REVIEW ARTICLE

Inflammatory serum markers in patients with

multiple trauma
CAN THEY PREDICT OUTCOME?
P. V. Giannoudis,
F. Hildebrand,
H. C. Pape
From St Jamess
University Hospital,
Leeds, England and
Hannover Medical
School, Hannover,
Germany

P. V. Giannoudis, BSc, MD,

EEC (Ortho), Professor in
Trauma and Orthopaedics
Department of Trauma and
Orthopaedic Surgery, St
Jamess University Hospital,
Beckett Street, Leeds LS9
7TF, UK.

F. Hildebrand, MD,
Resident

H. C. Pape, NMD, Professor
Department of Trauma
Surgery, Hannover Medical
School, Carl-NeubergStrasse 1, Hannover, D-30625
Hannover, Germany.
Correspondence should be
sent to Professor P. V.
Giannoudis.
2004 British Editorial
Society of Bone and
Joint Surgery
doi:10.1302/0301-620X.86B3.
15035 $2.00
J Bone Joint Surg [Br]
2004;86-B:313-23.
VOL. 86-B, No. 3, APRIL 2004

Clinical trials have provided information concerning the various immunological alterations
that occur after trauma which may cause the
multiple organ dysfunction syndrome (MODS)
and death. Once multiple systems are dysfunctional the rates of mortality are as high as
50%, the morbidity is severe and the costs of
health care are enormous.1,2 Although external
support such as ventilators, dialysis machines
and inotropic drugs, is provided to all patients
to compensate for the internal dysfunction
until they can support themselves, the end
result is often unpredictable.
Early evaluation of the prognosis in polytraumatised patients is difficult. Traditionally,
the clinical condition and management of the
patient are assessed by evaluation of cardiovascular, renal, liver and respiratory functions.
Nevertheless, the significance of such clinical
parameters as the urinary output, oxygen saturation, blood gases, C-reactive protein, base
excess, etc, is limited since only patients with a
clearly impaired organ function can be distinguished. Organ dysfunction which is not
detectable by these parameters does not guarantee a condition of the patient which is stable
enough for operation especially if in the borderline condition.3
The predictive value of several clinical
parameters is uncertain. For instance, initial
levels of lactate have been shown to correlate
with the development of MODS4 and prolonged hyperlactataemia is associated with
increasing mortality.4,5 By contrast, Rixen et
al6,7 demonstrated that the level of lactate was
not sufficiently sensitive to predict the probability of death but that age, the Glasgow Coma
Score (GCS), the Injury Severity Score (ISS),
base excess (BE) and the prothrombin time
were the most important predictors of the
development of post-traumatic complications
and death.
However, Tremblay, Feliciano and Rozycki8
reported that the value of BE depends on the
mechanism of injury since it appears to be
useful in assessing patients who have sustained

blunt trauma, but not in those with a penetrating injury. It was reported to be particularly
ominous in patients older than 55 years,9 without manifestation of base deficit in significant
injuries.
With advances in molecular medicine, new
information has emerged regarding the
response to injury and shock at the molecular
level. Several mechanisms have been proposed
for the development of post-traumatic complications including the macrophage theory, the
gut hypothesis, the two-hit theory and the
micro-environment theory.10 The last, namely
the theory of neutrophil-mediated tissue injury,
has gained much acceptance over the years
(Fig. 1).11 It suggests that adherence of activated neutrophils to the endothelium creates a
local environment between cells, isolated from
circulating oxidant scavengers and antiproteinases and unaffected by the dilutional flow
characteristics of luminally exposed cell surfaces. In the absence of these mechanisms
which would otherwise limit the accumulation
of toxic metabolites, neutrophil metabolites
secreted into this micro-environment quickly
reach very high concentrations allowing unopposed endothelial injury.12
As a result of the popularity of the microenvironment theory and the availability of
techniques to measure molecular mediators,
studies have been undertaken to search for
inflammatory markers which could detect
patients in the borderline condition and at
risk of developing post-traumatic complications. Alteration of treatment may then prevent the onset of adverse sequelae.
The purpose of this review is to highlight
our current knowledge on the effectiveness of
the existing inflammatory markers of immune
reactivity and evaluate their impact on our
clinical practice.

Molecular aspects of trauma

Under normal physiological conditions and
after surgical or accidental injury all cells in the
body constantly communicate with each other.
313

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P. V. GIANNOUDIS, F. HILDEBRAND, H. C. PAPE

Neutrophil adhesion to activated endothelium

Table I. The grouping of serum inflammatory markers

Group

Extravascular space
IL-6
Intravascular space

CD11b
TNF

Serum inflammatory markers

Acute-phase reactants LBP, CRP, procalcitonin

Mediator activity
TNF, IL-1, IL-6, IL-10, IL-18
Cellular activity
TNF-RI, TNF-RII, IL-1R-I, IL-1R-II, sIL-6-R, mIL-6-R,
ICAM-1
Eselectin, CD11b, elastase, HLA-DR class-II antigens, DNA

Leucocyte

IL-8
Leucocyte extravasation

ICAM

Release of elastase
Reactive oxygen species

Endothelium
Eselectin

Fig. 1
Diagram showing the micro-environment theory (leucocyte-endothelial cell interactions).

An array of regulatory proteins produced and secreted by

lymphocytes and other cells have a role in cascading the
immune response to trauma.13 Following trauma, patients
are subjected to dynamic alterations in the haemodynamic,
metabolic, and immune responses which are largely orchestrated by endogenous mediators referred to as cytokines.
An injured or a surgical patient is a stew of pulsating
cytokines.
The development of this inflammatory response which
may have fatal consequences is part of the normal response
to injury. This gives rise to the systemic inflammatory
response syndrome (SIRS) followed by a period mediated
by a compensatory anti-inflammatory response syndrome
(CARS).14 Within this inflammatory process there is a fine
balance between the beneficial effects of inflammation and
the potential for the process itself to cause remote tissue
injury leading to the adult respiratory distress syndrome
(ARDS) and MODS.15
According to the one-hit model the initial massive
injury and shock give rise to an intense systemic inflammation which causes activation of the innate immune system,
including macrophages, leukocytes, natural killer cells and
inflammatory cell migration enhanced by the production of
interleukin (IL)-8 and complement components (C5a and
C3a).16 In the two-hit model, the stimulus is less intense
and normally resolves, but the patient is vulnerable to secondary inflammatory insults which can reactivate SIRS and
precipitate late MODS.6 The second insult may take many
forms, among them sepsis and surgical operations. The clinician may have to make difficult decisions as to when and
how much to do for the borderline patients with polytrauma. Hyperstimulation of the inflammatory system,
either by single or multiple hits, is considered by many to
be the key element in the pathogenesis of ARDS and
MODS.17

Numerous studies have shown that stimulation of a variety of inflammatory mediators takes place immediately
after trauma.18,19 This response initially corresponds to the
first-hit phenomenon. Several investigators have also highlighted the issue of secondary surgical procedures acting as
additional inflammatory insults or second-hit phenomena.20,21 These models of first- and second-hit biological
responses to different stimuli have now become the basis of
our treatment plans and investigations. At the molecular
level, a variety of inflammatory mediators has been implicated in the pathogenesis of organ dysfunction. Serum
markers of immune reactivity can be selectively grouped
into markers of acute-phase reactants, mediator activity,
and cellular activity (Table I).

Markers of acute-phase reactants

This group of markers is usually of hepatic origin and
includes the lipopolysaccharide-binding protein (LBP), procalcitonin and C-reactive protein.
Lipopolysaccharaide-binding protein (LBP). This is a 58kDa
class-I acute-phase protein of mainly hepatic origin with the
ability to bind bacterial lipopolysaccharide (LPS).22 LPSactivated macrophages release proinflammatory cytokines,
such as IL-6, IL-1 and tumour-necrosis-factor (TNF)-.
LPB is synthesised in hepatocytes and released into the
blood. During the acute-phase response, this synthesis and
release can increase up to 30-fold.22 LBP was found to
block the effects of LPS in vitro and protect mice from
lethal outcome.23 LPS-induced production of cytokines by
macrophages was decreased by high concentrations of LBP.
Increased levels of LBP also prevented liver injury and
reduced mortality after an injection of LPS in mice.23 However, low concentrations of LPB enhanced production of
TNF- in macrophages induced by LPS.22 In general terms,
LBP enhances the effects of LPS when it is present in small
quantities, whereas it suppresses them in high concentrations.22,24
Levels of LBP rise in patients during the acute phase of
trauma or sepsis, with maximum values occurring on the
second and third days.22 In patients with MODS significantly higher concentrations of LBP were found in those
with documented infection.22 By contrast, no significant
differences were found between patients with SIRS and the
non-septic MODS group, suggesting that LBP may be used
as a marker to differentiate between SIRS and ongoing bacterial sepsis in the early post-traumatic course.25
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INFLAMMATORY SERUM MARKERS IN PATIENTS WITH MULTIPLE TRAUMA

In patients with severe sepsis or septic shock, concentrations of LBP were found to be significantly increased and it
may have prognostic significance in such patients26 although
Blairon, Wittebole and Laterre27 reported that LBP is only a
non-specific marker for sepsis, and that the response was not
clearly correlated with the severity of infection.
C-reactive protein (CRP). This is one of the acute-phase
response proteins produced by hepatocytes and is usually
found in concentrations of 0.3 to 1.7 mg/1.28 Increased production is due to cytokine-dependent induction of synthesis
and elevated levels may be detected within eight hours of a
stimulus and can reach 500 mg/1.28 Besides trauma, elevated levels of CRP may be seen in other conditions such as
autoimmune disease, infection and malignancy. The level of
CRP normally peaks within 48 hours of the stimulus. A fall
in serial measurements usually indicates resolution of the
underlying process, while persisting elevated levels may
indicate ongoing inflammation or infection.28
While the level of CRP has been used in clinical practice
for many years, it is relatively non-specific and not predictive of septic complications after major trauma.29 Clinical
studies have shown that there is no correlation between the
serum CRP and the severity of injury or prediction of survival in polytraumatised patients.29,30 In patients who are
critically ill neither the absolute level of CRP nor the rate of
its change has been found to relate to proven infection.31
Despite widespread clinical use, the level of CRP is not considered to be an ideal marker of the inflammatory mediator
response after trauma.
Procalcitonin (PCT). This is derived from a precursor protein preprocalcitonin, proteolysis of which results in the
formation of calcitonin. The latter is normally produced in
the C-cells of the thyroid. PCT is not normally detected in
the plasma of healthy individuals. A study of hepatocyte
tissue culture treated with TNF-a or IL-6 has shown detectable levels of PCT after 24 hours of culture, suggesting that
the liver is a potential source of production of PCT.32
In polytraumatised patients injury leads to increased
plasma levels of PTC dependent on the severity of injury,
with peak values on the first and third days. Increased concentrations of PCT during the first days after trauma have
been shown to predict severe SIRS, sepsis and MODS.33
Thus, PCT may be a useful marker for monitoring the
inflammatory status in these patients.33 There are significantly increased plasma levels of both PCT and IL-6 in
males compared with females.34
In a prospective study of 175 patients admitted to a surgical intensive-care unit the correlation between TNF-a,
IL-6 and various markers of inflammation was determined.
PCT proved to be the best of these.35 In contrast to the concentration of CRP a secondary increase in that of PCT
seemed to be an adequate indicator of severe infection
during late SIRS.36 However, not all studies have shown
PCT to be better than CRP as a marker of infection. In a
study of 205 medical and surgical patients a cut-off level
was described for the diagnosis of infection as 0.6 ng.ml-1
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315

for PCT and 7.9 mg.dL-1 for CRP. The latter showed a
higher sensitivity (71.8% v 67.6%) and specificity (66.6%
v 61.3%) for infection. Confirming these findings, a study
of 60 patients showed a higher level of PCT in infected than
in non-infected patients but the level of CRP was more predictive of infection.37
Post-operative plasma concentrations of PCT in patients
without signs of infection are largely influenced by the type
of surgical procedure. It has been shown that during the
first two post-operative days, the concentration of PCT is
elevated in all patients. Higher levels are found after major
surgical procedures compared with minor operations.38 In
severe lung contusion the level of PCT in bronchoalveolar
lavage (BAL) was not considered to be a reliable parameter
for assessing the extent of lung contusion.39
In general terms, the absolute level of PCT and the cutoff points for diagnosis appear to vary significantly, and the
prognostic value of PCT in trauma is as yet unclear. However, the correlation between PCT and the severity of sepsis
appears to be constant in all the available studies.

Markers of mediator activity

Tumour necrosis factor (TNF). This is an autocoid which
exists in multimers of two or three identical subunits and
contains several potential sites of glycosylation. TNF- and
TNF- may be distinguished. Both have almost the same
biological effects. TNF- is only synthesised by lymphocytes. In inflammatory reactions, TNF- is mainly
involved.40 It may also exist as an integral membrane protein. Membrane bound TNF- is a 26kD polypeptide
which includes a residual signal sequence, and is usually
cleaved to yield the soluble form. TNF- is a central regulator in the immunoinflammatory response after trauma
and is produced by monocytes, lymphocytes, Kupffer cells,
macrophages, endothelial cells and glial cells. It has a short
half time in the plasma of 14 to 18 minutes and is cleared by
binding to soluble TNF- receptors and natural TNF-
binding proteins. Release of TNF- is stimulated by such
triggers as IL-1, 2 and 12, interferon-, platelet aggregating
factor (PAF), complement protein C5a as well as by endotoxing and can induce its own release.41 Stimulation of
TNF- can be down-regulated by IL-4, 10 and 13, TNF-,
cortisol and agents which increase intracellular cyclic adenosine monophosphate.41
The effects of TNF- on endothelial cells are diverse. It
increases both the permeability of endothelial cells and the
expression of adhesion molecules like intercellular adhesion molecule 1 (ICAM-1) or Eselectin, leading to the activation and adhesion of granulocytes. It also increases the
procoagulated activity of endothelial cells.42
Most of the available studies of TNF on patients with
multiple injuries have been focused on the clinical course of
patients in intensive-care units. Despite the initiation of
treatment continually high levels of TNF- have been
reported to correlate with a poor outcome, although there
was no significant difference in the levels of TNF- when

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P. V. GIANNOUDIS, F. HILDEBRAND, H. C. PAPE

study commenced.43 This is not a consistent finding since

high levels of TNF- have been associated with increased
survival in patients with septic shock secondary to intraabdominal pathology.44 In patients with meningococcal
meningitis serum levels of TNF- have been shown to correlate with mortality45 but other authors have failed to
show significant increases in TNF- in septic patients.46
The results of using TNF- as a marker of sepsis and a predictor of mortality have been disappointing. This is partly
due to the pharmacokinetics of TNF-; single-point measurements have proved to be unhelpful since peak concentrations of TNF- are reached in one or two hours and may
have significantly decreased by four to six hours. Somewhere between these times systemic signs of sepsis occur. By
the time a septic event is recognised, concentrations of TNF
may have returned to the baseline level. The origin of the
sepsis also determines the magnitude of changes in circulating TNF. Increased concentrations of TNF have also been
observed in BAL of polytraumatised patients with posttraumatic ARDS.47
Efforts to modify the effect of TNF- have included the
use of anti-TNF- antibodies and the use of recombinant
soluble TNF-a receptors. In a rodent model, mortality after
an injection of endotoxin was signifantly decreased by pretreatment with TNF-a antibodies.48 In another experimental study, applications of TNF-a antibodies prevented the
development of organ dysfunction in E. coli sepsis.49 However, application of TNF antibodies before the induction of
experimental peritonitis resulted in increased rates of mortality. Furthermore, the antibody approach has produced
little evidence of benefit in man although improvements in
the haemodynamics of patients have been observed.50 The
use of recombinant soluble TNF-a receptors has been
shown to improve survival in mice,51 but at present there
have been no similar results in man. So far the use of TNF-a
or its soluble receptors has not proved to be useful as a
diagnostic marker.
Interleukin-1 (IL-1). This family of peptides consists of three
structurally related polypeptides: IL-1, IL- and IL-1
receptor antagonist (IL-1ra), produced primarily by monocytes. It is primarily induced in the presence of ischaemia or
sepsis by activated macrophages and activated endothelial
cells. Biologically, IL-1 has a similar activity to that of
TNF- and acts synergistically with it with induction of
fever, hypotension, endothelial cell adhesion as a procoagulant, and the chemotaxis of polymorphonuclear leucocytes (PMNs) and macrophages. In addition, IL-1 induces
release of TNF-, IL-6, IL-8, platelet activating factor
(PAF) and eicosanoids.52 The circulating half-life of IL-1 is
six minutes. This makes its detection after injury much less
likely than that of TNF-. Most studies carried out to
assess the efficacy of this marker have been done in septic
patients in whom it was found that the levels of IL-1 did
not correlate with death or MODS.53,54
IL-1ra is a cytokine-like molecule which binds to the IL-1
receptor but does not induce a response. Concentrations of

IL-1ra increase significantly in volunteers exposed to

endotoxin55 and concentrations are higher in survivors
than in non-survivors of animal models of sepsis.56 It has
been given to patients with SIRS and the clinical picture of
septic shock. In an initial trial in 99 patients,57 but not in a
subsequent phase-III trial,58 those receiving IL-1ra showed
a dose-dependent improvement in their clinical course and
rates of mortality. This improvement correlated with a
reduction in levels of IL-6 consistent with the control of
IL-6 by IL-1 and the correlation of the severity of the disease and outcome with levels of IL-6. Perhaps because of
the dynamic interaction between IL-1 and IL-1ra, there is
no clear relationship between the levels of IL-1 and the
severity of sepsis. This limits its usefulness as a clinical indicator or in research.
Interleukin-6 (IL-6). This is a glycoprotein with a molecular
weight of 22 to 29 kD. It is produced by a variety of cells
including T- and B-cells and endothelial cells. Production of
IL-6 is induced by viruses, LPS, IL-1 and TNF. It induces a
proliferation of B-lymphocytes with increased synthesis of
immune globulins and a proliferation of T-lymphocytes.
Furthermore, IL-6 causes an enhanced differentiation of
cytotoxic T-cells and an increased activity of natural killer
(NK) cells. Its main effect is to induce hepatic synthesis of
acute-phase proteins such as CRP, fibrinogen, 1-antitrypsin or complement factors.59
IL-6 is less transient and therefore more readily measurable than either IL-1 or TNF-. It appears to be one of the
best prognostic markers regarding the outcome of patients
with SIRS, sepsis or MODS. The association between early
increased plasma levels of IL-6, high ISS and a late adverse
outcome has been well documented in several studies.60-62
An early marked elevation of the concentration of IL-6
(>500 pg/dl) will distinguish injured patients who later
develop MODS.61 Hack et al63 reported a direct correlation
between IL-6 and levels of lactate in septic shock and IL-6
appeared to have prognostic significance in the differentiation of survivors from those who died (p = 0.0003).
Increased concentrations of IL-6 in BAL were associated
with a poor outcome in patients with ARDS.64 Dofferhoff
et al65 found a correlation of IL-6 with severity of illness in
septic patients as measured by the APACHE II score, but
there was no correlation between IL-6 and outcome. In
trauma patients, IL-6 is not predictive of septic complications but rather correlates with the magnitude of the
injury.66 It appears to be a marker of traumatic insult, with
significantly elevated levels occurring within one to four
hours after trauma, and correlates with the severity of illness.
Interleukin-10 (IL-10). This is an 18 kD protein mainly synthesised by T-lymphocytes but also produced by B-lymphocytes, monocytes and macrophages. Liberation of IL-10
is at least partially induced by TNF. It is an anti-inflammatory cytokine and reduces the synthesis of TNF- and IL-1
by mononuclear cells after application of endotoxin, both
in vitro67 and in vivo.68 Therefore it is also called a macroTHE JOURNAL OF BONE AND JOINT SURGERY

INFLAMMATORY SERUM MARKERS IN PATIENTS WITH MULTIPLE TRAUMA

phage-deactivation factor. It decreases cytokine production

of Th1 cells, which synthesise IL-2, IL-3, IFN- and TNF-.
These cells induce a type-IV immune reaction, a delayed
type of hypersensitivity reaction (DTH), via cytotoxic
T-cells while cytokines of the Th2 clone cause a humoral
immune response. Moreover, IL-10 inhibits both the antigen-presenting function of macrophages and the subsequent proliferation of T-cells.68
The plasma concentrations of IL-10 are elevated in
patients with polytrauma69,70 and after major surgery71 and
a correlation with the severity of the injury has been
reported.
An increased plasma concentrations of IL-10 has been
observed in septic patients and even higher values have
been found in patients who had suffered septic shock.72
However, other studies have shown an unchanged secretion
of IL-10 in patients undergoing major surgical procedures73
and even depressed concentrations in trauma patients.74
In animal studies neutralisation of IL-10 by specific antibodies increased the rates of survival after burns and subsequent sepsis.75 In a sepsis model, the neutralisation of IL-10
reduced the vulnerability to secondary infections such as
Pseudomonas pneumonia.76 In a peritonitis model, the
early blockade of IL-10 by antibodies led to elevated concentrations of TNF in the plasma and increased rates of
mortality. In the same model, both the therapeutic and the
prophylactic application of IL-10 reduced the early liberation of the proinflammatory cytokines IL-1, IL-6 and
TNF-.77 A later neutralisation of IL-10, 12 hours after the
induction of sepsis, seemed to be beneficial for survival.77 In
another study, application of IL-10, six hours after the
induction of peritonitis, improved the rates of survival in a
murine model.78 However, Remick et al79 reported no significant decrease of the rate of mortality after the application of human recombinant IL-10.
While the above studies support the CARS hypothesis80
it is as yet not clear whether early application of exogenous
IL-10 reduces mortality. The results appear to be inconsistent even in animal studies.
Interleukin-18 (IL-18). This was formerly termed interferon- (IFN-)-inducing factor, and shares structural features with the IL-1 family. It amplifies the development of
Th1 cells and it is an important factor for enhancing the
activity and proliferation of NK cells.81
IL-18 is primarily secreted by activated macrophages,
dendritic cells and Kupffer cells.82 It is synthesised as an
inactive precursor protein which is processed into the
mature form by IL-1-converting enzyme/caspase-1 (ICE).83
Secretion of active IL-18 into whole blood from healthy
humans and trauma patients can be effectively controlled by
blockade of the activity of ICE.83 By contrast, during sepsis,
alternative mechanisms are supposed to regulate the secretion of IL-18 since ICE inhibitors do not influence the
release of IL-18 into whole blood and septic patients.84,85
Most studies which characterise the expression of IL-18
have been performed in septic patients.86,87 Hochholzer et
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317

al88 found in an experimental study that IL-18 played an

important role in the induction of IFN- and lethality in
response to LPS. Values of IL-18 were significantly correlated with their APACHE II scores in septic patients and a
strong correlation between these and the levels of inflammatory cytokines was observed.89
In a clinical study in patients with post-operative sepsis,
the level of IL-18 was significantly higher in both survivors
and non-survivors than in the non-septic control group.86 It
was also observed that the level was significantly increased
in patients with lethal sepsis compared with septic survivors, with the conclusion that serum IL-18 represents an
early predictive factor for the lethal outcome of post-operative sepsis. The release of IL-18 seemed to depend on the
presence of micro-organisms because trauma-induced systemic inflammation without circulating bacteria did not
increase the plasma concentration of IL-18.84,85

Markers of cellular activity

Cytokine receptors. Cytokines exert their effects by inter-

action with receptor systems and mediate intercellular

events which often involve transcription of DNA.90
The effect of TNF- is mediated by the binding to two
membrane-bound receptors (TNF-RI, 55kD and TNF-RII,
75kD), which are found on nearly all cell types. In polytraumatised patients, increased concentrations of membrane-bound receptors were found to correlate with
increased rates of MODS.91 Each TNF receptor has a corresponding soluble molecule (sTNF-RI and sTNF-RII),
which competes with membrane receptors for binding free
TNF-, thus eliminating the bioactivity which would occur
with membrane-bound receptor ligation. In a clinical study,
early post-traumatic MODS and SIRS coincided with an
increase in the concentration of soluble TNF receptor protein.92 In severely burned patients, an early increase in
sTNF-RI and sTNF-RII was reported to be associated with
a higher risk for poor outcome.93 Major surgery resulted in
elevated sTNF-RI and sTNF-RII expression and high
sTNF-RII levels were associated with increased post-operative rates of mortality.94,95 It was concluded that high levels
of antagonists to TNF- represented persisted activation of
SIRS in critically ill patients.94,95
IL-1 and IL- bind to two classes of cell-surface receptor (IL-1-R1 and IL-1-RII). It has been postulated that
IL-1-RII prevents IL-1 from binding to type-1 receptors,
thus inhibiting signal transduction.96 IL-1 bioactivity is also
inhibited by the presence of IL-1 receptor antagonist
(IL-1ra). This inhibits IL-1 by binding to IL-1-RI without
agonist activity.96 Again, each membrane-bound IL-1
receptor has a soluble form (sIL-1R-I and sIL-1R-II). In
polytraumatised patients, increased levels of IL-1ra were
correlated with post-traumatic MODS.95 Mokart et al97
showed that raised concentrations of IL-1ra were associated with post-operative shock.
There are two forms of the IL-6 receptor, one soluble
(sIL-6-R) and the other membrane-bound (mIL-r-R). IL-6

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P. V. GIANNOUDIS, F. HILDEBRAND, H. C. PAPE

bound to sIL-6-R exerts its function independently from

mIL-6-R.98 In a clinical study, it was shown that serum
levels of sIL-6-R remained in the normal range after severe
head injury.98 They were elevated in the CSF, but no correlation with the extent of the cerebral lesion or the clinical
outcome was observed. Serum concentrations were significantly higher than those in the CSF and a pivotal role for
the IL-6/ sIL-r-R ratio in the injured brain was suggested.98
After major thoracoabdominal surgery, serum levels of
SIL-6-R were found not to be influenced by surgical
trauma.99 Concentrations of sIL-6-R in the drainage fluid
were significantly lower compared the serum levels. It was
concluded, that sIL-6-R is being constantly produced in
areas other than the operative field, while the level of
sIL-6-R is reduced by consumption (binding to IL-6) in the
operative field.99
Adhesion molecules. The adhesion of polymorphonuclear
leucocytes (PMNs) to capillary endothelial cells is the decisive step for their migration to the site of inflammation and
the hallmark of the microenvironment theory. The adhesion is provided by adhesion molecules, which are present
both on the PMN and on the surface of endothelial cells.100
Three major groups of adhesion molecule are distinguished: selectins, immune globulins and integrines. Both
selectins and immune globulins also appear in a soluble
form (sEselectin and sICAM-1).
All these proteins provide a specific attachment between
the ligand and the receptor, so that a selective accumulation
of PMNs occurs in the inflammatory tissue. The adhesion
of PMNs to endothelial cells and the migration of PMNs
through the endothelium are characterised by rolling,
attachment and diapedesis. These interactions of PMNs
and endothelial cells are associated with different adhesion
molecules. The neutrophil L-selectin and the endothelial Pand Eselectin provide the so-called tethering and rolling
of PMNs to endothelial cells.101 This transient adhesion
between PMNs and the endothelial cells is associated with
a lowered flow rate of PMNs and increased shearing forces.
Furthermore, selectins are characterised by their adhesion
being partially mediated via Lewisx antigen and sulphate
glycoconjugates.102 After activation of PMs, a so-called
shedding of L-selectin appears. This soluble form of
L-selectin (s-L-selectin) can be detected in blood serum.103
s-PMN-bound L-selectin was significantly reduced in
deceased septic patients and in trauma patients with posttraumatic MODS.104 A blockade of L-selectin has been
shown to exert protective effects in haemorrhagic shock.105
After adhesion the PMNs are attached to endothelial
cells by a stable cell-to-cell contact via integrines. The
integrin molecules consists of an - and a -subunit. The
latter is subdivided in 1, 2 and 3, from which the 2subunit is the most important. In addition, three different
2 integrines can be distinguished, CD18/CD11a (leucocyte function associated molecule-1, LFA-1), CD18/CD11b
(macrophage antigen-1, Mac-1) and CD18/CD11c. In an in
vitro model, the blockade of the common CD18 complex

led to a significantly reduced adhesion of stimulated PMNs

to activated endothelial cells.106 In vivo investigations in
rabbits showed that bacteraemia-induced accumulation of
PMNs in the lung was significantly lowered by a monoclonal CD18 antibody.107 Furthermore, the protective
effect of this antibody was also demonstrated in different
ischaemia-reperfusion models. The blockade of CD18
caused a significant reduction of accumulation of PMNs in
a model of haemorrhagic shock, in mesenteric ischaemia in
cats and in ischaemia of the limbs in the rat.108,109
The third group of adhesion molecules are the immune
globulins. Intercellular adhesion molecule-1 (ICAM-1) is
the best known. ICAM-1 is mainly expressed by endothelial
cells. Cells which have not been activated show a continuous baseline expression and a maximum expression occurs
approximately eight hours after activation. Expression of
ICAM-1 was shown to be induced by TNF-, IL- and
endotoxin. ICAM-1 was identified as the endothelial ligand
for 2 integrines.110
Several studies have assessed the expression and kinetics
of adhesion molecules in multiply-injured patients.15,111,112
While the levels of the CD11b receptor were elevated on
admission, the degree of upregulation did not correlate
with the ISS.15 However, levels of soluble Eselectin and soluble ICAM-1 were found to be significantly raised in the
plasma by the third day after injury, the magnitude of the
increase being related to the degree of injury.18 Law et al112
reported a significant correlation between the elevated
levels of soluble ICAM-1 and the later occurrence and
severity of MODS. Post-traumatic MODS has also been
associated with an increased number of CD11b/CD18
receptors on the leucocytes and an upregulation of the oxidative burst.112 In another study, both pulmonary endothelial cells and leucocytes showed a marked expression of
ICAM-1 in patients who had died from septic complications.113 The levels of sICAM-1 also increased in sepsis and
MODS, and death was predicted by serum concentrations
of sICAM-1.114 However, it is not clear whether sICAM-1
is split off or secreted by endothelial cells since the soluble
form of this molecule was also demonstrated in ICAM-1
knockout mice.115
In general terms the available results in the literature are
not consistent in assessing the predictive value of these molecules.
Elastase. Reactive oxygen species and proteases are neutrophil-derived toxic enzymes and have been considered to
be important in the development of acute lung injury for
some time.116 Mature neutrophils are unable to manufacture and store one such molecule, elastase, which has the
capacity to degrade most proteins in the extracellular
matrix and important plasma proteins.117 Besides its proteolytic activity, neutrophil elastase induces the release of
pro-inflammatory cytokines (IL-6, IL-8).118 Under normal
conditions the activity of neutrophil elastase is regulated by
endogenous protease inhibitors.119 The release of neutrophil elastase, assessed as plasma elastase-1 proteinase
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INFLAMMATORY SERUM MARKERS IN PATIENTS WITH MULTIPLE TRAUMA

inhibitor complex, is a sensitive marker of neutrophil cellular activity.

Significantly raised levels of E-1PI have been noted on
admission after multiple injuries and these were seen to be
higher in those patients with greater degrees of injury and
with the development of sepsis, being significantly elevated
on the first and third days compared with patients without
sepsis.15 One other study has reported a similar pattern of
release of elastase but was unable to correlate this with the
severity of injury, probably because of the small group of
patients studied.120 Repetitive increases in the level of
plasma elastase have been seen after trauma in patients who
developed MODS.121 Nuytinck et al122 also noted that
raised plasma levels of elastase correlated well with the
severity of injury and the occurrence of multiple organ failure. Moreover, these authors were also able to discriminate
between later survival or mortality at an early stage in the
clinical course using this parameter.
More recent studies have also shown consistently that
neutrophil elastase is increased in acute lung injury and in
patients with ARDS with, in some cases, a positive correlation between the severity of illness, the respiratory index
and organ failure.123,124
Overall, the levels of netrophil elastase are increased in
both clinical and animal models of acute lung injury. However, there has been no validation of a cut-off point which
could identify early patients at risk of developing post-traumatic complications. Further studies are needed to validate
the efficacy of this marker of immune reactivity.
Human leucocyte antigens; HLSA-DR class-II molecules. In
recent years, studies of the changes in the expression of
some leucocyte cell-surface antigens early in the postinjury
phase have been found to correlate with the development of
subsequent septic complications and death. In particular,
the level of expression of class-II major histocompatibility
antigens (MHC class II) on mononuclear cells in the peripheral blood has correlated most consistently with septic morbidity and mortality after surgery or trauma.125
MHC-class-II antigens play an indispensable role in the
presentation of processed antigen by antigen-presenting
cells to T-cells for the elaboration of a specific immune
response. Failure of this antigen-presenting capacity results
in an abortive or non-existent response. This may indicate
why some patients develop and succumb to septic complications after trauma, although it does not explain why
some recover their monocyte HLA-DR expression whereas
others suffering the same degree of trauma fail to do so,
going on to develop septic complications and ARDS. ClassII major histocompatibility molecules are expressed on the
surface of antigen-presenting cells, namely macrophages,
dendritic cells and B cells.
In one study, when an initial low level of expression rose
to normal levels within three to four days, the clinical
course was uneventful.126 When the rise in MHC class-II
expression was delayed, but still occurred, the post-operative course was complicated by the development of sepsis
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319

from which the patient recovered. However, when the level

of monocyte MHC class-II expression failed to recover
after trauma, patients often developed sepsis from which
they died.126
A study carried out in patients undergoing major resection of the digestive tract also showed that the operation
was associated with a major fall in the level of expression of
monocyte MHC class II, maximally on the first post-operative day, and that the pattern of recovery of this expression
and the relationship to clinical outcome were similar to
those seen after non-surgical trauma.127
More recently, after multiple injuries, release of IL-10
was reported to regulate monocyte HLA-DR expression.70
In another study assessing the expression of HLA-DR on
monocytes and T cells in 77 patients who had sustained
blunt trauma, the HLA-DR on T cells was reduced on the
day after admission in 20 patients with subsequent severe
sepsis, compared with 40 who did not develop infection.128
Several studies have described the delayed or complete
failure of recovery of the expression of the HLA-DR antigen on circulating monocytes in patients destined to
develop sepsis and who eventually died.129-131
The HLA-DR is considered to be the only marker of
immune reactivity which correlates accurately with morbidity and mortality after trauma. However, the need for a
dedicated laboratory for flow cytometric studies has not
made the use of this marker very attractive in clinical practice.
DNA. The mechanisms by which cell-free DNA is liberated
into the circulation of human subjects are unknown. One
possibility is that DNA is released after cell death. Plasma
DNA has been found to increase after major trauma and
has also been suggested as a potentially valuable prognostic
marker for patients at risk.132 In a clinical study in 83 multiply-injured patients the level of plasma DNA predicted
organ failure and MODS with an overall sensitivity and
specificity of 93% and 83%, respectively.133 In a further
clinical study in 84 patients with multiple injuries, it was
also confirmed that the plasma DNA was increased after
trauma.134 The concentrations were 12-fold higher in
patients with adverse outcome compared with those who
did not develop such complications.134
The prognostic value of free DNA has only recently been
investigated. The existing preliminary studies are promising. Nevertheless, more studies are required to validate the
sensitivity and specificity of such a marker in the clinical
setting.

Conclusions
Understanding of the pathophysiology and the immunobiology of both traumatic and surgical injury have contributed considerably to the debate surrounding the aetiology
of post-traumatic complications. Trauma and shock lead to
an activation of multiple humoral and cellular cascade
mechanisms, such as inflammatory mediators, as well as
complex mechanisms of host defence. These immuno-

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P. V. GIANNOUDIS, F. HILDEBRAND, H. C. PAPE

logical defence mechanisms may become insufficient and

allow further complications. Generalised capillary damage,
increase in permeability, and subsequent multiple organ
dysfunction or failure belong to the clinical picture of
MODS. Despite the application of conventional and supportive treatment in the intensive-care unit, the clinical
results in patients with ARDS and MODS remain disappointing.135
Several attempts at inhibiting components of the mediator cascade to influence the outcome after trauma and
sepsis have failed.136-138 The reason for this lack of success
is because of poor understanding of the critical mechanisms, the difficulty identifying those patients who may
benefit and the delay between the onset of complications
and the administration of the therapeutic agent. Clearly,
more evidence regarding the underlying biological problem
is needed. This issue may be resolved by the use of inflammatory and molecular markers and genetic technology to
allow the early identification of patients at high risk of
developing complications.
However, for a marker to be clinically useful it must be
easily and reliably measured, usually from a blood sample.
Its usefulness is tested by examining its sensitivity and its
specifity. An ideal marker will accurately discriminate
between those with a given condition and those without. In
the clinical setting, however, especially in the early posttraumatic course, the end-points are not always clearly
defined. The clinical signs of the patients may not allow
accurate evaluation of the clinical condition of the patient.
The ideal marker should be able to distinguish between the
inflammatory response and infection allowing earlier identification of patients with a specific disease process.
The CRP is probably the most widely used marker in
clinical practice at present, despite its inability to distinguish infection from inflammation, its response in proportion to the initial injury, and its inability to predict
outcome. Its use persists due to the inability to furnish an
acceptable alternative. The search for inflammatory markers initially concentrated on the initiators and promoters
of the inflammatory cascade-endotoxin, TNF- and
IL-1.44,45,53,54 These investigations proved to be disappointing. They appear to be transient and most likely it is the
response that they initiate which alters outcome. Recent
studies have shown promising results in other markers of
the inflammatory cascade. From all the available cytokines,
IL-6 seems to be the most reliable marker for systemic
inflammation, and LBP appears to be an accurate and early
marker of infection. The use of these two markers together
may offer the ability to detect the onset of SIRS and allow
early intervention to prevent MODS, to distinguish
between inflammation and infection and to monitor the
response to standard and innovative therapies.
The other marker of immune reactivity which appears to
have a high sensitivity and specifity is the HLA-DR antigens
on human peripheral mononuclear cells. However, because
of the laboratory work requiring antibody staining and

flow cytometric analysis they have not found great clinical

acceptance over the years.
In this article the existing evidence of the most investigated markers of immune reactivity has been evaluated. It
appears that at present the ideal marker, which could be
assessed easily with the best predictive outcome, is not
available. This is not surprising. The idea that single markers can predict the outcome of multiple causes of sepsis and
MODS in diverse populations ignores the complexities of
the molecular mechanisms behind the inflammatory processes. The multiple-injured population is diverse and has a
wide range of underlying and often multiple pathology.
Further studies are indicated to provide solid evidence
regarding the efficacy of any of the mediators of the inflammatory cascade. Parameters may have to be combined,
since no single marker may be able accurately to predict the
clinical course and outcome. The completion of the human
genome project and the exciting developments anticipated
in clinical genetics will promote the introduction of new
diagnostic tests and the identification of new candidate
genes and will hopefully provide innovative therapies to
assist the treatment of the multiple-injured patient.

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