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Intensive Care Nephrology 2000 |
Klinik und Poliklinik für Anästhesiologie und Spezielle Intensivmedizin, Friedrich-Wilhelms-Universität Bonn, Bonn, Germany.
Correspondence to Dr. Frank Stüber, Klinik und Poliklinik für Anästhesiologie und Spezielle Intensivmedizin, Friedrich-Wilhelms-Universität Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany. Phone: 49-228-287-6446; Fax: 49-228-287-6754; E-mail: fstuber{at}uni-bonn.de
Abstract
Abstract. Sepsis and its sequelae are still a major cause of morbidity and mortality on today's intensive care units. The evidence that endogenous mediators actually mediate the individual's response to infection has led to various approaches to assess the individual's contribution to the course of the disease. The role of an individual's genetic background and predisposition for the extent of inflammatory responses is determined by variabilities of genes encoding endogenous mediators that constitute the pathways of inflammation. Primary responses in inflammation are mediated by proinflammatory cytokines such as tumor necrosis factor and interleukin 1. Conversely, anti-inflammatory mediators are released and may induce a state of immunosuppression in sepsis. Pro- and anti-inflammatory responses contribute to the outcome of patients with systemic inflammation and sepsis. Therefore, all genes encoding proteins involved in the transduction of inflammatory processes are candidate genes to determine the human genetic background that is responsible for interindividual differences in systemic inflammatory responses to injury. The genetically determined capacity of cytokine production and release, heat shock protein expression, nitric oxide synthase activity, gene polymorphisms of coagulation factors or factors of the innate immune system-like defensins, and other genes involved in inflammation may contribute to a wide range of clinical manifestations of an inflammatory disease. Genomic information may be used to identify groups of patients with a high risk of developing severe sepsis and multiple organ dysfunction, and determining which patients will benefit from antimediator strategies because of their genetic determination to high cytokine release in the inflammatory response will be the subject of future trials.
The clinical course of sepsis exhibits great interindividual variation (1). Comparable numbers of infectious units of microbial organisms induce infectious diseases with a wide range of severities. The immune response to injury involves a complex pattern of primary, secondary, and tertiary humoral and cellular responses. In this system, the role of the genetic background in inflammatory responses is determined by genetic variabilities in endogenous mediators that constitute the pathways of inflammation.
Primary responses in inflammation are mediated by proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1) (2). Secondary proinflammatory mediators such as IL-6 and IL-8 and anti-inflammatory cytokines such as IL-1 receptor antagonist (IL-1ra) and IL-10 are induced by TNF and IL-1 (2). Tertiary mediators are factors of different (even noncytokine) origin, such as proteases, coagulation factors, kinins, eicosanoids, nitric oxide, and others factors that have effects in the distal portions of mediator cascades (3).
Recent evidence suggests that anti-inflammatory mediators have important effects on the immune system of the host (4). Anti-inflammatory mediators induce a state of immunosuppression in sepsis, which has also been termed immunoparalysis (5). This state of decreased immunoreactivity is accompanied by high levels of anti-inflammatory cytokines such as IL-10 and IL-1ra (6). Symptoms of immunosuppression include a decreased number of expressed surface HLA class II molecules on circulating monocytes/macrophages and impaired ex vivo responses of peripheral blood leukocytes to lipopolysaccharide (LPS) (7).
Pro- and anti-inflammatory responses contribute to the outcomes of patients with systemic inflammation and sepsis. Therefore, all genes that encode proteins involved in the transduction of inflammatory processes are candidate genes for determination of the human genetic background that is responsible for interindividual differences in systemic inflammatory responses to injury.
Which groups of patients have high risks of developing severe sepsis and multiple organ dysfunction in situations involving systemic inflammatory reactions? Are there groups at high risk for death? Will certain patients benefit more than others from antimediator strategies, because of their genetic determination for high levels of cytokine release in inflammatory responses?
These questions demonstrate that knowledge of the role of the genetic background in human systemic inflammation may yield patient benefits in the context of continuously high mortality rates.
Studies of the structure and function of single genes that obviously contribute to specific diseases have been conducted, particularly in the field of cytokine research. Members of the cytokine family, such as TNF, are highly conserved throughout evolution (8). Few genomic polymorphisms are known, and the functional relevance of most genomic variations remains obscure.
One goal in determining the role of the genetic background in inflammatory responses is to identify genomic markers suitable for clinical use and risk stratification of patients. Another goal is to understand the effects of genomic variations on gene regulation and protein expression.
Primary proinflammatory cytokines such as TNF and IL-1 induce secondary pro- and anti-inflammatory mediators such as IL-6 and IL-10. These cytokines have been shown to contribute substantially to the primary response of the host to infection. Both TNF and IL-1 are capable of inducing the same symptoms and the same severity of septic shock and organ dysfunction as endotoxin in experimental settings and in human subjects (9). Genetic variations in the TNF and IL-1 genes are of major interest with respect to genetically determined differences in the response to endotoxin.
Tumor Necrosis Factor
TNF is considered to be one of the most important mediators of endotoxin-induced effects. Interindividual differences in TNF release have been described (10,11).
The TNF locus consists of three functional genes. The gene for TNF
is positioned between that for lymphotoxin 
(LT) in the upstream
direction and that for LT
in the downstream direction. Genomic
polymorphisms within the TNF locus have been under intense
investigation.
Genetic variation within the TNF locus is rare, because the TNF gene is well conserved throughout evolution (8). The coding region in particular is highly conserved.
Interest has been primarily focused on the genomic variations of the TNF locus. Biallelic polymorphisms defined by restriction enzymes (NcoI and AspHI) and other single-base changes (at positions -308 and -238), as well as multiallelic microsatellites (TNFa to -e), have been investigated in experimental in vitro studies and in various diseases, with TNF being considered an important or possible pathogen. Functional importance for regulation of the TNF gene has been suggested for two polymorphisms within the TNF promoter region. Single-base changes have been detected at positions -850, -376, -308, and -238 (12,13,14,15).
A guanine to adenine transition at position -308 has been associated with susceptibility to cerebral malaria (16). Those results could not be confirmed by another malaria study, which showed fewer fever episodes among heterozygous carriers of the TNF2 allele (17). The rare allele TNF2 (with an adenine at position -308) was thought to be linked to strong TNF promoter activity (16). Studies of autoimmune diseases such as diabetes mellitus and lupus erythematosus did not demonstrate differences in allele frequencies or genotype distributions between patients and control subjects (18,19). In addition, patients with severe sepsis and a high proportion of Gram-negative infections did not display altered allele frequencies with respect to the two biallelic promoter polymorphisms (positions -238 and -308) (20). Analyses of the TNF promoter using reporter gene constructs revealed contradictory results. A first report proposed a functional importance for the guanine to adenine transition at position -308 (16). Two studies could not confirm differences in TNF promoter activity in relation to the position -308 polymorphism (20,21). A recent article reported a possible effect on TNF promoter activity of the position -308 guanine to adenine transition in a B cell line (22). Genotyping of this polymorphism in patients with severe sepsis does not contribute to risk assessment. The position -308 polymorphism is not a marker for susceptibility to or outcomes of severe sepsis caused by Gram-negative infections (20). TNF plasma levels do not seem to be affected by this polymorphism in patients with severe abdominal sepsis.
In contrast to these findings are the results of a recent study that suggest an association of the rare allele TNF2 with non-survivors of septic shock (23). That publication reopens the discussion regarding the functionality of the position -308 TNF promoter polymorphism and its possible relevance for routine clinical use. In addition to the discussion regarding the relevance of association, formal standards for genotyping techniques must be established. Are there typing techniques, such as allele-specific amplification, that indicate over- or underestimation of certain alleles and genotypes?
In contrast to genomic variations located in the promoter region, intronic
polymorphisms are more difficult to associate with a possible functional
relevance. Two biallelic polymorphisms located within intron 2 of
LT have been studied in autoimmune disease
(24,25).
One polymorphism is characterized by the presence or absence of a
NcoI restriction site. First reports demonstrated genomic blots
revealing characteristic 5.5- or 10.5-kb bands after genomic NcoI
digestion, which hybridized to TNF-specific probes
(26). These bands correspond
to the presence and absence, respectively, of a NcoI restriction site
within intron 1 of LT.
The TNFB2 allele, with this NcoI polymorphism (10.5-kb
band), was shown to be associated with high levels of TNF- release
ex vivo (27). Other
studies showed no differences between genotypes in other models of ex
vivo TNF induction, whereas another study suggested an increased
LT response in TNFB2 homozygotes
(11). The question of which
genotype is clearly associated with strong proinflammatory responses in
clinical situations involving severe Gram-negative infections and severe
sepsis cannot yet be answered by ex vivo studies. Different cell
culture and cytokine induction conditions contribute to differing results. In
addition, the genomic NcoI polymorphism within intron 1 of the
LT gene may represent a genomic marker without evidence for
its own functional importance in gene regulation. This genomic marker may
coincide with thus far undetected genomic variations that are responsible for
genetic determination of strong proinflammatory responses to infection.
Results from studies in patients with severe intra-abdominal sepsis suggest
that TNFB2 homozygotes are associated with strong TNF responses. In
contrast, genotyping for another biallelic polymorphism within intron 1 of
LT (AspHI) did not demonstrate significant
association with plasma TNF levels.
Several studies of chronic inflammatory autoimmune diseases suggest an association between the TNFB2 allele and the incidence or severity and outcome of the disease (24,25,28). Studies of acute inflammatory diseases such as severe sepsis in patients in surgical intensive care units demonstrated a correlation between TNFB2 homozygosity and mortality rates (20) or the incidence of septic states among traumatized patients (29). TNFB2 homozygotes displayed a relative risk of 2.9 of dying as a result of severe sepsis, compared with corresponding genotypes.
Interleukin-1
In addition to TNF, IL-1 is a potent proinflammatory cytokine released by
macrophages in systemic inflammatory responses. IL-1 is capable of inducing
the symptoms of septic shock and organ failure in animal models and is
regarded as a primary mediator of systemic inflammatory responses. Antagonism
of IL-1 in endotoxin-challenged animals, including primates, abrogates the
lethal effects of endotoxin
(30). A biallelic
TaqI polymorphism has been described within the coding region (exon
5) of IL-1
(31,32).
Despite the finding that a homozygous TaqI genotype was correlated
with high levels of IL-1 secretion
(31), genotyping of patients
with severe sepsis did not reveal any association with the incidence or
outcome of the disease.
IL-1 Receptor Antagonist
Proinflammatory mediators represent the hyperinflammatory side of the systemic inflammatory reaction. At the same time, anti-inflammatory mediators are induced by proinflammatory cytokines and attempt to counterbalance the overshooting of inflammatory activity. This physiologic process limiting the extent of inflammation via the release of anti-inflammatory proteins may breach the physiologic boundaries for local and systemic concentrations of these mediators. Proteins such as IL-4, IL-10, IL-11, IL-13, and IL-1ra contribute to a very powerful downregulation of cellular and humoral proinflammatory activities. This downregulation results in decreased expression of class II molecules on antigen-presenting cells, as well as in low ex vivo responses of immunocompetent cells to inflammatory stimuli. This state of immunosuppression has also been termed immunoparalysis (5). It results in a state of areactivity, with diminished capabilities for fighting infectious pathogens. A new term for this state, which is a consequence of the systemic inflammatory response, is compensatory anti-inflammatory response syndrome (33). The outcomes of patients with severe sepsis not only are influenced by hyperinflammation in fulminant cases of progressing organ dysfunction but also may be affected by immunosuppression and lack of restoration of immune function. In this view, overwhelming anti-inflammatory responses, with a possible genetic background of interindividual differences in the release of anti-inflammatory mediators, contribute to the human systemic inflammatory reaction to a similar extent, compared with proinflammatory responses.
A genomic polymorphism of the anti-inflammatory cytokine IL-1ra is located within intron 2 and consists of variable numbers of an 86-bp tandem repeat. This 86-bp motif contains at least three known binding sites for DNA-binding proteins (34). Ex vivo experiments suggested greater IL-1ra responses associated with alleles containing small numbers of the 86-bp repeat. Ex vivo studies also demonstrated higher levels of IL-1ra protein expression and protein release for A2 homozygous individuals, compared with heterozygotes, after stimulation with LPS (35).
The A2 allele has been associated with the incidence of autoimmune
diseases such as lupus erythematosus and insulin-dependent diabetes mellitus
(36,37).
In acute systemic inflammation, there is no difference between surviving and
nonsurviving patients with severe sepsis. This finding is in contrast to the
results concerning the biallelic NcoI polymorphism within intron 1 of
LT. Homozygotes for the TNFB2 genotype exhibited a
high mortality rate, compared with heterozygotes and TNFB1
homozygotes. The overall group of patients with severe sepsis did not exhibit
an increase in the TNFB2 allele frequency. For the IL-1ra
polymorphism, however, an increase in the A2 allele frequency in the
patients with severe sepsis was detected. Patients with the homozygous
TNFB2 and homozygous A2 haplotype did not survive in that
study.
Toll-Like Receptors
Transduction of the LPS signal into cells has been an unknown mechanism until recently. An analog of the so-called toll receptor in Drosophila species, which transduces signals for the elaboration of innate immune responses directed against bacteria and fungi, has been identified in mice and other species (38). A 1-bp change, resulting in an amino acid change in murine toll-like receptor 4 (TLR4), renders the extensively studied mouse strain CH3/HeJ highly resistant to LPS challenge (39). Nine TLR (TLR1 to -9) (40) have been identified in mammals to date. TLR1, -3, and -5 to -9 have been found to be orphan receptors, whereas TLR2 has been demonstrated to transduce peptidoglycan stimulation by Gram-positive organisms (41,42,43). In contrast, TLR4 seems to play a key role in the LPS-induced signaling pathway (44,45,46,47).
Studies of sepsis have used animal models as surrogates for septic human patients. These models are conveniently divided into toxic and microbial types. Each model type provides evidence for a genetic component. The canonical toxic (or "endotoxic") model involves administration of bacterial LPS endotoxin to animals. The fidelity of the model with respect to authentic sepsis is disputed. The acute inflammatory response initiated by endotoxemia is one of several responses to clinical infection. From the historical perspective, the first evidence for genetic predisposition to sepsis emerged from an experiment of nature, namely the discovery of endotoxin-resistant mice. The genetic locus for this endotoxin resistance (denoted lps) has been mapped to mouse chromosome 4 (reference), and a functionally significant gene and product have been identified (TLR4).
The presence of a functional TLR4 gene and gene product appears to be one of several determinants of outcomes in endotoxemia. A survey of inbred laboratory mice demonstrated a broad range of sensitivity to endotoxin, and genetic analysis of inbred recombinant mice (genetically defined crosses of the most and least sensitive of the laboratory strains) demonstrated five loci that appear to regulate endotoxin sensitivity. High-resolution mapping is in progress. The data suggest that the response to endotoxemia is a definable multigenic characteristic of laboratory mice. In human subjects, the work of Beutler and colleagues has revealed several genomic variations within the coding region of human TLR4. Whether the function of human TLR4 is affected by these polymorphisms remains to be evaluated. If it is, then the gene for human TLR4 is an important candidate gene for determination of the genomic contribution to innate immunity variability.
Concept of Gene Therapy
Gene therapy has made considerable progress in the past 5 yr. Vector technology has improved, and therapeutic approaches for severe systemic inflammatory disease have thus become available (48). The diagnostic methods required for gene therapy, however, are similar to the diagnostic tools used for other sepsis therapies. The following questions must be considered. What approach should be chosen, i.e., anti-inflammatory therapy or restoration of immune function? In what phase of the disease does the patient needs which therapy? Is the individual patient suitable for this therapeutic approach, i.e., does his or her genomic determination favor high proinflammatory activity (i.e., high TNF responder) or anti-inflammatory states associated with immunosuppression (i.e., high levels of IL-10 release). Gene therapy promises immuno-modulation of a broad range of mediators (49) or may be strictly mediator focused (50). The risks of uncontrolled induction of target genes must be explored (51). No treatment of critically ill patients using gene therapy has been documented to date. The combination of genomic testing for subpopulations at high risk for severe sepsis and gene therapy for certain risk groups may prove to be effective in the future.
Conclusions
Elucidation of the genetic determination of inflammatory processes would provide the possibility of developing valuable diagnostic tools and new therapeutic approaches for severe sepsis. Evaluation of possible genomic markers for risk stratification of patients with sepsis and individuals at high risk of developing organ failure has just begun. Many candidate genes remain to be studied, and the clinical significance of genomic markers must be tested. In addition, this new approach may provide a valuable inclusion criterion for studies testing immunomodulatory agents in the treatment of severe sepsis. Extension of single genomic markers to combined haplotypes, including relevant alleles, may reveal greater informativeness and diagnostic relevance. The combination of these promising new diagnostic tools with the "finetuning" immunomodulatory capabilities of gene therapy may lead to highly focused sepsis therapy in the future.
References
This article has been cited by other articles:
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  C. Hermann Review: Variability of host pathogen interaction Innate Immunity, August 1, 2007; 13(4): 199 - 218. [Abstract] [PDF]
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