Cytokines are supposed to be mediators in diarrhoeal diseases. The aim of this study is to characterize the effect of tumor necrosis factor-alpha (TNFalpha) on epithelial barrier function in the colonic epithelial cell line HT-29/B6. Active ion transport and barrier function were measured as short-circuit current and transepithelial electrical resistance (Rt), respectively. In parallel, freeze-fracture electron microscopy (EM) of tight junctions (TJ) and immunofluorescence microscopy of the zonula occludens protein-1 (ZO-1) were performed. Serosal addition of TNF(alpha) (100 ng/ml) decreased Rt by 81%. This effect was dose-dependent and could be mimicked by antibodies against the p55 form of the TNF receptor. Cytotoxic effects were excluded by a negative lactate dehydrogenase (LDH) assay. Immunofluorescence localization with anti-ZO-1 antibodies revealed no evidence for disruption of the monolayer after TNFalpha treatment. In freeze-fracture EM, TJ complexity was decreased by TNFalpha, as indicated by a decrease in the number of strands from 4.7 to 3.4. The tyrosine kinase blocker genistein and the protein kinase A inhibitor H-8 reduced the effect of TNFalpha. A combination of TNFalpha with interferon-gamma acted synergistically on the epithelial barrier. In conclusion, TNFalpha impairs epithelial barrier function by altering structure and function of the tight junction, which could be of pathogenic relevance in intestinal inflammation.
REFERENCES
Aggarwal
B. B.
, Eessalu
T. E.
(1987
). Induction of receptors for tumor necrosis factor-by interferons is not a major mechanism for their synergistic cytotoxic response.
J. Biol. Chem
262
, 10000
–10007
Barnes
D. A.
, Huston
M.
, Holmes
R.
, Benveniste
E. N.
, Yong
V. W.
, Scholz
P.
, Perez
H. D.
(1996
). Induction of RANTES expression by astrocytes and astrocytoma cell lines.
J. Neuroimmunol
71
, 207
–214
Bentzel
C. J.
, Hainau
B.
, Ho
S.
, Hui
S. W.
, Edelman
A.
, Anagnostopoulos
T.
, Benedetti
E. L.
(1980
). Cytoplasmic regulation of tight junction permeability: effect of plant cytokinins.
Am. J. Physiol
239
, 75
–.Braegger
C. P.
, MacDonald
T. T.
(1994
). Immune mechanisms in chronic inflammatory bowel disease.
Ann. Allergy
72
, 135
–141
Brett
J.
, Gerlach
H.
, Nawroth
P.
, Steinberg
S.
, Godman
G.
, Stern
D.
(1989
). Tumor necrosis factor/cachectin increases permeability of endothelial cell monolayers by a mechanism involving regulatory G proteins.
J. Exp. Med
169
, 1977
–91
Burke-Gaffney
A.
, Keenan
A. K.
(1993
). Modulation of human endothelial cell permeability by combinations of the cytokines interleukin-1 alpha/beta, tumor necrosis factor-alpha and interferon-gamma.
Immunopharmacology
25
, 1
–9
Campbell
W. N.
, Ding
X.
, Goldblum
S. E.
(1992
). Interleukin-1 alpha and-beta augment pulmonary artery transendothelial albumin flux in vitro.
Am. J. Physiol
263
, 128
–136
Chang
E. B.
, Musch
M. W.
, Mayer
L.
(1990
). Interleukins 1 and 3 stimulate anion secretion in chicken intestine.
Gastroenterology
98
, 1518
–1524
Chiossone
D. C.
, Simon
P. L.
, Smith
P. L.
(1990
). Interleukin-1: effects on rabbit ileal mucosal ion transport in vitro.
Eur. J. Pharmacol
180
, 217
–228
Claude
P.
(1978
). Morphological factors influencing transepithelial permeability: a model for the resistance of the zonula occludens.
J. Membrane Biol
39
, 219
–232
Dawson
D. C.
(1977
). Na and Cl transport across the isolated turtle colon: parallel pathways for transmural ion movement.
J. Membrane Biol
37
, 213
–233
Engelmann
H.
, Holtmann
H.
, Brakebusch
C.
, Avni
Y. S.
, Sarov
I.
, Nophar
Y.
, Hadas
E.
, Leitner
O.
, Wallach
D.
(1990
). Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity.
J. Biol. Chem
265
, 14497
–14504
Fasano
M.
, Baudry
B.
, Pumplin
D. W.
, Wasserman
S. S.
, Tall
B. D.
, Ketley
J. M.
, Kaper
J. B.
(1988
). Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions.
Proc. Natl. Acad. Sci. USA
88
, 5242
–5246
Goldblum
S. E.
, Sun
W. L.
(1990
). Tumor necrosis factor-alpha augments pulmonary arterial transendothelial albumin flux in vitro.
Am. J. Physiol
258
, 57
–.Heyman
M.
, Darmon
N.
, Dupont
C.
, Dugas
B.
, Hirribaren
A.
, Blaton
M. A.
, Desjeux
J. F.
(1994
). Mononuclear cells from infants allergic to cow's milk secrete tumor necrosis factor, altering intestinal function.
Gastroenterology
106
, 1514
–1523
Kandil
H. M.
, Berschneider
H. M.
, Argenzio
R. A.
(1994
). Tumour necrosis factor a changes porcine intestinal ion transport through a paracrine mechanism involving prostaglandins.
Gut
35
, 934
–940
Kaufmann
S. H.
, Desnoyers
S.
, Ottaviano
Y.
, Davidson
N. E.
, Poirier
G. G.
(1993
). Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis.
Cancer Res
53
, 3976
–3985
Kotler
D. P.
, Reka
S.
, Clayton
F.
(1993
). Intestinal mucosalinflammation associated with human immunodeficiency virus infection.
Dig. Dis. Sci
38
, 1119
–1127
Kreusel
K. M.
, Fromm
M.
, Schulzke
J. D.
, Hegel
U.
(1991
). Clsecretion in epithelial monolayers of mucus-forming human colon cells (HT-29/B6).
Am. J. Physiol
261
, 574
–.Lähdevirta
J.
, Maury
C. P. J.
, Teppo
A. M.
, Repo
H.
(1988
). Elevated levels of circulating cachectin/tumor necrosis factor in patients with acquired immunodeficiency syndrome.
Am. J. Med
85
, 289
–291
Madara
J. L.
, Stafford
J.
(1989
). Interferon-directly affects barrier function of cultured intestinal epithelial monolayers.
J. Clin. Invest
83
, 724
–727
Marano
C. W.
, Laughlin
K. V.
, Russo
L. M.
, Mullin
J. M.
(1995
). The protein kinase C inhibitor, bisindolylmaleimide, inhibits the TPA-induced but not the TNF-induced increase in LLC-PK1 transepithelial permeability.
Biochem. Biophys. Res. Commun
209
, 669
–76
Mullin
J. M.
, Laughlin
K. V.
, Marano
C. W.
, Russo
L. M.
, Soler
A. P.
(1992
). Modulation of tumor necrosis factor-induced increase in renal (LLC-PK1) transepithelial permeability.
Am. J. Physiol
263
, 915
–.Ott
M.
, Lembcke
B.
, Staszewski
S.
, Helm
E. B.
, Caspary
W. F.
(1991
). Intestinale Permeabilität bei Patienten mit erworbenem Immundefekt-Syndrom (AIDS).
Klin. Wochenschr
69
, 715
–721
Peralta Soler
A.
, Mullin
J. M.
, Knudsen
K. A.
, Marano
C. W.
(1996
). Tissue remodeling during tumor necrosis factor-induced apoptosis in LLC-PK1 renal epithelial cells.
Am. J. Physiol
270
, 869
–.Riecken
E. O.
, Zeitz
M.
, Ullrich
R.
(1990
). Non-opportunistic causes of diarrhoea in HIV infection.
Baill. Clin. Gastroenterology
4
, 385
–403
Rodriguez
P.
, Heyman
M.
, Candalh
C.
, Blaton
M. A.
, Bouchaud
C.
(1995
). Tumour necrosis factor-induces morphological and functional alterations of intestinal HT29 cl. 19A cell monolayers.
Cytokine
7
, 441
–448
Sandle
G. I.
, Higgs
N.
, Crowe
P.
, Marsh
M. N.
, Venkatesan
S.
, Peters
T. J.
(1990
). Cellular basis for defective transport in inflamed human colon.
Gastroenterology
99
, 97
–105
Sato
N.
, Goto
T.
, Haranaka
K.
, Satomi
N.
, Nariuchi
H.
, Mano-Hirano
Y.
, Sawasaki
Y.
(1986
). Actions of tumor necrosis factor on cultured vascular endothelial cells: morphologic modulation, growth inhibition and cytotoxicity.
J. Natl. Cancer Inst
136
, 1113
–1121
Schmitz
H.
, Fromm
M.
, Bode
H.
, Scholz
P.
, Riecken
E. O.
, Schulzke
J. D.
(1996
). Tumor necrosis factor-alpha induces Cland K+secretion in human distal colon driven by prostaglandin E2.
Am. J. Physiol
271
, 669
–674
Schulzke
J. D.
, Fromm
M.
, Bentzel
C. J.
, Zeitz
M.
, Menge
H.
, Riecken
E. O.
(1992
). Epithelial ion transport in the experimental short bowel syndrome of the rat.
Gastroenterology
102
, 497
–504
Schulzke
J. D.
, Fromm
M.
, Menge
H.
, Riecken
E. O.
(1987
). Impaired intestinal sodium and chloride transport in the blind loop syndrome of the rat.
Gastroenterology
92
, 693
–698
Schulzke
J. D.
, Fromm
M.
, Schmitz
H.
, Barmeyer
C.
, Riecken
E. O.
(1995
). Barrier impairment in gastrointestinal diseases: ulcerative colitis and celiac sprue.
Z. Gastroenterol
33
, 571
–572
Sinicco
A.
, Biglioni
A.
, Sciandra
M.
, Forno
B.
, Pollono
A. M.
, Raiteri
R.
, Gioannini
P.
(1993
). Cytokine network and acute primary HIV-1 infection.
AIDS
7
, 1167
–1172
Staddon
J. M.
, Herrenknecht
K.
, Smales
C.
, Rubin
L. L.
(1995
). Evidence that tyrosine phosphorylation may increase tight junction permeability.
J. Cell Sci
108
, 609
–19
van Dullemen
H. M.
, van Deventer
S. J.
, Hommes
D. W.
, Bijl
H. A.
, Jansen
J.
, Tytgat
G. N.
, Woody
J.
(1995
). Treatment of Crohn's disease with anti-tumor necrosis factor chimeric monoclonal antibody (cA2).
Gastroenterology
109
, 129
–135
Vyarkarnam
A.
, Matear
P.
, Meager
A.
, Kelly
G.
, Stanley
B.
, Weller
I.
, Beverley
P.
(1991
). Altered production of tumour necrosis factors alpha and beta and interferon gamma by HIV-infected individuals.
Clin. Exp. Immunol
84
, 109
–115
This content is only available via PDF.
© 1999 by Company of Biologists
1999
(update)-MotorCFPExtended.jpg?versionId=3964)
-bioRxiv.jpg?versionId=3964)
(update)-CSTW.png?versionId=3964)
-FocalPlaneNetwork.png?versionId=3964)
-Mole.png?versionId=3964)
