CHAPTER 1

INTRODUCTION AND SCOPE OF THIS THESIS

Introduction

2

INTRODUCTION

Wounds, healing and inflammationWhen the skin is damaged, for example by a small cut, a blood clot will start forming within seconds,and within minutes bleeding will stop. We should realize that the process of wound repair has thenalready started. Under normal circumstances, healing will be the end result of overlapping processes:inflammation, tissue formation and tissue remodelling [1]. Inflammation is a response of the immunesystem, since the immune system guards the individual against infection. Inflammation is defined inWebster's Medical Dictionary as "A local response to cellular injury that is marked by capillarydilatation, leukocyte infiltration, redness, heat, pain, swelling, and often loss of function and thatserves as a mechanism initiating the elimination of noxious agents and damaged tissue[2]."

Since minor wounds are sustained very regularly, the local inflammatory responses initiated by theimmune system are as crucial part of body homeostasis as is feeding or sleeping. Inflammation in anextremely complex homeostatic response that involves neutrophils, platelets, macrophages,endothelial cells and the coagulation and complement systems. The immune system can be dividedinto the innate immune system and the adaptive immune system. Furthermore, the system has beendivided into a humoral and a cellular system. Combined, these two divisions result in the four majorparts (Table 1.1) of the immune system.

This thesis focuses on a number of aspects of the innate humoral immune response. The earlyinflammatory response is part of the innate immune system. In contrast to the adaptive immunesystem, which provides improved responses upon repeated infection, the innate response is not basedon immune memory, but on evolutionary highly preserved mechanisms of pattern recognition ofpotential pathogens. For example, bacterial endotoxin (lipopolysaccharide; LPS) is recognized bythe innate immune system in even very low concentrations, and thus serves as a very strong stimulusof subsequent responses. Acting together, CD-14 and so-called Toll-like receptors (e.g. TLR-4)present on myeloid cells, are believed to form a single pathway common to all mammals totransduct the LPS-signal [3]. The importance of these Toll-like receptors is underscored by thediscovery that the receptors are coupled to a pathway that activates genes mediating innateimmune defenses in mammals, insects, and even plants [4,5].

Table 1.1. Major divisions in the immune system

Innate immune system Adaptive immune system

Humoral Cytokines, complement,

acute phase proteins

Immunoglobulins

Cellular Phagocytes, Natural killer

cells

T-lymphocytes

Chapter 1

Whereas local inflammation inat a certain size of the wounds,

Clinical signs of systemic inflamThe systemic manifestations oadmission are the subject of cause systemic effects (Table will develop. SIRS has been dmanifestations listed in Table intensive care unit nearly all pa

Grades and definitions of syste

Figure 1.1. To preserve a stabimmune system reacts to disturesponses is to eliminate the ditissue damage or infection (thesignals that direct the response

III

Cellula

Hu

C

II

Amplification

I

Induction

Homeostasis

disturbancerecovery

stimulus

response

Immune response phases

r

III

moral

ellular

3

the setting of tiny wounds may not have significant systemic effects, systemic effects will become evident.

mation f the innate immune response that result after trauma, burns or ICU-this thesis. When an inflammatory stimulus is sufficiently strong to1.2), the so-called systemic inflammatory response syndrome (SIRS)efined by a consensus conference [6] as present if two of the clinical1.3 are observed. At the ward many patients may have SIRS, at thetients have SIRS.

mic inflammation

le healthy situation (homeostasis), like other physiologic systems, therbances such as a trauma with phased responses. The purpose of thesesturbance and restore homeostasis. The induction phase the presence of stimulus) is recognized and transducted into cytokine or chemokine

that eliminates the injury. (Adapted from T.H.The et al. 1995)

Introduction

4

The American consensus statement contained, apart from the definition of SIRS three additionaldefinitions. The main purpose of these definitions has been to illuminate the distinction betweeninflammation and infection:

SIRS= fever + leukocytosis; Sepsis= SIRS + infection; Severe Sepsis= Sepsis + multiorgan dysfunction; Septic shock= Severe Sepsis + refractory hypotension [6].

Table 1.3. SIRS (Systemic Inflammatory Response Syndrome) criteria[6]

Temperature >380 or <360 CHeart rate > 90 /minRespiratory rate > 20

Hyperventilation (PaCO2 < 4.3 kPa)Leukocytes > 12 109/l or < 4 109/l

Immature neutrophils > 10%

Organ failureMultiple organ failure has been defined in 1985 by Goris as 'generalized, autodestructive inflam-mation' [7]. As more organs are affected by the systemic inflammatory process, the chances ofsurvival decrease.Two examples of how organ failure has been graded are shown in table 1.4, a minimal total score hasa high probability of survival, a maximal score has a very low probability of survival.

Biochemical signs of systemic inflammation - acute phase proteinsAlthough not strictly defined, the acute phase response can be considered to consist of the initialsystemic clinical and biochemical responses that follow for trauma or infection. Fever is the clinicalhallmark of the acute phase response, and it is usually accompanied by tachycardia [8]. Theseclinical signs are paralleled by many biochemical changes. Many of the proteins induced by theinnate immune response are called acute phase proteins.An elevated erythrocyte sedimentation rate (ESR) [9] was the first widely used laboratory

Table 1.4. Examples of two multiple organ failure scoresOrgan/system Goris’ Multiple organ failure

score [7]Sequential organ failureassessment (SOFA) [52]

Cardiovascular 0 – 2 0 – 4Pulmonary 0 – 2 0 – 4Renal 0 – 2 0 – 4Neurologic 0 – 2 0 – 4Liver 0 – 2 0 – 4\aHematologic/ coagulation 0 – 2 0 - 4Gastrointestinal 0 – 2Total score 0 – 14 0 - 24

Chapter 1

5

parameter of inflammation. Increased levels of the acute phase protein fibrinogen are a maindeterminant of an elevated ESR [10]. A large number of proteins have turned out to be acute phaseproteins (APP). What constitutes an acute phase protein has not clearly been defined. Anincreased levels after inflammation and some sort of effector function (as opposed to signallingcytokines) are the most important characteristics of acute phase proteins. Kushner [11] somewhatsweepingly defined APP as those proteins whose plasma concentration rises 25% or morefollowing an inflammatory stimulus. Albumin, the most abundant protein in the circulation, issometimes called a negative acute phase protein since it is down-regulated during inflammation.The metabolic effects and the reasons why albumin levels are decreased, are only partlyunderstood [12]. Although not subject of this thesis, decreased albumin levels are an importantillustration of the relation between inflammation and metabolism.

Nowadays, C-reactive protein (CRP) is the most widely directly measured acute phase protein. Inhealth it is hardly detectable in plasma, but with inflammation it can rise rapidly to 10- or 100-foldlevels. Although CRP’s function is not clearly identified, CRP is probably involved in early non-specific antibacterial defences [13]. Thanks to reliable assays and CRP’s half-life which is in theorder of one day, CRP has gained wide popularity for monitoring inflammation in many diseasestates. On the basis of the rapidity and magnitude of increase in concentration during the acute phaseresponse, acute phase proteins can be divided into 3 groups (Table 1.5).

Source of acute phase proteinsThe liver is quantitatively and qualitatively the major source of acute phase proteins. In severalhepatocyte models for different species, the induction of many acute phase protein-production hasbeen demonstrated. For example in 1966 Hurlimann et al. already performed in vitro studies thatshowed that hepatocytes produce CRP [15].

Table 1.5. Classification of some acute phase proteins

Increase by factor 1.5 Increase by factor 2 to 4 Increase by up to factor 1000Slow response Intermediate response Rapid response

Antitrombin III α1-proteinase inhibitor C-reactive protein (CRP)Complement C3, C4 α1 acid glycoprotein Serum Amyloid A (SAA)Ceruloplasmin α1 anti-chymotrypsinC1-inhibitor Haptoglobinα2-antiplasmin FibrinogenAdapted from Heinrich et al. [14]

Introduction

6

Function of acute phase proteinsA close look at the functions - as far as understood - of the acute phase proteins illustrates thepurpose of the acute phase response: provide the organism with those proteins that are necessary tokeep up both an adequate and a restrained inflammatory response. Table 1.5 shows that proteins forthe complement system, the coagulation system and the fibrinolytic system are provided by the acutephase response. Most acute phase proteins have larger molecular weights (>50,000 Dalton) andhigher concentrations (mg/L to g/L range) when compared to cytokines which have a lowermolecular weight and much lower concentrations. This is due to fact that acute phase proteins areoften effector molecules (e.g. thrombus formation, protease inhibitor, bactericidal factor) asopposed to cytokines that are hormone-like proteins. Understandably, effector functions requiremuch higher concentrations than necessary for signalling functions.

Function unknown or no functionCurrently it is possible to detect many biological substances and study their interactions with manycell types. This results in a huge number of possible relations that can be studied. It is helpful to tryto understand these mechanisms in a teleological manner. Teleology is the use of design or purposeas an explanation of natural phenomena [2]. In understanding inflammation this implies thatevolution should have provided the organism with defenses that can plausibly by expected to help theorganism survive. A mammal will certainly have a survival benefit from adequate protection againstminor trauma, since every animal will continuously sustain such traumas during its life. But ananimal cannot logically be expected to have an elaborate defense against the results of septic shock.An animal with septic shock or severe trauma will in all probability not survive anyway, so evolutionhas no interest in providing for defenses in ‘lost cases’. Of course the animal (or man) with septicshock may survive with medical intervention, but at this stage we cannot rely on inflammationresponses behaving in a ‘logical’ way. Thus, we may expect every protein found near a smalluncomplicated wound to have a relevant function, and search aggressively for this function if we donot know it yet. We also may understand why TNFα levels are very high in septic patients, but weshould not expect that these high levels have a specific function. In fact in these patients manymolecules with elevated or depressed levels will not have a function, may have lost their function ormay even function inappropriately.That a distinct degree of systemic inflammation might be beneficial to the host, was already believedcenturies ago [16,17], when patients who did not properly recover from wounds or infection weretreated by inducing additional inflammation. The widely practiced cauterization of wounds with hotirons or hot oil was also performed on healthy skin to enhance recovery of wounds elsewhere (seeillustration). This practice of counter-irritation was also performed in the 20th century, albeit in theslightly more humane form of turpentine abscesses. In fact turpentine in still used in animal models toinduce a sterile acute phase response [18].

Chapter 1

7

History of endogenous pyrogen and IL-6The genesis of fever has long been a subject of investigation. Decades ago it became apparent that an'endogenous pyrogen' had to exist. This substance is made by the body in response to tissue damageor infection and it induces fever by changing the temperature setpoint in the hypothalamus - a processthat can be inhibited by prostaglandin inhibitors like aspirin. With modern molecular biologicaltechnology is became clear that several cytokines not only induce fever but also induce acute phaseproteins. They can thus be considered not only endogenous pyrogens but turned out to be true

hormones of inflammation.In the 1980’s interleukin-1 (IL-1) became the first contender for the role of endogenous pyrogen.Experiments by Aarden et al. showed that hybridoma growth factor (HGF) was active in the IL-1

Figure 1.2. Patient who is cauterized after injury. Drawing by JohannesWechtlin in Feldtbuch der Wundarznei by Hans von Gersdorff, 1540.

Introduction

8

assay [19]. A very sensitive bio-assay for HGF was developed, and this assay did not respond to IL-1,indicating that HGF had to be another cytokine. After cloning of cDNA, HGF was discovered to beidentical with a new cytokine named interleukin-6 [20]. In many in vitro and in vivo studies thatfollowed, the B9.9 bioassay for HGF/IL-6 has been very important in defining the pleiotropic role ofIL-6, to be later replaced by ELISAs [21].The cytokines TNFα, IL-6 and IL-1 are elevated within hours after a major inflammatory stimulus[22]. TNFα peaks earlier than IL-6 and also induces IL-6. For example van Gameren andcolleagues [23] showed that administration of IL-6 to patients induced most of the responses listed inTables 1.2 and 1.3. Several studies have assessed the predictive value of cytokine levels for clinicaloutcome. In general, cytokine levels in patients with sepsis are much higher than in trauma patients[24]. In addition it was found by Hack et al. that higher IL-6 levels were associated with mortality insepsis patients [25]. Partly due to the fact that cytokine levels can rapidly change, and due todifficulty in routinely assaying cytokines, these measurements are not used in clinical practice. IL-6levels measured at the bedside (positive at > 1000 pg/ml) have been used to triage patients for anti-TNFα sepsis trials [26].

Methodological advantages of studying patients with mechanical trauma or burns The pathophysiology of inflammation has received intensive attention in patients that are criticallyill, but it is useful as well to study the physiology of inflammation in patients that are not critically ill,e.g. in healthy persons that sustain a nonlethal trauma. Trauma patients usually are healthy before theinjury and show a responses roughly proportional to the severity of the injury [27]. Since the time ofthe injury is a well-defined moment as opposed to sepsis, the timing and order of the elicitedresponses can be studied in more detail. Concerning inflammatory responses in trauma patients, patients with burns represent a special group.Thermal injury is a one-dimensional trauma, with the percentage of burned total body surface(TBSA) being the key parameter. With only the age of the patient and TBSA it is possible to make agood prediction of mortality and hospital stay [28]. Severely burned patients can have extensivetissue damage without accompanying organ damage. Early after sustaining burn injury, patientsshow a pronounced inflammatory response. This occasionally extreme response has made burnspatients a subject of many studies on virtually all known aspects of host responses.

Differentiating infectious from non-infectious causes of inflammation.Both trauma and infection can induce an acute phase reaction, clinically characterized by SIRS.When SIRS persists for a number of days after admission to the hospital, this can be due to thetrauma itself or secondary infection. In the first case a wait-and see policy is often justified. In thesecond case it may be crucial to search for a focus, or to start antibiotics and even perform anoperation. Since a majority of trauma patients at the ICU develop fever, with only proveninfection in half the these patients [29] - it is very useful to possess additional tools to recognizebacterial infection. Especially in immune suppressed patients (e.g. after transplantation) it wouldbe very useful to possess a rapid assay to detect bacterial infection, since in these patients themargins of error are small. Now that many inflammatory markers can be measured in thecirculation, researchers have looked for those markers that can discriminate bacterial infection

Chapter 1

9

from other causes of systemic inflammation. But discriminating causes of inflammation onpatterns of cytokine or acute phase protein responses have proven an elusive goal. Measuring thebacterial product endotoxin itself would seem to be an obvious solution to identify bacteremia andendotoxemia early [30]. Unfortunately circulating endotoxin assays are difficult to use andreproduce [31].In accordance with the concept of the spectrum of increasing systemic inflammatory responseswith increasing infection: SIRS → sepsis → severe sepsis → septic shock, systemic inflammationwith infection is associated with higher cytokine levels and higher levels of CRP. Thus, serialquantitative measurements of the extent of the acute phase response, usually in the form of CRPhave been a major clinical tool in detecting infection. The acute phase response appears tomodulate mainly in intensity and duration, not in the relative enhancement or inhibition ofcomponents of the response. This is of course in agreement with the non-specific nature of theacute phase reaction.

Procalcitonin to differentiate infection and non-infection?The recent introduction of a convenient assay for procalcitonin (PCT) and the first experienceswith measurements of circulating PCT have been promising. Some authors have proposed that apractical parameter that differentiates bacterial infection from other causes of inflammation is nowat hand. Although PCT is biochemically a precursor of calcitonin (a hormone involved in calciumhomeostasis) it is functionally not related to calcitonin. Both the cellular origin and function ofPCT are unknown. Nevertheless, circulating PCT measurements in many patients groups [32]have shown that:- Circulating PCT is elevated proportional to the inflammatory stimulus. - PCT can be induced by infusing endotoxin in volunteers. Elevated PCT-levels are detectable

at 6 hours and disappear with a half-life of about a day [33].- The dynamic range of PCT is very large: PCT rises at least a factor 400 within 6 hours in

volunteers receiving endotoxin. Where one report [34] claims that PCT levels are <0.01 ug/l innormal persons, PCT can reach 1000 ug/l in sepsis [35]. Thus PCT's dynamic range may beeven greater than that of CRP or SAA.

The most interesting aspect of PCT is the evidence that PCT compared to CRP is superior indiscriminating bacterial from non-bacterial inflammation. De Werra [36] compared patients withseptic shock, cardiogenic shock, and bacterial pneumonia. The best predictive value for septicshock was found for PCT in septic shock, with PCT varying from 72 to 135 ng/mL, comparedwith approximately 1 ng/mL in the other groups. The monograph on PCT by Meisner [32]contains a number of early studies that indicate that PCT is better than CRP in discriminatingserious infection from other causes of inflammation. More recently Gendrel et al. reported thatPCT was more specific and sensitive than CRP for the differentiation of bacterial and viralinfections in children [37]. In 236 children with viral infections they found a median PCT of 0.2ug/l and a median CRP of 10 mg/l. In 46 children with bacterial sepsis or meningitis PCT was 18ug/l and CRP 144 mg/l. Thus PCT is about a 100-fold higher in sepsis compared to viral infection,whereas CRP is 'only' 15 times higher.

Introduction

10

Still the literature is not conclusive on this very important issue. Those disease categories wheredetermining PCT may not be reliable need to be better defined. For example de Bont [38] foundthat in neutropenic patients PCT responses are much lower than would be expected on the basis ofother signs and elevated CRP-levels. The various results that suggest that preferential induction ofPCT by bacterial infection exists, have led some authors to hypothesize that endotoxin can inducePCT directly. A logical question is whether cytokines like TNFα or IL-6 are necessary andsufficient for the induction of PCT. And although increased levels of IL-6 and TNFα were alsoobserved after endotoxin infusion, this does not prove that TNFα and IL-6 are necessary for theinduction of PCT synthesis, as they are for the acute phase protein induction. Does PCT originatein the liver? Or more general, should PCT be viewed as an acute phase protein?

Platelets and endothelium in inflammation Platelets can be considered inflammatory agents as much as coagulatory agents. In systemicinflammation platelets first disappear from the circulation (thrombocytopenia), and as the patientrecovers uneventfully platelets will reappear in increased amounts [39] (thrombocytosis). Severalcytokines can induce this sequence. After administration to humans, IL-6 will induce a completeacute phase response, including the characteristic sequence of a nadir platelet count at day 2 or 3and a maximal platelet count at day 12 [23]. Concerning the causes of premature disappearance ofplatelets from the circulation, two important possibilities exist. The platelet end up as part of a clotor the platelet can (temporarily) adhere to the endothelium. In both cases platelets can amplifyinflammation through the release of powerful mediators. Consumption of platelets is an integral part of the syndrome of diffuse intravascular coagulation(DIC). The reverse, that DIC always accompanies platelet consumption, is not true. DIC ischaracterized by extensive intravascular formation of fibrin, in severe cases resulting in vascularocclusion and organ failure. DIC occurs secondary to diverse range of serious diseases, like sepsisor trauma [40]. It is important to note that significant decreases in platelet count are observed invirtually all patients with trauma or sepsis. Although a significant proportion of these patients mayhave indicators of 'low-grade' DIC, many do not have DIC, indicating that platelet sequestrationand DIC are not the same [41]. Endothelium, the single cell layer that separates the blood from the organs and tissues, is a veryactive cell system. Endothelium regulates hemodynamics and the transport of molecules and cellsbetween blood and the tissues. Activation of endothelium occurs early and universally [42] in theprocess of inflammation. Endothelial cell activation [43] is characterized by the loss of vascularintegrity, expression of adhesion molecules, transition from an antithrombotic to a prothromboticstate and cytokine production. Exposure of the subendothelium with tissue factor and vonWillebrand Factor and rapid expression of P-selectin can initiate the binding of platelets as well asthe adhesion molecules such as GPIIb/IIIa, P-selectin, CD-31, LFA-1, CD-36 [44] and CD-87(urokinase plasminogen activator receptor; uPAR) [45]. Cardiovascular research has producedextensive evidence of the importance of platelet-endothelium interaction. This is underscored bythe clinical effectiveness of aspirin and the recently introduced GPIIb/IIIa-inhibitors in limiting orpreventing coronary thrombosis [46]. Despite the evidence of the importance of the platelet-endothelial interaction in inflammation, direct studies on this interaction are methodologically

Chapter 1

11

very difficult. Ex vivo, endothelial cells and platelets are not the same as in vivo. Whereas theerythrocyte has a circulating precursor that can be clearly identified and quantified (thereticulocyte), no such equivalent exists for the circulating platelet. Therefore, in thrombocytopeniait is difficult to decide if decreased synthesis or increased aggregation or adhesion is present, evenwhen one resorts to a bone marrow biopsy or radioactively labeled platelet studies [47]. Re-injected radioactively labeled autologous platelets are not identical to native circulating platelets.

Serial platelet counts as an indicator of endothelial activationInstead of performing highly specialized platelet studies in a limited set of patients, in this thesisis was preferred to study serial platelet counts and study them in larger patient groups. Theassumption was that changes in the platelet count are to a large extent correlated to the magnitudeof endothelial activation, which in turn is related to systemic inflammation. A low admissionplatelet count is known to be a relatively strong predictor of adverse outcome in a variety ofdisease states. In meningococcal sepsis [48], after ruptured aortic aneurysm [49], at admission tothe intensive care [50] the early platelet count is one of the strongest predictors of outcome. As aresult the platelet count has been introduced in several intensive care scoring systems, while theleukocyte count has been eliminated from some scoring systems. The multi-organ dysfunctionscore (MODS) [51], the sequential organ failure assessment score (SOFA) [52] and the pediatricrisk of mortality score (PRISM-III) [53] use the platelet count and not the leukocyte count as oneof its component parameters. In contrast to the well-described importance of initial plateletcounts, the relevance of subsequent changes has received little attention. Within the healthyindividual the platelet count is quite stable with an intra-individual variation that is only 20% to30% (or 60 ·109/L) of the inter-individual variation [54,55]. Thus serial platelet counts might inprinciple provide additional information. As long as the platelet count is within the normal range(i.e. 150 to 350 ·109/L) it has been implicitly assumed that such a count is normal. Yet, in patientswith an uneventful clinical recovery after a moderate trauma, thrombocytosis normally developsduring the second week. Thrombopoietin has been identified as a major regulator ofthrombopoiesis [56]. After trauma IL-6 is released, which is also capable of inducingthrombocytosis [23]. In critically ill patients thrombocytosis is often not present, although suchpatients typically have cytokine levels (including IL-6) much higher than those observed afteruncomplicated trauma. Thus despite the fact that many ICU patients are in a more advanced stateof inflammation (ranging from SIRS to sepsis, severe sepsis or even septic shock) compared touncomplicated trauma patients (usually SIRS), they have lower platelet counts. Since in manyinstances these decreased platelet counts are still in the 'normal range' these changes have receivedlittle attention. That platelet counts decrease as a result of increased endothelial activation may bethe explanation of the inverse relation between inflammation and platelet count in critically illpatients.

Introduction

12

Fat embolism syndromeThis thesis originated in studies on the incidence and causes of the fat embolism syndrome (FES).FES is typically seen in young patients who sustain isolated long bone fractures [57]. After aninterval of 8 hours to 2 days the three cardinal symptoms of the syndrome appear: • petechiae with a typical upper-body distribution, unlike that seen in severe thrombocytopenia• respiratory distress• cerebral disturbances

As the name of the syndrome denotes, fat is involved in these three cardinal symptoms, since fatemboli of bone marrow origin can be recovered from skin lesions, the lung and the brain duringpathological examination. In the process of embolization, the venous fat apparently easily(by)passes the lungs. Aggressive, early operative stabilization of fractures, and the improvedlevel of supportive care are assumed to have contributed to the decrease in FES-incidence.Although FES in seen less frequently than for example 25 years ago, both its causes and thereasons for its decreased incidence are unclear. Fever and tachycardia are among the 'minorsymptoms' described for FES [57], indicating a link of FES with inflammatory responses. Insteadof being a result of FES, systemic inflammation might be a factor in inducing FES. CRP canagglutinate fat globules into emboli [58] - a property of CRP that has even led to the developmentof a bed-side CRP-test based on fat-agglutination [59]. Here we looked at the relation of earlyinflammatory signs with the subsequent development of FES. Prolonged increases in pressures in (closed) wounds around the fractured bone may assist theprocess of fat intravasation and subsequent embolization. Early operation may decompress thefractured bone and prevent further embolization of fat. The potential detrimental effect of delayedtreatment on the incidence of FES, and the relevance of a persisting open foramen ovale [60] as ashunt for fat globules are explored in this thesis.

Chapter 1

13

OUTLINE AND AIMS OF THIS THESISIn this thesis the systemic counterparts of acute local inflammation were studied. As a part of theinnate immune response, systemic manifestations of inflammation were primarily studied intrauma patients HypothesisThe various stages of the systemic inflammatory response are expressed by distinct markers (pro-inflammatory cytokines as well as acute phase proteins and platelet counts) in a typical timesequence. They reflect the underlying pathophysiological mechanism and may offer possibilitiesfor monitoring (new) intervention strategies by providing better clinical prognostic and diagnostictests. The clinical model used to study these marker kinetics were trauma patients since this patientcategory allows to note the starting point of the (pathophysiologic) chain of events.Trauma patients are young and in general have no comorbity. By a better understanding of thetime sequence of the marker responses in trauma patients with single injuries physician maysubsequently better interpret responses in critically ill patients, most of whom have preexistent co-morbidity.

Aims of this thesisTo study the various stages of the systemic inflammatory response, we selected a number of(circulating) markers that we assumed would adequately reflect and be correlated with(patho)physiological signs and symptoms. The protein responses of IL-6, PCT, CRP, SAA andα1-antiproteinase as well as the platelet count and leukocyte count and physiological parametersas temperature or heart rate were studied.The first studies concern:- Fat embolism syndrome (chapters 2 and 3) The subsequent chapters are ordered according to the sequence of inflammatory events: - Interleukin-6 (chapters 4 and 5)- Procalcitonin (chapter 6)- Platelet counts (chapters 7, 8 and 9)

Two studies (chapters 7 and 9) were an intervention studies; all studies were retrospective indesign, although in chapters 4, 5, 6 and 9 some samples and data were prospectively collected.Not studied in this thesis were: animals, local responses, adaptive immune responses.

Fat embolism syndromeChapter 2 is a retrospective study of the incidence of FES in 172 patients with an isolated fractureof the femoral shaft. The goal was to find associations of the incidence of FES with the type offracture, the timing of operation and an early inflammatory response.Chapter 3 verifies if a right-to-left shunt in the heart has a causal role in FES. Such a shunt couldexplain the transit of large fat globules from the venous to the arterial circulation.

Introduction

14

Interleukin-6 and acute phase responsesChapter 4 describes IL-6 levels in patients with burns at a time that IL-6 had never beenmeasured in patients with acute systemic inflammation. The aim was to correlate IL-6 levels withbasic acute phase responses: i.e. fever, CRP-levels and α1-antiproteinase levels, and address thequestion if IL-6 was the long sought endogenous pyrogen. Chapter 5 examines IL-6 and acute phase responses in burns patients in detail. The goal was tostudy time dependent changes of phenomena in which IL-6 could play a causal role. Alsocorrelations of these parameters with IL-6 and the potential causal relations on the basis ofpublished evidence (as it was published at that time) are formulated.

ProcalcitoninChapter 6 it is assumed that endotoxin is not necessary for the induction of PCT. Thus, the directeffects of TNFα and IL-6 on the expression of PCT, SAA and CRP were measured. In vitro,human liver slices were used. In vivo, two groups of cancer patients, treated with TNFα and IL-6respectively, were studied.

Primary and secondary thrombocytopenia Early and late decreased platelet counts in trauma and ICU patients may reflect increasedsequestration due to systemic endothelial activation.The aim in chapter 7 was to examine early changes in platelet count as they occur during the first 2days after trauma. We also investigated if high-dose methylprednisolone administered shortly afterthe injury affected platelet consumption. This may indicate if the pleiotropic effects steroids are ableto affect inflammation related platelet sequestration.In chapter 8 the aim was relate late changes (i.e. > 2 days) in platelet count with mortality, sincepersisting systemic inflammation is known to be associated both with platelet sequestration andmortality. Patients from one surgical ICU were studied. The value of platelet counts was alsocompared to leukocyte counts.The goal in Chapter 9 was to generalize the observations of the previous study and address changesin platelet counts and outcome in a very large heterogeneous European multi-center population ofICU patients. The predictive power for mortality of early and late changes in platelet countrespectively, was investigated. For this purpose a simple mathematical model to describe time-dependent changes in platelet counts was used. We also investigated the behavior of platelet countsaccording to admission groups (medical, surgery scheduled or unscheduled.)

Finally Chapter 10 attempts to integrate the results and give suggestions for further research. Inparticular the timing of all responses observed in the various studies is combined into one scheme.The question what would constitute an ideal inflammatory marker is discussed, as well if such amarker exists.

Chapter 1

15

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