Systemic regulation of bone marrow stromal cytokines following severe trauma

Abstract

Background

Traumatic injury generates a prolonged hypercatecholamine state that is associated with reduced growth of bone marrow erythroid progenitors mediated by the bone marrow stroma. The bone marrow stroma is made up of many cells including fibroblasts, which respond to inflammatory stimuli and alter the cytokine profile. We hypothesized that trauma plasma would increase bone marrow stromal fibroblast expression of interleukin-6 (IL-6), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), stem cell factor (SCF), and activation of nuclear factor kappa-light-chain-enhancer of activated B cells (Nf-κB) and correlate with injury severity and anemia.

Materials and Methods

Plasma from fifteen trauma patients was cultured with bone marrow fibroblast cells and compared with that from healthy volunteers. At 6, 24, and 48 hours the expression of IL-6, G-CSF, EPO, SCF and the activation of NF-κB were measured using quantitative polymerase chain reaction. The influence of trauma plasma on cytokine expression was further stratified by injury severity score (ISS).

Results

The average hemoglobin significantly decreased from admission to discharge (10.7±2.5 to 9.2±1.1 g/dL, p<0.04). The discharge hemoglobin significantly decreased 14% from the admission hemoglobin. After 48 hours, trauma plasma significantly increased IL-6, G-CSF and EPO bone marrow fibroblast expression when compared to normal plasma. When stratified by ISS, IL-6, G-CSF and EPO bone marrow fibroblast expression was highest in the trauma plasma ISS 27–41 group and was significantly elevated compared to normal plasma. When SCF expression was stratified by ISS, there was a significant increase in expression in ISS 27–41. Higher ISS was also associated with a larger decrease in hemoglobin despite no difference in total blood transfusions.

Conclusions

Severe trauma can systemically increase IL-6, G-CSF and EPO expression in bone marrow stroma. Increased hematopoietic cytokine expression following traumatic injury correlated with a hypercatecholamine state, anemia and injury severity.

Introduction

Severe traumatic injury can lead to a persistent injury-associated anemia despite cessation of bleeding. Regardless of the treatment of major injuries, many patients suffer from persistent anemia for weeks to months after injury. Following the first week after injury and control of bleeding, patients continue to be transfused, on average, one to two units of packed red blood cells per week while in the intensive care unit.^{1, 2, 5} These blood transfusions are an independent risk factor for mortality and systemic inflammatory response syndrome in the trauma setting.³

Previous studies have characterized this persistent injury-associated anemia as norepinephrine-mediated bone marrow dysfunction resulting in impaired proliferation of erythroid progenitor cells, an abnormal erythropoietin response, reduced erythroid commitment, prolonged mobilization of hematopoietic progenitor cells, and dysregulation of iron homeostasis.^4–17 Successful erythropoiesis is a complex process in which pluripotent stem cells proliferate and differentiate into mature red blood cells. Red blood cell formation requires the interaction between bone marrow progenitor cells and the microenvironment, which includes stromal cells, extracellular matrix proteins, and other soluble factors.¹⁸ The bone marrow stroma is made up of osteoblasts, osteoclasts, endothelial cells, stromal cells, mesenchymal progenitor cells, adipocytes, fibroblasts and several other hematopoietic niche cells.¹⁸ Previous studies have demonstrated a predominance of fibroblasts in human bone marrow stroma after severe trauma.² Fibroblasts are known to respond to inflammatory stimuli and alter the pro- and/or anti-inflammatory cytokine profile with respect to these stimuli.^{19, 20} Interleukin-6 (IL-6) is a pro-inflammatory cytokine that upregulates hepcidin expression, leading to the sequestration of iron in macrophages, and a myriad of additional downstream effects.^{21, 22} Granulocyte colony-stimulating factor (G-CSF) induces the mobilization of hematopoietic stem cells from the bone marrow to the peripheral blood.^{23, 24} Erythropoietin (EPO) is a major factor in the differentiation and activation of erythroid progenitor cells.²⁵ Stem cell factor (SCF) promotes the proliferation, differentiation and maturation of hematopoietic stem cells, and acts to retain hematopoietic stem cells in the bone marrow niche.^{24, 26} Nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) is a family of transcription factors central to the inflammatory process.^{27, 28} Therefore, following trauma the changes that occur in bone marrow stroma and cytokine milieu directly impact erythropoiesis.

The impact of severe trauma on bone marrow stromal expression of hematopoietic cytokines is difficult to elucidate. Trauma results in the prolonged mobilization of hematopoietic progenitor cells to the site of injury and a multitude of cytokines have been found in the bone marrow and systemic circulation.^{5, 9–14} The purpose of this study was to investigate the systemic effects of trauma on bone marrow stromal hematopoietic cytokine expression. We hypothesized that trauma plasma would increase bone marrow stromal fibroblast expression of interleukin-6 (IL-6), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), stem cell factor (SCF), and activation of nuclear factor kappa-light-chain-enhancer of activated B cells (Nf-κB) and correlate with injury severity and anemia.

Materials and Methods

Subjects

Peripheral blood samples were collected prospectively from fifteen adult trauma patients admitted with an injury severity score (ISS) greater than 15 and/or in hemorrhagic shock, and with an orthopedic injury requiring fixation. Hemorrhagic shock was defined as one or more of the following: systolic blood pressure ≤ 90mm Hg, mean arterial pressure ≤ 60mm Hg, lactate ≥ 2 mM, or base deficit ≥ 5 mEq/L. Detailed inclusion and exclusion criteria are listed in Figure 1. This study was reviewed and approved by the University of Florida Institutional Review Board (IRB201601386). Patients’ written consent was obtained before samples were collected.

Figure 1.

Inclusion and exclusion criteria for trauma patient participation.

Sample collection and processing

At the time of fracture fixation, one sodium heparin tube of whole blood was collected. Immediately after collection, plasma was aliquoted from the whole blood after centrifugation and immediately frozen at −80°C. For comparison, plasma was obtained from four healthy control patients and processed in the same manner.

The time from injury to fracture fixation ranged from 0 days to 6 days, with a mean of 2.1 days and a median of 2 days.

Adrenergic state, injury severity and outcomes

Patient charts were reviewed for demographic data on age, sex, ISS, presence of hemorrhagic shock on admission and initial blood transfusion requirement. In addition, initial laboratory values including hemoglobin and lactate were collected. Patients were followed during their hospital stay and outcome data were collected including hemoglobin trend, intensive care unit (ICU) length of stay, total number of packed red blood cell transfusions, and total hospital length of stay.

ISS ranged from 10 to 41 in the trauma patient group with a median of 26. For subgroup analysis, ISS was stratified at the median ISS to provide an ISS 10–26 (n=9) and ISS 27–41 (n=6) group. ISS was not stratified by mild, moderate and severe as some groups would have inadequate power for analysis. In addition, plasma norepinephrine was measured in healthy control and trauma patients by enzyme linked immunosorbent assay (MyBiosource Inc.) according to manufacturer instructions. Optical densities were measured with a microplate reader (BioTek Instruments, Winooski, VT).

Human bone marrow fibroblasts

A transformed cell line of human bone marrow fibroblasts (HS-5) from a healthy 30-year-old male was purchased from American Type Culture Collection (ATCC, Manassas, VA). The cells were thawed rapidly to 37°C from liquid nitrogen. The thawed cells were then added to 0.9mL of complete growth medium (Dulbecco’s Modified Eagle’s Medium) and spun to retrieve the cell pellet. The cell pellet was re-suspended in complete growth medium and added to a T-75 flask (USA Scientific, Ocala, FL). This cell culture was grown in a CO₂ incubator at 37°C until growth to 3,000,000 fibroblasts cells was achieved, then cells were dissociated from the flask and 300,000 cells were added to each well of a 6-well plate (Corning, Corning, NY).

Fibroblast culture with human plasma

Fibroblasts cells in a 6-well plate were grown for 48 hours at 37°C. The growth medium was removed and fresh growth medium with either 2% human control plasma or 2% human trauma plasma was added to each well. The cells were then incubated for 6 hours, 24 hours and 48 hours at 37°C. At the end of incubation time, the cells were gently scraped from the wells and added to RNA lysis buffer (Invitrogen, Carlsbad, CA) for RNA isolation.

Polymerase chain reaction

Fibroblast expression of IL-6, G-CSF, EPO, SCF, and activation of NF-κB subunit 1 was measured using real-time polymerase chain reaction (RT PCR). RT PCR was performed using SYBR Green Real-Time PCR Master Mix (Applied Biosystems, Foster City, CA). Primers were designed from specific mRNA sequences using the OligoPerfec Designer (ThermoFisher Scientific, Waltham, MA), ideally with 80–140 base pairs and a melting temperature of 60°C (Table 1). RNA isolation was performed using PureLink RNA Mini Kit (Invitrogen). RNA concentrations and purity were determined by measuring the ratio of UV absorbence at 260nm and 280nm. RNA (1 pg) was reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Amplification was performed using the Mx3005P QPCR System (Agilent Technologies, Barcelona, Spain).

Table 1.

Polymerase chain reaction primers and conditions

Gene	Forward Primer	Reverse Primer	Base Pair Position in mRNA sequence	qPCR Product Size (bp)	Annealing Temp (°C)
EPO	5’-AGCCATCTCCCCTCCAGATG -3’	5’-CCCCGGAGGAAATTGGAGTA-3’	613–714	101	56
G-CSF	5’-CACTCTGGACAGTGCAGGAA-3’	5’-GCACTTGAGCAGGAAAGCTCT-3’	182–255	74	56
IL-6	5’-GAAAGCAGCAAAGAGGCACT-3’	5’-TTTCACCAGGCAAGTCTCCT-3’	356–463	108	56
SCF	5’-GAAGCAGGGACAGTGGAGAG-3’	5’-TCCAGCACAAACAGTGGTGT-3’	44–175	131	56
NF-κB	5’-TGGAGTCTGGGAAGGATTTG-3’	5’-CGAAGCTGGACAAACACAGA-3’	1340–1468	128	56
Beta-actin	5’-TGAGACCTTCAACACCCCAGCCATG-3’	5’-CGTAGATGGGCACAGTGTGGGTG-3’	456–583	127	56

Statistical Analysis

Statistical analysis was performed in GraphPad Prism version 6.05 (GraphPad Software, La Jolla, CA). Significance was set at α=0.05. Data are illustrated in figures created in GraphPad Prism, presented as mean±standard deviation. Differences between two groups were analyzed by an unpaired Student’s t-test. Differences between three or more groups were analyzed by one-way analysis of variance.

Results

Patient population

Peripheral blood was collected from fifteen blunt trauma patients between 18 and 90 years of age, with a mean age of 43 years and a median age of 46 years. Seven of these patients were male and eight were female. Peripheral blood was collected from four male controls between 30 and 60 years of age, with a mean age of 41 years and a median age of 32 years.

The average lactic acid on admission after traumatic injury was 3.5 mmol/L. ISS ranged from 10 to 41, with a mean of 24 and a median ISS of 26. Trauma patients had an average hospital length of stay of 14 days and a mean ICU length of stay of 6 days. Patients received an average of 1.8 pre-operative packed red blood cell (pRBC) transfusions, 1.9 intra-operative pRBC transfusions and 2.0 post-operative pRBC transfusions (in relation to the orthopedic fixation). They received an average of 5.7 pRBC transfusions during their total hospital stay. The average hemoglobin significantly decreased from admission to discharge (10.7±2.5 to 9.2±1.1 g/dL, p<0.04). The discharge hemoglobin significantly decreased 14% from the admission hemoglobin despite the use of pRBC transfusions. Norepinephrine plasma concentration increased nearly seven-fold in the trauma group when compared to the normal plasma (Figure 2).

Figure 2.

Trauma was associated with elevated norepinephrine levels (**p=0.02). **p <0.05 between normal and trauma plasma

When examining this data stratified by ISS, there were no significant differences in age, admission lactate, hospital or ICU length of stay (Table 2). There were no significant differences in pre-operative, post-operative, or total pRBC transfusions when stratified by ISS (Table 2). There was a greater decrease in hemoglobin from admission to discharge in those patients with a higher injury severity (11.4±2.7 to 9.0±0.9 g/dL). Those with a higher ISS had a 21% decrease in hemoglobin compared to only 10% for those with a lower ISS, despite no difference in ICU or hospital length of stay and total number of pRBC transfusions.

Table 2.

Trauma patient outcome data stratified by ISS. ISS = injury severity score; ICU = intensive care unit

	ISS 10–26	ISS 27–41	P-value
N	9	6
ISS	18 ± 6	32 ± 5	<0.01
Age (years)	48 ± 19	35 ± 18	0.21
Admission lactate (mmol/L)	3.9 ± 1.8	2.9 ± 1.2	0.29
Hospital length of stay (days)	16 ± 8	11 ± 4	0.19
ICU length of stay (days)	6 ± 6	6 ± 2	0.97
Pre-operative pRBC transfusion (units)	1.9 ± 1.7	1.7 ± 1.9	0.82
Intra-operative pRBC transfusion (units)	1.9 ± 1.5	2.0 ± 1.9	0.90
Post-operative pRBC transfusion (units)	2.6 ± 3.9	1.2 ± 1.5	0.43
Total hospital stay pRBC transfusion (units)	6.3 ± 5.4	4.8 ± 4.7	0.59
Admission hemoglobin (g/dL)	10.3 ± 2.5	11.4 ± 2.7	0.43
Discharge hemoglobin (g/dL)	9.3 ± 1.3	9.0 ± 0.9	0.71
Percent decrease in hemoglobin at discharge	10	21	0.03

Interleukin-6

Trauma plasma increased IL-6 expression over time, reaching its peak at 48 hours with a nearly five-fold increase from 6 hours (Figure 3A). At 24 and 48 hours, IL-6 expression was significantly increased with trauma plasma as compared to normal plasma by 103% and 111% respectively (p=0.04 and p=0.02, respectively). IL-6 expression was significantly increased at 48 hours by trauma plasma when compared to bone marrow fibroblast expression alone (p=0.01).

Figure 3A-B. Trauma increased stromal IL-6 expression.

3A: Trauma plasma increased stromal fibroblast IL-6 expression reaching a peak at 48 hours. 3B: Stromal fibroblast IL-6 expression reached its peak in the trauma ISS 27–41 group at 48 hours and was significantly elevated to normal plasma (**p<0.01) and untreated fibroblasts (*p<0.01). *p <0.05 vs. untreated HS-5 fibroblasts, **p <0.05 between normal and trauma plasma. ISS = injury severity score

When trauma plasma was stratified by ISS, IL-6 expression significantly increased 169% (p<0.01) and 187% (p<0.01) at 48 hours when compared to normal plasma and bone marrow fibroblasts alone, respectively (Figure 3B).

Granulocyte Colony Stimulating Factor

Trauma plasma increased G-CSF expression at each time point reaching its peak expression at 48 hours with a six-fold increase from 6 hours (Figure 4A). At 48 hours, G-CSF expression was significantly increased by trauma plasma compared to the normal patient plasma (p=0.03) and to bone marrow fibroblast expression alone (p=0.01).

Figure 4A-B. Trauma increased stromal G-CSF expression.

4A: Trauma plasma increased stromal fibroblast G-CSF expression reaching a peak at 48 hours. 4B. Stromal fibroblast G-CSF expression reached its peak in the trauma ISS 27–41 group at 48 hours and was significantly elevated compared to normal plasma (**p<0.01) and untreated fibroblasts (*p<0.01). *p <0.05 vs. untreated HS-5 fibroblasts, **p <0.05 between normal and trauma plasma. G-CSF = granulocyte-colony stimulating factor; ISS = injury severity score

When trauma plasma was stratified by ISS, G-CSF expression increased 249% (p<0.01) and 130% (p<0.01) at 48 hours when compared to normal plasma and bone marrow fibroblasts alone, respectively (Figure 4B).

Erythropoietin

EPO expression increased at each time point after exposure to trauma plasma, reaching its peak at 48 hours, which is a 30% increase from 6 hours (Figure 5A). After 24 and 48 hours of exposure to trauma plasma, trauma EPO expression was significantly increased by 82% and 108% respectively, compared to bone marrow fibroblast expression alone (p=0.03 and p=0.02, respectively). Trauma EPO expression was only significantly elevated compared to normal plasma at 48 hours (p=0.04).

Figure 5A-B. Trauma increased stromal EPO expression.

5A: Trauma plasma increased stromal fibroblast EPO expression reaching a peak at 48 hours. 5B: EPO expression reached its peak in the trauma ISS 27–41 group at 48 hours and was significantly elevated compared to normal plasma (**p=0.03) and untreated fibroblasts (*p=0.04). *p <0.05 vs. untreated HS-5 fibroblasts, **p <0.05 between normal and trauma plasma. EPO = erythropoietin; ISS = injury severity score

Stratifying trauma plasma by ISS, EPO expression reached its peak at 48 hours in the ISS 27–41 group with a significant 141% increase in expression compared to bone marrow fibroblasts alone (p=0.04) and 221% in the when compared to the normal plasma (p=0.03) (Figure 5B).

Stem Cell Factor

SCF expression was significantly elevated after exposure to trauma plasma compared to bone marrow fibroblasts alone at 6, 24, and 48 hours (p<0.01, p<0.01, and p=0.04 respectively) (Figure 6A). However, trauma plasma did not significantly alter SCF expression at any time point when compared to normal plasma.

Figure 6A-B. Trauma did not alter stromal SCF expression.

6A: Trauma and normal plasma increased stromal SCF expression compared to untreated fibroblasts at 6, 24 and 48 hours. 6B: SCF expression reached its peak in the trauma ISS 27–41 group and was significantly elevated compared to normal plasma (**p=0.01) and untreated fibroblasts (*p<0.01). *p <0.05 vs. untreated HS-5 fibroblasts, **p <0.05 between normal and trauma plasma. SCF = stem cell factor; ISS = injury severity score

When trauma plasma was stratified by ISS, SCF expression increased by 129% (p=0.01) and 554% (p<0.01) at 48 hours when compared to normal plasma and bone marrow fibroblasts alone, respectively (Figure 6B).

Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells

NF-κB activation increased after exposure to trauma plasma over time with a 73% increase from 6 hours to 48 hours (Figure 7A). However, when comparing normal plasma to trauma plasma, NF-κB activation was not significantly altered at any time point. NF-κB activation was significantly increased at 48 hours with trauma plasma compared to bone marrow fibroblasts alone (p=0.01). When trauma plasma was stratified by ISS, there was no significant change in NF-κB activation at any time points (Figure 7B).

Figure 7A-B. Trauma did not significantly alter stromal NF-kB expression.

7A: Trauma and normal plasma did not significantly alter stromal NF-κB expression at any time point. 7B: Stromal NF-κB expression was not significantly altered when stratified by ISS. *p <0.05 vs. untreated HS-5 fibroblasts, **p <0.05 between normal and trauma plasma.

Discussion

Persistent injury-associated anemia has been demonstrated after trauma in both animal models and humans.^{2, 5, 7, 9, 11} The primary focus of these studies has been on the suppressed growth of erythroid progenitor cells after injury and its relationship to a prolonged hypercatecholamine state and persistent anemia. Plasma obtained from trauma patients has increased levels of cytokines that have been shown to play a role in erythropoiesis. The data presented here clearly show that the plasma from severely injured trauma patients systemically regulates bone marrow stromal cytokine expression and that the severity of trauma direct impacts stromal cytokine expression. Fonseca et al. demonstrated that the bone marrow stroma is under adrenergic control and is a critical regulator of erythropoiesis. ^{12, 29} Wu et al. demonstrated that the bone marrow stroma mediated the inhibition of erythroid progenitor colony growth in trauma patients.^{12, 29}

In this study, norepinephrine concentration was increased in the trauma plasma compared to normal plasma. Previous work has shown that norepinephrine is elevated following trauma, and that there is persistent elevation of norepinephrine that is associated with persistent injury-associated anemia.^{5, 6, 9, 11, 12, 15, 16} Our findings also suggest that hypercatecholaminemia has a direct relationship with erythropoietic dysfunction and anemia. The anemia is more severe in those patients with a higher ISS despite no statistical difference in transfusion, ICU and hospital length of stay.

Bone marrow stromal cells have reduced growth after severe traumatic injury and also have been shown to be made of primarily fibroblasts.^{2, 29} Fibroblasts also participate in cytokine regulation.^{19, 20} The data presented here demonstrate that trauma plasma increased bone marrow fibroblast IL-6 expression over time. This correlates with studies demonstrating higher circulating levels of IL-6 in both trauma patients and rodent trauma models.^30,31 In this study, trauma plasma produced a peak in IL-6 expression from stromal fibroblasts at 48 hours. When stratified by severity of injury, IL-6 expression was higher than with normal plasma and controls. Similarly, Qiao et al. demonstrated in their meta-analyses that trauma patients with higher IL-6 plasma levels were more likely to experiences complications or death which may have correlated with ISS.³²

Trauma plasma increased stromal G-CSF expression when compared to the control plasma and untreated fibroblasts. Trauma plasma from patients with higher injury severity had significant elevation of G-CSF expression from stromal fibroblasts when compared to control plasma. It has been previously shown that following severe trauma, G-CSF levels increase which led to the release of hematopoietic progenitor cells to the peripheral blood and that this increase correlated with ISS.^{33, 34} Cook et al. demonstrated in trauma patients that the severity of traumatic injury directly correlated with plasma G-CSF concentration and prolonged mobilization of hematopoietic progenitor cells from the bone marrow.³⁵ This increased G-CSF concentration, prolonged hematopoietic progenitor cell mobilization, and reduced bone marrow cellularity were associated with persistent injury-associated anemia.³⁵ G-CSF and norepinephrine have been linked to play a role in hematopoietic progenitor cell mobilization, therefore, it is likely that this link contributes to abnormal erythropoiesis after severe trauma.

In addition to IL-6 and G-CSF, trauma plasma also increased EPO stromal fibroblast expression when compared to control plasma. Previous studies have demonstrated that plasma EPO levels are increased following trauma, yet hemoglobin levels were reduced.^{2, 36} To explain this dichotomy, a murine model found that systemic inflammation suppressed bone marrow erythropoiesis by inducing apoptosis of immature erythroblasts and that administration of exogenous EPO did not improve erythropoiesis.³⁷ Despite elevation of circulating EPO following trauma, persistent anemia is seen following trauma. This persistent anemia is likely related to bone marrow’s inability to respond to EPO demonstrated by a reduced expression of the bone marrow EPO receptor. This correlates with our finding that trauma plasma increased expression of EPO in stromal fibroblasts as ISS worsens despite the presence of a moderate anemia.

Following culture with control and trauma plasma, SCF expression by bone marrow fibroblasts was elevated in both groups at all time points. When stratified by ISS, SCF expression was significantly elevated at 48 hours in the high ISS group. The increased SCF fibroblast expression by control and trauma plasma may correlate with the differing roles of SCF in the bone marrow niche. During times of homeostasis, SCF is a retention molecule to preserve hematopoietic progenitor cells within the bone marrow niche.²⁴ Alternately, SCF is also associated with progenitor cell mobilization which is necessary for healing following injury.^{17, 38, 38} Its role in bone marrow mobilization is consistent with its increased expression in stromal fibroblasts following traumatic injury, however, its increased expression with control plasma is not well understood. Further research on the role of SCF in the bone marrow niche is needed to fully understand its expression during times of both homeostasis and stress.

Fibroblast NF-κB activation was only significantly increased by trauma plasma at 48 hours, although the increase in normal plasma at 48 hours had a similar mean. These findings were not expected given that the NF-κB pathway is considered a prototypical pro-inflammatory pathway.³⁹ NF-κB has also been implicated in chronic inflammatory disease by inducing the transcription of pro-inflammatory cytokines, chemokines and matrix metalloproteinases.^{39, 40} It is possible that bone marrow fibroblasts do not play an integral role in this pathway.

Limitations

The limitations of this study include that a single bone marrow fibroblast cell line was used to study the bone marrow response to trauma plasma and may not fully replicate the bone marrow niche. However, fibroblasts are a major component of the bone marrow stroma and have been shown to participate in cytokine regulation. Further research will continue to expand on these results by investigating the effects of trauma plasma on the cytokine expression of whole bone marrow and the causal relationship between this induced pro-inflammatory state and persistent injury-associated anemia.

In addition, the trauma patient group was almost evenly divided into male and female sub-sections with the females varying from 19 years to 90 years old, while the untreated fibroblasts were obtained from a 30-year-old male. Schneider et al. demonstrated in a murine trauma and hemorrhagic shock model that reproductively mature females have an increase in differentiation of bone marrow cells towards monocyte/macrophage lineage, independent of age.³⁹ They also state that female sex hormones likely have a critical role in the bone marrow environment following trauma and hemorrhagic shock.³⁹ Given these findings and the present study’s sex breakdown, then further breakdown by pre- and post-menopausal females, no significant additional conclusions can be made in this trauma group as the sub-group sample size would be small. Further research with a larger sample size is needed to investigate the role of age and sex.

Conclusions

Circulating plasma from trauma patients activated bone marrow stromal fibroblasts, inducing an inflammatory state associated with elevation of norepinephrine. Trauma plasma increased stromal fibroblast IL-6, G-CSF and EPO expression, but did not significantly affect SCF expression or NF-κB activation compared to control plasma. The anemia is more severe in those patients with a higher ISS despite no statistical difference in transfusion, ICU and hospital length of stay, thus supporting a link between elevation of norepinephrine and erythropoietic dysfunction.