EAST 2014 PLENARY PAPER
In vitro transfusion of red blood cells results in decreased cytokine production by human T cells Kristin Long, MD, Jerold Woodward, PhD, Levi Procter, MD, Marty Ward, MS, Cindy Meier, MS, Dennis Williams, MD, and Andrew Bernard, MD, Lexington, Kentucky
BACKGROUND: Transfusion-related immunomodulation consists of both proinflammatory and anti-inflammatory responses after transfusion of blood products. Stored red blood cells (RBCs) suppress human T-cell proliferation in vitro, but the mechanism remains unknown. We hypothesized that cytokine synthesis by T cells may be inhibited when stored RBCs are present and that suppression between fresh and stored RBCs would be different. METHODS: Purified human T cells were stimulated to proliferate with anti-CD3/anti-CD28 and then exposed to stored or fresh RBCs. Cells were placed in culture for 5 days. Cell culture supernatants were analyzed for the production of typical T-cell cytokines using multianalyte ELISArray kits. RESULTS: Stimulated T cells proliferated. RBC exposure markedly suppressed this proliferation. Interleukin 10, interleukin 17a, interferon F, tumor necrosis factor >, and granulocyte macrophage colony-stimulating factor were increased in response to stimulation but depressed in the presence of stored RBCs. The use of fresh RBCs also resulted in depression of these cytokines when compared with stimulated T cells with no RBCs; however, this depression was less pronounced. CONCLUSION: T-cell activation is associated with both proinflammatory and anti-inflammatory cytokine release, comparable with patterns seen in trauma and acute injury. All of these responses are depressed by an exposure to stored RBCs. Decreased levels of these cytokines after RBC transfusion represents a potential contributor to the immunosuppressive complications seen in trauma patients after transfusion. This provides insight for future mechanistic studies to delineate the role of RBC transfusion in transfusion-related immunomodulation. (J Trauma Acute Care Surg. 2014;77: 198Y201. Copyright * 2014 by Lippincott Williams & Wilkins) KEY WORDS: Transfusion; immunomodulation; cytokine; blood.
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ransfusion of packed red blood cells (PRBCs) results in immunomodulation, including both suppressive and stimulatory responses. Postoperative patients receiving as little as 1 U of PRBC transfusion have immunosuppressive sequelae, including increased infection, length of stay, mechanical ventilator days, and mortality.1 At the cellular level, PRBC transfusion has been shown to suppress T-cell proliferation.2 The major focus of clinical research into this immunomodulation has been the duration of RBC storage and increased morbidity and mortality with older units.3 Storage duration has been shown to enhance both the immunologic derangements and the harmful postoperative outcomes in clinical practice.4Y6 Effects of PRBC less than 7 days old in comparison with those stored 7 days to 30 days have been reported; however, these studies used blood that has been processed by traditional blood bank protocols.7Y9 Murine studies have shown that fresh, unprocessed blood may abrogate some of the negative immune effects of stored RBCs, and in vitro Submitted: December 9, 2013, Revised: April 21, 2014, Accepted: April 28, 2014. From the Section on Trauma and Acute Care Surgery (L.P., C.M., A.B.), Division of General Surgery (K.L.), Department of Microbiology, Immunology and Molecular Genetics (J.W., M.W.), and Department of Pathology (D.W.), University of Kentucky, Lexington, Kentucky. This study was presented in the Raymond Alexander Paper Competition at the 27th Eastern Association for the Surgery of Trauma Annual Scientific Assembly, January 14Y18, 2014, in Naples Florida. Address for reprints: Kristin Long, MD, Department of General Surgery, University of Kentucky, 800 Rose St, Lexington KY 40536; email: [email protected]. DOI: 10.1097/TA.0000000000000330
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studies with human T cells lend support to these data.10 Previous data from this laboratory have shown that proliferation of human T cells is markedly suppressed after exposure to stored RBCs and that production of interleukin 2 (IL-2), an autocrine growth factor necessary for T-cell proliferation, is diminished after PRBC transfusion. Addition of exogenous IL-2 did not rescue T-cell proliferation in these studies.11 A multihit hypothesis for transfusion-related immunomodulation (TRIM) suggests that the combination of leukocyte contamination, storage time, and cytokine derangements all contribute to the immunosuppressive effects.12 This led us to hypothesize that cytokine production by stimulated human T cells would be inhibited by the addition of RBCs, as was noted in our previous experiments with IL-2. Likewise, given the ability of fresh RBCs to mitigate this suppression, we hypothesized that a difference in cytokine production by T cells would be noted between stored and fresh RBCs. This study sought to analyze profiles of prototypical T-cell cytokines after in vitro transfusion of RBCs (both fresh and stored) in coculture with stimulated human T cells, to further identify immunosuppressive and immunoinflammatory cytokine patterns and differences between cells exposed to fresh or stored RBCs.
MATERIALS AND METHODS T-cell Isolation and Culture Human peripheral blood monocytes obtained from volunteer blood donors (Kentucky Blood Center, Lexington, J Trauma Acute Care Surg Volume 77, Number 2
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
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Kentucky) using an institutional review board (IRB)Yexempt protocol were separated using Ficoll-Hypaque gradient. T cells were isolated from peripheral blood monocyte by depletion of all nonYT cells using a magnetic bead Pan T cell kit (Miltenyi Biotec, Auburn, CA) and an autoMACS Separator and plated at 1.0 106 cells/mL. Cells were cultured in RPMI 1640 media (Roswell Park Memorial Institute, RPMI, Life Technologies, Carlsbad, CA) supplemented with penicillin/streptomycin, 10% heat-inactivated fetal bovine serum, and further supplemented with MEM nonessential amino acid solution, sodium pyruvate, and glutamine because of longer continuous incubation periods, hence forth termed CRPMI [complete RPMI]. Cells were stimulated with plate bound anti-CD3 (1-Kg/mL OKT-3, Ortho Pharmaceutical, Raritan, NJ) and anti-CD28 (100 ng/mL, Becton Dickinson, San Jose, CA). All cultures were maintained at 37-C and 5% CO2.
Packed RBC Cultures To simulate transfusion units, allogeneic human PRBCs (O negative) were obtained from the University of Kentucky Hospital blood bank under an IRB-exempted protocol. All units obtained, both autologous and allogeneic, were leukoreduced and stored in the standard solutions of citrate-phosphate-dextrose and Optisol (crystalloid solution of sodium chloride, dextrose, adenine, and mannitol). None of the units were irradiated. PRBC (2-week to 3-week storage duration unless specified otherwise) were added to T-cell cultures. Acceptable initial hematocrits for blood bank PRBC specimens used for experiments were 60% to 80%, and samples were further diluted in CRPMI to 0.0365% to 2% by volume as shown in figure legends. The number of PRBC per well were standardized such that 2% hematocrit equated to 193 T 17 PRBCs per 1 T cell and followed by serial dilutions. For carboxyfluorescein diacetate succinimidyl ester (CFSE) staining and culture, RBCs were counted by light microscopy using a hemocytometer and diluted to an initial concentration of 2 108/mL (100 PRBCs per 1 T cell), followed by serial dilutions.
Fresh RBC Cultures Fresh RBCs were obtained from volunteer donors through an IRB-approved protocol (12-0966-P6H) by peripheral venipuncture using sterile technique. Approximately 2.5 mL of venous blood was collected, centrifuged to remove plasma, separated over a Ficoll-Hypaque density gradient, and washed of residual Ficoll to isolate erythrocytes. The RBCs were then counted by light microscopy using a hemocytometer and diluted to initial concentrations of 2 108/mL (100 RBCs per 1 T cell), followed by serial dilutions as in the PRBC experiments.
Long et al.
For CFSE experiments, T cells were initially suspended in phosphate-buffered saline/0.1% fetal bovine serum at a concentration of 5.0 106 cells/mL. CFSE 5 mM was prepared by dissolving one vial of stock CFSE solution (CellTrace, Grand Island, NY) with 18-KL DMSO. CFSE was then added at a volume of 0.25 KL per millimeter of T cells, rapidly inverted, and stained for 10 minutes at room temperature in the dark. Staining was quenched by the addition of excess CRPMI. Cells were centrifuged at 300 G for 10 minutes at 18-C, supernatant was removed, and cells were recounted before suspension at a final concentration of 2.0 106 cells/mL. CFSE-stained T cells were stored in the dark at room temperature until ready for plating.
Flow Cytometry CFSE-labeled T cells, after treatment and incubation, were harvested and stained with anti-CD4 PerCP (eBioscience, San Diego, CA) for 30 minutes at room temperature in the dark. Cells were then washed, resuspended in phosphate-buffered saline, and placed on ice for immediate flow cytometry analysis.
Cytokine Analysis After 5 days in culture, the supernatant from each well of 24-well plates from the experiments was aspirated into 1-mL sterile tubes and frozen at j80-C until the time of analysis. Multiple donors, both fresh and stored, were used in these experiments (three separate stored PRBC donors, five separate fresh donors). For the analysis, the samples were thawed on ice and then analyzed for a series of inflammatory cytokines (IL-10, IL-17a, interferon F [IFN-F], tumor necrosis factor > [TNF->], and granulocyte macrophage colony-stimulating factor [GM-CSF]) using a Multi-Analyte ELISArray Kit (Catalog Number MEH-004A, SA Biosciences, Valencia, CA) according to the manufacturer’s instructions, and the absorbance at 450 nm was read within 30 minutes of stopping the reaction.
Data Analysis Absorbance values at 450 nm for each sample are presented in their corrected form (original value with negative standard value subtracted). Data were analyzed using Microsoft Excel (Redmond, WA). Statistical differences were calculated using two-tailed Student’s t tests, with p G 0.05 representing statistical significance.
RESULTS
Proliferation Assay
RBC Exposure Suppresses Human T-cell Proliferation Stimulated Human T cells Produce IL-10, IL-17a, TNF->, GM-CSF, and IFN-F
Purified human T cells (1 106/mL) with PRBC cultures were incubated in 96-well or 24-well plates for a total of 72 hours. 3H-thymidine was added for the final 18 hours to 20 hours of incubation at 1 KCi per well, cells were harvested on Perkin Elmer unifilter plates, and cell proliferation was determined by scintillation counting.
Five-day supernatants from a CFSE dye dilution T-cell proliferation assay previously published10 were harvested and stored at j80-C. Purified human T cells were stimulated to proliferate with anti-CD3/CD28. Supernatants from these experiments were analyzed for cytokine production and demonstrated an expected increase in production of several cytokines
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Figure 1. Cytokine production by stimulated human T cells decreases after exposure to RBCs. Cell culture supernatants from proliferation assays were analyzed for levels of proinflammatory and anti-inflammatory cytokine production. Supernatants harvested from the experiment are depicted here. x-axis represents individual cytokines tested, and bars demonstrate average absorbance values. y-axis represents absorbance at 450 nm. Unstimulated human T cells produced essentially no detectable cytokines. Stimulated T cells produced near-maximal amounts of each tested cytokine (relative to each positive control in enzyme-linked immunosorbent assay). With the exception of IFN-F, all cytokines tested showed a dramatic decrease in production after addition of stored RBCs. The use of fresh, unprocessed RBCs partially mitigated this suppression.
(IL-10, IL-17a, TNF->, GM-CSF, and IFN-F) when compared with unstimulated human T cells (Fig. 1).
Transfusion of Stored RBCs Decreases Production of T-cell Cytokines in Stimulated Human T cells The addition of stored RBCs to stimulated human T cells results in severely suppressed proliferation (Fig. 1).10 Using cell culture supernatants from proliferation assays with stimulated human T cells exposed to stored RBCs, we were able to identify distinct differences in the production of several cytokines. Compared with stimulated human T cells, which produced near-maximal amounts of each cytokine, stimulated T cells exposed to stored RBCs were found to produce significantly less IL-10, IL-17a, TNF->, and GM-CSF (Fig. 1).
Use of Fresh RBCs Mitigates the Suppression of Cytokine Production When Compared With Stored Blood Bank RBCs Cell culture supernatants also included samples of stimulated T cells exposed to fresh, unprocessed RBCs. In proliferation assays, exposure to fresh RBCs did not suppress the proliferation of stimulated T cells (Fig. 1).10 Cytokine studies from these samples revealed that stimulated T cells in coculture with fresh RBCs demonstrated decreased levels of cytokines when compared with stimulated T cells alone; however, suppression with fresh RBCs was less than in conditions using stored RBCs (Fig. 1). IL-10 production was higher in cells transfused with fresh RBCs compared with stored RBCs (p = 0.035), as was IL-17a (p = 0.029). TNF-> production was higher in coculture with fresh RBCs compared with stored RBCs (p = 0.0356). GM-CSF production was higher in T cells 200
exposed to fresh RBCs as well (p = 0.0125). Only IFN-F did not show a statistical difference in the production between stimulated T cells exposed to stored RBCs and stimulated T cells exposed to fresh RBCs (p = 0.158).
DISCUSSION Exposure to stored RBCs depresses both proliferation by stimulated human T cells and their production of common cytokines, including IL-10, IL-17a, TNF->, and GM-CSF. This may affect cellular function in vivo. Although some of the suppressive effects of RBCs on T-cell proliferation can be abrogated by the use of fresh, nonprocessed RBCs, overall T-cell production of the studied cytokines was diminished after exposure to either stored or fresh RBCs. This finding lends support to the theory that storage induced-changes in banked blood are at least partially responsible for some of the immunomodulation seen after transfusion.11 TRIM is of particular interest in trauma patients because they constitute a significant portion of patients requiring emergent RBC transfusions. Higher circulating levels of TNF-> and other inflammatory cytokines have been associated with increased survival in severe trauma and hemorrhagic shock.13 Our data indicate that this proinflammatory response may be dampened by the transfusion of stored RBCs, and TNF-> suppression may be one mechanism for TRIM. A vigorous early inflammatory response to acute injury may have survival benefit for trauma patients, and this posttraumatic priming of the immune system could be affected by RBC transfusion.14 Comparable with studies of immune dysfunction and chronic, consistent levels of inflammation in obese patients, suppression of the inflammatory response to trauma or acute injury by RBC * 2014 Lippincott Williams & Wilkins
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transfusion may predispose these patients to a depressed acute phase response and subsequent increased risks of infectious complications.15 Other synergistic inflammatory cytokines such as IL-17a and GM-CSF are suspected to play similar roles in the response to trauma, and their suppression after RBC transfusion in acute injury could contribute to this immunomodulation.16,17 Higher levels of the anti-inflammatory cytokine IL-10 have also been linked to the development of sepsis after major trauma.18 We have also shown suppression of IL-10 in this in vitro transfusion model. The high levels of IFN-F found in these studies and the lack of suppression after transfusion are confounding and could be related to the maximal production and subsequent saturation of the assay. Repeating the study with diluted samples could provide more insight and possibly reveal a true suppression in conditions with RBCs. The cascade of cytokine production after injured cell activation is clearly affected by the transfusion of RBCs. Determining the time course of that alteration and the precise mechanism, whether related to T-cell signaling, RNA synthesis, or protein translation, will require further investigation. Notable limitations to our study include a relatively small donor population for both stored and fresh blood cells and the inability to obtain complete donor information that, in much larger series, could help delineate differences among populations. We show one time point, so we are unable to appreciate the course of cytokine release. In addition, only one concentration of RBC transfusion was used; however, it is one used extensively in past projects to suppress proliferation. Both stored and fresh RBCs similarly suppress cytokine production in human T cells, while fresh RBCs inhibit proliferation less than stored. These findings add to the unraveling of the ‘‘storage lesion.’’ Proliferation is obviously dependent on more than cytokine synthesis and autoactivation, and clinical effects from decreased cytokine production by T cells exposed to PRBCs require further study, specifically with regard to bacterial killing, activation of macrophages, and other effects on host immunity. Further in vitro and in vivo studies will be helpful, especially in acutely injured trauma patients. AUTHORSHIP K.L. designed the study, performed the experiments, interpreted the results, and wrote or revised the manuscript. L.P. interpreted the results and revised the manuscript. M.W. performed the experiments. C.M. designed the study. D.W. supervised the study and data analysis and interpretation. J.W. interpreted the results, supervised study design and experiments, and edited and revised the manuscript. A.B. supervised the study, interpreted the results, and edited the manuscript.
DISCLOSURE A.B. has received previous grant support from Eastern Association for the Surgery of Trauma and ongoing support for travel to meetings.
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REFERENCES 1. Bernard AC, Davenport DL, Chang PK, Vaughn TB, Zwischenberger JB. Intraoperative Transfusion of 1 U to 2 U packed red blood cells is associated with increased 30-day mortality, surgical site infection, pneumonia, and sepsis in general surgery patients. J Am Coll Surg. 2009;208:931Y937. 2. Bernard A, Meier C, Ward M, Browning T, Montgomery A, Kasten M, Snow C, Manning E, Woodward J. Packed red blood cells suppress T-cell proliferation through a process involving cell-cell contact. J Trauma. 2010;69:320Y329. 3. Wang D, Sun J, Solomon SB, Klein HG, Natanson C. Transfusion of older stored blood and risk of death: a meta-analysis. Transfusion. 2012;52: 1184Y1195. 4. Middleburg RA, van de Watering LM, Briet E, van der Bom JG. Storage time of red blood cells and mortality of transfusion recipients. Transfus Med Rev. 2013;27(1):36Y43. 5. Koch CG, Li L, Sessler DI, Figueroa P, Hoeltge GA, Mihaljevic T, Blackstone EH. Duration of red-cell storage and complications after cardiac surgery. N Engl J Med. 2008;358(12):1229Y1239. 6. Sparrow RL. Red blood cell storage and transfusion-related immunomodulation: a second hit in an inflammatory cascade? Vox Sang. 2010;95:261Y271. 7. Hendrickson JE, Hod EA, Hudson KE, Spitalnik SL, Zimring JC. Transfusion of fresh murine red blood cells reverses adverse effects of older stored red blood cells. Transfusion. 2011;51:695Y702. 8. Sparrow R. Time to revisit red blood cell additive solutions and storage conditions: a role for ‘‘omics’’ analyses. Blood Transfus. 2012;10:s7Ys11. 9. Rajashekharaiah V, Koshy AA, Koushik AK, Kaur H, Kumari K, Agrawal M, Priyanka, Ramya, Khatai S, et al. The efficacy of erythrocytes isolated from blood stored under blood bank conditions. Transfus Apher Sci. 2012;47(3):359Y364. 10. Long KL, Meier C, Ward M, Williams D, Woodward J, Bernard A. Immunologic profiles of red blood cells using in-vitro models of transfusion. J Surg Res. 2013;184:E1YE5. 11. Long K, Meier C, Bernard A, Williams D, Davenport D, Woodward J. T-cell suppression by red blood cells is dependent on intact cells and is a consequence of blood bank processing. Transfusion. 2014;54:1340Y1347. 12. Biedler AE, Schneider SO, Seyfert U, Rensing H, Greener S, Girndt M, Bauer I, Bauer M. Impact of alloantigens and storage-associated factors on stimulated cytokine response in an in-vitro model of transfusion. Anesthesiology. 2002;97:1102Y1109. 13. Namas R, Ghuma A, Torres A, Polanco P, Gomez H, Barclay D, Gordon L, Zenker S, Kim HK, Hermus L, et al. An adequately robust early TNF-alpha response is a hallmark of survival following trauma/hemorrhage. PLoS One. 2009;4(12):1Y10. 14. Stegmaier JC, Kirchhoff C, Bogner V Matz M, Kanz KG, Mutschler W, Biberthaler P. Dynamics of neutrophilic NF-kB translocation in relation to IL-8 mRNA expression after major trauma. Inflamm Res. 2008;57: 547Y554. 15. Winfield RD, Delano MJ, Cuenca AG, Cendan JC, Lottenberg L, Efron PA, Maier RV, Remick DG, Moldawer LL, Cuschieri J. Obese patients show a depressed cytokine profile following severe blunt injury. Shock. 2012; 37(3):253Y256. 16. Frangen TM, Bogdanski D, Schinkel C, Roetman B, Kalicke T, Muhr G, Koller M. Systemic IL-17 after severe injuries. Shock. 2008;29(4):462Y467. 17. Kimura F, Shimizu H, Yoshidome H, Ohtsuka M, Miyazaki M. Immunosuppression following surgical and traumatic injury. Surg Today. 2010;40:793Y808. 18. Giannoudis PV, Smith RM, Perry SL, Windsor AJ, Dickson RA, Bellamy MC. Immediate IL-10 expression following major orthopedic trauma: relationship to anti-inflammatory response and subsequent development of sepsis. Intensive Care Med. 2000;26(8):1076Y1081.
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In vitro transfusion of red blood cells results in decreased cytokine production by human T cells Kristin Long, MD, Jerold Woodward, PhD, Levi Procter, MD, Marty Ward, MS, Cindy Meier, MS, Dennis Williams, MD, and Andrew Bernard, MD, Lexington, Kentucky
BACKGROUND: Transfusion-related immunomodulation consists of both proinflammatory and anti-inflammatory responses after transfusion of blood products. Stored red blood cells (RBCs) suppress human T-cell proliferation in vitro, but the mechanism remains unknown. We hypothesized that cytokine synthesis by T cells may be inhibited when stored RBCs are present and that suppression between fresh and stored RBCs would be different. METHODS: Purified human T cells were stimulated to proliferate with anti-CD3/anti-CD28 and then exposed to stored or fresh RBCs. Cells were placed in culture for 5 days. Cell culture supernatants were analyzed for the production of typical T-cell cytokines using multianalyte ELISArray kits. RESULTS: Stimulated T cells proliferated. RBC exposure markedly suppressed this proliferation. Interleukin 10, interleukin 17a, interferon F, tumor necrosis factor >, and granulocyte macrophage colony-stimulating factor were increased in response to stimulation but depressed in the presence of stored RBCs. The use of fresh RBCs also resulted in depression of these cytokines when compared with stimulated T cells with no RBCs; however, this depression was less pronounced. CONCLUSION: T-cell activation is associated with both proinflammatory and anti-inflammatory cytokine release, comparable with patterns seen in trauma and acute injury. All of these responses are depressed by an exposure to stored RBCs. Decreased levels of these cytokines after RBC transfusion represents a potential contributor to the immunosuppressive complications seen in trauma patients after transfusion. This provides insight for future mechanistic studies to delineate the role of RBC transfusion in transfusion-related immunomodulation. (J Trauma Acute Care Surg. 2014;77: 198Y201. Copyright * 2014 by Lippincott Williams & Wilkins) KEY WORDS: Transfusion; immunomodulation; cytokine; blood.
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ransfusion of packed red blood cells (PRBCs) results in immunomodulation, including both suppressive and stimulatory responses. Postoperative patients receiving as little as 1 U of PRBC transfusion have immunosuppressive sequelae, including increased infection, length of stay, mechanical ventilator days, and mortality.1 At the cellular level, PRBC transfusion has been shown to suppress T-cell proliferation.2 The major focus of clinical research into this immunomodulation has been the duration of RBC storage and increased morbidity and mortality with older units.3 Storage duration has been shown to enhance both the immunologic derangements and the harmful postoperative outcomes in clinical practice.4Y6 Effects of PRBC less than 7 days old in comparison with those stored 7 days to 30 days have been reported; however, these studies used blood that has been processed by traditional blood bank protocols.7Y9 Murine studies have shown that fresh, unprocessed blood may abrogate some of the negative immune effects of stored RBCs, and in vitro Submitted: December 9, 2013, Revised: April 21, 2014, Accepted: April 28, 2014. From the Section on Trauma and Acute Care Surgery (L.P., C.M., A.B.), Division of General Surgery (K.L.), Department of Microbiology, Immunology and Molecular Genetics (J.W., M.W.), and Department of Pathology (D.W.), University of Kentucky, Lexington, Kentucky. This study was presented in the Raymond Alexander Paper Competition at the 27th Eastern Association for the Surgery of Trauma Annual Scientific Assembly, January 14Y18, 2014, in Naples Florida. Address for reprints: Kristin Long, MD, Department of General Surgery, University of Kentucky, 800 Rose St, Lexington KY 40536; email: [email protected]. DOI: 10.1097/TA.0000000000000330
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studies with human T cells lend support to these data.10 Previous data from this laboratory have shown that proliferation of human T cells is markedly suppressed after exposure to stored RBCs and that production of interleukin 2 (IL-2), an autocrine growth factor necessary for T-cell proliferation, is diminished after PRBC transfusion. Addition of exogenous IL-2 did not rescue T-cell proliferation in these studies.11 A multihit hypothesis for transfusion-related immunomodulation (TRIM) suggests that the combination of leukocyte contamination, storage time, and cytokine derangements all contribute to the immunosuppressive effects.12 This led us to hypothesize that cytokine production by stimulated human T cells would be inhibited by the addition of RBCs, as was noted in our previous experiments with IL-2. Likewise, given the ability of fresh RBCs to mitigate this suppression, we hypothesized that a difference in cytokine production by T cells would be noted between stored and fresh RBCs. This study sought to analyze profiles of prototypical T-cell cytokines after in vitro transfusion of RBCs (both fresh and stored) in coculture with stimulated human T cells, to further identify immunosuppressive and immunoinflammatory cytokine patterns and differences between cells exposed to fresh or stored RBCs.
MATERIALS AND METHODS T-cell Isolation and Culture Human peripheral blood monocytes obtained from volunteer blood donors (Kentucky Blood Center, Lexington, J Trauma Acute Care Surg Volume 77, Number 2
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
J Trauma Acute Care Surg Volume 77, Number 2
Kentucky) using an institutional review board (IRB)Yexempt protocol were separated using Ficoll-Hypaque gradient. T cells were isolated from peripheral blood monocyte by depletion of all nonYT cells using a magnetic bead Pan T cell kit (Miltenyi Biotec, Auburn, CA) and an autoMACS Separator and plated at 1.0 106 cells/mL. Cells were cultured in RPMI 1640 media (Roswell Park Memorial Institute, RPMI, Life Technologies, Carlsbad, CA) supplemented with penicillin/streptomycin, 10% heat-inactivated fetal bovine serum, and further supplemented with MEM nonessential amino acid solution, sodium pyruvate, and glutamine because of longer continuous incubation periods, hence forth termed CRPMI [complete RPMI]. Cells were stimulated with plate bound anti-CD3 (1-Kg/mL OKT-3, Ortho Pharmaceutical, Raritan, NJ) and anti-CD28 (100 ng/mL, Becton Dickinson, San Jose, CA). All cultures were maintained at 37-C and 5% CO2.
Packed RBC Cultures To simulate transfusion units, allogeneic human PRBCs (O negative) were obtained from the University of Kentucky Hospital blood bank under an IRB-exempted protocol. All units obtained, both autologous and allogeneic, were leukoreduced and stored in the standard solutions of citrate-phosphate-dextrose and Optisol (crystalloid solution of sodium chloride, dextrose, adenine, and mannitol). None of the units were irradiated. PRBC (2-week to 3-week storage duration unless specified otherwise) were added to T-cell cultures. Acceptable initial hematocrits for blood bank PRBC specimens used for experiments were 60% to 80%, and samples were further diluted in CRPMI to 0.0365% to 2% by volume as shown in figure legends. The number of PRBC per well were standardized such that 2% hematocrit equated to 193 T 17 PRBCs per 1 T cell and followed by serial dilutions. For carboxyfluorescein diacetate succinimidyl ester (CFSE) staining and culture, RBCs were counted by light microscopy using a hemocytometer and diluted to an initial concentration of 2 108/mL (100 PRBCs per 1 T cell), followed by serial dilutions.
Fresh RBC Cultures Fresh RBCs were obtained from volunteer donors through an IRB-approved protocol (12-0966-P6H) by peripheral venipuncture using sterile technique. Approximately 2.5 mL of venous blood was collected, centrifuged to remove plasma, separated over a Ficoll-Hypaque density gradient, and washed of residual Ficoll to isolate erythrocytes. The RBCs were then counted by light microscopy using a hemocytometer and diluted to initial concentrations of 2 108/mL (100 RBCs per 1 T cell), followed by serial dilutions as in the PRBC experiments.
Long et al.
For CFSE experiments, T cells were initially suspended in phosphate-buffered saline/0.1% fetal bovine serum at a concentration of 5.0 106 cells/mL. CFSE 5 mM was prepared by dissolving one vial of stock CFSE solution (CellTrace, Grand Island, NY) with 18-KL DMSO. CFSE was then added at a volume of 0.25 KL per millimeter of T cells, rapidly inverted, and stained for 10 minutes at room temperature in the dark. Staining was quenched by the addition of excess CRPMI. Cells were centrifuged at 300 G for 10 minutes at 18-C, supernatant was removed, and cells were recounted before suspension at a final concentration of 2.0 106 cells/mL. CFSE-stained T cells were stored in the dark at room temperature until ready for plating.
Flow Cytometry CFSE-labeled T cells, after treatment and incubation, were harvested and stained with anti-CD4 PerCP (eBioscience, San Diego, CA) for 30 minutes at room temperature in the dark. Cells were then washed, resuspended in phosphate-buffered saline, and placed on ice for immediate flow cytometry analysis.
Cytokine Analysis After 5 days in culture, the supernatant from each well of 24-well plates from the experiments was aspirated into 1-mL sterile tubes and frozen at j80-C until the time of analysis. Multiple donors, both fresh and stored, were used in these experiments (three separate stored PRBC donors, five separate fresh donors). For the analysis, the samples were thawed on ice and then analyzed for a series of inflammatory cytokines (IL-10, IL-17a, interferon F [IFN-F], tumor necrosis factor > [TNF->], and granulocyte macrophage colony-stimulating factor [GM-CSF]) using a Multi-Analyte ELISArray Kit (Catalog Number MEH-004A, SA Biosciences, Valencia, CA) according to the manufacturer’s instructions, and the absorbance at 450 nm was read within 30 minutes of stopping the reaction.
Data Analysis Absorbance values at 450 nm for each sample are presented in their corrected form (original value with negative standard value subtracted). Data were analyzed using Microsoft Excel (Redmond, WA). Statistical differences were calculated using two-tailed Student’s t tests, with p G 0.05 representing statistical significance.
RESULTS
Proliferation Assay
RBC Exposure Suppresses Human T-cell Proliferation Stimulated Human T cells Produce IL-10, IL-17a, TNF->, GM-CSF, and IFN-F
Purified human T cells (1 106/mL) with PRBC cultures were incubated in 96-well or 24-well plates for a total of 72 hours. 3H-thymidine was added for the final 18 hours to 20 hours of incubation at 1 KCi per well, cells were harvested on Perkin Elmer unifilter plates, and cell proliferation was determined by scintillation counting.
Five-day supernatants from a CFSE dye dilution T-cell proliferation assay previously published10 were harvested and stored at j80-C. Purified human T cells were stimulated to proliferate with anti-CD3/CD28. Supernatants from these experiments were analyzed for cytokine production and demonstrated an expected increase in production of several cytokines
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Figure 1. Cytokine production by stimulated human T cells decreases after exposure to RBCs. Cell culture supernatants from proliferation assays were analyzed for levels of proinflammatory and anti-inflammatory cytokine production. Supernatants harvested from the experiment are depicted here. x-axis represents individual cytokines tested, and bars demonstrate average absorbance values. y-axis represents absorbance at 450 nm. Unstimulated human T cells produced essentially no detectable cytokines. Stimulated T cells produced near-maximal amounts of each tested cytokine (relative to each positive control in enzyme-linked immunosorbent assay). With the exception of IFN-F, all cytokines tested showed a dramatic decrease in production after addition of stored RBCs. The use of fresh, unprocessed RBCs partially mitigated this suppression.
(IL-10, IL-17a, TNF->, GM-CSF, and IFN-F) when compared with unstimulated human T cells (Fig. 1).
Transfusion of Stored RBCs Decreases Production of T-cell Cytokines in Stimulated Human T cells The addition of stored RBCs to stimulated human T cells results in severely suppressed proliferation (Fig. 1).10 Using cell culture supernatants from proliferation assays with stimulated human T cells exposed to stored RBCs, we were able to identify distinct differences in the production of several cytokines. Compared with stimulated human T cells, which produced near-maximal amounts of each cytokine, stimulated T cells exposed to stored RBCs were found to produce significantly less IL-10, IL-17a, TNF->, and GM-CSF (Fig. 1).
Use of Fresh RBCs Mitigates the Suppression of Cytokine Production When Compared With Stored Blood Bank RBCs Cell culture supernatants also included samples of stimulated T cells exposed to fresh, unprocessed RBCs. In proliferation assays, exposure to fresh RBCs did not suppress the proliferation of stimulated T cells (Fig. 1).10 Cytokine studies from these samples revealed that stimulated T cells in coculture with fresh RBCs demonstrated decreased levels of cytokines when compared with stimulated T cells alone; however, suppression with fresh RBCs was less than in conditions using stored RBCs (Fig. 1). IL-10 production was higher in cells transfused with fresh RBCs compared with stored RBCs (p = 0.035), as was IL-17a (p = 0.029). TNF-> production was higher in coculture with fresh RBCs compared with stored RBCs (p = 0.0356). GM-CSF production was higher in T cells 200
exposed to fresh RBCs as well (p = 0.0125). Only IFN-F did not show a statistical difference in the production between stimulated T cells exposed to stored RBCs and stimulated T cells exposed to fresh RBCs (p = 0.158).
DISCUSSION Exposure to stored RBCs depresses both proliferation by stimulated human T cells and their production of common cytokines, including IL-10, IL-17a, TNF->, and GM-CSF. This may affect cellular function in vivo. Although some of the suppressive effects of RBCs on T-cell proliferation can be abrogated by the use of fresh, nonprocessed RBCs, overall T-cell production of the studied cytokines was diminished after exposure to either stored or fresh RBCs. This finding lends support to the theory that storage induced-changes in banked blood are at least partially responsible for some of the immunomodulation seen after transfusion.11 TRIM is of particular interest in trauma patients because they constitute a significant portion of patients requiring emergent RBC transfusions. Higher circulating levels of TNF-> and other inflammatory cytokines have been associated with increased survival in severe trauma and hemorrhagic shock.13 Our data indicate that this proinflammatory response may be dampened by the transfusion of stored RBCs, and TNF-> suppression may be one mechanism for TRIM. A vigorous early inflammatory response to acute injury may have survival benefit for trauma patients, and this posttraumatic priming of the immune system could be affected by RBC transfusion.14 Comparable with studies of immune dysfunction and chronic, consistent levels of inflammation in obese patients, suppression of the inflammatory response to trauma or acute injury by RBC * 2014 Lippincott Williams & Wilkins
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J Trauma Acute Care Surg Volume 77, Number 2
transfusion may predispose these patients to a depressed acute phase response and subsequent increased risks of infectious complications.15 Other synergistic inflammatory cytokines such as IL-17a and GM-CSF are suspected to play similar roles in the response to trauma, and their suppression after RBC transfusion in acute injury could contribute to this immunomodulation.16,17 Higher levels of the anti-inflammatory cytokine IL-10 have also been linked to the development of sepsis after major trauma.18 We have also shown suppression of IL-10 in this in vitro transfusion model. The high levels of IFN-F found in these studies and the lack of suppression after transfusion are confounding and could be related to the maximal production and subsequent saturation of the assay. Repeating the study with diluted samples could provide more insight and possibly reveal a true suppression in conditions with RBCs. The cascade of cytokine production after injured cell activation is clearly affected by the transfusion of RBCs. Determining the time course of that alteration and the precise mechanism, whether related to T-cell signaling, RNA synthesis, or protein translation, will require further investigation. Notable limitations to our study include a relatively small donor population for both stored and fresh blood cells and the inability to obtain complete donor information that, in much larger series, could help delineate differences among populations. We show one time point, so we are unable to appreciate the course of cytokine release. In addition, only one concentration of RBC transfusion was used; however, it is one used extensively in past projects to suppress proliferation. Both stored and fresh RBCs similarly suppress cytokine production in human T cells, while fresh RBCs inhibit proliferation less than stored. These findings add to the unraveling of the ‘‘storage lesion.’’ Proliferation is obviously dependent on more than cytokine synthesis and autoactivation, and clinical effects from decreased cytokine production by T cells exposed to PRBCs require further study, specifically with regard to bacterial killing, activation of macrophages, and other effects on host immunity. Further in vitro and in vivo studies will be helpful, especially in acutely injured trauma patients. AUTHORSHIP K.L. designed the study, performed the experiments, interpreted the results, and wrote or revised the manuscript. L.P. interpreted the results and revised the manuscript. M.W. performed the experiments. C.M. designed the study. D.W. supervised the study and data analysis and interpretation. J.W. interpreted the results, supervised study design and experiments, and edited and revised the manuscript. A.B. supervised the study, interpreted the results, and edited the manuscript.
DISCLOSURE A.B. has received previous grant support from Eastern Association for the Surgery of Trauma and ongoing support for travel to meetings.
Long et al.
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