TNF-alpha disruption of lung endothelial integrity: reduced integrin mediated adhesion to fibronectin

Robert F. Rotundo, Theresa M. Curtis, Melissa D. Shah, Baochong Gao, Anthony Mastrangelo, Susan E. LaFlamme, and Thomas M. Saba

Department of Physiology and Cell Biology, Neil Hellman Medical Research Building, Albany Medical College, Albany, New York 12208


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tumor necrosis factor-alpha (TNF-alpha ) causes an increase in transendothelial protein permeability of confluent monolayers of calf pulmonary artery endothelial (CPAE) cells, and the addition of plasma fibronectin (pFn) to the culture medium can attenuate this increase in permeability. We determined if reduced integrin function had a role in decreased endothelial cell adhesion to immobilized Fn after exposure of the endothelial monolayers to TNF-alpha . TNF-alpha also causes a reorganization of the subendothelial Fn rich matrix and a significant loss in RGD-dependent adhesion of TNF-alpha treated CPAE cells to pFn coated surfaces. However, flow cytometry revealed no decrease in alpha 5beta 1 or total beta 1 integrin expression on the surface of the CPAE cells after TNF-alpha . Reduced CPAE adhesion to immobilized Fn was, in part, due to a loss of beta 1-integrin function since the beta 1-integrin blocking antibody mAb 13 significantly (P < 0.05) prevented the adhesion of normal control CPAE cells but did not further reduce the adhesion of TNF-alpha -treated cells. In addition, antibodies which activate beta 1 integrins restored (P < 0.05) adhesion of TNF-alpha -treated cells to immobilized pFn but did not alter the adhesion of control cells. Despite reduced ability to adhere to immobilized Fn, TNF-alpha -treated CPAE monolayers demonstrated increased binding and incorporation of fluid-phase pFn into the subendothelial extracellular matrix (ECM) as measured by the analysis of the deoxycholate (DOC) detergent insoluble pool of 125I-Fn in the cell layer. In contrast to the RGD-mediated adhesion of CPAE cells to matrix Fn, the increased binding of soluble pFn after TNF-alpha was not inhibited by RGD peptides or mAb 13. Thus reduced integrin-dependent adhesion of the CPAE cells to matrix Fn as well as disruption of the Fn matrix may contribute to the increased protein permeability of previously confluent endothelial monolayer after TNF-alpha . In addition, increased ability for the monolayer to incorporate fluid-phase Fn into the ECM after TNF-alpha via a non-beta 1- integrin dependent mechanism may be a compensatory response to stabilize the Fn matrix and the endothelial barrier.

fibronectin; vascular permeability; integrins; lung permeability


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ADULT RESPIRATORY DISTRESS syndrome as a consequence of increased lung endothelial protein permeability is often observed in critically ill surgical, trauma, or burn patients with gram-negative sepsis (43). Tumor necrosis factor-alpha (TNF-alpha ), an inflammatory cytokine released from endotoxin-activated monocytes and macrophages sequestered in the lung microcirculation and interstitium during sepsis or lung inflammation, is believed to contribute to disruption of the lung vascular barrier leading to the increase in protein permeability (46, 47). Intravenous injection of recombinant human TNF-alpha has been shown to elicit cardiovascular and pulmonary disturbances similar to those observed during endotoxemia or gram-negative septic shock (38, 45), whereas the intravenous infusion of neutralizing antibodies to TNF-alpha reduces lethality in both endotoxemia and bacteremia in subhuman primates (46). Moreover, mice devoid of the 55-kDa TNF-alpha receptor are more resistant to the development of septic shock after endotoxin or bacterial challenge (34).

Endothelial monolayer barrier integrity is influenced by both cell-cell and cell-matrix interactions (13, 15, 24). The concept that TNF-alpha can alter vascular integrity is directly supported by various in vitro studies measuring protein permeability of cultured confluent endothelial monolayers after TNF-alpha exposure (10, 41, 48, 49). Defilipi et al. (12-14) showed that exposure of endothelial cells to a combination of TNF-alpha and interferon-gamma (IFN-gamma ) reduced endothelial cell adhesion to specific matrix proteins. For example, exposure of human umbilical vein endothelial cells (HUVECs) to TNF-alpha decreased the expression of alpha 6beta 1-integrins, resulting in reduced adhesion to substrate laminin, whereas combined treatment with both TNF-alpha and IFN-gamma decreased alpha vbeta 3-integrin surface expression, leading to reduced adhesion to vitronectin (12-14). Changes in integrin activity have also been implicated in a loss of endothelial cell adhesion, since parallel exposure of human endothelial cells with both TNF-alpha and IFN-gamma reduced the ligand-binding activity of alpha vbeta 3-integrins, resulting in a loss of adhesion to either denatured collagen, vitronectin, or fibrinogen (42).

Integrin ligation is important in transducing signals from the extracellular matrix (ECM) which in turn can influence cytoskeleton-mediated cell motility and gene expression (1, 15, 23). Endothelial cells express several integrins on their surface, including alpha 5beta 1- and alpha vbeta 3-integrins, which interact with a three-amino acid cell attachment sequence, Arg-Gly-Asp (RGD), found in fibronectin (Fn), fibrinogen, and vitronectin (13, 24, 35). Both alpha 5beta 1- and alpha vbeta 3-integrins recognize an RGD site accessible in the III-10 module of immobilized or matrix-incorporated Fn (35, 36). However, Dejana et al. (16) demonstrated that alpha 5beta 1 is the only integrin found clustered in focal contacts when HUVEC cells are plated on Fn. In addition to influencing cell adhesion, several studies have also suggested that alpha 5beta 1-integrins may facilitate cell-mediated assembly of soluble Fn in the ECM (1, 18, 51), since the binding of the NH2-terminal end of Fn to cell-associated matrix assembly sites on adherent cells (22) may require the participation of alpha 5beta 1-integrins (1, 18, 23, 27, 51).

We previously reported that the addition of TNF-alpha to confluent calf pulmonary artery endothelial (CPAE) monolayers causes disruption of the underlying fine fibrillar Fn matrix and an increase in the endothelial protein permeability (10, 11, 48, 49). This increase in endothelial protein permeability can be attenuated by the addition of purified human plasma Fn (pFn), presumably through its incorporation in the ECM (10, 48, 49). The current study was designed to determine if exposure of lung pulmonary artery endothelial monolayers to TNF-alpha would alter their adhesion to immobilized Fn and to determine if such reduced CPAE adhesion to Fn was related to either reduced surface expression of the alpha 5beta 1-integrins or a change in their activity or functional state.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelial cell culture. CPAE cells (CCL-209; American Type Culture Collection, Rockville, MD) were grown in MEM (GIBCO, Grand Island, NY) supplemented with 20% FBS (Hyclone, Logan, UT), nonessential amino acids (10 mM; GIBCO), penicillin (100 U/ml; GIBCO), and streptomycin (100 µg/ml; GIBCO). Experiments were performed between passages 17 and 23. MEM containing 5% FBS was used during the experimental treatments with and without 200 U/ml (~9 ng/ml) of recombinant human TNF-alpha (Cellular Products, Buffalo, NY). The specific activity of TNF-alpha is 2.1 × 107 AU/mg. All chemicals were purchased from Sigma Chemical (St. Louis, MO) unless otherwise specified.

Determination of endothelial protein permeability using the dual-chamber monolayer system. A dual-chamber monolayer assay was used to evaluate transendothelial 125I-labeled albumin clearance as a measure of endothelial protein permeability, as previously described (9, 10, 48, 49). CPAE cells were seeded (75,000 cells/well) in the luminal chamber of tissue culture transwell filters (6.5 mm diameter, 0.4 µM pore size; Nucleopore Polycarbonate Membrane; Costar, Cambridge, MA), grown to confluence, and exposed to 200 U/ml TNF-alpha for 2, 4, 6, or 18 h. The BSA tracer was prepared by iodination with Na 125I (ICN, Irvine, CA) using chloramine-T, as previously described (10, 48, 49).

Styrofoam collars fitted around luminal chambers were floated in larger abluminal chambers such that an equal fluid level is maintained in both chambers and convective flux across the monolayers is eliminated. Both chambers were kept at 37°C using thermostatically controlled water baths with the medium in the abluminal chamber constantly stirred. The volume of the luminal chamber that is cleared of the 125I-albumin tracer as it passes into the abluminal chamber represents the total activity of the abluminal chamber. The clearance volume during 5-min intervals was calculated by dividing the amount of albumin flux during the interval by the tracer concentration in the luminal chamber. Albumin clearance (VAlbt) was calculated by summing the incremental clearance volumes as described by the equation
V<SUB>Albt</SUB> = <LIM><OP>∑</OP><LL><IT>i</IT>=1</LL><UL><IT>t</IT></UL></LIM> <FR><NU>V<SUB>A<IT>i</IT></SUB> × &Dgr;[A]<SUB><IT>i</IT></SUB></NU><DE>[L]<SUB><IT>i</IT></SUB></DE></FR>
where VAi is the volume in the abluminal chamber at each time point (i), Delta [A]i is the increase in tracer concentration between time points, and [L]i is the tracer concentration in the luminal chamber at each time point. The change in VAlb over time (dVAlb/dt) was determined by weighted least-squares nonlinear regression for all time intervals over the 1-h assay period.

Dual-label immunofluorescence and differential interference contrast microscopy. CPAE cells (280,000 cells/well) were seeded on glass coverslips in 12-well tissue culture dishes and grown to confluence. After treatment with TNF-alpha , the cells were fixed in 3% formaldehyde in PBS (GIBCO) for 15 min, permeabilized with 0.5% Triton X-100 at 4°C for 5 min, blocked with 2% BSA + 50 mM glycine + 0.2% Tween 20 in PBS for 1 h, incubated with primary antibody (Ab) for 1 h at room temperature, and washed with PBS before being incubated with secondary Ab for 1 h. Primary Abs included rabbit polyclonal Ab against bovine Fn (1:100 dilution; Calbiochem, La Jolla, CA) and mouse monoclonal antibody (mAb) against the human alpha 5beta 1-integrin complex (mAb HA5; 1:500 dilution; Chemicon, Temecula, CA). Secondary Abs included rhodamine-isothiocyanate conjugated goat anti-rabbit IgG (Cappel Organon Teknika, Durham, NC) and FITC goat anti-mouse IgG (Boehringer Mannheim, Indianapolis, IN). Coverslips (Fisher Scientific, Pittsburgh, PA) were mounted on slides using AntiFade (Molecular Probes, Eugene, OR) and were viewed using a Nikon Microphot SA fluorescent microscope.

Immunoprecipitation of total cell surface alpha 5beta 1-integrins. Confluent CPAE cell layers were trypsinized, washed with trypsin inhibitor (1 mg/ml) in PBS, and prelabeled with 0.5 mg/ml Sulfo-NHS-biotin (Pierce, Rockford, IL) for 30 min at 4°C, and 0.05 mM Tris · HCl (pH 7.4), 0.15 mM NaCl, 0.1 mM MgCl2, and 0.1 mM CaCl2 (TBS) was added to stop the reaction. The mAb 9EG7 or mAb HA5 against the alpha 5beta 1-complex (4 µg/ml) was added to 106 suspended cells in 1 ml of TBS. Pellets were treated with extraction buffer [1% Triton X-100, 150 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA (pH 8.0), 0.2 mM phenylmethylsulfonyl fluoride, and 0.5% Nonidet P-40], and protein-G agarose was added to the supernatants for precipitation. Pelleted samples were washed with extraction buffer, resuspended with gel sample buffer, and boiled for 5 min. Total cell lysates, immunoprecipitated proteins, and IgG controls were separated using 7.5% SDS-PAGE and transferred to nitrocellulose (Schleider and Schuell, Keene, NH). Blots stained with streptavidin-horseradish peroxidase (HRP; Amersham Pharmacia Biotech, Piscataway, NJ) or alpha 5- or beta 1-polyclonal Abs (Chemicon, Temecula, CA) and goat anti-rabbit IgG-HRP were then developed using chemiluminescence (Amersham Pharmacia Biotech). Equal loading was determined by cell number before extraction.

Flow cytometry. Confluent CPAE monolayers treated with or without TNF-alpha for 18 h were trypsinized, washed, and resuspended in PBS (1 × 106 cells/ml). Cells were incubated at 4°C with either mAb clones 9EG7, HA5, or AIIB2 (a gift from Caroline Damsky at the University of California-San Francisco) for 30 min, washed with PBS, and labeled for 30 min at 4°C with either FITC-conjugated goat anti-rat IgG (Boehringer Mannheim Biochemical, Indianapolis, IN) or FITC-conjugated goat anti-mouse IgG, respectively. Samples were fixed with 3% paraformaldehyde, and 104 cells were analyzed using a Becton-Dickinson FACScan.

Binding of soluble pFn to CPAE cells. Confluent CPAE monolayers grown in 12-well culture plates were treated for 18 h with or without TNF-alpha . pFn used for binding and incorporation experiments was iodinated using the chloramine-T method, as previously described (37). The cell layers were washed, and 1 ml of MEM containing 1% BSA and 1 µg/ml of human 125I-pFn [2.4 × 106 counts · min-1 (cpm) · 1 µg-1] was added to each well at 4°C. The cell layers were incubated with 125I-pFn, washed with cold PBS, dissolved with 1 N NaOH, and counted. Unlabeled pFn (300 µg/ml) was added to control and TNF-alpha -treated cells with 125I-pFn to quantify nonspecific binding, which was subtracted from experimental values. Samples were quantitated using a Wallac 1470 Wizard gamma counter.

Assay of 125I-pFn incorporation in ECM of CPAE monolayers. CPAE monolayers grown to confluence in 12-well culture plates were exposed for 18 h to TNF-alpha or diluent medium alone, washed with PBS, and supplemented with 1 ml of MEM containing 5% FBS (Fn depleted) and 1 µg/ml of human 125I-pFn (2.4 × 106 cpm/µg). The cell layers were incubated at 37°C for 1, 3, 6, or 24 h. To evaluate the incorporation of 125I-Fn in the ECM, cells were detergent extracted with deoxycholate (DOC), and the DOC insoluble pool of 125I-pFn (covalently incorporated Fn) was quantitated as previously described (37). Fn-depleted FBS was obtained by passing the serum over a gelatin-Sepharose column.

Microscopic analysis of cell adhesion to substrate Fn. Fn (0.5-2 µg/ml) diluted in PBS was adsorbed on 24-well cell culture plates and blocked with 1% BSA at 4°C. Confluent CPAE cell layers treated with TNF-alpha for 18 h were trypsinized, and 5 × 104 CPAE cells suspended in 0.5 ml of MEM were added to each well for 30 min at 37°C. Thereafter, wells were washed three times with PBS, and the number of adherent cells per 1 mm2 was counted using an inverted microscope. Depending on the protocol, cell layers were preincubated with either a RGD-containing peptide (GRGDSP; GIBCO), a control RGE-containing peptide (GRGESP; GIBCO), mAb 13 (Becton-Dickinson, Franklin Lakes, NJ), mAb 12G10 (Chemicon), or mAb 9EG7 for 30 min at 4°C before analysis in the adhesion assay.

Electrical cell impedance sensor analysis of cell adhesion to substrate Fn. Cell adhesion was also studied with an electrical cell impedance sensor (ECIS) from Applied Biophysics (Troy, NY). This system can analyze the dynamic behavior of cells in culture by continuously recording changes in electrical resistance resulting from altered cell attachment and/or spreading on gold-plated sensors fixed within the ECIS culture wells (19). Confluent CPAE monolayers were pretreated with TNF-alpha for 18 h, lifted by trypsinization, suspended (5 × 104 cells) in 400 µl of MEM supplemented with 1% BSA, and added to eight-well ECIS plates precoated with human pFn (25 µg/ml) or 1% BSA. The electrical resistance in ohms was recorded over a 60-min interval. To verify RGD-dependent adhesion to the Fn-coated surface, 250 µg/ml of the RGD peptide or the control RGE peptide was added.

Statistical methods. Data are presented as means ± SE. Data were analyzed by a Student's t-test or a two-way ANOVA, with significance from controls determined by a Tukey test. A confidence level of 95% (P < 0.05) was used to establish statistical significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Protein permeability increase, Fn rearrangement, and intercellular gap formation after TNF-alpha exposure to CPAE monolayers. Figure 1 presents the endothelial protein permeability of the CPAE monolayers after TNF-alpha treatment. Protein clearance across control lung endothelial monolayers was 0.031 µl/min. This was not significantly elevated during the initial 6 h but did increase significantly (P < 0.05) by 18 h post-TNF-alpha . We used immunofluorescence microscopy to study the endogenous bovine Fn-rich subendothelial matrix (Fig. 2, left) and differential interference contrast (DIC) microscopy to visualize intercellular gap formation (Fig. 2, right). Rearrangement or disruption of the Fn matrix was readily apparent at 18 h, with a loss of the fibrillar Fn network from the ECM. Parallel DIC analysis demonstrated a tight and uniform monolayer with slight changes in morphology by 6 h. Gap formation was evident at 18 h after exposure to TNF-alpha , consistent with the significant increase in protein permeability. These results suggest a temporal relationship between the structural rearrangement of the Fn matrix, increased protein permeability, and gap formation of the endothelial monolayer, all which seemed to occur in parallel after TNF-alpha treatment. Based on these data, we used an 18-h TNF-alpha treatment period for the remainder of this study.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1.   Temporal effect of tumor necrosis factor-alpha (TNF-alpha ) on protein permeability of calf pulmonary artery endothelial (CPAE) monolayers. Confluent monolayers were exposed to 200 U/ml of purified human recombinant TNF-alpha for 2, 4, 6, or 18 h. Control wells were incubated with medium alone for 18 h. Integrity of the endothelial cell barrier was determined using transendothelial protein clearance (µl/min). Values for protein clearance represent means ± SE with 4-10 wells/group. *Significantly greater than control (P < 0.05).



View larger version (131K):
[in this window]
[in a new window]
 
Fig. 2.   Temporal effect of TNF-alpha on the organization of fine fibronectin (Fn) fibers in matrix and monolayer morphology. CPAE monolayers were treated with 200 U/ml TNF-alpha for 0, 2, 4, 6, or 18 h, fixed, permeabilized, and stained for endogenous Fn. TNF-alpha treatment disrupted the organization of Fn in the subendothelial matrix and caused gap formation between endothelial cells (black arrows), as demonstrated by immunofluorescent examination of endogenous bovine Fn (left) and differential interference contrast (DIC) microscopic analysis (right), respectively.

Loss of alpha 5beta 1-integrin association with fine Fn fibers of the ECM after TNF-alpha exposure to CPAE monolayers. We next examined the effect of TNF-alpha exposure on the presence of the Fn alpha 5beta 1-integrins in relation to the fine Fn fibers from the ECM by costaining for both the alpha 5beta 1-integrins and endogenous Fn fibers and visualizing them by fluorescence microscopy. In confluent control monolayers (Fig. 3, top left), alpha 5beta 1-integrins are seen to align (Fig. 3, top right) with fine Fn fibers at the periphery of the cells. With higher magnification, cells could be seen within the center of these dense bundles (data not shown). In contrast, after treatment with TNF-alpha the presence of both fine Fn fibers (Fig. 3, bottom left) and the alpha 5beta 1-integrins (Fig. 3, bottom right) within the bundles was lost although cells were still present. Another integrin found on the surface of endothelial cells that is capable of interacting with the RGD site of Fn is the alpha vbeta 3-integrin (13). Costaining for both the alpha vbeta 3-integrins with mAb LM609 and the bovine Fn fibers did not show colocalization within circular Fn bundles, but rather diffuse staining of the alpha vbeta 3-integrins around the periphery of cells (data not shown). The reduced colocalization of the fine Fn fibers with alpha 5beta 1-integrins after TNF-alpha suggested that the increase in protein permeability may reflect decreased alpha 5beta 1-mediated cell adhesion to Fn in the matrix.


View larger version (135K):
[in this window]
[in a new window]
 
Fig. 3.   TNF-alpha treatment disrupts localization of alpha 5beta 1-integrins to the fine Fn fibers of the subendothelial matrix of CPAE monolayers. Confluent endothelial cells were treated with either medium alone (Control) or medium containing 200 U/ml of TNF-alpha for 18 h at 37°C. Cell layers were fixed, permeabilized, and stained for dual-labeling fluorescence microscopy to detect the localization of endogenous Fn (top) and alpha 5beta 1-integrins (middle) using a polyclonal antibody (Ab) to bovine Fn and HA5, a monoclonal antibody (mAb) to alpha 5beta 1-integrins, respectively. Overlay of Fn and alpha 5beta 1-integrin is shown at bottom; arrows indicate examples of colocalization of alpha 5beta 1-integrins and fine Fn fibers, and dotted ovals demonstrate areas with a loss of such localization as seen throughout TNF-alpha -treated monolayers.

Effect of TNF-alpha on adhesion of endothelial cells to immobilized Fn. We confirmed a decrease in adhesion of TNF-alpha -treated CPAE cells to pFn-coated wells by direct cell counting via microscopic analysis. No significant differences occurred between control and TNF-alpha -treated cells plated on wells coated with 0.5 µg/ml pFn or BSA alone (Fig. 4A). However, a significant (P < 0.05) decrease of 35 and 50% in adhesion of TNF-alpha -treated CPAE cells to wells coated with 1.0 and 2.0 µg/ml pFn, respectively, was observed compared with control cells. The assay was repeated with the addition of RGD- and RGE-containing peptides to evaluate the role of integrins in the observed adhesion to the immobilized Fn surface. Control and TNF-alpha -treated CPAE cells were plated on wells coated with pFn (2 µg/ml) after preincubation with RGD or RGE peptide (500 µg/ml). RGD peptide caused a significant (P < 0.05) loss of adhesion to Fn in both the control and TNF-alpha -treated groups (Fig. 4B).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of TNF-alpha exposure on CPAE cell adhesion to Fn-coated surfaces. Confluent monolayers were pretreated with medium alone (Control) or medium containing 200 U/ml of TNF-alpha for 18 h, trypsinized, added to culture wells coated with 0, 0.5, 1.0, or 2.0 µg/ml plasma fibronectin (pFn), incubated for 30 min at 37°C, and washed with PBS. Adherent cells were counted using an inverted microscope. A: pretreatment of CPAE monolayers with purified TNF-alpha decreased the ability of CPAE cells to adhere to immobilized pFn substrate. Data are represented as the average of adherent cells in 3 × 1 mm2 areas ± SE (3 experiments performed in triplicate). *Significantly less than controls within same Fn concentration group (P < 0.05). B: suspended cells were pretreated with 500 µg/ml RGD or control RGE peptide for 30 min at 4°C before addition to culture wells coated with 2.0 µg/ml of pFn. The decrease in the number of adherent cells with RGD peptide demonstrates that binding of immobilized Fn by CPAE cells is RGD dependent. Data are presented as the average of adherent cells in 3 × 1 mm2 areas ± SE (2 experiments performed in triplicate). **Significantly greater than RGE control; ***significantly less than TNF-treated RGE control (P < 0.05).

Temporal effect of TNF-alpha on adhesion of endothelial cells to immobilized Fn as measured by ECIS. ECIS (19) was used to examine the temporal aspects of cell adhesion to wells coated with 25 µg/ml pFn over 60 min after addition of the cells to the culture medium. Resistance began to increase in both control and TNF-alpha -treated groups by 20-25 min after plating (Fig. 5), with significance (P < 0.05) observed by 30 min. Thereafter, the TNF-alpha -treated group remained significantly lower for the remainder of the 1-h incubation. The presence of cells on the unwashed electrodes from all groups was confirmed by visualization using an inverted microscope after the ECIS recordings were completed. As measured by ECIS (Fig. 5), the addition of RGD peptides inhibited both control and TNF-alpha -treated CPAE cell attachment and spreading on the Fn surface, whereas RGE peptides had no effect (data not shown). Using an inverted microscope, we observed that control cells were spread on the electrode, whereas those exposed to the RGD peptide were found to be rounded in appearance (data not shown). Thus exposure of CPAE cells to TNF-alpha caused a reduction in their RGD-dependent adhesion to Fn.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5.   Temporal effect of RGD-dependent adhesion of CPAE cells to immobilized pFn as measured by electrical cell impedance sensor (ECIS). Confluent CPAE monolayers were treated with medium alone (Control) or medium containing 200 U/ml TNF-alpha for 18 h, trypsinized, and reapplied to ECIS wells precoated with 25 µg/ml of pFn, and electrical resistance was measured for 1 h. A significant decrease (P < 0.05) in resistance of TNF-treated cells compared with control cells occurred after 30 min and continued until 1 h, as indicated by arrow. RGD peptide (250 µg/ml) was added to control (control + RGD) and TNF-treated (TNF + RGD) wells, demonstrating an RGD dependence of adhesion of CPAE cells to immobilized pFn as the increase in electrical resistance is inhibited. Control RGE peptide (250 µg/ml) did not block adhesion to Fn-coated wells (data not shown). Data are presented as means ± SE of 21-22 wells for control or TNF-alpha and 3 wells for samples with RGD or RGE peptides.

Influence of integrin-blocking Abs on the adhesion of TNF-alpha -treated CPAE cells to immobilized pFn. We examined the role of integrins in adhesion of CPAE cells to immobilized pFn using integrin-blocking Abs. Control and TNF-alpha -treated cells were preincubated with 10 µg/ml mAb 13 (beta 1-integrin blocking), 10 µg/ml mAb LM609 (alpha vbeta 3-integrin blocking), or no mAb at all and were applied to wells coated with 2.0 µg/ml pFn. Significant (P < 0.05) losses in adhesion to Fn were observed with control cells preincubated with mAb 13 and mAb LM609 (Fig. 6). Each Ab alone reduced cell adhesion by ~40%, down to the level of TNF-alpha treated cells. In contrast, adhesion of TNF-alpha -treated cells to immobilized pFn was not inhibited (P > 0.05) by mAb 13 or mAb LM609 (Fig. 6). Interestingly, preincubation with both antibodies together did not further reduce adhesion in either group (data not shown). These data further suggested that a loss of integrin-mediated adhesion to surface Fn occurs in CPAE cells treated with TNF-alpha .


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of integrin-blocking Abs on the adhesion of TNF-alpha -treated CPAE cells to surface-immobilized pFn. Confluent CPAE monolayers were treated with either medium alone (Control) or medium containing 200 U/ml of TNF-alpha for 18 h and trypsinized. Suspended cells were pretreated for 30 min at 4°C with 10 µg/ml of the beta 1-blocking Ab (mAb 13), 10 µg/ml of the alpha vbeta 3-integrin blocking Ab (mAb LM609), or no Ab before cells were added to culture wells coated with 2.0 µg/ml pFn. Both the beta 1- and alpha vbeta 3-blocking Abs reduced adhesion of control cells to immobilized pFn to that of TNF-alpha treated cells, whereas with blocking mAb and TNF-alpha treatment, there was no further reduction. Pretreatment with both mAbs together also demonstrated no further reduction (data not shown). Data are recorded as averages of adherent cells in 3 × 1-mm2 areas ± SE of 3 experiments performed in quadruplicate. *Significantly (P < 0.05) less than control (No Ab).

Surface expression of alpha 5beta 1-integrin by flow cytometry. We then speculated that the loss of integrin-mediated adhesion to Fn by CPAE cells treated with TNF-alpha may be the result of a loss of alpha 5beta 1-surface expression. To measure surface expression on CPAE cells, we performed flow cytometry using mAb HA5 to detect the alpha 5beta 1-complex. First, the specificity of mAb HA5 and cross-reactivity for the bovine alpha 5beta 1-complex was confirmed by Western blot analysis of biotinylated surface proteins immunoprecipitated with mAb HA5. Staining blots with streptavidin-HRP revealed two bands consistent with the molecular masses of the alpha 5- and beta 1-integrin subunits (Fig. 7A), and reprobing with a polyclonal Ab against alpha 5-integrin confirmed the upper band was alpha 5.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of TNF-alpha exposure on the surface expression of alpha 5beta 1-integrins on CPAE cells. A: trypsinized CPAE cells were surface biotinylated and incubated with mAb HA5 before extraction. Samples were separated by 7.5% SDS-PAGE, blotted, and stained with streptavidin to detect the labeled surface proteins precipitated by mAb HA5. The blot was reprobed with polyclonal anti-alpha 5 Ab to confirm the indicated band (data not shown). mAb HA5 precipitates bovine alpha 5- and beta 1-integrin subunits. IP, immunoprecipitation. B: after 3 days of culture, CPAE monolayers treated with medium alone (Control) or medium containing 200 U/ml TNF-alpha for 18 h were trypsinized, washed, and counted. Samples containing 1 × 106 cells were analyzed by flow cytometry after labeling with 4 µg of the mouse mAb clone HA5 and an FITC-conjugated secondary Ab (2°Ab). Data are representative of 7 experiments. C: mean fluorescence intensity. Exposure to TNF-alpha for 18 h did not significantly decrease (P > 0.05) the surface expression of alpha 5beta 1-integrins in confluent CPAE cells.

Flow cytometry was performed using mAb HA5 on cells harvested from control and TNF-alpha -treated monolayers. A graph of a representative analysis is shown in Fig. 7B. The averages of mean fluorescence intensity from combined experiments showed no significant (P > 0.05) difference between the control and TNF-alpha -treated groups (Fig. 7C). In addition, no loss of alpha 5beta 1-integrin expression was seen when flow cytometry was performed on cells lifted with trypsin compared with cells lifted with 5 mM EDTA (B. Gao, K. Powell, T. M. Saba, unpublished results), which confirmed that the trypsinization procedure used to lift CPAE cells did not change alpha 5beta 1-integrin surface expression. In addition, recent flow cytometric analysis using mAb LM609 actually showed an approximate twofold increase in the expression of alpha vbeta 3-integrin on the surface of TNF-alpha -treated CPAE cells compared with controls (B. Gao, K. Powell, T. M. Saba, unpublished results). Thus the reduced adhesion of TNF-alpha -treated CPAE cells to immobilized Fn was not the result of reduced alpha 5beta 1- or alpha vbeta 3-surface expression.

Surface expression of 9EG7-detectable epitope by flow cytometry. The beta 1-integrin subunit can play a major role in the activation of the integrin complex and exists in distinct conformations or activity states that influence their interactions with ligand (2, 32). Moreover, expression of the mAb 9EG7-dependent epitope on the beta 1-integrin reflects a high affinity or ligand-bound state that can be influenced by cations, chelating agents, antibodies, inside-out signaling, or ligand occupation (2, 26, 32). Mastrangelo et al. (26) demonstrated a decrease in mAb 9EG7-dependent expression associated with a loss of Fn-dependent adhesion in MG-63 cells transfected with chimeric receptors containing the beta 1-cytoplasmic domain. Since we hypothesized that reduced adhesion of TNF-alpha -treated cells to immobilized pFn might correlate with reduced surface expression of the mAb 9EG7-dependent epitope, we next investigated the possibility that TNF-alpha treatment had altered the activity state of alpha 5beta 1-integrins. First, cross-reactivity to bovine beta 1-subunits was confirmed by probing Western blots of immunoprecipitated alpha 5beta 1-integrin with mAb 9EG7 (data not shown). Flow cytometry performed using mAb 9EG7 and mAb AIIB2, an Ab against beta 1-integrins, revealed a slight but nonsignificant (P > 0.05) increase in both fluorescence intensity for the mAb 9EG7-dependent epitope and the beta 1-integrin on TNF-alpha -treated CPAE cells (Fig. 8). Thus the decreased adhesion of TNF-alpha -treated cells to Fn was not caused by a significant decrease in beta 1-integrin surface expression.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 8.   Effect of TNF-alpha on CPAE cell surface expression of the beta 1-integrin epitope detectable by mAb clone 9EG7. A: confluent CPAE monolayers were treated with medium alone (green) or medium containing 200 U/ml TNF-alpha (red) for 18 h. Samples containing 1 × 106 cells were lifted by trypsinization and analyzed by flow cytometry after labeling with 4 µg of the rat clones mAb 9EG7 or mAb AIIB2 (for total beta 1-integrin determination) and an FITC-conjugated secondary Ab (2°Ab) against rat Ab. Data are representative of 10 experiments. B: mean fluorescence intensity. Exposure to TNF-alpha for 18 h did not significantly (P > 0.05) increase the expression of mAb 9EG7-detectable epitope or total beta 1-integrins on cell surface.

Effect of Mn2+ on the mAb 9EG7-detectable epitope in bovine endothelial cells. Bazzoni et al. (3) demonstrated that phorbol myristate acetate (PMA) increased the adhesion of K562 cells to pFn without an increase in mAb 9EG7-dependent epitope expression. The same study showed that mAb 9EG7 stimulated the adhesion to Fn via alpha 5beta 1-integrins in the presence of Mn2+, a divalent cation that increases mAb 9EG7-dependent epitope expression (2, 3, 25). To verify the existence of a functional mAb 9EG7-dependent epitope in bovine endothelial cells, biotinylated CPAE cells were incubated with mAb 9EG7 in the presence or absence of 5 mM Mn2+. The results of immunoprecipitation with this Ab showed two major bands after staining with streptavidin (Fig. 9A, lanes 2 and 3) that aligned with bands of alpha 5- and beta 1-subunits immunoprecipitated with mAb HA5 (lane 1). These two bands were the only major bands present above 67.5 kDa on the blot, suggesting that alpha 5beta 1-integrin was a major complex immunoprecipitated by mAb 9EG7. The increased band intensity with Mn2+ treatment (lane 3) confirmed that the mAb 9EG7-dependent epitope exists on alpha 5beta 1-integrins expressed on the surface of CPAE cells. Reprobing with a polyclonal Ab against the alpha 5-subunit confirmed the upper band (Fig. 9B). Accordingly, Mn2+ induced the surface expression of the mAb 9EG7-dependent beta 1-epitope in bovine endothelial cells, thus verifying the existence of a functional mAb 9EG7 epitope in our bovine cells.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 9.   Analysis of the beta 1-integrin epitope detectable by mAb 9EG7 on CPAE cells. A: suspended cells were biotinylated, labeled with mAb 9EG7 or mAb HA5 in Tris-buffered saline (TBS) ± 5 mM Mn2+, extracted, and immunoprecipitated. Samples were separated by 7.5% SDS-PAGE, Western blotted, and stained with streptavidin-horseradish peroxidase. Major bands from the 9EG7 immunoprecipitate (lanes 2 and 3) aligned with alpha 5- and beta 1-integrin bands from HA5-immunoprecipitated samples (lane 1). B: blot in A was stripped and reprobed with a polyclonal Ab to alpha 5, confirming that the addition of 5 mM Mn2+ increases the coprecipitation of alpha 5 by mAb 9EG7 (lanes 2 and 3).

Effect of beta 1-activating antibodies on cell adhesion. Addition of mAb 9EG7 can activate the alpha 5beta 1-integrin without Mn2+, presumably by shifting the beta 1-subunit from an inactive to an active state (33). If true, then the TNF-alpha treatment of CPAE cells may have caused inactivation of alpha 5beta 1-integrins, resulting in reduced adhesion to pFn, which should be recoverable by beta 1-activating Abs. To test this concept, suspended control and TNF-alpha -treated CPAE cells were preincubated with 10 µg/ml of the beta 1-activating antibodies mAb 12G10 and mAb 9EG7 before being plated on pFn-coated wells. Neither activating Ab significantly (P < 0.05) increased adhesion of control cells to immobilized pFn, yet both Abs significantly (P < 0.05) increased adhesion of TNF-alpha -treated cells by 30-40%, thus reversing the adhesion deficit (Fig. 10). The restoration of TNF-treated CPAE cells by the beta 1-activating antibodies was, in turn, prevented by the beta 1-blocking mAb AIIB2 (data not shown). These beta 1-activating antibodies rescue adhesion of TNF-alpha -treated but not control cells, supporting the concept that TNF-alpha treatment causes a significant population of beta 1-integrins to become inactive.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 10.   Effect of activating Abs against beta 1-integrin on the adhesion of TNF-alpha -treated CPAE cells to surface-immobilized pFn. Confluent CPAE monolayers were treated with either medium alone (Control) or medium containing 200 U/ml of TNF-alpha for 18 h and trypsinized. Suspended cells were pretreated for 30 min at 4°C with 10 µg/ml of the beta 1-integrin-activating Abs mAb 12G10, mAb 9EG7, or no Ab before cells were added to culture wells coated with 2 µg/ml of pFn. Activation of the beta 1-integrin did not significantly increase (P > 0.05) adhesion of control cells but did significantly increase (P < 0.05) that of TNF-treated cells (*) compared with cells treated with TNF-alpha but preincubated with no Ab. The restoration of TNF-treated cells activated by mAb 9EG7 was blocked with 10 µg/ml of the beta 1-blocking mAb AIIB2 but not with a nonblocking control mAb HA5. Data are expressed as the average of adherent cells in 3 × 1-mm2 areas ± SE of 3 experiments performed in quadruplicate.

Soluble 125I-pFn binding and ECM incorporation to CPAE monolayers after TNF-alpha treatment. Because the alpha 5beta 1-integrin has been shown to mediate soluble Fn binding to human fibroblasts (18), we then explored the possibility that the binding of soluble pFn to the cell layers may also be reduced in TNF-alpha -treated monolayers in which alpha 5beta 1-integrin activity was reduced. 125I-pFn binding at 4°C was measured after 5, 10, 20, 30, or 60 min of incubation with TNF-alpha . Contrary to our expectations, the binding of soluble Fn was significantly (P < 0.05) increased five- to sixfold after TNF-alpha treatment at 60 min (Fig. 11A).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 11.   TNF-alpha treatment increases binding of soluble 125I-pFn to CPAE monolayers. CPAE monolayers were treated with medium alone (Control) or medium containing 200 U/ml TNF-alpha for 18 h. A: confluent cells were washed, and 1 ml of MEM containing 1% BSA and 1 µg/ml of human 125I-pFn [2.4 × 106 counts · min-1 (cpm) · µg-1] was added to each well and incubated for 5, 10, 20, 30, or 60 min at 4°C. Monolayers were washed with cold PBS, dissolved with 1 N NaOH, and quantitated. B: at 24, 48, or 72 h of growth, cell layers were washed, and 1 ml of MEM containing 1% BSA and 1 µg/ml of human 125I-pFn (2.4 × 106 cpm/µg) were added to each well. Cell layers were incubated for 60 min at 4°C, washed with cold PBS, dissolved with 1 N NaOH, and quantitated. In both experiments, counts obtained from samples incubated with unlabeled human pFn (300 µg/ml) were subtracted from experimental values as nonspecific binding. TNF-alpha exposure resulted in increased binding of soluble pFn to both preconfluent and confluent CPAE monolayers. Each point represents 3 experiments performed in triplicate (mean ± SE). *Significantly greater (P < 0.05) than controls within same incubation interval groups (A) or postseeding interval groups (B).

Kowalczyk et al. (21, 22) demonstrated that the restrictive barrier formed by endothelial cell-cell interactions can limit the accessibility of soluble pFn to matrix assembly sites. To determine if the unexpected increase in soluble 125I-pFn binding to the CPAE cell layer after TNF-alpha exposure could be explained by increased accessibility to the basolateral region resulting from loss of barrier function after TNF-alpha , binding experiments were repeated using preconfluent CPAE monolayers studied at 24 and 48 h postseeding to remove the restrictive nature of the confluent endothelial barrier. Binding of fluid-phase 125I-pFn to both control and TNF-alpha -treated monolayers decreased as the monolayer reached confluency. However, at all time points (24, 48, and 72 h), the TNF-alpha -treated endothelial cells bound significantly (P < 0.05) more 125I-pFn than untreated controls, even at 24 h postseeding (~2.5-fold increase). These observations suggest that, while accessibility can affect binding of 125I-pFn to endothelial cell layers, it cannot fully explain the increased binding of soluble pFn observed after TNF-alpha treatment.

Because TNF-alpha -treated cells bound more soluble fluid-phase pFn, we predicted increased incorporation of soluble pFn in the matrix by TNF-alpha -treated monolayers. To test this prediction, we measured the DOC-insoluble pool of 125I-pFn in the monolayers as an index of Fn matrix assembly, as previously described (10, 11, 28, 29, 37). Significantly (P < 0.05) more 125I-pFn was detected in the DOC-insoluble fraction of TNF-treated CPAE cell layers than for controls by 3 h, with an approximate threefold increase in incorporation at 24 h (Fig. 12). Thus both an increase in binding and incorporation of soluble pFn exists after exposure of endothelial cells to TNF-alpha .


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 12.   Increased incorporation of 125I-pFn in the ECM of CPAE monolayers exposed to TNF-alpha . Confluent CPAE monolayers were treated with medium alone (Control) or medium containing 200 U/ml TNF-alpha for 18 h at 37°C and washed, and 1 ml of MEM containing 5% FBS (Fn-depleted) and 1 µg/ml of human 125I-pFn (2.4 × 106 cpm/1 µg) was added to each well. Samples were incubated at 37°C for 1, 3, 6, or 24 h. To evaluate the incorporation of the 125I-pFn in the ECM, cells were detergent-extracted with deoxycholate (DOC), and the DOC-insoluble 125I-pFn was quantitated. Each point represents 2 experiments, performed in triplicate wells. Data are expressed as means ± SE in units of ng/106 cells. *Significantly greater (P < 0.05) than controls within same incubation interval group.

Effect of RGD peptides and mAb 13 on fluid-phase pFn binding to TNF-alpha -treated CPAE monolayers. It has been suggested that alpha 5beta 1-integrins can facilitate pFn incorporation in the ECM (1, 18, 51). However, despite the reduction of beta 1-integrin function, we observed an increase in soluble pFn binding and ECM incorporation after TNF-alpha treatment. Because it is possible that beta 1-integrins, although losing adhesive activity for immobilized Fn, may still play a role in soluble ligand binding, we repeated the binding studies using control and TNF-alpha -treated CPAE monolayers exposed to RGD peptides and mAb 13 to investigate a role for beta 1-integrins in soluble pFn binding. TNF-alpha -treated monolayers again showed increased binding of 125I-pFn compared with controls, but only the control cells were inhibited (P < 0.05) when incubated with the RGD peptide compared with RGE peptide (Fig. 13A).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 13.   RGD peptide and mAb 13 do not block binding of soluble 125I-pFn by TNF-alpha -treated CPAE monolayers. Confluent CPAE monolayers were treated with medium alone (Control) or medium containing 200 U/ml TNF-alpha for 18 h and washed, and 1 ml of MEM containing 1% BSA and 1 µg/ml of human 125I-pFn (2.4 × 106 cpm/µg) was added to each well at 4°C in the presence of 500 µg/ml of RGE or RGD peptide (A) or 10 µg/ml of clone mAb 13 (B). Cell layers were incubated for 60 min at 4°C or 37°C, washed with cold PBS, dissolved with 1 N NaOH, and quantitated. RGD peptides could not be used at 37°C because of lifting of the CPAE monolayers. Counts obtained from samples incubated with unlabeled human pFn (300 µg/ml) were subtracted from experimental values as nonspecific binding. Each point represents 3 experiments performed in duplicate (mean ± SE). *Significantly less than RGE or No Ab controls (P < 0.05).

Using mAb 13 to specifically block beta 1-integrins, Fogerty et al. (18) observed an inhibition of soluble Fn binding to human fibroblast monolayers. Surprisingly, soluble pFn binding to control and TNF-alpha -treated monolayers was not blocked at 4°C by the addition of 10 µg/ml mAb 13 (Fig. 13B, left). Because we were unsure whether inhibition at 4°C influenced the possible allosteric-based inhibition of mAb 13 (13), we repeated the binding study at 37°C (Fig. 13B, right). In this case, the control group showed a decrease (P < 0.05) in pFn binding after incubation with mAb 13 while the TNF-alpha -treated group did not display such a decrease with mAb 13. Blocking the alpha vbeta 3-integrin with mAb LM609 did not inhibit pFn binding to control or TNF-alpha -treated groups at either 4°C or 37°C (data not shown). These data further support the conclusion that TNF-alpha treatment of CPAE monolayers leaves beta 1-integrins in an inactive state, resulting in reduced cell adhesion to pFn-coated surfaces. In response, there appears to be an increase in integrin-independent binding and ECM incorporation of soluble Fn, perhaps as a compensatory mechanism.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously shown that the addition of either recombinant TNF-alpha , soluble RGD-containing peptides, or polyclonal antibodies against alpha 5beta 1-integrins to the culture medium of previously confluent CPAE monolayers increased their protein permeability in association with disruption of the fine fibrillar Fn-rich matrix (10, 48, 49). The addition of soluble human pFn can prevent and reverse the increase in monolayer protein permeability caused by TNF-alpha , and specificity was apparent, since other RGD-containing proteins such as fibrinogen and vitronectin will not work (10, 48, 49). In the current study, we observed reduced colocalization of alpha 5beta 1-integrins with fine Fn fibers in the ECM after TNF-alpha exposure, which correlated with reduced adhesion of the CPAE cells to immobilized Fn. Curtis et al. (11) demonstrated that the protein permeability increase and Fn matrix rearrangement is not the result of proteolysis of Fn within the subendothelial ECM after TNF-alpha treatment, since no Fn degradation could be detected in the medium or cell layer/matrix. In addition, protease inhibitors did not prevent the increase in protein permeability after TNF-alpha (11). Thus we hypothesized that this reduced ability of CPAE cells to adhere to matrix Fn may be because of changes in either the surface expression of alpha 5beta 1-integrins or the activation state of the beta 1-subunit. Cytokines such as TNF-alpha , interleukin-1beta , and IFN-gamma have been shown to modulate the surface expression of integrins on a variety of cells (12, 14, 17, 31). Although exposure of HUVECs (40) to 100 ng/ml TNF-alpha can decrease the expression of the alpha 5beta 1-integrin, we were unable to detect any significant reduction in the expression of alpha 5beta 1-integrins after exposure of CPAE monolayers to ~9 ng/ml TNF-alpha , as used in our experimental protocol.

Although mAb 13, a blocking Ab against the beta 1-subunit, significantly inhibited adhesion of control cells to pFn, only partial adhesion blocking occurred. Other integrins on the cell surface may also be involved in the adhesion of CPAE cells to immobilized Fn such as alpha vbeta 3, alpha vbeta 5, and alpha IIBbeta 3. We observed that blocking the alpha vbeta 3-integrin with mAb LM609 also inhibited adhesion to immobilized Fn. Defilippi et al. (13, 14) showed that cotreatment of HUVECs with TNF-alpha and IFN-gamma decreased the surface expression of alpha vbeta 3-integrin, whereas B. Gao, K. Powell, and T. M. Saba (unpublished results) demonstrated that surface expression of alpha vbeta 3-integrins on CPAE cells doubled after exposure to TNF-alpha for 18 h, as used in the current study. There was also no additive blocking effect against adhesion when cells were coincubated with mAb 13 and mAb LM609 together, raising the possibility that the alpha 5beta 1- and alpha vbeta 3-integrins work in concert in mediating the adhesion of CPAE cells to immobilized Fn. In support of this concept is the finding that M21 melanoma cells require surface expression of both alpha 5beta 1- and alpha vbeta 3-integrins to attach and spread on Fn-coated surfaces (7). Although alpha 5beta 1- and alpha vbeta 3-integrins may both play a role in maintaining pulmonary endothelial monolayer barrier function, the increased protein permeability caused by TNF-alpha appears not to be the result of decreased surface expression of these receptors. Furthermore, unlike alpha 5beta 1-integrins, alpha vbeta 3-integrins were not colocalized with Fn in the matrix, suggesting a specific role for alpha 5beta 1-integrins in the adhesion of CPAE monolayers to Fn-rich matrix. Other RGD-dependent integrins, such as alpha vbeta 5 and alpha IIBbeta 3, may also play a role in CPAE cell adhesion to immobilized Fn, and their role may be addressed in future studies.

The ability of beta 1-integrin-activating Abs to restore the adhesion of TNF-alpha -treated CPAE cells to Fn surfaces, without affecting the adhesion activity of control cells, suggests that a loss of alpha 5beta 1-integrin activity may be responsible for the observed increased protein permeability. The role for alpha 5beta 1 is further supported by the ability of mAb 9EG7 to immunoprecipitate alpha 5beta 1-integrin subunits. One must also consider the possibility that TNF-alpha treatment may have caused a shift of a population of alpha 5beta 1-integrins from an active to an inactive state, incapable of binding immobilized pFn. Mould et al. (33) have shown that an inactive pool of alpha 5beta 1-integrins incapable of binding ligand can indeed exist and that mAb 9EG7 can rescue this inactive pool with respect to ligand binding. An association between decreased surface expression of the mAb 9EG7-dependent epitope and decreased cell adhesion to Fn has also been previously suggested (26). We did not observe decreased expression of this epitope after TNF-alpha in the current study. Despite a lack of changes in surface expression in our study, this epitope appeared to be functionally responsive to Mn2+, a well-known characteristic of this epitope (2, 3). However, a direct relationship between mAb 9EG7-dependent epitope expression and cell adhesion to substrate Fn does not always hold true. For example, PMA treatment of K562 cells increased the ability to adhere to Fn without increased surface expression of the mAb 9EG7-dependent epitope (3).

The possibility that TNF-alpha -induced loss of adhesion may be mediated by the inactivation of integrins is also supported by several other studies. Ruegg et al. (42) cotreated human endothelial cells with 200 ng/ml TNF-alpha and 330 ng/ml IFN-gamma and observed a loss of adhesion to immobilized vitronectin, denatured collagen, and fibrinogen, but not to Fn. This loss of adhesion was the result of inactivation of alpha vbeta 3-integrins, even in the presence of their increased surface expression. The mechanism by which beta 1 deactivation can be caused by TNF-alpha , as suggested by our current data, can only be speculated. Blystone et al. (4) demonstrated that alpha vbeta 3-integrins can negatively influence alpha 5beta 1-integrin activity with respect to Fn-mediated phagocytosis by suppressing calcium/calmodulin-dependent protein kinase II (CaMKII) activity. Furthermore, Bouvard et al. (5) have shown that CaMKII mediates CHO cell adhesion to Fn by regulating the affinity state of the alpha 5beta 1-integrin. Based on these findings, one could speculate that the twofold increase in surface expression of alpha vbeta 3-integrin on CPAE cells after TNF-alpha exposure may have caused a reduction of CaMKII activity, thereby reducing alpha 5beta 1-integrin activity and contributing to the decrease in adhesion of CPAE cells to the Fn matrix.

In addition to their role in cell adhesion to ECM, alpha 5beta 1-integrins may facilitate the binding of soluble Fn to the cell layer and its matrix assembly (1, 18, 23, 27, 51). Our findings of decreased beta 1-integrin-mediated binding of soluble pFn after TNF-alpha are consistent with the belief that a population of alpha 5beta 1-integrins expressed on the surface of TNF-alpha -treated CPAE cells are in a low-affinity or inactive state. However, the reduction we observed in beta 1-mediated pFn binding was greatly offset by a dramatic increase in pFn binding to the cell layer, which could not be blocked by mAb 13 or RGD peptide. The alpha 5beta 1-integrins have the ability to assist in the assembly of soluble Fn in ECM when transfected in CHO cells (50), but mAb LM609, which blocks such matrix assembly of Fn, did not inhibit binding of soluble pFn to the cell layer in our current study. Thus the increased binding of soluble pFn does not appear to be integrin mediated; however, it may be mediated by tissue transglutaminase, since its extracellular activity is increased in CPAE cells after TNF-alpha , causing nonreducible Fn multimer formation in the matrix (8).

In response to binding an immobilized ligand, such as Fn, major integrins will cluster within focal adhesion complexes, resulting in interactions with actin-associated cytoskeletal proteins (6). The structural link between the ECM, integrins, and the actin cytoskeleton stabilizes cell adhesion and contributes to the mechanical basis by which integrins can influence endothelial cell shape. Changes in the ability of the cells to bind to the ECM results in cytoskeletal reorganization and global changes in cell shape (30, 44). Sims et al. (44) showed that changes in cell shape can result from cytoskeletal tension, generated by an actomyosin filament sliding mechanism, and that this tension can be physically resisted by cell surface integrins bound to immobilized adhesion sites within the ECM, including the RGD or cell attachment site in Fn. Thus the ability of integrins to bind to matrix proteins may exert a unique control on cell shape. Loss of this binding could result in a loss of monolayer confluence and the development of intercellular gaps, with an increase in endothelial protein permeability like we observed.

In summary, our findings demonstrate that TNF-alpha , which increases protein permeability and reorganizes the Fn matrix, also reduces the ability for CPAE cells to adhere to immobilized Fn. This loss of adhesion to an Fn substrate appears to be related to inactivation or a reduced function of alpha 5beta 1-integrins. However, in parallel, TNF-alpha treatment caused both an increase in beta 1-integrin-independent binding of soluble pFn to the CPAE cell layer and its ECM incorporation. This suggests that TNF-alpha released in the plasma during postoperative bacterial sepsis or lung inflammatory injury may have a dual role. It may initially increase the permeability of the lung vascular barrier to facilitate the transvascular flux of plasma proteins essential to pulmonary host defense but, thereafter, lead to enhanced incorporation of pFn in the subendothelial matrix to rapidly stabilize the endothelial barrier.


    ACKNOWLEDGEMENTS

The technical assistance of Edward Lewis, Van Fronhofer, Eshin Cho, and Kara L. Powell and the secretarial assistance of Debbie Moran is acknowledged. We thank Caroline Damsky at the University of California-San Francisco for the generous gift of the mAb clone AIIB2 originally provided to the laboratory in support of the thesis research of T. M. Curtis.


    FOOTNOTES

This study was supported primarily by National Institutes of Health Grants GM-21447 (T. M. Saba) and in part by GM-51540 (S. E. LaFlamme). Trainee support was provided by T32-HL-07194 and F32-HL-010201 (T. M. Curtis), T32 HL-07529 (R. F. Rotundo), and T32-GM-07033 (A. Mastrangelo).

Address for reprint requests and other correspondence: T. M. Saba, Center for Cell Biology and Cancer Research, MC-165, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208 (E-mail: sabat{at}mail.amc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00145.2000

Received 5 May 2000; accepted in final form 2 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Akiyama, SK, Yamada SS, Chen WT, and Yamada KM. Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization. J Cell Biol 109: 863-875, 1991[Abstract].

2.   Bazzoni, G, Ma L, Blue ML, and Hemler ME. Divalent cations and ligands induce conformational changes that are highly divergent among beta 1 integrins. J Biol Chem 273: 6670-6678, 1998[Abstract/Free Full Text].

3.   Bazzoni, G, Shih DT, Buck CA, and Hemler ME. Monoclonal antibody 9EG7 defines a novel beta 1 integrin epitope induced by soluble ligand and manganese, but inhibited by calcium. J Biol Chem 270: 25570-25577, 1995[Abstract/Free Full Text].

4.   Blystone, SD, Slater SE, Williams MP, Crow MT, and Brown EJ. A molecular mechanism of integrin crosstalk: alpha vbeta 3 suppression of calcium/calmodulin-dependent protein kinase II regulates alpha 5beta 1 function. J Cell Biol 145: 889-897, 1999[Abstract/Free Full Text].

5.   Bouvard, D, Molla A, and Block MR. Calcium/calmodulin-dependent protein kinase II controls alpha 5beta 1 integrin-mediated inside-out signaling. J Cell Sci 111: 657-665, 1998[Abstract/Free Full Text].

6.   Burridge, K, Fath K, Kelly T, Nuckolls G, and Turner C. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol 4: 487-525, 1988[ISI].

7.   Charo, IF, Nannizzi L, Smith JW, and Cheresh DA. The vitronectin receptor alpha vbeta 3 binds fibronectin and acts in concert with alpha 5beta 1 in promoting cellular attachment and spreading on fibronectin. J Cell Biol 111: 2795-2800, 1990[Abstract].

8.   Chen, R, Gao B, Huang C, Olsen B, Rotundo RF, Blumenstock, and Saba TM. Transglutaminase-mediated fibronectin multimerization in lung endothelial matrix in response to TNF-alpha . Am J Physiol Lung Cell Mol Physiol 279: L161-L174, 2000[Abstract/Free Full Text].

9.   Cooper, JA, Del Vecchio PJ, Minnear FL, Burhop KE, Selig WM, Garcia JGN, and Malik AB. Measurement of albumin permeability across endothelial monolayers in vitro. J Appl Physiol 62: 1076-1083, 1987[Abstract/Free Full Text].

10.   Curtis, TM, McKeown-Longo PJ, Vincent PA, Homan SM, Wheatley EM, and Saba TM. Fibronectin attenuates increased endothelial monolayer permeability after RGD peptide, anti-alpha 5beta 1, or TNF-alpha exposure. Am J Physiol Lung Cell Mol Physiol 269: L248-L260, 1995[Abstract/Free Full Text].

11.   Curtis, TM, Rotundo RF, Vincent PA, McKeown-Longo PJ, and Saba TM. Matrix Fn disruption and increased endothelial permeability after TNF-alpha is independent of Fn proteolysis. Am J Physiol Lung Cell Mol Physiol 275: L126-L138, 1998[Abstract/Free Full Text].

12.   Defilippi, P, Silengo L, and Tarone G. alpha 6beta 1 Integrin (Laminin receptor) is down-regulated by tumor necrosis factor alpha  and interleukin-1beta in human endothelial cells. J Biol Chem 267: 18303-18307, 1992[Abstract/Free Full Text].

13.   Defilippi, P, Silengo L, and Tarone G. Regulation of adhesion receptors expression in endothelial cells. Curr Top Microbiol Immunol 184: 87-98, 1993[Medline].

14.   Defilippi, P, Truffa G, Stefanuto G, Altruda F, Silengo L, and Tarone G. Tumor necrosis factor alpha  and interferon gamma  modulate the expression of the vitronectin receptor (integrin beta 3) in human endothelial cells. J Biol Chem 266: 7638-7645, 1991[Abstract/Free Full Text].

15.   Dejana, E. Endothelial cell adhesive receptors. J Cardiovasc Pharmacol 21: S18-S21, 1993[ISI][Medline].

16.   Dejana, E, Collela S, Conforti G, Abbadini M, Gaboli M, and Marchisio PC. Fibronectin and vitronectin regulate the organization of their respective Arg-Gly-Asp adhesion receptors in cultured human endothelial cells. J Cell Biol 107: 1215-1223, 1988[Abstract].

17.   Dekker, SK, Vink J, Jan Vermeer B, Bruijn JA, Mihm MC, Jr, and Byers HR. Differential effects of interleukin 1-alpha (IL-1alpha ) or tumor necrosis factor-alpha (TNF-alpha ) on motility of human melanoma cell lines on fibronectin. J Invest Dermatol 102: 898-905, 1994[Abstract].

18.   Fogerty, PJ, Akiyama SK, Yamada KM, and Mosher DF. Inhibition of binding of fibronectin to matrix assembly sites by anti-integrin (alpha 5beta 1) antibodies. J Cell Biol 111: 699-708, 1990[Abstract].

19.   Giaever, I, Keese CR, and Mitra P. Electrical measurement can be used to monitor the attachment and spreading of cells in tissue culture. Res Report BioTechniques 11: 504-510, 1991.

20.   Jin, HM, Vincent PA, Charash WE, Saba TM, McKeown-Longo PJ, Blumenstock FA, and Lewis E. Incorporation of circulating fibronectin into various tissues during sepsis: colocalization with endogenous tissue fibronectin. Exp Mol Pathol 55: 203-216, 1991[ISI][Medline].

21.   Kowalczyk, AP, and McKeown-Longo PJ. Basolateral distribution of fibronectin matrix assembly sites on vascular endothelial monolayers is regulated by substratum fibronectin. J Cell Physiol 152: 126-134, 1992[ISI][Medline].

22.   Kowalczyk, AP, Tulloh RH, and McKeown-Longo PJ. Polarized fibronectin secretion and localized matrix assembly sites correlate with subendothelial matrix formation. Blood 75: 2335-2342, 1990[Abstract].

23.   LaFlamme, SE, Thomas LA, Yamada SS, and Yamada KM. Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly. J Cell Biol 126: 1287-1298, 1994[Abstract].

24.   Lampugnani, MG, Resnati M, Dejana E, and Marchisio PC. The role of integrins in the maintenance of endothelial monolayer integrity. J Cell Biol 112: 479-490, 1991[Abstract].

25.   Lenter, M, Uhlig H, Hamann A, Jeno P, Imhof B, and Vestweber D. A monoclonal antibody against an activation epitope on mouse integrin chain beta 1 blocks adhesion of lymphocytes to the endothelial integrin alpha 6beta 1. Proc Natl Acad Sci USA 90: 9051-9055, 1993[Abstract].

26.   Mastrangelo, AM, Homan SM, Humphries MJ, and LaFlamme SE. Amino acid motifs required for isolated beta  cytoplasmic domains to regulate "in trans" beta 1 integrin conformation and function in cell attachment. J Cell Sci 112: 217-229, 1999[Abstract/Free Full Text].

27.   McDonald, JA, Quade BJ, Broekelmann TJ, LaChance R, Forsman K, Hasegawa E, and Akiyama S. Fibronectin's cell adhesive domain and an amino terminal matrix assembly domain participate in its assembly into fibroblast pericellular matrix. J Biol Chem 262: 2957-2967, 1987[Abstract/Free Full Text].

28.   McKeown-Longo, PJ, and Mosher DF. Interaction of the 70,000 molecular weight amino terminal fragment of fibronectin with the matrix assembly receptor of fibroblasts. J Cell Biol 100: 364-374, 1985[Abstract].

29.   McKeown-Longo, PJ, and Mosher DF. The assembly of the fibronectin matrix in cultured human fibroblast cells. In: Fibronectin, edited by Mosher DF.. San Diego, CA: Academic, 1989, p. 163-179.

30.   Mooney, DJ, Ranger R, and Ingber DE. Cytoskeletal filament assembly and the control of cell spreading and function by the extracellular matrix. J Cell Sci 108: 2311-2320, 1995[Abstract/Free Full Text].

31.   Mortarini, R, Anichini A, and Parmiani G. Heterogeneity for integrin expression and cytokine-mediated VLA modulation can influence the adhesion of human melanoma cells to extracellular matrix proteins. Int J Cancer 47: 551-559, 1991[ISI][Medline].

32.   Mould, AP. Getting integrins into shape: recent insights into how integrin activity is regulated by conformational changes. J Cell Sci 109: 2613-2618, 1996[Free Full Text].

33.   Mould, AP, Akiyama SK, and Humphries MJ. The inhibitory anti-beta 1 integrin monoclonal antibody 13 recognizes an epitope that is attenuated by ligand occupancy. J Biol Chem 271: 20365-20374, 1996[Abstract/Free Full Text].

34.   Pfeffer, K, Matsuyama T, Kundig TM, Wakeham A, Kishihara K, Shahinian A, Wiegmann K, Ohashi PS, Kronke M, and Mak TW. Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection. Cell 73: 457-467, 1993[ISI][Medline].

35.   Pierschbacher, MD, and Ruoslahti E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 309: 30-33, 1984[ISI][Medline].

36.   Pytela, R, Pierschbacher MD, and Ruoslahti E. Identification and isolation of a 140 kilodalton cell surface glycoprotein with properties expected of a fibronectin receptor. Cell 40: 191-196, 1985[ISI][Medline].

37.   Rebres, RA, McKeown-Longo PJ, Vincent PA, Cho E, and Saba TM. Extracellular matrix incorporation of normal and NEM-alkylated fibronectin: liver and spleen deposition. Am J Physiol Gastrointest Liver Physiol 269: G902-G912, 1995[Abstract/Free Full Text].

38.   Redl, H, Schlag G, and Lamche H. TNF- and LPS-induced changes of lung vascular permeability: studies in unanesthetised sheep. Circ Shock 31: 183-192, 1990[ISI][Medline].

39.   Resnikoff, M, Brien TP, Vincent PA, Rotundo RF, Lewis E, McKeown-Longo PJ, and Saba TM. Lung matrix incorporation of fibronectin reduces vascular permeability in postsurgical bacteremia. Am J Physiol Lung Cell Mol Physiol 277: L749-L759, 1999[Abstract/Free Full Text].

40.   Romer, LH, and Polin RA. Endotoxin, tumor necrosis factor, and dexamethasone effects on human endothelial cell fibronectin dynamics: synthesis, matrix assembly, and receptor expression. Biochem Cell Biol 73: 515-524, 1995[ISI][Medline].

41.   Royall, JA, Berkow RL, Beckman JS, Cunningham MK, Matalon S, and Freeman BA. Tumor necrosis factor and interleukin 1alpha increase vascular endothelial permeability. Am J Physiol Lung Cell Mol Physiol 257: L399-L410, 1989[Abstract/Free Full Text].

42.   Ruegg, C, Yilmaz A, Bieler G, Bamat J, Chaubert P, and Lejeune FJ. Evidence for the involvement of endothelial cell integrin alpha vbeta 3 in the disruption of the tumor vasculature induced by TNF and IFN-gamma . Nat Med 4: 408-414, 1998[ISI][Medline].

43.   Saba, TM. Fibronectin: role in phagocytic host defense and lung vascular integrity. In: Fibronectin in Health and Disease, , edited by Carsons S.. Boca Raton, FL: CRC, 1989, p. 49-68.

44.   Sims, JR, Karp S, and Ingber DE. Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape. J Cell Sci 103: 1215-1222, 1992[Abstract/Free Full Text].

45.   Tracey, KJ, Beutler B, Lowry SF, Merryweather J, Wolpe S, Milsark IW, Hariri RJ, Fahey TJ, III, Zentella A, Albert JD, Shires GT, and Cerami A. Shock and tissue injury induced by recombinant human cachectin. Science 234: 470-474, 1986[ISI][Medline].

46.   Tracey, KJ, Fong Y, Hess DG, Manogue KR, Lee AT, Kuo GC, Lowry SF, and Cerami A. Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteremia. Nature 330: 662-664, 1987[ISI][Medline].

47.   Van der Poll, T, and Lowry SF. Tumor necrosis factor in sepsis: mediator of multiple organ failure or essential part of host defense? Shock 3: 1-12, 1995[ISI][Medline].

48.   Wheatley, EM, McKeown-Longo PJ, Vincent PA, and Saba TM. Incorporation of fibronectin into matrix decreases the TNF-induced increase in endothelial monolayer permeability. Am J Physiol Lung Cell Mol Physiol 265: L148-L157, 1993[Abstract/Free Full Text].

49.   Wheatley, EM, Vincent PA, McKeown-Longo PJ, and Saba TM. Effect of fibronectin on permeability of normal and TNF-treated lung endothelial cell monolayers. Am J Physiol Regulatory Integrative Comp Physiol 264: R90-R96, 1993[Abstract/Free Full Text].

50.   Wu, C, Hughes PE, Ginsberg MH, and McDonald JA. Identification of a new biological function for the integrin alpha vbeta 3: initiation of fibronectin matrix assembly. Cell Adhes Commun 4: 149-158, 1996[ISI][Medline].

51.   Wu, CY, Keivens VM, O'Toole TE, McDonald JA, and Ginsberg MH. Integrin activation and cytoskeletal interaction are essential for the assembly of a fibronectin matrix. Cell 83: 715-724, 1995[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 282(2):L316-L329
1040-0605/02 $5.00 Copyright © 2002 the American Physiological Society