TY - JOUR
AU - Pallister, I
AB - Abstract Background Changes in human bone marrow associated with the systemic inflammatory response to injury are little understood. It was hypothesized that major trauma results in an altered bone marrow leucocyte progenitor profile, with either uniform depletion or the balance between multipotent and committed progenitors varying, depending on whether self-renewal is favoured over differentiation. Methods Bone marrow aspirate and peripheral blood samples were obtained at definitive surgery in adults with pelvic fractures from blunt trauma (major trauma with Injury Severity Score (ISS) at least 18, or isolated fractures) and control patients undergoing iliac crest bone grafting. ISS, interval to surgery and transfusion in the first 24 h were recorded. Bone marrow aspirate flow cytometry was used to identify haemopoietic progenitor cells (CD34+), multipotent cells (CD34+ CD45+ CD38−) and oligopotent cells (CD34+ CD45+ CD38lo/+ and CD34+ CD45+ CD38BRIGHT(++ +) subsets). Peripheral blood levels of inflammatory markers were measured, and the ratio of immature to mature (CD35−/CD35+) granulocytes was determined. Results The median (range) interval between injury and sampling was 7 (1–21) and 5 (1–21) days in the major trauma and isolated fracture groups respectively. The CD34+ pool was significantly depleted in the major trauma group (P = 0·017), particularly the CD34+ CD45+ CD38BRIGHT(++ +) oligopotent pool (P = 0·003). Immature CD35− granulocytes increased in bone marrow with increasing injury severity (P = 0·024) and massive transfusion (P = 0·019), and in peripheral blood with increasing interval to surgery (P = 0·005). Conclusion Major blunt trauma resulted in changes in the bone marrow CD34+ progenitor pool. At the point in recovery when these samples were obtained, oligopotent progenitors were lost from the bone marrow, with continued release of immature cells. Surgical relevance Major trauma and haemorrhagic shock leads to dysregulation of the systemic inflammatory response, which is central to the development of acute respiratory distress syndrome and multiple organ failure. Bone marrow failure results in an inability to maintain haemopoietic cell production, leading to persistent anaemia and an increased susceptibility to infection. The present study documents a significant change in leucopoiesis following major trauma, with a reduction in multipotent progenitors, a significant loss of oligopotent progenitors and release of immature cells into the peripheral circulation. Although changes occurring during the recovery phase following injury are documented, questions are raised about modulation of bone marrow activity following major trauma to preserve self-renewal, differentiation and the production of functional mature end cells. A more detailed study of the CD34+ progenitors within the resuscitation phase can now be justified to define further the effect of trauma and haemorrhagic shock on leucopoiesis with subsequent clinical progress. Introduction Dysregulation of the systemic inflammatory response to injury1–3 is central to the development of acute respiratory distress syndrome, multiple organ failure and sepsis. A substantial body of work has determined the key effector cells and mediators in this response4–6, and surgical strategies have evolved in the light of this understanding7–12. Despite this, there is a major gap in knowledge. The stem cell pool in human bone marrow is the point of origin of the cellular components of blood. Although the importance of primed and activated circulating leucocytes has long been recognized, their bone marrow origins have eluded detailed study, possibly because of the relative inaccessibility of this tissue for research. The endothelial precursor cells involved in both healing of the endothelium and vasculoneogenesis in wounds originate from the same stem cell pool13,14. The role of the vascular endothelium is increasingly being recognized for its importance in the shocked state, coagulopathy and orchestrating the recruitment of circulating leucocytes, both in initiation of the inflammatory response and in its resolution. The few data that exist relating to the response to injury in human leucopoietic bone marrow point towards suppression (both haemopoietic and stromal cell) following major trauma15, supported by studies of progenitor cells isolated from peripheral blood and subsequent colony-forming unit assays16,17. Haemopoietic stem cells (HSCs) give rise to multipotent progenitors, which in turn give rise to oligopotent and then lineage-restricted progenitors, and ultimately to mature circulating leucocytes (Fig. 1)18. Commitment, proliferation and differentiation are believed to be mediated by extrinsic factors, such as cytokines. Differentiation is characterized by changing patterns of CD expression. Fig. 1 Open in new tabDownload slide Schematic representation of human leucopoiesis18. The CD34+ cell pool includes cell types up to the stage of lineage-restricted progenitors. Multipotent progenitors may also give rise to common lymphoid progenitors in an alternative route of lineage commitment, but this has been omitted for the sake of clarity. Similarly, the granulocyte (CD15+ CD35−) progenitor population may also give rise to eosinophils and basophils, but this again has been omitted for clarity Given the sustained nature of the systemic inflammatory response to injury and the associated dysfunction of circulating leucocytes, it was hypothesized that the pattern of self-renewal/differentiation among HSCs/bone marrow leucocyte progenitors would be significantly altered following trauma. This may be manifest as uniform depletion of the whole CD34+ pool. Alternatively, if the bone marrow favoured differentiation at the expense of self-renewal, then depletion of the multipotent progenitor pool would be seen. Conversely, oligopotent progenitor depletion may reflect self-renewal at the stem cell/multipotent progenitor level being favoured over differentiation. Any changes in the balance of self-renewal/differentiation are likely to be manifest as alterations in markers of effector cell maturity. A study was conducted to elucidate changes in human bone marrow activity after pelvic injury, exploiting the opportunity of definitive fracture fixation to obtain bone marrow samples. Methods The setting of the study was a major acute university hospital providing tertiary-level (level 1) trauma care. Ethical approval was obtained for a prospective observational study from the local research ethics committee (LREC). Consent (or assent from the next of kin) was sought from injured patients as soon as was reasonable after admission, and before surgery in the control group, in accordance with LREC approval. All adult patients with fractures of the pelvis (pelvic ring or acetabulum) after blunt trauma were eligible for the study. The patients were divided into those with major blunt trauma (Injury Severity Score (ISS) at least 18)19 and those with isolated fractures. Control samples were obtained from patients without acute trauma undergoing iliac crest bone graft harvesting. Exclusion criteria were pre-existing conditions (connective tissue disease, diabetes, malignancy) or use of medication (steroids/cytotoxic agents) that may impair the inflammatory response. Sample collection Bone marrow aspirates and peripheral blood samples were obtained at the time of definitive fracture surgery. Patients who had sustained major trauma underwent surgery when judged (on clinical grounds) to be well enough to withstand a major operative procedure. The bone marrow aspirate and peripheral blood samples from these patients represent a profile of bone marrow leucocyte progenitors at a time when the patients were beginning to recover from their injuries, but still required critical care support. Patients with an isolated fracture were operated on as soon as possible, allowing for interhospital transfer in approximately half of this group. Anticoagulated (EDTA) peripheral blood samples were drawn from freshly inserted arterial or venous cannulas in the anaesthetic room, before transfer to the operating theatre. An anticoagulated (EDTA) bone marrow aspirate was obtained from the pelvis using a bone marrow aspiration needle (Mana-Tech, Burton on Trent, UK) as soon as possible during the operative fracture fixation for injured patients, and at the very beginning of surgery for controls. Flow cytometric analysis Flow cytometry was performed within 2 h of sample collection. Antibodies and associated isotype controls, conjugated to various fluorochromes (FITC, PE, APC, PerCP), were purchased from BD Biosciences (Oxford, UK; CD35-FITC, CD15-APC, CD45-FITC, CD34-PerCP) and Caltag (Buckingham, UK; CD38-PE). Whole peripheral blood or bone marrow aspirate (100 µl) was incubated on ice in the dark for 30 min with the relevant antibodies and isotype controls at preoptimized titrations, before undergoing red blood cell lysis (EasyLyse™; Biostat, Stockport, UK) in accordance with the manufacturer's protocol. The cells were washed in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline with 0·2 per cent bovine serum albumin and 0·05 per cent sodium azide), fixed in FACSfix™ (BD Biosciences), and the data were acquired on a flow cytometer (FACSAria™; BD Biosciences) within 24 h. The target for analysis was 5000 CD34+ events, but this was not always achieved; however, a minimum of 2000 events was always acquired. Analysis of the CD38 subset was performed on low-side-scatter CD45+ CD34+ cells. Immature CD15+ CD35− granulocytes and mature CD15+ CD35+ granulocytes were measured within the bone marrow aspirate and peripheral blood, and the ratio of immature to mature granulocytes was calculated. The data were analysed using Diva™ software and FlowJo version 7.5 (BD Biosciences). Enzyme-linked immunosorbent assay Plasma was collected by centrifugation (1000g for 20 min) of anticoagulated (EDTA) whole blood samples within 2 h of collection and stored at − 80 °C until analysis. Supernatant samples from the bone marrow aspirates were processed in the same manner. The plasma and bone marrow aspirate supernatants were thawed rapidly and centrifuged (1000g for 10 min) immediately before analysis. Interleukin (IL) 6, soluble IL-6 receptor (sIL-6R), IL-8 (PeliKine™; Sanquin/CLB, Raamsdonksveer, The Netherlands) and granulocyte colony-stimulating factor (G-CSF) (Quantikine®; R&D Systems, Minneapolis, Minnesota, USA) were measured according to the manufacturer's protocol. The sensitivity of the assays was: 0·2 pg/ml for IL-6, 10 pg/ml for sIL-6R, 3 pg/ml for IL-8 and 20 pg/ml for G-CSF. Definitions Data relating to the mechanism and pattern of injury were recorded, as well as the interval from injury to the samples being obtained. In the major trauma group, the need for massive transfusion (defined as hypotension and a transfusion requirement of more than 10 units in 24 h) during initial resuscitation was also recorded20, along with admission Glasgow Coma Scale (GCS) score, systolic blood pressure and base deficit. Comparisons were made between the control, isolated fracture and major trauma groups. The impact of interval between injury and sampling was studied, along with ISS and transfusion requirements in the major trauma group. Within the major trauma group the interval to surgery was classified into three groups (1–4, 5–8 and 9 or more days) and the ISS into two groups (18–25, 26 or more). Sex and the need for transfusion were always discrete variables and naturally split the data into subsamples (men and women; massive transfusion and no massive transfusion). Flow cytometric analysis included the bone marrow multipotent cell fraction, defined as CD34+ CD45+ CD38− with low side scatter, and the oligopotent cell fraction, defined as CD34+ CD45+ CD38lo/+ with low side scatter. The more mature proportion of this oligopotent cell fraction, before lineage-restricted progenitors, was termed CD34+ CD45+ CD38BRIGHT(++ +) (Fig. 1). Lineage-restricted granulocyte progenitors were CD15+ CD35−, whereas mature granulocytes were CD15+ CD35+. Statistical analysis Continuous data are presented as median (range). The graphics and statistical analyses were carried out using SPSS® (IBM, London, UK). Most variables were skewed and therefore log-transformed before analysis. This was necessary for the analysis of CD surface markers, CD35 ratios and G-CSF data. After transformation the variables were well matched to the normal distribution as measured by the Kolmogorov–Smirnov goodness-of-fit test (P = 0·440 was the smallest test statistic encountered). As a result, all comparisons, unless stated otherwise, were conducted using ANOVA. Comparison of group characteristics (interval to surgery, age and sex) was performed by means of the Mann–Whitney U test and χ2 test of association. Results Fifteen surgical control patients, 25 with an isolated fracture and 17 patients who had sustained major trauma were included in the analysis. Patient details are shown in Table 1. The interval from injury to bone marrow aspiration/peripheral blood sampling was lower in the isolated fracture group than in the major trauma group, but not significantly so (P = 0·164). The age and sex distribution were similar in the trauma groups (P = 0·682 and P = 0·391 respectively). All patients with major injuries required transfusion but only 12 were massively transfused (more than 10 units packed red blood cells; median 14·5 (11–29) units). Thirteen patients in the major trauma group received fresh frozen plasma (median 8 (4–20) units) and nine received platelets (median 2 (1–3) units). The median stay in the intensive care unit in this group was 9 (2–26) days. Table 1 Patient details . Surgical control (n = 15) . Isolated fracture (n = 25) . Major trauma (n = 17) . Age (years)* 34 (22–61) 42 (17–63) 40 (17–65) Sex ratio (M : F) 7 : 8 16 : 9 13 : 4 Blood pressure on admission (mmHg)* NA 124 (94–156) 100 (60–154) GCS score on admission* NA 15 (14–15) 14 (3–15) Base deficit on admission (mmol/l)* NA − 0·02 (−2·0 to 2·0) − 4·9 (−0·8 to − 15·6) Injury Severity Score* NA 9 (9–17) 27 (18–50) Mechanism of injury  MCRTC NA 4 7  VRTC NA 14 7  Crush NA 2 1  Fall NA 5 2 Interval from injury to sampling (days)* NA 5 (1–21) 7 (1–21) White blood cell count at sampling (×109/l)* 6·2 (5–7·2) 7·8 (5·2–10·3) 9·98 (3·02–26·6) Absolute neutrophil count at sampling (×109/l)* 3·4 (2·4–4·5) 5·1 (3·8–7·9) 7·6 (2·31–22·9) . Surgical control (n = 15) . Isolated fracture (n = 25) . Major trauma (n = 17) . Age (years)* 34 (22–61) 42 (17–63) 40 (17–65) Sex ratio (M : F) 7 : 8 16 : 9 13 : 4 Blood pressure on admission (mmHg)* NA 124 (94–156) 100 (60–154) GCS score on admission* NA 15 (14–15) 14 (3–15) Base deficit on admission (mmol/l)* NA − 0·02 (−2·0 to 2·0) − 4·9 (−0·8 to − 15·6) Injury Severity Score* NA 9 (9–17) 27 (18–50) Mechanism of injury  MCRTC NA 4 7  VRTC NA 14 7  Crush NA 2 1  Fall NA 5 2 Interval from injury to sampling (days)* NA 5 (1–21) 7 (1–21) White blood cell count at sampling (×109/l)* 6·2 (5–7·2) 7·8 (5·2–10·3) 9·98 (3·02–26·6) Absolute neutrophil count at sampling (×109/l)* 3·4 (2·4–4·5) 5·1 (3·8–7·9) 7·6 (2·31–22·9) * Values are median (range). NA, not applicable; GCS, Glasgow Coma Scale; MCRTC, motorcycle road traffic collision; VRTC, vehicle road traffic collision occupant/pedestrian. Open in new tab Table 1 Patient details . Surgical control (n = 15) . Isolated fracture (n = 25) . Major trauma (n = 17) . Age (years)* 34 (22–61) 42 (17–63) 40 (17–65) Sex ratio (M : F) 7 : 8 16 : 9 13 : 4 Blood pressure on admission (mmHg)* NA 124 (94–156) 100 (60–154) GCS score on admission* NA 15 (14–15) 14 (3–15) Base deficit on admission (mmol/l)* NA − 0·02 (−2·0 to 2·0) − 4·9 (−0·8 to − 15·6) Injury Severity Score* NA 9 (9–17) 27 (18–50) Mechanism of injury  MCRTC NA 4 7  VRTC NA 14 7  Crush NA 2 1  Fall NA 5 2 Interval from injury to sampling (days)* NA 5 (1–21) 7 (1–21) White blood cell count at sampling (×109/l)* 6·2 (5–7·2) 7·8 (5·2–10·3) 9·98 (3·02–26·6) Absolute neutrophil count at sampling (×109/l)* 3·4 (2·4–4·5) 5·1 (3·8–7·9) 7·6 (2·31–22·9) . Surgical control (n = 15) . Isolated fracture (n = 25) . Major trauma (n = 17) . Age (years)* 34 (22–61) 42 (17–63) 40 (17–65) Sex ratio (M : F) 7 : 8 16 : 9 13 : 4 Blood pressure on admission (mmHg)* NA 124 (94–156) 100 (60–154) GCS score on admission* NA 15 (14–15) 14 (3–15) Base deficit on admission (mmol/l)* NA − 0·02 (−2·0 to 2·0) − 4·9 (−0·8 to − 15·6) Injury Severity Score* NA 9 (9–17) 27 (18–50) Mechanism of injury  MCRTC NA 4 7  VRTC NA 14 7  Crush NA 2 1  Fall NA 5 2 Interval from injury to sampling (days)* NA 5 (1–21) 7 (1–21) White blood cell count at sampling (×109/l)* 6·2 (5–7·2) 7·8 (5·2–10·3) 9·98 (3·02–26·6) Absolute neutrophil count at sampling (×109/l)* 3·4 (2·4–4·5) 5·1 (3·8–7·9) 7·6 (2·31–22·9) * Values are median (range). NA, not applicable; GCS, Glasgow Coma Scale; MCRTC, motorcycle road traffic collision; VRTC, vehicle road traffic collision occupant/pedestrian. Open in new tab All women in the surgical control group underwent fusion procedures to the pelvic ring for postpartum pelvic pain, caused by mechanical instability and not osteitis. One man underwent pelvic ring fusion for residual pain following ligamentous pelvic injury 4 months after the original injury. The remainder all required iliac crest bone grafting for long bone non-unions. All of these patients had previously had isolated fractures, but more than 3 months had elapsed since the original injury and none had active infection. Flow cytometric analysis The CD34+ pool was significantly depleted in the bone marrow aspirate of the major trauma group (P = 0·005) and was considerably smaller in the isolated fracture group (P = 0·091) in comparison with controls. No significant differences were found in the percentage of multipotent progenitors (CD34+ CD45+ CD38−) between the three groups (Table 2), but significantly fewer oligopotent progenitors (CD34+ CD45+ CD38BRIGHT(++ +)) were observed in the isolated fracture (P = 0·029) and major trauma (P < 0·001) groups than in the controls (Fig. 2). Fig. 2 Open in new tabDownload slide CD34+ CD45+ cells with low side scatter were selected and analysed for CD38-PE or matched isotype control expression in surgical control, isolated fracture and major trauma groups. a Dot plots show a highly CD34+ population with no or low CD38 expression, moving upwards to a CD38BRIGHT(++ +) population (ringed); this is strongly positive in the surgical controls, reduced in the isolated fracture group and absent from the major trauma population. b Histograms showing relative frequencies of CD34+ CD38BRIGHT(++ +) cells; the smaller CD38 peak represents the CD38BRIGHT(++ +) population Table 2 Analysis of cell surface markers in bone marrow aspirate samples and circulating granulocytes . P (ANOVA) . . SC versus IF versus MT . Interval to surgery (IF and MT) . Massive transfusion requirement (MT only) . ISS (MT only) . Bone marrow  Multipotent cells   CD34+ 0·017 (< 0·001) 0·752 0·387 0·079   CD34+ CD45+ CD38− 0·502 0·375 0·148 < 0·001  Oligopotent cells   CD34+ CD45+ CD38lo/+ 0·815 0·858 0·340 0·049   CD34+ CD45+ CD38BRIGHT(++ +) 0·003 (< 0·001) 0·469 0·889 0·999  CD35−/CD35+ ratio 0·028 0·417 0·019 0·024 Circulating granulocytes  CD35−/CD35+ ratio 0·564 0·005 0·153 0·167  CD35− cells standardized for absolute neutrophil count 0·668 0·072 0·070 0·026 . P (ANOVA) . . SC versus IF versus MT . Interval to surgery (IF and MT) . Massive transfusion requirement (MT only) . ISS (MT only) . Bone marrow  Multipotent cells   CD34+ 0·017 (< 0·001) 0·752 0·387 0·079   CD34+ CD45+ CD38− 0·502 0·375 0·148 < 0·001  Oligopotent cells   CD34+ CD45+ CD38lo/+ 0·815 0·858 0·340 0·049   CD34+ CD45+ CD38BRIGHT(++ +) 0·003 (< 0·001) 0·469 0·889 0·999  CD35−/CD35+ ratio 0·028 0·417 0·019 0·024 Circulating granulocytes  CD35−/CD35+ ratio 0·564 0·005 0·153 0·167  CD35− cells standardized for absolute neutrophil count 0·668 0·072 0·070 0·026 P values are shown for ANOVA of percentage expression (log-transformed) of CD cell surface markers in bone marrow aspirates (reflecting multipotent and oligopotent progenitors) and circulating granulocytes. P values in parentheses are for analysis of mean fluorescence intensity. SC, surgical control; IF, isolated fracture; MT, major trauma; ISS, Injury Severity Score. Open in new tab Table 2 Analysis of cell surface markers in bone marrow aspirate samples and circulating granulocytes . P (ANOVA) . . SC versus IF versus MT . Interval to surgery (IF and MT) . Massive transfusion requirement (MT only) . ISS (MT only) . Bone marrow  Multipotent cells   CD34+ 0·017 (< 0·001) 0·752 0·387 0·079   CD34+ CD45+ CD38− 0·502 0·375 0·148 < 0·001  Oligopotent cells   CD34+ CD45+ CD38lo/+ 0·815 0·858 0·340 0·049   CD34+ CD45+ CD38BRIGHT(++ +) 0·003 (< 0·001) 0·469 0·889 0·999  CD35−/CD35+ ratio 0·028 0·417 0·019 0·024 Circulating granulocytes  CD35−/CD35+ ratio 0·564 0·005 0·153 0·167  CD35− cells standardized for absolute neutrophil count 0·668 0·072 0·070 0·026 . P (ANOVA) . . SC versus IF versus MT . Interval to surgery (IF and MT) . Massive transfusion requirement (MT only) . ISS (MT only) . Bone marrow  Multipotent cells   CD34+ 0·017 (< 0·001) 0·752 0·387 0·079   CD34+ CD45+ CD38− 0·502 0·375 0·148 < 0·001  Oligopotent cells   CD34+ CD45+ CD38lo/+ 0·815 0·858 0·340 0·049   CD34+ CD45+ CD38BRIGHT(++ +) 0·003 (< 0·001) 0·469 0·889 0·999  CD35−/CD35+ ratio 0·028 0·417 0·019 0·024 Circulating granulocytes  CD35−/CD35+ ratio 0·564 0·005 0·153 0·167  CD35− cells standardized for absolute neutrophil count 0·668 0·072 0·070 0·026 P values are shown for ANOVA of percentage expression (log-transformed) of CD cell surface markers in bone marrow aspirates (reflecting multipotent and oligopotent progenitors) and circulating granulocytes. P values in parentheses are for analysis of mean fluorescence intensity. SC, surgical control; IF, isolated fracture; MT, major trauma; ISS, Injury Severity Score. Open in new tab The was some evidence of depletion of the CD34+ pool with increasing injury severity (P = 0·079). In particular, increasing injury severity had a significant impact on multipotent (CD34+ CD45+ CD38−; P < 0·001) and oligopotent (CD34+ CD45+ CD3810/+; P = 0·049) subpopulations in the major trauma group. The need for massive transfusion and the interval to sampling had no influence on the bone marrow subpopulations in the major trauma group. Cytokine expression IL-6 levels were higher in the major trauma (median 18·6 (2·7–1697) pg/ml; P = 0·007) and isolated fracture (9·8 (1·8–119·1) pg/ml; P = 0·028) groups than in controls (7·0 (0–26·9) pg/ml. Concentrations of IL-6 appeared to decline with increasing interval between injury and sampling after major trauma (1–4 days: 67·5 (22·9–1697) pg/ml; 5–8 days: 6·1 (4·7–234) pg/ml; at least 9 days: 4·7 (2·7–71·1) pg/ml; P = 0·068). There was no significant difference in IL-8 and sIL-6R levels between the major trauma, isolated fracture and control groups, although there was evidence of a higher sIL-6R level in the major trauma compared with the isolated fracture group (36 172 (14 823–76 302) versus 25 787 (11 304–122 347) pg/ml; P = 0·046). The G-CSF level in peripheral blood was similar in the three groups, and there was only weak evidence that it was affected by either the delay to surgery (P = 0·101), the need for transfusion (P = 0·100) or the severity of injury as measured by ISS (P = 0·074). Immature granulocytes There was a significant difference in the ratio of immature to mature (CD35−/CD35+) granulocytes in the bone marrow between the major trauma, isolated fracture and control groups (P = 0·028), and strong evidence that this was influenced by the need for massive transfusion (P = 0·019) and injury severity (P = 0·024), but not the interval from injury to sampling (Table 2). In contrast, there was strong evidence that only the interval from injury to sampling influenced the ratio of immature to mature (CD35−/CD35+) granulocytes in the peripheral blood (P = 0·005), with a shorter delay being associated with relatively fewer immature cells. Further analysis of the CD35− cells standardized with respect to the absolute granulocyte count in peripheral blood indicated that injury severity had an influence (P = 0·026). G-CSF levels correlated well with peripheral blood granulocyte CD35 expression (rS = − 0·873, P < 0·001). There was some evidence of an association between G-CSF and the ratio of immature to mature (CD35−/CD35+) granulocytes in the peripheral blood (rS = − 0·591, P = 0·056). No correlation was found between the profile of the ratio of immature to mature (CD35−/CD35+) granulocytes in the peripheral blood and the changes in CD34+ subpopulations. Discussion Previous studies have described changes in circulating CD34+ cell numbers in relation to surgical trauma21 and in the bone marrow months after severe spinal cord injury22. However, data describing the relationship between the bone marrow hierarchy and acute major trauma are lacking. The schematic representation of leucopoiesis (Fig. 1) reveals the complexity of differentiation and maturation. This is not a linear process, as may be suggested by such a diagram. This representation is simplified, excluding as it does other possible routes of differentiation for the same CD34+ pool, including the lymphoid cell line, endothelial precursor cells and other mature cells from granulocyte (CD15+ CD35−) progenitors (eosinophils and basophils). The present results indicate a significant change to leucopoiesis following major trauma, with suppression in maturation. This is summarized in Table 3, along with an outline of the time course of relevant features in the systemic inflammatory response syndrome, along with some mediators, markers and cellular changes described in previous studies. Suppression of bone marrow activity is well recognized in critical illness, burns, hypovolaemic shock and trauma15,16, along with evidence of migration of HSCs to the site of direct trauma and shock-induced lung injury23,24. Table 3 Summary of main findings in relation to previously published data Time after injury . SIRS . Circulating markers and mediators . Bone marrow and circulating progenitors and granulocytes . Present findings . Minutes SIRS reflecting base deficit and shock1 Rapid increase in levels of IL-6 and IL-8, and generation of alarmins through cellular  injury5,10,12 Increased granulocyte numbers16 Hours Declining mature granulocyte numbers and increasing proportion of immature cells16 Days Persisting SIRS associated with nosocomial infection2,3 Raised levels settle towards normal in absence of ‘second hit’ Decreased colony-forming activity in bone marrow aspirates compared with increased activity in peripheral blood after major trauma15 CD34+ cell pool significantly reduced in MT Particularly the oligopotent CD34+ CD45+ CD38BRIGHT(++ +) pool Multipotent progenitor depletion associated with increasing ISS CD34+ cell numbers increased in relation to surgical trauma21 Lineage-restricted (granulocyte) progenitor CD15+ CD35− in bone marrow increased in association with ISS and massive transfusion Immature granulocytes CD15+ CD35− increased in peripheral blood with increasing interval to surgery Weeks Resolution Resolution Mature granulocytes CD15+ CD35+ G-CSF levels associated with CD35 expression Months Resolved Resolved Increased CD34+ cell numbers in the bone marrow months after severe spinal cord injury22 Time after injury . SIRS . Circulating markers and mediators . Bone marrow and circulating progenitors and granulocytes . Present findings . Minutes SIRS reflecting base deficit and shock1 Rapid increase in levels of IL-6 and IL-8, and generation of alarmins through cellular  injury5,10,12 Increased granulocyte numbers16 Hours Declining mature granulocyte numbers and increasing proportion of immature cells16 Days Persisting SIRS associated with nosocomial infection2,3 Raised levels settle towards normal in absence of ‘second hit’ Decreased colony-forming activity in bone marrow aspirates compared with increased activity in peripheral blood after major trauma15 CD34+ cell pool significantly reduced in MT Particularly the oligopotent CD34+ CD45+ CD38BRIGHT(++ +) pool Multipotent progenitor depletion associated with increasing ISS CD34+ cell numbers increased in relation to surgical trauma21 Lineage-restricted (granulocyte) progenitor CD15+ CD35− in bone marrow increased in association with ISS and massive transfusion Immature granulocytes CD15+ CD35− increased in peripheral blood with increasing interval to surgery Weeks Resolution Resolution Mature granulocytes CD15+ CD35+ G-CSF levels associated with CD35 expression Months Resolved Resolved Increased CD34+ cell numbers in the bone marrow months after severe spinal cord injury22 The median interval from injury to sampling for the major trauma (MT) group was 7 (range 1–21) days. SIRS, systemic inflammatory response syndrome; IL, interleukin; ISS, Injury Severity Score; G-CSF, granulocyte colony-stimulating factor. Open in new tab Table 3 Summary of main findings in relation to previously published data Time after injury . SIRS . Circulating markers and mediators . Bone marrow and circulating progenitors and granulocytes . Present findings . Minutes SIRS reflecting base deficit and shock1 Rapid increase in levels of IL-6 and IL-8, and generation of alarmins through cellular  injury5,10,12 Increased granulocyte numbers16 Hours Declining mature granulocyte numbers and increasing proportion of immature cells16 Days Persisting SIRS associated with nosocomial infection2,3 Raised levels settle towards normal in absence of ‘second hit’ Decreased colony-forming activity in bone marrow aspirates compared with increased activity in peripheral blood after major trauma15 CD34+ cell pool significantly reduced in MT Particularly the oligopotent CD34+ CD45+ CD38BRIGHT(++ +) pool Multipotent progenitor depletion associated with increasing ISS CD34+ cell numbers increased in relation to surgical trauma21 Lineage-restricted (granulocyte) progenitor CD15+ CD35− in bone marrow increased in association with ISS and massive transfusion Immature granulocytes CD15+ CD35− increased in peripheral blood with increasing interval to surgery Weeks Resolution Resolution Mature granulocytes CD15+ CD35+ G-CSF levels associated with CD35 expression Months Resolved Resolved Increased CD34+ cell numbers in the bone marrow months after severe spinal cord injury22 Time after injury . SIRS . Circulating markers and mediators . Bone marrow and circulating progenitors and granulocytes . Present findings . Minutes SIRS reflecting base deficit and shock1 Rapid increase in levels of IL-6 and IL-8, and generation of alarmins through cellular  injury5,10,12 Increased granulocyte numbers16 Hours Declining mature granulocyte numbers and increasing proportion of immature cells16 Days Persisting SIRS associated with nosocomial infection2,3 Raised levels settle towards normal in absence of ‘second hit’ Decreased colony-forming activity in bone marrow aspirates compared with increased activity in peripheral blood after major trauma15 CD34+ cell pool significantly reduced in MT Particularly the oligopotent CD34+ CD45+ CD38BRIGHT(++ +) pool Multipotent progenitor depletion associated with increasing ISS CD34+ cell numbers increased in relation to surgical trauma21 Lineage-restricted (granulocyte) progenitor CD15+ CD35− in bone marrow increased in association with ISS and massive transfusion Immature granulocytes CD15+ CD35− increased in peripheral blood with increasing interval to surgery Weeks Resolution Resolution Mature granulocytes CD15+ CD35+ G-CSF levels associated with CD35 expression Months Resolved Resolved Increased CD34+ cell numbers in the bone marrow months after severe spinal cord injury22 The median interval from injury to sampling for the major trauma (MT) group was 7 (range 1–21) days. SIRS, systemic inflammatory response syndrome; IL, interleukin; ISS, Injury Severity Score; G-CSF, granulocyte colony-stimulating factor. Open in new tab The global depletion of the CD34+ pool observed in this study reflects the loss of cells from the true stem cell to the oligopotent progenitors; however, more detailed examination showed that it was the CD34+CD45+CD38BRIGHT(++ +) subpopulation that was very significantly depleted after major trauma. This was a consistent finding, and was independent of the need for massive transfusion, and not associated with the time after injury. Although there was no similar depletion of the multipotent CD34+ CD45+ CD38− subpopulation, increasing injury severity within the major trauma group was associated with declining numbers of such cells. With the limited data available, and in particular only one sample per patient, it is difficult to interpret these findings with confidence. A decline in the oligopotent CD34+ CD45+ CD38BRIGHT(++ +) subpopulation may have resulted from an increased draw or demand on these progenitors to push out lineage-restricted progenitors, resulting in the observed increase in proportion of bone marrow granulocyte (CD15+ CD35−) progenitors. Alternatively, fewer cells in the multipotent CD34+ CD45+ CD38− subpopulation may have been committing to differentiate, possibly favouring renewal of the multipotent pool instead. Samples from seriously injured patients in this study were obtained at a range of time points following injury. Circulating IL-8 levels were comparable with those in controls at this time, IL-6 levels fell consistently with increasing interval following injury in the major trauma population, and sIL-6R levels were raised in the major trauma group compared with those in patients with isolated fractures. These data are consistent with the clinical picture of a resolving or beneficially evolving systemic inflammatory response to injury. Increasing numbers of immature granulocytes in peripheral blood have been documented in association with major trauma. Moore and colleagues16 showed an increase in circulating haemopoietic progenitor cells following abdominal trauma, along with an increase in colony-stimulating activity in the conditioned medium of lipopolysaccharide-stimulated peripheral blood monocytes from murine bone marrow, indicating a greater mobilization of immature cells from the bone marrow. Endogenous damage-associated molecular pattern molecules have been implicated in the release of haemopoietic progenitors by promoting the activity of the IL-17–G-CSF axis, which is central to the egress of granulocytes from the bone marrow25. In the present study, G-CSF levels correlated well with increased numbers of immature granulocytes in peripheral blood from patients who had undergone major trauma. In addition, an increased proportion of CD35− cells was observed with increasing time from injury. This finding may not be paradoxical, but a reflection of the magnitude and duration of bone marrow dysfunction in patients who had taken longer to recover sufficiently for surgery to be undertaken. Because of the difficulties in obtaining bone marrow aspirates in the relevant clinical setting, the data presented in this study essentially pertain to bone marrow from patients judged well enough to undergo major fracture surgery. As a consequence, the methodology of this study has placed an important limitation on its findings. Bone marrow aspiration was possible only at the time of definitive fracture surgery, and so the characteristics of the bone marrow described do not reflect leucopoiesis at the time of maximum bone marrow dysfunction. Instead, the bone marrow aspirates represent a recovery phase after major trauma. In spite of these limitations, important questions are raised, suggesting that it may be possible to modulate bone marrow activity following major trauma. Chronic anaemia is observed in critical care patients who are also predisposed to septic complications15,16,26. Data from randomized trials have indicated that the use of epoetin alfa, intended to combat the anaemia of critical care associated with major injuries, also decreases the risk of death26,27. Although the mechanism of action has yet to be defined, it may be that survival can be influenced by the modulation of bone marrow. Attempts at therapeutic manipulation of the systemic inflammatory response to injury28 would benefit from a more detailed understanding of the CD34+ progenitor pool, and the struggle to preserve self-renewal, differentiation and the production of competent mature end cells after major injury. Acknowledgements The authors thank the Arbeitsgemeinschaft für Osteosynthesefragen (AO) Foundation, the patients and their families, the staff of the Critical Care Unit and Haematology Laboratory, and other staff and colleagues in Morriston Hospital and within the Institute of Life Science at Swansea University, for their help and support. This study was supported financially by an AO Foundation Research Grant and Abertawe Bro Morgannwg University Health Board Research and Development funds. Disclosure: The authors declare no conflict of interest. References 1 Bochicchio GV , Napolitano LM, Joshi M, McCarter RJ, Scalea TM. 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TI - Altered leucocyte progenitor profile in human bone marrow from patients with major trauma during the recovery phase
JO - British Journal of Surgery
DO - 10.1002/bjs.8919
DA - 2012-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/altered-leucocyte-progenitor-profile-in-human-bone-marrow-from-5g2oaHI0SY
SP - 1591
EP - 1599
VL - 99
IS - 11
DP - DeepDyve
ER -