Abstract
Hemoglobin-based oxygen carriers (HBOCs) show potential as safe, efficacious, pre-hospital resuscitation fluids. The major criticism of HBOC-201 is its vasoactive property, attributed partially to low-molecular weight (low-MW) tetrameric/dimeric (TD) hemoglobin (Hb) in HBOC solution. Here we sought to determine whether resuscitation with decreasing concentrations of low-MW Hb component of HBOC affects immune responses in hemorrhagic swine. 28 anesthetized swine underwent a soft muscle crush and controlled hemorrhage of 55% blood volume, followed by resuscitation with HBOC containing 31%, 2%, or 0.4% low-MW Hb in four 10 ml/kg infusions at 20, 30, 45 and 60 minutes before hospital arrival at 75 minutes. IL-10, cell activation and adhesion markers and CD4:CD8 ratio remained unchanged in all 3 groups compared to baseline. Leukocyte apoptosis was equally elevated across all groups. Purification from 31% to 0.4% low-MW Hb in HBOC solution did not alter immune effects in a swine model of severe controlled hemorrhagic shock.
INTRODUCTION
Hemoglobin (Hb) based oxygen carriers (HBOCs) show potential as effective pre-hospital resuscitation fluids in delayed hospital evacuation scenarios in military and rural civilian arenas. HBOC-201 (Biopure Corporation, Cambridge, MA) is comprised of polymerized bovine hemoglobin, containing ≤ 2–3% low-MW TD Hb in Lactated Ringer's solution (LR) [Citation[1], Citation[2]]. We and other investigators have demonstrated efficacy of HBOC-201 compared to standard resuscitative fluids in improving tissue oxygenation, lowering fluid requirements, and significantly improving survival in swine animal models of hemorrhagic shock (HS) [Citation[3-8]]. Further, our previous studies strongly indicated that HBOC-201 triggered no or equal immunoactivation in blood compared to other fluids [Citation[9]].
The primary concern over HBOC-201 is its documented vasoactive property [Citation[10], Citation[11]]. Low-MW Hb scavenges nitric oxide, thus preventing smooth muscle relaxation and elevating blood pressure. Resta et al. found that vasoactive side effects improved upon lowering the concentration of low-MW Hb in HBOC [Citation[12]]. A recent study by Yu et al. demonstrated that HBOCs containing 38%, 78% and 16% low-MW Hb caused ST elevation in rats, whereas HBOCs containing 0.4% low-MW Hb did not [Citation[13]]. Further studies are required to fully examine immune effects of Hb components in HBOC solutions. In an in vitro study by McFaul et al., the incubation of isolated human monocytes with purified Hb resulted in an increase of IL-8 and TNF-α at 4 hours [Citation[14], Citation[15]]. Moreover, other components of HBOC-201, including glutaraldehyde (polymerization agent) and N-acetylcysteine (NAC) (anti-oxidant), also have immune activating properties [Citation[16]]. Hence, potential for activation of neutrophils and release of cytotoxic byproducts, including reactive oxygen species, support a theoretical basis for possible HBOC-mediated post-trauma tissue injury and MOF [Citation[17]].
The relationship between concentration of low-MW Hb in HBOC and potential immunomodulation in vivo is not well studied. Here we sought to determine whether resuscitation with decreasing concentrations of low-MW Hb components of HBOCs affects immune responses in a swine model of severe controlled hemorrhage.
MATERIALS AND METHODS
The experiments reported in the present study were conducted according to principles set in the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animals Resources, National Research Council, National Academy Press (1996). The study was approved by the WRAIR/NMRC Institutional Animal Care and Use Committee (IACUC); all procedures were performed in an animal facility approved by the Association for Assessment and Accreditation for Laboratory Animal Care International.
Hemorrhagic Shock (HS) Model
Details of the surgery and resuscitation of this HS model and resuscitation were previously published by Rice et al. [Citation[5]]. Twenty-eight Yorkshire Pigs (weight 20–30 kg) were sedated with ketamine and anesthetized with isoflourane. At the start of experiment (time 0) the Kocher clamp was closed over the abdominus rectus muscle to simulate a soft tissue injury following 55% estimated blood volume (EBV) withdrawal via right external jugular vein introducer. Pigs were hemorrhaged over 15 min, after which they were randomized to the treatment groups (10 pigs in 31TD; 9 pigs in 2TD; 9 pigs in 0.4TD) and resuscitated at time 20, 30, 45 and 60 min with 10 ml/kg of high low-MW content of 31% tetrameric/dimeric Hb (Oxyglobin, HBOC-301, [31 TD]); low low-MW content 2% tetrameric/dimeric Hb (Hemopure, HBOC-201, [2TD]); and very low-MW Hb content 0.4% dimeric/tetrameric Hb (0.4TD). At 75 minutes hospital arrival and care was simulated. EDTA-treated blood samples were collected for bioassays at times 0 (before injury), 75 and 240 minutes. At 240 minutes the pigs were euthanized and taken to necropsy.
Immunophenotyping
Peripheral blood mononuclear cells (PBMC) were isolated from whole blood using a Ficoll density-gradient and centrifugation; cells were cryopreserved in liquid nitrogen for future analysis.
Cell surface staining was performed on either 100 µl fresh blood or 1 × 106 cryopreserved PBMCs. Direct conjugation of samples was performed using the following anti-porcine antibodies: CD3-FITC, CD4-PE, (BD Biosciences, Palo Alto, CA), or CD8-PE (Southern Biotechnology, Birmingham, AL). Indirect conjugation was performed using purified anti-porcine CD25, CD11b, and CD49d (Serotec, Raleigh, NC), together with PE conjugated anti-mouse F(ab')2 (Jackson Immunoresearch Laboratories, West Grove, PA). Details of the protocol were previously published [Citation[9]].
Acquisition and analysis of all samples were performed on a Beckman Coulter flow cytometer (Beckman Coulter, Fullerton, CA) with Cytonics CTMJ RXP software. Forward and side scatter determined lymphocyte, monocyte, and granulocyte populations. A minimum of 10,000 events was acquired within lymphocyte and granulocyte gates and a minimum of 1,000 events within the monocyte gate. Expression of CD4, CD8, and CD25 was measured as percentages of gated lymphocytes. Expression of CD11b and CD49d was quantified using mean fluorescence intensity (MFI) of the appropriate cell population and all time points were normalized for the value of time 0.
Annexin V Staining
Evaluation of apoptosis was determined using an Annexin-V assay kit (R&D Systems, Minneapolis, MN). The assay was performed per the manufacturer's protocol on either 100 µl whole blood treated with 1 × RBC lysis buffer (eBiosciences, San Diego, CA) or cryopreserved PBMCs, adjusted to 1 × 106 cells per 100 µl PBS. Samples were washed in PBS and resuspended in Annexin-V incubation reagent. Samples were incubated in the dark and at room temperature for 15 minutes, washed in 1 × binding buffer. Acquisition was performed within two hours of staining on a Coulter flow cytometer. Annexin + /PI + cells were excluded from the final analysis.
Plasma Cytokines
IL-10 levels were detected using porcine-specific ELISA kits (R&D Systems, Minneapolis, MN, and Biosource International, Inc., Camarillo, CA) according to manufacturer's protocol. Details are reported by Dong et al. [Citation[9]]. Assays were performed in duplicate on frozen undiluted plasma. Due to sample concentration discrepancy at time 0 IL-10 was normalized for time 0.
Statistical Analysis
Data are presented as mean±standard error of the mean (SEM). Analysis of variance (ANOVA) was used to compare groups at each time point whereas mixed models were used to compare groups over time. P-values of ≤ 0.05 (two-sided) were considered statistically significant.
RESULTS
Survival and Hemodynamics
Survival was 100% (10/10), 100% (9/9) and 89% (8/9) with 31TD, 2TD and 0.4TD, respectively (p > 0.05). Mean arterial pressure (MAP) was highest with 31TD (p < 0.001) and similar in 2TD vs. 0.4TD. Heart rate, MPAP and central venous pressure increased in all groups, but were higher in 31TD (p < 0.001); 2TD and 0.4TD were similar. All pigs had elevated MPAP after the 2nd infusion and remained above baseline. Detailed physiology results are reported by Rice et al. [Citation[5]].
Apoptosis
The level of apoptosis in the total leukocyte populations (neutrophils, monocytes and lymphocytes analyzed together in a total cell gate) ranged between 2.4–3.2% at time 0 (A). At time 75 min and 4 hours the level of apoptosis persistently increased in all groups two- and three-folds ranging between 5–8% and 6–8.8%, respectively. No statistically significant group differences were noted at any time of the experiment. There was, however, a statistically significant (p = 0.0001) increase over time, yet this increase was similar for all groups at 75 and 240 minutes, p = 0.22 and p = 0.55, respectively.
Figure 1 Apoptosis in total leukocytes and lymphocytes before and after HS and resuscitation. 1A. Apoptosis in total leukocyte population. Apoptosis data is presented as mean percentages of Annexin + /PI − cells±SEM within “total cells” gate. Neutrophils, monocytes and lymphocytes were included in the flow cytometry analysis. All groups exhibited an overtime increase of apoptosis, however, no group differences were observed. 1B. Apoptosis in lymphocyte population. Apoptosis data is presented as mean percentages of Annexin + /PI − cells±SEM within “lymphocytic cells” gate. All groups exhibited an overtime increase of apoptosis, however, no group differences were observed. Black bar − 31TD; grey bar − 2TD; open bar − 0.4 TD.
Similar trends were seen in the lymphocytic gate. At time 0, the background of apoptosis in the lymphocyte population ranged between 1–1.6% (B). At 75 minutes and 4 hours, the level of apoptosis persistently increased in all groups, reaching 2.3–3% and 3–4.6%, respectively. Apoptosis was elevated in all groups and no statistically significant group differences were noted at any time point (p = 0.88 and p = 0.74 at 75 and 240 minutes, respectively); however, there was a statistically significant increase over time across all groups (p = 0.005).
Cellular and Plasma Immune Markers
summarizes data on immune effects in blood and plasma following HS and resuscitation with HBOCs. The concentration of IL-10 trended to increase overtime compared to time 0 in all groups, but there was no group difference.
Table 1. Immune responses of 31 TD, 2 TD and 0.4 TD treatment groups before and after HS and resuscitation
CD4+ and CD8 + T lymphocyte sub-populations and CD4/CD8 ratios were stable in all treatment groups throughout the experiment. No changes overtime and between groups were also noted in lymphocytes expressing the activation marker CD25.
There was a mild enhancement of CD11b MFI in lymphocytes at 75 minutes, both in the 31TD and 0.4TD groups. Again, no significant differences between groups and overtime were observed. At 4 hours CD11b MFI was similar to time 0. No changes overtime and between groups were observed in CD11b MFI on monocytes and neutrophils in any of the groups.
The expression of CD49d MFI on neutrophils, monocytes and lymphocytes remained stable in all treatment groups throughout the experiment.
DISCUSSION
Here we report that decreased concentration of low-MW Hb component of HBOCs from 31% to 0.4% induced nearly equal immune effects in blood and plasma following resuscitation in a swine model of 55% EBV severe controlled hemorrhage. The CD4/CD8 ratios, cell surface marker of lymphocytes activation CD25; leukocyte adhesion marker CD49d and monocytic, and neutrophylic CD11b remained unchanged throughout the experiment. The lymphocytic CD11b adhesion marker was, briefly, not significantly elevated at 75 minutes after four infusions of HBOCs; however, no group differences were observed. Apoptosis was highly elevated in total leukocytes and lymphocyte sub-population across all treatment groups following both, pre- and post-hospital phases of resuscitation. Previously, Dong et al. reported that apoptosis was not increased in lymphocytes following five infusions of HBOC-201 in a moderate severe 40% EBV controlled hemorrhage swine model [Citation[9]]. Such inconsistency may be due to decreased severity of HS model, 40% versus 55% EBV, and lower number of animals examined for apoptosis (5–6 in 40% HS and 9–10 in 55% HS). In addition, in the study by Dong et al., apoptosis was examined only in lymphocytes and not in the total leukocyte population, which could have revealed an apoptosis enhancement. Since no-resuscitation group and standard fluid group controls were not included in the current study, it remains to be determined whether the elevation of apoptosis was triggered by the severity of the injury or HBOCs infusions. In any case, the level of apoptosis did not correlate with the concentration of low-MW Hb in HBOC and, perhaps, can be attributed to the severity of the HS model.
The current literature is conflicting as to the role of apoptosis following shock. Several studies link increased apoptosis levels to immunosuppression and to worse patient outcome following septic shock, hemorrhagic shock, and resuscitation [Citation[18-20]]. However, work by Giamarellos et al. indicated that early monocyte apoptosis may actually have a protective function in septic shock. In this study, patients with > 50% monocyte apoptosis on the day of symptoms onset survived significantly longer than those with < 50% monocyte apoptosis. Additionally, this group had lower serum IL-6, IL-8 and TNF-α levels at the end of a seven-day monitoring period. The authors suggested that monocytes undergoing apoptosis may lose ability to release pro-inflammatory cytokines, thus inhibiting a hyperactive immune response and contributing to their survival outcome [Citation[21]]. In the present study no IL-6 responses were detected in plasma. At 75 minutes and 4 hours, incremental enhancement of IL-10 was noted across all groups. Our data is in agreement with that of Dong et al., showing modest enhancement of IL-10 following five HBOC-201 infusions in a 40% HS swine model [Citation[9]].
Despite the strong apoptosis increase, CD4 + and CD8 + T cells, and the CD4/CD8 ratio remained stable across all groups throughout the experiment. Perhaps, the enhanced apoptosis can be attributed to dying B cells, suggesting that evaluation of the T/B ratio should be carefully examined in future experiments. Little is known about HBOC effects on the function of immune cells. Humoral responses of B cells as well as CD4 + and CD8 + T cells proliferative and cytotoxic activity should be carefully examined in future experiments in swine with HS.
CD11b, a B2 integrin, and CD49d, an α4 integrin, are adhesion markers involved in the migration and binding of leukocytes to endothelium; both are characteristically up-regulated in shock. Here we show no CD49d upregulation on neutrophils, monocytes and lymphocytes across all groups throughout the experiment. Similar results were demonstrated previously by Dong et al. in their 40% HS model, which demonstrated no elevation of CD49d MFI on lymphocytes with HBOC-201 at any time-point after HS and resuscitation [Citation[9]]. Here, we detected no upregulation of CD11b MFI on neutrophils and monocytes at any time across all groups. At 75 minutes after HBOCs resuscitation, all treatment groups exhibited a brief, not significant enhancement of CD11b MFI in lymphocytes, primarily NK cells, which decreased to a background level by 4 hours. Failure to detect persistent, positive upregulation of adhesion markers on leukocytes in blood might be attributed to the quick migration and sequestration of these activated cells to the site of tissue injury—abdominus rectus muscle. In this regard, activation and adhesion markers should be analyzed in injured tissue and local draining lymph nodes.
We conclude that the decrease in concentration of low-MW of Hb in HBOC formulations did not alter immune effects in a swine model of controlled HS. Overall cellular markers exhibited no or minimal upregulation, except apoptosis, which was equally elevated following all formulated infusions of HBOC. Our data suggest that varying the concentration of low-MW Hb in HBOC insignificantly attenuates nor intensifies its immunomodulating properties.
The mainly neutral results from this study and the 40% HS study reported previously by Dong et al. complete the safety reports, providing animal data suggesting that HBOC-201 resuscitation is unlikely to adversely affect innate immunity in humans with HS.
The first two authors equally contributed to this work.
The authors thank HM1 Benjamin Esperat and Ms. Noemy Carballo for their excellent technical support. The studies described herein were entirely funded by the U.S. Government, Office of Naval Research, Work Unit Number 602236N.4426.W26.A0241. Dr. L. Bruce Pearce is an employee of Biopure Corp. and has financial interest in the subject material, HBOC-201. Dr. Pearce's contribution to the manuscript was limited to editing of the study design, protocol and manuscript. NMRC and Biopure Corp. have a Cooperative Research and Development Agreement for the evaluation of HBOC-201 in trauma clinical trials and a Materials Transfer Agreement for the supply of HBOC for pre-clinical studies. There are no funds transferred from Biopure to NMRC in either of these agreements.
Notes
Dr. Carrie Hall and Dr. Daniel Freilich are military service members. This work was prepared as part of their official duties. Title 17 U.S.C. § 105 provides that “Copyright protection under this title is not available for any work of the United States Government.” Title 17 U.S.C. § 101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person's official duties.
The opinions expressed in this article are those of the authors, and do not necessarily reflect the official policy or opinions of the Departments of the Navy, Army, and Defense, or the U.S. Government.
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