Lung matrix deposition of normal and alkylated plasma fibronectin: response to postsurgical sepsis

Thomas P. Brien, Pramod P. Reddy, Peter A. Vincent, Edward P. Lewis, Jeffrey S. Ross, and Thomas M. Saba

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

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Plasma fibronectin (Fn) can both enhance phagocytic clearance of microparticulate debris by macrophages as well as incorporate it into the lung extracellular matrix (ECM). The goal of this study was to document that N-ethylmaleimide (NEM)-treated human plasma Fn (HFn) would lose its ability to incorporate into the lung ECM in vivo even though it would retain its ability to stimulate test particle phagocytosis and bind to fibrin. Using dual-label immunofluorescence, we compared the lung deposition of purified normal HFn and NEM-alkylated HFn (NEM-HFn) after their intravenous injection into postoperative nonbacteremic and bacteremic sheep in relationship to the localization of endogenous sheep Fn. Two days after a sterile surgical thoracotomy, sheep were infused with either 5 × 108 Pseudomonas aeruginosa (postsurgical bacteremic model) or the diluent (nonbacteremic model). They also received a bolus 100-mg injection (5 min) of either HFn or NEM-HFn. Analysis of serial lung biopsies harvested at 2-h intervals demonstrated little deposition of NEM-HFn compared with HFn in the lung interstitial matrix of postoperative nonbacteremic sheep. In contrast, enhanced deposition of both HFn and NEM-HFn was observed in the lungs of postoperative bacteremic sheep. However, in the lungs of bacteremic sheep, HFn displayed a diffuse fibrillar deposition pattern in the lung characteristic of ECM incorporation, whereas the enhanced NEM-HFn deposition, especially in the interstitial ECM region of the lung, was primarily focal and punctate, with very little fibrillar incorporation. Immunofluorescent analysis with antibodies specific to fibrinogen, Fn, and lung macrophage surface antigens coupled with immunoperoxidase staining for HFn antigen revealed that the punctate fluorescence pattern was due to both the binding of HFn to fibrin and its colocalization with inflammatory cells. Thus treatment of plasma Fn with low concentrations of NEM will limit its normal in vivo fibrillar incorporation into the interstitial ECM region of the lung.

extracellular matrix

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

FIBRONECTIN (Fn) can influence macrophage phagocytic function as well as lung endothelial cell adhesion to the subendothelial extracellular matrix (ECM) (5). The plasma pool of Fn (pFn) is synthesized primarily by hepatocytes, and pFn can be incorporated into the insoluble tissue pool of Fn in the ECM (10, 11, 16, 24, 32). Accordingly, the ECM pool of Fn consists of Fn locally synthesized as well as Fn derived from the plasma (30). Patients who become septic after major surgery, trauma, or burn often manifest low levels of pFn (36) in temporal association with lung vascular failure and pulmonary edema. Because pFn deficiency will amplify the increase in lung endothelial protein permeability in sheep during postoperative sepsis (23), it has been postulated that soluble pFn may influence the integrity of the lung vascular barrier. This concept is supported by the observation that an infusion of Fn-rich cryoprecipitate or purified human pFn (HFn) to elevate the plasma level of Fn can attenuate the increase in lung vascular permeability in sheep during postoperative bacteremia (12).

The mechanism by which infusion of pFn reduces lung protein permeability with posttrauma or postoperative bacteremia is unknown. One concept relates to its opsonic role (5) in that a sustained decrease in pFn after trauma, burn, or major surgery will limit liver and spleen phagocytic clearance of abnormal nonbacterial bloodborne particulates formed due to injury, sepsis, and/or intravascular coagulation (17, 30, 31, 36). Their delayed removal is believed to contribute to lung microvascular embolization and leukosequestration (34, 35) as well as the subsequent release of proteases and reactive oxygen metabolites capable of altering endothelial integrity.

Another potential mechanism is related to the ability of pFn to incorporate into the ECM of the lung (10, 11, 15, 16, 24) where it may influence endothelial integrity due to the binding of endothelial cell surface alpha 5beta 1-integrins with RGD (Arg-Gly-Asp) cell attachment sites in matrix-localized Fn (14, 42). This concept is supported by the finding that incorporation of pFn into the subendothelial matrix of cultured lung endothelial monolayers can attenuate as well as reverse the increase in monolayer protein permeability after exposure to either human tumor necrosis factor-alpha (42) or soluble RGD peptides (14).

N-ethylmaleimide (NEM) treatment of pFn prevents its in vitro incorporation into the ECM of cultured cell layers (20). Thus one approach to determine the relative importance of its in vivo incorporation into the lung matrix versus its opsonic influence on phagocytic function in terms of its protective effect on lung vascular integrity is to compare the effect of normal versus alkylated (NEM-treated) HFn (NEM-HFn) on lung protein permeability in postoperative bacteremic sheep. However, this experimental approach assumes that NEM-treated HFn will not incorporate in vivo into the interstitial lung matrix of sheep after intravenous infusion.

We determined whether exogenously administered normal pFn, but not NEM-treated pFn, would incorporate into a normal lung interstitial ECM even though both would retain opsonic activity. The experimental model used was the postoperative sublethal bacteremic sheep because this has been previously employed to study lung endothelial protein permeability in vivo. HFn was used as an "immunologic tracer" because its deposition in the lung could be selectively detected with species-specific antibodies to HFn that do not cross-react with endogenous sheep Fn (SFn) (12).

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Purified HFn and its alkylation. We used purified HFn prepared in a pasteurized and lyophilized form (Revlon Health Care Group/USV Armour Pharmaceuticals). This preparation has been previously used for both in vitro and in vivo studies, including a phase II clinical study in surgical patients (31). It enhances the phagocytosis of gelatin-coated particles by macrophages (31) and can attenuate the increase in lung vascular permeability in postsurgical bacteremic sheep when injected intravenously at a dose of 500 mg/sheep. Purified HFn was alkylated by treatment with 10-20 mM NEM for 30-60 min at pH 11 (26). It was then dialyzed overnight against 10 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS)-0.15 M NaCl (CAPS-buffered saline) at pH 11 and subsequently dialyzed overnight against normal saline. Before infusion, the pH of the NEM-treated Fn solution was adjusted to 7.4 with 1 M phosphate buffer.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Fn preparations. The NEM-treated HFn was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to verify that it had not undergone fragmentation. Samples were diluted 1:5 with buffer containing 3.2% sodium dodecyl sulfate and 16% glycerol in 0.5 M tris(hydroxymethyl)aminomethane (Tris), pH 6.8. A discontinuous Tris-glycine buffer system was used in vertical slab minigels (Bio-Rad, Richmond, CA) with 3% stacking gels (pH 6.8) and 6% separating gels (pH 8.8). Protein was visualized with Coomassie brilliant blue. Both the normal HFn and NEM-HFn had a molecular mass of ~440,000 Da, with no significant fragmentation observed (data not shown).

Iodination of purified HFn. Iodination of purified HFn was accomplished by mixing 400 µl of Fn (1 mg/ml in 0.2 M phosphate buffer) with 1 mCi of Na125I and 35 µl of chloramine T (4 mM in 0.4 M phosphate buffer, pH 7.4). After a 1-min reaction period, 35 µl of sodium metabisulfite [8 mM in phosphate-buffered saline (PBS)] were added to stop the reaction, and the mixture was added to a G-25 gel-filtration column (PD-10, Pharmacia). Fractions containing the labeled protein were pooled and dialyzed against PBS. All 125I-HFn preparations were stored at -80°C until use.

Competitive binding assay of HFn and NEM-HFn. Before intravenous injection of HFn and NEM-HFn, we analyzed each preparation in terms of its ability to competitively block the incorporation of 125I-HFn into the ECM of cultured fibroblasts (21). Fibroblasts were used in these competitive binding assays because their ability to assemble an Fn-rich fibrillar matrix is well documented (19, 20). A1-F human foreskin fibroblasts were cultured in Ham's F-12 medium supplemented with 10% fetal bovine serum (Hyclone, Logan UT), 100 U/ml of penicillin, and 100 µg/ml of streptomycin (GIBCO BRL, Life Technologies, Grand Island, NY) (19, 21, 26). Briefly, at 5-8 days postseeding, the cells were washed with PBS and supplemented with medium containing 0.2% bovine serum albumin, 1-3 µg of 125I-HFn, and the unlabeled HFn or NEM-HFn at concentrations of either 0, 5, 25, or 100 µg/ml. After a 30-min incubation at 37°C, the cell layers were again washed with PBS and extracted with 1 N NaOH before isotopic measurement of 125I-HFn bound to the cell layers (19, 21, 26).

Liver slice bioassay of opsonic activity. Normal HFn and NEM-HFn were evaluated for the retention of opsonic ability with a rat liver tissue slice bioassay with the gelatinized reticuloendothelial test lipid emulsion as the target particles (4, 5, 31). Fn-mediated opsonic activity relative to Kupffer cell uptake of the test emulsion is expressed as a percentage of the 2.0-mg test particle dose removed per 100 mg of liver (31).

Kupffer cell and peritoneal macrophage monolayer assays of opsonic activity. Opsonic activity of purified HFn and NEM-HFn was also evaluated by both a hepatic Kupffer cell and a peritoneal macrophage monolayer bioassay with gelatinized 51Cr-labeled Formalin-fixed sheep red blood cells (RBCs) as the target particles as previously described (4, 9, 31). In both monolayer assays with isolated rat macrophages (4, 9, 31), 51Cr-RBC uptake is expressed as a percentage of the target particle dose removed per 2 × 106 macrophages. Fn-mediated internalization of the gelatinized fixed sheep RBC target particles by these macrophage monolayers as used in these bioassays has been verified by electron microscopy (9).

Surgical thoracotomy and lung lymph fistula model. Our surgical protocol, approved by the Institutional Animal Care and Use Committee, involved the cannulation of the lymphatic drainage of the lung typically done for assessment of lung protein clearance in sheep (10-12, 23). Male sheep (22-30 kg) were fasted of solid food for 48 h before surgery. General anesthesia was induced with 2.5% sodium pentothal (15 mg/kg). The sheep were mechanically ventilated throughout the surgical procedure at 18 breaths/min, with a tidal volume of 15 ml/kg. Anesthesia was maintained with 50% N2O, 50% O2, and 1-1.5% halothane.

Each sheep received 2 liters of normal saline intravenously during the surgical procedure. Under sterile conditions, a right thoracotomy was performed, and the efferent duct of the caudal mediastinal lymph node was cannulated (12, 23, 29). The posterior portion of the lymph node was ligated at the level of the inferior pulmonary ligament to minimize contamination of lung lymph by diaphragmatic lymphatics. The Silastic catheter was tunneled outside the thorax and anchored at the level of the caudal mediastinal lymph node when the sheep is standing, and the incision was closed with O-silk. The right common carotid artery was cannulated with a polyethylene catheter (PE-205) impregnated with tridodecylmethylammonium chloride-heparin for blood sampling. The right internal jugular vein was cannulated with an 8.5-Fr Cordis Introducer (Cordis Laboratories, Miami, FL) to facilitate test solution infusion and placement of a Swan-Ganz catheter.

Experimental protocol and serial lung biopsy. After surgery, the sheep recovered in metabolic cages with free access to food and water after 24 h. All sheep received sterile normal saline intravenously for 2 days after surgery. At 48 h postsurgery, anesthesia was again induced, and biopsies of the left lung were obtained at time 0 and then at 2-h intervals, with an emphasis on the 4- to 8-h interval when the pulmonary pressures had normalized (10-12). For biopsy, the left lung was inflated to a pressure of 35 cmH2O, and two curved clamps were placed around a small (~3 × 2 × 1-cm) distal segment of the lung. The tissue was cut between the clamps, and the proximal lung was closed with a running, interlocking 5-0 prolene suture. The chest incision was closed with towel clamps between biopsies to prevent evaporative water loss. All groups underwent serial biopsies.

After the time 0 lung biopsy, the sheep received 100 mg of either HFn or NEM-HFn (2 mg/ml) over 5 min via the introducer catheter. At time 0, the sheep received a 60-min intravenous infusion of either 5 × 108 live, washed Pseudomonas aeruginosa (American Type Culture Collection no. 27853, Rockville, MD) suspended in 50 ml of sterile saline (10-12) or 50 ml of sterile diluent saline. The small 100-mg dose of HFn will only raise the total pFn antigen level by ~5-10% (10) and is much less than the 500-mg dose of HFn used to attenuate the increase in lung protein clearance with postsurgical bacterial infusion (33). However, as an immunologic tracer, this 100-mg dose provides enough HFn antigen for its clear detection (distinct from endogenous SFn) in the tissues with immunofluorescence and species-specific antibodies.

Hemodynamic measurements. Mean systemic arterial pressure and pulmonary arterial pressure were measured with Statham P23AC pressure transducers and a Grass model 7B polygraph. Central venous pressure and pulmonary capillary wedge pressure were also recorded. Cardiac output was determined by thermodilution utilizing a cardiac output computer (Edwards model 7510A). Systemic vascular resistance was calculated using the pressure and cardiac output measurements.

Plasma sample collection. Serial 9.0-ml blood samples were collected from the sheep in 0.4 ml of an anticoagulant-antiprotease mixture containing 7.5% EDTA, 2.0 mM iodoacetate, and 3.0 mM benzamidine to prevent clotting and Fn fragmentation. Leukocyte counts and hematocrits were determined at all intervals. To obtain plasma, the samples were centrifuged at 6,000 g for 10 min. An Abbott biochromatic automated analyzer (ABA-100) was used to measure the total protein concentration in plasma using a modified Biuret method (10, 12).

Fn concentration. The concentrations of SFn and HFn in the plasma samples were determined by a Laurell rocket electroimmunoassay with species-specific antibodies (10, 12). Rabbit monospecific antibodies against SFn as well as goat monospecific antibodies against HFn were used. Purified SFn, HFn, or NEM-HFn standard was included on each plate, which was electrophoresed at 80 V for 20 h with an LKB multiphor system.

Dual-label immunofluorescence. Dual-label immunofluorescence with species-specific antibodies was used to determine the localization of both the endogenous SFn and purified HFn injected into the sheep (10, 11). The specificity of the antibodies was verified by both enzyme-linked immunosorbent assay and immunostaining of cultured human fibroblasts adherent to their Fn-rich matrix versus sheep lung tissue (10). Direct immunofluorescence was used for detection of HFn with fluorescein isothiocyanate (FITC)-conjugated goat anti-HFn (Cappel, Durham, NC) as the primary antibody. This antibody does not react with SFn (10). In contrast, indirect immunofluorescence was used to detect SFn. The primary antibody was rabbit anti-SFn, which was rendered species specific by passage over HFn affinity columns. Rhodamine isothiocyanate (RITC)-conjugated goat anti-rabbit immunoglobulin (Ig) G (Cappel, Durham, NC) served as the secondary antibody.

Tissue specimens taken for immunofluorescent analysis were quick-frozen in liquid nitrogen and stored at -80°C. Then, 5-µm cryostat sections were cut from the frozen tissue, placed on microscope slides, and air-dried. The tissue sections were washed with either 0.05 M PBS or Bray's extraction buffer that contained 2 M urea and 10 mg/ml of heparin for 20 min, followed by blocking of nonspecific binding with 3% bovine serum albumin for 1 h. The sections were then incubated with the primary antibodies for 1 h. After four washes with PBS, the secondary antibody was added for 1 h. The tissue samples were again washed four times, air-dried, mounted under N-propylgallate, and then examined with a Nikon microscope equipped for epifluorescence. Barrier filters for rhodamine and fluorescein were employed to allow differentiation between endogenous SFn and exogenous HFn.

Colocalization of injected HFn with lung macrophages in sheep. Lung tissue was also studied by dual-label staining to detect colocalization of the intravenously injected HFn or NEM-HFn with lung-localized sheep macrophages. The technique was identical to that described in Dual-label immunofluorescence except that mouse monoclonal antibody to sheep alveolar macrophage (Biosource International, Camarillo, CA) and RITC-labeled sheep anti-mouse IgG (Cappel) were used along with the FITC-labeled goat anti-HFn.

Extraction of noncovalently bound Fn in lung tissue sections. Immunofluorescent analyses were also done before and after extraction of the lung sections with a heparin-urea extraction buffer (6) used previously in our laboratory (10, 33). The 0.05 M phosphate extraction buffer (pH 7.2) was supplemented with an antiproteinase mixture consisting of 3 mM benzamidine HCl, 7.5% EDTA, and 2 mM iodoacetic acid. Each 1.0 ml of buffer was also supplemented with 2 M urea and 10 mg of bovine heparin (Sigma, St. Louis, MO). Tissue sections were subjected to a 20-min incubation at room temperature (20-25°C) in the extraction buffer. This extraction will remove soluble Fn or Fn noncovalently bound to collagen, fibrin, or proteoglycans (10, 11), but it will not remove Fn cross-linked to fibrin or Fn covalently incorporated in the matrix (10, 11, 33).

Fn incorporation into a fibrin clot. Because postsurgical bacteremia induces intravascular coagulation in sheep (23), we also verified that both 125I-HFn and 125I-NEM-HFn were capable of covalently binding to any fibrin within a clot. Five micrograms of either purified 125I-HFn or purified 125I-NEM-HFn were added to 2 ml of whole sheep blood, allowed to clot for 2 h at 37°C, centrifuged at 8,000 revolutions/min for 15 min, and then washed three times with normal saline and counted. After 30 s of sonication, the clot was extracted with deoxycholate (DOC) detergent for 30 min before analysis of pool I (DOC-soluble) and pool II (DOC-insoluble) 125I radioactivity.

Colocalization of human Fn and human fibrin in the lung. Because antibodies to human fibrinogen (HFb), but not to sheep fibrinogen, are commercially available, we used fresh human cryoprecipitate that is enriched with both Fn and fibrinogen to determine whether circulating HFn would bind to fibrin deposited in the lung during postsurgical bacteremia. Lung samples harvested from a separate group of sheep given six units of fresh human cryoprecipitate intravenously were studied by dual-label immunofluorescence. RITC-labeled goat anti-HFb (E-Y Laboratories, San Mateo, CA) was used to detect human fibrin. FITC-labeled sheep anti-HFn (The Binding Site, Birmingham, UK) was used to detect HFn.

Immunoperoxidase staining for endogenous sheep Fn. Lung sections were analyzed for detailed localization of endogenous SFn by immunoperoxidase staining. Unstained 5-µm sections from Formalin-fixed paraffin-embedded random lung samples were deparaffinized, rehydrated, and then positioned in slide holders within the reaction chamber of a Ventana ES automated immunohistochemistry system (Ventana Medical Systems, Tucson, AZ). The commercially available Ventana Medical Systems DAB detection kit, negative control reagent, and protein block (BioGenese, Ramon, CA) were mounted on the reagent carousel, and the instrument was programmed for manual titration of the primary antibody. A protein block was first applied to limit endogenous peroxidase activity and reduce nonspecific binding of the antibody. The primary antibody (species-specific rabbit Ig against SFn) was then applied at a dilution of 1:500 over a 24-min incubation interval at 37°C. Negative controls were simultaneously incubated with the negative control reagent.

Slides were sequentially incubated with universal biotinylated secondary Ig antibody, avidin-horseradish peroxidase conjugate, and diaminobenzidine substrate followed by standard copper sulfate enhancement. After each incubation step, the slides were washed to both stop the reaction and remove any unbound reagent. Slides were counterstained with hematoxylin and dehydrated, and coverslips were applied.

Immunoperoxidase staining for exogenously injected HFn. Localization of the intravenously injected HFn in the lung was also studied by immunoperoxidase staining. Thus, after deparaffinization, the slides were first pretreated with 10 mM citrate (pH 6) for the purpose of antigen retrieval. In this process, slides were placed in citrate buffer and heated to boiling (microwave) three times at a duration of 5 min each, followed by a 30-min cooling period. No protein block or negative control reagent was used. The primary antibody was again sheep anti-HFn biotinylated with a standard kit. It was applied at a dilution of 1:1 over a 6-h incubation at 37°C. The universal biotinylated secondary antibody was removed from the reagent carousel.

Analysis of data. Data are expressed as means ± SE. A confidence level of 95% was used for significance.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

To document that NEM-treated HFn would not bind and incorporate into the ECM of cell layers under in vitro conditions, we first compared soluble unlabeled HFn and NEM-HFn for their ability to competitively inhibit the binding of 125I-HFn to fibroblast cell layers (Fig. 1). Increasing concentrations of HFn added to the medium resulted in a progressive inhibition in the binding of soluble 125I-HFn to the cell layer. This inhibition was not observed with the NEM-HFn. In contrast, HFn treated with NEM at concentrations of 10-20 mM retained its ability to support in vitro macrophage phagocytosis of gelatin-coated target particles (Table 1). However, recent studies in our laboratory (Lewis and Saba, unpublished data) indicate that higher concentrations of NEM (50-100 nM) can also reduce the opsonic activity of HFn.


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Fig. 1.   Inability of N-ethylmaleimide (NEM)-treated human plasma fibronectin (HFn) to competitively block 125I-HFn incorporation into matrix. Fibroblasts were coincubated with 125I-HFn for 30 min in presence of excess amounts of either HFn or NEM-HFn [unlabeled fibronectin (Fn)] at concentrations of either 0, 5, 25, or 100 µg/ml. Cells were then washed with phosphate-buffered saline and extracted with 1 N NaOH before amount of 125I-HFn bound to cell layers was measured. Counts/min in cell layer without addition of unlabeled Fn equaled 100%.

                              
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Table 1.   Ability of NEM-treated HFn to enhance particle phagocytosis as measured by liver slice and macrophage monolayer bioassays

To employ HFn as an immunologic tracer to study the lung incorporation of plasma-derived Fn, it was important to document that the hemodynamic pattern and neutrophil depletion response typically seen in the postoperative bacteremic sheep model (10-12) were similar in both the HFn- and NEM-HFn-injected sheep. As shown in Table 2, the various hemodynamic parameters were similar and relatively stable throughout the 8-h interval in nonbacteremic sheep infused with either HFn or NEM-HFn. In contrast, postsurgical sheep infused with bacteria demonstrated an early elevation in pulmonary arterial pressure over 1-2 h and a delayed decline in systemic arterial pressure over 6-8 h (P < 0.05), but this response pattern was again similar in sheep given the 100-mg dose of either HFn or NEM-HFn. In nonbacteremic sheep, the peripheral leukocyte count did not change over 8 h (P > 0.05; Fig. 2), but bacterial infusion resulted in a similar acute leukopenia in sheep given either HFn or NEM-HFn (P < 0.01; Fig. 2). As already documented (10, 11, 28; M. Resnikoff, T. P. Brien, E. Lewis, P. A. Vincent, P. J. McKeown-Longo, and T. M. Saba, unpublished observations), injection of only the saline diluent into the sheep does not alter the hemodynamic variables or the white blood cell levels.

                              
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Table 2.   Hemodynamic parameters in postoperative control and bacteremic sheep infused with small doses of normal or NEM-alkylated HFn


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Fig. 2.   Circulating white blood cell (WBC) levels after infusion of HFn or NEM-HFn in nonbacteremic (A) and bacteremic (B) sheep. Blood samples were diluted 1:20 in 0.1% crystal violet-3% acetic acid, and total WBC counts were performed. Values are means ± SE.

Figure 3 depicts the alterations in endogenous SFn in the presence of HFn or NEM-HFn. In nonbacteremic sheep, the endogenous mass of SFn in the plasma (SFn concentration × calculated plasma volume) was relatively constant and unaltered by the intravenous infusion of HFn or NEM-HFn. Bacteremic sheep showed a progressive and similar decline in the mass of SFn in the plasma by 4 h (P < 0.05), which was similar to that in sheep given the low dose (100 mg) of either NEM-HFn or HFn. Thus a 100-mg dose of HFn or NEM-HFn injected intravenously into the sheep as an immunologic marker does not alter the typical hemodynamic, neutropenic, or endogenous pFn depletion response to postoperative bacteremia. This is different than the therapeutic dose of HFn (500 mg/sheep) previously used experimentally to prevent the increase in lung vascular permeability with postoperative bacteremia in sheep (12), which elevates the total pFn concentration by ~50% (to 900-950 µg/ml).


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Fig. 3.   Endogenous sheep plasma Fn (SFn) mass over time. SFn concentrations were determined by Laurel rocket electroimmunoassay with species-specific antibodies and appropriate standards for each of the 4 experimental groups: nonbacteremic+HFn (n = 2 sheep), nonbacteremic+NEM-HFn (n = 3 sheep), bacteremic+HFn (n = 2 sheep), and bacteremic+NEM-HFn (n = 3 sheep). SFn mass was calculated as product of SFn plasma concentration × plasma volume. Initial plasma volume was calculated as product of animal's weight × a constant (58 ml/kg) × baseline hematocrit. Changes in plasma volume over time were calculated from changes in hematocrit, assuming red blood cell mass remained constant.

Figures 4 and 5 depict the immunofluorescent analysis of lung tissue harvested from the sheep 4 h after an infusion of either HFn (A and C) or NEM-HFn (B and D). Each section was stained for both endogenous SFn (A and B) and infused exogenous HFn (C and D) and analyzed with dual-label immunofluorescence microscopy. As shown in Fig. 4, A and B, abundant amounts of endogenous SFn were detected in the lung matrix of postoperative nonbacteremic sheep. This endogenous Fn was not removed when the thin lung tissue sections were subjected to Bray's extraction buffer. In the postoperative nonbacteremic sheep infused with normal HFn, we detected positive staining for HFn in the lung tissue using antibodies specific to HFn (Fig. 4C), but little or no NEM-HFn was detected in the lung tissue after NEM-HFn infusion (Fig. 4D).


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Fig. 4.   Micrographs of lung in nonbacteremic sheep 4 h after HFn or NEM-HFn infusion. Lung tissue was stained with a dual-label immunofluorescence technique. A and B: rhodamine fluorescence for endogenous SFn. C and D: fluorescein fluorescence for exogenous HFn or NEM-HFn. Magnification, ×400.


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Fig. 5.   Micrographs of lung in bacteremic sheep 4 h after HFn or NEM-HFn infusion. Lung tissue was stained with a dual-label immunofluorescence technique. A and B: rhodamine fluorescence for endogenous SFn. C and D: fluorescein fluorescence for exogenous HFn or NEM-HFn. Magnification, ×400.

In the postoperative bacteremic sheep (Fig. 5), the deposition pattern of HFN and NEM-HFn was very different. Again, endogenous SFn was clearly detectable in a fibrillar pattern in the lung matrix (Fig. 5, A and B). Bacterial challenge increased the amount of normal HFn deposited in the lung (Fig. 5C) compared with the nonbacteremic sheep, and this HFn could not be removed by extraction with Bray's buffer (10) (data not shown), consistent with its incorporation into the ECM. The HFn pattern was primarily fibrillar, reflecting ECM incorporation, with some HFn observed in punctate areas (Fig. 5C), whereas the NEM-HFn fluorescence had much less fibrillar deposition with some punctate distribution (Fig. 5D). However, this punctate pattern seen with NEM-HFn was again not removed by extraction in Bray's buffer, suggesting that the NEM-HFn was covalently bound but not actually incorporated into the ECM.

Micrographs showing background staining for Fn antigen in lung tissue harvested from sheep before septic challenge with no primary or secondary antibody added as well as the staining for both endogenous SFn and HFn antigen in septic sheep not previously injected with either HFn or NEM-HFn are shown in Fig. 6. The background fluorescence was undetectable (Fig. 6A). Specificity of the antibody is clearly apparent, with very little nonspecific FITC fluorescence of the HFn antigen when HFn or NEM-HFn was not injected (Fig. 6C) but intense RITC fluorescence staining for endogenous SFn (Fig. 6B).


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Fig. 6.   Representative micrographs depicting background nonspecific control staining of lung tissue harvested from sheep that were not injected with either HFn or NEM-HFn (saline control). A: background control fluorescence for Fn antigen before septic challenge with no primary or secondary antibody added. B: rhodamine fluorescence staining for endogenous SFn antigen with both primary and secondary antibodies added in septic sheep. C: fluorescein isothiocyanate fluorescence staining for HFn antigen with both primary and secondary antibodies added in septic sheep.

Our observations suggested that the NEM-HFn detected in the lungs of the postoperative bacteremic sheep was either associated with inflammatory macrophages ingesting Fn-coated particulates or perhaps covalently bound to fibrin microaggregates (22). To investigate this possibility, the lung tissue was then dually stained for both sheep pulmonary macrophages and HFn. Although only a small number of pulmonary macrophages could be detected in the lung sections from postoperative nonbacteremic sheep, neither HFn nor NEM-HFn was colocalized with them (Fig. 7). In contrast, in the postoperative bacteremic sheep, the injected HFn and NEM-HFn (Fig. 8) were both colocalized with macrophages in the lung.


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Fig. 7.   Micrographs of nonbacteremic lung stained for colocalization of sheep pulmonary macrophages (Mphi ) and HFn. Lung biopsies were stained with a dual-label immunofluorescence technique. A and B: rhodamine fluorescence for sheep macrophages. C and D: fluorescein fluorescence for HFn or NEM-HFn. Magnification, ×400.


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Fig. 8.   Micrographs of bacteremic lung stained for colocalization of Mphi and HFn. Lung biopsies were stained with a dual-label immunofluorescence technique. A and B: rhodamine fluorescence for sheep macrophages. C and D: fluorescein fluorescence for HFn or NEM-HFn. Magnification, ×400.

To look for direct evidence of fibrin-Fn complexes, tissue biopsies were harvested from a separate group of postoperative bacteremic sheep after they were first infused intravenously with fresh human plasma cryoprecipitate enriched with both HFn and HFb. In this experiment, both the lung and liver were examined (Fig. 9) because these two organs are major sites for the deposition of fibrin microaggregates formed with gram-negative bacteremia or endotoxemia. Colocalization of HFn and HFb in both the lung and liver tissue was apparent (Fig. 9).


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Fig. 9.   Micrographs of lung and liver in postoperative bacteremic sheep infused with 6 units of fresh human plasma cryoprecipitate over a 4.5-h time span. A 60-min bacterial infusion was started 2.5 h after start of cryoprecipitate infusion. Sheep were unanesthetized during 8-h experiment but were then anesthetized before lung and liver biopsies were harvested. Tissues were stained with fluorescent antibodies to human fibrinogen (HFb) and HFn. A and B: rhodamine fluorescence for HFb. C and D: fluorescein fluorescence for HFn. Magnification, ×400.

Fn binds to the alpha -chain of fibrin, and this covalent interaction is not disrupted by Bray's extraction buffer. The Factor XIIIa acceptor site that mediates fibrin-Fn cross-linking and the site of NEM alkylation are both at the NH2 terminus of Fn. To confirm that NEM-HFn alkylation did not alter the ability of HFn to still covalently bind to fibrin, 5 mg of 125I-radiolabeled HFn or NEM-HFn were added to whole sheep blood that was then allowed to clot at 37°C. The clot was initially counted for total 125I radioactivity and then extracted with DOC detergent to allow for quantification of 125I-HFn or 125I-NEM-HFn in the pool I (DOC-soluble) and pool II (DOC-insoluble) portions of the clot. When this happens, ~5-6% of the total protein load in the clot is Fn covalently linked to fibrin (22). Our analysis (data not shown) demonstrated that 93% of both HFn (n = 3 samples) and NEM-HFn (n = 3 samples) present in the clot was covalently bound and remained in the DOC-insoluble pool II fraction. Thus some of the intense NEM-HFn punctate staining observed in the lungs of the bacteremic sheep was likely due to its binding to fibrin-containing microthrombi.

We then performed immunoperoxidase staining for both SFn and HFn separately to localize Fn within the microscopic architecture of the lung. Endogenous SFn was clearly abundant in the ECM of both nonbacteremic and bacteremic sheep (Fig. 10). Under high magnification, SFn was detected in the subepithelial and subendothelial regions as well as abundantly in the interstitial matrix area in a fibrillar staining pattern. The intensity of the staining for SFn in the lung interstitial matrix area was much reduced in the postsurgical bacteremic sheep (Fig. 10). With regard to the injected HFn, we found small amounts of HFn in the nonbacteremic lungs, primarily in a fibrillar pattern in the interstitial matrix area (Fig. 11). In contrast, in the bacteremic lungs, there was a marked increase in the interstitial fibrillar deposition of HFn (Fig. 11, brown staining pattern), with a small amount observed in association with sequestered inflammatory cells. Deposition of HFn in the subendothelial and subepithelial ECM regions was also increased during bacteremia. In the nonbacteremic sheep infused with NEM-HFn, we detected very little or no NEM-HFn antigen in the lung interstitial matrix. More importantly, in contrast to the fibrillar deposition pattern with limited punctate deposition of HFn in the bacteremic sheep, the NEM-HFn appeared mainly in a punctate, nonfibrillar pattern in association with both fibrin and inflammatory macrophages.


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Fig. 10.   Immunoperoxidase staining of lung for endogenous SFn. A and B: nonbacteremic sheep. C and D: bacteremic sheep. Magnification, ×400.


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Fig. 11.   Immunoperoxidase staining of lung for exogenous HFn or NEM-HFn. A and B: nonbacteremic sheep. C and D: bacteremic sheep. Magnification, ×400.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Previous studies (10, 16, 24, 25, 37) have shown that Fn is an integral component of the normal interstitial, subendothelial, and subepithelial matrices in the mature lungs of rats, sheep, and humans. Indeed, this has been validated by in vivo isotopic kinetic experiments (15) and organ extraction experiments (33) as well as immunofluorescent analysis (10, 16) in normal rats and sheep. McKeown-Longo and Mosher (19-21) first characterized the in vitro deposition of HFn into the detergent-insoluble pool of proteins within the ECM of cultured human fibroblasts. Matrix assembly of soluble HFn or non-HFn by cells in culture apparently requires the initial binding of HFn to cell-associated matrix assembly sites via the 29-kDa NH2-terminal region of the molecule (21). Once bound, Fn is believed to participate in a disulfide exchange reaction with previously assembled Fn to yield large fibrillar Fn polymer complexes (20). Treatment of pFn with NEM prevents its binding to matrix assembly sites and incorporation into the ECM in cultured cell layers (20). NEM can react with thiol groups on proteins and, under certain experimental conditions, it can also cause alkylation of epsilon -amino and imidazole groups (3, 7). Early studies on the effect of NEM on Fn by Wagner and Hynes (38) showed that treatment of Fn with 10 mM NEM for 30 min in CAPS buffer (pH 11) resulted in the blocking of free sulfhydryl groups that was assumed to be the basis for the 10-fold decrease in the binding of Fn to cultured cells. Subsequently, McKeown-Longo and Mosher (20) documented that it was the alkylation of lysine and histidine residues by NEM that also occurs at pH 11 that was apparently responsible for the inability of NEM-treated Fn to bind to the matrix. When only sulfhydryl groups were alkylated by either iodoacetomide or iodoacetic acid at pH 7, Fn retained its cell-binding activity (20). Thus this NEM-mediated inhibition of Fn matrix incorporation appears to be due to the alkylation of specific residues in the NH2-terminal domain of Fn, which is the same region of Fn that binds to matrix assembly sites.

In the present study, we documented that HFn treated with low doses of NEM (10-20 nM) retains its ability to enhance target particle phagocytosis but displays a reduced ability to be incorporated in vivo into the lung matrix in the typical fibrillar pattern characteristic of the Fn matrix assembly. The concentration of NEM is very important to dissociate these two functions because NEM used at a >50 nM concentration will reduce the opsonic activity of HFn as well as its ability to be incorporated into the ECM (Lewis and Saba, unpublished data). Our competitive binding assays also demonstrated in vitro that NEM-HFn was unable to inhibit binding of 125I-HFn to fibroblasts, whereas untreated HFn competitively inhibited such binding in a dose-dependent fashion, similar to previous findings by McKeown-Longo and Mosher (20).

Our dual-label immunofluorescent observations indicated that endogenous SFn, viewed under RITC fluorescence, had a diffuse fibrillar pattern in the lung. Exogenously injected HFn, viewed under FITC fluorescence, showed a much stronger signal in bacteremic than in nonbacteremic sheep and was present mainly in a fibrillar pattern, with some intense focal punctate areas also detected. We initially assumed that if NEM-HFn could not undergo the process of assembly into the matrix, then it should not be present in lung tissue. Yet, we observed an unexpected intense focal punctate staining for the HFn antigen in the lung sections biopsied from bacteremic sheep receiving NEM-HFn. At first glance, such immunofluorescent results appeared not to be consistent with the in vitro evidence that NEM-HFn was unable to incorporate into the matrix. However, this was found to be due, in part, to the association of NEM-HFn with lung macrophages in the bacteremic sheep, perhaps reflecting their removal of Fn-coated microparticulates. NEM-HFn was also bound to fibrin in the lung, an observation consistent with a previous study (23) documenting fibrinogen consumption and low-grade intravascular coagulation in postsurgical bacteremic sheep.

In a series of classic studies, Warner and colleagues (39-41) characterized pulmonary intravascular macrophages in sheep as a unique component of the mononuclear phagocytic system. Pulmonary intravascular macrophages (PIMs) are especially present in large numbers in sheep and goats (39). PIMs reside in the lung capillaries and are avidly phagocytic. However, they are also believed to potentially contribute to acute lung injury and ongoing pulmonary inflammation, especially in sheep with gram-negative bacteremia, due to their ability to rapidly clear bloodborne bacteria (41), endotoxin (40), and other particulates (39). As reviewed by Warner and Brain (39), inflammatory mediators released by activated PIMs may trigger the accumulation of neutrophils, fibrin, and platelets in the lung after bacterial or endotoxin injection. Our present findings suggest that some of the Fn observed in a punctate pattern in the lungs is likely localized to these macrophages, especially in the postoperative bacteremic sheep.

There is much evidence that fibrin microaggregates can deposit in the lungs. Examination of the microvasculature of patients after severe injuries revealed fibrin microaggregates in the pulmonary arterioles and capillaries and microscopic evidence of pulmonary edema (18). In patients who develop adult respiratory distress syndrome, radiolabeled fibrinogen injected shortly after injury accumulates in the lung, suggesting the trapping of fibrin in the pulmonary circulation (8). In sheep with pulmonary permeability edema secondary to sepsis caused by cecal ligation and perforation, electron microscopy data revealed interstitial edema, aggregation of neutrophils, and fibrin deposition in the lungs (13). Fibrin microaggregates have also been found throughout the vascular bed of the lungs, kidneys, and livers in dogs given intravenous thrombin that were temporally coupled with the inhibition of fibrinolysis (1) and in the pulmonary capillaries of dogs infused with a prothrombin activator to trigger fibrin microembolism (34). Indeed, covalently linked fibrin-Fn complexes would be expected to fluoresce with the antibody system used in the present study but would not be removed by Bray's extraction buffer (6). Thus some of the focal punctate areas of intense staining we observed may represent fibrin-Fn complexes due to microthrombosis.

pFn is covalently cross-linked to the alpha -chain of fibrin by Factor XIIIa, also known as plasma transglutaminase (22). This can occur both in whole plasma and with the coincubation of purified pFn and fibrinogen (22). Incorporation of pFn into the fibrin clot is dependent on both temperature and Factor XIIIa activity (22). Factor XIIIa can also cross-link Fn to itself, especially at sites of matrix assembly with cultured fibroblasts, thus accelerating the accumulation of Fn into the matrix (2). Our in vitro data confirmed that alkylation of HFn with NEM does not block its ability to covalently bind to fibrin, thus providing an essential characteristic required to explain the punctate pattern in association with fibrin that we unexpectedly observed, especially in the bacteremic sheep.

Immunofluorescent analysis of lung tissue harvested from the bacteremic sheep receiving human plasma cryoprecipitate showed significant colocalization of the injected HFn and HFb, presumably reflecting Fn-fibrin complexes at sites of vascular injury. Because the amount of Fn or fibrinogen in the plasma cryoprecipitate that was injected only raised plasma levels in our sheep by 10-15%, then theoretically 85-90% of the HFn should have been bound to endogenous sheep fibrin and only 10-15% to human fibrin. Thus the fact that we were able to detect any significant HFn colocalized with the deposited human fibrin(ogen) is very strong evidence that fibrin-Fn complexes were present in the lungs of the postoperative bacteremic sheep.

Immunoperoxidase staining confirmed that very little NEM-HFn was present in a fibrillar pattern in the interstitial matrix in bacteremic sheep, although it was clearly detected in a punctate pattern in association with inflammatory macrophages. Nonbacteremic sheep receiving NEM-HFn showed minimal, if any, HFn in the lung matrix. This was expected because NEM-HFn cannot incorporate into the matrix and the infusion of only the sterile saline diluent does not cause fibrin microembolization of the lung in sheep. Charash et al. (10) observed a fibrillar deposition as well as a punctate distribution of the intravenously infused HFn in postoperative septic sheep. Based on our present findings, it is likely that the intense fibrillar pattern of HFn in the lung of postsurgical bacteremic sheep observed by Charash et al. actually represented matrix incorporation, as originally concluded, as well as some focal punctate staining due to fibrin-Fn complexes and/or HFn associated with lung macrophages.

In summary, our findings document that alkylation of HFn with low millimolar concentrations of NEM can yield a molecule that retains its ability to increase particle phagocytosis by macrophages but loses its ability to incorporate normally into the lung ECM in vivo, even though it retains its dimeric structure, molecular mass, and ability to bind to gelatin and fibrin. Accordingly, comparing the effect of intravenous infusion of large doses of normal HFn with NEM-HFn on lung protein clearance may determine whether the incorporation of plasma-derived HFn into the lung matrix is essential for it to attenuate the increase in lung protein permeability in postoperative bacteremic sheep.

    ACKNOWLEDGEMENTS

This study was supported by National Institute of General Medical Sciences Grant GM-21447.

    FOOTNOTES

T. P. Brien was a National Institutes of Health Postdoctoral Research Fellow supported by National Institute of General Medical Sciences Grant T32-GM-07033 and is currently a Resident in Pathology. P. P. Reddy was a National Insitutes of Health Postdoctoral Research Fellow supported by National Heart, Lung, and Blood Institute Grant T32-HL-07529 and is currently a Resident in Surgery.

Address for reprint requests: T. M. Saba, Dept. of Physiology and Cell Biology (A-134), Albany Medical College, 47 New Scotland Ave., Albany, NY 12208.

Received 19 May 1997; accepted in final form 17 December 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Arfors, K.-E., C. Busch, S. Jakobson, O. Lindquist, P. Malmberg, L. Rammer, and T. Saldeen. Pulmonary insufficiency following intravenous infusion of thrombin and AMCA (transexamic acid) in the dog. Acta Chir. Scand. 138: 445-452, 1972[Medline].

2.   Barry, E. L. R., and D. F. Mosher. Factor XIIIa-mediated crosslinking of fibronectin in fibroblast cell layers. J. Biol. Chem. 264: 4179-4185, 1989[Abstract/Free Full Text].

3.   Bednar, R. A. Reactivity and pH dependence of thiol conjugation to N-ethylmaleimide: detection of a conformational change in chalcone isomerase. Biochem. J. 29: 3684-3690, 1990.

4.   Blumenstock, F. A., T. M. Saba, E. Roccario, E. Cho, and J. E. Kaplan. Opsonic fibronectin after trauma and particle injection as determined by a peritoneal macrophage monolayer assay. J. Reticuloendothel. Soc. 30: 61-71, 1981[Medline].

5.   Blumenstock, F. A., T. M. Saba, P. Weber, and R. Laffin. Biochemical and immunological characterization of human opsonic alpha 2SB glycoprotein: its identity with cold-insoluble globulin. J. Biol. Chem. 253: 4287-4291, 1978[Abstract].

6.   Bray, B. A., I. Mandl, and G. M. Turno. Heparin facilitates the extraction of tissue fibronectin. Science 214: 793-795, 1981[Medline].

7.   Brewer, C. F., and J. P. Riehm. Evidence for possible nonspecific reactions between N-ethylmaleimide and proteins. Anal. Biochem. 18: 248-255, 1967.

8.  Busch, C., S. Dahlgren, S. Jakobson, B. Jung, J. Modig, and T. Saldeen. The use of 125I-labelled fibrinogen for determination of fibrin trapping in the lungs in patients developing the microembolism syndrome. Acta Anaesthesiol. Scand. 19, Suppl. 57: 46-54, 1975.

9.   Cardarelli, P. M., F. A. Blumenstock, T. M. Saba, and F. J. Rourke. Fibronectin-enhanced attachment of gelatin-coated erythroyctes to isolated hepatic Kupffer cells. J. Leukoc. Biol. 36: 477-492, 1984[Abstract].

10.   Charash, W. E., P. A. Vincent, P. J. McKeown-Longo, T. M. Saba, E. Lewis, and M. A. Lewis. Kinetics of plasma fibronectin: increased lung tissue incorporation after postoperative bacteremia. Am. J. Physiol. 260 (Regulatory Integrative Comp. Physiol. 29): R553-R562, 1991[Abstract/Free Full Text].

11.   Charash, W. E., P. A. Vincent, T. M. Saba, F. L. Minnear, P. J. McKeown-Longo, J. A. Migliozzi, M. A. Lewis, E. Lewis, and C. Giunta. Immunofluorescent analysis of plasma fibronectin incorporation into the lung during acute inflammatory vascular injury. Am. Rev. Respir. Dis. 148: 467-476, 1993[Medline].

12.   Cohler, L. F., T. M. Saba, E. P. Lewis, P. A. Vincent, and W. E. Charash. Plasma fibronectin therapy and lung protein clearance with bacteremia after surgery. J. Appl. Physiol. 63: 623-633, 1987[Abstract/Free Full Text].

13.   Craig, I., D. Judges, A. Guidec, M. Leflue, N. Paterson, R. Finky, and W. Sibbald. Pulmonary permeability edema in a large animal model of nonpulmonary sepsis. A morphologic study. Am. J. Pathol. 128: 1241-1251, 1987.

14.   Curtis, T. M., P. J. McKeown-Longo, P. A. Vincent, S. M. Homan, E. M. Wheatley, and T. M. Saba. Fibronectin attenuates increased endothelial monolayer permeability after RGD peptide, anti-alpha 5beta 1, or TNF-alpha exposure. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L248-L260, 1995[Abstract/Free Full Text].

15.   Deno, D. C., T. M. Saba, and E. P. Lewis. Kinetics of endogenously labeled plasma fibronectin: incorporation into tissues. Am. J. Physiol. 245 (Regulatory Integrative Comp. Physiol. 14): R564-R575, 1983[Medline].

16.   Jin, H.-M., P. A. Vincent, W. E. Charash, T. M. Saba, P. J. McKeown-Longo, F. A. Blumenstock, and E. Lewis. Incorporation of circulating fibronectin into various tissues during sepsis: colocalization with endogenous tissue fibronectin. Exp. Mol. Pathol. 55: 203-216, 1991[Medline].

17.   La Celle, P., F. A. Blumenstock, C. McKinley, T. M. Saba, P. A. Vincent, and V. Gray. Blood-borne collagenous debris complexes with plasma fibronectin after thermal injury. Blood 75: 470-478, 1990[Abstract].

18.   Lindquist, O., L. Rammer, and T. Saldeen. Pulmonary insufficiency, microembolism, and fibrinolysis inhibition in a post-traumatic autopsy material. Acta Chir. Scand. 138: 545-549, 1972[Medline].

19.   McKeown-Longo, P. J., and D. F. Mosher. Binding of plasma fibronectin to cell layers of human skin fibroblasts. J. Cell Biol. 97: 466-472, 1983[Abstract].

20.   McKeown-Longo, P. J., and D. F. Mosher. Mechanism of formation of disulfide-bonded multimers of plasma fibronectin in cell layers of cultured human fibroblasts. J. Biol. Chem. 259: 12210-12215, 1984[Abstract/Free Full Text].

21.   McKeown-Longo, P. J., and D. F. Mosher. 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].

22.   Mosher, D. F. Action of fibrin-stabilizing factor on cold insoluble globulin and alpha 2-macroglobulin in clotting plasma. J. Biol. Chem. 251: 1639-1645, 1976[Abstract].

23.   Niehaus, G. D., P. T. Schumacker, and T. M. Saba. Influence of opsonic fibronectin deficiency on lung fluid balance during bacterial sepsis. J. Appl. Physiol. 49: 693-699, 1980[Abstract/Free Full Text].

24.   Oh, E., M. Pierschbacher, and E. Ruoslahti. Deposition of plasma fibronectin in tissues. Proc. Natl. Acad. Sci. USA 78: 3218-3221, 1981[Abstract].

25.   Rebres, R. A., E. Cho, R. F. Rotundo, and T. M. Saba. Reduced incorporation of plasma fibronectin into the lung matrix during postoperative sepsis. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L409-L418, 1996[Abstract/Free Full Text].

26.   Rebres, R. A., P. J. McKeown-Longo, P. A. Vincent, E. Cho, and T. M. Saba. Extracellular matrix incorporation of normal and NEM-alkylated fibronectin: liver and spleen deposition. Am. J. Physiol. 269 (Gastrointest. Liver Physiol. 32): G902-G912, 1995[Abstract/Free Full Text].

28.   Resnikoff, M., T. P. Brien, E. P. Lewis, D. E. MacNaughton, and T. M. Saba. Lung permeability with postoperative gram-negative bacteremia as influenced by matrix incorporation of plasma fibronectin. Surg. Forum 47: 51-53, 1996.

29.   Rizk, T., R. Rebres, P. Vincent, E. Lewis, P. J. McKeown-Longo, and T. M. Saba. ED1-containing cellular fibronectin release into lung lymph during vascular injury with postoperative bacteremia. Am. J. Physiol. 264 (Lung Cell. Mol. Physiol. 8): L66-L73, 1993[Abstract/Free Full Text].

30.   Saba, T. M. Kinetics of plasma fibronectin: relationship to phagocytic function and lung vascular injury. In: Fibronectin, edited by D. F. Mosher. San Diego, CA: Academic, 1989, p. 395-439.

31.   Saba, T. M., F. A. Blumenstock, D. M. Shah, R. H. Landaburu, M. E. Hrinda, D. C. Deno, J. M. Holman, Jr., E. Cho, C. Phelan, and P. M. Cardarelli. Reversal of opsonic deficiency in surgical, trauma, and burn patients by infusion of purified human plasma fibronectin. Am. J. Med. 80: 229-240, 1986[Medline].

32.   Saba, T. M., and E. Cho. Reticuloendothelial (RE) response to surgery as modified by intravenous administration of plasma cryoprecipitate or cold-insoluble globulin (plasma fibronectin) purified by affinity chromatography. Adv. Shock Res. 3: 251-271, 1980[Medline].

33.   Saba, T. M., and E. Cho. Effect of acute plasma fibronectin depletion on tissue fibronectin levels: analysis by a new fluorescent immunoassay. Exp. Mol. Pathol. 41: 81-95, 1984[Medline].

34.   Schaeffer, R. C., Jr., M. I. Barnhart, and R. W. Carlson. Pulmonary fibrin deposition and increased microvascular permeability to protein following fibrin microembolism in dogs---a structure-function relationship. Microvasc. Res. 33: 327-352, 1987[Medline].

35.   Schumacker, P. T., and T. M. Saba. Pulmonary gas exchange abnormalities following intravascular coagulation: reticuloendothelial involvement. Ann. Surg. 192: 95-102, 1980[Medline].

36.   Scovill, W. A., S. J. Annest, T. M. Saba, F. A. Blumenstock, J. C. Newell, H. H. Stratton, and S. R. Powers. Cardiovascular hemodynamics after opsonic alpha -2-surface binding glycoprotein therapy in injured patients. Surgery 86: 284-293, 1979[Medline].

37.   Stenman, S., and A. Vaheri. Distribution of a major connective tissue protein, fibronectin, in normal human tissues. J. Exp. Med. 147: 1054-1064, 1978[Abstract].

38.   Wagner, D. O., and R. O. Hynes. Domain structure of fibronectin and its relation to function. J. Biol. Chem. 254: 6746-6754, 1979[Abstract].

39.   Warner, A. E., and J. D. Brain. The cell biology and pathogenic role of pulmonary intravascular macrophages. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L1-L12, 1990[Abstract/Free Full Text].

40.   Warner, A. E., M. M. DeCamp, Jr., R. M. Molina, and J. D. Brain. Pulmonary removal of circulating endotoxin results in acute lung injury in sheep. Lab. Invest. 59: 219-230, 1988[Medline].

41.   Warner, A. E., R. M. Molina, and J. D. Brain. Uptake of bloodborne bacteria by pulmonary intravascular macrophages and consequent inflammatory responses in sheep. Am. Rev. Respir. Dis. 136: 683-690, 1987[Medline].

42.   Wheatley, E. M., P. J. McKeown-Longo, P. A. Vincent, and T. M. Saba. Incorporation of fibronectin into matrix decreases the TNF-induced increase in endothelial monolayer permeability. Am. J. Physiol. 265 (Lung Cell. Mol. Physiol. 9): L148-L157, 1993[Abstract/Free Full Text].


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