Regular ArticleLymphokine Activated Killer Cells Enhance IL-2 Prevention of Sepsis-Related Death in a Murine Model of Thermal Injury
Abstract
It has previously been shown by this laboratory that immunomodulation of thermally injured animals with low-dose interleukin-2 (IL-2) and indomethacin (Indo) improves survival following septic challenge. Lymphokine-activated killer (LAK) cells have been shown to be effective in certain viral infections and to act in synergy with IL-2 in the treatment of certain types of cancer. We have studied the effect of LAK cells in combination with IL-2 and Indo in a murine model of thermal injury and sepsis. Male A/J mice received a 25% scald burn injury or sham burn and were randomized into five groups: (a) sham/vehicle, (b) burn/vehicle, (c) burn/IL-2 (250 U) + Indo (5 μg), (d) burn/LAK cells (2 × 106 cells), or (e) burn/LAK cells + IL-2 + Indo and were treated accordingly for 6 days following injury. LAK cells were generated by in vitro IL-2 treatment of syngeneic spleen cells for 72 hr and cytotoxic activity was confirmed by standard 51Cr release assay using natural killer (NK)-sensitive and NK-resistant targets. In the groups receiving LAK cells they were administered on Day 1 and Day 6 postinjury. On Day 10, septic challenge by cecal ligation and puncture (CLP) or splenectomy, for in vitro studies, was performed. Five-day survival after CLP was 80% in the sham/vehicle group compared to 0% in the burn/vehicle group (P < 0.01). IL-2/Indo and LAK/IL-2/Indo improved survival to 25% (P < 0.05) and 57.1% (P < 0.01), respectively. In vitro lymphocyte IL-2 production in response to mitogens was increased in the LAK/IL-2/Indo group compared with the burn/vehicle group (3 U/ml vs 2 U/ml, respectively, P < 0.05). IL-2/Indo alone had no significant effect on these in vitro parameters. As shown previously, monokine (IL-1, IL-6, and TNF) secretion by adherent splenocytes in response to LPS was markedly increased following thermal injury; no change was observed with any of the therapeutic protocols. We conclude that combined LAK/IL-2/Indo therapy is an effective regime for reduction of sepsis-related mortality and that LAK cells act synergistically with IL-2 to improve in vitro parameters of immune function in this model.
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Immuno-suppression secondary to sepsis
1999, Reanimation UrgencesThe effector component of the cytotoxic T-lymphocyte response has a biphasic pattern after burn injury
1998, Journal of Surgical ResearchIntroduction.Burn injury delays allograft rejection and impairs the host defense against infection. These functions are mediated via the cytotoxic T-lymphocyte (CTL) response. The CTL response is divided into antigen recognition/processing and effector phases. Presensitization allows selective analysis of changes, induced by burn injury, in the effector limb of the CTL response in relation to time and burn size.
Methods.Anesthetized CBA mice were primed with either a flank allograft from C57BL/6 (B6) mice or an autograft (negative control). Five weeks after grafting, animals were anesthetized and received either a 0, 20, or 40% burn. Spleens were harvested 3, 7, 10, and 14 days after burn injury (n= 96), cocultured with B6 stimulator splenocytes, and assessed for CTL response to radiolabeled allogeneic targets in a51Cr release assay. In experiment 2, spleens were harvested from unburned and 40% burned animals on Postburn Days 3 and 14. After triple staining, cells were analyzed by flow cytometry for CD4, CD8, and CD25 antigens. In experiment 3, splenocytes from 0 and 40% burned animals on Postburn Days 3 and 14, were cocultured with B6 stimulators for 5 days. Supernatants were evaluated for interleukin (IL)-2, IL-5, and interferon-γ (IFN-γ) using ELISA.
Results.The CTL response for 20 and 40% burned animals decreased 3 days postburn (−11.9 and −30.1%,P< 0.05), returned to baseline in 7–10 days, and was increased by 14 days postburn (15.8 and 22.6%,P< 0.05). The T-helper lymphocyte population (CD4) from 40% burn animals was significantly decreased on Postburn Days 3 and 14 (10.12 ± 0.45% vs 11.78 ± 0.29% and 10.19 ± 0.24% vs 14.21 ± 0.97%, respectively,P< 0.05). The CTL effector (CD8) splenocyte population was significantly higher in the burned animals on Postburn Day 14 (4.55% vs 3.71%,P< 0.05). On Postburn Day 3, average IL-5 production was higher in the burned animals (1.80 pg/ml vs 0.59 pg/ml, respectively,P< 0.05). The burn group, on Postburn Days 3 and 14, showed a decrease in mean IL-2 production (212.81 pg/ml vs 263.6 pg/ml and 342.7 pg/ml vs 421.4 pg/ml, respectively,P< 0.05). Mean IFN-γ production on Postburn Days 3 and 14 was decreased in burned mice (263.75 pg/ml vs 285.57 pg/ml and 218.16 pg/ml vs 263.42 pg/ml,P< 0.05).
Conclusions.Burn injury impairs the effector limb of the CTL response as a function of burn size in the immediate postburn period. CTL activity returns to baseline within 7–10 days postburn and has a rebound increase by Day 14. Early CTL suppression, after burn injury, may be due to a decrease in the T-helper subpopulation. The late increase in cytotoxicity may be secondary to an increase in the effector CTL population in the late postburn period. Burn injury causes a T-helper-2 phenotype as demonstrated by depressed IL-2 and IFN-γ production and increased IL-5 production.
Immunosuppression following thermal injury: The pathogenesis of immunodysfunction
1997, British Journal of Plastic SurgeryBackground. Cytotoxic lymphocytes (CTLs) are an important component of immune function, involved in antigen recognition and resistance to viral infection. Burn injury suppresses cell-mediated immunity, induces allograft tolerance, and increases the risk of viral infection, but the mechanisms are not well understood. This study analyzes the effect of burn size and burn wound excision on CTL activity.
Methods. Anesthetized CBA mice (n=12) received a 0%, 20%, or 40% body surface area contact burn. Additional mice (n=16) received a 40% burn that was totally, partially, or not excised 72 hours after burn. Excised areas were covered with normal, syngeneic skin. Two weeks later harvested splenocytes were cocultured with allogeneic stimulators. CTL activity was determined by a 51Cr release assay, in which CTL effectors were tested on allogeneic, radiolabeled targets. Dilution curves of CTL activity were compared by ANOVA.
Results. Both 20% and 40% burns significantly inhibited CTL activity (p<0.05). Total but not partial excision of a 40% burn restored CTL activity (p<0.01). Both total and partial wound excision also improved survival (p<0.05).
Conclusions. Burn injury inhibits CTL activity in a size-dependent manner, and total wound excision significantly improves both CTL function and survival after injury. This study suggests a mechanism for the immunosuppressive effects of burn injury and provides an immunologic rationale for early, complete burn wound excision.
Compartmentalization of the inflammatory response in sepsis and SIRS
2006, Journal of Endotoxin Research