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(Journal of Leukocyte Biology. 2002;72:339-346.)
© 2002 by Society for Leukocyte Biology

Nitric oxide reduces bacterial superantigen-immune cell activation and consequent epithelial abnormalities

Alisa Rachlis, James L. Watson, Jun Lu and Derek M. McKay

Intestinal Disease Research Programme, McMaster University, Hamilton, Ontario, Canada

Correspondence: Derek M. McKay, Ph.D., Intestinal Disease Research Programme, HSC-3N5C, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5. E-mail: mckayd{at}fhs.mcmaster.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibition of the inducible form of nitric oxide (NO) synthase prolonged the murine enteropathy evoked by the bacterial superantigen, Staphylococcus aureus enterotoxin B (SEB). We examined the ability of NO to alleviate SEB-induced epithelial dysfunction and immune cell activation. Human peripheral blood mononuclear cells (PBMC) were activated by SEB for 24 h ± the NO donors, S-nitroso-N-acetylpenicillamine and spermine-NONOate. The conditioned medium (CM) was collected and applied to T84 epithelial monolayers, and permeability [i.e., transepithelial resistance (TER)] and stimulated ion transport (i.e., short-circuit current responses to carbachol and forskolin) were assessed 24 h later. Exposure to CM led to an ~40% drop in TER and hyporesponsiveness to both secretagogues. CM made in the presence of NO donors (10-4 M) had no significant effect on epithelial barrier or ion transport parameters. NO donors alone had no effect on naive epithelia, and addition of the NO donors to previously made CM did not affect the ability of this CM to alter epithelial function. Moreover, the NO donors dose-dependently reduced SEB-evoked PBMC proliferation and cytokine production (i.e., interferon-{gamma}, tumor necrosis factor {alpha}) but did not affect viability. These findings suggest a beneficial role for NO in inflammation by reducing immune cell activation and thus ameliorating consequent physiological abnormalities, in this instance, perturbed epithelial permeability and active ion transport.

Key Words: SNAP • conditioned medium • PBMC • transepithelial resistance


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial superantigens (SAgs) are potent T cell stimuli that act by binding as an unprocessed protein to domains of the major histocompatibility complex class II molecule and the Vß region of the T cell receptor outside of the antigen-specific groove [1 ]. This can result in activation of up to 25% of T cells, leading to significant cytokine synthesis, which often follows the pattern of increases in tumor necrosis factor {alpha} (TNF-{alpha}), interferon-{gamma} (IFN-{gamma}), and interleukin (IL)-2, followed by IL-10 [2 , 3 ]. SAgs have been implicated in a number of inflammatory and autoimmune diseases, particularly psoriasis and multisystem vasculitis [4 ]. There has been, until recently, only fragmentary evidence in support of SAg participation in enteric inflammation and/or epithelial dysfunction [5 ]. However, a novel bacterial SAg has been identified in a cohort of patients with Crohn’s disease [6 ], and the putative role for SAgs in the initiation or exaggeration of enteric inflammation has recently been reviewed [7 ].

Using Staphylococcus aureus enterotoxin B (SEB) as a model SAg, we showed that immune cell activation in a coculture model composed of monolayers of the human colonic T84 epithelial cell line and peripheral blood mononuclear cells (PBMC) resulted in diminished epithelial ion transport responsiveness and increased permeability [8 ]. Similarly, systemic administration of SEB to mice led to a self-limiting enteropathy characterized by a reduced ability of colonic and jejunal epithelium to respond to pro-secretory stimuli [9 , 10 ]. In the latter instance, nitric oxide (NO) appeared to be necessary for the recovery of normal secretory responses, as administration of an inhibitor of inducible NO synthase (iNOS) prolonged the epithelial hyporesponsiveness [11 ].

NO has been the subject of extensive research efforts, and yet the role of this molecule in inflammation and the regulation of epithelial function is controversial. Opinion is divided, with advocates for NO being a proinflammatory molecule and equally convincing data being presented in favor of NO exerting anti-inflammatory influences in a variety of model systems [12 , 13 ]. Similarly, in terms of the regulation of enteric epithelial ion transport, the paradoxical situation exists where NO has been found to be pro-secretory and pro-absorptive [14 ]. Based on our analysis of SEB-induced murine enteropathy, we hypothesized that NO would be beneficial in reducing or preventing the epithelial ion transport and barrier abnormalities evoked by SAg-immune cell stimulation.

To test this postulate, we conducted in vitro studies in which T84 epithelial monolayers were cultured with conditioned medium (CM) from PBMC activated by SEB in the presence or absence of NO donors. Epithelial function was subsequently assessed in Ussing chambers. The data presented herein show that the NO donors, S-nitroso-N-acetylpenicillamine (SNAP) and spermine-NONOate, significantly inhibit SEB-induced PBMC proliferation and cytokine production (i.e., IFN-{gamma} and TNF-{alpha}) in the absence of any significant cytotoxic effects and that this suppression of immune activity was accompanied by a reduced ability of the SAg-exposed immune cells to affect epithelial ion transport and barrier functions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and experimental design
T84 cells (human colonic crypt-like epithelial cells) were maintained in growth medium composed of equal parts Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium supplemented with 10% fetal calf serum, 10% (vol:vol) HEPES, and 2% (vol:vol) penicillin-streptomycin (all from Invitrogen/Life Technologies, Grand Island, NY). T84 cells were seeded (106) onto tissue culture-treated, semipermeable transwell filter supports (0.4 µm pore size, 1 cm2 surface area; Costar Inc., Cambridge, MA) in 12-well plates and cultured for 7 days (37°C, 5% CO2), at which time monolayers consistently displayed transepithelial resistances of >=1000 {Omega}•cm2 [8 ]. PBMC were isolated from the buffy coat interface after ficoll density centrifugation (Ficoll-Paque®, Amersham Pharmacia Biotech, Piscataway, NJ) of blood from healthy volunteers (male and female, age range, 20–50 years, excluding those with illness). Cells were resuspended in culture medium at 106/ml and used within 30 min of isolation. We have reported that PBMC isolated by this procedure consist of ~75% CD3+ T cells and 5–15% CD14+ monocytes, with the remainder B cells (based on size and granularity), as determined by fluorescence-activated cell sorter analysis [15 ].

PBMC (106 cells/ml) were treated as follows: 1) no treatment (i.e., nonactivated); 2) exposed to the NO donors SNAP (Research Biochemical International, Natick, MA) and spermine-NONOate (Calbiochem-Novabiochem Corp., La Jolla, CA) at 10-4–10-6 M (SNAP has a half-life of 5 h, releasing one molecule of NO per molecule of donor, and spermine-NONOate has a half-life of 39 min and releases two molecules of NO per molecule of donor); activated with SEB (1 µg/106 PBMC; Sigma Chemical Co., St. Louis, MO; 3) 1 µg/ml was identified as an optimal dose of SEB in our previous dose-response studies [8 ]); 4) activated with SEB in the presence of the NO donors added 1 h before or 1 h or 4 h after SEB addition to the PBMC. The PBMC were exposed to SEB (±NO donors) for 24 h, and the cell-free CM was collected after centrifugation (1000 rpm for 10 min). CM was stored at -70°C prior to use in physiological studies and cytokine determinations. In some experiments, NO donors (10-4 M) were subsequently added to CM, and the ability of this "spiked" CM to affect epithelial function was assessed. Nitrite/nitrate levels in CM were measured by the Griess reaction following a published methodology [16 ].

In additional studies, PBMC were separated into plastic adherent (i.e., monocytes) and nonadherent (i.e., T and B cells) cells by overnight incubation at 37°C in 6-well plates. The adherent cells were rinsed (x3) in media and pretreated with NO donors for 1 h (10-4 M). Simultaneously, the nonadherent cells were fixed by a 30-min exposure to 4% paraformaldehyde, rinsed (x3), resuspended in culture media, and then added back onto the adherent monocytes from the appropriate blood donor ± SEB at a final concentration of 1 µg/ml. The CM was collected 24 h later.

In a final series of experiments, PBMC (106/ml) were activated with the mitogen concanavalin A (Con A; 1 µg/ml) or Escherichia coli-derived lipopolysaccharide (LPS; 10 ng/ml; both from Sigma Chemical Co.) ± NO donors (both at 10-4 or 10-5 M), and the CM was collected 24 h later.

Epithelial permeability and ion transport
Filter-grown T84 monolayers were cultured with 25% conditioned media (diluted with fresh medium and added into the basal compartment of the transwell plate) from the various PBMC cultures for 24 h at 37°C and were then mounted into modified Ussing chambers [8 ]. Additional experiments were conducted in which confluent T84 monolayers were exposed to the NO donors only (10-5 or 10-4 M; placed in the basal compartment of the culture well) for 24 h.

Permeability
Transepithelial resistance (TER in {Omega}•cm2) reflects the barrier property to passive ion movement that we and others have used as an accepted index of paracellular permeability [8 , 17 ]. TER was measured in the transwell before and after the 24-h experimental period using a voltmeter and chopstick electrodes (Millicel-ERS, Millipore, Bedford, MA), and the percent increase or decrease was calculated.

Ion transport
Briefly, T84 monolayers were mounted in Ussing chambers (Medical Research Apparatus, Clearwater, FL) and bathed in oxygenated Krebs buffer [8 ]. The spontaneous, potential difference across the epithelium was maintained at 0 V by an automated voltage clamp (WPI Instruments, Narco Scientific, Missisauga, Ontario, Canada), and the injected short-circuit current (Isc in µA/cm2) required to maintain 0 V was continuously monitored as an indication of net active ion transport. Baseline Isc was recorded after a 15-min equilibration period. The maximum change in Isc to occur within 10 min of addition of forskolin (FSK; 10-5 M) and then the cholinergic agonist carbachol (CCh; 10-4 M added 30 min after FSK; both from Sigma Chemical Co.) to the buffer bathing the serosal side of the epithelia was recorded [8 ]. These secretagogues raise intracellular Ca2+ and cyclic adenosine monophosphate, respectively, and evoke electrogenic chloride secretion from T84 cells.

PBMC proliferation and cytokine synthesis
[3H]-Thymidine incorporation
PBMC were plated into sterile 96-well plates (105 cells/well) ± SEB (0.1 µg/well) ± NO donors and were incubated at 37°C for 72 h. Subsequently, each well was pulsed with 1 µCi [3H]-thymidine (DuPont-New England Nuclear, Wilmington, DE), and 18 h later, the cells were harvested onto glass-fiber filters and radioactivity was determined in a scintillation counter (Becton Dickinson, Mississauga, Ontario, Canada). All determinations were performed in quadruplicate.

Cytokine production
TNF-{alpha} and IFN-{gamma} were measured by enzyme-linked immunosorbent assay (ELISA) in nonactivated CM and in CM from SEB-activated PBMC, Con A-activated PBMC, and LPS-activated PBMC (DuoSet, ELISA Development System, R&D Systems, Inc., Minneapolis, MN) ± NO donors. Both assays had a sensitivity of 8 pg/ml, and cytokine determinations were performed in duplicate serial dilutions. The use of neutralizing antibodies has shown that these cytokines are important in this model of immune-mediated epithelial dysfunction [8 ].

Viability analysis
All PBMC were >95% viable immediately after purification. Following SEB ± NO donor treatment, PBMC viability was determined by trypan blue dye exclusion, measurement of lactate dehydrogenase (LDH) in the CM [8 ], and Western blotting for poly ADP ribose polymerase (PARP), an end-stage enzyme in apoptosis [18 ]. Briefly, whole-cell lysates were prepared by standard procedures and samples (20–30 µg protein), electrophoresed through 4–10% (29:1 acrylamide/bisacrylamide) sodium dodecyl sulfate gels. Separated proteins were electroblotted to immobilon nitrocellulose membrane (Millipore), 100 V for 1.5 h at 4°C, and were blocked in 5% low-fat Carnation powdered milk/Tris-buffered saline/Tween 20 (0.1% Tween) for 1 h. Washed blots were incubated overnight with a mouse anti-PARP monoclonal antibody (1:1000; BD PharMingen, San Diego, CA), washed, then incubated with goat anti-mouse immunoglobulin G conjugated to horseradish peroxidase (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. Following washing, the immunoreactive proteins were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech) and by exposing the membrane (20 s–5 min) to Kodak XBL film.

Statistical analysis and data presentation
Data are presented as mean ± SEM. When appropriate, data were normalized against time-matched control epithelial preparations or in the case of TNF-{alpha} and IFN-{gamma}, presented as the percent increase compared with nonactivated PBMC from the same blood donor. N values are defined as the number of experiments (i.e., number of blood donors) in which two to three T84 monolayers were examined per condition. Data were analyzed by Student’s paired t-test (for cytokine levels) or one-way ANOVA, followed by Newman-Keuls multiple comparisons test (for epithelial physiology) and a level of statistically significant difference accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Nitrite/nitrate levels in CM from PBMC cultures exposed to SEB + NO donors were significantly greater than in media from PBMC + SEB only, which in turn was slightly but statistically and significantly greater than medium retrieved from nonactivated PBMC: Nonactivated PBMC = 1.5 ± 0.1 µM, PBMC + SEB = 6.2 ± 0.2 µM*, and PBMC + SEB + NO donors = 238.2 ± 19.1{dagger} µM nitrite/nitrate (n=3; *<0.05; {dagger}<0.0001). These data confirm activity of the NO donors.

NO reduces SEB-immune, cell-mediated, epithelial functional abnormalities
Permeability
TER across T84 monolayers cultured in 25% CM from nonactivated PBMC or nonactivated PBMC + NO donors for 24 h was not significantly different from pretreatment TER values (n=3; data not shown). In contrast, exposure to CM from SEB-activated PBMC consistently resulted in ~40% drop in TER. SEB-CM made in the presence of NO donors was unable to disrupt epithelial barrier function: NO donors (10-4 M), added as a pre- or post-treatment, completely prevented any change in T84 TER (Fig. 1 ; n=3–5). A 1 h pretreatment with 10-6 M did not affect the SEB-CM-induced drop in TER, whereas 10-5 M NO donor pretreatment did result in a small, but statistically significant improvement in TER (data not shown).



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  		Figure 1. NO prevents epithelial barrier dysfunction caused by exposure to CM from SEB-activated immune cells. Graph shows the change in T84 TER 24 h after culture with 25% CM from SEB-activated PBMC (SEB CM) ± NO donors (10-4 M; SEB CM + NO-d) added as a pre (1h pre-T)- or post-treatment (1h or 4h post-T; mean±SEM; *, P<0.05 compared with pretreatment TER values; n=3–5 experiments; 2–3 T84 monolayers/condition per experiment; TER pretreatment values ranged from 1000 to 2150 {Omega}•cm2).

 
In separate studies, culture with NO donors only (i.e., no CM) for 24 h did not significantly affect TER, with 10-5 M and 10-4 M NO donors causing ~2% drop and ~15% increase in TER compared with pretreatment values, respectively (n=4; see also Table 1 ). Similarly, the addition of NO donors to CM did not alter the ability of the CM to elicit a drop in epithelial TER and reduce secretory responsiveness (Table 1) . Both pieces of data indicate that the liberated NO affected the immune cells and not the epithelium directly.


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  		Table 1. Addition of NO Donors to Conditioned Medium Does Not Affect Its Ability to Alter Epithelial Function

 
Ion transport
Exposure to nonactivated PBMC CM or NO donors only for 24 h affected neither CCh- nor FSK-evoked Isc responses in T84 monolayers (data not shown; n=3). In contrast and consistent with our previous publications [8 ], T84 monolayers cultured for 24 h with SEB-CM displayed significantly reduced Isc responses to both secretagogues, which were completely normalized if the CM was obtained from SEB-activated PBMC that had first received a 1-h pretreatment with NO donors (10-4 M; Fig. 2 ; n=3–5); lower doses of NO donors did not affect the response (Fig. 2) . Addition of NO donors 1 h after SEB treatment of PBMC resulted in a CM with a significantly ablated ability to cause reduced epithelial Isc responsiveness to CCh and FSK (Fig. 3 ). In contrast, PBMC exposed to NO donors 4 h after SEB produced a CM, which when added to T84 monolayers for 24 h, did result in a significantly reduced response to CCh (Fig. 3A) . However, this 4 h post-treatment, NO-donor regimen was sufficient to prevent the reduced response to FSK (n=4; Fig. 3B ).



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  		Figure 2. NO reduces the T84 epithelial ion transport abnormalities caused by exposure to CM from SEB-activated immune cells. Secretory responses to (A) carbachol (10-4 M) and (B) forskolin (10-5 M) were observed 24 h after culture with a 25% CM ± NO donors added to the PBMC 1 h before SEB exposure (mean±SEM; data are normalized to responses in time-matched, naïve, control T84 monolayers; control {Delta}Isc range, carbachol=58–110 µA/cm2; forskolin=60–80 µA/cm2; *, P<0.05 compared with naïve controls; n=3–5 experiments; 2–3 T84 monolayers/condition per experiment).

 


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  		Figure 3. NO inhibition of T84 epithelial ion transport abnormalities is time-dependent. Bar graphs show the effects of a 24-h culture with CM ± NO donors (10-4 M) added to the PBMC 1 h or 4 h after SEB (i.e., post-treatment) on changes in Isc evoked by (A) carbachol (10-4 M) and (B) forskolin (10-5 M) (mean±SEM; *, P<0.05 compared with naïve, time-matched controls; n=4 experiments; 2–3 epithelial monolayers/condition per experiment).

 
NO inhibits PBMC proliferation
SEB treatment led to a significant increase in PBMC proliferation that was significantly and dose-dependently inhibited by a 1-h pretreatment with the NO donors (Fig. 4 ; n=3–5). Furthermore, SEB-induced PBMC proliferation was significantly reduced when the NO donors were introduced to the culture 1 h or 4 h after the SEB (Fig. 4) .



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  		Figure 4. NO inhibits SEB-induced PBMC proliferation. NO donors (NO-d) were added as a 1-h pretreatment (pre-T.) or a 1- or 4-h post-treatment (post-T.) to the PBMC + SEB cultures (mean±SEM; n=3–5; * and #, P<0.05 compared with control and SEB-only, respectively).

 
NO reduces SEB-stimulated PBMC secretion of TNF-{alpha} and IFN-{gamma}
Nonactivated PBMC showed negligible, spontaneous production of TNF-{alpha} and IFN-{gamma}. There was significant variability between stimulated cytokine production from individual PBMC donors. Nevertheless, by 6 h post-SEB treatment, both cytokines were increased [TNF-{alpha}=1168±517 and IFN-{gamma}=565±291 pg/ml (n=3)]. Levels of both cytokines were further increased in CM-collected, 18 h, post-SEB treatment [TNF-{alpha}=4139±973 and IFN-{gamma}=2715±1265 pg/ml (n=3)], and there were no further increases at 24- or 30-h post-SEB treatment (data not shown). Figure 5 shows that the SEB-stimulated cytokine production was dose-dependently reduced by a 1-h pretreatment of the PBMC with NO donors. In fact, 10-4 M NO donors completely blocked the ability of SEB to elicit TNF-{alpha} or IFN-{gamma} synthesis, independent of whether the NO donor was given as a 1 h pre- or post-treatment (n=4–6). The same dose of NO donors, when added to the PBMC cultures 4 h after SEB challenge, reduced the levels of cytokine detectable in the CM 18 h later by 70–80%.



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  		Figure 5. NO inhibits SEB-induced PBMC TNF-{alpha} and IFN-{gamma} production. Cytokine levels in medium were measured by ELISA 18 h after exposure to SEB ± NO donors (NO-d.) added as a 1-h pretreatment (pre-T.) or a 1- or 4-h post-treatment (post-T.) to the PBMC + SEB cultures (mean±SEM; n=4–6; *, P<0.05 compared with PBMC+SEB).

 
Finally, the presence of NO donors (i.e., 1-h pretreatment) dose-dependently reduced PBMC production of TNF-{alpha} and IFN-{gamma} in response to Con A and LPS (Table 2 ; n=3).


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  		Table 2. NO Donors Reduce Cytokine Secretion by Con A- and LPS-Activated PBMC

 
TNF-{alpha} is not produced in the absence of functional T cells in the PBMC
Fixed T and B cells were recombined with the adherent monocytes ± SEB for 24 h, and the CM was collected. Under these conditions, TNF-{alpha} was undetectable in CM from nonactivated cells only or the recombined PBMC, independent of whether they were activated with SEB ± NO donors (n=4).

NO does not decrease PBMC viability
Trypan blue dye exclusion showed that PBMC viability was not affected by 24 h exposure to the NO donors only (10-6–10-4 M), was slightly increased by SEB treatment, and there was no additive or synergistic effect of NO donors + SEB cotreatment. Thus, at the highest dose of NO donors, PBMC trypan blue positivities after 24 h of culture were 3.0 ± 0.8, 3.9 ± 0.7, 5.5 ± 1.2, and 5.9 ± 1.8% for control, NO donor only (10-4 M), SEB only, and NO donors + SEB, respectively. Similarly, the level of LDH in CM was not significantly increased by NO donor + SEB treatment (data not shown; n=2). Finally, Western blotting for PARP, an enzyme cleaved following caspase activation, revealed no significant increase of the cleaved p85 form of the molecule in extracts from NO donors + SEB-treated PBMC compared with SEB-only-treated cells (Fig. 6 ).



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  		Figure 6. NO does not lead to a significant increase in PBMC death. Representative Western blot showing intact PARP (p116) and the p85 cleavage product (indicative of apoptosis) in extracts of PBMC (from two separate donors) treated with SEB ± NO donors for 24 h [positive control, verotoxin 1 (+VE; 10 ng/ml, 8 h)-treated HEp2 cell extract; negative control (-VE), HEp2 cell extract]. No obvious differences occur between the treatment groups.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Representing the body’s first line of defense, the barrier and ion transport functions of the enteric epithelium are important components of innate immunity. These roles of the epithelium are influenced by immune cells and characteristically perturbed in many conditions including inflammatory bowel disease [15 , 19 , 20 ]. We adopted a reductionist approach to explore immune modulation of epithelial permeability and ion transport in vitro by culturing T84 epithelial monolayers with human immune cells. Using this model, we reported that polycolonal T cell stimulation by bacterial SAgs resulted in significant impairment of the barrier property of the epithelium and reduced its ability to respond to prosecretory stimuli [8 , 21 ]. The data from the current study indicate a protective role for NO, as inclusion of NO donors dampened the immune response and completely prevented SEB-driven epithelial dysfunction.

Proinflammatory and anti-inflammatory roles for NO have been presented with equal conviction [12 ]. It is likely that the role of NO is organ/system-specific and will depend on many variables including whether the NO donors or inhibitors of NO synthesis are delivered systemically or locally [22 ]. Having defined the effects of SEB-immune cell activation on T84 epithelial function, we turned to this in vitro model to specifically address the impact of NO on immune-mediated disruption of epithelial permeability and stimulated active ion transport. We have found that SEB-CM from PBMC that were activated in the presence of NO donors (10-4 M) had no discernable effects on epithelial permeability or ion transport. Moreover, this was independent of whether the NO donors were added as a 1-h pretreatment or 1 h or 4 h after the SEB addition to the PBMC cultures [with the exception of the {Delta}Isc to CCh (Fig. 3A) , which likely reflects subtle differences in the sensitivity of the ion transport mechanism(s) to the mixed mediator milieu produced by SEB-evoked immune cell activation]. These findings are compatible with SEB-induced murine enteropathy, where inhibition of iNOS activity prolonged the diminished secretory capacity of jejunal segments examined ex vivo [11 ] and are complementary to other studies suggesting that NO is protective against toxic shock. For instance, protection against TNF-{alpha}-induced lethal shock required functional iNOS [23 , 24 ], E. coli LPS-induced jejunal damage was reduced by cotreatment with the NO donor SNAP [25 ], and inhibition of NO synthesis resulted in the increased mortality of SEB-treated mice [26 ].

Epithelial cells have been identified as target and source of NO [27 28 29 30 ]. NO has been implicated and refuted as a cause of increased permeability in tissue [31 , 32 ] and epithelial monolayers [33 , 34 ]. Similarly, NO has been suggested as a cause of the ion transport hyporesponsiveness that accompanies chemical-induced colitis and radiation-induced epithelial dysfunction in mice [35 , 36 ] and, conversely, in stimulating active chloride secretion [37 ]. In the present study, we found that T84 monolayers assessed in Ussing chambers 24 h after exposure to the NO donors only were not functionally different from naïve monolayers. Also, spiking CM with NO donors did not ameliorate the effects of the CM on epithelial barrier and ion transport functions. These data suggested that the NO donors affected immune cell activation and not the epithelial layer directly. Subsequent analyses revealed that NO donors dose-dependently inhibited SEB-evoked PBMC proliferation and the production of TNF-{alpha} and IFN-{gamma}—key cytokines in this model of SAg-immune cell-mediated epithelial dysfunction [8 ], and cytokines known to directly affect epithelial ion transport and permeability [15 , 17 , 28 ]. Indeed, CM, which contained no detectable TNF-{alpha} and IFN-{gamma}, was ineffective in altering epithelial function. These data are consistent with the observation that in vivo administration of the NO donor, isosorbide dinitrate, blocked SEB-induced T cell proliferation [38 ]. Also, using a murine model of toxic shock, Kumins et al. [39 ] showed that those animals treated with the NO donor, molsidomine, had reduced serum TNF-{alpha} levels. Similarly, iNOS-deficient mice infected with S. aureus have an exaggerated polarization of the T helper-1 (Th1) cell response compared with control littermates [40 ]. Collectively, these studies underscore the potential of NO to minimize Th1-dominated events.

Using the mixed PBMC population allows the possibility that the NO donors could affect the T cells or the antigen-presenting cells (APC). Addressing this, we first fixed the T cells, preserving their surface structures while preventing activation, then added SEB to the recombined PBMC, and subsequently measured TNF-{alpha}, as monocytes will not make IFN-{gamma}. Under these conditions, TNF-{alpha} was undetectable in CM from nonactivated cells or those activated by SEB ± NO donors. Although this does not unequivocally exclude the APC as a target for NO effects, it does underscore the need for functional T cells in this model and the likelihood that at least part of the beneficial effects of NO in this model system are mediated through direct action on the T cells.

In addition, the NO donors also dose-dependently inhibited PBMC cytokine synthesis evoked by Con A or E. coli LPS. These data are not unprecedented: NO has been shown to reduce the stimulated synthesis of TNF-{alpha}, IFN-{gamma}, IL-1, and IL-3 in other in vitro culture systems [41 42 43 ]. Thus, we speculate that NO at the site of inflammation may be generally protective via an ability to dampen immune cell activation and cytokine production. Indeed, in our earlier report, we found that intraperitoneal delivery of the NO donor SNAP did not ablate the jejunal ion transport abnormalities evoked by SEB [11 ], supporting the contention that anti-inflammatory effects of NO may occur with local rather than systemic delivery.

One interpretation of the data presented here is that these levels of NO are toxic to the immune cells; however, we have no evidence to support this. All three indices of cell viability used (i.e., trypan blue dye exclusion, release of LDH, PARP cleavage) failed to show any significant differences between immune cells activated with SEB in the presence or absence of the NO donors. Similarly, decreased cell viability has not been identified as significant in other studies where NO inhibition of immune cell cytokine synthesis was reported [41 , 43 , 44 ]. Thus, another mechanism to explain NO inhibition of cytokine production must be sought. NO may induce an anergic response, and preliminary studies from our laboratory show that TNF-{alpha} and IFN-{gamma} production by PBMC in response to SEB is reduced by 80% in cells treated 48 h earlier with the NO donors (personal observance). Moreover, NO nitrosates and affects the activity of many proteins and thus a multitude of possible target candidates exist. Given the requirement for SAgs to bind the T cell receptor, NO interference with signaling from the T cell receptor needs to be addressed. Also, NO has been shown to affect nuclear factor-{kappa}B and Jak3/STAT5 signaling [44 , 45 ], and obstruction of these or related intracellular pathways could mediate the effect of NO. All of these possibilities remain to be rigorously tested.

Finally, independent of the exact intracellular mechanism, our data show that NO can inhibit SEB-evoked immune cell cytokine synthesis and that one physiological consequence of this is amelioration of the perturbed epithelial ion transport and barrier functions induced by exposure to SEB-activated immune cells. Our findings add to a growing body of evidence that support the contention that targeted delivery of NO to the site of inflammation would exert an immunosuppressive influence, potentially limiting the extent of the inflammation and concomitant abnormalities, such as increased epithelial permeability and perturbed ion secretion.


   ACKNOWLEDGEMENTS
 
Funding was provided by a Canadian Institutes Health Research (CIHR) operating grant (MT-13421) to D. M. M., a CIHR scholar. We thank Dr. M. Akhtar (McMaster University) for assistance with aspects of this work and J. C. Y. Ching (University of Toronto), who provided control protein extracts for the PARP Western blot.

Received January 19, 2002; revised March 13, 2002; accepted March 14, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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I. V. Pinchuk, E. J. Beswick, J. I. Saada, G. Suarez, J. Winston, R. C. Mifflin, J. F. Di Mari, D. W. Powell, and V. E. Reyes
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