Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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The study of
mucosal immunity has revealed that complex reciprocal interactions
occur between intestinal intraepithelial lymphocytes (IEL) and
intestinal epithelial cells (IEC). The present study focuses on the
induction of inducible nitric oxide (NO) synthase in cocultures of
freshly isolated rat IEL and the rat epithelial cell line IEC-18 after
the addition of interleukin-1 (IL-1
), tumor necrosis factor-
,
or lipopolysaccharide. When IEL and IEC were separated using Transwell
chambers, NO synthesis was not induced, indicating that cell-cell
contact was required. Culture of IEC-18 with IEL, even in the absence
of inflammatory stimuli such as IL-1
, resulted in upregulation of
class I and II antigens on IEC-18, due to the interferon-
(IFN-
)
that is constitutively produced by IEL. Addition of anti-IFN-
antibody to the NO-producing cocultures resulted in inhibition of NO
synthesis as well as the upregulation of class I and II antigen
expression. These data indicate that IFN-
production by IEL
conditions IEC for the expression of other components of the
inflammatory process.
cell-cell interaction; intestine; inflammation
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INTRODUCTION |
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INTRAEPITHELIAL LYMPHOCYTES (IEL) and
intestinal epithelial cells (IEC) display multiple reciprocal
interactions because of their intimate association at various mucosal
sites. One facet of this interaction is production of soluble
mediators, and another aspect is cognate IEL-IEC interaction. IEL
produce a wide variety of cytokines (see Ref. 1 for review), and
investigators have shown that these cytokines modulate IEC function,
including promotion of decreased barrier function and
Cl secretion mediated by interleukin (IL)-4 and
interferon-
(IFN-
) (7) and modulation of epithelial cell
restitution and IL-6 secretion by transforming growth factor-
(TGF-
) and IL-1
(12). It is also well documented that epithelial
cells produce many cytokines (6), as well as a variety of chemokines
(34), which can act as comitogens and chemotactic factors for
lymphocytes. It is likely that cytokines produced by IEC influence IEL
function in many ways that are yet to be defined. The proof of the
importance of a fine balance in cytokine levels necessary for the
maintenance of intestinal physiology is illustrated by the various
intestinal pathologies described in cytokine-deficient mice such as
IL-2 (24) and IL-10 (20) knockout mice.
Another aspect of IEL-IEC communication is via cell-cell contact, and
several different cell surface molecules have been shown to mediate
IEL-IEC interaction. Cepek et al. (5) have described a mechanism for
adhesion of IEL to IEC via an integrin molecule on the T cell CD103 and
the cadherin molecule on the epithelial cell. Another example of
cognate interaction is the constitutive lytic activity displayed by
CD8+ IEL (see Ref. 1 for review). Groh et al. (18) have
identified the nonclassical major histocompatibility complex (MHC)
class I chain-related gene A (MICA) and MICB as target molecules on IEC
for the cytotoxic action of T cell receptor (TCR) +
CD8
IEL. Recently, the receptor for MICA, NKG2D, has
been detected on natural killer cells as well as
TCR+ cells, indicating a role for these antigens in tumor
surveillance (2, 32). Yio and Mayer (35) have described yet another cognate interaction mediated by the CD8 molecule on the IEL and the
integrin-like molecule gp180 on the IEC. Recently, Yamamoto et al. (33)
described the inhibitory effect of IEC membranes on anti-CD3-induced
proliferation of IEL but not of peripheral T lymphocytes.
Because IEC constitutively express class I and II antigens, IEC also function as antigen-presenting cells (APC) in the classical interaction of the TCR on the T cell with either class I or II molecules on the APC. Recently, Nakazawa et al. (21) have shown that human IEC can express low levels of the costimulatory molecule CD86 on appropriately stimulated IEC, indicating that IEC could also invoke a primary immune response. Intestinal epithelial cells have also been shown to express nonclassical MHC molecules. Because these molecules display much less polymorphism than classical histocompatibility molecules and have a somewhat restricted tissue distribution, the T cells that are resident in epithelial sites may interact with these nonpolymorphic molecules (see Ref. 3 for review).
A major role of the intestinal mucosa is to provide a barrier against invasion of the host by pathogens present in the intestinal lumen. The production of nitric oxide (NO) by epithelial cells might function as a host defense mechanism because NO displays microbicidal properties (11). Thus the in vivo finding that inducible NO synthase (iNOS) is upregulated in the intestine after administration of an inflammatory agent such as lipopolysaccharide (LPS) (9, 25) is in accord with a host defense mechanism initiated by the many cytokines that are produced by an inflammatory stimulus such as invasion by a pathogen. Furthermore, Witthoft et al. (31) have shown that enteroinvasive bacteria directly induce NO production in the human colon epithelial cell lines Caco-2 and HT-29. This laboratory (19) has recently shown that iNOS is constitutively present in the epithelial cells of the ileal mucosa of untreated rats and mice and that SCID mice, devoid of mature T cells, express decreased levels of constitutive ileal iNOS. This finding led to the investigation of the role of IEL in iNOS expression in intestinal epithelial cells. The current study finds that in the presence of an inflammatory stimulus, IEL influence iNOS expression in epithelial cells through a mechanism that utilizes both soluble and cell-cell interaction.
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MATERIALS AND METHODS |
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Animals. Sprague-Dawley, Lewis, and ACI male rats, weighing 150-174 g, were purchased from Harlan Sprague Dawley (Indianapolis, IN) and used for experimentation between 4 and 6 mo of age. Animals were housed in a specific pathogen-free animal facility and provided rodent chow and acidified water ad libitum.
Antibodies.
For fluorescence-activated cell sorting (FACS) analysis,
TCR-phycoerythrin (PE)(R73),
TCR-FITC(V65),
CD3-FITC(G4.18), CD4-FITC and PE(OX-35), CD8-FITC and PE(OX-8),
CD45-FITC(OX-1), RT1A-FITC(OX-18), RT1B-FITC(OX-6), and CD54-FITC(1A29)
were purchased from Pharmingen (San Diego, CA). For blocking studies,
unlabeled CD4, CD8, RT1A, RT1B, CD54, and CD11a antibodies were
obtained in the no-azide/low-endotoxin form or in the purified form and dialyzed to remove the azide before use in vitro. For IFN-
neutralization studies, a polyclonal antibody was utilized (Biosource
International, Camarillo, CA). Polyclonal anti-human IL-15 (Biosource
International) was used as a control antibody for the polyclonal
IFN-
antibody.
Preparation of intraepithelial lymphocytes.
Small IEL were harvested according to established procedures (8). The
small intestine was flushed to remove luminal contents, the Peyer's
patches were removed, and the intestine was cut longitudinally and into
1-cm pieces that were stirred for 30 min at 37°C in 1 mM
dithioerythritol-10% FCS in Ca2+-Mg2+-free
PBS. The cell population was filtered over a nylon wool column, and the
lymphocytes were recovered from the interface of a 44%/67% Percoll
gradient. The recovered cell population contained 75-85% cells
that displayed the forward by side scatter properties characteristic of
lymphocytes, and 95% of this lymphoid population was
CD45+, indicating cells of hematopoetic origin (see Table
1 for further phenotypic analysis). For
positive and negative sorting, the cells were stained with PE-labeled
antibodies and sorted using a Becton Dickinson FACStar Plus. Medium
used for cocultures was DMEM supplemented with 5% FCS, 1 mM
L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/ml
penicillin, 100 µg/ml streptomycin, 13.6 µM folic acid, 0.3 mM
L-asparagine, and 5× 105 M
2-mercaptoethanol.
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Epithelial cell line IEL cocultures.
IEC-18 was obtained from American Type Culture Collection and
maintained in culture by weekly passage in DMEM (4.5 g/l glucose) containing 5% FCS, 1 mM L-glutamine, and 0.1 U/ml insulin.
New lines were established from frozen aliquots at 3- to 4-mo
intervals. Cocultures were established by the addition of 0.5-2.0 × 105 IEL to microtiter wells, 2 × 106 IEL to 22-mm wells, and 2 × 107 to
100-mm wells containing confluent monolayers of IEC-18. Confluent microtiter wells contain ~1-1.5 × 105 IEC-18,
22-mm wells contain 4-6 × 106 IEC-18, and 100-mm
petri dishes contain 10-15 × 106 IEC-18. Various
concentrations of recombinant human IL-1 (Dupont De Nemours,
distributed by NCI), recombinant murine tumor necrosis factor-
(TNF-
) (Genzyme, Cambridge, MA), recombinant rat IFN-
(GIBCO BRL,
Gaithersburg, MD), or LPS (Escherichia coli 011B4, Sigma
Chemical, St. Louis, MO) were added, and supernatant NO2 levels were determined on various days after initiation of
coculture. Where indicated, the monolayers of IEC-18 were
trypsinized and surface markers were assayed by staining with
FITC-labeled antibodies and examined by FACS. For selected experiments,
IEL were separated from IEC-18 using Transwell culture wells (Corning
Costar, Cambridge, MA) with a 0.4-µM pore size membrane.
Preparation of peritoneal macrophage cultures. The peritoneal cavity of the rat was lavaged with 5% FCS-MEM. The cells were washed once, counted, and plated at 0.25, 1, and 2.0 × 105 peritoneal cells/well. After a 4-h incubation at 37°C, the monolayers were washed vigorously to remove nonadherent cells and used for experimentation.
Supernatant NO2 assay. A microplate assay was utilized, combining 0.05 ml of culture supernatant and 0.05 ml of Greiss reagent (17). NaNO2 (Sigma Chemical) was used as the standard, and samples were analyzed with a maximum velocity microplate reader. NO2 is a stable product in culture supernatants, and values are an indication of total amount of NO synthesized during the culture period. Supernatant NO2 levels were assayed at various times after initiation of culture, depending on whether the initiation of NO synthesis or total amount of NO synthesized was being determined in a particular experiment.
Western blot. Cell lysates were prepared by three rapid freeze-thaw cycles and centrifuged. The supernatant protein was quantitated using the Pierce bicinchoninic acid kit. Forty micrograms of protein were loaded per lane of a 8% polyacrylamide gel. The gel was transferred to a nitrocellulose membrane, incubated with rabbit polyclonal anti-MAC iNOS antibody (Transduction Laboratories, Lexington, KY), and then incubated with horseradish peroxidase-conjugated anti-rabbit IgG.
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RESULTS |
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Induction of NO synthesis by cytokines.
To ascertain which stimuli or combinations of stimuli, added
exogenously, induced NO synthesis in IEC-18, 25 ng/ml IFN-, 100 pg/ml TNF-
, 5 ng/ml IL-1
, and 10 µg/ml LPS were added singly or
in combination to microtiter wells containing confluent monolayers of
IEC-18. Supernatant NO2 levels from 48-h cultures are
depicted in Fig. 1. Under the influence of
any single stimulus, NO synthesis was not induced. The combination of
IL-1
and IFN-
resulted in induction of modest amounts of NO,
whereas other double combinations of the stimuli were not effective.
Under the influence of several combinations of triple stimuli, NO
synthesis was variably induced, with the combination of IL-1
,
TNF-
, and IFN-
being the most effective and the combination of
TNF-
, LPS, and IL-1
the least effective.
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Induction of NO synthesis in IEL-IEC coculture.
Various numbers of freshly isolated IEL were added to confluent
monolayers of IEC-18 in microtiter wells. In the presence of 5 ng/ml
IL-1, a cell concentration-dependent increase in supernatant NO2 levels was seen (Fig.
2A). It can be seen that addition
of IEL alone to the IEC-18 monolayer did not result in NO synthesis. In
cocultures with a constant number of IEL (2 × 105/microtiter well), a concentration-dependent increase in
supernatant NO2 was seen in the presence of increasing
concentrations of IL-1
(Fig. 2B). When spleen or mesenteric
lymph node lymphocytes were substituted for IEL in the cocultures, NO
was not induced, indicating that a specific interaction between IEL and
IEC was necessary for induction of NO synthesis. Similarly, when the
IEL population was cocultured with hepatocytes freshly harvested from
Sprague-Dawley rats, NO synthesis was not induced (Fig. 2B,
inset), even though stimulation with a cytokine mixture of
IFN-
, LPS, IL-1
, and TNF-
did result in NO synthesis by the
hepatocytes (59.0 µM NO2, data not shown).
These data indicate that IEL, but not mesenteric lymph node lymphocytes
or splenocytes, coinduce NO synthesis in IEC and that another
epithelial cell such as the hepatocyte does not serve as a target
NO-producing cell when cocultured with IEL.
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IEL coinduction of NO synthesis in peritoneal macrophages.
Because the IEC-18 cell line was derived from an outbred rat (23) and
the Sprague-Dawley rats used as a source of IEL in these experiments
are also outbred, it was important to confirm that IEL could induce NO
synthesis in a completely syngeneic system. Therefore, various
concentrations of peritoneal macrophages from a Lewis rat were allowed
to adhere to culture dishes for 3-4 h and washed free of
nonadherent cells, and freshly harvested Lewis IEL were added to these
cultures. As can be seen in Fig. 3,
addition of IEL resulted in enhanced supernatant NO2
levels. Because IEL have been shown to constitutively produce IFN-
and peritoneal macrophages have been shown to produce NO in response to
IFN-
, NO synthesis by peritoneal macrophages in the presence of
anti-IFN-
antibody was examined. A modest decrease (from 33 to 22 µM NO2) in NO synthesis was observed in cultures where 50 µg/ml of antibody were added, indicating that some of the NO
synthesis in the cocultures could be attributed to IFN-
(Fig. 3,
inset). Addition of 125 ng/ml of IFN-
to peritoneal
macrophage culture resulted in 13.0 µM NO2 in the
supernatant (data not shown), illustrating that peritoneal macrophages
synthesized less NO in response to addition of exogenous IFN-
than
when cocultured with IEL.
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Influence of other inflammatory stimuli on coinduction of NO
synthesis in IEL-IEC cultures.
To determine whether stimuli other than IL-1 resulted in coinduction
of NO synthesis, IEL-IEC-18 cocultures were established in the presence
of various concentrations of TNF-
, LPS, or IFN-
. As can be seen
in Fig. 4, at concentrations of 100 pg/ml
TNF-
and 10 µg/ml LPS, NO synthesis was also induced. However, in
the presence of IFN-
, even at high concentrations such as 125 ng/ml, NO synthesis was not induced. This experiment revealed that IFN-
alone was an insufficient stimulus for induction of NO synthesis in
IEC-18.
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Western blot for iNOS protein.
To ascertain that iNOS protein was present in the induced cultures,
IEC-18 monolayers were established in 100-mm petri dishes and
stimulated with 5 ng/ml IL-1 and/or 25 ng/ml IFN-
or by the
addition of 2 × 107 IEL in the presence of IL-1
and/or IFN-
. On day 2 of culture, supernatant
NO2 levels were determined, lysates of the cultured cells
were prepared, and a Western blot was performed (Fig.
5). It is clear that iNOS protein was not
present in IEC-18 control cell cultures or in cultures that were
treated with IL-1
, IFN-
, IEL, or IEL plus IFN-
. Supernatant
NO2 levels of 1.3 µM or less were measured in these
cultures. When IEC-18 was stimulated with a combination of IL-1
and
IFN-
, an NO2 level of 10.1 µM was measured and a faint
band at 130 kDa was present. However, when IEC-18 was cultured with IEL
and IL-1
, a band migrating at 130 kDa was detected along with
enhanced supernatant NO2 levels (43.0 µM). Therefore, the
amount of iNOS protein present in cocultures of IEL and IEC-18 plus
IL-1
correlated well with the supernatant NO2 levels.
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Effect of separation of IEL from IEC-18 on NO synthesis.
To determine whether cell-cell interaction was required for coinduction
of NO synthesis, IEL were added to the upper compartment of a Transwell
culture system that was inserted into a 22-mm well containing a
confluent IEC-18 monolayer. IL-1 (5 ng/ml) was added to the culture
system, and supernatant NO2 was determined on day 2 of coculture. As can be seen in Table 2, NO
was definitely not induced in cultures where IEL were separated from
IEC-18, even though IL-1
came in contact with both cell types. This
result clearly demonstrated that cell contact between the IEL and IEC was required for NO synthesis to ensue.
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Determination of which IEL subpopulation is responsible for inducing
NO synthesis.
The IEL population is a complex mixture of CD3+ IEL that
express either CD4 or CD8 (Table 1). To determine whether a
subpopulation of IEL was responsible for iNOS coinduction, IEL were
treated with CD4-PE antibody and sorted by flow cytometry techniques. The sorted populations were then added to IEC-18 cultures in the presence of IL-1 and supernatant NO2 assessed on day
4. As depicted in Fig. 6, depletion of
CD4+ IEL resulted in almost complete abrogation of NO
synthesis. Addition of positively sorted CD4+ IEL resulted
in induction of NO synthesis at cell concentrations that were
ineffective with an unseparated population of IEL. Clearly, these
results showed that the CD4+ IEL subpopulation was capable
of coinducing NO synthesis.
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Effect of IFN- antibody on IEL-mediated coinduction
of NO synthesis and class I and II expression.
Because IFN-
mediated upregulation of class II molecules on IEC-18
and the CD4+ subpopulation of IEL was an effective
coinducer of iNOS in IEC-18, blocking of class II upregulation may
possibly prevent NO synthesis in this coculture system. Therefore,
confluent monolayers of IEC-18 were established in 22-mm culture wells
and 2 × 106 IEL were added, along with 5 ng/ml
IL-1
, in the presence of various concentrations of polyclonal
IFN-
antibody. Cohort cultures received the control antibody,
polyclonal anti-human IL-15. On day 2 of culture, supernatant
NO2 levels were determined, cells were trypsinized, and
expression of class I and II antigens on IEC-18 was determined by FACS
analysis (expressed as median fluorescence intensity, MFI). As depicted
in Fig. 7A, addition of IL-15
antibody had little effect on class I and II expression, whereas
addition of IFN-
antibody (Fig. 7B) resulted in ~50%
reduction in class I MFI and 80% reduction in class II MFI. As seen in
Fig. 7B, inset, anti-IL-15 did not inhibit NO
synthesis, whereas anti-IFN-
completely inhibited NO synthesis.
These results suggest that IFN-
plays a crucial role in the
coinduction of NO synthesis by IEL.
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Effect of class I and II blocking antibodies on coinduction of NO
synthesis by IEL and mixed lymphocyte reaction.
To determine whether blocking of class II antigen with antibody would
prevent induction of NO synthesis in this culture system, various
concentrations of class I and class II antibodies were added to
IEL-IEC-18 plus IL-1 cocultures as well as to a mixed lymphocyte
reaction consisting of Lewis responding lymph node lymphocytes and
irradiated ACI lymph node lymphocytes. As depicted in Table
3, addition of class I antibody induced a
modest inhibition of the mixed lymphocyte reaction while having no
effect on NO synthesis, whereas addition of class II antibody resulted
in complete inhibition of the mixed lymphocyte reaction but also no
effect on NO synthesis. Various other antibodies, including
intercellular adhesion molecule-1, lymphocyte function-associated
antigen-1 (LFA-1), CD4, and CD8 antibodies, were also added to the
IEL-IEC-18 cocultures, and similarly, no inhibition of NO synthesis was
observed. Therefore, at this point, the cell surface molecules involved in mediating upregulation of NO synthesis in this coculture system have
not been identified.
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DISCUSSION |
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Previous study has shown that iNOS is upregulated in various inflammatory bowel diseases including colitis and Crohn's disease (26) as well as in animal models of colitis (13), although the exact mechanism of induction is unknown. It is assumed that NO synthesis is triggered by exposure of the epithelial cell to the multiple inflammatory cytokines characteristic of these disease processes, much like that observed in vitro on stimulation of various cell types with cytokines. The current study provides evidence that IEL can coinduce iNOS in epithelial cells in the presence of a single added inflammatory cytokine, indicating that induction of iNOS may occur under more subtle inflammatory conditions as well as under the influence of multiple inflammatory cytokines. Because we have also observed that IEL can coinduce NO synthesis in peritoneal macrophages, iNOS coinduction by CD4+-activated lymphocytes may be relevant in inflammatory conditions at various sites, in addition to the intestinal epithelial layer.
Although much effort has been focused on the functions of
CD8+ IEL, including their constitutive lytic function,
proliferation in response to gp180 on the epithelial cell (35) and
production of keratinocyte growth factor by the TCR +
subset (4), relatively little is known about the functions of the
CD4+ IEL. However, attention has been focused on the
CD4+ T cell by experiments that describe a role for
CD4+ memory Th1 cells in the mediation of intestinal
inflammatory disease. It has been demonstrated that the
CD4+, CD45RBhigh, Th1-like subpopulation of
lymphocytes obtained from lymph nodes causes wasting disease and
hyperplasia of the colonic mucosa when injected into SCID mice. In
contrast, the CD4+, CD45RBlow, Th2-like lymph
node population did not cause inflammatory bowel disease in SCID mice
and could actually abrogate the effects of the CD45RBhigh
population via the production of TGF-
(22). The present study shows
the ability of the CD4+ IEL subpopulation to coinduce NO
synthesis in epithelial cells, revealing a proinflammatory role for the
CD4+ IEL subset utilizing an in vitro system. More study is
needed to determine whether subpopulations of IEL have different
functions, for example the CD4+CD8
vs. the CD4+CD8
+ subsets. Recent
experiments (unpublished observations) staining for intracellular
cytokines reveal that both CD4+ and CD8+ IEL
stain for IFN-
, indicating that either cell population could be
producing the IFN-
required for NO synthesis in this coculture system. Another role for CD4+CD8
as well
as CD4+CD8+ IEL is that of provision of B cell
help via production of IL-4 and IL-5 in an IEL-B cell culture system
(15, 16). Recent study by Todd et al. (30) indicates that different
subpopulations of IEL are recovered depending on the isolation
procedure used, demonstrating that IEL populations utilized for study
must be carefully characterized.
The cell surface molecules on IEL and IEC that mediate the cell-cell
interaction required for iNOS induction remain unidentified. The
splenocyte and mesenteric lymph node lymphocyte populations, which
contain ~30% CD4+ lymphocytes, did not serve to coinduce
NO synthesis in IEC-18, indicating that a molecule present on activated
lymphocytes such as IEL is necessary for iNOS-coinducing function. As
far as the contribution of the target NO producing cell in this system
of cell-cell interaction, there must also be a requisite molecule on
this cell population because hepatocytes, cells that readily produce NO
in response to multiple cytokine stimuli (10), did not produce NO when
cocultured with IEL plus IL-1 (Fig. 2B, inset). The
fact that peritoneal macrophages can serve as target NO-producing cells
indicates that the molecule that the IEL reacts with on the
NO-producing cell may not be restricted to the IEC or perhaps more than
one molecule can serve as a target molecule for the IEL. The complete
lack of effect of the blocking antibodies on NO synthesis in the
IEL-IEC-18 cocultures, although the same antibodies (Table 3) very
effectively block the rat mixed lymphocyte reaction, indicates that
different cell surface molecules are mediating these two interactions.
This experimental coculture system also provides the important
indication that iNOS may be coinduced in many cell types via the
combination of a cytokine that by itself does not induce iNOS but in
the presence of an activated lymphocyte will induce iNOS. In a similar
system using mouse macrophages as target NO-producing cells, Tao and
Stout (28) have shown that coculture of resting Th1 lymphocyte clones
but not Th2 clones with IFN--primed macrophages will induce NO in
the macrophages in an antigen-specific manner (i.e., in the presence of
the protein to which the clones have been sensitized and an H-2
compatible macrophage), presumably due to the cytokines produced by the
Th1 clone on antigen recognition. However, plasma membrane fractions
obtained from either activated Th1 or Th2 clones, but not resting
clones, could induce NO in IFN-
-primed macrophages in an
antigen-nonspecific manner. Tian et al. (29) also demonstrated that
plasma membranes from activated T cells enhanced NO production by
IFN-
- and TNF-
-stimulated macrophages, and this enhancement was
partially inhibited by antibodies to CD40L and LFA-1. These data
provide evidence that activated T cells, in particular CD4+
T cells, have a membrane determinant that can promote NO synthesis in
an appropriately primed macrophage. The CD4+ IEL are an
activated population and may display a determinant similar to that
which others have found on activated T cell clones.
These experiments have illustrated the complex interactions between IEL
and IEC that presumably are necessary to preserve optimal intestinal
function. This paradigm has been promoted by Fujihasi et al. (14) to
describe interactions between components of gut-associated lymphoid
tissue and the parenchymal cells of the gut. In the presently described
system, coculture of IEL with the IEC-18 epithelial cell line results
in upregulation of class I and II antigen expression on IEC, a
condition that reflects normal intestinal physiology (27). Because of
the activated status of IEL, this cell population may be constitutively
producing other mediators that may maintain levels of antigens on IEC
that facilitate IEL-IEC interaction. In this setting, on exposure to an
inflammatory stimulus such as IL-1 or TNF-
, NO is produced, perhaps by both the pure cytokine pathway and the CD4+ IEL
coinduction pathway.
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ACKNOWLEDGEMENTS |
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I thank Dr. Richard L. Simmons for guidance and support.
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FOOTNOTES |
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This work was supported by National Institute of Allergy and Infectious Diseases Grants AI-14032 and AI-16869 (both to R. L. Simmons).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. A. Hoffman, Dept. of Surgery, W1545 BST, 200 Lothrop St., Pittsburgh, PA 15261 (E-mail: hoffmanr3{at}msx.upmc.edu).
Received 14 October 1999; accepted in final form 14 January 2000.
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