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Published online before print June 14, 2004
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T cells in the regulation of neutrophil-mediated tissue damage after thermal injury
Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham
1 Correspondence: The University of Alabama at Birmingham, Center for Surgical Research, Department of Surgery, G094 Volker Hall, 1670 University Blvd., Birmingham, AL 35294-0019. E-mail: schwacha{at}uab.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T cells have been shown to regulate the inflammatory responses in tissues that are prone to neutrophil-mediated injury post-burn, their role in the induction of secondary tissue injury post-burn remains unknown. To study this, T cell-deficient ( TCR–/–) and wild-type (WT) mice were subjected to thermal injury or sham procedure, and tissue samples were isolated 1–24 h thereafter. Burn injury induced neutrophil accumulation in the lung and small intestines of WT mice at 1–3 h post-injury. No such increase in neutrophil tissue content was observed in TCR–/– mice. An increase in tissue wet/dry weight ratios was also observed in these organs at 3 h post-burn in WT but not in TCR–/– mice. A parallel increase in plasma and small intestine levels of the chemokines macrophage-inflammatory protein-1ß (chemokine ligand 4) and keratinocyte-derived chemokine (CXC chemokine ligand 1) were observed in injured WT mice but not in injured TCR–/– mice. Increased activation (CD120b expression) of the circulating T cell population was also observed at 3 h post-burn in WT mice. These results indicate the T cells, through the production of chemokines, play a central role in the initiation of neutrophil-mediated tissue damage post-burn.
Key Words: chemokine • CD120b • lung • small intestine • myeloperoxidase • liver • inflammation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-), interleukin-6, prostaglandin E2, and nitric oxide]. These mediators interact with the host to induce secondary tissue damage that contributes to the development of a systemic inflammatory response (SIRS) and subsequent multiple organ failure [1
2
3
4
]. These studies also indicate that immune dysfunction contributes to the subsequent development of SIRS, sepsis, and multiple organ failure. Experimental studies have been directed at elucidating a better understanding of the mechanisms responsible for thermal injury-induced immune dysfunction [5
6
7
8
9
]. Recent studies from our laboratory [10
] as well as early findings by others [11
, 12
] suggest an important role for T cells in the immune response to thermal injury. Under nonpathological conditions, T cells are present at low levels in peripheral lymphoid tissues (i.e., spleen); however, they are the predominant or even exclusive T lymphocyte population in epithelial-rich tissues [13
14
15
]. T cells of the T cell receptor (TCR) lineage are part of the innate immune system and have multiple functions including immune surveillance, wound repair, inflammation, and protection from malignancy [16
, 17
].
It has been established in clinical and experimental studies that thermal injury can induce remote organ injury at sites such as the lung, liver, and small intestines [18
19
20
21
22
23
24
25
]. Moreover, these burn-induced organ injuries appear to be primarily mediated by neutrophils [18
, 19
, 24
, 26
]. Boismenu et al. [27
] have shown that intraepithelial T cells produce a number of chemokines for recruitment of immune cells, such as neutrophils, to injured tissue and appear to play an important role in the maintenance of epithelial homeostasis. Experimental studies have documented that T cell-deficient mice have dysfunctional regulation of inflammatory responses in various tissues including the skin, gastrointestinal tract, and lung [28
29
30
]. Moreover, T cell-deficient mice are incapable of mounting a normal TNF- response in vitro or in vivo [31
]. Proinflammatory cytokines, such as TNF-, are potent activators of T cells [32
], and systemic levels of this cytokine as well as other proinflammatory mediators are elevated following thermal injury [33
, 34
]. The effect of systemic neutralization of TNF- post-burn was investigated by O’Riordain et al. [35
], who described improved survival in a model of thermal injury and sepsis. Thus, based on the role of T cells in the immunopathologic response to burn injury, the ability of T cells to produce chemokines and recruit immune cells to injured tissue, and the presence of remote organ injury post-burn, the present study was undertaken to determine the role of T cells in the induction of tissue damage following thermal injury.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene ( TCR–/–) and C57BL/6J wild-type (WT) mice (18–22 g; 8–10 weeks of age; Jackson Laboratories, Bar Harbor, ME) were used for all experiments. The mice were allowed to acclimatize in the animal facility for at least 1 week prior to experimentation. Animals were randomly assigned into a thermal-injury group or a sham-treatment group. The experiments in this study were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham and were performed in accordance with the National Institutes of Health (NIH) guidelines for the care and handling of laboratory animals.
Thermal injury procedure
Mice received a scald burn as described elsewhere [10
, 36
, 37
]. Briefly, the mice were anesthetized by intraperitoneal (i.p.) injection of ketamine/xylazine, and the dorsal surface was shaved. The animal was placed in a custom-insulated mold exposing 12.5% of their total body surface area (TBSA) along the right dorsum. The mold was immersed in 70°C water for 7 s to produce a full-thickness burn [38
]. An injury site covering 25% TBSA was induced by exposing the right and left dorsal surfaces. The mice were then resuscitated with 1 ml Ringer’s lactate solution administered by i.p. injection and returned to their cages. The cages were placed on a heating pad for 2 h until the mice were fully awake, at which time they were returned to the animal facility. Sham treatment consisted of resuscitation with Ringer’s lactate solution only. Lethality in this thermal injury model was not significant.
Myeloperoxidase (MPO) assay
At 1, 3, 6, 12, and 24 h after thermal injury, tissue samples were collected (i.e., lung, intestine, liver) and snap-frozen in liquid nitrogen. The accumulation of neutrophils was assessed by measuring MPO activity, as described previously with minor modifications [39
, 40
]. Samples from sham animals were collected at 3 h post-procedure. In brief, frozen tissue samples were thawed and suspended in 10 vol phosphate buffer (pH 6.0) containing 1% of hexadecyltrimethylammonium bromide. The samples were homogenized on ice (Wheaton overhead stirrer, Wheaton Instruments, Millville, NJ), sonicated (15 s) at maximum power, and freeze-thawed two times. The homogenates were then centrifuged at 12,000 g for 20 min at 4°C. An aliquot of the tissue homogenate (30 µl) was added to 180 µl phosphate buffer (pH 6.0) containing 0.167 mg/ml o-dianisidine dihydrochloride (INC Biomedicals Inc., Aurora, OH) and 0.0005% hydrogen peroxide. The change in absorbance at 460 nm at 25°C was measured spectrophotometrically for 10 min using an enzyme-linked immunosorbent assay (ELISA) plate-reader (Bio-tek Instruments, Inc., Winooski, VT). MPO activity was calculated using a standard curve generated with human MPO (Sigma Chemical Co., St. Louis, MO). Results are expressed as the change in absorbance (
mOD)/min/mg protein. Protein determination in the final homogenate was determined using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA).
Determination of wet-to-dry weight ratios
Wet-to-dry weight ratios of lung, liver, and small intestines were used as a measure of tissue edema and damage. Tissue samples were weighed (wet weight) and then subjected to desiccation for 48 h using a SpeedVac Plus (Savant, Framingdale, NY) until a stable dry weight was achieved, and wet-to-dry weight ratios were determined.
Determination of chemokine levels
Plasma and tissue samples were collected from mice at 3 h after thermal injury or sham procedure. Tissue samples were snap-frozen in liquid nitrogen and stored along with plasma at –80°C until analysis. Tissue samples were homogenized in protease inhibitor cocktail (Calbiochem, La Jolla, CA) as described by Faunce et al. [41
] prior to analysis of keratinocyte-derived cytokine (KC; CXC chemokine ligand 1), macrophage inflammatory protein (MIP)-1 [chemokine ligand 3 (CCL3)], MIP-1ß (CCL4), and lymphotactin (XCL-1) content by ELISA, according to the manufacturer’s recommendations (R&D Systems, Minneapolis, MN). Chemokine levels in the tissues were normalized to mg total protein, as determined using the Bio-Rad protein assay (Bio-Rad Laboratories). Plasma samples were diluted 1:5 in assay buffer, and chemokine content was determined by ELISA.
Determination of hematological parameters
Anticoagulated whole blood was obtained from WT mice and 3 h after thermal injury or sham procedure, by cardiac puncture. Samples were subjected to hematological analysis with a Hemavet® multispecies hematology system counter 1500R (CDC Technologies Inc., Oxford, CT) as described elsewhere [42
].
Determination of T cell activation
Anticoagulated whole blood was stained with a combination of antibodies conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE) to assess T cell activation. The manufacturer’s lysed whole blood method was used (BD Biosciences PharMingen, San Diego, CA). In brief, 100–200 µl hepranized whole blood was added per tube and incubated with 10–20 µl antibody for 30 min at room temperature in the dark. Nonspecific binding was prevented by the prior addition of 10 µl Fc-Block® (BD Biosciences PharMingen) to the tube. Erythrocytes were lysed with FACSLyse solution (BD Biosciences PharMingen), and the remaining cells were pelleted by centrifugation (200 g for 5 min). The cells were washed with 2 ml washing solution [phosphate-buffered saline (PBS) containing 1% fetal bovine serum and 0.1% sodium azide], pelleted by centrifugation, and stored at 4°C in the dark in 100 µl PBS containing 2% formaldehyde until analysis by fluorescein-activated cell sorter. Cells were stained with FITC-conjugated anti-mouse TCR [clone GL3, Armenian hamster immunoglobulin G2 (IgG2), 
, BD Biosciences PharMingen], PE-conjugated anti-mouse CD120b [TNF receptor type II (TNFRII)/p75, clone TR75-89, Armenian hamster IgG1, 
, BD Biosciences PharMingen), or PE-conjugated CD3
(clone 500A2, Syrian hamster IgG2, , BD Biosciences PharMingen) alone or in combination. Appropriate isotype controls were included to assess nonspecific staining. The entire lymphocyte and monocyte population (as determined for forward- and side-scatter) was gated to include the presumptive large granular lymphocyte (LGL) population in the analysis. Location of the T cell-positive populations in forward- and side-scatter plots was determined by back-gating on CD3-positive cells. FITC and PE were analyzed with a Becton Dickinson FACSort flow cytometer (San Jose, CA). A minimum of 10,000 events was collected, and WinMDI 2.8 software (Joseph Trotter, Scripps Research Institute, La Jolla, CA) was used to analyze the results.
Statistical analysis
Data are expressed as mean ± SEM unless noted otherwise, and comparisons were analyzed using ANOVA and Tukey’s test for multiple comparisons. A P value of <0.05 was considered to be statistically significant for all analyses.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T cell-deficient mice following thermal injury. Thermal injury did not alter MPO activity in the liver (Fig. 1C)
. Based on these preliminary findings in the lung and small intestines, all subsequent studies were conducted at 3 h after thermal injury.
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T cells did not display an increase in lung MPO activity post-injury. Similarly, a threefold increase in MPO activity in the small intestines was observed in WT mice, whereas T cell-deficient mice displayed no such increase in MPO content post-burn (Fig. 2B) . No change hepatic MPO content at 3 h post-burn was observed, irrespective of the presence or absence of T cells (data not shown).
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20% (P<0.05) in lung wet/dry ratio was observed in WT mice after thermal injury, whereas a 10% increase (P<0.05) in small intestine wet/dry weight ratio was observed under those conditions. Mice lacking T cells did not display tissue edema in the lung and small intestines post-burn.
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/ T cells on plasma and tissue chemokine levels T cell-deficient mice. In contrast, a significant (P<0.05) elevation in plasma levels of MIP-1ß and KC was observed in injured WT mice. Plasma levels of MIP-1ß were not altered in T cell-deficient mice post-injury, whereas KC levels were elevated under those conditions (P<0.05). Nonetheless, the increased plasma levels of KC in the T cell-deficient mice post-injury were significantly less (P<0.05) than that of WT mice that were thermally injured.
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, MIP-1ß, KC) levels were not altered by burn injury or T cell deficiency (Fig. 3A
3B
3C
3D)
. In contrast, a significant elevation (P<0.05) in small intestine levels of chemokines was observed in WT mice post-injury (Fig. 3E
3F
3G
3H)
. Mice deficient in T cells did not express elevated chemokine levels in the small intestines post-burn.
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T cell activation in WT mice post-burn
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TCR and CD120b was significantly greater in injured mice (P<0.05; Fig. 4C
4D
4E
). Back-gating on CD3-positive cells verified that the population of cells positive for TCR and CD120b was also in the region positive for the T cell marker CD3 (Fig. 4A)
. The percentage of gated cells positive for TCR significantly increased (P<0.05) from 3.2 ± 0.8% in shams to 12.0 ± 4.5% in burns (mean±SEM; n=five to six mice/group). The ratio of CD120b+ T cells to CD120b– T cells increased from 3.2 to 5.8 following burn injury; however, this increase was not statistically significant.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T cells produce a number of chemokines for recruitment of such cells [27
, 44
]. Chemokines are low molecular weight (8–10 kDa) proinflammatory cytokines that participate in at least three phases of leukocyte recruitment. They foster adhesion to the vascular endothelium via activation of integrin receptors, they induce diapedis through the endothelial junctions and underlying tissue to the inflammatory foci, and they activate leukocyte effector functions including production of reactive oxygen intermediates and exocytosis of degradative enzymes [45
]. The potential roles of T cells in this process post-burn were addressed in the present study using mice genetically deficient in T cells. Neutrophil accumulation and tissue injury (i.e., edema) were not observed following thermal injury in the lungs and small intestine of mice lacking T cells. Similarly, at 3 h post-injury, an increase in plasma and small intestine chemokine (MIP-1ß, KC) levels was observed in normal mice, which were markedly attenuated in mice lacking T cells. In WT mice, the circulating T cell population was increased at 3 h post-burn and displayed an elevated activation status (i.e., increased CD120b expression).
The involvement of T cells in a wide variety of disease processes is indicative of an important role for this T cell subset in innate and acquired immunity [14
, 46
]. The data presented here suggest a causative relationship between T cell activation and chemokine production and the post-burn inflammatory response. T cell activation is a complex process that not only involves engagement of the TCR with ligand but also myriad adhesion molecules, cytokines, and costimulatory ligands [47
]. TNF- has been shown to be a mediator of early T cell activation, suggesting that it may serve as a point of regulation of cellular function/activation [47
]. Cellular responses to TNF- are mediated by two functionally distinct receptors, p55 (TNFR1, CD120a) and p75 (TNFR2, CD120b), which are independently expressed on the cell surface [48
]. It is interesting that T cells of the TCR lineage respond more robustly to TNF- than ß T cells [32
]. The increased responsiveness of T cells to TNF- is dependent on the presence of CD120b, which is highly inducible on this T cell subset [32
, 48
]. CD120b is the high-affinity receptor that is primarily expressed by cells of hematopoietic lineage and signals thymocyte and peripheral T cell activation and proliferation as well as natural killer cell activation [49
, 50
]. In addition, studies have shown that increased CD120b expression lowers the threshold of T cell activation [47
]. Our findings demonstrate that circulating T cells expressed elevated levels of CD120b at 3 h post-burn, suggesting an increased state of activation. Although it is unclear whether tissue T cells in the lung and small intestine are in an activated state, the findings of tissue injury and in part elevated chemokine levels are highly suggestive of this notion.
Studies in humans have shown an increased number of T cells in the spleens of patients with hairy-cell leukemia [51
]. This would appear to indicate that circulating T cells are susceptible to chemotactic gradients and can be targeted to damaged tissue [52
]. In addition, circulating and resident T cells express chemokine receptors that may allow them to extravasate and migrate to sites of tissue damage as proposed by Ferrarini et al. [52
]. As T cells, upon activation, can produce chemokines, they may contribute to the recruitment of additional circulating T cells to sites of tissue damage [27
, 52
]. These capacities of this unique T cell subset are consistent with their role in immune surveillance [15
, 46
, 52
]. Nonetheless, it remains to be determined definitively whether trafficking of T cells from the circulation to damaged tissue occurs early post-burn.
The role of chemokines as mediators of neutrophil recruitment into sites of inflammation is an area of intensive interest [53
]. Boismenu et al. [27
] demonstrated that T cells, upon activation, produce the chemokines MIP-1, MIP-1ß, and XCL-1. More recently, studies by Cardona et al. [54
] demonstrated a strong correlation between chemokine expression and the magnitude of the inflammatory response in a murine model of neurocysticercosis. These studies showed that TCR–/– mice displayed attenuated inflammation and decreased chemokine expression. Studies have also shown that T cells from macaques monkeys produce the ß chemokines MIP-1 and MIP-1ß in response to simian immunodeficiency virus infection [55
]. Our findings presented here indicate that in "nontraditional" disease states, such as traumatic injury, T cells are also central in orchestrating and mediating the inflammatory and immune response. This is not surprising, as this T cell subset has been shown previously to be involved in wound repair (i.e., tissue damage) [16
, 17
].
In the present study, it was somewhat unexpected that chemokine levels were not elevated in the lung at 3 h post-injury in parallel with elevated neutrophil/MPO levels and tissue edema. This observation may be a result of the time post-injury that chemokine levels were assessed (3 h), in that at earlier times post-injury (0–3 h), chemokine levels would be elevated, initiating immune cell recruitment to the lung. This concept would be consistent with the development of a chemotactic gradient early post-injury prior to the actual sequestration of immune cells to tissues [56
]. In addition, the observation that the gut responded differently than the lungs to thermal injury is not surprising as a result of bacterial translocation from the gut early post-burn and compartmentalization of the inflammatory response under such conditions [21
, 57
, 58
]. Nonetheless, studies have suggested a link between gut and lung injury post-burn [59
]. It remains to be determined what role, if any, T cells have in this phenomena. The elevation of MIP-1ß and KC levels in the systemic circulation post-burn was in part T cell-dependent, suggesting that these chemokines are likely to be the central mediators of T cell-dependent neutrophil sequestration. It is surprising that XCL-1 levels were suppressed post-burn in WT and TCR–/– mice. This may be related to multiple factors including transient expression of this chemokine by T cells [60
], suppression by elevated glucocorticoid levels post-burn [1
, 61
], and/or depletion from the circulation by activated neutrophils expressing XCR-1 [62
]. A limitation of the present study is that only an association between T cell activation and chemokine production post-burn was demonstrated. It cannot be excluded that T cells are also important in the regulation of chemokine production by other cell types post-injury. Nonetheless, as previous studies have clearly demonstrated that this unique T cell subset produces chemokines upon activation [27
, 44
, 63
], it is highly probable that T cells are the chemokine-producing cells in our murine burn model. Future studies will need to be directed at elucidating the direct and indirect relationships between T cells and chemokine production post-injury.
The observed changes in T cell activation appear to be independent of changes in the number of circulating lymphocytes post-burn. A trend toward increased WBC counts in the blood was observed post-burn, predominantly as a result of increased neutrophil counts in the blood, consistent with the inflammatory state observed post-burn in patients [64
, 65
]. Moreover, the murine burn model we used clearly demonstrated plasma leak post-burn with significant increases in Hct and RBC levels, consistent with clinical findings with regard to these changes in homeostasis post-burn [65
].
The findings presented here further support a role for T cell in post-burn immunopathology [10
]. Overall, activation of T cell early post-injury is likely to be protective, although it is associated with detrimental aspects, such as neutrophil-mediated tissue damage in the lung and gut as presented here. As T cells are central in the development of subsequent macrophage hyperactivity post-burn [1
, 10
], it can be speculated that this T cell-dependent tissue injury may be in part causative via the activation of inflammatory cascades or generation of other mediators. In conclusion, activation of T cells post-burn appears to lead to the production of chemokines in epithelial-rich tissues, such as small intestines, leading to neutrophil sequestration and subsequent secondary tissue damage post-burn. Clearly, additional studies are needed to develop a more comprehensive understanding of this unique T cell subset and their role in post-injury immunopathology. Such studies will likely contribute to improved therapeutic regimes for trauma and burn patients leading to decreased morbidity and mortality.
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ACKNOWLEDGEMENTS |
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T cell activation post-burn. Received April 2, 2004; revised May 6, 2004; accepted May 14, 2004.
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T cells by isopentenyl pyrophosphate and regulation by cytokines Blood 95,39-47This article has been cited by other articles:
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