Scientific Review

Surgical sepsis: dysregulation of immune function and therapeutic implications

P. Boontham P. Chandran B. Rowlands O. Eremin
Department of Surgery, Queens Medical Centre, University of Nottingham, Nottingham, NG7 2UH

Correspondence to: P Boontham, Department of Surgery, Queens Medical Centre, University of Nottingham, Nottingham, NG7 2UH

Introduction Definition of sepsis Host immune response Inducers of SIRS Dendritic cells Cytokine cascade

Proinflammatory cytokines Antiinflammatory cytokines Soluble receptors Multiple organ dysfunction syndrome Treatment Conclusions References

Keywords: Sepsis, SIRS, cytokines, dendritic cells, MODS, treatment, quorum sensing molecules
Surg J R Coll Surg Edinb Irel., 1 August 2003, 187-206

Sepsis is defined clinically as the systemic inflammatory response of the host to the documented systemic infection. The pathophysiological disturbance involves both the innate and adaptive immune systems encompassing cellular immunity, humoral components and the complement system. Dendritic cells (antigen-presenting cells) are key cells involved in the regulation of the immune response in sepsis, in particular in activating T cells and especially inducing the production and secretion of specific cytokines. These are the main mediators in establishing prominent disturbances of inflammation in patients with sepsis. The clinical features of the sepsis syndrome may vary from minor clinical disturbances to severe multiple organ failure and death of the host. Appropriate therapeutic strategies for patients with sepsis utilise conventional therapy and new novel forms of treatment, which are showing promise for the future

INTRODUCTION
The incidence of sepsis in hospitals (nosocomial infections) continues to increase despite improvements in antimicrobial therapy and supportive care; it remains a major cause of death in hospital. Sepsis and its sequelae represent progressive stages of severe infection that varies from mild physiological derangements to severe multiple organ dysfunction and death.

Incidence of sepsis
The Centre for Disease Control (CDC) in the USA has estimated that the incidence of sepsis is increasing, from 73.6 per 100,000 patients in 1979 to 175.9 per 100,000 in 1989. Recently published data (1997) have documented that over 250,000 patients in the USA developed a bacteraemia each year.¹ Similarly, the incidence of sepsis in the UK has increased from 17.7 per 1000 admissions to hospitals in 1985 to 80.3 in 1996.² A number of factors were thought to be responsible for this increased incidence; more use of invasive devices (e.g. central venous catheters) in clinical practice and enhanced reliability in diagnosing sepsis, the increased prevalence of patients with HIV/AIDS, prolonged survival of HIV/AIDS patients with a subsequent enhanced duration of risk.³

There are two different types of bacterial sepsis; nosocomial sepsis and communityacquired sepsis, which have different aetiologies, microbiological profiles and patient outcome. These differences are shown in Table 1.

Outcome of patients with sepsis
The CDC also estimated the mortality rate from sepsis, which showed a decrease from 31.0% in 1979 to 25.3% in 1989. The improvement in survival was attributed to the changes in the population of patients who developed nosocomial infections, the improvements in supportive care and new developments in pharmacological therapy.³ Recent and more up-to-date data (2000) have documented continuing high mortality rates in patients with sepsis. The overall mortality rate in the USA is 35% and in the UK is 40%, however, mortality rates for nosocomial sepsis was higher than community-acquired sepsis (31% and 14%, respectively).^2,4-6 Rangel-Frausto et al. (1995) demonstrated a stepwise progression in mortality in patients based on the severity of the condition; a 7% mortality from systemic inflammatory response syndrome (SIRS), 16% from sepsis, 20% from severe sepsis and 46% mortality from septic shock.⁷ However, in different patient populations, mortality rates may be difficult to determine using these criteria alone.

DEFINITION OF SEPSIS
In the past there has been much confusion regarding the definition of and terminology for sepsis. Many definitions of sepsis were published but there was no consensus. In 1991, an American College of Chest Physicians (ACCP) / Society of Critical Care Medicine (SCCM) Consensus Conference established a series of definitions for sepsis that could be easily applied to patients in different stages of sepsis.⁸ These definitions have been widely adopted, and are shown in Table 2.

The main concept of the ACCP/SCCM definitions is SIRS, which is recognised by a group of cardinal signs that include fever or hypothermia, tachycardia, tachypnoea and leucocytosis or leucopenia with a shift to the left of the differential white blood cell (WBC) count. The SIRS can be due to infectious and non-infectious causes. Non-infectious conditions that are associated with SIRS include trauma, burns, haemorrhagic or hypovolaemic shock and acute pancreatitis. The term ‘sepsis’ is defined as SIRS that results from overt infection, which can be bacterial, viral, fungal or protozoal. Gram-negative bacteria are the main cause of sepsis and are implicated in 50-60% of cases, whilst Gram-positive bacteria account for a further 35-40% of cases.⁹

The Consensus Conference also recognised a progression in the disease state from SIRS with sepsis to severe SIRS with sepsis, which was associated with single or multiple organ dysfunction, hypotension and tissue hypoperfusion. Septic shock occurs when there is systemic hypotension that does not respond to fluid resuscitation, which results in defective tissue perfusion and anaerobic metabolism. Multiple organ dysfunction syndrome (MODS) is associated with severely compromised organ function that is not capable of maintaining normal homeostatic mechanisms in the septic patient.

HOST IMMUNE RESPONSE: INNATE IMMUNITY
Normally, the host immune system provides many types of defence mechanisms and responses to invading organisms. Mechanical barriers are provided by the epithelial surfaces of the skin and the lining of the gut and respiratory tract. The difficulty of penetrating the epithelial barrier ensures that most pathogens do not gain entry into host tissues to establish a source of pathogens. In addition to providing a physical barrier to infection, the epithelial layer also produces a range of chemicals that are useful in preventing infection, e.g. secretion of hydrochloric acid and enzymes in the stomach, antibacterial peptides in the gut and respiratory tract.

Once organisms breach the protective barriers of the body (including the skin, mucous secretions and ciliary action of the respiratory tract, acid secretion in the stomach and the specialised epithelial barrier and gastro-intestinal lymphatic tissues in the gut) they come in contact with host defences in situ, in the blood and lymphatic circulations. There are two separate but closely integrated immune defence mechanisms. The innate (non-specific) and adaptive (specific) immunity, which come into play in an integrated manner with the intention of localising the further spread and in situ destruction of the invading pathogen(s).

The cells involved in innate immunity include ‘professional’ phagocytes (polymorphonuclear neutrophils), those which have antigen (Ag) presenting capacity (monocytes/macrophages and dendritic cells [DCs]) and ‘non-professional’ phagocytes (endothelial cells and Kupffer cells). The phagocytic cells ingest invading microorganisms, destroy and digest them intracellularly. Once the organism has been internalised by the phagocyte, it is exposed to an array of killing mechanisms. For example: reactive oxygen intermediates, which involve an enzyme in the phagocyte membrane that produces oxygen free radicals (O2-) and reactive nitrogen intermediates, which induce the formation of nitric oxide (NO). Both O2- and NO metabolites are toxic for intracellular bacteria. For optimal triggering and activation of this mechanism, the phagocytes require contact with cytokines such as interferon gamma (IFN-.) and tumour necrosis factor alpha (TNF-a). There are some other mechanisms for killing intracellular bacteria such as acidification of lysosomal enzymes after the formation of phagolysosomes.^10,11

Macrophages occur in the subepithelial tissues of the skin and the intestinal epithelium and epithelial cells lining alveoli of the lungs. Organisms, which penetrate an epithelial surface, will encounter these local tissue macrophages (histiocytes). If invasion by microorganisms occurs via the blood stream or lymphatic circulation, then anti-microbial defence is provided by fixed macrophages lining the blood sinusoids of the liver (Kupffer cells), the spleen and the subcapsular sinuses of lymph nodes. The interaction of macrophages with certain bacterial components leads to production of a range of macrophage-derived cytokines, which non-specifically amplify immunological and inflammatory reactions. Macrophages are able to engulf opsonized organisms as well as directly bind to certain pathogens by virtue of expression of microbial receptors. For example, CD14 acts as a receptor for bacterial lipopolysaccharide (LPS); the integrin molecules CD11b/ CD18, CD11c/CD18 recognise several microbes including Leishmania, Bordetella, Candida as well as LPS.¹²

The polymorphonuclear leucocytes form a large circulating pool of phagocytic cells with reserves in the bone marrow. Invading micro organisms trigger an inflammatory response with the release of cytokines and chemotactic factors. As a result, circulating polymorphonuclear leucocytes adhere to vascular endothelium, squeeze out of blood vessels and migrate towards the focus of infection, where phagocytosis occurs.¹³

Even in normal conditions, polymorphonuclear leucocytosis are short-lived; in infection, the increased output from the bone marrow results in a polymorphonuclear leucocytosis in the blood. If a particularly rapid response is needed, immature cells may also be released - described as ‘a shift to the left’ on a blood film. Natural killer (NK) cells are another type of innate immune cells that are very effective in controlling infections. Natural killer cells kill target cells by releasing perforin, which damages the target cell membrane and results in the death of the cell; however, the exact mechanism in infection remains unclear. The activity of NK cells is enhanced by many cytokines such as interleukin-12 (IL-12) and IFN- These cytokines activate NK cell function and provide an important mechanism for focusing and activating cells at sites of infection.¹⁴ In conclusion, a prompt response to infection by the innate immune response is achieved by concentrating phagocytes at likely sites of infection and having a population of cells that can be rapidly mobilised during an inflammatory response.

Soluble factors such as lysozyme and phagocytosis with intracellular digestion, generates the signals to promote the adaptive immune response. Dendritic cells are believed to play a key role in this transition. Adaptive immunity is the major and specific defense mechanism, in which humoral and cell-mediated immune (CMI) mechanisms come into play to inactivate and remove the pathogens. The relative importance of humoral versus CMI mechanisms varies from infection to infection. Certain components of the immune response are essential for controlling specific infections.

The complement system is a multi-component triggered enzyme cascade that involves both the innate and adaptive immunity. The invading microorganisms and their metabolic products activate the complement system via both the classical and alternative pathway. This process is aided by the recognition of complement receptors on phagocytes. Following activation, complement can remove or destroy the invading organisms by a number of different processes, such as direct intercalation of complement proteins into the membrane of the organism, release of split products that act as chemotactic factor for neutrophils, activation and/or opsonization via phagocytic cells bearing receptors specific for the split products of complement, thereby resulting in the lysis of the organisms. The complement activation products (e.g. C3a and C5a) also promote a local inflammatory response by inducing local mast cell degranulation and, thus, vasodilatation and the extravasation of lymphocytes and neutrophils from the blood into tissues spaces. They are also involved in the modulation of adaptive immune responses that lead to antibody production.¹⁵

INDUCERS OF SIRS/SEPSIS
In infection, microorganisms show pathogen-associated molecular patterns (PAMPs), which can be recognised, by so-called ‘pattern-recognition receptors’ (PRRs) of different specificities by the host. Pathogen-associated molecular patterns are classified by the presence of defined bacterial and viral components on microorganisms: for example, the LPS of Gram-negative bacteria, the glycolipids of mycobacteria, the peptidoglycans (PGNs) and lipoteichoic acids (LTAs) of gram-positive bacteria, the mannans of yeast or fungi, the double-stranded RNAs of viruses and the unmethylated CpG motifs of bacterial DNA. Protein-carbohydrate interactions have significant roles in pathogen recognition, for example lectins (sugar-binding glycoproteins). Several membrane bound lectins are also involved in the recognition and uptake of pathogens.¹⁶ In mammals, the PAMPs stimulate the immune cells of the host to release various mediators and cytokines including IL-1 and TNF-a. These inflammatory cytokines are important in the response to infection, but inappropriate or excessive production of these cytokines leads to septic shock, a leading cause of death in patients with bacterial infections.¹⁷ 

*Th1: T helper (type 1) cells; Th2: T helper (type 2) cells

Lipopolysaccharide or endotoxin, the principal component of the outer membrane of Gram-negative bacteria is recognised by the innate immune system. The LPS molecule is a complex glycolipid that contains a highly conserved disaccharide core known as lipid A. Lipid A is a hexacylated diglucosamine-1, 4-bisphosphate.  Lipoproteins are commonly found in both Gram-positive and Gram-negative bacteria, and have profound immunoregulatory functions, including activation of monocytes and/or macrophages. The portion of lipoprotein responsible for its immunologic activity is located in the amino-terminal triacylated lipopeptide region.¹⁹ A recent study has demonstrated that synthetic lipopeptides can activate B cells and macrophages, and removal of this lipid element from bacteria renders them non-activating.²⁰ There is now clear evidence that LPS and other microbial ligands, including LTAs and PGNs, are able to activate cells via the mammalian Toll-like receptors (TLRs).¹⁶

Mammalian TLRs are thought to be part of the innate response against microbial pathogens. The TLR family members are transmembrane proteins containing repeated leucine-rich motifs in their extracellular domains, similar to other pattern-recognition proteins of the innate immune system. They also contain a cytoplasmic portion that is homologous to the IL-1R and, for this reason, can trigger intracellular signalling pathways.^20,21 Like IL-1R, TLR triggers the activation of various transcription factors, including nuclear factor kappa B (NF-.B), which is involved in the expression of many proinflammatory cytokines. The sequence of events that occurs after activation of TLRs is thought to be mediated by three protein complexes: a complex on the receptor, a complex that phosphorylates the inhibitor protein I.B and a complex that degrades I.B. Once I.B is degraded, the nuclear localisation signal of NF-.B is revealed and NF-.B moves to the nucleus, where it can activate target genes17 (Figure 1). Key proinflammatory and immunomodulatory cytokines such as IL-1, IL-6, IL-8, IL-10, IL-12 and TNF-a, are induced after activation of TLRs by microbial ligands.²⁰

The TLR family has been recently recognised as a central component of the innate immune system. Two important responses of the innate immune system to pathogen invasion include phagocytosis and gene modulation. The first TLR family member to be identified, TLR4, was later shown to induce the expression of proinflammatory cytokines and costimulatory molecules, which in turn are required for naive T cell activation. Subsequently, several TLRs have been identified, mainly by sequence homology. Currently, at least nine human members have been partially characterised, namely TLR1 to TLR9. 

These TLRs are expressed in the cell types involved in first-line host defences such as macrophages, neutrophils, epithelial cells lining the gut and respiratory tract, dermal endothelial cells, B and T cells and DCs.²² Because these cells migrate into or are resident at sites that interface with the environment, TLR-expressing cells are well-placed to defend against invading microbes.²³ Dendritic cells express many kinds of TLRs, which probably reflects their important role in detecting microorganisms and activating the adaptive immune system.²⁴

The expression of TLRs on cells of the monocyte/macrophage lineage is consistent with the role of TLRs in modulating inflammatory responses via cytokine release and expression of co-stimulatory molecules. Expression of TLR4 and TLR2 can be differentially regulated at sites of infection or inflammation, either directly by bacterial components, or indirectly by primary cytokines. Expression of TLR4 is important for detecting LPS and, thus, the host’s response to Gram-negative bacteria. After exposure to LPS or to IFN-y, TLR4 was shown to increase the production of the proinflammatory cytokines IL-1 and IL-6 and the costimulatory molecules, CD80 and CD86, on DCs.²⁴  In contrast, TLR2 is unaffected by LPS but transduced by signals evoked by LTAs, PGNs, bacterial lipopeptides and whole Gram-positive bacteria. ^25-27 Brightbill et al (1999) have shown that microbial lipoproteins and lipopeptides activate cells in a TLR2-dependent manner, requiring the lipid portion of the ligand for optimal activity. This has been further confirmed using the TLR2 gene knockout mouse, which did not respond to microbial lipoproteins.

DENDRITIC CELLS AND INFECTION
Dendritic cells, first described by Steinman and his colleagues in 1972, are potent antigen-presenting cells (APCs) that play a crucial role in providing the essential components for initiating and developing the primary T cell response of the host to invading organisms.²⁸ Dendritic cells are derived from the CD34+ bone marrow progenitors that separate into lymphoid and myeloid lineages to develop lymphoid and myeloid DCs, respectively.²⁹ Dendritic cells are located in most tissues of the body, where they readily encounter invading microorganisms, take up and process microbial and viral Ags. They subsequently undergo maturation and migrate via afferent lymphatics to paracortical areas of secondary lymphoid organs where they interact with naive T cells.³⁰  While migrating to the lymph nodes, they change from an immature stage, which has phagocytic and endocytic activities, to a mature and activated stage for efficient T cell interaction and stimulation. This activation and maturation is accompanied by morphological, phenotypic and functional changes. Only mature DCs have high levels of surface expression of the major histocompatability complex (MHC) HLA-DR, costimulatory molecules (CD80 and CD86) and the adhesion molecule CD40, all of which are essential for effective Ag presentation on the T cell receptor (TCR) and subsequent CD8+ and CD4+ T cell stimulation.²⁸

The recognition of PAMPs by DCs is a critical step in the generation of an acquired immune response. Recently, it has been shown that human DCs express TLR1-5, whilst TLR4 and TLR5 are present on monocytes and polymorphonuclear cells. The TLR3 appears to be only expressed by DCs, but absent in all the other leukocytes analysed.²² However, a recent article documented that naive monocytes express high levels of TLR1, TLR2, TLR4 and TLR5, all of which decreased as monocytes differentiated into immature DCs. The only TLR, which increased during immature DC formation was TLR3, which was undetectable in monocytes but clearly expressed in immature DCs. As DCs are activated and mature, they lose expression of TLRs, except for TLR1 and TLR6, which correlates with a loss of responsiveness to LPS, as assessed by TNF-a production.³¹ It has been estimated that immature DCs express at most 150 TLR4 molecules per cell.³¹

The maturation of immature DCs requires the activation of NF-.B and results in the up-regulation of HLA-DR, CD40, CD80, CD83, and CD86, and the production of cytokines such as IL-12 and TNF-a. Two TLRs appear to play a key role in inducing maturation of immature DCs; LPS-induced DC maturation is dependent on TLR4 whilst PGN-induced maturation of DCs is dependent on TLR2. (Figure 1) In animal models, neither TLR4-deficient animals nor mutation of genes that control TLR4 expression in DCs demonstrated any responses to LPS.^32,33 Others have demonstrated that PGN could not induce DC maturation in the absence of TLR2.27 Besides induction by bacterial stimuli, DC maturation can also be induced by proinflammatory cytokines such as TNF-a and INF-a, and CD40 cross-linking by CD40 ligand on activated T cells.

In peripheral blood, two DC subsets differing in their expression of CD 11b and CD 11c have been identified and termed the lymphoid and myeloid types of DCs.²⁸ In mice, DCs  expressing CD8a (CD8a+) with low or dull CD11b and CD11c expression are of the lymphoid lineage and DCs with CD8a-CD11c+ and CD11b+ phenotype are of the myeloid lineage. The lymphoid DCs (LDCs) are able to induce a T helper 1 (Th1) response, whereas the myeloid DCs (MDCs) induce a T helper 2 (Th2) type of response. In contrast to mouse DCs, the human MDCs (CD11c+) and LDCs (CD11c-) induce Th1 and Th2 responses, respectively.^34,35 Dendritic cells that induce a Th1 and Th2 response are termed DC1 and DC2, respectively.³⁶

Typically, DC1 induces Th1 cells to secrete IL-2, IFN-. and IL-12, whereas DC2 induces Th2 cells to secrete IL-4, IL-5 and IL-10, whilst IL-3 and TNF-a are produced by both Th1 and Th2 cells.^37,38  Dendritic cells not only induce T cells to secrete cytokines but also are important sources of cytokines. Human DCs produce TNF-a, IL-1a/ß, IL-6, IL-8, IL-10, IL-12, IL-15, transforming growth factor-ß1 (TGF-ß1) and IFN-a/ß.^28,34,38-40

Dendritic cells are essential for an effective anti-pathogen immune response to infection. Normally, DCs reside in an immature state in peripheral tissues, acting as sensors for the presence of pathogens. As soon as the immature DCs recognise the PAMPs on the microorganisms though their TLRs, an integrated and sequential immune response is induced and developed. This leads to activation and maturation of DCs, processing of the microorganisms by both the innate and subsequently adaptive immune responses in a seamless manner. There are two different types (intracellular, extracellular) of infection by microorganism. The intracellular infections (e.g. viruses, Mycobacteria, Salmonella spp and Listeria spp) mainly induce CMI responses in which DCs are the key APCs, while extracellular infections (e.g. most common bacteria) induce a predominantly humoral immune response. However, DCs are the crucial APCs for both types of infections. On occasions, the host immune system cannot eliminate the invading organisms and the DCs may act as a reservoir for the invading organism; for example, HIV and Salmonella infection. In these latter instances, the host cannot kill the organisms and they will replicate in the host DCs, which act as the source of organisms that infect other cells of the host.^41-43

Dendritic cells are normally active and effective against infection by pathogens but in some clinical conditions, such as in critically ill and elderly patients, their function may be limited, suppressed and defective. In patients with sepsis, there is evidence of reduced numbers of DCs.⁴⁴  Uyemura et al (2002) recently documented reduced expression of costimulatory molecules and IL-12 production^.45 The causes for this dysfunction and decreased number of DCs are poorly defined and inadequately studied.

CYTOKINE CASCADE
The systemic inflammatory response is propagated and the severity modulated by a number of humoral mediators such as lipid metabolites, reactive nitrogen and oxygen metabolites, nucleotides and cytokines. These mediators interact with each other as a molecular network to regulate the inflammatory response.

Cytokines are a group of soluble, low molecular weight glycoproteins, which act to regulate both the amplitude and duration of the systemic immuno-inflammatory response. Cytokines are highly active at very low concentrations and may act on cells in a paracrine and autocrine manner. The SIRS and the clinical variants of sepsis appear to result from inappropriate, excessive and/or prolonged release of mediators into the systemic circulation, in particular certain key regulatory cytokines.

Normally cytokines are not stored in intracellular compartments; they are synthesised de novo and released in response to tissue and cell damage. This regulation of cytokine production and release is predominantly at the level of gene transcription. One crucial transcription factor is NF-.B, which is composed of a p50 and a p65 subunit, and appears to play an important role in regulating the cytokine response (Figure 1). It is retained in an inactive cytoplasmic complex by binding to the inhibitory subunit I.B and regulates the transcription of various proinflammatory and immunoregulatory cytokines, genes encoding for immunoreceptors, cell adhesion molecules, haematopoietic growth factors, acute phase proteins, and enzymes such as cyclooxygenase-2 and inducible NO synthase.⁴⁶ NF-kB is activated in many cell types by a range of stimuli such as PAMPs, TNF-a and IL-1ß. NF-.B activation is important for the transcription of many cytokines such as TNF-a, IL-1ß, IL-6 and IL-8.⁴⁷

Figure 1: A schematic diagram of the network of intracellular signalling pathways showing the interaction of external inflammatory stimuli inducing cytokine synthesis. Bacterial products (LPS and PG), bind to specific cell surface receptors (TLRs) and induce a complex downstream intracellular signalling pathway. The first induced complex is an adaptor molecule such as MyD88, and a serinethreonine innate immunity kinase such as IRAK. The activated TLR complex in turn activates the NIK complex in a step mediated by TRAF6. The NIK complex, then, phosphorylates target serines in the amino-terminal domain of I.B, which is known as nuclear factor-.B (NF-.B) inhibitory sub-unit. Once I.B is degraded, the nuclear localisation signal of NF-kB is revealed and NF-kB moves to the nucleus, where it can activate target genes, resulting in enhanced production of pro- and antiinflammatory cytokines (LPS: lipopolysaccharide; PG: peptidoglycan; TLR: Toll-like receptor; MyD88: myeloid differentiation factor; IRAK: interleukin-1 receptor accessory protein kinase; TRAF6: tumour necrosis factor-receptor-associated factor; NIK: NF-kB-inducing kinase; I.B: inhibitory subunit kB; NF-kB: nuclear factor-kB)

Figure 2: Plasma levels of inflammatory cytokines in patients with sepsis

In humans and animal experimental models of sepsis, cytokines are released in a sequential manner resulting in a “cytokine cascade”. The cytokine cascade is initiated when the pathogenic organisms are exposed to the host immune system that induces production and secretion of early or “proximal” cytokines such as TNF-a and IL- 1ß. Both TNF-a and IL-1ß are believed to be the main cytokines mediating most of the pathophysiological disturbances of sepsis. They act synergistically to stimulate the release of “distal” cytokines, such as IL-6 and IL-8 and antiinflammatory cytokines, such as IL-4, IL-10 and IL-13.⁴⁷

These various cytokines (TNF-a, IL-1ß, IL-6, IL-8, IL-4, IL-10 and IL-13) have synergistic and antagonistic effects in the immuno-inflammatory response of sepsis.⁴⁸

PROINFLAMMATORY CYTOKINES

Tumour necrosis factor-a (TNF-a)
Tumour necrosis factor-a is considered to be a central mediator of immune activation and inducer of the pathophysiological disturbances associated with bacteraemia and sepsis. Tumour necrosis factor-a is a 157 amino acid polypeptide, 17-kDa in weight, produced primarily by monocytes, macrophages, lymphocytes, neutrophil granulocytes, mast cells, fibroblasts and endothelial cells in response to bacterial toxins, inflammatory products and other invasive stimuli.

Tumour necrosis factor-a activates neutrophils, up-regulates the endothelial adhesion molecules such as E-selectin and ICAM-1, increases capillary permeability and has a direct cellular toxic effect. Moreover, its metabolic effects include stimulating the release of triglycerides from adipose tissue, promoting the release of amino acids from proteins, and causing the catabolism of skeletal muscle. It also induces anaerobic glycolysis and, accordingly, increases production of lactate. Infusion of TNF-a into animal models or humans results in SIRS including fever, haemodynamic abnormalities, leucopaenia, elevated liver enzymes, coagulopathy and septic shock.^37,47,49 Tumour necrosis factor-a production is rapidly activated with systemic release (detected within one hour) following entry of pathogens into the host or significant tissue damage (Figure 2).⁵⁰

Tumour necrosis factor-a plays a major role in coordinating the inflammatory response and activating the cytokine cascade. Studies in vitro and in vivo demonstrate that TNF-a is a potent inducer of other cytokines, including IL-1ß, IL-6, IL-8 and IL-10.⁴⁷ Tumour necrosis factor-a can be detected in the plasma of many septic patients, and its concentration generally correlates with the severity of the illness and patient outcome. Animal studies have demonstrated that the concentration of TNF-a in plasma was significantly increased in severe sepsis and in animals who subsequently died.^51,52 Infusion of neutralising antibodies to TNF-a can improve the survival in animal models of septic shock.^53-55 Similarly, in humans, TNF-a levels in plasma are raised in patients with SIRS. These levels increased substantially in severe sepsis and in patients who died.^49,56-58 Although the serum concentration of TNF-a is usually associated with clinical outcome in patients with sepsis, persistently elevated TNF-a levels may be a better predictor of outcome than a single measurement.

Interleukin-1ß (IL-1ß)
Another acute phase cytokine is IL-1ß. It is one of the two IL-1 subsets, which consist of IL-1a and IL-1ß. Both IL-1a and IL-1ß activate the same IL-1 receptors and, therefore, share various biological activities; however, IL-1ß is the predominant form of these cytokines produced in patients with sepsis.⁵⁹ (Figure 2) Like TNF-a, IL-1ß is a potent proinflammatory cytokine, which can activate both neutrophils and endothelial cells by up-regulation of all classes of adhesion molecules. In addition, it has several other physiological effects including induction of fever through activity of the hypothalamus, increasing capillary permeability, hypotension and appetite suppression. In animal models and human volunteers, infusion of IL-1ß causes fever, anorexia, malaise, arthralgia, headache and haemodynamic abnormalities (shock-like state with hypotension).⁶⁰ Furthermore, IL-1ß activates the production of other cytokines, including IL-6, IL-8 and TNF-a. Like TNF-a, IL-1ß appears to be a predictor of the severity of sepsis but levels of IL-1ß cannot predict mortality in patients with sepsis.⁶¹

Interleukin-6 (IL-6)
Interleukin-6 is a 21-kDa glycoprotein produced by many cell types, including lymphocytes, fibroblasts and monocytes. It has a variety of biological effects including activation of B and T lymphocytes, induction of acute phase protein production in the liver and modulation of haematopoiesis.⁶² The exact role of  IL-6 in sepsis is still unclear. However, infusion of IL-6 into human volunteers and experimental animals does not induce a sepsis-like state, as seen with infusion of TNF-a and IL-1ß.⁶⁰ The appearance of IL-6 in the plasma of patients with sepsis is delayed (four to eight hours) and may be related directly to TNFa and IL-1ß production.

Many studies in humans have demonstrated that plasma IL-6 concentrations in sepsis correlate closely with severity and outcome of the sepsis.

 These two cytokines are synthesised by monocytes/macrophages, polymorphonuclear leucocytes and hepatocytes in response to infection. Normally, IL-1ß is not present in human plasma, but it has been detected in the plasma of patients in the early phases (two to three hours) of sepsis. septic patients, the plasma concentration of IL-8 peaks at three to four hours. Infusion of IL-8 into human volunteers ^58,63 In severe sepsis and septic shock, a poor outcome is correlated with circulating IL-6 concentrations of >1000 pg/mL.⁶⁴

Interleukin-8 (IL-8)
Interleukin-8 is a small basic protein produced by mononuclear phagocytes, polymorphonuclear leucocytes, lymphocytes, endothelial cells, epithelial cells and a variety of mesothelial cell types in response to various stimuli, including endotoxin, IL-1ß and TNF-a. The primary function of IL-8 is to activate and chemo-attract neutrophils to the site of tissue damage and inflammation. Several functional responses of the neutrophil occur following exposure to IL-8 including change in shape, adherence to endothelial cells via neutrophil integrin activation, directed movement, enzyme secretion and an increased production of reactive oxygen metabolites.³⁷ 

In septic patients, the plasma concentration of IL-8 peaks at three four hours. Infusion of  IL-8 into human volunteers does not cause septic shock, but can induce recruitment and activation of neutrophils into specific sites, which may lead to tissue injury, such as adult respiratory distress syndrome (ARDS).⁶⁵ Patients with sepsis syndrome have detectable concentrations of circulating IL-8, and the concentrations in those who are in shock or those who die are significantly higher than in survivors or those with a mild form of the syndrome.⁶⁶

ANTIINFLAMMATORY CYTOKINES

Interleukin-10 (IL-10)
Interleukin-10 is a non-covalently linked homodimeric cytokine (160 amino-acid protein) that is produced by a large variety of cells including monocytes, macrophages, B and T lymphocytes, DCs and NK cells. Interleukin-10 has many antiinflammatory and immunosuppressive activities. In vitro, IL-10 down-regulates monocyte/macrophage and DC effect or functions including Ag-presenting capacity (inhibits expression of MHC class II HLA-DR and costimulatory molecules) and the production of cytokines such as TNF-a, IL-1ß, IL-6, IL-8 and IL-12. The cytokine also strongly inhibits CD4+ T cell proliferation and cytokine production.^67,68

In contrast, IL-10 has stimulatory effects on CD8+ T cells and induces their recruitment, cytotoxic activity and proliferation.⁶⁷ The main function of IL-10, therefore, is that of a potent inhibitor of the production of proinflammatory cytokines. In addition, the inhibitory effects of IL-10 on TNF-a and IL-1 production are crucial to its antiinflammatory activities because these two cytokines often have synergistic activities on the inflammatory response. In animal studies, IL-10 prevents endotoxin-induced morbidity and mortality and diminishes TNF-a release during endotoxaemia.^69,70

In sepsis, high plasma concentrations of IL-10 have been detected both in animal and human studies.^{51,57,58,71,72} However, maximal levels of IL-10 in patients with sepsis were detected after 24 hours.^68,71 (Figure 2) Significantly higher levels of plasma IL-10 have been detected in patients with severe sepsis, compared with patients having the less severe form of sepsis.^57,58,73

Interleukin-4 (IL-4)
Interleukin-4, a 20-kDa glycoprotein is produced by activated Th2 cells, basophils and mast cells. Interleukin-4 has the same functions as IL-10; for example, both IL-4 and IL-10 inhibit the secretion of proinflammatory cytokines by monocytes/macrophages and neutrophils. It is a dominant mediator of Th2 cell differentiation and proliferation but few studies of IL-4 activity have been published.

Interleukin-13 (IL-13)
Interleukin-13 has some of the activities of IL-4 and IL-10. However, in a recent human study, plasma IL-13 secretion was not induced by endotoxin and remained undetectable in plasma in the majority of the septic patients studied.⁷²

SOLUBLE RECEPTORS AND RECEPTOR ANTAGONISTS

Soluble TNF receptors (sTNFR)
There are two cell surface TNF soluble receptors: p55 (sTNFR-p55) and p75 (sTNFR-p75). These soluble receptors are produced by proteolytic cleavage of the extracellular binding domain of TNFRs from the cell surface. The TNF-a bound to sTNFR exists in equilibrium with the unbound form and the balance determines the biological activities of TNF-a concentration.⁶⁰

Plasma concentrations of sTNFR are present in normal humans and are increased in the circulation of patients with endotoxaemia. In healthy human volunteers, endotoxin injection induces the release of the sTNFRs (increased five to seven-fold) into the circulation.^47,60 In human sepsis, sTNFRs are increased and correlate with the severity and mortality of the disease. There were higher plasma concentrations of sTNFRs in patients with severe sepsis, and were associated with a higher mortality as well.^57,74,75 However, the role of sTNFRs in sepsis is poorly understood.

Interleukin-1 receptor antagonist (IL-1Ra)
Interleukin-1 receptor antagonist is a 23 kDa glycoprotein and is produced mainly by macrophages. Interleukin-1 receptor antagonist blocks IL-1 activity by competing for binding to IL-1Rs, thereby, modulating cell signal transduction.⁷⁶ In patients with sepsis, the plasma concentration of IL-1Ra is higher in the more severe forms of sepsis.⁵⁷ In a human volunteer study, there were no detrimental clinical effects when endotoxin was injected at the same time as IL-1Ra.⁷⁷

CYTOKINE CASCADE AND CLINICAL OUTCOME
During sepsis, the balance between the biological effects of the proinflammatory and antiinflammatory cytokines is thought to determine the clinical outcome of the disease. Proinflammatory cytokines (TNF-a, IL-1ß, IL-6, IL-8 and IL-12) are necessary for initiating an effective inflammatory process against infection by pathogens and tissue damage, whereas excessive and inappropriate production has a deleterious effect, resulting in defective tissue microcirculation and hypoxia with essential systemic circulatory malfunction, ARDS, multiple organ-system dysfunction and death. In contrast, antiinflammatory cytokines (IL-4, Il-10 and IL-13) appear important in controlling and down-regulating the inflammatory response through depression of the host immune system.

In human studies, serum TNF-a, IL-6, IL-8 and IL-10 levels were raised in patients with SIRS, even in those cases in which an overt infection could not be documented; these levels were high in patients with severe sepsis and in those who subsequently died.⁵⁸ Another study documented that the IL-10: TNF-a ratio was associated with eventual outcome in human sepsis; IL-10: TNF-a ratio was high in patients with severe sepsis and was markedly higher in those who subsequently died.⁵⁷ Production and secretion of cytokine antagonists (e.g. IL-1Ras) and soluble receptors (e.g. sTNFRs) also mirror clinical outcome. Both IL-1Ras and sTNFRs are elevated in patients with sepsis and have antiinflammatory effects.^57,60 The interaction between the pro- and antiinflammatory cytokines secreted is an important factor determining the clinical outcome in patients with sepsis.

SEQUENTIAL STAGES IN SIRS AND SEPSIS
In patients with significant tissue damage and/or infection, the host defences undergo a series of sequential changes resulting in the immunoinflammatory response. (Modified from: Bone, RC et al Chest 1997; 112: 235-43.)⁷⁸

Firstly: The host defences release a variety of mediators into the site of tissue damage and/or infection. The body’s initial response is to induce a proinflammatory state in which mediators have multiple overlapping effects designed to limit the proliferation and invasion of pathogenic organisms. Thereafter, the antiinflammatory response is produced to limit the possible harmful effects of the proinflammatory mediators. For example, they reduce monocytic MHC class II expression, impair DC Ag uptake and presenting activity, and reduce the ability of cells (lymphocytes, DCs, monocytes/ macrophages) to produce inflammatory cytokines. The levels of both the proinflammatory and antiinflammatory mediators are much higher in situ than in the systemic circulation.

Secondly: If the infection and/or tissue damage is sufficiently severe, first proinflammatory and later antiinflammatory mediators will appear in the systemic circulation. The presence of proinflammatory mediators in the circulation is part of the normal host response and serves as a warning signal that the initial infection cannot be controlled in situ. The proinflammatory mediators help recruit neutrophils, T cells and B cells, platelets, and coagulation factors to the site of the infection and/or tissue damage. This cascade stimulates a compensatory systemic antiinflammatory response, which rapidly down-regulates the proinflammatory response. The clinical and laboratory features of SIRS may become demonstrable.

Thirdly: Loss of regulation of the proinflammatory response, which becomes excessive and dysregulated, (e.g. in severe poorly controlled sepsis) results in a significant systemic reaction manifested by the clinical and laboratory features of SIRS. The pathophysiological changes include the following:

• Progressive endothelial cell malfunction, leading to increased microvascular permeability

• Platelet sludging which blocks the microcirculation, causing maldistribution of blood flow and possibly tissue ischaemia and cellular hypoxia which, in turn, may cause reperfusion injury

• Activation of the coagulation system and impairment of the protein C/protein S inhibitory pathway

• Profound vasodilatation, fluid transudation, and maldistribution of blood flow resulting in circulatory malfunction and shock

If homeostasis is not rapidly restored, significant organ dysfunction and eventual failure may result.

Fourthly: If the compensatory antiinflammatory reactions are inappropriate and excessive, immunosuppression will result. The persistence of immunosuppression can lead to further prolongation of sepsis and organ dysfunction. The final stage of MODS is called ‘immunologic dissonance’. It is an inappropriate, dysfunctional immunoinflammatory response by the host and is associated with a significant morbidity and mortality.

MULTIPLE ORGAN DYSFUNCTION SYNDROME (MODS)

Respiratory dysfunction
Pulmonary dysfunction is common in the patient with SIRS or sepsis, and is manifested as tachypnoea, hypoxaemia and respiratory alkalosis. The primary pathophysiological disturbance is pulmonary capillary endothelial dysfunction resulting in interstitial and alveolar oedema. The exudate is protein rich and contains numerous mononuclear and polymorphonuclear cells. Endothelial permeability is increased in response to proinflammatory cytokines (e.g. TNF-a) with progression to alveolar exudation and basement membrane destruction. Neutrophils are sequestrated into the lung in response to IL-8 and subsequently produce O₂ free radicals, which in turn, lead to alveolar tissue damage. If severe enough, it may progress to acute lung injury and ARDS. The latter may complicate up to 60% of cases of septic shock.⁷⁹

Cardiovascular dysfunction
The response to the fall in blood pressure is an increase in cardiac output. Consequently, baro-receptors induce a pronounced tachycardia, stroke volume is increased due to decreased afterload, but hypovolaemia may decrease preload and cardiac output as well. Independent of the effects of preload and afterload, intrinsic myocardial muscle depression is present within 24 hours of the onset of sepsis. Both endotoxin and proinflammatory cytokines have been shown to induce myocardial dysfunction. These effects are mediated through NO.^80,81 Nitric oxide is synthesised by inducible NO synthase (iNOS) in vascular endothelium, smooth and cardiac muscle in response to proinflammatory cytokines. Nitric oxide reduces myocardial contractility and responsiveness to ß-adrenergic agents, for this reason, the hypotension may be refractory to treatment with fluids, inotropes and conventional vasoconstrictors.^82-84

Renal dysfunction
Acute renal failure complicates 50% of cases of septic shock and significantly increases mortality, occurring as part of the MODS. Several mechanisms have been proposed for the pathogenesis of acute renal failure occurring in sepsis. The cytokine-induced systemic vasodilatation and relative hypovolaemia in sepsis result in renal hypoperfusion. The kidney produces intrinsic vasoconstrictors in response to cytokines and the rennin-angiotensin-aldosterone system. The vasoconstrictors disturb the normal auto-regulation of blood flow to the kidney. There are other metabolites, such as thromboxane and leukotrienes that also reduce renal blood flow in sepsis. Like other tissues, the kidney is also susceptible to leukocyte-mediated tissue injury with neutrophil aggregation in response to cytokines and free radicals released in situ.^85,86

Haematological dysfunction
Sepsis is often associated with a disorder of coagulation secondary to the cytokine-mediated activation of the coagulation pathways. Disseminated intravascular coagulation (DIC) produces both bleeding and microvascular thrombi, which have been proposed as mechanisms inducing MODS. The cytokine-mediated activation of coagulation in sepsis occurs via the tissue factor-dependent extrinsic pathway. Tissue factor acts as cofactor and activator of factor VIIa that in turn activates factors IX and X of the extrinsic pathway.

Reduction of the anticoagulant system is another reason for the coagulation disorder observed in patients. Normally antithrombin III (ATIII) is an inhibitor of the serine proteases, which are responsible for activity of the coagulation clotting factors IXa, Xa, XIa, XIIa and thrombin. In sepsis and shock, the circulating levels of ATIII are reduced and this fall has been correlated with increased mortality. In one human study, ATIII replacement has been shown to improve survival in patients with septic shock.⁸⁷

Thrombomodulin is an endothelial cell-derived inhibitor of clotting and activator of fibrinolysis. It acts as a thrombin binding protein, reducing the effect of thrombin. The thrombinthrombomodulin complex has further anti-coagulant properties as an activator of protein C and protein S, which inactivates factors V and VIII. The production of thrombomodulin by endothelial cells is down-regulated by proinflammatory cytokines. Hence, the thrombomodulin level is reduced in sepsis, and circulating free levels of protein S are also reduced.⁸⁸

Hepatic dysfunction
The liver is believed to play a major role in the initiation of MODS, the most serious complication in the clinical course of sepsis. The hepatic dysfunction occurs in the first hours after the initial onset of sepsis. This is usually due to shock and hepatic hypoperfusion, leading to severe alterations in various aspects of hepatic function, with DIC and bleeding ensuing. However, the early hepatic dysfunction may be reversed with adequate supportive treatment. Thereafter, additional injury to endothelial cells and associated parenchymal cells occurs with initial resuscitation. A number of mediators of reperfusion injury are produced and released, and contribute to the enhanced hepatic tissue injury.^89,90 Nitric oxide is the inflammatory mediator which is thought to play an important role in hepatic injury. In a recent human study, there was a higher level of NO in patients with severe sepsis, compared with patients with less severe clinical sepsis.⁹¹

Kupffer cells, the hepatic macrophages, account for approximately one-third of nonparenchymal cells of the liver and constitute 80-90% of the fixed-tissue mononuclear cell population. Kupffer cells are located adjacent to hepatic sinusoids so they interface with both the portal and systemic circulations. Their functions include phagocytosis of pathogens and foreign substances, Ag presentation and secretion of numerous inflammatory mediators. Once activated, Kupffer cells produce cytokines, which subsequently are released into the systemic circulation and activate the immunoinflammatory cascade. Kupffer cells are a principal source of TNF-a and IL-6, which are important cytokines involved in the SIRS in patients with sepsis.⁸⁹ However, in patients with sepsis, both phagocytosis and killing activity of Kupffer cells are impaired. Therefore, impairment of hepatic clearance of endotoxins and bacteria may result.⁹² This defect of Kupffer cell function may promote and/or worsen the MODS and the hepatic dysfunction. In patients with sepsis, hyperbiluribinaemia may be a result of direct toxic effect on liver parenchymal cells causing inflammation in the portal triads which result in disturbance of the total bile canalicular system (bile secretion) and canalicular contraction. In addition, progressive cholestasis would be an important clinical sign, which reflects an irreversible change in hepatic cellular function.⁹³

Neurological dysfunction
As with other organs, the neurological dysfunction in sepsis is thought to be associated with excess release of inflammatory mediators. There are two clinical entities of neurological dysfunction, septic encephalopathy and critical illness polyneuropathy. Septic encephalopathy happens early in the course of sepsis and the diagnosis is established by the typical clinical features and electro-encephalogram findings. Critical illness polyneuropathy is a predominantly motor axonal dysfunction, which is manifest as transient neuromuscular blockade, an axonal motor neuropathy or a thick filament myopathy. Treatment of both neurological dysfunctions is supportive and full recovery can occur.^94-96

Gut barrier dysfunction
The gastro-intestinal tract provides a number of important functions; its large digestive and absorptive area, important barrier preventing the systemic absorption of intraluminal microorganisms and their toxic products and the largest reservoir of lymphocytes in the body. The barrier function is an important characteristic of the gut. Its workings are complex and it involves epithelial, molecular, and immune components. Moreover, the gut-associated lymphoid tissue (GALT) and liver are also involved in these immune defence mechanisms, called the ‘gut-liver axis’.⁹⁷ Derangements in intestinal barrier function can lead to systemic invasion by gutderived microbes and/or activation of inflammatory cells in the GALT and the liver. In an animal study of bacterial pneumonia gut barrier dysfunction was associated with gut mucosal and microvascular injury.⁹⁸

Critically ill patients are susceptible to injury of the intestinal mucosa, changes in gut permeability and failure of GALT defense mechanisms. Two distinct forms of gutbarrier dysfunction have been described. The first, called translocation, in which particulate Ags and viable microbes, are transported across enterocytes into the submucosal compartment. Bacterial translocation is defined as the passage of viable bacteria, usually Gram-negative aerobes and/or their endotoxins from the gut lumen to normally sterile tissues such as the mesenteric lymph nodes and beyond.⁹⁹ The second type is an increase in the paracellular permeability of the intestinal epithelium, which permits increased transmucosal absorption of water-soluble macromolecules. In disease states when the mucosal barrier is compromised (e.g. severe tissue damage and/or sepsis) microorganisms and their toxic products gain access to the portal and systemic circulations of the host. These substances, subsequently produce the host generalised inflammatory response. Under these circumstances, SIRS and MODS develop leading to clinical deterioration and eventual death of the patient.⁹⁷

ROLE OF QUORUM-SENSING SIGNAL MOLECULES IN SEPSIS
Billions of bacteria (400-500 different species) normally inhabit the gastrointestinal tract. These bacteria are believed to be critical for normal nutrition and well-being of the host. The bacteria produce and secrete many kinds of substances into the gut lumen, including small molecules called quorum sensing signal molecules (QSMs). Quorum-sensing signal molecules are produced by many Gram-positive and Gramnegative bacteria, some of which are present in the gut. These molecules are involved in coordinating responses of bacterial populations to environmental changes as well as generating communication signals between neighbouring bacteria by freely diffusing across bacterial cell membranes. In addition, QSMs modulate immune functions in the host, thereby, enhancing bacterial survival.^100-102 To date, many types of QSMs have been documented. Some of the earliest and best described QSMs are those belonging to the family of N-acylhomoserine lactones produced by Gram-negative bacteria. Amongst these, N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL or OHHL) and N-(3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-HSL or OdDHL) have been identified in pathogenic bacteria. These molecules bind to and activate transcriptional regulators in bacteria, which in turn activate virulence gene expression that controls the virulence in bacteria, and production of secondary metabolites, in a cell density-dependent manner.¹⁰³ Moreover, these molecules can regulate biofilm formation in bacteria, which protects them from host defences and provides enhanced resistance to antibiotics.^104-106

During infection, pathogens employ various strategies to enhance survival and multiplication. Also, various environmental signals induce the expression of virulence determinants in bacteria, such as temperature, pH, osmolarity and nutrient availability. One of the most important factors determining the infection of the host is a critical cell population density sufficient to overwhelm the host immune defences, particularly by opportunistic pathogens in the gut.¹⁰⁷ In man and animals, it has been postulated that gut-associated bacteria are important for health and nutritional homeostasis. This is thought to be mediated through secreted bacterial products in the gut, such as QSMs, which induce tolerance in the GALT (possibly through inhibition of activation and maturation of DCs), resulting in selective uptake of beneficial nutritional peptides and a barrier to uptake of toxic molecules. In patients with sepsis, there is alteration in number and activity of gut microflora and the increased production of QSMs is likely to occur with disruption of gut homeostasis and GALT function.

In vitro studies have demonstrated that OdDHL, which is secreted by Pseudomonas aeruginosa, can suppress the secretion of TNF-a and IL-12 by murine macrophages.¹¹⁰ Another animal study has demonstrated that OdDHL is a potent inducer of inflammation in the skin of mice.¹¹¹ However, there are very little data about their effects on the human immune system.

In patients with sepsis, the gut barrier function is impaired. As a result, toxic substances including QSMs and/or endotoxins from both pathogenic and non-pathogenic bacteria freely enter the host systemic circulation.⁹⁷ These circulatory substances induce and/or modulate the subsequent host inflammatory response. It has been postulated that very high levels of QSMs may be immunosuppressive and/or dysregulate key elements of CMI, in particular DC function and the concurrent cytokine cascade.¹¹²

TREATMENT OF PATIENTS WITH SEPSIS

Conventional therapy
At present, the morbidity and mortality of sepsis continues to be significant and these have not been reduced despite the improvements of clinical care in the intensive care setting. Not only is the control of infection and appropriate supportive therapy important in the treatment of sepsis but also the early recognition of sepsis, thereby, allowing earlier intervention. Conventional therapy for sepsis consists of eradication of infections with antibiotics, elimination of septic foci by surgical procedures (e.g. drainage of pus and tissue debridement) and supportive intensive care management of cardiovascular abnormalities and other organ failure.

The aim of initial resuscitation and supportive therapies is to maintain organ perfusion and oxygenation. Hypoxaemia should be treated with increased concentrations of inspired oxygen and monitoring by pulse oximetry and regular blood gas analysis. Mechanical ventilation is often required to support the poor respiratory function and reduce the work of breathing due to the non-compliant lungs. Cardiovascular support is another mainstay of treatment of sepsis. Hypovolaemia should be corrected with appropriate fluid replacement and guided by monitoring of pulmonary capillary wedge pressure to ensure adequate filling pressures and optimal use of the preload reserve. Inappropriate systemic vasodilation may be treated by vasoconstrictors, such as noradrenaline, although it is important to avoid excessive falls in stroke output due to after load mismatch. Catecholamine-based positive inotropic agents such as dobutamine and adrenaline may be needed. Dobutamine administration can improve myocardial function in sepsis, enhancing cardiac output and oxygen delivery.^113,114 Vasopressure therapy should be limited once arterial pressure has been restored. Repeated clinical and haemodynamic evaluation is necessary to guide therapy, and the serial measurement of blood lactate levels assessing the adequacy of tissue oxygenation.

Although the documentation of endotoxin levels in the blood of patients with sepsis is frequent in Gram-negative bacterial infection there is no relationship between detectable levels of endotoxin and the severity of the infection.^115,116 Microbiological assessment of samples collected (urine, sputum, necrotic tissues/pus and blood) should be carried out for identification of responsible pathogens. Antibiotic therapy is initiated on an empirical basis with broad-spectrum antibiotics; this is then changed to the most appropriate one as microbiological results become available. Failure to choose appropriate antibiotic therapy promptly is associated with increased mortality.

There may be need for the transfusion of blood and/or coagulation factors and platelets as sepsis may be associated with DIC. Treatment with specific anticoagulation agents may decrease morbidity and mortality in patients with sepsis (see below).^87,117,118 Despite the full gamut of treatment, many patients fail to respond and die.

Novel therapies
Conventional therapies, although crucial, are not the only treatments used in sepsis. Specific therapies are being used more frequently and have been shown to play an important role in the treatment of sepsis. Specific therapies have been introduced as a result of the increase in knowledge and better understanding of the pathophysiological disturbances of sepsis, and are being targeted against the different mediators thought to be involved. These therapies have included antibodies directed at endotoxin, anti-TNF-a antibody, restoration of normal anticoagulation pathways (infusing activated protein C or antithrombin III concentrates), the exogenous administration of cytokine antagonists (e.g. IL-1Ra and sTNFRs) and corticosteroids. Many studies have been carried out to evaluate the effects of these various treatments.

The first target for immunotherapy was endotoxin but, in early trials using anti-endotoxin antibodies, no overall benefit was demonstrated.^119,120 However, one study in animals showed an improvement in outcome in an animal model of sepsis.¹²¹ Monoclonal antibodies (mAbs) against TNF-a are one of the most frequent forms of novel therapy being used and studied in sepsis. The results in animal models have demonstrated that mAbs to TNF-a protected animals from septic shock and improved survival.^53-55 In humans, however, phase III studies could not demonstrate improvement in survival in patients with sepsis.^122,123 A recent study in humans has demonstrated that mAbs to TNF-a significantly reduced plasma levels of IL-6, which correlated with overall mortality in patients with sepsis; there was, however, no significant reduction in the 28-day mortality rate.¹²⁴ Other cytokine manipulations have involved IL-10 and IL-12; high circulating levels of both cytokines have been shown to be associated with reduced mortality in animal studies but no published data support this concept in human studies.^70,125 In humans, many factors such as age, genetic background and type of infection are all thought to be confounding variables.¹²⁶

Another novel therapy is aimed at improving the haemostatic defect that is thought to be a main cause of immune system dysfunction in patients with sepsis. Recombinant human activated protein C (rbAPC or drotrecogin alpha) is an example of such a treatment. The rbAPC promotes the anticoagulant, antiinflammatory and profibrinolytic properties of patients with sepsis. In phase II studies, confirmed efficacy in coagulation control was not shown to lead to improvement in mortality. In phase III studies, an improvement in 28-day mortality rates (24.7%) was seen in treated patients, compared with those receiving placebo (30.8%). However, the rbAPC is not a recommended treatment in patients with sepsis due to lack of cost-effectiveness and the need for further studies.¹²⁷ To date, neither tissue factor pathway inhibitor (TFPI or tifacogin) nor antithrombin III have shown any statistically significant reduction of 28-day mortality in phase III studies.^128,129

Both IL-1Ra and sTNFR are soluble receptors for IL-1 and TNF, respectively. They have been shown to block the interaction of these cytokines with their receptors in tissues. Human phase II and III studies, using infusions of IL-1Ra have demonstrated decreased mortality, compared with control groups.^130-132 However, subsequent phase III studies did not show significant reduction in mortality rate, when compared with standard treatment.¹³³ There have been studies of sTNFR in animals, which have shown improvement in survival in animals with sepsis that received sTNFR treatment.^134,135 While, in human studies, there was no significant improvement in overall survival.^136,137

Corticosteroid treatment in sepsis is still controversial. Many recent studies have demonstrated that low dose hydrocortisone (physiological concentration) administration to patients with septic shock induced a significant rise in blood pressure and decreased the plasma levels of proinflammatory cytokines. This was associated with a modulation of SIRS and a substantial reduction in mortality rate, although this did not reach statistical significance.^138-142

CONCLUSIONS
Sepsis/SIRS is a complex disease and clinical entity that encompasses a large variety of local and systemic inflammatory responses. Many types of cells and mediators have been involved in the pathophysiology of the disease process. Proinflammatory and antiinflammatory mediators secreted by a range of immune cells are thought to play a crucial role in inducing the inflammatory responses documented. Although there are many in vitro and in vivo studies published relating to sepsis, our knowledge of the pathophysiology of sepsis and SIRS is still poorly defined and in need of urgent clarification. In SIRS and sepsis, orthodox therapies and good supportive care are still the mainstay of treatment. However, novel therapies based on our understanding of the pathophysiological disturbances underlying SIRS/sepsis, are being introduced into clinical practice, albeit with little clinical benefit. The reason for this may be the complexity of the immune response elicited and any associated dysfunction in SIRS/sepsis and MODS. A multimodal approach may be a better strategy than a single therapeutic option. The high mortality of sepsis may be improved if combined agents were used concurrently or sequentially for treatment. Some preliminary studies in animal models offer hope for improved outcomes in the future.

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Copyright: 24 March 2003