0013.7227/92/1312-0710%03.00/0 Endocrinology Copyright 0 1992 by The Endocrine
IL-6 NAOKO
Increases MARUO,
Vol. 131, No. 2 Printed in ll.9.A.
Society
IKUO
Endothelial MORITA,
Section of Physiological Chemistry, Bunkyo-ku, Tokyo, Japan
MIKA
SHIRAO,
Faculty of Dentistry,
Permeability AND
SEI-ITSU
Tokyo Medical
in Vitro
MUROTA
and Dental
University,
Yushima,
ABSTRACT The effect of interleukin 6 (IL-6) on endothelial permeability was examined by measuring fluorescein isothiocyanate-labeled albumin flux across an endothelial cell monolayer. Bovine vascular endothelial cells (BVEC) were cultured up to confluency on collagen-coated polycarbonate micropore filters and then the filters were-mounted on modified Bovden chambers. Treatment of the BVEC with IL-6 at 100 ne/ml for 21 h caused a remarkable increase in the permeability of fluorescein isothiocyanate-labeled albumin across the endothelial monolayer. This effect of IL-6 was concentration dependent, in the range from lo-200 rig/ml of IL-6. The effect of IL-6 was also time dependent, the maximal level being reached at 21 h from the beginning of the treatment. This stimulatory effect of IL-6 on albumin clearance was completely abol-
ished by the addition of anti-IL-6 antibody. Light microscopic observation of a cross-section of a monolayer showed that the IL-6-induced increase in the permeability was correlated with changes in cell shape and rearrangement of intracellular actin fibers. IL-6 did not show any cytotoxicity toward or growth inhibition of endothelial cells, even at more than 200 rig/ml. The enhancing effect of IL-6 on the increase in the permeability was reversible; when IL-6 was removed by a medium change and the cells were incubated for a further 24 h without IL-6, the permeability was restored to the control level. These results suggest that IL-6 can induce an increase in endothelial permeability in uitro by rearranging actin filaments and by changing the shape of endothelial cells. (Endocrinology 131: 710-714, 1992)
I
from their ability of taking up acetylated low density lipoprotein (5). The endothelial cells were cultured in an Eagle’s minimum essential medium (MEM) (GIBCO) containing 10% fetal calf serum (Whittacker, MA). The cells were seeded onto collagen-coated micropore membranes, using a Transwell apparatus (Costar, Cambridge, MA) and grown to confluency (usually for 2-3 days).
T IS well known that endothelial cells function as a barrier against high molecular plasma protein and lipid influx acrossthe blood vesselwall. Breakdown of this barrier, which is often seenin inflammation, leads to an increasein vascular permeability and the development of edema. For example, in the case of endothelial cells exposed to either histamine, bradykinin, or peptide leukotrienes, their intracellular junctions open, which results in local leakage of serum proteins (1). Interleukin 6 (IL-6) was originally identified as a B cell differentiating factor, and recently many investigators demonstrated that IL-6 showed a wide variety of biological activities toward various tissuesand cells (2). In autoimmune diseases,such as rheumatoid arthritis, abnormal overproduction of IL-6 was observed. For example, high levels of IL6 were detected in synovial fluid taken from the joints of patients. At the same time, increasesin transvascular fluid and serumprotein flux were also observed in the lesions(3). In this study, to clarify the involvement of IL-6 in these inflammatory diseases,we examined the effect of IL-6 on endothelial permeability in vitro. It is demonstrated here that IL-6 has the capacity to increase endothelial permeability through its induction of gap formation between adjacent cells as a result of rearrangement of actin filaments.
Reagents Human recombinant IL-6 was kindly provided by TOSOH Corporation (Tokyo, Japan). One unit was defined as the half-maximal activity required to induce IgM production of the IL-6-responsive human B lymphoblastoid cell line SKWCL4. The specific activity was determined to be 1.0 X lo6 U/mg using the same line (6). Fluoresce& isothiocyanatelabeled BSA (FITC-BSA) was obtained from Nordic Immunoloev fNetherlands). FITC-labeled Rhalloidin was from Sigma (St. Louis, 66): [5’Cr] Sodium chromate (250-500 Mci/mg, 1 Ci = 37 Gbq) was purchased from Amersham (UK).
Measurement
Bovine previously
of
endothelial
endothelial
cell permeability
The amount of FITC-labeled albumin that passed across an endothelial monolayer was measured. Endothelial monolayers grown on the surface of membrane filters were washed twice with a Hank’s balanced salt solution (HBSS), and then placed on 24 well plates (Costar). One hundred microliters of HBSS containing 10 rg/ml FITC-BSA were put into the luminal chamber and then the apparatus was placed in a COz incubator. After incubation, the fluorescence of FITC-BSA cleared into the abluminal chamber was determined with a spectrophotofluorometer, RF-540 (Shimadzu, Japan). The data are expressed as follows: permeability index(%) = [experimental clearance] - [spontaneous clearance]/[clearance of filter alone] - [spontaneous clearance] X 100
Materials and Methods Preparation
of
cell monoluyers
vascular endothelial cells (BVEC) were prepared as described (4). The cells used here were identified as endothelial cells
Morphological
observation
Endothelial cells were seeded onto sterilized 13-mm coverslips on a 24-well plate and grown to confluency, and then incubated with IL-6 (100 rig/ml) for a further 24 h. The cell layers were washed twice with 5% glucose and then stained with 0.4% silver nitrate in 5% glucose. Phase-contrast pictures were taken with a Nikon 35-mm camera im-
Received January 30, 1992. Address correspondence and requests for reprints to: Ikuo Morita, Section of Physiological Chemistry, Faculty of Dentistry, Tokyo Medical and Dental University, l-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan. 710
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IL-6
0
permeablllty 10
5
INCREASES
Index
ENDOTHELIAL TABLE
(%I 15 ,
20
25 1
control
IL-6
PERMEABILITY
711
1. Wr release from endothelial cells treated with IL-6
Culture conditions “‘Cr release (cpm) Control 13,691+ 233 IL-6 (20 rig/ml) 13,620 + 276 (100 rig/ml) 12,240 + 164 (200 rig/ml) 12,211 rt 232 Cells were incubated in medium containing 37 kBq Wr for 24 h before IL-6, treatment. After washing with medium to remove extracellular ‘ICr, medium containing various concentrations of IL-6 was added. After IL-6 treatment for 21 h, 5’Cr release from the cells was measured with a y-counter. The values are means f SE for four samples. The total uptake of 51Cr into this endothelial monolayer was 186,308 + 2,054 cpm.
FIG. 1. IL-6 induces a permeability increase in endothelial monolayers. Endothelial cells were cultured on membrane filters, as described under Materials and Methods, and then treated with IL-6 (100 rig/ml) only or IL-6 (100 rig/ml) + MH166 (1 fig/ml) for 21 h. After the incubation, FITC-BSA cleared across the filter was measured. The values are means f SE for fOUr SampleS.
permeability 10
0
1
Index
(%I
20 I
30 1
40
control
IL-6
20nglml
40nglml
1OOnglml
200nglml
FIG. 2. Dose-dependent study on IL-6 treatment of endothelial monolayers. Cells were incubated for 21 h in the presence of various concentrations of IL-6 and then subjected to the FITC-BSA assay. The values are means + SE for fOUr samples.
0
5
permeability 10 I
Index 16 I
(%) 20 I
25 ‘
control
IL-6
FIG. 4. Silver nitrate staining. Endothelial monolayers cultured with or without 100 rig/ml IL-6 for 21 h were fixed and stained as described under Materials and Methods. a, Control endothelial monolayer; b, treated with 100 rig/ml IL-6 for 21 h.
3hr
6hr
21hr
FIG. 3. Time course study on IL-6 treatment of endothelial monolayers. Endothelial monolayers were treated with 100 rig/ml IL-6, and then incubated for 3,6, and 21 h. After the incubation, the endothelial permeability was measured as described under Materials and Methods.
mediately after the incubation with silver nitrate. F-a&in was stained with FITC-conjugated phalloidin. After the treatment with IL-6, the cell layers on the coverslips were rinsed twice with PBS and then fixed for 20 min with 1% paraformaldehyde in PBS at room temperature. Twenty milligrams per ml FITC-phalloidin were then added to the cell layers, followed by incubation for 45 min at 37 C. After washing twice with PBS, the monolayers were viewed and photographed under a phasecontrast and fluorescent microscope.
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712
IL-6 INCREASES
ENDOTHELIAL
Endo Voll31.
PERMEABILITY
l
1992 No 2
FIG. 5. Cross-section of an endothelial monolayer. Endothelial cells were treated as in Fig. 4, and then fixed and embedded to obtain thin sections. a and c, Control endothelial monolayer; b and d, endothelial monolayer, treated with 100 rig/ml IL-6 (magnification: a and b, x520; c and d, X1,250).
Light microscopy of fixed sections For examination by light microscopy, the endothelial cell monolayers were fixed with 2.5% glutaraldehyde, postfixed with 2% osmium tetroxide, dehydrated in an ascending ethanol series, and then embedded in an EPON 812 mixture (7). Sections of approximately 2.0 pm thickness were stained with 1% toluidine blue in 1% sodium borate solution. After the thin sectioning, the specimens were stained with 25% uranyl acetate in methanol and lead titrate, and then photographed. 51Cr release assay Endothelial cell injury was estimated as the release of chromium from prelabeled cells, as previously described (8). In brief, confluent monolayer cells were prelabeled with 2 PCi [Wrlsodium chromate for 18 h in a MEM supplemented with 10% fetal bovine serum. After the labeling, the cells were washed twice with the MEM and then treated with IL-6 for a further 24 h. The culture supematant was collected in tubes, and centrifuged to remove cells and cell debris, and then the radioactivity in the supernatant was measured with a y-scintillation spectrophotometer (Packard Auto-Gamma, Packard Instrument Co., Downers Grove, IL).
Results Incubation of IL-6 at the concentration of 100 r&ml with an endothelial cell monolayer for 21 h causedan increasein FITC-labeled albumin permeability through the monolayer. To confirm that this effect on albumin clearancewas caused by IL-6 itself, we investigated the effect of anti-IL-6 antibody MH166 (9) on it. The stimulatory effect of IL-6 on the monolayer permeability was almost completely inhibited in the presenceof the anti IL-6 antibody (1 &ml) in the assay system (Fig. 1). However, nonspecific mouse immunoglobulin G itself at 1 pg/ml had no such inhibitory effect at all (data not shown). This finding confirmed that the increase in albumin clearancewas causedby IL-6 itself. This effect of IL-6 was concentration and time dependent (Figs. 2 and 3). Significant stimulation of albumin clearance was observed with a dose of 20 rig/ml (Fig. 2) and after 3 h (Fig. 3) of IL-
6 treatment. The increased albumin permeability with IL-6 was not caused by injury to the endothelial cell monolayer by IL-6, becauseunder the sameconditions, no more release of 51Crfrom an IL-6 treated endothelial monolayer than in the control was observed (Table 1). We also confirmed that IL-6 did not affect cell growth at all, at least during the experimental period, by measuring the incorporation of 3Hthymidine (data not shown). To elucidate the mechanism of this increasein endothelial permeability caused by IL-6, we examined whether or not morphological changesoccurred during the treatment of cells with IL-6. In an IL-6 treated cell monolayer stained with silver nitrate, the cell border of each cell was irregular and had blebs, as compared with a control monolayer (Fig. 4). Furthermore, rounding of the cells and an increase in cell thickness were observed in cross-sectionalviews of an IL-6treated endothelial monolayer (Fig. 5). As there was a possibility that IL-6 could causethe contraction of endothelial cells, we next examined whether or not IL-6 affected F-actin, one of the proteins causing contraction, using FITC-phalloidin (10). As expected, in cells treated with IL-6 for 21 hours, a decreasein the peripheral band of F-actin and rearrangement of actin around the perinuclear area were observed (Fig. 6). Moreover, in some IL-6-treated cells the actin filaments appeared to be aligned irregularly within the cells. Both the permeability increase and the rearrangement of actin filaments on IL-6 treatment were reversible. Removal of IL-6 from the medium and further incubation of a monolayer for 24 h without IL-6 restored the monolayer permeability to the control level (permeability index: IL-6, 21.8%; removal of IL-6, 2.2%) and the distribution of the actin fibers returned to normal (Fig. 6). Discussion In the present study we demonstrated that IL-6 directly influenced the albumin permeability of bovine endothelial
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IL-6
FIG. 6. Fig. 4. staining b, cells medium
INCREASES
ENDOTHELIAL
FITC-phalloidin staining. Endothelial cells were treated as in After fixation, the cells were subjected to FITC-phalloidin as described under Materials and Methods. a, Nontreated cells; treated with 100 rig/ml IL-6; c, cells cultured with the fresh without IL-6 for 24 h after treatment of IL-6 (100 rig/ml).
cells. As IL-6 did not increase 51Crrelease, an indicator of cell lysis, and did not inhibit cell proliferation, its enhancing effect of IL-6 on the permeability was not caused by its toxicity. This conclusion was also supported by data indicating that the effect of IL-6 was reversible. Thus, we showed that IL-6 in the range of lo-200 rig/ml increasedthe albumin permeability of a confluent monolayer of bovine aortic endothelial cells without cytotoxicity. Associated with this increasein permeability, a considerablemorphological change in cell shape, gap formation between adjacent cells and
PERMEABILITY
713
rearrangement of F-actin were also observed in IL-6 treated cells. Wysolmerski and Lagunoff reported that the mechanism of the permeability increase was triggered by myosin light chain phosphorylation mediated by myosin light chain kinase (11). It is suggestedthat there is a possibility that IL6 may induce endothelial cell contraction by affecting this myosin light chain kinase. On the other hand, several investigators suggestedthat CAMP may be one of the important regulators of the endothelial barrier, on the basis of the results of in vim and in vitro studies. For example, it has been reported that isoproterenol, which is known to be an enhancer of the cellular CAMP level, reduced the endothelial permeability induced by thrombin [12]. Also, in preliminary experiments, we found that (BuhcAMP could reduce transendothelial permeability and could antagonize the effect of IL-6 (permeability index: IL-6 28.1%; IL-6+(Bu),cAMP, 16.0%). We have observed that the production of prostaglandin 12(PGI1), which is well known to increase the CAMP level, in endothelial cells is down regulated by IL-6 (13). These data suggest that there may be some relationship between the reduction in PGIz production and the increase in permeability induced by IL-6, but further investigation is necessaryto confirm this. There is a possibility that IL-l enhances the biosynthesis of IL-6 in endothelial cells aspreviously reported (14). Therefore, we examined whether or not IL-l affected the endothelial permeability. However, IL-l could not increase the permeability (data not shown). The reason was unclear, but there are two possibilities.One is IL-l could not induce IL-6 production in the endothelial cells used in this experiment. Another is IL-l stimulated PGIl production in the endothelial cells as described previously (13), and this PGIl abolished the effect of IL-6 produced by IL-l on the endothelial permeability. Concerning the effects of cytokines on the barrier function, Mullin and Snack (15) recently reported that tumor necrosis factor caused the breakdown of the barrier function in an epithelial cell sheet and suggestedthat this might be related to cachexia. Because there has been little study about the presence or functioning of motility-promoting cytokines in tissuemorphogenesisor neoplasia, it would be interesting to study the involvement of tumor necrosis factor and IL-6 in these phenomena. It has been reported that a large amount of IL-6 is produced during acute and chronic inflammation. Moreover, some inflammatory mediators, for example, lipopolysaccharide, could induce IL-6 production in fibroblasts and endothelial cells (14, 16). In the synovial fluid of patients with rheumatoid arthritis, in fact, 20-100 rig/ml IL-6 were detected. In addition, 50 rig/ml IL-6 were produced in a case of widespread burns (17). According to our results, the amount of IL-6 detected in these pathological conditions should be enough for it to have a direct action on endothelial cells, for it to enhance the permeability, and it can be easily considered that IL-6 plays an important role in the generation of such pathological conditions asedemain the inflammatory process.Our results also showed that the anti-IL-6 antibody could prevent the increase in endothelial permeability. To
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714
IL-6
INCREASES
ENDOTHELIAL
clarify the interaction of IL-6 production with edema and related inflammatory conditions, we must investigate the effects of the anti IL-6 antibody in an inflammatory model
converting
References permeability of microcarrier-cultured endothelial monolayers in response to histamine and thrombin. Am J Path01 122:50-61 Kishimoto K 1989 The biology of interleukin 6. Blood 74:1-10
2. 3. Hirano T, Matsuda T, Turn& M, Miyasaka N, Buchan G, Tang B, Sato K, Shimizu M, Maini R, Feldmann M, Kishimoto T 1988 Excessive’production rheumatoid arthritis.
5.
of interleukin 6/B cell stimulatory factor-2 in Eur J lmmunol 18:1797-1801 Morita I, Kanayasu T, Murota S 1984 Kallikrein stimulates prostacvclin production in bovine vascular endothelial cells. Biophys _ _ Biochim Acta 792:304-309 Vovta IC. Netland PA. Via DP. Zetter BR 1984 Suecific labeling of endothelial cells using fluorescent acetylated-low density lipoprotein. J Cell Biol99:81A J
,
,
1
6. Hirano T, Taga T, Nakano N, Yasukawa K, Kashiwamura S, Shimizu K, Nakajima K, Pyun KH, Kishimoto T 1985 Purification 7.
activities.
Biochem
1992 No 2
J 186:605-608
9.
10.
does not play a cytoprotective role in endothelial cell injury induced by 15-hydroxyperoxyeicosatetraenoic acid, an arachidonate lipoxygenase product. Prostagl Leukotr Essential Fatty Acids 41:157-161 Matsuda T, Hirano T, Kishimoto T 1988 Establishment of an interleukin 6(IL6)/B cell stimulatory factor 2-dependent cell line and preparation of anti-IL6 monoclonal antibodies. Eur J Immunol
18:951-956 Wulf E, Deboben
A, Bautz FA, Faulstich H, Wieland
T 1979
Fluorescent phallotoxin, a tool for the visualization of cellular actin. Proc Nat1 Acad Sci USA 76:4498-4502 11. Wysolmerski RB, Lagunoff D 1990 Involvement of myosin light chain kinase in endothelial cell retraction. Proc Nat1 Acad Sci USA
1, Killacley JJF,Johnston MG, Movat HZ 1986 Increased
4.
enzyme
Endo. Voll31.
8. Ochi H, Morita I, Murota S 1990 Prostaglandin
in viva. The present report is the first on a contractile action of IL6 on endothelial cells. Further investigation is necessary to clarify the physiological role of IL-6 in normal and inflammatory states.
PERMEABILITY
to homogeneity and characterization of human B cell differentiation factor (BCDF or BSF-~2). Proc Nat1 Acad Sci USA 82:5490-5494 Del Vecchio PJ, Ryan JW, Chung A, Ryan US 1980 Capillaries the adrenal cortex possess aminopeptidase A and angiotensinogen-
of
87:16-20 12. Minnear FC, DeMichele MAA, Moon DG, Rieder CL, Fenton11 JW 1989 Isoproterenol reduces thrombin-induced pulmonary endothelial
permeability
in vitro.
Am J Physiol
13. Maruo N, Morita I, Ishizaki of interleukin 6 on prostaglandin vascular endothelial’cells. Arch
257:H1613-HI623
Y, Murota S 1992 Inhibitory
effects IZ production in cultured bovine Biochem Biophys 292:600-604
14. Sironi M, Breviario F, Proseruio P, Biondi A. Vecchi A. Van Damme J, Dejana E, Maantoiani k 1989 IL-1 stimulates IL-6 production
15. Mullin
in endothelial
cells. J Immunol 142:549-553 of tumor necrosis and transepithelial permeability.
JM, Snack KV 1990 Effect
epithelial tight Res 50:2172-2176
junctions
factor on Cancer
16. Jirik FR, Podor TJ, Hirano T, Kishimoto T, Loskutoff DJ, Carson DA, Lotz M 1989 Bacterial lipopolysaccharide and inflammatory mediators Immunol
augment 142:144-147
IL-6
secretion
by
human
endothelial
cells.
J
17. Nijsten MWN, DeGroot ER, TenDuis HJ, Klasen HJ, Hack CE, Aarden LA 1987 Serum levels of interleukin-6 and acute phase responses.
Lancet
ii:921
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 May 2015. at 07:55 For personal use only. No other uses without permission. . All rights reserved.
IL-6 NAOKO
Increases MARUO,
Vol. 131, No. 2 Printed in ll.9.A.
Society
IKUO
Endothelial MORITA,
Section of Physiological Chemistry, Bunkyo-ku, Tokyo, Japan
MIKA
SHIRAO,
Faculty of Dentistry,
Permeability AND
SEI-ITSU
Tokyo Medical
in Vitro
MUROTA
and Dental
University,
Yushima,
ABSTRACT The effect of interleukin 6 (IL-6) on endothelial permeability was examined by measuring fluorescein isothiocyanate-labeled albumin flux across an endothelial cell monolayer. Bovine vascular endothelial cells (BVEC) were cultured up to confluency on collagen-coated polycarbonate micropore filters and then the filters were-mounted on modified Bovden chambers. Treatment of the BVEC with IL-6 at 100 ne/ml for 21 h caused a remarkable increase in the permeability of fluorescein isothiocyanate-labeled albumin across the endothelial monolayer. This effect of IL-6 was concentration dependent, in the range from lo-200 rig/ml of IL-6. The effect of IL-6 was also time dependent, the maximal level being reached at 21 h from the beginning of the treatment. This stimulatory effect of IL-6 on albumin clearance was completely abol-
ished by the addition of anti-IL-6 antibody. Light microscopic observation of a cross-section of a monolayer showed that the IL-6-induced increase in the permeability was correlated with changes in cell shape and rearrangement of intracellular actin fibers. IL-6 did not show any cytotoxicity toward or growth inhibition of endothelial cells, even at more than 200 rig/ml. The enhancing effect of IL-6 on the increase in the permeability was reversible; when IL-6 was removed by a medium change and the cells were incubated for a further 24 h without IL-6, the permeability was restored to the control level. These results suggest that IL-6 can induce an increase in endothelial permeability in uitro by rearranging actin filaments and by changing the shape of endothelial cells. (Endocrinology 131: 710-714, 1992)
I
from their ability of taking up acetylated low density lipoprotein (5). The endothelial cells were cultured in an Eagle’s minimum essential medium (MEM) (GIBCO) containing 10% fetal calf serum (Whittacker, MA). The cells were seeded onto collagen-coated micropore membranes, using a Transwell apparatus (Costar, Cambridge, MA) and grown to confluency (usually for 2-3 days).
T IS well known that endothelial cells function as a barrier against high molecular plasma protein and lipid influx acrossthe blood vesselwall. Breakdown of this barrier, which is often seenin inflammation, leads to an increasein vascular permeability and the development of edema. For example, in the case of endothelial cells exposed to either histamine, bradykinin, or peptide leukotrienes, their intracellular junctions open, which results in local leakage of serum proteins (1). Interleukin 6 (IL-6) was originally identified as a B cell differentiating factor, and recently many investigators demonstrated that IL-6 showed a wide variety of biological activities toward various tissuesand cells (2). In autoimmune diseases,such as rheumatoid arthritis, abnormal overproduction of IL-6 was observed. For example, high levels of IL6 were detected in synovial fluid taken from the joints of patients. At the same time, increasesin transvascular fluid and serumprotein flux were also observed in the lesions(3). In this study, to clarify the involvement of IL-6 in these inflammatory diseases,we examined the effect of IL-6 on endothelial permeability in vitro. It is demonstrated here that IL-6 has the capacity to increase endothelial permeability through its induction of gap formation between adjacent cells as a result of rearrangement of actin filaments.
Reagents Human recombinant IL-6 was kindly provided by TOSOH Corporation (Tokyo, Japan). One unit was defined as the half-maximal activity required to induce IgM production of the IL-6-responsive human B lymphoblastoid cell line SKWCL4. The specific activity was determined to be 1.0 X lo6 U/mg using the same line (6). Fluoresce& isothiocyanatelabeled BSA (FITC-BSA) was obtained from Nordic Immunoloev fNetherlands). FITC-labeled Rhalloidin was from Sigma (St. Louis, 66): [5’Cr] Sodium chromate (250-500 Mci/mg, 1 Ci = 37 Gbq) was purchased from Amersham (UK).
Measurement
Bovine previously
of
endothelial
endothelial
cell permeability
The amount of FITC-labeled albumin that passed across an endothelial monolayer was measured. Endothelial monolayers grown on the surface of membrane filters were washed twice with a Hank’s balanced salt solution (HBSS), and then placed on 24 well plates (Costar). One hundred microliters of HBSS containing 10 rg/ml FITC-BSA were put into the luminal chamber and then the apparatus was placed in a COz incubator. After incubation, the fluorescence of FITC-BSA cleared into the abluminal chamber was determined with a spectrophotofluorometer, RF-540 (Shimadzu, Japan). The data are expressed as follows: permeability index(%) = [experimental clearance] - [spontaneous clearance]/[clearance of filter alone] - [spontaneous clearance] X 100
Materials and Methods Preparation
of
cell monoluyers
vascular endothelial cells (BVEC) were prepared as described (4). The cells used here were identified as endothelial cells
Morphological
observation
Endothelial cells were seeded onto sterilized 13-mm coverslips on a 24-well plate and grown to confluency, and then incubated with IL-6 (100 rig/ml) for a further 24 h. The cell layers were washed twice with 5% glucose and then stained with 0.4% silver nitrate in 5% glucose. Phase-contrast pictures were taken with a Nikon 35-mm camera im-
Received January 30, 1992. Address correspondence and requests for reprints to: Ikuo Morita, Section of Physiological Chemistry, Faculty of Dentistry, Tokyo Medical and Dental University, l-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan. 710
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 May 2015. at 07:55 For personal use only. No other uses without permission. . All rights reserved.
IL-6
0
permeablllty 10
5
INCREASES
Index
ENDOTHELIAL TABLE
(%I 15 ,
20
25 1
control
IL-6
PERMEABILITY
711
1. Wr release from endothelial cells treated with IL-6
Culture conditions “‘Cr release (cpm) Control 13,691+ 233 IL-6 (20 rig/ml) 13,620 + 276 (100 rig/ml) 12,240 + 164 (200 rig/ml) 12,211 rt 232 Cells were incubated in medium containing 37 kBq Wr for 24 h before IL-6, treatment. After washing with medium to remove extracellular ‘ICr, medium containing various concentrations of IL-6 was added. After IL-6 treatment for 21 h, 5’Cr release from the cells was measured with a y-counter. The values are means f SE for four samples. The total uptake of 51Cr into this endothelial monolayer was 186,308 + 2,054 cpm.
FIG. 1. IL-6 induces a permeability increase in endothelial monolayers. Endothelial cells were cultured on membrane filters, as described under Materials and Methods, and then treated with IL-6 (100 rig/ml) only or IL-6 (100 rig/ml) + MH166 (1 fig/ml) for 21 h. After the incubation, FITC-BSA cleared across the filter was measured. The values are means f SE for fOUr SampleS.
permeability 10
0
1
Index
(%I
20 I
30 1
40
control
IL-6
20nglml
40nglml
1OOnglml
200nglml
FIG. 2. Dose-dependent study on IL-6 treatment of endothelial monolayers. Cells were incubated for 21 h in the presence of various concentrations of IL-6 and then subjected to the FITC-BSA assay. The values are means + SE for fOUr samples.
0
5
permeability 10 I
Index 16 I
(%) 20 I
25 ‘
control
IL-6
FIG. 4. Silver nitrate staining. Endothelial monolayers cultured with or without 100 rig/ml IL-6 for 21 h were fixed and stained as described under Materials and Methods. a, Control endothelial monolayer; b, treated with 100 rig/ml IL-6 for 21 h.
3hr
6hr
21hr
FIG. 3. Time course study on IL-6 treatment of endothelial monolayers. Endothelial monolayers were treated with 100 rig/ml IL-6, and then incubated for 3,6, and 21 h. After the incubation, the endothelial permeability was measured as described under Materials and Methods.
mediately after the incubation with silver nitrate. F-a&in was stained with FITC-conjugated phalloidin. After the treatment with IL-6, the cell layers on the coverslips were rinsed twice with PBS and then fixed for 20 min with 1% paraformaldehyde in PBS at room temperature. Twenty milligrams per ml FITC-phalloidin were then added to the cell layers, followed by incubation for 45 min at 37 C. After washing twice with PBS, the monolayers were viewed and photographed under a phasecontrast and fluorescent microscope.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 May 2015. at 07:55 For personal use only. No other uses without permission. . All rights reserved.
712
IL-6 INCREASES
ENDOTHELIAL
Endo Voll31.
PERMEABILITY
l
1992 No 2
FIG. 5. Cross-section of an endothelial monolayer. Endothelial cells were treated as in Fig. 4, and then fixed and embedded to obtain thin sections. a and c, Control endothelial monolayer; b and d, endothelial monolayer, treated with 100 rig/ml IL-6 (magnification: a and b, x520; c and d, X1,250).
Light microscopy of fixed sections For examination by light microscopy, the endothelial cell monolayers were fixed with 2.5% glutaraldehyde, postfixed with 2% osmium tetroxide, dehydrated in an ascending ethanol series, and then embedded in an EPON 812 mixture (7). Sections of approximately 2.0 pm thickness were stained with 1% toluidine blue in 1% sodium borate solution. After the thin sectioning, the specimens were stained with 25% uranyl acetate in methanol and lead titrate, and then photographed. 51Cr release assay Endothelial cell injury was estimated as the release of chromium from prelabeled cells, as previously described (8). In brief, confluent monolayer cells were prelabeled with 2 PCi [Wrlsodium chromate for 18 h in a MEM supplemented with 10% fetal bovine serum. After the labeling, the cells were washed twice with the MEM and then treated with IL-6 for a further 24 h. The culture supematant was collected in tubes, and centrifuged to remove cells and cell debris, and then the radioactivity in the supernatant was measured with a y-scintillation spectrophotometer (Packard Auto-Gamma, Packard Instrument Co., Downers Grove, IL).
Results Incubation of IL-6 at the concentration of 100 r&ml with an endothelial cell monolayer for 21 h causedan increasein FITC-labeled albumin permeability through the monolayer. To confirm that this effect on albumin clearancewas caused by IL-6 itself, we investigated the effect of anti-IL-6 antibody MH166 (9) on it. The stimulatory effect of IL-6 on the monolayer permeability was almost completely inhibited in the presenceof the anti IL-6 antibody (1 &ml) in the assay system (Fig. 1). However, nonspecific mouse immunoglobulin G itself at 1 pg/ml had no such inhibitory effect at all (data not shown). This finding confirmed that the increase in albumin clearancewas causedby IL-6 itself. This effect of IL-6 was concentration and time dependent (Figs. 2 and 3). Significant stimulation of albumin clearance was observed with a dose of 20 rig/ml (Fig. 2) and after 3 h (Fig. 3) of IL-
6 treatment. The increased albumin permeability with IL-6 was not caused by injury to the endothelial cell monolayer by IL-6, becauseunder the sameconditions, no more release of 51Crfrom an IL-6 treated endothelial monolayer than in the control was observed (Table 1). We also confirmed that IL-6 did not affect cell growth at all, at least during the experimental period, by measuring the incorporation of 3Hthymidine (data not shown). To elucidate the mechanism of this increasein endothelial permeability caused by IL-6, we examined whether or not morphological changesoccurred during the treatment of cells with IL-6. In an IL-6 treated cell monolayer stained with silver nitrate, the cell border of each cell was irregular and had blebs, as compared with a control monolayer (Fig. 4). Furthermore, rounding of the cells and an increase in cell thickness were observed in cross-sectionalviews of an IL-6treated endothelial monolayer (Fig. 5). As there was a possibility that IL-6 could causethe contraction of endothelial cells, we next examined whether or not IL-6 affected F-actin, one of the proteins causing contraction, using FITC-phalloidin (10). As expected, in cells treated with IL-6 for 21 hours, a decreasein the peripheral band of F-actin and rearrangement of actin around the perinuclear area were observed (Fig. 6). Moreover, in some IL-6-treated cells the actin filaments appeared to be aligned irregularly within the cells. Both the permeability increase and the rearrangement of actin filaments on IL-6 treatment were reversible. Removal of IL-6 from the medium and further incubation of a monolayer for 24 h without IL-6 restored the monolayer permeability to the control level (permeability index: IL-6, 21.8%; removal of IL-6, 2.2%) and the distribution of the actin fibers returned to normal (Fig. 6). Discussion In the present study we demonstrated that IL-6 directly influenced the albumin permeability of bovine endothelial
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IL-6
FIG. 6. Fig. 4. staining b, cells medium
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FITC-phalloidin staining. Endothelial cells were treated as in After fixation, the cells were subjected to FITC-phalloidin as described under Materials and Methods. a, Nontreated cells; treated with 100 rig/ml IL-6; c, cells cultured with the fresh without IL-6 for 24 h after treatment of IL-6 (100 rig/ml).
cells. As IL-6 did not increase 51Crrelease, an indicator of cell lysis, and did not inhibit cell proliferation, its enhancing effect of IL-6 on the permeability was not caused by its toxicity. This conclusion was also supported by data indicating that the effect of IL-6 was reversible. Thus, we showed that IL-6 in the range of lo-200 rig/ml increasedthe albumin permeability of a confluent monolayer of bovine aortic endothelial cells without cytotoxicity. Associated with this increasein permeability, a considerablemorphological change in cell shape, gap formation between adjacent cells and
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rearrangement of F-actin were also observed in IL-6 treated cells. Wysolmerski and Lagunoff reported that the mechanism of the permeability increase was triggered by myosin light chain phosphorylation mediated by myosin light chain kinase (11). It is suggestedthat there is a possibility that IL6 may induce endothelial cell contraction by affecting this myosin light chain kinase. On the other hand, several investigators suggestedthat CAMP may be one of the important regulators of the endothelial barrier, on the basis of the results of in vim and in vitro studies. For example, it has been reported that isoproterenol, which is known to be an enhancer of the cellular CAMP level, reduced the endothelial permeability induced by thrombin [12]. Also, in preliminary experiments, we found that (BuhcAMP could reduce transendothelial permeability and could antagonize the effect of IL-6 (permeability index: IL-6 28.1%; IL-6+(Bu),cAMP, 16.0%). We have observed that the production of prostaglandin 12(PGI1), which is well known to increase the CAMP level, in endothelial cells is down regulated by IL-6 (13). These data suggest that there may be some relationship between the reduction in PGIz production and the increase in permeability induced by IL-6, but further investigation is necessaryto confirm this. There is a possibility that IL-l enhances the biosynthesis of IL-6 in endothelial cells aspreviously reported (14). Therefore, we examined whether or not IL-l affected the endothelial permeability. However, IL-l could not increase the permeability (data not shown). The reason was unclear, but there are two possibilities.One is IL-l could not induce IL-6 production in the endothelial cells used in this experiment. Another is IL-l stimulated PGIl production in the endothelial cells as described previously (13), and this PGIl abolished the effect of IL-6 produced by IL-l on the endothelial permeability. Concerning the effects of cytokines on the barrier function, Mullin and Snack (15) recently reported that tumor necrosis factor caused the breakdown of the barrier function in an epithelial cell sheet and suggestedthat this might be related to cachexia. Because there has been little study about the presence or functioning of motility-promoting cytokines in tissuemorphogenesisor neoplasia, it would be interesting to study the involvement of tumor necrosis factor and IL-6 in these phenomena. It has been reported that a large amount of IL-6 is produced during acute and chronic inflammation. Moreover, some inflammatory mediators, for example, lipopolysaccharide, could induce IL-6 production in fibroblasts and endothelial cells (14, 16). In the synovial fluid of patients with rheumatoid arthritis, in fact, 20-100 rig/ml IL-6 were detected. In addition, 50 rig/ml IL-6 were produced in a case of widespread burns (17). According to our results, the amount of IL-6 detected in these pathological conditions should be enough for it to have a direct action on endothelial cells, for it to enhance the permeability, and it can be easily considered that IL-6 plays an important role in the generation of such pathological conditions asedemain the inflammatory process.Our results also showed that the anti-IL-6 antibody could prevent the increase in endothelial permeability. To
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714
IL-6
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clarify the interaction of IL-6 production with edema and related inflammatory conditions, we must investigate the effects of the anti IL-6 antibody in an inflammatory model
converting
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PERMEABILITY
to homogeneity and characterization of human B cell differentiation factor (BCDF or BSF-~2). Proc Nat1 Acad Sci USA 82:5490-5494 Del Vecchio PJ, Ryan JW, Chung A, Ryan US 1980 Capillaries the adrenal cortex possess aminopeptidase A and angiotensinogen-
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