = 88 +/- 2 (SD) mmHg], pulmonary arterial pressure and pulmonary interstitial pressure were 16 +/- 8 and -9.6 +/- 2 cmH(2)O, respectively. After 6 h of hypoxia (arterial PO(2) = 39 +/- 16 mm Hg), the corresponding values were 30+/-8 and 3.5+/-2.5 cm H(2)O (P<0.05). Pulmonary interstitial proteoglycan extractability, evaluated by hexuronate assay after 0.4 M guanidinium hydrochloride extraction, was 12.3, 32.4, and 60.6 microg/g wet tissue in normoxia and after 3 and 6 h of hypoxia, respectively, indicating a weakening of the noncovalent bonds linking proteoglycans to other extracellular matrix components. Gel filtration chromatography showed an increased fragmentation of chondroitin sulfate- and heparan sulfate-proteoglycans during hypoxic exposure, accounting for a loss of extracellular matrix native architecture and basement membrane structure. Gelatin zymography demonstrated increased amounts of the proteolytically activated form of gelatinase B (matrix metalloproteinase-9) after hypoxic exposure, providing evidence that the activation of proteinases may play a role in hypoxia-induced lung injury.     

Ambient particulate matter induces alveolar epithelial cell cycle arrest: role of G1 cyclins

We hypothesized that the ambient air pollution particles (particulate matter; PM) induce cell cycle arrest in alveolar epithelial cells (AEC). Exposure of PM (25microg/cm(2)) to AEC induced cells cycle arrest in G1 phase, inhibited DNA synthesis, blocked cell proliferation and caused decrease in cyclin E, A, D1 and Cyclin E- cyclin-dependent kinase (CDK)-2 kinase activity after 4h. PM induced upregulation of CDK inhibitor, p21 protein and p21 activity in AEC. SiRNAp21 blocked PM-induced downregulation of cyclins and AEC G1 arrest. Accordingly, we provide the evidence that PM induces AEC G1 arrest by altered regulation of G1 cyclins and CDKs.     

Alveolar epithelial surface area-volume relationship in isolated rat lungs.

In vitro studies of the alveolar epithelial response to deformation require knowledge of the in situ mechanical environment of these cells. Because of the presence of tissue folding and crumpling, previous measurements of the alveolar surface area available for gas exchange are not equivalent to the epithelial surface area. To identify epithelial deformations in uniformly inflated lungs representative of the in vivo condition, we studied isolated Sprague-Dawley rat lungs (n = 31) fixed by perfusion with glutaraldehyde on deflation after cycling three times at high lung volume (10-25 cmH2O). The epithelial basement membrane in 45 electron micrographs (x12,000)/rat was traced, digitally scanned, and analyzed. Epithelial basement membrane surface area (EBMSA) was computed from a morphometric relationship. EBMSA was found to increase 5, 16, 12, and 40% relative to EBMSA at 24% total lung capacity at lung volumes of 42, 60, 82, and 100% total lung capacity, respectively. The increases in EBMSA suggest that epithelial cells undergo significant deformations with large inflations and that alveolar basement membrane deformation may contribute to lung recoil at high lung pressures.     

Anti-inflammatory effect of pre-elafin in lipopolysaccharide-induced acute lung inflammation

The aim of the present study was to evaluate the anti-inflammatory activity of pre-elafin, an elastase-specific inhibitor, in lipopolysaccharide (LPS)-induced acute lung inflammation. C57BL/6 mice were pre-treated intranasally with recombinant human pre-elafin or vehicle only. One hour later, they were instilled intranasally with LPS (2 microg/mouse). Animals were sacrificed 6 hours after LPS instillation and bronchoalveolar lavage (BAL) was performed with three 1-ml aliquots of saline. LPS induced a lung inflammation characterised by a 100-fold increase in BAL neutrophils compared to control animals (265.8 +/- 54.5 x 10(3) and 2.4 +/- 1.3 x 10(3) neutrophils/ml, respectively). Pre-elafin dose-dependently reduced the neutrophil influx in the lung alveolar spaces by up to 84%. No elastase activity was detectable in all BAL fluids tested. Pre-elafin also reduced significantly LPS-induced gelatinase activity, as shown by zymography, and BAL macrophage inflammatory protein-2 (MIP-2) and KC levels, two potent neutrophil attractants and activators. Moreover, pre-elafin also significantly reduced mRNA levels of the three members of the IL-1 ligand family, namely IL-1alpha, IL-1beta and IL-1 receptor antagonist (IL-1Ra), type II IL-1 receptor, and TNFalpha as assessed in whole lung tissue by RNase protection assay. Thus, pre-elafin may be considered as a potent anti-inflammatory mediator.     

Effects of elastase and cigarette smoke on alveolar epithelial permeability

To determine whether instilled porcine pancreatic elastase (PPE) increases alveolar epithelial permeability, we measured alveolar epithelium permeability X surface area (PS) for [14C]sucrose and 125I-bovine serum albumin (125I-BSA) in isolated perfused lungs from hamsters previously exposed to PPE and/or cigarette smoke. Saline (0.5 ml) with 0, 5, or 20 units PPE was instilled intratracheally in anesthetized hamsters. Those exposed to smoke for 4-6 wk received 0 or 5 units; PS was measured 3 h later. Nonsmokers received 0, 5, or 20 units; PS was measured 3 h, 24 h, or 5 days later. Control PS values were (cm3/s X 10(-4), +/- SE) 0.84 +/- 0.11 for sucrose and 0.030 +/- 0.006 for BSA. Three and 24 h following 20 units PPE, (PS)sucrose was twice the control valve. (PS)BSA was four times control at 3 h but not significantly increased at 24 h. Five days after PPE both were back to control levels. Five units PPE or smoke exposure alone caused no PS changes. Smoke exposure and 5 units PPE caused (PS)sucrose to increase markedly (1.85 +/- 0.32); (PS)BSA was not significantly increased (0.076 +/- 0.026). Thus instilled PPE causes reversible increases in alveolar epithelial PS; cigarette smoking potentiates this effect.