Download Differential stimulation of IL-6 secretion following apical and

Biochemical Soclety Transactlons ( 1 996) 24
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Differential stimulation of IG6secretion following apical
and basol.ted presentation of IL-1 on epithelial cdl lines.
CATHETUNA
O’SHAUGHNESSY,ENAPROSSm TRACY KEANE
AND LUKEO’NEILL’
Elan Corporation Research In?i.tute, Trinity College, Dublin 2.
’BiochemistryDepartment, T m t y College, Dublin 2, Ireland.
IL-1 is a classic pro-inflammatory cytokine[l). Its
biological effects are shared by two functionally active forms ILl a and IL-Ip[l]. In humans the primary source of IL-1 is the
macro@age although a variety of other cell types can make IL-1
includmg endothelial cells, astro es, NK cells and dendritic
cells[2]. IL-1 has a diverse range g a r g e t tissues and manifests a
range of activitiesinclud+g actvation of T cells to express CD25
and produce IL-2, stmulation of fibroblast prolifmon,
indu.ction, of PG and LT synthesis, indu@on of B cell
prolferaaon and maturation and uprepiahon of adhesion
molecule expression on vascular endothehal cells[2]. Equivocal
results on the
ression of II,- 1 mRNA in epithelial cells may be
explained b d x e n t antigenic stimuli 3,4]. Its role has not yet
been fuuy (retermined in epithelial cell iology. A key feature of
the ro lnflammato effects of IL-1 is the induction of other
C y t O L i including% -6. In addition to its importance in B cell
growth and differentiation IL-6 plays a major role in the acute
phase response elicited by +sue trauma. IL-6 d i r e l y stimulates
acute hase rotem synthesis by hepatocytes both in VIVO and in
vitro
In tks study we have examined three human carcinoma
cell lines for IL-6 secretion in response to IL-1 stimulation:
ECV304 is y endothelial cell line, Calu-3 is a lun epithelialdenved cell hne and CaCo-2. an intestmal epithelial c$ h e .
AU three cell pries were cultured on Costar tissue culture
24-well plates (1x10 celldwell). The epithelial cell lines Calu-3
and CaCo-2, were also cultured in the Costar Transwell” system.
When grown at hi density in the Transwelle stem, they form
a structura~yan f~nctiona~y
polarized epigeliai mawlayer
(Fig. 1), characterised by having distinguishable membranes:
apical and basolateral (basal-lateral). The apical membrane is
covered by microvilli and contains ion-transporters, glycolipids
and GPI linked proteins. The basal membrane is involved in cellsubstratum interactions and has basement membrane receptors
and hemi-desmosornes. The lateral membrane is involved in cell@ *teractions and contains desmosomes,, gap, juneons, tight
junctrons and cell adhesion molecules. The ti JunctrOn forms a
structural boundary between the apical and asolateral domains
and plays a role in the restriction of lipid diffusion and
s e p a a o n of some,etegral membrane ,proteins.,The polarized
ce monolaver exhlbits a transepithehai electrical rmstance
which can be measured as an indication of how ‘tight’ a
monolayer is. Both CaCo-2 and Calu-3 show transepithelial
resistances in excess of 300Ncm‘. An added advantage of the
Transwell* s stem is that it allows access to both apical and basal
membranes &r stimulation and sampling. It is the classic system
used to studv drug and ion transport bv cells.
Fiqurc 1. Costar Tnnrwcll’ S-.
i
4.
P
f?
~
Cells gown on 24-well lates were stmulated wth
vanous concentrations of IL-I d e r 24 hours the supemtams
were collected and assayed for IL6 usmg the ‘Duoset ELISA
Kit’ (&myme) IL-6 secretion was found to be dosedependent.
eakmg when stmulated with 50n ml IL-I and decreasng wth
stmulation concentrations (fig 2) Both ECV304 secreted
I- ngml IL-6 in response to 30ngml IL-1 Calu-3 however,
produced more than 10 times thts amount This r o w was
also shown to be IL-1 s p d c ie the effect was b l z e d by the
IL-l receptor antagorust (data not shown)
RIP
Zoo00
-
p\
83s
c m
cab-2
Ecv304
mm
00
20
40 60
80
loo 120 140 160
[IL-I] n g / d
Figure 2 Sub-confluent cells were samulated wth IL-I at the
m&cated conccnmaon The supernatants were collened
after 24 hours and assaved for IL-6 content
CONCENTRATION OF IL-6 DETECTED IN CULTURE
SUPERNATANT (pg/ml)
cab3
CnCe2
&/ basolateral
&I
Baselme
2559
5902
240
071
Samulaaon
59657
8427
16224
110
basolateral
Basolaieral
Sumulaaon
7808 7
2991 7
5232
474
Table 1 Cell monolavers were stundated wth L-ia
(30neJml) for 24 hours Supematants were then asMyod for
IL-6wntent
Cells grown in the Trapwell” system were stimulated
either apically or basolaterally w t h 30n ml IL-I. Mer 24 hours
!he supematants were assayed for -6 content using the
Quanfikine ELISA Kit’ (R&D Systems). This was crossvalidated with Genzyme’s assa Once again, Calu-3 were more
responsive to IL-I in term ofL-6 secretion. a-6secretion was
found to be redominantly from the a ical membrane irrapedve
of apical or L o t a t 4 F d a t i o n c f h l e 1). ~ntqresting~y,,
caly3 appear to constitutively secrete IL-6. %s
secretton is
differential ie. four times higher a ically than basolatdly and
may reflect endo enous levels of otRer aanpnatory mediators.
dif€erence was observed III both the levels of
A
constitutive IL-6 secretion and the response to IL-1 stimulation
by Calu-3 versus CaCo-2 cells. This may rdect the increased
sensitivity to a pro-inflammatory response which is F j a t e d
with pulmonary (Calu-3) rather than intestinal epithehal tissue
(CaCo-2).
T h e e results demonstrate for the first time IL-1 induced
IL-6 secretion by lung
thehaldenved cells (Calu-3 . The
higher levels of secreted r - 6 apically versus basolatmdy may
suggest compartmentalisationof the inflammatory response m the
lung.
fi
This work was supported by Elan Corporation
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