TWO-HIT MODEL

In the two-hit model, the inciting injury induces a systemic inflammatory response (Figure 1). This “first hit” primes the immune system for an exaggerated and potentially lethal inflammatory reaction to a secondary, otherwise nonlethal, stimulus (“second hit”). This secondary stimulus may be either endogenous or exogenous. Endogenous second hits include cardiovascular instability, respiratory distress, metabolic derangements, and ischemia/reperfusion injuries. In contrast, exogenous second hits include surgical interventions, blood product transfusions, and missed injuries. The two-hit model proposes that this second hit results in destructive inflammation leading to multiple organ failure (MOF) and potentially death. This model has been supported by the work of Moore and colleagues who linked postinjury opportunistic infections to SIRS and MOF.

Figure 1 Pathogenesis of postinjury multiple organ failure.

SYSTEMIC INFLAMMATORY RESPONSE SYNDROME

In 1991, a consensus conference of the American College of Chest Physicians and the American Society of Critical Care Medicine (ACCP/SCCM) defined SIRS as a generalized inflammatory response triggered by a variety of infectious and noninfectious events. They arbitrarily established clinical parameters through a process of consensus. Table 1 summarizes the diagnostic criteria for SIRS. At least two of the four criteria must be present to fulfill the diagnosis of SIRS. Note that this definition emphasizes the inflammatory process regardless of the presence of infection. The term sepsis is reserved for SIRS when infection is suspected or proven. Subsequent studies have validated these criteria as predictive of increased intensive care unit (ICU) mortality, and that this risk increases concurrent with the number of criteria present.

Table 1 Clinical Parameters of Systemic Inflammatory Response Syndrome

PaCO₂, Arterial CO₂ partial pressure.

Systemic inflammatory response syndrome is characterized by the local and systemic production and release of multiple mediators, including proinflammatory cytokines, complement factors, proteins of the contact phase and coagulation system, acute-phase proteins, neuroendocrine mediators and an accumulation of immunocompetent cells at the local site of tissue damage. The severity of trauma, duration of the insult, genetic factors, and general condition of the individual determine the local and systemic release of proinflammatory cytokines and phospholipids.

COMPENSATORY ANTI-INFLAMMATORY RESPONSE SYNDROME

Trauma not only stimulates the release of proinflammatory mediators, but also the parallel release of anti-inflammatory mediators. This compensatory anti-inflammatory response is present concurrently with SIRS (Figure 2). When these two opposing responses are appropriately balanced, the traumatized individual is able to effectively heal the injury without incurring secondary injury from the autoimmune inflammatory response. However, overwhelming CARS appears responsible for post-traumatic immunosuppression, which leads to increased susceptibility to infections and sepsis. With time, SIRS ceases to exist and CARS is the predominant force.

Figure 2 Postinjury multiple organ failure occurs as a result of a dysfunctional inflammatory response. CARS, Compensatory anti-inflammatory response syndrome; MOF, multiple organ failure; SIRS, systemic inflammatory response syndrome.

CYTOKINE RESPONSE

Cytokines exert their effects in both a para- and auto-crine manner. Proinflammatory cytokines, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) are released within 1–2 hours. Secondary proinflammatory cytokines are released in a subacute fashion and include IL-6, IL-8, macrophage migratory factor (MMF), IL-12, and IL-18. Clinically, IL-6 levels correlate with injury severity score (ISS) and the development of MOF, acute respiratory distress syndrome (ARDS), and sepsis.

Interleukin-6 also acts as an immunoregulatory cytokine by stimulating the release of anti-inflammatory mediators such as IL-1 receptor antagonists and TNF receptors which bind circulating proinflammatory cytokines. IL-6 also triggers the release of prostaglandin E2 (PGE2) from macrophages. Prostaglandin E2 is potentially the most potent endogenous immunosuppressant. Not only does it suppress T-cell and macrophage responsiveness, it also induces the release of IL-10, a potent anti-inflammatory cytokine that deactivates monocytes. Serum IL-10 levels correlate with ISS as well as the development of post-traumatic complications.

Following trauma, IL-12 production is decreased, stimulating a shift in favor of T_H2 cells and the subsequent production of antiinflammatory mediators IL-4, IL-10, IL-13, and transforming growth factor beta (TGF-β). This decrease in IL-12 and resultant increase in T_H2 cells correlates with adverse outcomes. A listing of pro and anti-inflammatory mediators appears in Tables 2 and 3.

Table 2 Proinflammatory Mediators

Ig, Immunoglobulin; IL, interleukin; MIF, migration inhibitory factor; TNF, tumor necrosis factor.

Table 3 Anti-Inflammatory Mediators

CSF, Colony-stimulating factor; IFN, interferon; Ig, immunoglobulin; IL, interleukin; MHC, major histo-compatibility complex; MSH, melanocyte stimulating hormone; TGF, transforming growth factor; T_H, T helper.

CELL-MEDIATED RESPONSE

Trauma alters the ability of splenic, peritoneal, and alveolar macrophages to release IL-1, IL-6, and TNF-α leading to decreased levels of these proinflammatory cytokines. Kupffer cells however, have an enhanced capacity for production of proinflammatory cytokines. Cell-mediated immunity not only requires functional macrophage and T cells but also intact macrophage–T-cell interaction. Following injury, human leukocyte antigen (HLA-DR) receptor expression is decreased leading to a loss of antigen-presenting capacity and decreased TNF-α production. Prostaglandin E2, IL-10, and TGF-β all contribute to this “immunoparalysis.”

T-helper cells differentiate into either T_H1 or T_H2 lymphocytes. T_H1 cells promote the proinflammatory cascade through the release of IL-2, interferon-γ (IFN-γ), and TNF-β, while T_H2 cells produce anti-inflammatory mediators. Monocytes/macrophages, through the release of IL-12, stimulate the differentiation of T-helper cells into T_H1 cells. Because IL-12 production is depressed following trauma, there is a shift toward T_H2, which has been associated with an adverse clinical outcome.

Adherence of the leukocyte to endothelial cells is mediated through the upregulation of adhesion molecules. Selectins such as leukocyte adhesion molecule-1 (LAM-1), endothelial leukocyte adhesion molecule-1 (ELAM-1), and P-selectin are responsible for polymorphonuclear leukocytes (PMNL) “rolling.” Upregulation of integrins such as the CD11/18 complexes or intercellular adhesion molecule-1 (ICAM-1) is responsible for PMNL attachment to the endothelium. Migration, accumulation, and activation of the PMNL are mediated by chemoattractants such as chemokines and complement anaphylotoxins. Colony-stimulating factors (CSFs) likewise stimulate monocyte- or granulocyto-poiesis and reduce apoptosis of PMNL during SIRS. Neutrophil apoptosis is further reduced by other proinflammatory mediators, thus resulting in PMNL accumulation at the site of local tissue destruction.

LEUKOCYTE RECRUITMENT

Proinflammatory cytokines enhance PMNL recruitment, phagocytic activity, and the release of proteases and oxygen-free radicals by PMNL. This recruitment of leukocytes represents a key element for host defense following trauma, although it allows for the development of secondary tissue damage. It involves a complex cascade of events culminating in transmigration of the leukocyte, whereby the cell exerts its effects. The first step is capture and tethering, mediated via constitutively expressed leukocyte selectin denoted L-selectin. L-selectin functions by identifying glycoprotein ligands on leukocytes and those upregulated on cytokine-activated endothelium.

Following capture and tethering, endothelial E-selectin and P-selectin assist in leukocyte rolling or slowing. P-selectin is found in the membranes of endothelial storage granules (Weibel-Palade bodies). Following granule secretion, P-selectin binds to carbohydrates presented by P-selectin glycoprotein ligand (PSGL-1) on the leukocytes. In contrast, E-selectin is not stored, yet it is synthesized de novo in the presence of inflammatory cytokines. These selectins cause the leukocytes to roll along the activated endothelium, whereby secondary capturing of leukocytes occurs via homotypic interactions.

The third step in leukocyte recruitment is firm adhesion, which is mediated by membrane expressed β₁– and β₂-integrins. The integrins bind to ICAM resulting in cell-cell interactions and ultimately signal transduction. This step is critical to the formation of stable shearresistant adhesion, which stabilizes the leukocyte for transmigration.

Transmigration is the final step in leukocyte recruitment following the formation of bonds between the aforementioned integrins and Ig-superfamily members. The arrested leukocytes cross the endothelial layer via bicellular and tricellular endothelial junctions in a process coined diapedysis. This is mediated by platelet–endothelial cell adhesion molecules (PECAM), proteins expressed on both the leukocytes and intercellular junctions of endothelial cells.

PROTEASES AND REACTIVE OXYGEN SPECIES

Polymorphonuclear lymphocytes and macrophages are not only responsible for phagocytosis of microorganisms and cellular debris, but can also cause secondary tissue and organ damage through degranulation and release of extracellular proteases and formation of reactive oxygen species or respiratory burst. Elastases and metalloproteinases which degrade both structural and extracellular matrix proteins are present in increased concentrations following trauma. Neutrophil elastases also induce the release of proinflammatory cytokines.

Reactive oxygen species are generated by membrane associated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is activated by proinflammatory cytokines, arachidonic acid metabolites, complement factors, and bacterial products. Superoxide anions are reduced in the Haber-Weiss reaction to hydrogen peroxide by superoxide dismutase located in the cytosol, mitochondria, and cell membrane. Hydrochloric acid is formed from H₂O₂ by myeloperoxidase, while the Fenton reaction transforms H₂O₂ into hydroxyl ions. These free reactive oxygen species cause lipid peroxidation, cell membrane disintegration, and DNA damage of endothelial and parenchymal cells. Oxygen radicals also induce PMNL to release proteases and collagenase as well as inactivating protease inhibitors.

Reactive nitrogen species cause additional tissue damage following trauma. Nitric oxide (NO) is generated from L-arginine by inducible nitric oxide synthase (iNOS) in PMNL or vascular muscle cells and by endothelial nitric oxide synthase in endothelial cells. Nitric oxide induces vasodilatation. Inducible nitric oxide synthase is stimulated by cytokines and toxins, whereas endothelial nitric oxide synthase (eNOS) is stimulated by mechanical shearing forces. Damage by reactive oxygen and nitrogen species leads to generalized edema and the capillary leak syndrome.

COMPLEMENT, KININS, AND COAGULATION

The complement cascade, kallikrein-kinin system, and coagulation cascade are intimately involved in the immune response to trauma. They are activated through proinflammatory mediators, endogenous endotoxins, and tissue damage. The classical pathway of complement is normally activated by antigen-antibody complexes (immunoglobulins [Ig] M or G) or activated coagulation factor XII (FXII), while the alternative pathway is activated by bacterial products such as lipopolysaccharide. Complement activation following trauma is most likely from the release of proteolytic enzymes, disruption of the endothelial lining, and tissue ischemia. The degree of complement activation correlates with the severity of injury. The cleavage of C3 and C5 by their respective convertases results in the formation of opsonins, anaphylotoxins, and the membrane attack complex (MAC). The opsonins C3b and C4b enhance phagocytosis of cell debris and bacteria by means of opsonization. The anaphylotoxins C3a and C5a support inflammation via the recruitment and activation of phagocytic cells (i.e., monocytes, polymorphonuclear cells, and macrophages), enhancement of the hepatic acute-phase reaction, and release of vasoactive mediators (i.e., histamine). They also enhance the adhesion of leukocytes to endothelial cells, which results in increased vascular permeability and edema. C5a induces apoptosis and cell lysis through the interaction of its receptor and the membrane attach complex (MAC). Additionally, C3a and C5a activate reparative mechanisms. C1-inhibitor inactivates C1s and C1r, thereby regulating the classical complement pathway. However, during inflammation, serum levels of C1-inhibitor are decreased via its degradation by PMNL elastases.

The plasma kallikrein-kinin system is a contact system of plasma proteases related to the complement and coagulation cascades. It consists of the plasma proteins FXII, prekallikrein, kininogen, and factor XI (FXI). The activation of FXII and prekallikrein occurs via contact activation when endothelial damage occurs exposing the basement membrane. Factor XII activation forms factor XIIa (FXIIa), which initiates the complement cascade through the classical pathway, whereas prekallikrein activation forms kallikrein, which stimulates fibrinolysis through the conversion of plasminogen to plasmin or the activation of urokinase-like plasminogen activator (u-PA). Tissue plasminogen activator (t-PA) functions as a cofactor. Additionally, kallikrein supports the conversion of kininogen to bradykinin. The formation of bradykinin also occurs through the activation of the tissue kallikrein-kinin system, most likely through organ damage as the tissue kallikrein-kinin system is found in many organs and tissues including the pancreas, kidney, intestine, and salivary glands. The kinins are potent vasodilators. They also increase vascular permeability and inhibit the function of platelets.

The intrinsic coagulation cascade is linked to the contact activation system via the formation of factor IXa (FIXa) from factor XIa (FXIa). Its formation leads to the consumption of FXII, prekallikrein, and FXI while plasma levels of enzyme-inhibitor complexes are increased. These include FXIIa-C1 inhibitor and kallikrein-C1 inhibitor. C1-inhibitor and α1-protease inhibitor are both inhibitors of the intrinsic coagulation pathway.

Although the intrinsic pathway provides a stimulus for activation of the coagulation cascade, the major activation following trauma is via the extrinsic pathway. Increased expression of tissue factor (TF) on endothelial cells and monocytes is induced by the proinflammatory cytokines TNF-α and IL-1β. The factor VII (FVII)–TF complex stimulates the formation of factor Xa (FXa) and ultimately thrombin (FIIa). Thrombin-activated factor V (FV), factor VIII (FVIII), and FXI result in enhanced thrombin formation. Following cleavage of fibrinogen by thrombin, the fibrin monomers polymerize to from stable fibrin clots. The consumption of coagulation factors is controlled by the hepatocytic formation of antithrombin (AT) III. The thrombin–antithrombin complex inhibits thrombin, FIXa, FXa, FXIa, and FXIIa. Other inhibitors include TF pathway inhibitor (TFPI) and activated protein C in combination with free protein S. Free protein S is decreased during inflammation due to its binding with the C4b binding protein.

Disseminated intravascular coagulation (DIC) may occur following trauma. After the initial phase, intra- and extra-vascular fibrin clots are observed. Hypoxia-induced cellular damage is the ultimate result of intravascular fibrin clots. Likewise, there is an increase in the interactions between endothelial cells and leukocytes. Clinically, coagulation factor consumption and platelet dysfunction are responsible for the diffuse hemorrhage. Consumption of coagulation factors is further enhanced via the proteolysis of fibrin clots to fibrin fragments. The consumption of coagulation factors is further enhanced through the proteolysis of fibrin clots to fibrin fragments by the protease plasmin.

ACUTE-PHASE REACTION

The acute-phase reaction describes the early systemic response following trauma and other insult states. During this phase, the biosynthetic profile of the liver is significantly altered. Under normal circumstances, the liver synthesizes a range of plasma proteins at steady state concentrations. However, during the acute phase reaction, hepatocytes increase the synthesis of positive acute-phase proteins (i.e., C-reactive protein [CRP], serum amyloid A [SAA], complement proteins, coagulation proteins, proteinase inhibitors, metal-binding proteins, and other proteins) essential to the inflammatory process at the expense of the negative acute-phase proteins. The list of acute-phase proteins is in Table 4.

Table 4 Acute-Phase Proteins

Note: Positive acute-phase proteins increase production during an acute-phase response. Negative acute-phase proteins have decreased production during an acute-phase response.

The acute-phase response is initiated by hepatic Kupffer cells and the systemic release of proinflammatory cytokines. Interleukin-1, IL-6, IL-8, and TNF-α act as inciting cytokines. The acute phase reaction typically lasts for 24–48 hours prior to its downregulation. Interleukin-4, IL-10, glucocorticoids, and various other hormonal stimuli function to downregulate the proinflammatory mediators of the acute-phase response. This modulation is critical. In instances of chronic or recurring inflammation, an aberrant acute-phase response may result in exacerbated tissue damage.

The major acute-phase proteins include CRP and SAA, the activities of which are poorly understood. C-reactive protein was so named secondary to its ability to bind the C-polysaccharide of Pneumococcus. During inflammation, CRP levels may increase by up to 1000-fold over several hours depending on the insult and its severity. It acts as an opsonin for bacteria, parasites, and immune complexes, activates complement via the classical pathway, and binds chromatin. Binding chromatin may minimize autoimmune responses by disposing of nuclear antigens from sites of tissue debris. Clinically, CRP levels are relatively non-specific and not predictive of post-traumatic complications. Despite this fact, serial measurements are helpful in trending a patient’s clinical course.

Serum amyloid A interacts with the third fraction of high-density lipoprotein (HDL3), thus becoming the dominant apolipoprotein during acute inflammation. This association enhances the binding of HDL3 to macrophages, which may engulf cholesterol and lipid debris. Excess cholesterol is then utilized in tissue repair or excreted. Additionally, SAA inhibits thrombin-induced platelet activation and the oxidative burst of neutrophils, potentially preventing oxidative tissue destruction.

SUMMARY

Injury triggers a tremendously complex response involving a multitude of systems. The individual’s immune system must balance the proinflammatory response, which is necessary to clear injured tissue, yet not cause overwhelming endogenous injury with a necessary downregulation of the inflammatory process in order to provide an environment of minimal inflammation that can nurture the cell proliferation and tissue remodeling needed for healing. A loss of this balance can cause additional tissue injury from the immune response itself, or leave the individual susceptible to infection and sepsis. As our understanding of the immune response following injury grows, the more likely that we will be able to monitor and effectively manage the traumatized, critically ill patient.