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#Post#: 64--------------------------------------------------
Intramedullary nailing of the femur and the systemic activation
of monocytes and
By: osmaniaortho Date: December 13, 2011, 8:56 am
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HTML http://www.wjes.org/content/6/1/34
Background
Trauma such as found patients with femur fractures, induces a
systemic inflammatory response, which ranges from mild SIRS to
ARDS. Neutrophils (i.e. PMN) play an important role in the
pathogenesis of this inflammatory condition. Additional
activation of PMNs during intramedullary nailing (IMN) is
thought to act as a second immunological hit. Damage control
orthopedics has been developed to limit this putative
exacerbation of the inflammatory response. The hypothesis is
tested that IMN exacerbates systemic inflammation, thereby
increasing the risk for ARDS.
Methods
Thirty-eight trauma patients who required IMN for femur fracture
were included. The development of SIRS and ARDS was recorded.
Blood samples were taken prior and 18 hours after IMN.
Inflammatory response was analyzed by changes in plasma IL-6
levels, monocyte (HLA-DR) and PMN phenotype (MAC-1 and
responsiveness for the innate immune stimulus fMLP in the
context of active FcγRII).
Results
Plasma IL-6 was significantly enhanced in severely injured
patients compared to patients with isolated femur fractures and
matched controls (P = 0.005; P = 0.018). This enhanced
inflammatory tone was associated with a lower percentage HLA-DR
positive monocytes (P = 0.002). The systemic PMN compartment was
activated, characterized by an increased MAC-1 expression and a
significantly decreased sensitivity for the innate stimulus fMLP
Interestingly the PMN compartment was not affected by IMN.
Conclusions
Multitrauma patients were characterized by a marked activation
of the systemic inflammatory response, associated with a
systemic activation of the monocyte and PMN compartments. IMN
particularly affected the monocyte arm of the systemic innate
immune system.
Keywords: Trauma; ARDS; neutrophils; tissue damage
Introduction
ARDS (Acute Respiratory Distress Syndrome) is a frequent
complication after trauma. Although mortality rates has been
reduced over the last decade by improved treatment strategies
and modalities, morbidity rates remain high, as the incidence of
ARDS has only slightly decreased [1]. Several risk factors have
been identified for the development of ARDS, such as
intramedullary osteosynthesis/nailing (IMN) of a femoral
fracture, massive blood transfusion and thoracic injury [2].
When IMN is performed in the presence of these risk factors, the
incidence of ARDS can be over 40%[3,4]. In this case, IMN is
seen as a second hit.
Systemic inflammation is key in the development of ARDS. The
amplitude of this systemic response is often measured by plasma
IL-6 levels. However, systemic activation of the cellular innate
immune system is essential in the development of ARDS [5]. When
extravasation of polymorphonuclear granulocytes (i.e. PMNs or
neutrophils) is blocked or animals are depleted of PMNs, no ARDS
occurs after a sufficient insult [6]. In addition, in patients
with sepsis, circulating HLA-DR negative monocytes were
identified, which point at a pro-inflammatory profile, as
described previously. These cells are thought to contribute to
additional tissue damage [7]. The role of these cells during IMN
has not been investigated yet.
This etiological study was designed to test the hypothesis
whether IMN contributes to a more pronounced systemic
inflammation, characterized by a change phenotype of cells of
the innate immune system. This hypothesis was tested in 2
subgroups of patients with different injury severity (isolated
femur fracture and femur fracture in multitrauma).
Patients and methods
Patients
Forty-five trauma patients were included in this study. They
were admitted to the Department of Traumatology, University
Medical Center Utrecht with a fracture of the femur, which
required primary or secondary intramedullary nailing. Exclusion
criteria were age < 16 years or > 80 years and patients with an
altered immunological status (e.g. use of corticosteroids or
chemotherapy). The local ethical committee approved the study
and written informed consent was obtained from all patients or
their spouses in accordance to the protocol.
Clinical parameters and sampling
The Injury Severity Score and APACHE II Score were calculated on
admission. During admission the occurrence of pulmonary
complications (i.e. acute lung injury [ALI] or acute respiratory
distress syndrome [ARDS]) were assessed according to their
clinical criteria as determined in the consensus conference [8].
Recently, we have shown that the extent of systemic inflammation
of innate immune cells can be visualized by measuring the
expression of activation markers on blood PMNs [9]. The most
sensitive marker turned out to be the responsiveness of active
FcγRII (CD32) on PMN's for the innate immune stimulus fMLP
[9,10]. The most commonly used marker is MAC-1 (CD11b), which
peaks between 6 and 18 hours after insult (i.e. trauma or
surgery)[11]. In contrast to PMN's, changes in activation of the
systemic monocyte compartment can be determined by analyzing the
percentage of circulating HLA-DR positive monocytes [7].
Blood samples were taken at two distinct time points: one hour
prior to IMN and 18 hours after the intramedullary nail was
introduced. To investigate the influence of IMN, patients were
stratified by isolated femur fracture and femur fractures in
multitrauma. Patients were compared with healthy, age and gender
matched controls as described previously (see Table 1)[9].
Table 1. Patient demographics.
Materials
For analysis of PMN receptor expression by flowcytometry the
following monoclonal antibodies were commercially purchased:
FITC-labeled IgG1 negative control (clone DD7, Chemicon,
Hampshire, United Kingdom), RPE-labeled IgG2a negative control
(clone MRC OX-34, Serotec, Dusseldorf, Germany) and RPE-labelled
CD11b (clone 2LPM19c, DAKO, Glostrup, Denmark). An antibody,
which recognizes an active FcyRII/CD32 (designated FcyRII*), is
manufactured at the Department of Pulmonary Science at the
University Medical Center Utrecht (MoPhab A27, UMCU, Utrecht,
The Netherlands)[12,13].
For analysis of monocyte HLA-DR expression by flowcytometry the
following monoclonal antibody was commercially purchased:
FITC-labeled HLA-DR (YE2/36-HLK, Serotec, Dusseldorf, Germany).
Pmn and monocyte receptor expression
The inflammatory status of a patient can be assessed by
analyzing the expression of active FcyRII (FcyRII*) on PMNs in
the peripheral blood [9]. A low expression of fMLP induced
FcyRII* correlates with increased inflammation. This approach
has been validated in a previous study [9]. The expression of
fMLP induced FcyRII* was compared with a more common activation
marker MAC-1 (CD11b)[14].
Blood was collected in a vacutainer® with sodium heparin as
anticoagulant, cooled immediately and kept on ice during the
whole staining procedure. The analysis of the PMN receptor
expression was started within two hours after the blood sample
was obtained. The expression of the above mentioned markers was
measured as described previously [9]. Expression of active
FcγRII by FITC-labeled MoPhab A27 was measured after 5
minutes of stimulation of whole blood at 37°C with
N-formyl-methionyl-leucyl-phenylalanine (fMLP 10-6M) to evaluate
the responsiveness of the cells for a bacterial derived
activating agonist. After stimulation, the samples were put on
ice again and analyzed.
Blood samples were stained with fluorescein isothiocyanate
(FITC) directly labeled antibodies (MoPhab A27) as described
previously [9]. The expression of CD11b and HLA-DR were
performed according the recommendations of the manufacturer. In
short, directly labeled antibodies were added 1:20 to whole
blood and incubated for 60 minutes on ice. After incubation, the
red cells were lysed with ice-cold isotonic NH4Cl. After a final
wash with PBS2+ (phosphate buffered saline with added sodium
citrate (0,38% wt/vol) and isotonic pasteurized plasma proteins
(10% vol/vol), the cells were analyzed in a FACScalibur
Flowcytometer (Becton & Dickenson, Mountain view. CA). The PMNs
and monocytes were identified according to their specific
side-scatter and forward-scatter signals. Data from individual
experiments are depicted as histograms of fluorescence intensity
in arbitrary units (AU) or summarized as the median channel
fluorescence (MCF) of at least 10000 events.
Interleukin-6
IL-6 was determined using a human IL-6 sandwich ELISA (Endogen,
Pierce Biotechnology, IL, United States) according to the
procedures prescribed by the manufacturer. Detection limit of
this ELISA was 5 pg/ml.
Statistical Analysis
All data were analyzed using SPSS version 15.0 software (The
Apache Software Production 2008, Chicago, Illinois). Results are
expressed by medians + range. Statistical analysis was performed
using a non-parametric Mann Whitney U Test for two groups and a
Kruskall Wallis H test for multiple comparisons. Paired analysis
(before and after surgery) was performed using Wilcoxon Signed
Ranks test. Statistical significance was defined as p < 0.05.
Results
Demographics
A total of 45 patients fulfilled the inclusion criteria in a
period of 1 year. Of these 45 patients, 3 patients were missed
due to logistical restrictions, 2 patients underwent external
fixation initially, but did not receive conversion to
intramedullary osteosynthesis, 1 patient did not give consent
and in 1 patients sampling was flawed. Thus, 38 patients were
adequately followed up (84%). Their median ISS was 13 (range
9-43) and their median APACHE II Score was 5 (range 0-25) at
admission. Intramedullary nailing was performed either directly
or in a staged damage control approach. Seven patients developed
ALI/ARDS, which indicates an adequate patient selection. Further
demographics are listed in Table 1.
Types Of Injury
Prior to surgery, multitrauma patients demonstrated increased
levels of plasma IL-6 (Figure 1) and a decreased expression of
fMLP induced FcγRII* on PMNs (Figure 2) compared to
patients with an isolated femur fracture (P = 0.005 and P <
0.0001 respectively) and matched control donors (P = 0.018 and P
= 0.004 respectively). In contrast, MAC-1 expression (Figure 3)
and the percentage HLA-DR positive monocytes (Figure 4) did not
demonstrate a difference between multitrauma patients and
patients with isolated femur fractures. The percentage HLA_DR
positive monocytes was decreased in all patients, compared to
matched control donors (P = 0.002). There was no significant
correlation between plasma IL-6 levels and cellular markers,
indicating that the measured markers identify different aspects
of the systemic inflammatory response.
Figure 1. Plasma IL-6 levels. Multitrauma patients demonstrated
increased levels of plasma IL-6 compared to patients with
isolated femur fracture (P = 0.018) or matched controls (P =
0.005). Pre-operative IL-6 levels ("black square") were
significantly increased in patients who developed respiratory
failure (P < 0.001). Eighteen hours after intramedullary nailing
("open triangle"), plasma IL-6 levels were significantly
increased in patients with isolated femur fractures (P = 0.030),
but not in multitrauma patients (P = 0.515), which could be due
to insufficient power.
Figure 2. PMN fMLP induced FcyRII expression. Multitrauma
patients demonstrated decreased expression of fMLP induced
FcyRII on PMNs compared to patients with isolated femur fracture
(P = 0.004) or matched controls (P < 0.001). Pre-operative fMLP
induced FcyRII* ("black square") was more decreased in patients
who developed ARDS (P < 0.001). Eighteen hours after
intramedullary nailing ("open triangle"),fMLP induced FcyRII*
did not change compared to pre-operatively.
Figure 3. PMN MAC-1 expression. No statistical significant
increased MAC-1 expression was seen in multitrauma patients. In
addition, no increased pre-operative expression ("black square")
was demonstrated in patients who developed respiratory failure
and no difference was seen 18 hours after intramedullary nailing
("open triangle").
Figure 4. HLA-DR positive monocytes. The percentage HLA-DR
positive monocytes was decreased in all patients compared to
controls (P = 0.002). The pre-operative ("black square") lowest
percentage was seen in patients who developed respiratory
failure (P = 0.002). Eighteen hours after intramedullary nailing
("open triangle"), a further decrease in HLA-DR positive
monocytes was seen in patients with isolated femur fracture (P <
0.001) and multitrauma patients (P = 0.047).
Symptoms Of Systemic Inflammation During Follow-Up
Seven patients developed respiratory failure and fulfilled the
ALI/ARDS criteria, whereas 17 patients only fulfilled the SIRS
criteria during the 48 hours after IMN. Pre-operative IL-6
levels were significantly increased in patients who developed
respiratory failure (P < 0.001). Pre-operative expression of
fMLP induced FcγRII* on PMNs (P < 0.001) and the percentage
of HLA-DR positive monocytes (P = 0.002) was lower in patients
with more severe inflammatory response. In contrast, MAC-1
expression did not demonstrate a significant difference in
patients with a more severe inflammatory response.
Impact of intramedullary nailing
Eighteen hours after intramedullary nailing, plasma IL-6 levels
were significantly increased in patients with isolated femur
fractures (P = 0.030), but not in multitrauma patients (P =
0.515, Figure 1). The activation markers of PMNs (fMLP induced
FcγRII* and MAC-1) did not change after intramedullary
nailing in either patients with isolated femur fracture or
multitrauma patients (Figure 2 and 3). In contrast, the
percentage HLA-DR positive monocytes decreased significantly in
both patient groups (P < 0.001 of isolated femur fractures and P
= 0.047 for multitrauma patients, Figure 4).
Discussion
This study confirms that multitrauma patients have a significant
inflammatory response measured by plasma levels of IL-6 and PMNs
phenotype. Furthermore, patients who developed ALI/ARDS
demonstrated severe systemic inflammation measured by plasma
IL-6 levels and PMN activation markers. This study is thereby
comparable with previous studies which measured plasma cytokine
levels and PMN phenotype. In addition, we measured PMN
activation towards the innate stimulus fMLP. Active inside-out
control of PMNs towards fMLP was significantly decreased in
patients with more severe injuries. However, with this sensitive
measurement, no additional activation of PMNs occurred after IMN
of femur fractures, in either patients with isolated femur
fractures or multitrauma patients.
Trauma induces inflammation and severe inflammation has been
related to the development of ALI/ARDS [15]. It has been
demonstrated that PMNs play an essential role in the
pathophysiology of ALI/ARDS, whereas the roles of cytokines
(such as IL-6) and monocytes are less clear, because these
cytokines often have multiple target cells and different
functions. IL-6 levels have often been used for their prognostic
importance, but no causal pathophysiological relation has been
identified [16,17]. It is true that more trauma results in more
systemic inflammation and thus in more cytokine release.
However, IL-6 does not cause damage to the pulmonary
endothelium. Products produced by PMNs cause this damage and our
data support the importance of PMNs. Severe trauma results in an
altered PMN phenotype patients who developed ARDS demonstrated
the most activated PMNs. In addition, our study suggest a role
for monocytes as well in the pathophysiology of ALI/ARDS.
Monocyte HLA-DR expression was decreased in multitrauma
patients, indicating a more pro-inflammatory type of monocytes
which has been suggested previously to contribute to the tissue
damage during a systemic inflammatory response.
We next studied the impact of intramedullary nailing on the
systemic inflammatory response. Additional inflammation caused
by surgery is seen as additional trauma and has been considered
as a possible risk factor for organ failure such as ARDS [18].
Much to our surprise the increased damage caused by IMN only
partly induced changes in the systemic inflammatory response
(only monocyte HLA-DR expression in patients with isolated femur
fractures). Most striking was the absence of additional PMN
activation after intramedullary nailing. This lack of change in
PMN phenotype during IMN is in line with suggestions from a
previously published report [19]. In that cohort, no increase
was seen in MAC-1 expression on PMNs after bilateral femur
fracture fixation. Thus, the extend of PMN activation appears
mainly determined by the severity of initial trauma and is
apparently not altered by intramedullary nailing. In contrast,
plasma IL-6 levels and monocyte HLA-DR were significantly
altered by intramedullary nailing. Thus, an impact of the
surgical procedure can be measured by cytokines and the monocyte
compartment.
The blood samples were taken 1 hour prior to IMN and 18 hours
after IMN, regardless of the interval between trauma and
surgery. Although this affects the reproducibility of the
results, it reflects daily care practice. 18 hours after IMN the
peak of plasma IL-6 levels will be passed (max at 6 hours
post-operatively), but the changes in PMN phenotype will be most
defined. PMN phenotype behaved similarly in all patients,
therefore, 38 patients were sufficient to state the conclusion.
Because we analyzed the functional phenotype of PMNs and
monocytes, more information was obtained than merely static
phenotypes. The inflammatory cellular response deficit to the
development of ARDS appears to be mainly determined by the
initial injuries and not the additional insult by IMN.
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