Matrix fibronectin disruption in association with altered endothelial cell adhesion induced by activated polymorphonuclear leukocytes

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

45, l-21

(1986)

Matrix Fibronectin Disruption in Association with Altered Endothelial Cell Adhesion Induced by Activated Polymorphonuclear Leukocytes’ PETERS.RICHARDS,* THOMAS M. SABA,~ PETERJ. DEL VECCHIO,~ PETER A. VINCENT,~ AND VERA C. GRAY Departments

of Physiology Medical Receiraed

and Ophthalmology, Neil College of Union University. November

Hellman Medical Research Albany, New York 12208

5. 1985; and in revised

form

Febraar?l21.

Building.

Albany

1986

Sequestration of activated polymorphonuclear leukocytes (PMN) within the lung microcirculation may contribute to pulmonary vascular injury following trauma, sepsis, or disseminated intravascular coagulation. In this study cultured rat endothelial ceils were utilized to evaluate the effect of PMN activation on endothelial cell attachment. The concept that disruption of the extracellular fibronectin matrix is associated with altered endothelial cell adhesion was also tested. Rat endothelial cells were grown in culture and identified by morphological techniques as well as immunofluorescent staining of Factor VIII R:Ag. Endothelial cells were labeled with “Cr in order to establish a cell injury assay based on release of free ?r or cell-associated 51Cr. PMN activation was verified microscopically and by chemiluminescence activity following phorbol myristate acetate (PMA) or opsonized zymosan exposure. Following incubation with PMA, the leukocytes aggregated. chemiluminesced vigorously. and caused endothelial cell injury and detachment as determined by release of 5LCr-labeled endothelial cells. PMNs exposed to serum-treated zymosan exhibited a more modest chemiluminescence burst which was consistent with their decreased activity to injure the endothelial monolayer. With PMA activation the degree of endothelial detachment from the monolayer increased as a function of time with a plateau observed by 3 hr. Microscopic immunofluorescent analysis of extracellular fibronectin in endothelial cell cultures revealed disruption of the fibrillar matrix fibronectin after incubation with PMAactivated neutrophils in association with endothelial cell disadhesion. Thus. exposure of activated rat PMN to rat endothelial cells in culture induces endothelial damage and an associated disruption of the fibronectin matrix which may contribute to endothelial cell detachment. rsJ 1986 Academic Press. Inc.

INTRODUCTION Pulmonary vascular injury, often associated with the post-traumatic adult respiratory distress syndrome (ARDS), may develop from a number of predisposing conditions that contribute to the margination of leukocytes in the pulmonary microcirculation. The presence of ongoing disseminated intravascular coagulation with microembolus formation (Saldeen, 1976). endotoxemia (Brigham et al., 1974), plasma fibronectin depletion (Niehaus et al., 1980). and reticuloendothelial * This study was primarily supported by USPHS Grant GM-21447 entitled “Systemic Host Defense Following Trauma” from the Institute of General Medical Sciences. Partial support was also provided by POl-HL-32418 grant. 2 Peter S. Richards, Ph.D.. was a NIH Postdoctoral Fellow in the Department of Physiology, supported by T32-GM-07033 from the Institute of General Medical Sciences during these studies. 3 To whom reprint requests should be addressed (Professor and Chairman) at the Department of Physiology, Albany Medical College, 47 New Scotland Avenue, Albany, N.Y. 12208. 4 P J. Del Vecchio, Ph.D., has a joint appointment in Ophthalmology and Physiology. Partial support of his laboratory by a grant from the “Research to Prevent Blindness Foundation” is acknowledged. 5 Peter A. Vincent is an NIH Predoctoral Trainee in the Department of Physiology, supported by HL-07194 from the Heart, Lung and Blood Institute. 0014-4800/86 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ET AL.

phagocytic failure (Saba and Jaffe, 1980) have all been implicated as etiologic factors in ARDS. Damage to the pulmonary endothelium following experimental endotoxin infusion or microembolization of the pulmonary microcirculation has been confirmed by measuring lung transvascular protein and fluid clearance as an index of lung vascular permeability (Heflin and Brigham, 1981; Johnson and Malik, 1980). Intraperitoneal sepsis and bacteremia will also initiate lung vascular injury (Brigham et al., 1974; Niehaus et al., 1980) and this response is amplified by parallel depletion of plasma libronectin and reticuloendothelial phagocytic depression (Niehaus et al., 1980). Septicemia induces acute peripheral granulocyte depletion with parallel localization of the polymorphonuclear leukocytes (PMN) in the lung (Lanser and Saba, 1981). The margination of neutrophils in the lung with bacteremia augments the pulmonary localization of blood-borne bacteria (Lanser and Saba, 1981) whose ingestion by such phagocytic cells provides the potential for further lung injury as a result of their activation during bacterial ingestion. That PMN may have a role in the full expression of acute lung vascular injury with sepsis, endotoxemia, or pulmonary embolization is further supported by the ability for granulocytopenia to minimize the injury observed in various experimental models (Heflin and Brigham, 1981; Johnson and Malik, 1980). Activated PMN generate oxygen metabolites and release proteases as part of their role in host defense and the inflammatory process (Baehner et al., 1968; Passo and Weiss, 1984; Weissman et al., 1972). It has been postulated that PMN sequestered in the lung microvascular compartment contribute to the pathogenesis of pulmonary injury and ARDS by releasing oxygen radicals and/or neutral proteases which can either damage the lung vascular endothelial barrier or the associated connective tissue structures (Brigham and Meyrick, 1984; Rinaldo and Rogers, 1982). Thus, direct membrane damage due to oxygen radical-induced lipid peroxidation as well as degradation of protease-sensitive subendothelial matrix components, such as fibronectin which is important to cell-cell interaction and cell adhesion to a substratum, may be important mechanisms in PMN-mediated endothelial injury. In the present study, an in vitro model of PMN-mediated endothelial injury/attachment was developed using rat endothelial cells and rat peripheral blood polymorphonuclear leukocytes to study the effect of activated PMN on endothelial cell attachment in monolayers. With this approach, we also evaluated the concept that exposure of endothelial cell cultures to activated PMNs would disrupt the distribution and organization of the fibronectin in the extracellular matrix. Cultured rat heart endothelial cells were tested for the presence of Factor VIII R:Ag and fibronectin by immunofluorescence. PMN activation by both soluble (phorbol myristate acetate) and particulate (serum-treated zymosan) activators was measured by luminol-enhanced chemiluminescence. To determine the extent of PMN-mediated endothelial cell injury/detachment, activated rat PMN were incubated with 51Cr-labeled endothelial monolayers and isotopic analysis was used to quantify cell disadhesion from the substratum. MATERIALS

AND METHODS Fibronectin isolation and antiserum preparation. Fibronectin associated with rat endothelial cells maintained in culture was identified by immunofluorescent microscopy utilizing monospecific antiserum against rat plasma fibronectin. Both circulating plasma fibronectin and insoluble cell-associated fibronectin are recog-

FIBRONECTIN

AND

ENDOTHELIAL

CELL

ADHESION

3

nized by antibodies generated against the plasma form of the molecule (Ruoslahti et al., 1981). Fibronectin was isolated from rat plasma by affinity chromatography on gelatin-Sepharose as previously described (Blumenstock et al., 1979). The purified fibronectin was homogeneous as judged by polyacrylamide gel electrophoresis. Antiserum to fibronectin was raised in rabbits as previously described (Richards and Saba, 1983). The monospecific nature of the antiserum was confirmed by immunoelectrophoresis of the antiserum against normal rat plasma as well as affinity-purified fibronectin (Richards and Saba, 1983). Isolation of polymorphonuclear leukocytes from whole blood. PMN were isolated from CPD-anticoagulated donor rat blood utilizing the technique of Boyum (1968). This involved dextran sedimentation of red cells (45 min, 4°C) followed by centrifugation of the plasma at 400g for 30 min at room temperature in the presence of Histopaque cell separation medium (Sigma). The PMNs, which were localized in the pellet following centrifugation in Histopaque. were separated from any residual red cell contamination by hypotonic lysis of the red cells followed by washing with 0.9% saline. In the present study, we obtained approximately 2 x 10’ leukocytes from each 350- to 400-g donor rat. PMN viability was assessed by trypan blue exclusion. PMN were further characterized microscopically (Meltzer, 1981). Luminol-enhanced PMN chemiluminescence assay. Chemiluminescence was performed in a Tracer Analytic Model G891 Delta 300 liquid scintillation counter equipped with two 2-in. bialkali photomultiplier tubes. In this procedure, the leukocytes (1 x 105) were suspended in 1 ml of 25 mM Hepes (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) buffered Hank’s balanced salt solution and incubated in the presence of lo-’ M luminol(5-amino-2,3-dihydro-1,4-phthalazinedione; Sigma) with the addition of either 1 mg serum-treated zymosan (STZ) or 200 ng phorbol myristate acetate (PMA). Chemiluminescence determinations were done in triplicate at 8-min intervals within each experimental group. Results were expressed in terms of counts per minute (cpm). Rat endothelial cell culture preparation. The differential adhesion technique documented by Kasten (1973) was slightly modified to obtain endothelial cells from rat heart tissue. These cells have been extensively characterized on a morphologic and cytochemical basis (Kasten, 1973; Wenzel et al., 1970) and have also been utilized previously in studies involving endothelial injury (Acosta and Li, 1979; Wenzel et al., 1970). Ventricles of 5-6 hearts were removed under either anesthesia from 4-day-old (newborn) donor rats. This tissue was washed four times with Hank’s balanced salt solution (HBSS), and finely minced with dissecting scissors. The tissue was then subjected to four successive trypsinization (10 ml 0.125% trypsin in calcium and magnesium free HBSS) steps under stirred conditions using a 50-ml trypsinization flask. After each trypsinization, free cells (myocardial and endothelial) were removed and supplemented with 2 ml of endothelial cell culture medium consisting of Dulbecco’s modified essential medium (DMEM) and 20% fetal bovine serum. The cells were recovered by centrifugation at lOOOg, resuspended in endothelial cell culture medium, and allowed to adhere to the bottom of gelatin (O.l%)-coated wells in a 24-well tissue culture cluster plate (Falcon) for 90 min. The culture medium was then removed by aspiration and the culture wells were washed once with DMEM to remove nonadherent (mostly myocardial) cells. Thereafter, culture medium supplemented with I,50 pg of endothelial cell growth factor (Sigma) and 1000 U penicillin- 1000 IJ-8 strepto-

4

RICHARDS

ET AL.

mycin (KC Biological) per milliliter as well as 20% fetal bovine serum was added to the remaining adherent cells. At 90 min, the adherent cells are primarily endothelial cells (Kasten, 1973), since the myocardial cells require a much greater time interval (approximately 24 hr) to adhere to a culture surface (Kasten, 1973). This differential rate of adherence allows for the efficient harvesting of endothelial cells. Phase contrast morphologic appearance coupled with positive staining for Factor VIII (F VIII R:Ag) were used to identify the cells as endothelial cells. Within 5 days, the rat endothelial cells grew to confluence and exhibited a cobblestone-like morphology as viewed by phase contrast microscopy. The average number of endothelial cells present in each 16-mm diameter tissue culture well at 5 days was 256,167 t 6,848 (SEM) cells per well as determined by trypsinization (0.125%) of six representative wells followed by direct cell counting in a hemocytometer. PMN-mediated endothelial cell injury assays were performed on these S-day-old cultures of confluent cells. Indirect immunofluorescence microscopic staining of F VIII R:Ag and fibronectin. Both F VIII R:Ag and fibronectin are produced in vitro by vascular endothelial cells (Birdwell et al., 1978; Darnule et al., 1983; Jaffe et al., 1973; Jaffe and Mosher, 1978; Johnson and Foss, 1983; Macarak et al., 1978). In addition, F VIII R:Ag is considered to be a specific marker of this cell type. To confirm the nature of the cell monolayer, immunofluorescent staining of endothelial cell-associated F VIII R:Ag and fibronectin was performed on confluent endothelial cultures. Endothelial cell cultures were grown on gelatin-coated glass multi-compartment tissue culture chamber slides (Lab-Tek) for these Factor VIII R:Ag immunofluorescence determinations. Prior to staining, the cultures were washed five times with phosphate-buffered saline containing 0.1% bovine serum albumin (PBS/O. 1% BSA) and then fixed with cold (- 10°C) acetone. Rabbit antiserum to rat F VIII R:Ag was provided by Dr. Robert Benson of the New York State Department of Health, Division of Laboratories and Research (Albany, N.Y.). It was pretested for monospecificity using crossed immunoelectrophoresis. Cultured endothelial cell monolayers were either untreated or exposed to nonactivated or activated leukocytes prior to immunofluorescence analysis. Depending on the goal of the experiment, the washed cells were fixed with either cold ( - 1O’C) methanol or 3% Formalin. Cold methanol ( - 20°C) was applied for 2 min at 2O”C, while 3% Formalin was applied for 10 min at 20°C when used as the fixative. Methanol fixation allows the antibody to gain access to the cell interior and permits visualization of the intracellular and extracellular fibronectin. In contrast, Formalin fixation does not permit antibody to enter the cell and allows visualization of only the extracellular fibronectin. Rabbit antiserum to rat fibronectin was applied for 60 min as the source of primary antibody. Normal rabbit serum served as the control. Following a series of brief washes of the cultures in PBS, we applied fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Cappel Laboratories) for 60 min. The cultures were again washed and then examined for fluorescence using a Nikon Labophot microscope equipped for epifluorescence with a xenon light source. Isotopic labeling ofendothelial cells. Endothelial cells were allowed to incubate for 18 hr in the presence of approximately 5 pCi of Na, 51Cr O4 (New England Nuclear) which was added to the culture medium. As previously documented, when present in the sodium chromate form, 51Cr is taken up from the media and becomes associated with macromolecules inside cells (Korst, 1973).

FIBRONECTIN

AND

ENDOTHELIAL

CELL

ADHESION

5

Under the present culture conditions, the monolayer of endothelial cells typically took up 30-35% of the label within the standard 18-hr incubation interval. PMN-mediated endothelial cell injury/detachment assay. The in vitro assay used to evaluate endothelial cells following exposure to activated PMNs was carried out under incubation conditions similar to those documented by Harlan et al. (1982). “Cr-labeled endothelial cells in 24-well tissue culture clusters were washed five times with PBS/O.l% BSA by pipetting and gentle aspiration to remove unbound 5iCr. Leukocytes (2.4 x 106) were then added and allowed to adhere for 10 min to the endothelial cell culture wells, each of which contained approximately 2.6 x 10s endothelial cells. Thereafter, serum-treated zymosan (prepared by incubating boiled and washed zymosan for 30 min with normal rat serum at 37°C) or phorbol myristate acetate was added at concentrations of 2 mg/ml and 200 rig/ml, respectively, to a final buffer (HBSS/O.S% BSA) volume of 0.5 ml. Following incubation at 37°C with exposure to 5% CO, for l-, 2-, 3-, or 4-hr intervals, the incubation medium was pipetted off and combined with five 0.5-ml PBS/O.l% BSA washes of the postincubation cultures. Aliquots (1 ml each) of this combined culture medium-wash fluid were assessed for 5*Cr radioactivity with a Tracer Analytic y scintillation counter equipped with a 2-in. NaI crystal. An additional l-ml aliquot of this fluid was also centrifuged at 12,000g for 5 min followed by separation of the cell pellet so that pelletable and nonpelletable supernatant Wr could be distinguished. Other investigators have shown that 51Cr is released from cells in the nonreutilizable chromic form (Korst, 1973) and that this released free 51Cr cannot be retained by phagocytes in assays of PMN-mediated target cell damage (Nathan et al., 1979). Thus, one can utilize pelletable radioactivity as an index of “Cr-labeled intact endothelial cells which may have detached from the endothelial monolayer due to some experimental intervention (Harlan et al., 1982). In the present study, we quantified the total “Cr in the supernatant as well as the relative contribution of cell-associated 51Cr (pelletable) and free 51Cr (nonpelletable) to this total radioactivity. The amount of free 5iCr was used as an index of cell injury or disruption while the amount of pelletable or cell-associated 51Cr released was used to denote disadhesion of intact endothelial cells from the monolayer. Statistical analysis ofdata. The experimental data were subjected to statistical analysis by using the Student t test with the confidence level set at 95%. Values are expressed as means _t SEM. RESULTS Indirect immunoj7uorescence forfibronectin andfactor VZZZantigen. Rat endothelial cells were evaluated by indirect immunofluorescence microscopy for the presence of F VIII R:Ag and tibronectin (intracellular and extracellular) as shown in Figs. 1 and 2, respectively. Consistent with expected morphology, the endothelial cells were polygonal in shape with large ovoid nuclei (Figs. lA, B). F VIII R:Ag staining (Fig. IA) was distinctive both for its perinuclear localization and for its punctate appearance which are characteristic of this endothelial cell-associated antigen (Del Vecchio and Lincoln, 1982). The control slide which used normal rabbit serum in the place of antiserum to FVIII showed minimal fluorescence (Fig. 1B). Thus, by this criteria these were clearly monolayers of endothelial cells with a high level of purity apparently achieved. Fibronectin is synthesized by several cell types, including endothelial cells. As

6

RICHARDS

ET AL.

FIG. 1. Indirect immunofluorescent staining of primary cultures of rat endothelial cells for Factor 8 antigen (F VIII R:Ag). The acetone-fixed endothelial cells were incubated for 60 min with rabbit anti-rat F VIII R:Ag antiserum (Al or normal rabbit serum (IS). This was followed by a 60-mitt incubation with FITC-conjugated goat anti-rabbit IgG at a 1: 10 dilution. The magnification was 300 x . The distinct presence of Factor VIII R:Ag in the endothelial cell monolayer is shown.

FIBRONECTIN

AND ENDOTHELIAL

CELL ADHESION

7

FIG. 2. Indirect immunofluorescent staining of tibronectin in a primary culture of rat endothelial cells. Methanol-fixed endothelial cells were incubated at room temperature for 60 min with rabbit anti-rat fibronectin (1: 10 dilution) (A) or normal rabbit serum (1: IO) (B) followed by a 60-min incubation with FITC-goat anti-rabbit IgG (1:20). The magnification was 300 x The presence of tibronectin is readily apparent.

RICHARDS ET AL.

FIG. 3. Effect of activating agents on rat peripheral blood polymorphonuclear leukocytes. Leukocytes (I x IOVml) were incubated for 90 min at 37°C in the presence of either buffer (HBSS/O.S% BSA) (A), serum-treated zymosan (1 mgiml) (B), or phorboi myristate acetate (200 ngiml) (C). DifQuik stain. The magnification was 375 x .

FIBRONECTIN

AND

ENDOTHELIAL

FIG.

CELL

ADHESION

9

3-Conrinrted.

shown in Fig. 2, fibronectin was found both within the endothelial cell as well as in association with the extracellular matrix in contrast to the F VIII R:Ag staining which was distinct in its perinuclear cytoplasmic localization. These cultures were washed thoroughly (5 x with PBS/O. I%, BSA) prior to staining, to minimize possible association of fetal bovine serum fibronectin with the endothelial monolayer (Hayman and Ruoslahti, 1979). Experimental stimulation of PMNs. The effect of exposure of the resting isolated PMN to serum-treated zymosan and phorbol myristate acetate was evaluated by microscopic observation as well as by chemiluminescence determination as an index of activation. The PMN preparation had a greater than 95% viability by trypan blue dye exclusion. The preparation of PMN was relatively pure although some contamination with small mononuclear cells was observed (Fig. 3A). In contrast to the cells in a resting state which were well dispersed (Fig. 3A), following exposure to STZ, the PMN appeared to have phagocytized the particles and to undergo substantial rapid clumping or aggregation (Fig. 3B). This effect was also apparent following exposure to PMA (Fig. 3C). Interestingly, this response appeared confined for the most part to the PMN and not to the small number of mononuclear cells. Examination of the cells under higher magnification (not shown) revealed that the PMA-activated PMN were also highly vacuolated when compared to nonactivated PMN. The PMN responded to STZ challenge with a modest chemiluminescent burst which exhibited a maximum of 87,101 k 7565 cpm and was sustained for about 24 min before beginning to decline toward baseline which was reached by about 1 hr (Fig. 4). In contrast, PMN incubated with PMA demonstrated a more pronounced and dramatic, short-lived burst of chemiluminescence (in excess of 200,000 cpm) which normalized within about 0.5 hr. Thus, activation as denoted by this param-

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RICHARDS

ET AL.

230 220

-

210

-

200

-

190

-

160

-

170

-

160

-

150

-

140

-

0

PMN AND PNtlRBOL MYRISTATE ACETATE (PMA) 0 PMN AND SERUM TREATED ZY MDSAN ( STZ) A PMN AND BUFFEA

130 ,120

-

110

-

100

-

90 60 ‘70 60 50 40 30 20 10 ‘Do”““““““” 0

6

16

24

32

40

TIME POST EXPOSURE

4U

56

OF PMN

64

72

BD

68

TO ACTIVATORS

96

104

112 120

(min)

FIG. 4. Luminol (10-7M)-enhanced chemiluminescent response following the exposure of rat PMN to serum-treated zymosan (2 mg/ml) or phorbol myristate acetate (200 &ml). Triplicate determinations were performed at 8-min intervals within each of the experimental groups. Values are expressed as means -+ SEM.

eter was more striking after PMA, but in both instances, however, activation was apparent when compared to the control PMN incubated with buffer. Injury andlor altered endothelial cell adhesion. The effects of PMN activation by STZ or PMA on the adherence of the SICr-labeled endothelial cells over a 4-hr incubation period are shown in Figs. 5 and 6. For comparative purposes, endothelial cells were incubated with 0.025% trypsin (which would be expected to cause endothelial cell disadhesion from substratum and little membrane damage) and with 0.5 M NaOH (which would loosen the cells as well as digest the cell membrane and cause extensive release of radioactivity). Over the 4-hr incubation period, STZ-activated PMN caused radioactivity to be released from endothelial cells at a level similar to that seen with the control endothelial cells incubated with either PMN, STZ, or the buffer alone (Fig. 5). A large release of total 5iCr was found in both the trypsin-treated and NaOH-treated groups as would be expected. When the collected supernatant was subjected to centrifugation at 12,OOOg (Table I), it was observed that nearly half (49.4 + 7.5%) of the 51Cr from

FIBRONECTIN

AND ENDOTHELIAL

BUFFER

PMN +

PMN

CELL ADHESION

ST2

TRYPSIN

11

NaOH

ST2

FIG. 5. Rat endothelial cell damage as reflected by 51Cr release following exposure of endothelial monolayer to PMN in the presence of serum-opsonized zymosan. 51Cr-labeled endothelial cells were incubated for 4 hr with buffer (HBSS/O.S% BSA) only, PMN + STZ (2 mg/ml), PMN alone, STZ alone, trypsin (0.025%). or 0.5 N NaOH. Postincubation media were combined with five PBS/O.I% BSA culture well washes and assessed for 5iCr radioactivity. Values are expressed as means 2 SEM. Asterisks indicate the presence of a significant (P < 0.05) difference when compared to j’Cr release from endothelial cells in buffer alone.

the trypsin-treated endothelial cell cultures was found to be pelletable (i.e., associated with disadhered endothelial cells) as anticipated. The greatest release was with NaOH and interestingly over 30% of the radioactivity released in this setting was still pelletable, suggesting prolonged association of jiCr with cell fragments and macromolecules. Although the total Wr release from PMN + STZ-treated cultures was similar to that seen in the control buffer group (Fig. 5), a significantly (P < 0.05) greater amount of the radioactivity released was pelletable upon centrifugation (Table I). Further experiments were performed on the 51Cr-labeled endothelial cells using PMN activated with PMA. After a 4-hr incubation with PMA-activated PMN, 20,341 + 3311 cpm were released compared with 5,087 + 776 cpm in the control buffer only group (Fig. 6). The majority of this 400% increase in 51Cr radioactivity was found in the pelletable fraction following centrifugation (Table II), pointing to significant disadhesion of intact labeled endothelial cells after exposure to the PMA-activated leukocytes. The time course of radioactivity release from Yr-labeled endothelial cells incubated with buffer alone, PMN + STZ, or PMN + PMA is depicted in Fig. 7. In the control buffer-exposed PMN group, maximal radioactivity release (9,072 2 2035 cpm) was achieved at 4 hr. In this second set of studies, when endothelial cells were incubated with PMN + STZ, less radioactivity was released at each time interval studied compared to the control buffer group. In contrast, incubation of the endothelial monolayer with PMN + PMA resulted in at least a 100%

12

RICHARDS

ET AL. * 18)

50-0r x c: v) 2 Y si E w”

f 40-

30-

:: t 2 2 9 2w dL

20-

d

lo-

BUFFER

PMN

PMN

PMA

VPSIN

NaOH

+ PMA

FIG. 6. Rat endothehal cell damage as reflected by Wr release following exposure of endotheliai monolayer to PMN in the presence of PMA. Wr-labeled endothelial cells were incubated for 4 hr with buffer (HBSS/O.S% BSA), PMN + PMA (200 ngiml), PMN alone, PMA alone, 0.025% trypsin, or 0.5 N NaOH. Postincubation media was combined with five PBS/O.l% BSA culture well washes and assessed for Wr radioactivity. Values are expressed as means -+ SEM. Asterisks indicate the presence of a significant (P < 0.05) difference when compared to 5’Cr release from endothelial cells in buffer alone.

increase in the release of “Cr above control levels at all times studied. Once again (Table III) a significantly (P < 0.05) greater amount of 51Cr was pelletable in the media-wash combinations of PMN + STZ- and PMN + PMA-treated endothelial cells when compared to the control buffer alone group. The fraction of pelletable radioactivity actually appeared to increase over time when SICr-labeled endotheTABLE 1 Effect of Exposure on Endothelial Monolayer to Leukocytes Treated with Serum Opsonized Zymosan (STZ) with Respect to Pelletable (Cell-Associated) WR Radioactivity” in Media following a 4-hr Incubation Period Experimental group:

Buffer (n = 8)

Both PMN + STZ (n = 8)

Only PMN (n = 8)

Only STZ (n = 7)

Trypsin (n = 7)

NaOH (n = 8)

16.9 f 0.8%

32.4 2 3.l%b

14.8 k 0.9%

14.9 k 1.1%

49.4 2 7.5%b

32.3 5 6.3Yob

0 Cr-labeled endothelial cells were incubated for 4 hr with buffer (HBSS/O.S% BSA) alone, PMN + STZ (2 mgiml), PMN. STZ, trypsin (0.025%), or 0.5 M NaOH. The incubation medium was removed, combined with five 0.5ml PBS/O.I% BSA washes of the cell culture, and centrifuged at 12,OOOgto separate pelletable from nonpelletable radioactivity. Data are expressed as percentage of total 5rCr released in the pelletable fraction (means ? SEM). b Significantly (Z’ < 0.05) greater when compared to endothelial cells incubated with buffer alone.

FIBRONECTIN

AND ENDOTHELIAL

CELL ADHESION

13

TABLE II Effect of Exposure of Endothelial Monolayer to Leukocytes Treated with Phorbol Myristate Acetate (PMA) with Respect to Pelletable (Cell-Associated) “Cr Radioactivity0 in Media following a 4-hr Incubation Period Experimental group:

Buffer (n = IO)

Both PMN + PMA (n = 7)

Only PMN (n = 7)

Only PMA (n = 8)

Trypsin (n = 8)

NaOH (n = 8)

9.9 e 0.7%

66.5 k 1.9%b

12.4 L 0.6%”

7.7 k 0.6!Xb

72.8 k l.8W

23.0 2 I.OW

0 Wr-labeled endothelial cells were incubated for 4 hr with buffer (HBSS/O.S% BSA) alone. PMN + PMA (200 rig/ml). PMN, PMA, trypsin (0.025%). or 0.5 M NaOH. The incubation medium was recovered, combined with five OS-ml PBS/O.I% BSA washes of the cell culture, and centrifuged at 12,OOOg. Data are expressed as percentage of total Wr released in the pelletable fraction (means + SEM). b Significantly (P < 0.05) different when compared to endothelial cells incubated with buffer alone.

lial cells were incubated with PMN + STZ or PMN + PMA, indicating a time dependence of the endothelial detachment response when exposed to activated PMN. Disruption of matrixfibronectin: fmmunofluorescence studies. A novel aspect of the current study was to evaluate the concept that the exposure of the endothelial cell monolayers to activated PMN would disrupt the normal fibrillar pattern of fibronectin in the extracellular matrix of the cells. Thus, immunofluorescence analyses of fibronectin in the endothelial cultures was examined 4 hr after incubation with nonactivated or PMA-activated PMN, a time corresponding to the release of the 51Cr-labeled endothelial cells from the monolayer. As noted previously, for this experiment, the endothelial cells were fixed with Formalin in . PMN AND PHORBOL ACETATE (PMA)

MVRISTATE

0 PMN AND SERUM TREATED ZVMOSAN (STZt n PMN AND BUFFER

0

1 INCUBATION

2

3 PERIOD

4 (hrs)

FIG. 7. Time course of PMN-mediated endothelial cell damage. Labeled (“0) endothelial cells were incubated for- 1, 2, 3, or 4 hr with buffer (HBSSIO.S% BSA) alone (n = 18 at each time point), PMN + STZ (2 mgiml) (n = 6 at each time point). or PMN + PMA (200 &ml) (n = 12 at each time point). Postincubation media were combined with five PBS/O.S% BSA culture well washes and assessed for Wr radioactivity. Values are expressed as means 2 SEM. Significant (P < 0.05) differences exist between the control PMN + buffer group 51Cr release when compared to Wr release by either the PMN + PMA or the PMN + STZ group at all four time intervals studied.

14

RICHARDS ET AL.

TABLE III Temporal Release of Pelletable (Cell-Associated) Wr Radioactivity’ in Media following Incubation of Rat Endothelial Cells with STZ-Activated Neutrophils or PMA-Activated Neutrophils Incubation interval (hr) Experimental

group

Endothelial cells + buffer (controls) alone Endothelial cells + PMN + STZ Endothelial cells + PMN + PMA

1 16.3 (n 20.0 (n 52.1 (n

f = f = 2 =

2 1.3% 18) 1.2% 6) 1.9%* 12)

18.3 (n 28.4 (n 74.3 (n

” = ” = e =

3 1.0% 18) 0.9%* 6) 1.9%* 12)

4

17.7 k 0.7% (n =

18)

27.3 2 2.5%* (n

= 6)

74.5 + 1.4%b (n = 12)

17.3 2 0.6%’ (n

29.6 (n 74.0 (n

=

18)

k = 2 =

I.l%* 6) 1.25X* 12)

n SiCr-labeled endothelial cells were incubated with or without activated PMN for I, 2, 3, or 4 hr. At these time intervals the media were removed, combined with five OS-ml PBS/O. I% BSA washes of the cell cultures, and centrifuged at 12,OOOg.Data are expressed as percentage of total “Cr released in the pelletable fraction (means 5 SEM). * Significantly (P < 0.05) more radioactivity was in pelletable form at the time interval studied when the endothelial cells were incubated with PMN + STZ or PMN + PMA as compared to endothehal cells in buffer alone. The 2-hr values are greater than the values at I hr for each of the experimental groups, PMN + STZ and PMN + PMA, but are the same on a temporal basis in the control group.

order to maintain membrane integrity and prevent entry of the antibody to the cell interior. As shown in Fig. 8, essentially no background immunofluorescence was observed in the absence of adding antibody to fibronectin (Fig. 8A) to the monolayer whose integrity is depicted by phase contrast microscopy (Fig. 8B). Immunofluorescence staining for fibronectin (Fig. 8C) revealed the anticipated fibrillar deposition of fibronectin in the extracellular matrix. Exposure to nonactivated rat PMN for 4 hr did not disturb the fibrillar appearance of the deposited extracellular fibronectin, although fluorescence was observed in association with the PMN (Fig. 8E). In contrast, after incubation of the endothelial cells with PMA (200 rig/ml) we observed disruption and rounding-up of the endothelial cells as viewed by phase contrast microscopy (Fig. 8H) with a dramatic loss of fibrillar matrix fibronectin in the cultures (Fig. 8G). Also, aggregation of the activated PMN was observed with intense staining for fibronectin associated with the PMN (Fig. 8G). Whether destruction and/or fragmentation of the proteolytic sensitive fibronectin matrix was followed by ingestion of the altered matrix protein by these phagocytic cells can only be speculated as the basis for this finding. Thus, exposure of confluent rat endothelial cell cultures to PMA-activated neutrophils results in injury to the endothelial monolayer, release of intact cells into the medium, and a parallel disruption of the extracellular fibrillar fibronectin matrix. Whether fibronectin fragments are released into the culture supernatant perhaps due to the action of proteolytic enzymes released from the activated PMN can only be speculated. DISCUSSION The sequestration of activated PMN in the lung microcirculation is believed to contribute to altered vascular permeability and acute lung vascular injury with septicemia after burn or trauma. Early studies by Craddock et al. (1977) demonstrated that complement activation and leukocyte aggregation were associated with respiratory distress following cellophane membrane hemodialysis. Experimental studies involving granulocyte depletion have subsequently emphasized

FIG. 8. Immunofluorescent and phase contrast microscopic analyses of rat endothelial cell cultures after incubation with nonactivated or activated rat PMNs. Groupings include controls without PMN used for background staining with normal rabbit serum (A,B), controls without PMN used for detecting normal tibrillar deposition of matrix tibronectin with rabbit anti-rat fibronectin (C.D). experimentals with nonactivated PMN that were subsequently treated with anti-rat fibronectin (E,F), and experimentals with activated PMN that were subsequently treated with anti-rat tibronectin (G,H). Cultures were evaluated 4 hr after exposure to the PMN. PMA was used at a concentration of 200 rig/ml. Addition of PMA alone produced no detectable alteration in either the matrix fibronectin as studied by immunofluorescence or the endothelial monolayer appearance as studied by phase contrast microscopy. 15

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that granulocytes may be involved in the pulmonary injury caused by endotoxin (Heflin and Brigham, 1981) and microembolization (Johnson and Malik, 1980). Indeed, the direct infusion of a large load of foreign l- to 2-pm microparticles, whether injected on the arterial or venous side of the circulation (especially in an animal like a sheep with limited reticuloendothelial phagocytic clearance capacity), can cause an acute leukopenia and lung vascular injury (Niehaus et al., 1984;1980). Leukocyte mediation of pulmonary vascular injury may be related to the capacity of this cell to generate toxic oxygen radicals and release its granule contents upon activation. Such substances can damage endothelial cell membranes and connective tissue proteins. Compounds such as PGE,! as well as endotoxin may potentiate the endothelial injury caused by activated PMN (Brigham and Meyrick, 1984; Wedmore and Williams, 1981). As reviewed by Weiss and Lo Buglio (1982), a number of in vitro studies involving human leukocytes and human umbilical vein endothelial cells have emphasized that activated leukocytes can cause endothelial damage by at least two different mechanisms. Sachs et al. (1978) showed that CSa-stimulated leukocytemediated endothelial cell damage (as assessed by “Cr release from cultured endothelial cells) could be prevented by treatment with a combination of superoxide dismutase (SOD) and catalase. Weiss et al. (1981) later showed that catalase completely prevented endothelial damage following exposure of leukocytes to phorbol myristate acetate, suggesting that activated leukocytes damaged endothelial cells by releasing hydrogen peroxide. Finally, Harlan et al. (1982) observed that detachment of viable intact endothelial cells from culture surfaces can occur following incubation with leukocytes exposed to serum-treated zymosan. This effect was inhibited by serum or soybean trypsin inhibitor but not by catalase. These earlier observations on PMA-activated cells were primarily done with human cells. Accordingly, a unique contribution of the present study was the use of both endothelial cells and leukocytes harvested from the same animal species, i.e., rats. However, the relationship between the stimulus used and the type of injury induced by PMN in vitro remains to be further defined, since Ayars et al. (1984) have recently shown that PMA-stimulated human leukocytes cause disadhesion of pneumocytes in vitro by a mechanism which can be blocked with neutral protease inhibitors. In addition, Fligiel et al. (1984) have reported that hydrogen peroxide pretreatment of certain proteins including libronectin renders them more sensitive to proteolytic degradation by leukocyte neutral proteases. Thus, toxic oxygen radicals and proteases have the potential to act in concert to damage endothelium or in essence potentiate or amplify the response. This may be due to fragmentation of the adhesive matrix fibronectin by proteolytic enzymes in the presence of oxygen metabolites which both block antiprotease activity and perhaps injure the cell with regard to its ability to synthesize and/or secrete new fibronectin. If so, then oxygen metabolite scavengers as well as large doses of protease inhibitors could independently and/or collectively modulate the injury, depending on relative fibronectin requirements of the endothelial cell line for adhesion to its substratum. Our observations of disruption of the fibrillar fibronectin extracellular matrix in the cultured endothelial cell monolayer after exposure to activated PMN add a new observation with regard to understanding the mechanism of endothelial cell injury and altered adhesion. Indeed, alteration of the fibronectin matrix has been

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previously suggested by our laboratory (Niehaus et al., 1980; Saba and Jaffe, 1980), but direct experimental support of this concept has not been available in the cultured cell model with cell-elaborated fibronectin matrices. We would speculate that proteases may fragment the adhesive tissue fibronectin localized around endothelial cells or between the endothelial cells and their collagenous substratum, resulting in a loss in adhesive ability. In parallel, oxygen metabolites may inactivate antiprotease activity and also injure the endothelial cell whose viability may be essential for synthesis and incorporation of new fibronectin on its basal-extracellular matrix surface. It is also possible that perturbed or damaged endothelial cells themselves may act to increasingly degrade basement membrane components as this has been demonstrated following migratory stimulation of cultured fetal bovine endothelial cells (Kalebic er al., 1983). The present study involved utilizing rat endothelial cells as identified morphologically and by F VIII R:Ag and tibronectin staining, in an assay of PMN-mediated endothelial injury. This model was chosen in order to ultimately allow for correlations with in viva models of lung injury with sepsis. It has the advantage of being comprised of two readily available cell populations (rat leukocytes and rat endothelial cells) and represents a totally autologous (animal) system. Interestingly, the rat leukocyte appears very similar to the human leukocyte in regard to the relative localization and quantity of granule-associated enzymes (Calami and Spitznagel, 1982; Varani et al., 1982). With respect to characterization of the rat endothelial cell cultures for constituents other than F VIII R:Ag and fibronectin, such studies have been done by other investigators using endothelial cells from various species including the rat (Madri et al., 1980; Sage et al., 1981; Sampson et al., 1975). On the basis of these findings, we expect the rat endothelial extracellular matrix in vitro to contain laminin, collagen types III, IV, and V, and proteoglycans (e.g., heparan sulfate) in addition to fibronectin. Differences exist in the amount and/or type(s) of collagens secreted in vitro by endothelial cells isolated from different areas of the vascular tree (Sage et al., 1981). Cultured vascular endothelial cells have also been shown to produce angiotension converting enzyme (Del Vecchio and Lincoln, 1982). and coagulation Factor V (Cerveny et al., 1984). Our finding that PMA-stimulated polymorphonuclear leukocytes cause endothelial damage in association with disruption of the fibronectin matrix adds a new dimension to the observation by Weiss ef al. (1981) using human leukocytes and cultured human endothelial cells which emphasize a role for hydrogen peroxide. We have also observed, according to the parameters of our assay system, some baseline level of disadhesion of intact endothelial cells from their substratum over the 4-hr period in the absence of exposure to activated PMN. The degree of disadhesion was amplified by the presence of PMA-activated neutrophils and began to plateau off by 3 hr following exposure of the endothelial monolayer to the activated cells, which was sooner than the 6-hr period observed by Weiss et al. (1981). The substantial amount of endothelial damage that we observed due to PMA stimulation of the rat leukocytes correlated with the strong chemiluminescent burst from the cell that PMA produced. It also temporally correlated with disturbance of the fibrillar weblike network of the insoluble fibronectin as detected by immunofluorescence analysis. Minimal release of intact 51Cr endothelial cells was observed in cultures which did not manifest destruction of the tibronectin network, suggesting a relationship be-

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tween these two events. On a temporal basis, the chemiluminescence activity peaks very early, while the endothelial damage appears to continue over a 3- to 4-hr period. Chemiluminescence activity is associated with the generation of reduction products of molecular oxygen (Rosen and Klebanoff, 1976). Accordingly, we would speculate that some of the leukocyte-mediated endothelial damage following exposure to PMA in our rat system should be inhibitable by oxygen radical scavengers such as SOD and catalase. A concept of considerable importance to the etiology of vascular injury is the possible role of degradation of extracellular matrix tissue fibronectin in the process of endothelial cell injury/detachment due to products such as proteases released from activated phagocytic cells. Fibronectin is a large molecular weight dimeric protein, which is highly susceptible to cleavage by leukocyte granule-derived proteases (McDonald et al., 1979; Wojteska-Lukosik ef al., 1984). This includes leukocyte elastase and cathepsin G. Fibronectin has unique globular domains with high affinity for fibrin, actin. collagen, and heparan sulfate which may be distinct from the cell attachment site and these domains are held together by flexible protease-sensitive polypeptide regions (Hynes and Yamada, 1982). Plasma fibronectin can be incorporated into various tissues with long-term retention as shown by Oh et al. (1981) and Deno et al. (1983), and recent observations by Bray (1985) point to the potential for tissue matrix tibronectin to exchange with tibronectin in plasma. Fibronectin is believed to play an attachment role in cell-cell interaction and cell adhesion to a substratum (Hynes and Yamada, 1982) and fragmentation of fibronectin may alter its adhesive or ligand properties. Preliminary immunoblot analysis on only a few samples to date (Vincent and Saba, unpublished data) reveals fibronectin fragments in the culture supernatants of endothelial cells incubated for 4 hr with activated PMN, which provides indirect support for this concept. Previous studies by Harlan et al. (1982) showed that neutral proteases from STZ-activated leukocytes appeared not to damage endothelial cell membranes but to mediate extensive detachment of viable human umbilical vein endothelial cells as well as the degradation of endothelial cell surface tibronectin in vitro. Our observations using STZ-stimulated rat leukocytes and cultured rat endothelial cells are consistent with this conclusion as we cannot demonstrate endothelial cell membrane damage based on release of radioactivity into the incubation and wash media. However, based on the pelletability of the released 51Cr, a modest amount of endothelial cells were released but yet they appear intact after such treatment. We have also considered the possibility that free 5’Cr released from endothelial cells may become subsequently associated with or ingested by phagocytizing/aggregating leukocytes following exposure to STZ or PMA. However, the literature suggests that this is not likely (Korst, 1973; Nathan et al., 1979). Species-related variations in endothelial cells may account for the difference between our data showing minimal endothelial cell detachment following exposure to STZ-stimulated leukocytes and those of Harlan et al. (1982) which showed extensive endothelial disruption under very similar conditions. The fact that endothelial cells cultured from human umbilical vein are unaffected by a dose of endotoxin that causes extensive lysis and detachment of bovine pulmonary artery endothelial cells (Harlan et al., 1983) further points out the possibility of endothelial cells from different species or perhaps different tissue beds can manifest distinctly unique responses. Thus, caution must be exercised in extrapolating conclusions from data based on cells obtained from different species or when using a

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mixture of cells from different species, i.e., human neutrophils and bovine endothelial cells. Furthermore, one must be careful in extrapolating findings on one type of endothelial cells to the endothelium of a different organ. It is possible that the smaller STZ-activated leukocyte to endothelial cell ratio utilized in our study as compared to Harlan et al. (1982) was not sufficient to generate the degree of endothelial injury as observed with large numbers of leukocytes. However, we selected this ratio to more closely approximate what might develop in vivo with sepsis after surgery or trauma. In summary, the current study utilizing rat leukocytes and rat endothelial cells provides the data base for a model to study the mechanisms by which activated leukocytes damage an endothelial barrier. In this context, parallel in viva studies in rats can be conducted to allow for correlation between in viva and in vitro observations in the same species. The possibility that disruption of the extracellular fibrillar tibronectin matrix contributes to the altered adhesion of endothelial cells observed after incubation with activated neutrophils warrants consideration.

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