Alcohol Research Program, Burn and Shock Trauma Institute, Department of Surgery, Loyola University Chicago Medical Center, Maywood, IL 60153
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ABSTRACT |
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The mechanism of
alcohol-mediated increased infection in burn patients remains unknown.
With the use of a rat model of acute alcohol and burn injury, the
present study ascertained whether acute alcohol exposure before thermal
injury enhances gut bacterial translocation. On day 2 postinjury, we found a severalfold increase in gut bacterial
translocation in rats receiving both alcohol and burn injury compared
with the animals receiving either injury alone. Whereas there were no
demonstrable changes in intestinal morphology in any group of animals,
a significant increase in intestinal permeability was observed in
ethanol- and burn-injured rats compared with the rats receiving either
injury alone. We further examined the role of intestinal immune defense
by determining the gut-associated lymphoid (Peyer's patches and
mesenteric lymph nodes) T cell effector responses 2 days after alcohol
and burn injury. Although there was a decrease in the proliferation and interferon- by gut lymphoid T cells after burn injury alone; the
suppression was maximum in the group of rats receiving both alcohol and
burn injuries. Furthermore, the depletion of CD3+
cells in healthy rats resulted in bacterial accumulation in mesenteric lymph nodes; such CD3+ cell depletion in alcohol- and
burn-injured rats furthered the spread of bacteria to spleen and
circulation. In conclusion, our data suggest that the increased
intestinal permeability and a suppression of intestinal immune defense
in rats receiving alcohol and burn injury may cause an increase in
bacterial translocation and their spread to extraintestinal sites.
T lymphocyte; infection immunity bacteria; inflammation; shock
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INTRODUCTION |
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DESPITE ADVANCES IN THE INTENSIVE care units, infection remains the leading cause of multiple organ failure following burn, trauma, and hemorrhage (14, 17). The management of these patients becomes much more difficult when these injuries are accompanied with prior alcohol consumption (27, 33, 37, 46, 49). Nearly 50% of all burn/trauma patients are shown to have alcohol in their blood. A number of previous studies have shown that prior alcohol exposure contributes significantly to the complications associated with burn and trauma (27, 33, 46). These patients exhibit higher incidence of infection and are likely to die compared with the patients with no alcohol exposure (39, 53). Similarly, alcohol consumption by experimental animals also results in increased susceptibility to infection following burn injury (7, 16, 28, 39). The mechanism of such alcohol-mediated increased incidence of infection and associated mortality remain unknown.
A few studies have indicated that hospitalized patients often succumb to opportunistic pathogens originating from their own gastrointestinal (GI) tracts (11, 12, 40, 50). The GI tract is normally very effective in keeping bacteria in the lumen. However, injury conditions such as burn or trauma may disrupt the normal intestinal barrier functions and thus may allow increased bacterial infiltration to mesenteric lymph nodes (MLN) and other extraintestinal sites including spleen, liver, lung, and blood (11-13, 40, 48). This passage of bacteria from the GI tract to extraintestinal sites has been termed bacterial translocation. Three major factors could contribute to bacterial translocation: 1) physical disruption of mucosal barrier, 2) intestinal overgrowth of bacteria, and 3) suppression of the immune defense.
A major complication in burn patients is their inability to mount an
appropriate host response to invading pathogens (3, 5, 6, 14, 23,
29, 36, 41, 51). Both clinical and experimental evidence
suggests that burn injury results in a state of immunosuppression. This
suppression is characterized by a decrease in splenic and peripheral
blood T cell proliferation (5, 6, 23, 29, 41, 51) and
macrophage dysfunction (36). Similar changes are noted in
cells of the immune system after acute and chronic alcohol consumption
(10, 24-26, 54, 56). Although clinical evidence
showing enhanced morbidity and mortality in burn patients with prior
alcohol exposure compared with patients with no alcohol exposure is
overwhelming, only a few studies have been carried out to address the
underlying mechanism. These studies have shown that acute alcohol
exposure before thermal injury produced a greater suppression of
mitogen-induced splenic-lymphocyte proliferation, serum immunoglobulin
levels, and neutrophil chemotaxis (28). In addition,
studies by Napolitano et al. (39) have suggested that
chronic alcohol exposure resulted in damage of both gut villi and
submucosal region. More recent studies, including our own (7,
16), suggest that mice receiving low doses of alcohol before
burn injury exhibit impaired delayed-type hypersensitivity, splenic T
cell proliferation, interleukin (IL)-2 production, and enhanced
susceptibility to infection. The damage in gut as shown by Napolitano
et al. (39) may contribute to enhanced bacterial translocation. However, the process of infection involves not only the
passage of bacteria from the GI tract to extraintestinal sites but also
survivability of translocated bacteria in the extraintestinal sites.
Thus the host immune defense becomes a much more critical component in
the bacterial translocation process. In the present, study we
investigated the effects of acute alcohol exposure before thermal
injury on intestinal integrity. Furthermore, we examined gut lymphoid
[mesenteric and Peyer's patches (PP)] T cell proliferation and
interferon (IFN)- production to determine whether alterations in
intestinal T cell effector functions are responsible for enhanced bacterial translocation.
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MATERIALS AND METHODS |
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Animals and reagents. Adult male Sprague-Dawley rats weighing 225-250 g were obtained from Harlan (Indianapolis, IN). The rats were maintained in accordance with the guidelines set forth by Loyola University Chicago Medical Center Animal Care and Use Committee. Concanavalin A (ConA) was purchased from Sigma (St. Louis, MO). Nylon wool was obtained from Polysciences (Warrington, PA). Reagents for cell culture were obtained from Biowhitaker (Walkersville, MD). Reagents for bacterial culture were purchased from Difco (Detroit MI). [3H]lactulose and [14C]mannitol were purchased from American Radiolabeled Chemicals (St. Louis, MO).
Rat model of acute alcohol and burn injury. We used the rat model of acute alcohol and burn injury as suggested by Kawakami et al. (28). Rats were randomly divided into four experimental groups: 1) sham + saline, 2) sham + alcohol, 3) burn + saline, and 4) burn + alcohol. In alcohol-treated groups, the levels of blood alcohol equivalent to 90-100 mg/dl were achieved by gavage feeding of 5 ml of 20% alcohol in saline. In some experiments, animals were also gavaged with 40% ethanol to achieve a blood alcohol level of 240-250 mg/dl to determine whether the absolute blood alcohol level was a determinate of the outcome. In saline groups, animals were gavaged with 5 ml saline. Four hours after alcohol gavaging, all animals except the control group were anesthetized with pentobarbital sodium (65 mg/kg). Hairs were shaved from their dorsal body surface. For burn procedure, animals were transferred into a template, which was fabricated to expose 25-30% of the total body surface area. Animals were then immersed in a hot water bath (95-97°C) for 10 s. Sham-burn animals were subjected to identical anesthesia and other treatments, except they were immersed in luke-warm water. The animals were dried immediately and given fluid resuscitation with ~10 ml physiological saline. Animals were allowed to recover from anesthesia and were returned to their cages. Animals were allowed food and water ad libitum.
Measurements of blood alcohol levels. Blood was drawn via cardiac puncture. The levels of blood alcohol were measured at various time points using the kit provided by Sigma.
Measurements of bacterial translocation. Two days (~48 h) after alcohol and burn injury, animals were anesthetized and killed. The abdominal cavity was exposed under aseptic conditions. Mesenteric lymph nodes, spleen, and blood were collected. The organs were weighed and homogenized in sterile glass tissue grinders (Fisher Scientific). Equal volume of blood (10 µl) or tissue homogenate (10 µl) from various experimental groups was cultured separately on Tryptic soy agar (Difco) plates. The agar plates were incubated for 24-48 h at 37°C for the growth of bacteria. Bacterial colony-forming units (CFU) were counted. If the plates did not show any bacterial growth up to 48 h, the organ was considered negative for the presence of bacteria.
Histochemical analysis of intestine. As described previously (13, 48), 1-cm-long pieces of small intestine were fixed in 10% formalin in PBS for 24 h and embedded in paraffin. These were then cut (4-5 µm) and mounted on glass slides. Using the standard hematoxylin-eosin staining procedure, the intestine sections were observed under the light microscope for changes in intestinal morphology and later photographed. For electron microscopy, intestinal rings (0.5-cm long) were fixed in a 4% glutaraldehyde solution and transferred to the Loyola University Medical Center Core Imaging Facility (www.meddean.luc.edu/cif) for further processing. Briefly, tissues were washed in buffer; postfixed in 1% osmium tetroxide and dehydrated in a graded series of alcohol followed by propylene oxide. Spurr low-viscosity resin (Electron Microscopy Sciences, Ft. Washington, PA.) was used for infiltration and embedding. Blocks were polymerized at 56°C overnight. Sections were cut at 1 µm and stained with 1% toluidine blue for light microscopic evaluation and orientation followed by thin sectioning at 80 nm with a diamond knife. Sections were stained with 5% uranyl acetate and Reynold's lead citrate, analyzed at 75 kV on a Hitachi H-600 transmission electron microscope, and photographed (×20,000 magnification).
Intestinal permeability. Rats' right femoral arteries were cannulated under anesthesia, using PE-50 tubing filled with heparin saline (10 U/ml), and midline laparotomy was performed (48). Renal artery and renal vein in both kidneys were ligated. A 20-cm-long ileum was isolated without damaging intestinal and mesenteric structures, and PE-10 tubing was cannulated into the isolated ileum from the proximal end. As described previously (48), trace quantities of [3H]lactulose and [14C]mannitol were mixed in 1 ml of saline and injected into the isolated ileum through the tubing. The abdominal wall was closed with suture. One milliliter of blood was collected at 90 min from a cannulated femoral artery. Plasma was collected and mixed with 5 ml of liquid scintillant (ICN Biomedical). [3H]lactulose and [14C]mannitol radioactivity were counted by using a two-channel liquid-scintillation counter (Beckman Coulter) and expressed as dpm.
T cell preparation. Rats were anesthetized, and via midline incision, the intestine was exposed. PP and MLN were removed aseptically. Isolated PP and MLN were gently crushed to prepare a single-cell suspension in Hanks' balanced salt solution (HBSS) supplemented with 10 mM HEPES and 50 µg gentamicin/ml. To obtain pure T cell preparation, cell suspensions were incubated with nylon wool-packed columns. These columns were prequiliberated with HBSS supplemented with 10 mM HEPES, 5% fetal calf serum (FCS), and 50 µg gentamicin/ml. The columns containing cells were incubated at 37°C for 50-60 min. T cells were obtained by eluting the columns with 30-40 ml HBSS at a flow rate of 1 drop/s.
T cell proliferation. For the measurements of T cell proliferation, mixed MLN and PP cells and isolated T cells were resuspended in RPMI 1640 supplemented with L-glutamine (2 mM), 2-mercaptoethanol (50 µM), HEPES (10 mM), gentamicin (50 µg/ml), and FCS (10%) at a density of 5 × 106 cells/ml. One hundred microliters of the cell suspensions were added to the wells of a 96-well plate (5). The cells were cultured at 37°C and in 5% CO2 in the presence or absence of ConA (5 µg/ml). After 66 h of culture, 0.5 µC [3H]thymidine were added to each well containing the cells. After an additional 6 h incubation, cells were harvested using a PHD cell harvester (Cambridge Technology, Watertown, MA). The incorporation of radiolabeled thymidine was quantified using a liquid-scintillation counter, and the data are expressed as dpm.
T cell IFN- production.
Nylon wool-purified T cells (5 × 105/well) were
cultured in a 96-well plate for 48 h in the presence of ConA at
37°C in the presence of 5% CO2. Supernatants were
harvested 48 h after culture. IFN-
in the supernatants was
measured using ELISA kits (Biosource International).
Depletion of T cells. Rats were intraperitoneally injected with a mixture of anti-CD4 (OX8; 5 mg/kg) and anti-CD8 (OX38; 5 mg/kg) to deplete the CD4 and CD8 T cells. These doses of antibodies were used to deplete CD4 and CD8 cells in previous studies (1). Immediately after the administration of antibody, rats were gavaged with saline or alcohol and underwent sham or burn injury. Rats were killed on day 2 postinjury. Blood was drawn via cardiac puncture. MLN and spleen were aseptically removed. The depletion of T cells was determined in circulation, spleen, PP, and MLN by counting CD3-positive cells using FITC-labeled antibodies to CD3 and flow cytometry.
Statistical analysis. These data, wherever applicable, are presented as means ± SE and were analyzed using ANOVA statistical program (Statistical Package for Social Sciences Software program, version 2.0; SigmaStat). A P value <0.05 between groups was considered as statistically significant.
The experiments described here were carried out in adherence to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. ![]() |
RESULTS |
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Circulating blood ethanol levels.
Two separate groups of rats (4 in each group) were gavaged
with a single dose of 5 ml of 20 and 40% alcohol. After 4 h, rats were killed by CO2 inhalation. Blood was drawn via cardiac
puncture, and serum levels of ethanol were measured. Administration of
5 ml of 20% alcohol resulted in a blood ethanol level in the range of
90-100 mg/dl, whereas the administration of 5 ml of 40% gave a
blood ethanol level in the range of 240-250 mg/dl. An additional series of experiments was performed to monitor the clearance of alcohol
from the bloodstream (Fig. 1). Rats were
killed at 0.5, 2, 4, 6, 8, and 24 h after alcohol administration,
and their blood alcohol levels were measured. An increase in blood
ethanol levels was noted within 30 min of alcohol administration. As
shown in Fig. 1, Rats gavaged with 5 ml of 20% alcohol achieved blood
ethanol levels in the range of 170-180 mg/dl, whereas 5 ml of 40%
alcohol resulted in values of 380-400 mg/dl within 30 min of
alcohol administration. Approximately 70-75% of the circulating
alcohol was metabolized within 8 h after administration and 100%
after 24 h. The sensitivity of the ethanol detection kit was 10 mg/dl; thus values <10 mg/dl were considered negative.
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Determination of bacterial number.
Bacterial numbers were determined by culturing blood and homogenates
prepared from MLN and spleen of various experimental groups. As shown
in Fig. 2, the number of bacteria
recovered in the MLN of sham animals was (1.5 ± 0.7 CFU).
Slightly more bacterial counts (3.8 ± 1.1) were noted in MLN of
sham animals receiving a single dose of alcohol. The number of bacteria
recovered from MLN of burn animals (10 ± 2.1) was significantly
higher (P < 0.05) compared with MLN of animals
receiving either sham or sham and alcohol injury. A severalfold
increase in bacteria (72 ± 17) was recorded in animals subjected
to combined alcohol and burn injury. No bacterial growth was noted in
the blood and in the homogenates prepared from spleens of animals from
any experimental group.
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Morphological analysis of intestine.
Animals were killed 2 days after alcohol and burn injury, and the
morphological analyses of intestines were carried out using both light
and electron microscopy. Photomicrographs of intestine from light
microscopy are presented in Fig. 3,
A (sham + vehicle), B (sham + ethanol),
C (burn + vehicle), and D (burn + ethanol). There was no evidence of any substantial damage to the villi
or submucosa of the intestine of animals following alcohol and burn injury (Fig. 3C) compared with sham animals (Fig. 3,
A or B). To further confirm these observations,
intestines from various experimental groups of animals were examined
under an electron microscope, and results from these analyses are
presented as Fig. 3, a (sham + vehicle), b
(sham + EtOH), c (burn + vehicle), and d (burn + EtOH). Similar to the light microscopy, there
were no demonstrable differences in the morphology of the intestines of alcohol- and burn-injured rats compared with the intestines of rats
receiving either burn + vehicle or sham injury. As can be seen in
the representative electron micrographs, there was no difference in the
tight junctions (shown by arrows) in the intestines of alcohol- and
burn-injured rats (Fig. 3d) compared with the intestine of
burn alone (Fig. 3c) as well as the intestines of sham-injured rats regardless of their treatment (Fig. 3, a
and b).
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Lactulose and mannitol permeability.
Figure 4 shows increases in plasma
concentrations of lactulose and mannitol after their infusion into the
intestinal lumen. Increases in plasma lactulose and mannitol
concentrations after their transfer from the intestinal lumen were not
significantly different in the sham group regardless of their
treatment. However, a significant (P < 0.05) increase
in plasma levels of lactulose and mannitol was observed on day
2 in burn rats receiving vehicle alone compared with sham animals.
A further increase in plasma levels of both lactulose and mannitol was
recorded in burn-injured rats receiving ethanol. However, the increase
in plasma lactulose was not found significantly different between burn
and burn + ethanol groups. In contrast, the plasma mannitol levels
in the burn + ethanol group of rats were significantly
(P < 0.05) higher compared with those observed in
burn-alone groups.
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Effect of alcohol exposure on intestinal T cell proliferation after
burn injury.
We determined ConA-mediated T cell proliferative responses both in
mixed as well as in nylon wool-purified MLN and PP cells. As shown in
Fig. 5, there was no change in the
proliferation of ConA-mediated mixed PP (Fig. 5A) and MLN
(Fig. 5B) cells obtained from sham animals receiving alcohol
compared with sham rats receiving saline. The proliferation of PP and
MLN cells obtained 2 days after burn injury was significantly decreased
compared with the cells derived from sham group of animals (Fig. 5,
A and B). The proliferation was further
suppressed in PP and MLN cells derived from rats receiving both alcohol
and burn injuries. In subsequent experiments, we purified T cells from
PP and MLN using nylon wool columns as described in MATERIALS AND
METHODS. Proliferative responses in nylon wool-purified T cells
were determined to delineate whether or not the observed ConA-mediated
proliferative disturbances in PP and MLN T cells were due to the
presence of other accessory cells. Results from these experiments are
shown in Fig. 5, C and D. Similar to mixed PP and
MLN cells, we found significantly more suppression in the proliferation
of isolated T cells from PP (Fig. 5C) and MLN (Fig.
5D) of rats receiving both alcohol and burn injury compared
with the T cells derived from rats receiving either injury alone. These
results suggest that ethanol exposure before burn injury exacerbates
the suppression of intestinal T cells.
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T cell IFN- production.
In these experiments, we examined the production of IFN-
by PP and
MLN T cells. As shown in the Table 2, the
levels of IFN-
produced by PP and MLN T cells obtained from sham
animals were not significantly different, regardless of their
treatment. The levels of IFN-
produced by PP and MLN T cells from
burn animals gavaged with saline were significantly suppressed
(P < 0.05) compared with the T cells from sham groups
of animals. The production of PP and MLN T cells IFN-
was further
suppressed in animals receiving both alcohol and burn injury. On
statistical analysis, the suppression of IFN-
production by PP and
MLN T cell of animals receiving alcohol and burn injury was found to be
significantly different (P < 0.05) from the T cells
obtained from burn animals receiving saline and sham-injured animals.
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Effect of T cell depletion on bacterial translocation.
To further substantiate the role of T cells in bacterial translocation,
more experiments were carried out in which we monitored bacterial
presence in MLN, spleen, and blood of animals depleted of T cells. The
depletion of T cells was monitored in circulation, spleen, PP, and MLN
by counting CD3-positive cells using FITC-labeled antibodies to CD3 and
flow cytometry. We selected CD3+ cells to monitor because
both CD4+ and CD8+ T cells also express CD3
receptor, and thus the depletion of CD4+ and
CD8+ may also result in the depletion of CD3+
cells. Furthermore, one potential problem in these experiments was that
the cells obtained from animals treated with anti-CD4 and anti-CD8
antibodies may not have any anti-CD4 and anti-CD8 binding sites
available for additional antibody binding. Thus flow cytometric
analysis of such cells with similar anti-CD4+ and
-CD8+ antibodies may not correctly represent the status of
cells present. By using anti-CD3 antibody, which recognizes different
receptors on T cells, we have overcome this problem. The presence of
CD3-positive cells in blood, spleen, PP, and MLN is shown in Table
3. We found that ~48% cells were CD3
positive in PP, 70% in MLN, 35% in spleen, and 54% were positive in
blood of the normal rats. Intraperitoneal administration of a mixture
of anti-CD4+ and anti-CD8+ antibodies in normal
healthy rats resulted in the depletion of control CD3+
values by 50% in PP and MLN, 80% in spleen, and 90% in blood (Table
3). As can be seen in the Table 3, such depletion of T cells caused
accumulation of bacteria in MLN of healthy rats. A significant increase
in bacterial numbers was observed in MLN of alcohol- and burn-injured
rats, which were depleted of T cells (Table
4). Furthermore, blood cultures from the
CD3-depleted animals receiving both alcohol and burn injury were
positive for bacterial growth. Blood cultures from other groups of rats
were found negative.
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DISCUSSION |
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This study demonstrates that ethanol exposure before thermal injury results in a severalfold increase in bacterial translocation. Whereas no demonstrable morphological changes in the intestine were noted in any group of animals, a significant increase in intestinal permeability was observed in ethanol- and burn-injured rats compared with the rats receiving either injury alone. Intestinal permeability was determined by monitoring the transfer of radiolabeled lactulose and mannitol into the blood after their infusion into a segment of the intestine. Although, in this study, we have not investigated the mechanism of lactulose and mannitol transfer into the bloodstream after their infusion into the intestinal lumen, others (48, 55) have shown that lactulose passes primarily through the paracellular route, whereas mannitol (4) is transported both transcellularly across the apical membrane as well as through paracellular spaces (34, 55). Previous studies have shown that the villar tips have many more junctions of relatively smaller diameter than in the crypt region, whereas the junctions of larger diameter are present in the crypts (34, 56). The junctions of larger diameter, albeit fewer in number, may allow for the passage of both the larger lactulose as well as the smaller mannitol molecules in the crypt surface, and the smaller diameter villar junctions may only allow the smaller mannitol to permeate and thus may provide some explanation for the increased transfer of mannitol compared with lactulose in ethanol- and burn-injured rats. Nevertheless, the finding in this study, which we are unable to explain, is the increased transfer of both lactulose and mannitol from intestinal lumen into the blood in ethanol- and burn-injured rats in absence of demonstrable differences in tight junctions and paracellular spaces. However, it is likely that ethanol and burn injury may have disturbed the physiological regulation of tight junctions and paracellular spaces without causing visible damage. Such regulatory alterations in tight junctions/paracellular spaces may contribute to the enhanced permeability to lactulose and mannitol in ethanol- and burn-injured rats and, therefore, may provide some explanation for enhanced bacterial translocation in ethanol and burn injury.
Another significant factor that contributes to the increased bacterial
translocation is the suppression of intestinal immune defense. Our
results suggest that although there was a decrease in the proliferation
in both PP and MLN T cells following burn injury alone, the suppression
was maximum in the group of animals receiving both alcohol and burn
injuries. The suppression of T cell proliferation is accompanied by a
significant suppression of IFN- production by PP and MLN T cells in
animals receiving alcohol and burn injury compared with the animals
receiving either injury alone. Finally, we found that the depletion of
CD3+ cells in healthy rats resulted in an increase in the
bacterial accumulation in MLN; such depletion of CD3+ cells
in burn- and alcohol-injured rats furthered the spread of bacteria to
spleen and circulation. These results corroborate earlier findings from
ours and other laboratories (7, 28, 54) and support the
concept that the effects of acute alcohol exposure are not restricted
to splenic or peripheral lymphocytes but are also present in intestinal
lymphoid T cells. Although, as noted by earlier investigators
(7, 28, 39), a single dose of acute alcohol exposure may
not be sufficient to suppress the T cell responses; however, in
combination with burn injury, the T cell response is dramatically
affected. Kawakami et al. (28) suggested that acute gavage
feeding of ethanol before burn injury suppressed splenic T cell
mitogenesis to a greater extent than the T cells from animals with no
alcohol exposure. Napolitano et al. (39) suggested that,
similar to acute alcohol consumption, chronic ethanol intake also may
have synergistic effects on splenic T cell proliferation following
thermal injury. Suppression of T cell proliferation has been a common
finding after burn, trauma, and sepsis (5, 6, 23, 29, 30, 41,
51). Similar decreases in T cell mitogenesis have been recorded
after alcohol exposure (10, 24, 26, 45, 54, 56). It is
interesting to note that intestinal lymphoid organs contain more
lymphocytes than the total lymphocytes from all other parts of the
immune system. According to an estimate, the population of T cells
found in the epithelium of the small intestine may alone account for almost 60% of all T cells in the body (38). Thus mucosal
lymphocytes are the most significant part of the immune system; hence
the disturbances in T cell effector functions may allow more bacteria to grow in MLN and result in their spill into blood circulation and spleen.
Whereas the mechanism underlying the intestinal T cell suppression in
alcohol and burn injury remains undefined, a number of previous studies
suggested that burn injury results in hyperactivation of some cells of
the immune system, leading to aberrant production of inflammatory
mediators such as tumor necrosis factor-, transforming growth
factor-
, PGE2, and corticosterone (3, 5, 12, 14, 28, 41, 51, 53). Other functions of the immune system, however,
are dramatically paralyzed. These include the suppression of macrophage
ability to present antigen (5, 14, 29, 36, 41, 51, 54), T
cell proliferation, IL-2 production, and IL-2 receptor expression
(5, 6, 23, 29). Similarly, the effects of ethanol on the
immune system, independent of trauma or burn injuries, are also
associated with a decrease in macrophage antigen-presenting ability, T
cell proliferation, and IL-2 production (10, 24-26, 45,
56). In addition to the functional deficits identified in the
cells of the immune system after alcohol or thermal injury, studies
have also suggested the loss of immune cells from both systemic and
intestinal lymphoid organs (22, 43). Previous studies by
Szabo et al. (54) and Miller-Graziano et al.
(36) suggested that the suppression of T cell function in
alcohol or burn injury was secondary to macrophage dysfunction. Their
studies showed T cell suppression only when T cells were cultured in
the presence of macrophages. Similarly, studies by Faunce et al.
(15) have implicated the role for macrophage-derived IL-6
in T cell suppression in ethanol- and burn-injured mice. In contrast,
previous studies including our own (5, 6, 23, 29) as well
as the data presented here suggest a suppression of T cell mitogenesis
both in the absence or presence of macrophages or other adherent cells.
Furthermore, we have shown attenuated anti-CD3-linked signaling
molecules in freshly isolated T cells (5, 8, 9). The
differences between our studies and others, however, are likely due to
factors such as the degree of burn or route of alcohol administration.
The studies by Faunce et al. (15) have used a mouse model
of 15% burn and intraperitoneal alcohol injection, whereas in our
studies, rats were orally gavaged with alcohol and received a 30% burn.
The intestinal immune system is the first line of defense against enteric bacteria. The lymphoid tissues associated with the intestine are exposed continuously to antigens in the lumen of gut. Under healthy conditions, a few indigenous bacteria are known to continuously translocate to MLN, but because of intact immune defense, these bacteria do not survive. Thus MLN from normal animals remains relatively sterile. However, injuries, such as alcohol and burn, disrupt the effective mucosal defense to intraluminal microorganisms, leading to the passage of viable bacteria across the luminal barrier to MLN and distant organs. The definitive pathways by which bacteria reach MLN and systemic organs are uncertain. Macrophages may transport bacteria from the gut to the MLN, and from the MLN they can enter systemic circulation. The fact that appropriate activation of intestinal T cells is critical in maintaining immunity against the translocation of enteric bacteria is derived from many sources (19-21, 31, 42, 44, 52). Owens and Berg (42) noted spontaneous gut bacterial translocation to MLN, spleen, and liver in athymic (nu/nu) mice, whereas no translocation was noticed in heterozygous (nu/+) or nude (+/+) mice grafted with thymus. Yet another study showed that depletion of CD4+ and CD8+ T cells also resulted in increased translocation of Salmonella typhimurium and other enteric bacteria (31, 37, 44). Additional studies showed the ability of adoptively transferred T cells to confer protection against a number of bacterial infections, including Escherichia coli (19, 20), Mycobacterium leprae (18), Listeria monocytogenes (47), S. typhimurium (37), or Bordetella pertusis (32).
We found a significant suppression in intestinal T cell IFN-
production. Such a decrease in IFN-
may affect the phagocytic ability of macrophages and phagocytic cells and thus allow bacteria to
multiply and transfer to extraintestinal sites. IFN-
produced by the
intestinal T cell helps in resolution of Yersinia
enterocolitia infection (30). In an another study
(2), IFN-
was shown to confer protection against
S. typhimurium invasion of epithelial cells and fibroblasts.
Moreover, the decrease in IFN-
enhanced the susceptibility to
infection. IFN-
-deficient mice showed impaired ability of
macrophages to produce nitric oxide and superoxide anion necessary to
kill bacteria (21, 43). Other cytokines or chemokines
produced by T cells help in maintaining humoral immunity such as T
cell-dependent antibody production, recruiting, and activating
microbicidal activities of cells such as macrophages and neutrophils
(31, 52). Cytolytic effector functions, as expressed by
CD8+ T cells, are also mediated by either direct
cell-to-cell contact or by secreting cytokines (21). These
studies together suggest that alterations in IFN-
or in other T cell
effector responses will potentially result in perturbation of the
complex network of host defense, resulting in immune dysfunction.
In summary, the results presented here suggest that enhanced intestinal
permeability in rats receiving alcohol and burn injury may cause an
increase in bacterial translocation, whereas the suppression in
intestinal T cell effector responses, such as the decrease in the
production of IFN-, may further their accumulation and spread to
extraintestinal sites.
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ACKNOWLEDGEMENTS |
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We thank E. J. Kovacs for helpful discussions and F. Haque for technical assistance.
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FOOTNOTES |
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This study was supported by National Institutes of Health through Grant R21AA-12901-01A1 (to M. A. Choudhry), and RO1 Grants GM-42577 (to R. L. Gamelli), and GM-53235 and GM-56865 (to M. M. Sayeed). Financial support from the Dept. of Surgery, Loyola University Chicago Medical Center (to M. A. Choudhry) is acknowledged.
Address for reprint requests and other correspondence: M. A. Choudhry, Burn and Shock Trauma Institute, Loyola Univ. Chicago Medical Center, 2160 South First Ave., Maywood, IL 60153 (E-mail: mchoudh{at}lumc.edu).
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. Section 1734 solely to indicate this fact.
First published January 30, 2002;10.1152/ajpgi.00235.2001
Received 8 June 2001; accepted in final form 18 January 2002.
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