OX62+OX6+OX35+ rat dendritic cells are unable to prime CD4+ T cells for an effective immune response following acute burn injury

Nadeem Fazal

doi:10.1016/j.rinim.2013.06.001

. 2013 Jun 29;3:64–72. doi: 10.1016/j.rinim.2013.06.001

OX62+OX6+OX35+ rat dendritic cells are unable to prime CD4+ T cells for an effective immune response following acute burn injury^☆

Nadeem Fazal ^1,^*

PMCID: PMC3908324 PMID: 24600560

Abstract

Co-stimulatory molecules expressed on Dendritic Cells (DCs) function to coordinate an efficient immune response by T cells in the peripheral lymph nodes. We hypothesized that CD4+ T cell-mediated immune suppression following burn injury may be related to dysfunctional DCs residing in gut associated lymphoid tissues (GALT), such as Mesenteric Lymph Nodes (MLN). Therefore, we studied co-stimulatory molecules expressed on burn rat MLN DCs as an index of functional DCs that would mount an effective normal CD4+ T cell immune response. In a rat model of 30% Total Body Surface Area (TBSA) scald burn, OX62+OX6+OX35+ DCs and CD4+ T cells were isolated from MLN of day 3 post-burn and sham control rats. DCs were tested for their expression of co-stimulatory molecules, and prime CD4+ T cell (DC:CD4+T cell co-culture assays) to determine an effector immune response such as CD4+ T cell proliferation. The surface receptor expressions of MLN DCs co-stimulatory molecules, i.e., MHC-II, CD40, CD80 (B7-1), and CD86 (B7-2) were determined by Flow cytometry (quantitatively) and confocal microscopy (qualitatively). Tritiated thymidine and CFDA-SE determined CD4+ T cell proliferation following co-incubation with DCs. Cytokine milieu of MLN (IL-12 and IL-10) was assessed by mRNA determination by RT-PCR. The results showed down-regulated expressions of co-stimulatory markers (CD80, CD86, CD40 and MHC-II) of MLN DCs obtained from burn-injured rats, as well as lack of ability of these burn-induced DCs to stimulate CD4+ T cell proliferation in co-culture assays, as compared to the sham rats. Moreover, anti-CD40 stimulation of affected burn MLN DCs did not reverse this alteration. Furthermore, a marked up-regulation of mRNA IL-10 and down-regulation of mRNA IL-12 in burn MLN as compared to sham animals was also observed. To surmise, the data indicated that dysfunctional OX62+OX6+OX35+ rat MLN DCs may contribute to CD4+ T-cell-mediated immune suppression observed following acute burn injury.

Keywords: Immunosuppression, Mesenteric lymph nodes, MHC-II, CD80, CD86, CD40

1. Introduction

Our previous studies have focused on gut-associated lymphoid tissues (GALT), precisely; the roles of Antigen-Presenting Cells (APC) and CD4+ T cells in immunosuppression following acute burn-injury [1,2]. We showed that naïve rat MLN CD4+ T cells exhibit markedly reduced IL-2 production and proliferation when stimulated in the presence of MLN APCs from burn-injured rats [2]. In the current study, we expanded our previous observations seen in rat burn MLN APCs to MLN DCs that are recognized to be unique in effectively activating naïve CD4+ T cells. The involvement of co-stimulatory molecules expressed on APCs such as DCs, which are responsible for T cell suppression in burn and septic injuries continues to be a subject of extensive studies in several laboratories [3–7]. Recent studies have emphasized the importance of DCs as exclusive antigen presenters and activators of naïve T cells in the draining lymph nodes [8,9]. Immature DCs with decreased expression of MHC-II, CD80/86, CD40, and their altered interactions with CD28, CTLA-4, and CD40L on the T cells, can adversely affect naïve T cell activation and their responses [10–12]. Studies have also shown that activated CD4+ T cells originating from MLN recirculate back to MLN, where they proliferate; the proliferation of CD4+ cells in the MLN was found to be greater than that of CD8+ cells [13]. Such altered interactions between DC and the T cells has been proposed to lead to an anergy-like state in T cells and/or active T cell suppression [14]. We hypothesized that disturbances in DC functions in burn-injury conditions could also contribute to impaired CD4+ T-cell mediated immunity. In the current study we limited our experiments to DCs and CD4+ T cells derived from MLN as they have been known to drain lymph, not only from intestinal wall, but also from burn-scalded skin, thus becoming a central place for initiating a competent immune response or suppressed immune response following acute burn injury.

2. Materials and methods

2.1. Animal model

Male Sprague Dawley rats (250–300 g) were housed and used in compliance with the regulation of the Animal Care Facility of the Chicago State University. This rat model has been previously described as a suitable model demonstrating the effector immune response associated with burn injury [1,2]. Rats were anesthetized with sodium pentobarbital (40–50 mg/kg, intraperitoneally), their dorsal body surface hair shaved off, and placed in an appropriately sized template device such that the shaved area of the skin on the animals’ back was exposed. Adequacy of anesthesia was tested by the absence of withdrawal response to toe pinching. The template device was then lowered into a hot water bath (95–97 °C) to immerse the exposed skin area in hot water for 8 s. With this technique, full thickness third degree burns comprising 30% of the total body surface was obtained. Sham rats were subjected to identical anesthesia and other treatments, except that they were immersed in 37 °C water. The animals were dried immediately, and given fluid resuscitation with 0.9% saline (3 cc/body surface area) to maintain urine output. Untreated and un-operated rats were used as controls.

2.2. Rat-specific antibodies

All rat specific antibodies, i.e., OX6 (MHC-II), OX35 (CD4+), OX52 (pan T cell), OX8 (T cells and NK cells), OX12 (Ig L chain), and OX33 (pan-B cells), CD40, CD86 (B7-2), CD80 (B7-1) and anti-CD3 were obtained from BioLegend, San Diego, CA.

2.3. Dendritic cell isolation

For DC separation, Anti-DC (OX62) Microbeads, rat (Miltenyi Biotech Inc., Auburn, CA) developed for the positive selection or depletion of OX62+ dendritic cells (DC) from lymphoid tissues was used as per instructions of the manufacturer. Lymphoid organs (MLN) were minced and digested in 2 mg/ml Collagenase D (Roche) in RPMI, 1% FCS for 30 min at 37 °C. EDTA at 10 nM was added during the last 5 min, and the cell suspension was pipetted several times and filtered. Cells were washed once in PBS/EDTA, 2 mM/1% FCS, and low-density cells were obtained after centrifugation on 14.5% Nycodenz gradient. When Nycodenz gradient was found not enough to remove dead cells, we employed Dead Cell Removal Kit (Miltenyi, Cat no. 130-090-101) to improve yield of live cells. To remove clumps that clogged the columns, cells were passed through 30 μm nylon mesh (Miltenyi). After centrifugation cells were re-suspended in 80 μl of buffer per 10⁷ cells. Twenty microlitre of Anti-rat DC (OX62) Microbeads per 10⁷ cells were added, mixed and incubated for 15 min at 6–12 °C. PE-conjugated Rat anti-OX62 monoclonal antibody was added at this time. Cells were re-suspended in 500 μl buffer for upto 10⁸ total cells. Cell suspension was applied to MS columns in a magnetic environment. One millilitre of buffer was pipetted onto the columns. Cell mix was removed away from magnetic field and firmly flushed out fraction with magnetically labeled cells using plunger supplied with the columns. For flow cytometric and confocal image analysis, cells were stained with fluorescent antibodies for OX62, MHC Class II (OX6) and CD4+ (OX35) co-stimulatory markers expressed on DCs.

2.4. Enrichment of CD4+ T cells

CD4+ T cells were isolated using IMMULAN goat anti-mouse IgG-coated beads (Biotech Laboratories, Houston, TX). The Immulan (TM) T cells subpopulation kits are affinity chromatography based for the separation and selection of rat T cell subpopulations. Ninety nine percent of macrophages and B cells were removed from enriched CD4+ T cell populations. Purity of T cells subpopulations range (89–98%). Single cell suspension were prepared and incubated for 2 h to get rid of adherent monocytes. Fifteen millilitre of rabbit anti-rat IgG coated Immulan beads were then introduced onto columns. Cell suspensions were layered onto columns for 10 min. Effluent were then collected with 10 ml of RPMI media at a flow rate of 1 drop/s. Cells were labeled with mouse anti-rat CD8 Ab for 1 h. Labeled cells were then loaded again on columns containing goat anti-mouse IgG coated beads. The columns were washed with 10 ml medium at a flow rate of 1 drop/s. Effluent was the enriched CD4+ T cells. We were able to obtain 8–10 × 10⁶ CD4+ T cells per rat MLN by this separation method.

2.5. CD4+ T cell:DC co-culture assays

CD4+ T cells and DCs at (10:1 ratio) were co-cultured in 96-well flat-bottomed microtiter plates (Falcon, Lincoln Park, NJ) for 72 h/37 °C/5% CO₂. Cells were activated with anti-CD3 (1 μg/ml) by immobilizing on the treated tissue culture 96-well plates that were coated with the antibodies (∼10 μg/ml) and incubated at 37 °C for 90 min, prior to cell cultures. Tritiated thymidine 1 μCi (37Bq) or CFDA-SE (Molecular probes, Life Technologies, Grand Island, NY) was added to each well in the last 16–18 h of culture. Cells were harvested at the end of culture period onto filtermats (Skatron, Sterling, VA) with a semi-automatic PHD cell harvester (Cambridge Technology Inc.). The counts per minute (cpm) of the filter membrane were measured in scintillation liquid on a Beckman LS 6500 liquid scintillation counter (Fullerton, CA).

2.6. Flow cytometry

FACS sorting core facilities of the Department of Biological Sciences Chicago State University was used to sort out DCs after labeling with fluorescent labels. Flow cytometry was also used for the determination of T cell type and activation cell markers labeling in the experiments. DCs were labeled with respective FITC/PE/APC-tagged rat-specific antibodies at 1–5 μg/ml per 10⁶ cells. Isotype control antibodies and unstained cells were used as controls. Cells were fixed in 0.7 ml of 1% paraformaldehyde solution and acquired on a FACScan flow cytometer (Becton Dickinson).

2.7. Confocal microscopy and image analyses

MLN DCs and CD4+ T cells were labeled with appropriate FITC/APC or PE-labeled antibodies and an aliquot was cytospun onto microscopic slides. The slides were then analyzed with a Zeiss LSM 510 laser-scanning microscope. C-Apochromat 40 × 1.20 water immersion was used for viewing and image was acquired with Zeiss LSM 510; version 4.2 SPI software. One hundred cells labeled with a fluorescent dye uptake were counted for each specimen. The cell, which had taken respective fluorescent-antibody label was considered as positive. Moreover, DCs and CD4+ T cells were also differentiated from each other by their size and morphology and only those cells that fulfilled the criteria were counted as positive. Isotype control antibodies and unstained cells were used as controls.

2.8. Reverse-transcription polymerase chain reaction (RT-PCR)

Gene sequences were obtained from Gene Bank for IL-12 and IL-10 and primers were custom-made. Isolation of total RNA was done aseptically from MLN tissues using RNA Easy kit (QIAGEN, Inc., Valencia, CA) as per manufacturers protocol. Primers used for the amplification of murine IL-12, IL-10 and GAPDH were as follows: IL-12, 5′-CTT GCC CTC CTA AAC CAC CTC AGT-3′ (forward) and 5′-CCA CCA GCA TGC CCT TGT CTA-3′ (reverse); IL-10, 5′-GAA GAC AAT AAC TGC ACC CAC TTC-3′ (forward) and 5′- ATG GCC TTG TAG ACA CCT TGG TCT-3′ (reverse); GAPDH, 5′-TGA TGA CAT CAA GAA GGT GGT GAA-3′ (forward) and 5′-TGG GAT GGA AAT TGT GAG GGA GAT-3′ (reverse). Polymerase chain reaction (PCR) was performed in a 25 μl of reaction volume containing 0.2 μmol/l primers, 1 U Taq DNA polymerase under the following conditions: 95 °C for 30 s, 58 °C for 30 s, and then 72 °C for 30 s (30 cycles). PCR products were visualized with ethidium bromide on 1.5% Agarose gel. After complete electrophoresis, the PCR gel images were captured and analyzed using NIH image software with a multiscan Sony computer. Data was expressed as a ratio of the measured calibrated pixel intensity of each cytokine band divided by the corresponding GAPDH band at each point.

2.9. Statistical analysis

All statistical analyses were carried out using the Statistical Package, Social Sciences Software Program (SPSS, SigmaStat version 2.0, Chicago, IL). To determine inter- and intragroup differences between variables, one-way repeated-measures ANOVA, followed by a pairwise multiple comparisons procedure (Tukey's post hoc test) was performed. Statistical significance was set at p < 0.05. The statistical analysis of the different experimental groups included the comparison of Sham, and Burn.

3. Results

3.1. Expression and phenotypic characterization of MLN DCs co-stimulatory molecules by flow cytometry and confocal microscopy

Expression and phenotypic characterization of MLN DCs were done quantitatively by Flow cytomtery and qualitatively/semi-quantitatively by confocal image analysis. Notably, first challenge was to get enriched cell populations of DCs since they constitute ∼1% of total cell population in rat MLN. Anti-DC (OX62+) Rat Dendritic Cell isolation kit MACS (Miltenyi) was used as described in the methods section. OX62 is a specific epitope of the rat integrin α_E2 subunit expressed on dendritic cells of the rat. Cells collected by using the positive selection method contained ∼84% OX62+ DCs. According to specifications provided by Miltenyi microbeads the cells collected by positive selection were all dendritic cells, with a presumable complete elimination of T cells, B cells, and macrophages. This technique yielded 80,000–100,000 DCs per rat MLN. The flow cytometry profile in Fig. 1 shows dendritic cells expressing OX62+ (84%) (Fig. 1A). Dendritic cells expressing (OX62) were also found positive for MHC Class II (OX6) (Fig. 1B). Scarcity of the yield of prospective DCs limited the number of flow cytometric analyses experiments, especially in burn-injured animals, therefore confocal microscopy visual image analyses was relied upon for subjective analysis and continuity of the proposed experiments in this study. Fig. 1(C and D) shows confocal images of DC expressing OX62+ PE-labeled, and MHC-II FITC-labeled surface molecules.

Fig. 1 — Phenotypic and morphological characterization of MLN DCs. DCs were obtained by Magnetic Activated Cell Separation (MACS) as given in methods section. DCs purity was assessed by flow cytometric analyses. Representative flow cytographs of OX62-PE-labeled DCs (A) and OX6-MHC-II-FITC-labeled DCs (B) obtained from Mesenteric lymph nodes of rat. Data is representative of three separate experiments showing similar results. Confocal microscopy samples were prepared as given in the methods. Representative figures showing confocal images of OX62+PE-red-labeled DCs (C). OX62+ marker appears to have stained the outer layers of DCs as shown in the figure. Z-stack images also showed the same staining pattern (data not shown). (D) OX62+PE-labeled DCs and MHC Class-II-FITC-labeled DCs appear as yellow in color because of mixture of PE-red and FITC-green labels (Yellow-label) (D). Dendrites of DCs are obvious in (D) and show typical features of DCs. Confocal image analyses were used to further characterize dendritic cells for qualitative analysis of cells as given in detail in methods section.

Furthermore, our ability to study isolated DCs by confocal microscopy documented that the surface receptor expressions of OX62/MHC-II/CD4+ were found in nearly 80–90% of the enriched cells. Fig. 2(A–D) shows representative figures of confocal images of surface expression of co-stimulatory markers. Based upon the uptake of particular marker we qualitatively assessed and verified our flow cytometry results by visually counting the cells that take the respective marker. One hundred co-stimulatory molecules labeled DCs were counted from three representative samples of experimental animal group to determine percentage of positive cells. The confocal results confirmed our flow cytometry observations. Fig. 2 shows our ability to successfully label and subjectively count surface expressions of all four co-stimulatory molecules used in this study. FACS analyses of DCs obtained from day 3 post-burn and sham controls MLN allowed us to quantitate surface expressions of CD40, CD80 (B7-1), CD86 (B7-2) and MHC-II. OX62 surface marker was used both to separate DCs by magnetic beads, and also to assess the purity of DCs in different assays so that surface expressions could be quantitatively compared within the experimental groups. Hence, equal numbers of OX62-expressing DCs were used to compare surface expression of co-stimulatory molecules in the following experiments.

Fig. 2 — Qualitative image analyses captured by confocal microscopy of rat MLN DCs co-stimulatory molecules, i.e., CD40-FITC-green- (A), CD80-PE-red- (B), CD86-PE-red- (C) and MHC-II-FITC-green (D). DCs were enriched by MACS separation as mentioned in methods and labeled with appropriate fluorescent antibody. Briefly, 100 MLN DCs expressing co-stimulatory activation marker were counted from three separate slides from three different samples of sham control and day 3 post-burn animals. Expression of the co-stimulatory molecules was determined by the uptake of the respective label and counted as positive cell.

3.2. Down-regulation of MLN DCs co-stimulatory molecules after burn injury

The results in Fig. 3 depict the representative flow cytographs of MLN DCs co-stimulatory molecules; CD80, CD86, CD40 and MHC-II from day-3 post-burn and sham controls. Fig. 3A and B shows pooled data from three separate experiments and presented as bar graphs showing reductions in Mean Channel Fluorescence (MCF) of CD40 and MHC-II of day 3 post-burn MLN DCs as compared to corresponding sham MLN DCs (Fig. 3A). Similarly, statistically significant (*p < 0.05) reductions were also found in the MCF expressions of CD80 (B7-1) and CD86 (B7-2) in DCs of day 3 post-burn rats, compared to the sham controls (Fig. 3B).

3.3. DC:CD4+ T cell co-culture assays

MLN CD4+ T cells (sham) were co-incubated with MLN DCs obtained from either, day 3 post-burn, or sham control animals at 10:1 ratio for 72 h, in the presence of αCD3 (10 μg/ml) as detailed in the methods section. After 64 h, the cell cultures were incubated with ³H Thymidine or CFDA-SE for another 8 h, and then assessed for CD4+ T cell proliferation by cell harvester (³H Thymidine) or flow cytometry (CFDA-SE). CD4+ T cells (sham) and allogeneic DCs (sham) 10:1 were used as controls along with CD4+ T cells obtained from sham and burn rats without DC co-stimulation. The ³H Thymidine results in Fig. 4 show statistically significant reduction (*p < 0.05) in the proliferative responses of CD4+ T cells, when incubated with DCs (10 CD4+ T cell:1 DC ratio) obtained from MLN of rats. CD4+ T cell proliferations with DCs obtained from sham rats were more than two times the proliferations of CD4+ T cell in co-cultures with burn rat MLN DCs. CD4+ T cell proliferations of sham or burn rats in the absence of DCs were also found to be higher than the CD4+ T cell proliferations when co-cultured with burn rat DCs.

CFDA-SE technique was later used to reconfirm the results found by ³H thymidine incorporation for CD4+ T cell proliferation (Fig. 4A and B). The cells were labeled with CFDA-SE as detailed in the methods section and cultured in the presence of anti-CD3 (10 μg/ml). At days 1 and 2 post culture, the cells were harvested and CFDA-SE labeling determined by Flow cytometry. The results showed that this labeling technique could efficiently determine upto 8–12 cell cycles of division, which is an index of cell proliferation. The peaks in Fig. 4B showed the data from a representative experimental sample. Similar results were found in three separate experiments. The CFDA-SE results also showed that CD4+ T cells from burn were significantly suppressed (*p < 0.05) as compared to sham CD4+ T cells at day 2, however such a difference in vitro proliferation could not be discerned at day 1 (Fig. 4B).

Confocal image analysis helped us determine not only the purity of cell population but also the phenotype of respective immune cells as exhibited by cell-surface markers. This also depicted the direct observation of cell to cell contact that is required for the stimulation of CD4+ T cells by DCs. Fig. 4C is an example of such a DC:CD4+ T cell co-culture experiment. Fig. 4C shows a representative DC depicting co-stimulatory molecule and structural phenotype of a typical DC with dendrites extending from the surface. The size of CD4+ T cell (5 μm) as compared to DC (10 μm) differentiates both immune cells.

3.4. Assessment of cytokine milieu of MLN

The cytokine milieu may also change the phenotype and effector function of immune cells following acute injury such as burn. Therefore to determine the cytokine milieu; a pro-inflammatory (IL-12) and anti-inflammatory (IL-10) cytokine mRNA was assessed that would indicate the in vivo environment where such an interaction between DCs and CD4+ T cell would likely take place. Lymphatic tissue, i.e., MLN was aseptically crushed and mRNA obtained by using RNA Easy kit. RT-PCR was performed as detailed in methods section. Fig. 5A (blots) and B (densitometric units) shows a marked down-regulation of mRNA IL-12 and up-regulation of mRNA IL-10 in day 3 post-burn burn MLN as compared to MLN of sham control animals. mRNA blots of the samples as shown in Fig. 5A were converted into densitometric units and plotted on bar graphs (Fig. 5B).

Fig. 5 — MLN samples were aseptically collected from day 3 post-burn and sham control rats as given in methods. Isolation of total RNA was done aseptically from MLN tissues using RNA Easy kit (QIAGEN, Inc., Valencia, CA) as per manufacturers protocol. Gene sequences were obtained from Gene Bank for IL-12 and IL-10 and primers were custom-made. (5A-mRNA gene expressions) IL-12 mRNA 766 bp (top row) and IL-10 mRNA 410 bp (bottom row) determined by RT-PCR from MLN of sham control and day-3 post-burn animals. First four samples in both upper and lower rows come from sham controls, and last three samples from day 3 post-burn MLN tissue samples. The results indicate up-regulation of IL-12 (766 bp) in sham and IL-10 (410 bp) in day 3 post-burn MLN tissue samples. (5B)Data shown in the histogram as densitometric units based upon the density of each band. The bar graph shows statistically significant upregulation of mRNA of IL-10 in burn samples, whereas IL-12 is downregulated in burn samples of MLN tissues. *p < 0.05 shows statistically significant values.

3.5. Anti-CD40 stimulation of DCs to up-regulate co-stimulatory molecules

Anti-CD40 stimulation is known to activate DCs and help them function as mature DCs. Therefore, following down-regulation observed in DCs of burn-injured rats we tried to stimulate DCs with anti-CD40 antibody to investigate if we could reverse the dysfunction observed in burn-derived DCs. MLN DCs were isolated from day 3 post-burn and sham rats as described in the methods section and stimulated with plate bound anti-CD40 antibody for 24 h to determine induction of CD80 and MHC-II ligands on DCs. The co-stimulatory molecules i.e., CD80 and MHC-II are known to be specifically activated and expressed following anti-CD40 stimulation of DCs. Confocal data analyses in Fig. 6A and B (the y-axis shows % of DCs labeled cells) showed that stimulation of sham control MLN DC with anti-CD40 resulted in demonstrable increase (*p < 0.05) of CD80 expression (5+2% to 18+5%), while there was no statistically significant (**p > 0.05) in MHC-II expression (32+7% to 35+11%). Un-stimulated burn MLN DCs did show CD80 receptor expression (4±1%) and MHC-II (25±10%), however stimulation of burn MLN DCs with anti-CD40 stimulation caused the loss of CD80 and MHC-II receptor expression (Fig. 6B). We were not able to spot any significant numbers of cells that exhibited CD80 (∼2±1%) and/or MHC-II (∼2±1%) receptor uptake. The experiments were repeated thrice with reproducible results.

Fig. 6 — (A and B) Effect of anti-CD40 stimulation on MLN DCs: MLN DCs were isolated from day 3 post-burn and sham rats as described in the methods section and stimulated with plate bound anti-CD40 antibody for 24 h to determine induction of CD80 and MHC-II ligands on DCs. Bar graphs shows data as percentage of DCs from image analyses of confocal microscopy of the surface expression of MLN DC co-stimulatory molecules, i.e., CD80 and MHC-II on sham control (6A) and day 3 post-burn animals (6B). DCs obtained were stimulated with and without anti-CD40 antibody. Dendritic cells were then labeled with FITC- or PE-anti CD80 or -MHC-II antibody and the images were captured and analyzed by a confocal microscope as given in the methods. Briefly, 100 labeled anti-CD40-stimulated MLN DCs were counted from three separate slides from three different samples of sham control and day 3 post-burn samples. Similarly, 100-labeled DCs from un-stimulated control samples were also counted on separate slides. Expression of the co-stimulatory molecules was determined by the appropriate uptake of the label and counted as positive. The expression of surface molecules CD80 and MHC-II MLN DCs were counted by confocal visualization of fluorescent dye and is given as percentage labeled cells in the y-axis of the figure (% positive DCs). Black bars depicts anti-CD40-stimulated and gray bars show un-stimulated MLN DCs. Statistical analyses were performed on these percentage values, *p < 0.05 represents statistically significant, whereas, **p > 0.05 as statistically insignificant values.

4. Discussion

In the current study we have shown that a specific subset of rat MLN DCs OX62+OX6+OX35+ is dysfunctional following acute burn injury. This DC dysfunction is exhibited by down regulation of co-stimulatory molecules namely, i.e., MHC-II, CD80, CD86, and CD40, and the lack of ability of DCs to induce proliferation of CD4+ T cells present in MLN. Burn MLN had a cytokine milieu of excess anti-inflammatory cytokine (IL-10) and lack of pro-inflammatory cytokine (IL-12). Moreover, anti-CD40 stimulation of dysfunctional burn-derived MLN DCs did not up-regulate co-stimulatory surface receptor expression rather there was loss of CD80 and/or MHC-II receptor expression. This perturbed DC:T-cell interaction following acute burn injury is indicative of a local effector immune response expressed in gut-associated lymphoid tissues (GALT) such as MLN and probably not a generalized effector immune homeostasis observed in animal models of injury. This DC-mediated adaptive immune response seems to contribute to T-cell immunosuppression observed by others and us in studies of intestinal immune response following acute injury such as acute burn injury. As mentioned earlier, our studies were limited to GALT depicting a local intestinal immune response; we did not expand our studies to a general more robust immune effector response, which may occur in splenic or circulatory DCs and/or CD4+ T cells. To our knowledge this is the first demonstration of a defined DC subset, i.e., OX62+OX6+OX35+ that in acute-burn injury model was able to alter an immune response in the MLN CD4+ T cells of rats. The data agrees with our previous published data where we observed a similar immunosuppressive effect in MLN APCs and T cells in acute burn injury model. Different subsets of DCs have been described in rats; OX62+CD4+ and OX62+CD4− DC subset are found in rats’ afferent mesenteric lymph, and locate themselves in the MLN [10–13]. Although, previous studies indicated that DCs in close proximity to T cells in rat MLNs were CD4− and that they carried self tissue antigen derived from intestinal apoptotic epithelial cell remnants and presumably represented ‘tolerogenic’ DCs that inhibit T cell expansion [13–15], recent studies [16–18] have shown that both rat splenic OX62+CD4− and OX62+CD4+ DCs induced an allogeneic proliferative response in resting T cells, but that CD4− DCs were the main producers of IL-12 and responsible for the differentiation of naïve T cells into Th1 type T cells. The CD4+ DCs did not significantly produce IL-12 and produced Th2 cytokines. Although precise origin of the DC subsets is much less established in the rat than in the murine species, rats’ DCs seem to have comparable functional subsets as human and murine DCs. In our studies, we focused on the OX62+CD4+ subsets of DCs as they are more closely implicated in the regulation of CD4+ T cell priming and differentiation under conditions of bacterial infections as opposed to viral/helminthic infections. Maturity-related DC deficits are discernable in down-regulations of MHC-II and co-stimulatory molecules such as, CD80/86, CD40, and ICOSL, and to be found in DC that have migrated to the draining lymph nodes. Expression of cell surface molecules, MHC-II, CD80/86, CD40, and ICOSL were presumed to be up-regulated in control mice intestinal wall DC during their migration from locations such as lamina propria, Peyer's patches, to the draining MLNs [19–21]. We found that co-stimulatory molecules which are indicative of maturation of DCs were down-regulated following day-3 post-burn in the presence of high IL-10 and low IL-12 levels in the mesenteric lymph nodes. Similar effects were also reported in splenic DCs at 10-days post-burn mice, specifically affecting CD4+ T cells (5, 6). Furthermore, others also report dysfunctional DCs in burn and sepsis model (13, 14 and 19). Our data however suggests a disturbance in DCs at a much earlier time i.e., day 3 post-burn and more so in the absence of any apparent infection. In our hands, rat burn model do not have a coexisting infection at day 3 post-burn (data not shown). Nevertheless we have tried to use LPS stimulation to mimic the effects of infection but our results were not inconclusive. Thus, our study stands in contrast to most of the previous studies where disturbances in adaptive immune response mediated by DCs were demonstrated in an infection or sepsis model. In the current study we report dysfunctional MLN DCs in a rat model of acute burn injury without sepsis.

To further our study we then attempted to reverse dysfunctional DCs following acute burn injury by stimulating with anti-CD40, which is known to stimulate maturity markers on DCs. Our results suggested that although the MLN DCs obtained from sham control animals do show a response but such a response was absent in burn MLN DCs. We report either absence of CD80 and/or MHC-II or propose receptor shedding following burn injury. The initial presence of a positive anti-CD40 effect on sham control DCs where the existing levels of co-stimulatory molecules were found to be responsive and showed an obvious up-regulation of co-stimulatory molecules and then absence of such an affect following burn injury is in agreement of findings where anti-CD40 stimulation in mice caused loss of immune cells via apoptosis (22). We have shown in our previous studies MLN CD4+ T cell following burn-plus-sepsis injury are vulnerable to be deleted via apoptosis (1). The CD40 ligand (CD40L), a member of TNF family, is expressed on activated T cells; its counterpart CD40, a member of TNF-receptor family, is expressed on APCs [20]. Interactions between T cells’ CD40L and B cells’ CD40 are important in humoral immunity [21]. In addition, CD40 and CD40L interactions between T cells and other APCs (macrophages, DCs) lead to APCs’ up-regulation of CD80/86 molecules, and production of IL-12 which is a potent cytokine for generation of Th1 type of T cell responses [22]. T cells’ response subsequent to CD40L ligation by anti-CD40 was an early/short-term priming of TCR followed by cytokine production and proliferation, and a later induction of T cell unresponsiveness with up-regulation of cytokines, TGFβ and IL-10, and cell cycle disruption [23]. Several previous studies producing blockade of CD80/86 ligands by treatment of experimental animals with a soluble CTLA-4-Ig fusion protein, or of blockade in experimental animals of CD40L that prevents CD40 mediated induction of CD80/86 ligands, have shown a resulting hypo-responsiveness of T cells allowing for acceptance of allotransplanted solid organs by these animals [23]. Recent investigations have indicated that T cell unresponsiveness produced by CTLA-4-Ig treatment of animals, receiving solid organ transplantation, may not necessarily be due to CD80/86 blockade affecting CD28 co-stimulation but that it could be due to CTLA-4-Ig acting as a CTLA-4 mimic that binds and activates CD80/86 molecules on dendritic cells [17]. Such activation apparently leads to activation of dendritic cell intracellular enzyme, indoleamine 2,3-dioxygenase (IDO), which causes breakdown of tryptophan, and accumulation of the breakdown product kynurenine. Since tryptophan is required in the microenvironment of the T cells for their proliferation and their possible survival, and kynurenine could inhibit T cell proliferation, and promote their apoptosis [17], the CTLA-4-Ig mediated induction of IDO could effectively disturb T cell expansion. This action of CTLA-4-Ig on the dendritic cells appears to be mediated by induction of IFNγ, and IFNγ-mediated transcriptional up-regulation of IDO. IFNγ presumably acted on DCs in an autocrine/paracrine manner, and activated the STAT-1/NF-κB/p38 MAPK signaling pathway. As can be surmised from above discussion, studies now support the concept that APC/T cell receptor–ligand molecules allow for a bi-directional transmission of signals between APCs and T cells; CD80/86/CTLA-4 and CD40L/CD40 appear to be operating in this manner. Potential disturbances in CD80/CD86/CTLA-4 and CD40L/CD40 interactions may play role(s) in the T cell hypo-responsiveness in burn/sepsis injury conditions. CD40-triggerring in DCs occurs with the induction of CD40L expressed on activated T cell following stimulations from mature DCs; CD40L ligation of CD40 allows for feed-back reinforcement of the state of DC maturity in terms of optimum expression of CD80/86 and production of IL-12 to lead to CD4+T cell polarization to Th1 cells. Our data has suggested that there is a relative deficiency of CD40 expression on MLN DCs harvested from burn-injured rats; this may be a cause for attenuated IL-12 and possibly up-regulated expression and release of IL-10 from the DCs. In these experiments, OX62+CD4+ DCs (∼2–3 x 10⁴ cells) were isolated from MLNs of burn-injured rats. However, we determined IL-12 and/or IL-10 mRNA in MLN tissue samples of burn-injured and sham control animals. We tested use of anti-CD40 antibody-stimulation to reverse the dysfunction observed both in phenotypic expression of co-stimulatory molecules of MLN DCs as well as their ability to cause proliferation of CD4+ T cells. On the contrary our results showed absence or total loss of co-stimulatory molecules on burn rat MLN DCs as assessed by confocal microscopy image analysis. However anti-CD40-stimulation showed an effect on sham control MLN DCs where both basal and anti-CD40 caused levels of co-stimulatory molecules seemed to be up-regulated.

Dendritic cells are rare, heterogeneous population of hematopoietic cells. All enriched DC isolated from MLN may not be of lymphoid origin alone and there may be heterogeneity in the population studied. Previous investigators had co-cultured DC and T cells without additional growth media and found morphologically and functionally similar DC to freshly isolated DC. It is possible and is known that depletion of a subset of DC may occur at earlier time points post injury and this also affects T cell activation. The inhibitory effects of injured rat DCs on naïve T cells could be related to inadequacy of DCs’ maturation following their capturing/processing injury-related antigens in peripheral tissues. Under appropriate immune response conditions DC maturation is known to be triggered by microbial signals such as LPS/TLR ligands. However, a failure of maturation in burn injury conditions may result from potentially pathogenic microbial signals as well as tissue injury-related products, and/or inappropriate generation of inflammatory mediators. Maturity-related DC deficits are discernable as down regulations of MHC-II and co-stimulatory molecules such as, CD80/CD86, and CD40, and are found in DCs that have migrated to the draining lymph nodes. In addition to the possibility of DC immaturity, a triggering of CD80/86 on DCs via CTLA-4 molecules expressed on injured host T cells could lead to induction of indoleamine 2,3-dioxygenase (IDO) in DCs that could in turn inhibit T cell growth and promote apoptosis [16,23–27]. Thus, IDO induction in burn-injured rats’ DCs may be another or alternative cause of inhibition of T cells. Under the injury conditions, the lymph nodes may house not only those DCs that do not mature adequately and/or express IDO, but also DCs which might be damaged by their prior interactions with anergic T cells and/or regulatory T cells in peripheral tissues; anergic as well as regulatory T cells have been shown to interfere with the differentiation of DCs such that they do not adequately support IL-12 production and polarization of naïve T cells to Th1 type CD4+T cells [21,28]. An important mechanism of action of anergic/regulatory T cells on DCs is exerted through imprecise interactions between T cell's CD40L and DC’ CD40 [21]. Thus, there are at least three possibilities for burn-injury-related derangements in DCs: (1) immaturity leading to down-regulation of MHC-II/CD80, CD86/CD40/ICOS, (2) IDO induction and (3) defective DC response to CD40 triggering in terms of down regulation of IL-12 expression/production. In this study we have followed the first possibility, i.e., DC immaturity due to down-regulation of MHC-II/CD80/CD86 and CD40. IDO studies are currently in progress in our laboratory and we are investigating potential role of IDO in immunosuppression. We conclude, (1) burn injured rat MLN DCs have decreased surface expressions of co-stimulatory molecules, i.e., MHC-II, CD40, CD80 and CD86. (2) Decreases in co-stimulatory molecules may be responsible for impaired ability of burn DCs to prime MLN CD4+ T cell proliferation. (3) MLN DC dysfunction may be related to absence of co-stimulatory molecules especially CD80 and/or MHC-II. Overall, the results suggest burn injury causes disturbances in interactions between CD4+ T cells and DCs’ through primary and secondary co-stimulatory receptor/ligand molecules, contributing to CD4+T cell deficits in burn injured animals. This in turn may contribute to immunosuppression observed in burn-injury.

Acknowledgments

The author wishes to acknowledge generous support from the College of Pharmacy, and the CTRE Chicago State University grant to the author. Dr. Ashraf Ali, Director, FACS Facility of Biological Sciences Department of Chicago State University and also College of Pharmacy students doing APPE research electives in my laboratory.

Footnotes

^☆

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

References

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[bib2] 2.Fazal N, Raziuddin S, Khan S, Al-Ghoul WM. Antigen presenting cells (APCs) from thermally injured and/or septic rats modulate CD4 +T cell responses of naïve rat. Biochimica et Biophysica Acta. 2005;1762(1):46–53. doi: 10.1016/j.bbadis.2005.07.005. [DOI] [PubMed] [Google Scholar]

[bib3] 3.Shen H, de Almeida PE, Kang KH, Yao P, Chan CW. Burn injury triggered dysfunction in dendritic cell response to TLR9 activation and resulted in skewed T cell functions. PLoS One. 2012;7(11):e50238. doi: 10.1371/journal.pone.0050238. doi: 10.1371/journal.pone.0050238. Epub 2012 Nov 26. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib4] 4.Williams KN, Szilagyi A, He LK, Conrad P, Halerz M, Gamelli RL, Shankar R, Muthumalaiappan K. Dendritic cell depletion in burn patients is regulated by MafB expression. Journal of Burn Care and Research. 2012;33(6):747–758. doi: 10.1097/BCR.0b013e318250457f. [DOI] [PubMed] [Google Scholar]

[bib5] 5.Patenaude J, D'Elia M, Hamelin C, Bernier J. Selective effect of burn injury on splenic CD11c (+) dendritic cells and CD8alpha (+) CD4 (−) CD11c (+) dendritic cell subsets. Cellular and Molecular Life Sciences. 2010;67(8):1315–1329. doi: 10.1007/s00018-009-0251-8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib6] 6.Patenaude J, D'Elia M, Hamelin C, Garrel D, Bernier J. Burn injury induces a change in T cell homeostasis affecting preferentially CD4+ T cells. Journal of Leukocyte Biology. 2005;77(2):141–150. doi: 10.1189/jlb.0703314. [DOI] [PubMed] [Google Scholar]

[bib7] 7.Fujimi Satoshi, Peter H. Lapchak, Zang Yan, Malcolm P. MacConmara, Adrian A. Maung, Adam J. Delisle, John A. Mannick, James A. Lederer. Murine dendritic cell antigen-presenting cell function is not altered by burn injury. Journal of Leukocyte Biology. 2009;85(5):862–870. doi: 10.1189/jlb.0408257. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib8] 8.Shortman K, Liu YJ. Mouse and human dendritic cell subtypes. Nature Reviews Immunology. 2002;2(3):151–161. doi: 10.1038/nri746. [DOI] [PubMed] [Google Scholar]

[bib9] 9.Kapsenberg ML. Dendritic-cell control of pathogen-driven T-cell polarization. Nature Reviews Immunology. 2003;3(12):984–993. doi: 10.1038/nri1246. [DOI] [PubMed] [Google Scholar]

[bib10] 10.Yrlid U, Macpherson G. Phenotype and function of rat dendritic cell subsets. Acta Pathologica, Microbiologica et Immunologica Scandinavica. 2003;111(7–8):756–765. doi: 10.1034/j.1600-0463.2003.11107807.x. [DOI] [PubMed] [Google Scholar]

[bib11] 11.Turnbull E, MacPherson G. Immunobiology of dendritic cells in the rat. Immunological Reviews. 2001;184:58–68. doi: 10.1034/j.1600-065x.2001.1840106.x. [DOI] [PubMed] [Google Scholar]

[bib12] 12.Liu LM, MacPherson GG. Rat intestinal dendritic cells: immunostimulatory potency and phenotypic characterization. Immunology. 1995;85(1):88–93. [PMC free article] [PubMed] [Google Scholar]

[bib13] 13.Hsieh Chi-Hsun, Hsu Jun-Te, Hsieh Ya-Ching, Frink Michael, Raju Raghavan, J Hubbard William, I Bland Kirby, H Chaudry Irshad. Suppression of activation and costimulatory signaling in splenic CD4+ T Cells after trauma-hemorrhage reduces T-cell function. American Journal of Pathology. 2009;175(4):1504–1514. doi: 10.2353/ajpath.2009.081174. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib14] 14.Jobin N, Garrel DR, Bernier J. Increased burn-induced immunosuppression in lipopolysaccharide-resistant mice. Cellular Immunology. 2000;200(2):65–75. doi: 10.1006/cimm.2000.1619. [DOI] [PubMed] [Google Scholar]

[bib15] 15.Ramirez F, Mason D. Recirculatory and sessile CD4+ T lymphocytes differ on CD45RC expression. Journal of Immunology. 2000;165(4):1816–1823. doi: 10.4049/jimmunol.165.4.1816. [DOI] [PubMed] [Google Scholar]

[bib16] 16.Wesa A, Galy A. Increased production of pro-inflammatory cytokines and enhanced T cell responses after activation of human dendritic cells with IL-1 and CD40 ligand. BMC Immunology. 2002;3(1):14. doi: 10.1186/1471-2172-3-14. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib17] 17.Steinman RM, Nussenzweig MC. Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance. Proceedings of the National Academy of Sciences of the United States of America. 2002;99(1):351–358. doi: 10.1073/pnas.231606698. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib18] 18.Barlow Y. T lymphocytes and immunosuppression in the burned patient: a review. Burns. 1994;20(6):487–490. doi: 10.1016/0305-4179(94)90002-7. [DOI] [PubMed] [Google Scholar]

[bib19] 19.Duan X, Yarmush D, Leeder A, Yarmush ML, Mitchell RN. Burn-induced immunosuppression: attenuated T cell signaling independent of IFN-gamma- and nitric oxide-mediated pathways. Journal of Leukocyte Biology. 2008;83(2):305–313. doi: 10.1189/jlb.0407228. [DOI] [PubMed] [Google Scholar]

[bib20] 20.Finnerty CC, Przkora R, Herndon DN, Jeschke MG. Cytokine expression profile over time in burned mice. Cytokine. 2009;45(1):20–25. doi: 10.1016/j.cyto.2008.10.005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib21] 21.Schwacha MG. Macrophages and post-burn immune dysfunction. Burns. 2003;29(1):1–14. doi: 10.1016/s0305-4179(02)00187-0. [DOI] [PubMed] [Google Scholar]

[bib22] 22.Shigeta A, Tada Y, Wang JY, Ishizaki S, Tsuyusaki J, Yamauchi K, Kasahara Y, Iesato K, Tanabe N, Takiguchi Y, Sakamoto A, Tokuhisa T, Shibuya K, Hiroshima K, West J, Tatsumi K. CD40 amplifies Fas-mediated apoptosis: a mechanism contributing to emphysema. American Journal of Physiology - Lung Cellular and Molecular. 2012;303(2) doi: 10.1152/ajplung.00337.2011. [DOI] [PubMed] [Google Scholar]

[bib23] 23.Ni Choileain N, MacConmara M, Zang Y, Murphy TJ, Mannick JA, Lederer JA. Enhanced regulatory T cell activity is an element of the host response to injury. Journal of Immunology. 2006;176(1):225–236. doi: 10.4049/jimmunol.176.1.225. [DOI] [PubMed] [Google Scholar]

[bib24] 24.Cairns B, Maile R, Barnes CM, Frelinger JA, Meyer AA. Increased Toll-like receptor 4 expression on T cells may be a mechanism for enhanced T cell response late after burn injury. Journal of Trauma. 2006;61(2):293–298. doi: 10.1097/01.ta.0000228969.46633.bb. [discussion 298–9] [DOI] [PubMed] [Google Scholar]

[bib25] 25.Munn DH, Sharma MD, Mellor AL. Ligation of B7-1/B7-2 by human CD4 (+) T cells triggers indoleamine 2,3-dioxygenase activity in dendritic cells. Journal of Immunology. 2004;172(7):4100–4110. doi: 10.4049/jimmunol.172.7.4100. [DOI] [PubMed] [Google Scholar]

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PERMALINK

OX62+OX6+OX35+ rat dendritic cells are unable to prime CD4+ T cells for an effective immune response following acute burn injury^☆

Nadeem Fazal

Abstract

1. Introduction