Intraepithelial lymphocytes coinduce nitric oxide synthase in intestinal epithelial cells

Rosemary A. Hoffman

Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-1beta (IL-1beta ), tumor necrosis factor-alpha , 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-1beta , resulted in upregulation of class I and II antigens on IEC-18, due to the interferon-gamma (IFN-gamma ) that is constitutively produced by IEL. Addition of anti-IFN-gamma 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-gamma production by IEL conditions IEC for the expression of other components of the inflammatory process.

cell-cell interaction; intestine; inflammation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma (IFN-gamma ) (7) and modulation of epithelial cell restitution and IL-6 secretion by transforming growth factor-beta (TGF-beta ) and IL-1beta (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) gamma delta + CD8- IEL. Recently, the receptor for MICA, NKG2D, has been detected on natural killer cells as well as gamma delta 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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, alpha beta TCR-phycoerythrin (PE)(R73), gamma delta 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-gamma 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-gamma 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× 10-5 M 2-mercaptoethanol.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Phenotypic analysis of Sprague-Dawley rat small intestinal IEL

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-1beta (Dupont De Nemours, distributed by NCI), recombinant murine tumor necrosis factor-alpha (TNF-alpha ) (Genzyme, Cambridge, MA), recombinant rat IFN-gamma (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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma , 100 pg/ml TNF-alpha , 5 ng/ml IL-1beta , 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-1beta and IFN-gamma 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-1beta , TNF-alpha , and IFN-gamma being the most effective and the combination of TNF-alpha , LPS, and IL-1beta the least effective.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   Induction of nitric oxide (NO) synthesis in intestinal epithelial cells (IEC-18) by various inflammatory stimuli. Stimuli, including 5 ng/ml interleukin-1beta (IL-1beta ), 100 pg/ml tumor necrosis factor-alpha (TNF-alpha ), 10 µg/ml lipopolysaccharide (LPS), and 25 ng/ml interferon-gamma (IFN-gamma ) were added singly (A) or in double (B) or triple (C) combinations to confluent cultures of IEC-18 in microtiter wells. Supernatant NO2 levels were assayed on day 2 of culture and are expressed as means ± SD of triplicate wells. Data are representative of 5 independent experiments.

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-1beta , 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-1beta (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-gamma , LPS, IL-1beta , and TNF-alpha 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.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   Induction of NO synthesis in intraepithelial lymphocyte (IEL)-IEC-18 coculture. A: various numbers of freshly isolated rat IEL (0.125, 0.25, 0.5, and 1.0 × 105) were added to confluent monolayers of IEC-18 in microtiter wells in the presence of 5 ng/ml IL-1beta . B: a constant number of IEL, splenocytes (spleen), or mesenteric lymph node lymphocytes (MLN) (2 × 105) were added to confluent monolayers of IEC-18 in microtiter wells in the presence of 0.05, 0.5, and 5 ng/ml IL-1beta . B, inset: NO2 levels in cocultures of hepatocytes and IEL plus various concentrations of IL-1beta . Supernatant NO2 levels were assayed on day 3 of culture and are depicted as means ± SD of triplicate wells. Data are representative of 5 independent experiments.

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-gamma and peritoneal macrophages have been shown to produce NO in response to IFN-gamma , NO synthesis by peritoneal macrophages in the presence of anti-IFN-gamma 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-gamma (Fig. 3, inset). Addition of 125 ng/ml of IFN-gamma 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-gamma than when cocultured with IEL.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3.   Induction of NO synthesis in IEL-peritoneal macrophage cultures. Various numbers of Lewis peritoneal cells (0.5, 1.0, and 2.0 × 105) were plated in microtiter wells and allowed to adhere for 4 h at 37°C. Monolayers were vigorously washed free of nonadherent cells, and 2 × 105 freshly harvested Lewis IEL were added to macrophage cultures. Supernatant NO2 levels were determined on day 2 of culture and are depicted as means ± SD of triplicate determinations. Data are representative of 2 independent experiments. Inset: NO2 levels in IEL-peritoneal macrophage cultures in the presence of various concentrations of IFN-gamma antibody (aby).

Influence of other inflammatory stimuli on coinduction of NO synthesis in IEL-IEC cultures. To determine whether stimuli other than IL-1beta resulted in coinduction of NO synthesis, IEL-IEC-18 cocultures were established in the presence of various concentrations of TNF-alpha , LPS, or IFN-gamma . As can be seen in Fig. 4, at concentrations of 100 pg/ml TNF-alpha and 10 µg/ml LPS, NO synthesis was also induced. However, in the presence of IFN-gamma , even at high concentrations such as 125 ng/ml, NO synthesis was not induced. This experiment revealed that IFN-gamma alone was an insufficient stimulus for induction of NO synthesis in IEC-18.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of other inflammatory stimuli on induction of NO synthesis in IEL-IEC-18 cultures. Freshly harvested rat IEL (2 × 105) were added to confluent monolayers of IEC-18 in microtiter wells in the presence of 10-fold dilutions of TNF-alpha (A), LPS (B), or IFN-gamma (C). Supernatant NO2 levels were assayed on day 2 of culture and are expressed as means ± SD of triplicate wells. Gray bars, NO2 levels in presence of IEL. Black bars, levels in absence of IEL. Data are representative of 3 individual experiments.

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-1beta and/or 25 ng/ml IFN-gamma or by the addition of 2 × 107 IEL in the presence of IL-1beta and/or IFN-gamma . 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-1beta , IFN-gamma , IEL, or IEL plus IFN-gamma . Supernatant NO2 levels of 1.3 µM or less were measured in these cultures. When IEC-18 was stimulated with a combination of IL-1beta and IFN-gamma , 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-1beta , 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-1beta correlated well with the supernatant NO2 levels.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   Western blot for inducible NO synthase protein. Confluent monolayers of IEC-18 were established in 100-mm petri dishes. Cultures were stimulated with either 5 ng/ml IL-1beta or 25 ng/ml IFN-gamma or both in the presence and absence of 2 × 107 freshly harvested IEL in a final culture volume of 10 ml of complete medium. On day 2 of culture, supernatant NO2 levels were assayed, lysates of the cultured cells were prepared, and a Western blot was performed. Lysates prepared from RAW 264.7 cells stimulated with LPS served as the positive control, and lysates from the EL4 lymphoma cell line served as the negative control.

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-1beta (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-1beta 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.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Characterization of IEL-IEC-18 cocultures using the Transwell culture system

Because IEL have been shown to constitutively express IFN-gamma , we measured the level of this cytokine in the culture supernatants and found picogram levels of IFN-gamma , regardless of whether the IEL were in contact with or separated from the IEC-18. Similarly, class I expression was upregulated on IEC-18 obtained from cultures with exogenously added IFN-gamma as well as IEC-18 cocultured with IEL, regardless of whether cell-cell contact between IEC-18 and IEL was established. Upregulation of class II antigen was also observed, although higher levels of class II were seen in those cultures where cell-cell contact between IEL and IEC-18 was established, especially in the presence of IL-1beta . Treatment of IEC-18 with IL-1beta alone does not result in increased expression of class I or II on IEC-18 (unpublished observation). This experiment separated the upregulation of class I and, to a lesser extent, class II on IEC-18, which did not require cell-cell contact, from the NO synthesis that was not observed unless IEC-18-IEL contact was established.

To rule out the possibility that a soluble factor was present in a culture system containing IEL, IEC-18, and IL-1beta that was not produced when IEL and IEC-18 were separated from each other by a membrane, supernatants were transferred from cultures where NO synthesis had been induced to naive IEC-18 cultures. NO synthesis was monitored in the secondary culture and an increase in supernatant NO2 was not observed, indicating that a soluble mediator capable of inducing NO synthesis was not produced in the cultures where NO was coinduced by IEL (data not shown).

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-1beta 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.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of CD4+ and CD4-depleted IEL on coinduction of NO synthesis. Freshly harvested rat IEL were treated with phycoerythrin-labeled CD4 antibody and sorted. CD4-depleted and CD4-positively sorted IEL were added to confluent monolayers of IEC-18 in the presence and absence of 5 ng/ml IL-1beta . Control IEL consisted of unmanipulated IEL population. Supernatant NO2 levels were determined on day 4 of culture and are represented as means ± SD of triplicate determinations. Data are representative of 4 individual experiments.

Effect of IFN-gamma antibody on IEL-mediated coinduction of NO synthesis and class I and II expression. Because IFN-gamma 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-1beta , in the presence of various concentrations of polyclonal IFN-gamma 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-gamma 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-gamma completely inhibited NO synthesis. These results suggest that IFN-gamma plays a crucial role in the coinduction of NO synthesis by IEL.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of IFN-gamma antibody on NO synthesis and class I and II expression. Confluent monolayers of IEC-18 were established in 22-mm tissue culture wells, and 2 × 106 IEL were added in the presence of 5 ng/ml IL-1beta and various concentrations of either anti-IL-15 or -IFN-gamma antibody. On day 2 of culture, supernatant NO2 levels were assayed, and cell monolayers were trypsinized and stained for class I (filled bars) and II (open bars) antigen expression. A and B: median fluorescence intensity (MFI) of antigen expression. B, inset: supernatant NO2 levels are depicted. MFI levels of antibody isotype control were always <2 on a log scale. Data are representative of 3 separate experiments. Class I and II MFI with 12.5 µg/ml anti-IL-15 vs. anti-IFN-gamma were significantly different (P <=  0.01) by unpaired Student's t-test.

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-1beta 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.

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Comparison of blocking effect of class I and II antibodies on IEL coinduced NO synthesis and MLR


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma delta + 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-beta (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+CD8alpha alpha - vs. the CD4+CD8alpha alpha + subsets. Recent experiments (unpublished observations) staining for intracellular cytokines reveal that both CD4+ and CD8+ IEL stain for IFN-gamma , indicating that either cell population could be producing the IFN-gamma 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-1beta (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-gamma -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-gamma -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-gamma - and TNF-alpha -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-1beta or TNF-alpha , NO is produced, perhaps by both the pure cytokine pathway and the CD4+ IEL coinduction pathway.


    ACKNOWLEDGEMENTS

I thank Dr. Richard L. Simmons for guidance and support.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abreu-Martin, MT, and Targan SR. Regulation of immune responses of the intestinal mucosa. Crit Rev Immunol 16: 277-309, 1996[ISI][Medline].

2.   Bauer, S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL, and Spies T. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285: 727-729, 1999[Abstract/Free Full Text].

3.   Blumberg, RS, Simister N, Christ AD, Israel EJ, Colgan SP, and Balk SP. MHC-like molecules on mucosal epithelial cells. In: Essentials of Mucosal Immunology, edited by Kagnoff MF.. San Diego: Academic, 1996, p. 85-89.

4.   Boismenu, R, and Havran WL. Modulation of epithelial cell growth by intraepithelial gamma-delta T cells. Science 266: 1253-1255, 1994[ISI][Medline].

5.   Cepek, KL, Shaw SK, Parker CM, Russell GJ, Morrow JS, Rimm DL, and Brenner MB. Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the alpha Ebeta 7 integrin. Nature 372: 190-193, 1994[ISI][Medline].

6.   Christ, AD, and Blumberg RS. The intestinal epithelial cell: immunological aspects. Springer Semin Immunopathol 18: 449-461, 1997[ISI][Medline].

7.   Colgan, SP, Resnick MB, Parkos CA, Delp-Archer C, McGuirk D, Bacarra AE, Weller PF, and Madara JL. IL-4 directly modulates function of a model human intestinal epithelium. J Immunol 153: 2122-2129, 1994[Abstract/Free Full Text].

8.   Coligan, JE, Kruisbeek AM, Margulies DH, and Shevach EM. Current Protocols in Immunology. New York: Wiley, 1994, p. 3.19.1.

9.   Cook, HT, Bune AJ, Jansen AS, Taylor GM, Loi RK, and Cattell V. Cellular localization of inducible nitric oxide synthase in experimental endotoxic shock in the rat. Clin Sci (Lond) 87: 179-186, 1994[ISI][Medline].

10.   Curran, RD, Billiar TR, Stuehr DJ, Hofmann K, and Simmons RL. Hepatocytes produce nitrogen oxides from L-arginine in response to inflammatory products of Kupffer cells. J Exp Med 170: 1769-1774, 1989[Abstract].

11.   De Grotte, MA, and Fang FC. NO inhibitions: antimicrobial properties of nitric oxide. Clin Infect Dis 21, Suppl2: S162-S165, 1995[ISI][Medline].

12.   Dignass, AU, and Podolsky DK. Cytokine modulation of intestinal epithelial cell restitution: central role of transforming growth factor beta. Gastroenterology 105: 1323-1332, 1993[ISI][Medline].

13.   Eutamene, H, Theodorou V, Fioramonti J, and Bueno L. Implication of NK1 and NK2 receptors in rat colonic hypersecretion induced by interleukin 1beta : role of nitric oxide. Gastroenterology 109: 483-489, 1995[ISI][Medline].

14.   Fujihasi, K, Kweon M, Kiyono H, VanCott JL, van Ginkel FW, Yamamoto M, and McGhee JR. A T cell/B cell/epithelial cell internet for mucosal inflammation and immunity. Springer Semin Immunopathol 18: 477-494, 1997[ISI][Medline].

15.   Fujihashi, K, Yamamoto M, McGhee JR, Beagley KW, and Kiyono H. Function of alpha beta TCR+ intestinal intraepithelial lymphocytes: Th1- and Th2-type cytokine production by CD4+CD8- and CD4+CD8+ T cells for helper activity. Int Immunol 5: 1473-1481, 1993[Abstract].

16.   Fujihashi, K, Yamamoto M, McGhee JR, and Kiyono H. alpha beta T cell receptor-positive intraepithelial lymphocytes with CD4+, CD8- and CD4+, CD8+ phenotypes from orally immunized mice provide Th2-like function for B cell responses. J Immunol 151: 6681-6691, 1993[Abstract/Free Full Text].

17.   Green, LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, and Tannenbaum SR. Analysis of nitrate, nitrite and [15N] nitrate in biological fluids. Anal Biochem 126: 131-137, 1882.

18.   Groh, V, Steinle A, Bauer S, and Spies T. Recognition of stress-induced MHC molecules by intestinal epithelial gamma delta T cells. Science 279: 1737-1740, 1998[Abstract/Free Full Text].

19.   Hoffman, RA, Zhang G, Nussler NC, Gleixner SL, Ford HR, Simmons RL, and Watkins SC. Constitutive expression of inducible nitric oxide synthase in the mouse ileal mucosa. Am J Physiol Gastrointest Liver Physiol 272: G383-G392, 1997[Abstract/Free Full Text].

20.   Kühn, R, Löhler J, Rennick D, Rejewsky K, and Müller W. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75: 263-274, 1993[ISI][Medline].

21.   Nakazawa, A, Watanabe M, Kanai T, Yajima T, Yamazaki M, Ogata H, Ishii H, Azuma M, and Hibi T. Functional expression of costimulatory molecule CD86 on epithelial cells in the inflamed colonic mucosa. Gastroenterology 117: 536-545, 1999[ISI][Medline].

22.   Powrie, F, Carlino J, Leach MW, Mauze S, and Coffman RL. A critical role for transforming growth factor-beta but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RBlow CD4+ T cells. J Exp Med 183: 2669-2674, 1996[Abstract].

23.   Quaroni, A, Wands J, Trelstad RL, and Isselbacher KJ. Epithelioid cell cultures from rat small intestine. J Cell Biol 80: 248-265, 1979[Abstract].

24.   Sadlack, B, Merz H, Schorle H, Schimpl A, Feller AC, and Horak I. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 75: 253-261, 1993[ISI][Medline].

25.   Salter, M, Knowles RG, and Moncada S. Widespread tissue distribution, species distribution and changes in activity of Ca2+-dependent and Ca2+-independent nitric oxide synthases. FEBS Lett 291: 145-149, 1991[ISI][Medline].

26.   Singer, II, Kawka DW, Scott S, Weidner JR, Mumford RA, Riehl TE, and Stenson WF. Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease. Gastroenterology 111: 871-885, 1996[ISI][Medline].

27.   Steiniger, B, Falk P, Lohmuller M, and Van Der Meide PH. Class II MHC antigens in the rat digestive system. Normal distribution and induced expression after interferon-gamma treatment in vivo. Immunology 68: 507-513, 1989[ISI][Medline].

28.   Tao, X, and Stout RD. T cell-mediated cognate signaling of nitric oxide production by macrophages. Requirements for macrophage activation by plasma membranes isolated from T cells. Eur J Immunol 23: 2916-2921, 1993[ISI][Medline].

29.   Tian, L, Noelle R, and Lawrence D. Activated T cells enhance nitric oxide production by murine splenic macrophages through gp39 and LFA-1. Eur J Immunol 25: 306-309, 1995[ISI][Medline].

30.   Todd, D, Singh AJ, Greiner DL, Mordes JP, Rossini AA, and Bortell R. A new isolation method for rat intraepithelial lymphocytes. J Immunol Methods 224: 111-127, 1999[ISI][Medline].

31.   Witthoft, T, Eckmann L, Kim JM, and Kagnoff MF. Enteroinvasive bacteria directly activate expression of iNOS and NO production in human colon epithelial cells. Am J Physiol Gastrointest Liver Physiol 275: G564-G571, 1998[Abstract/Free Full Text].

32.   Wu, J, Song Y, Bakker ABH, Bauer S, Spies T, Lanier LL, and Phillips JH. An activating immunoreceptor complex formed by NKG2D and DAP10. Science 285: 730-732, 1999[Abstract/Free Full Text].

33.   Yamamoto, M, Fujihashi K, Kawabata K, McGhee JR, and Kiyono H. A mucosal intranet: intestinal epithelial cells down-regulate intraepithelial, but not peripheral, T lymphocytes. J Immunol 160: 2188-2196, 1998[Abstract/Free Full Text].

34.   Yang, S-K, Eckmann L, Panja A, and Kagnoff MF. Differential and regulated expression of c-x-c, c-c, and c-chemokines by human colon epithelial cells. Gastroenterology 113: 1214-1223, 1997[ISI][Medline].

35.   Yio, XY, and Mayer L. Characterization of a 180-kDa intestinal epithelial cell membrane glycoprotein, gp180. J Biol Chem 272: 12786-12792, 1997[Abstract/Free Full Text].


Am J Physiol Gastrointest Liver Physiol 278(6):G886-G894
0193-1857/00 $5.00 Copyright © 2000 the American Physiological Society