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The Journal of Immunology, 2006, 177: 4514-4520.
Copyright © 2006 by The American Association of Immunologists, Inc.

Trauma-Hemorrhage Induces Depressed Splenic Dendritic Cell Functions in Mice1

Takashi Kawasaki, William J. Hubbard, Mashkoor A. Choudhry, Martin G. Schwacha, Kirby I. Bland and Irshad H. Chaudry2

Center for Surgical Research and Department of Surgery, University of Alabama, Birmingham, AL 35294


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although Kupffer cell, splenic, and peritoneal macrophage functions are markedly altered following trauma-hemorrhage (T-H), it remains unclear whether T-H also affects splenic dendritic cell (sDC) functions. We hypothesized that sDC functions will also be compromised following T-H. Male C3H/HeN (6- to 8-wk) mice were randomly assigned to sham operation or T-H. T-H was induced by midline laparotomy and ~90 min of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (four times the shed blood volume in the form of Ringer’s lactate). Two hours later, the mice were sacrificed; sDC were isolated; and the changes in their apoptosis, MHC class II expression, and ability to produce costimulatory cytokines and Ag presentation were measured. The results indicate that sDC Ag presentation capacity was significantly decreased and MHC class II expression was also significantly decreased following T-H. Moreover, LPS-induced IL-12 production and LPS- or IL-12-induced IFN-{gamma} production following T-H were significantly decreased. Thus, the markedly decreased MHC class II expression and cytokine (IL-12, IFN-{gamma}) production following T-H may be the cause for the depressed sDC Ag presentation under those conditions. This depression in Ag presentation could contribute to the host’s enhanced susceptibility to sepsis following T-H.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It is well known that traumatic injury-induced immunosuppression is associated with an increased susceptibility to sepsis, organ failure, and mortality (1, 2). The impairment in host defense following trauma could result from a defect in cell-mediated immune responses (3, 4) and impaired microbicidal activity by cells of the innate immune system. A number of studies have demonstrated that trauma-hemorrhage (T-H)3 induces a marked alteration of many immune functions, including T cell activation, proliferation and cytokine release, macrophage Ag presentation function, and cytokine release (5, 6, 7, 8, 9). Furthermore, our previous studies have shown that following hemorrhage, splenic macrophage, peritoneal macrophage, and Kupffer cell Ag-presenting capacity are depressed, and this depression is associated with decreased MHC class II expression (10, 11).

Professional APCs such as dendritic cells (DC), macrophages, and B lymphocytes are able to ingest, process, and present Ag in the context of MHC molecules (12, 13). These cells have different immunologic functions. DC, originally described by Steinman and Cohn (14) in 1973 and of increased interest recently, are the most potent APCs that are intimately involved in initiation of innate and adaptive immunity (15, 16). Immature DC are strategically located in tissues that represent pathogen entry routes, where they continuously monitor the environment via the uptake of both particulate and soluble products. DC maturation is associated with enhanced production of inflammatory cytokines and chemokines, reduced endocytic and phagocytic capacity, and acquisition of migratory functions that allow Ag-loaded DC to move from the marginal zones to the T cell areas or from nonlymphoid to lymphoid tissues (17). Mature DC highly express MHC and costimulatory molecules, including CD40, CD80, CD83, and CD86, on their surface (18, 19). They also have the ability to activate both Th1 and Th2 cell responses.

Murine and human studies have shown that the significant loss of DC during sepsis and following LPS administration could result from an increased apoptosis of DC (17, 20, 21). In patients with sepsis, it has been reported that DC were depleted in the spleen (21). When compared with trauma patients, the decrease in splenic DC (sDC) population in septic patients was significant (21). Furthermore, it has been reported that monocyte conversion to immature DC is impaired in trauma patients and this may contribute to postinjury immune alterations (22). Similar to septic patients, an apoptotic loss of sDC in septic mice has also been demonstrated (23, 24). However, it remains unknown whether T-H has any effect on sDC function. The aim of our study, therefore, was to determine whether T-H has any effect on sDC functions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Male C3H/HeN mice (Charles River Laboratories or The Jackson Laboratory), 6–8 wk old and weighing 20–25 g, were used in the experiments. These mice were allowed to acclimatize to the animal facility for 1 wk before the experiments. Animal experiments were conducted in accordance with guidelines set forth in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health and approved by the Institutional Animal Care and Use Committee at the University of Alabama.

Trauma-hemorrhage

Animals were anesthetized with isoflurane (Attane; Minrad) and restrained in supine position. A 2.0-cm midline laparotomy (i.e., induction of soft tissue trauma) was performed and then closed aseptically in two layers using 6-0 Ethilon sutures (Ethicon). Subsequently, both femoral arteries were aseptically catheterized with polyethylene-10 tubing (Clay-Adams), and the animals were allowed to awaken. Blood pressure was monitored continuously through one of the femoral catheters using a blood pressure analyzer (Digi-Med BPA-190; Micromed). Upon awakening, the animals were bled through the other catheter to mean arterial pressure of 35 ± 5 mmHg that was maintained for 90 min. At the end of that period, animals were resuscitated with 4 times the shed blood volume in the form of lactated Ringer’s solution over 30 min. Lidocaine was applied to the groin incision sites, the catheters were removed, the vessels were ligated, and the incisions were closed. Sham-treated animals underwent the same anesthetic and surgical procedures, but neither hemorrhage nor fluid resuscitation was performed. The animals were anesthetized by isoflurane administration 2 h after T-H, at which time blood and spleen were collected for analysis.

Plasma collection and storage

Blood was obtained by cardiac puncture and centrifuged at 400 x g for 10 min at 4°C. Plasma was immediately frozen and stored at –80°C until analyzed.

Splenocyte preparation and flow cytometric analysis

The spleen was placed in ice-cold, 4°C PBS and gently ground between frosted slides to produce single-cell suspension. The suspension was centrifuged at 300 x g for 15 min, the pellet was resuspended in PBS, erythrocytes were hypotonically lysed, and the remaining cells were washed with PBS by centrifugation at 300 x g for 15 min. Splenocyte viability was consistently >95%, as determined using trypan blue exclusion procedure. The splenocytes were resuspended in RPMI 1640 medium (Invitrogen Life Technologies) with 10% heat-inactivated FBS (Invitrogen Life Technologies) to yield a final concentration of 1 x 105 cells/ml. Cell suspensions were first blocked with 1 µg/ml Fc block (clone 93) Ab for 15 min on ice and then stained with Ab against surface markers CD11c (clone N418) or 33D1 (clone 33D1). Cells were acquired using a BD LSRII (BD Biosciences), and 10,000 events were collected for analysis.

Isolation of sDC and flow cytometric analysis

Spleens were digested by Liberase CI (Roche) and teased apart by repeated pipetting in PBS containing 5% FCS and 5 mM EDTA. The RBC were osmotically lysed, and splenocytes were blocked with 1 µg/ml Fc block (clone 93) Ab for 15 min on ice. Cell suspensions were enriched with anti-CD11c magnetic beads and positive selection columns MS+, according to manufacturer’s instructions (Miltenyi Biotec). Flow cytometric analysis demonstrated that cells contained >90% CD11c-positive cells. Cell suspensions were stained with Ab against surface markers MHC II (clone M5/114.15.2), CD80 (clone 16-10A1), CD83 (clone Michel-17), or CD86 (clone GL1) for DC (eBioscience). Cells were acquired using a BD LSRII (BD Biosciences), and 10,000 events were collected for analysis.

Determination of cytokine level in plasma and CD11c-positive cell culture supernatants

Purified CD11c-positive cells (1 x 105 cells/well) from spleen were cultured in 96-well tissue culture plates in RPMI 1640 medium containing 10% FCS and 50 ng/ml gentamicin with or without LPS (10 µg/ml) or murine rIL-12 (10 ng/ml). After 24-h incubation at 37°C, 5% CO2, the plate was centrifuged at 400 x g for 10 min. Supernatants were collected and frozen at –80°C until use. Plasma samples were collected and clarified by centrifugation, as described above. The levels of IL-12p70, IL-10, IFN-{gamma}, IL-6, MCP-1, and TNF-{alpha} in the plasma and DC supernatant were measured using cytometric bead array (CBA) mouse inflammation kit (BD Biosciences), according to the manufacturer’s instructions. The sensitivity of IL-12p70 assay is 10.7 pg/ml, according to the information provided by the manufacturer. IL-12p40 levels in cell supernatants were measured by ELISA (BD Pharmingen), as described previously in our laboratory (25).

Apoptosis assay

Apoptotic cells were detected using the annexin V-FITC kit (BD Biosciences). Briefly, a total of 1 x 105 purified CD11c-positive cells was incubated with 5 µl of annexin V-FITC and 5 µl of propidium iodide (PI) in binding buffer for 15 min. Apoptotic DC were identified by staining with annexin V positive and PI negative (early apoptotic cells) or annexin V positive and PI positive (late apoptotic cells). Quantitative analysis was performed by BD LSRII (BD Biosciences) with 10,000 events acquired.

Ag presentation

The capacity of purified splenic CD11c-positive cells to present Ag to D10.G4.1-cloned Th cells (American Type Culture Collection) was conducted according to the method of Kaye et al. (26) and Ayala et al. (10). In brief, the CD11c-positive cells were incubated for 30 min (37°C, 5% CO2, in the dark) with 50 µg/ml mitomycin C (Sigma-Aldrich), following which the cells were washed (four times) in PBS, and a series of dilutions was made. The DC were then cocultured with 2 x 104 cells of D10.G4.1 in the presence or absence of 300 µg/ml conalbumin (Sigma-Aldrich) for 48 h (37°C, 5% CO2). BrdU was added to the cells, and cells were reincubated for 24 h. After incubation, T cell proliferation was determined using Cell Proliferation Biotrak ELISA System (Amersham Biosciences).

Statistical analysis

The data are presented as mean ± SE. One-way ANOVA and Tukey’s test were used for the comparison between groups, and the differences were considered to be significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Plasma cytokines

Plasma TNF-{alpha}, IL-6, MCP-1, and IL-10 concentrations were significantly increased at 2 h following T-H (Fig. 1). However, plasma IL-12p70 and IFN-{gamma} concentrations were not detectable in both sham and T-H groups.


Figure 1
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  		FIGURE 1. Plasma TNF-{alpha} (A), IL-6 (B), IL-10 (C), and MCP-1 (D) levels following T-H. Male C3H/HeN mice were subjected to sham or T-H. At 2 h following resuscitation, plasma was collected for analysis. Data are shown as mean ± SEM of six animals in each group. *, p < 0.05 compared with sham.

 
Percentage of DC after sham operation and T-H

To determine whether T-H affects the percentage of sDC, splenocytes were stained with DC-specific marker anti-CD11c Ab and assessed using flow cytometry. The percentage of sDC was found to be significantly decreased following T-H when compared with sham mice (Fig. 2A). To further confirm this, we used another mouse DC-specific surface marker anti-33D1 Ab. As with anti-CD11c Ab, a significantly decreased population of sDC was found following T-H (Fig. 2B).


Figure 2
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  		FIGURE 2. Changes of percentages of CD11c-positive splenocytes (A) and 33D1-positive splenocytes (B) following T-H. At 2 h following resuscitation, splenocytes were isolated and stained with DC-specific markers for analysis. Data are shown as mean ± SEM of six animals in each group. *, p < 0.05 compared with sham.

 
MHC-II and costimulating factor expression on sDC

The maturation of DC is closely involved in its activation of immune functions. The mature DC highly express MHC-II and costimulatory factors, such as CD40, CD80, CD83, and CD86, on their surface (18, 19). To examine whether T-H affects DC maturation, we analyzed expression of MHC-II, CD80, CD83, and CD86 on sDC surface after T-H. Fig. 3 shows the effect of T-H on the expression of MHC-II and costimulating factors on the surface of sDC. Following T-H, MHC-II (Fig. 3A) and CD83 (Fig. 3C) expression were significantly decreased. However, there were no significant differences between sham and T-H mice in CD80 (Fig. 3B) and CD86 (Fig. 3D) expression.


Figure 3
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  		FIGURE 3. Surface phenotype of CD11c-positive sDC following T-H. At 2 h following resuscitation, sDC were purified from splenocytes by MACS sorting; stained with Abs against MHC-II (A), CD80 (B), CD83 (C), and CD86 (D); and analyzed by flow cytometry. For flow cytometry data, a representative example of six independent analyses is shown (dotted line, isotype control; black line, sham; gray line, T-H). Data are shown as mean ± SEM of six animals in each group. MFI, mean fluorescence intensity. *, p < 0.05 compared with sham.

 
Apoptosis of sDC

As shown in Fig. 4, the percentage of both annexin V-positive and PI-negative cells (early phase of apoptosis) and annexin V-positive and PI-positive cells (late phase of apoptosis) was significantly increased following T-H. These results, along with the previous reports (23, 24) and our results shown in Fig. 2, demonstrate that apoptosis is a possible cause of sDC loss following T-H.


Figure 4
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  		FIGURE 4. Changes of apoptotic rate of sDC following T-H. At 2 h following resuscitation, sDC were purified from splenocytes. Purified CD11c-positive cells were incubated with annexin V-FITC and PI. A representative example of six independent analyses is shown (A). Percentage of late apoptotic DC (B). Late apoptotic DC were identified by staining with annexin V positive and PI positive. Percentage of early apoptotic DC (C). Early apoptotic DC were identified annexin V positive and PI negative. Data are shown as mean ± SEM of six animals in each group. *, p < 0.05 compared with sham.

 
Cytokine production by sDC

There were no significant differences between sham and T-H DC production of TNF-{alpha}, IL-6, and IL-10 without LPS stimulation. After incubation with LPS, these cytokine concentrations increased significantly in both sham and T-H mice. However, LPS-induced production of TNF-{alpha} and IL-6 was significantly suppressed in sDC following T-H. In contrast, there was no significant difference in LPS-induced IL-10 production between T-H and sham groups (Fig. 5). MCP-1 production by sDC was below detectable concentrations in both sham and T-H mice with or without LPS stimulation.


Figure 5
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  		FIGURE 5. TNF-{alpha}, IL-6, and IL-10 production by CD11c-positive cells following T-H. At 2 h following resuscitation, sDC were purified from splenocytes. Purified CD11c-positive cells were cultured in 96-well tissue culture plates with LPS (10 µg/ml) for 24 h. The levels of TNF-{alpha} (A), IL-6 (B), and IL-10 (C) in DC supernatant were measured using CBA. Data are shown as mean ± SEM of six animals in each group. *, p < 0.05 compared with the equivalent sham group. #, p < 0.05 compared with nonstimulation group (–).

 
IL-12 and IFN-{gamma} are important cytokines in DC maturation (27, 28). Activated DC produce IL-12, and IL-12 up-regulates the production of IFN-{gamma} from Th1 and NK cells. IL-12 also activates DC autocrine production of IFN-{gamma}. These processes in DC could play a key role against early infections after T-H by enhancing immunity against pathogens. We therefore assessed the effect of T-H on DC production of IL-12 and IFN-{gamma}. The results indicated that LPS- or IL-12-induced IFN-{gamma} production by sDC decreased following T-H (Fig. 6A). Furthermore, the release of IL-12p40 and IL-12p70 by DC after LPS stimulation was also significantly decreased following T-H (Fig. 6B). These results suggest that suppressed IFN-{gamma} and IL-12 production may be the cause of inhibition of DC maturation following T-H.


Figure 6
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  		FIGURE 6. IFN-{gamma} and IL-12 production by CD11c-positive cells following T-H. At 2 h following resuscitation, sDC were purified from splenocytes. Purified CD11c-positive cells were cultured in 96-well tissue culture with LPS (10 µg/ml) or human rIL-12 (10 ng/ml) for 24 h. The levels of IFN-{gamma} (A) and IL-12p70 (B) in DC supernatant were measured using CBA. IL-12p40 levels (B) in cell supernatants were measured by ELISA. Data are shown as mean ± SEM of six animals in each group. *, p < 0.05 compared with the equivalent sham group. #, p < 0.05 compared with nonstimulation group (–).

 
Ag-presenting capacity of DC following T-H

The results indicate that CD11c-positive cells isolated from the spleen of both sham and T-H mice stimulated more D10.G4.1 T cell line proliferation in the presence of conalbumin (the Ag to which the D10.G4.1 cell responds following appropriate Ag processing and presentation by the DC) than in its absence (Fig. 7). Furthermore, the CD11c-positive cells isolated from T-H mice showed a lower capacity to stimulate T cell proliferation than those from sham controls (Fig. 7).


Figure 7
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  		FIGURE 7. Changes of Ag-presenting capacity of CD11c-positive cells following T-H. Ag-presenting capacity was determined using Ag conalbumin specifically to stimulate D10.G4.1 cloned Th cell proliferation. At 2 h following resuscitation, sDC were purified from splenocytes. DC were then cocultured with 2 x 104 cells of D10.G4.1 in the presence or absence of 300 µg/ml conalbumin for 48 h. BrdU was added to the cells, and cells were reincubated for 24 h. After incubation, T cell proliferation was measured using ELISA. Data are shown as mean ± SEM of six animals in each group. *, p < 0.05 compared with the equivalent sham group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study demonstrates that the percentage of sDC decreased following T-H. In addition to the loss of DC, expression of surface Ag studies demonstrated that T-H down-regulated the activation of DC. Furthermore, sDC Ag presentation function was also suppressed following T-H. In addition to Ag presentation, DC also produce cytokines such as IFN-{gamma} and IL-12 that can directly influence the generation of immune responses to injury such as T-H. In this regard, the decreased IFN-{gamma} and IL-12 production of sDC after T-H demonstrated in the present study may contribute to the profound immunosuppression that is known to occur in trauma and shock patients.

The loss of sDC after T-H may be due to down-regulation of DC-specific markers, as suggested by the loss of several molecules such as CD11c and 33D1. However, previous studies have demonstrated that bacterial stimuli such as LPS induce the migration of sDC from the marginal zone to the T cell area within a few hours, and the redistribution of DC in the T cell area was rapidly followed by a dramatic decrease in the number of DC in the spleen (17). Indeed, several studies suggest that the loss of sDC occurs both in patients with sepsis and in mouse model of sepsis (21, 23, 24). Although it has been shown that circulating endotoxin levels increase following hemorrhagic shock (29) causing the loss of sDC due to their redistribution, other studies have failed to show any increase in endotoxin at any time point during or following hemorrhage (30, 31). Thus, the precise cause for the loss of DC following T-H remains unknown.

Studies in murine and human subjects have increasingly focused on the importance of the loss of DC and apoptosis of DC during sepsis. In this regard, studies have shown a significant loss of sDC within 24 h of LPS administration, and LPS has been demonstrated to induce apoptosis of DC in vivo (17, 20, 21). Previous studies have also illustrated a specific decrease in the sDC population in septic human patients when compared with patients who had suffered trauma (21). It has also been reported that in trauma patients, monocyte conversion to immature DC is impaired and thus may contribute to postinjury immune alterations (22). However, to the best of our knowledge, studies examining the functions of sDC in trauma patients have not yet been conducted. In addition, an apoptotic loss of sDC in septic mice 24–48 h after the onset of polymicrobial sepsis (cecal ligation and puncture) has also been demonstrated (23, 24). However, studies have shown that the apoptosis of DC is not Fas ligand dependent (32, 33, 34). Cytokines are important mediators of sepsis, and the apoptotic cell death program can be triggered by TNF-{alpha}. Therefore, one potential mechanism for the increase of sDC apoptosis in T-H mice could be due to increased level of TNF-{alpha} in the tissue. Although we did not measure spleen tissue concentration of TNF-{alpha}, circulating TNF-{alpha} levels were markedly elevated. Furthermore, although spontaneous TNF-{alpha} production by sDC was not increased following T-H, it is possible that tissue cytokine concentration is close to that of plasma. Taken together, it is possible that increased TNF-{alpha} induces DC apoptosis following T-H.

Mature DC are potent stimulators of cellular and humoral immune responses (18). To acquire naive T cell stimulatory ability, DC must undergo maturation. This involves up-regulation of surface MHC-II and costimulatory molecules during their migration from the periphery to T cell areas of secondary lymphoid tissue (35). There are many stimuli that can initiate this maturation process in vitro. These include the proinflammatory cytokines (TNF-{alpha} and IL-1beta) and bacterial products such as LPS (36). LPS has also been shown to stimulate the maturation of DC in vivo (17). Mature DC have potent Ag-presenting function with their high expression of costimulating factors and MHC-II Ag and with increased cytokine production capacity (18). Although there was no significant difference in the expression of DC surface CD80 and CD86 between cells from sham and T-H animals, CD83 and MHC-II were significantly suppressed under those conditions. Furthermore, proinflammatory cytokine (TNF-{alpha}) production was also decreased following T-H. In contrast, Paterson et al. (37) have shown that CD11c-positive cells from thermally injured mice had high intracellular levels of IL-1beta, IL-6, and TNF-{alpha} after LPS stimulation. The reason for the differences in our results and those of Paterson et al. (37) may be due to the fact that different injury models were used. Moreover, they stimulated CD11c-positive cells at 7 days after burn injury, whereas we stimulated those cells at 2 h after resuscitation. Because only a single time point was used in our study, it remains to be determined whether CD11c-positive cells were depressed at 7 days after resuscitation. Nonetheless, previous studies have shown that depressed immune functions return toward normal by 7–10 days following resuscitation (6, 11), and it is therefore likely that the depression in DC also returned to normal by that time. This aspect, however, remains to be determined. The importance of the MHC-II-TCR system in T cell responses has been well documented, and CD83 is well known as the marker for maturation of DC (19, 38). Decreased expression of costimulatory CD83 and MHC-II on T-H mouse DC may contribute to suppression in T cell proliferation because these costimulatory molecule signals are required for full T cell activation and proliferation. Therefore, our results strongly suggest that DC maturation is impaired after T-H and this may be the cause for the depressed sDC Ag presentation.

It can also be argued that IL-12p70 levels we measured were very low, and thus it is unclear whether or not they should be taken into account. In this regard, IL-12 is secreted in three main forms: the p70 heterodimer, p40 homodimers, and p40 monomers. The function of the p40 moieties is not yet established following T-H. However, the studies of Trinchieri et al. (39, 40) have shown that IL-12p40 is frequently secreted in large excess over the p70 heterodimer. Studies have also shown that p35 is only secreted as a part of the heterodimer when p40 is also produced in the same cell (39, 40). Furthermore, IL-12p70 is known as the active form of IL-12, and it stimulates IFN-{gamma} production from DC, T cells, NK, and other cells (27). Additionally, IL-12 has been shown to be a potent inducer of differentiation of Th1 cells, i.e., Th1 cells (40). Studies have also shown that IL-12p40 associates with not only the IL-12 p35 chain, but also with a p19 chain to form a novel cytokine, IL-23 (41). Moreover, p19 has homology with IL-6 and G-CSF, like p35, and is secreted only when it is associated with IL-12p40 (41). Thus, one would expect IL-12p70 concentration to be very low under our experimental conditions. Cytokine secretion by DC initiates and enhances both innate and acquired immunity. Activation of DC by infectious agents leads to secretion of IL-12, which subsequently induces IFN-{gamma} production by NK cells and directs Th1 development. IFN-{gamma}, in turn, acts on monocytes to augment IL-12 secretion (27). Thus, IL-12 and IFN-{gamma} comprise a positive feedback system, which is probably required for optimal production of IL-12 in vivo (28). Studies using neutralizing Ab against IFN-{gamma} in IL-12- or IFN-{gamma}-deficient mice have confirmed the importance of these cytokines for innate immunity and Th1 development and for controlling intracellular pathogens (42, 43). In addition to expressing high levels of Ag-presenting and costimulatory molecules, mature DC release large amounts of IL-12 that can stimulate preferential Th1 immune responses (44). However, release of IL-10 blocks the DC maturation process by interfering with the up-regulation of costimulatory molecules and production of IL-12, subsequently limiting the ability of DC to initiate a Th1 response (45, 46). IL-10 impairs cellular immunity and leads to induction of Th2 responses. In our study, production of IL-12 and IFN-{gamma} by DC following T-H was significantly suppressed; however, IL-10 production was not altered under those conditions. Because mature DC resist the suppressive effects of IL-10 (18), it suggests that IL-10 may have more potent ability to block the maturation process in mice following T-H. Furthermore, it has been reported recently that MCP-1 is associated with the development of polarized Th2 (47). Because the concentration of MCP-1 increased significantly following T-H, this raises the possibility that circulating MCP-1 regulates sDC maturation. Taken together, it appears that T-H blocks innate immunity and Th1 immune responses of DC rather than the Th2 process.

In summary, our studies indicate that the stimulatory potential of DC against Ag is impaired following T-H. Furthermore, T-H suppresses DC maturation and Th1 responses. Additionally, T-H depresses Ag presentation function of DC. Our findings thus shed additional light on the role of DC in the alteration of immune functions following T-H.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant RO1 GM37127. Back

2 Address correspondence and reprint requests to Dr. Irshad H. Chaudry, Center for Surgical Research, University of Alabama, 1670 University Boulevard, Volker Hall, Room G094, Birmingham, AL 35294-0019. E-mail address: Irshad.Chaudry{at}ccc.uab.edu Back

3 Abbreviations used in this paper: T-H, trauma-hemorrhage; CBA, cytometric bead array; DC, dendritic cell; PI, propidium iodide; sDC, splenic DC. Back

Received for publication May 11, 2006. Accepted for publication July 11, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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