The immunology of trauma

The immune system is complex, with numerous mechanisms to confront a myriad of both foreign pathogens and even nonforeign neoplasms. The immune response to injury is mediated by the innate and adaptive arms of the immune system. The innate response is nonspecific and includes cellular components such as polymorphonuclear leukocytes (PMNLs), eosinophils, natural killer cells, and noncellular components such as complement, lysozyme, coagulation proteins, and neutrophil extracellular traps. The adaptive response is pathogen- and antigen-specific and is exemplified by T and B cells and antibodies. Critically, however, the cross talk between the innate and adaptive arms is robust, and several cell populations seem to share properties with both arms. This cross talk is essential for up- and downregulation of immune responses and helps provide context for aspects of the immune response to interpret whether or not a specific antigen represents a threat.

Classically, immune-mediated responses were thought to be based on self and nonself interactions, but because this concept does not adequately describe other immunologic situations such as tumors, autoimmunity, or trauma, another model has been proposed by Matzinger et al. The danger model theorizes that danger, not “foreignness,” is what initiates an immune response. To recognize danger, the immune system employs a system of pattern recognition that identifies both frequent patterns seen with infection and patterns recognized by cell damage. Evolutionarily primitive pattern recognition receptors (PRRs) recognize and bind conserved microbial constituents called pathogen-associated molecular patterns (PAMPs). PAMPs allow for differentiation between infectious and noninfectious antigens. Additionally, other PRRs recognize molecular patterns associated with cellular damage. The mechanism by which a cell dies determines whether an immune response is initiated. Under typical no-traumatic cell death, apoptosis occurs and apoptotic cells are scavenged with no subsequent immune response. When injury or infection causes cell damage, alternative mechanisms of cell death such as necrosis, necroptosis, or pyroptosis may occur, and both innate and adaptive responses are triggered. These endogenous controlling signals are termed alarmins. They are the alarm signals that emanate from stressed or injured tissues. Both PAMPs (which respond to exogenous signals) and alarmins (which respond to endogenous signals) have similar conserved hydrophobic portions that are able to engage the same PRRs and elicit a comparable inflammatory response. Because of their similarities, PAMPs and alarmins are classified as danger-associated (or sometimes damage-associated) molecular patterns (DAMPs).

The immune response to trauma is markedly similar to that seen with a microbial infection ( Fig. 1 ). The PAMPs/DAMPs released in response to trauma and infection elicit an intrinsic inflammatory immune response through similar PRRs such as the toll-like receptor (TLR) family. The TLRs are a key molecular link between tissue injury, infection, and inflammation. TLRs are membrane-bound receptors that are known to activate two distinct signaling pathways in inflammation. The first is a myeloid differentiation factor 88 (MyD88)-dependent pathway that is activated by all TLRs with the exception of TLR3. This signaling cascade is propagated through a number of interleukin (IL) l receptor–associated kinases with subsequent quick activation of nuclear factor κB (NF-κB) cells and mitogen-activated protein kinase (MAPK), leading to the production of proinflammatory cytokines (IL-1α/β, IL-6, IL-8, macrophage inflammatory protein [MIP]-1α/β, tumor necrosis factor [TNF]-α). The second signaling pathway is a MyD88-independent pathway and is activated through binding of TLR3 and TLR4. This pathway culminates with the induction of interferon (IFN).

Because there are numerous TLRs (12 human TLRs are currently described), and each recognizes PAMPs/DAMPs through diverse mechanisms, functional responses differ depending upon the TLR signaling pathway activated. Following trauma, tissue injuries, hypoxia, and hypotension, as well as secondary insults such as ischemia/reperfusion, compartment syndromes, operative interventions, and infections, all TLRs contribute to a response that is characterized by local and systemic release of proinflammatory cytokines, arachidonic acid metabolites, activation of complement factors, kinins, and coagulation factors as well as release of hormonal mediators. Clinically, this is the systemic inflammatory response syndrome (SIRS). Paralleling SIRS is an anti-inflammatory response referred to as the compensatory anti-inflammatory response syndrome (CARS). The injured patient must strike a fine balance between SIRS and CARS for healing and recovery to occur. When these processes are unbalanced, the patient is at risk for increased susceptibility to infection and multiple-organ failure (MOF) ( Fig. 2 ).

In this chapter we will discuss the main components of the immune response following trauma including examples of specific DAMPs: cytokine response; leukocyte recruitment; protease and reactive oxygen species; complements, kinins, and coagulation; acute phase reactants; SIRS; CARS; and the two-hit model.

Danger (damage)-associated molecular patterns

Many of the molecules identified as DAMPs are proteins released from the cell following injury. Intracellular proteins include high-mobility group box 1 (HMGB1) and heat shock proteins (HSPs); extracellular matrix proteins include hyaluronan fragments. Nonprotein DAMPs include deoxyribonucleic acid (DNA), adenosine 5′-triphosphate (ATP), and uric acid. Two examples of DAMPs are discussed next.

High-mobility group

HMGB1 is a nuclear, nonhistone chromosomal DNA-binding protein that functions as a structural cofactor for proper DNA transcriptional regulation and gene expression. When released extracellularly from injured or necrotic cells, it stimulates both innate and adaptive immune responses. It drives the initiation and potentiation of proinflammatory mediators, inducing a cell-mediated (T helper [TH]1 type) response and serves as a chemoattractant for immature dendritic cells. In apoptotic cells, HMGB1 is irreversibly bound to the chromatin and does not stimulate an immune response. If large numbers of apoptotic cells are cleared by macrophages, then HMGB1 is passively released. Active secretion of HMGB1 occurs from a variety of cell types following an inflammatory stimulus, thus potentiating the inflammatory response to trauma, burn, and infection while initiating tissue regeneration.

Heat shock proteins

HSPs are highly conserved intracellular proteins that are constitutively expressed and function as molecular chaperones that facilitate the synthesis and folding of proteins. Under stressful conditions such as heat shock, pH shift, or hypoxia, their expression is increased, protecting the cell by stabilizing unfolded proteins and allowing the cell to repair or synthesize replacement proteins. When HSP production is upregulated or they are released extracellularly, they act as danger signals and elicit immune responses, at least some of which are mediated through the interaction with TLRs.

Cytokine response

Once the immune response is initiated a multitude of mediators are released. Cytokines exert their effects in both a para- and autocrine manner. Proinflammatory cytokines TNF-α and IL-1β are released within 1 to 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.

IL-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. PGE2 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 Injury Severity Score as well as the development of posttraumatic complications.

Following trauma, IL-12 production is decreased, stimulating a shift in favor of T-helper (TH) 2 cells and the subsequent production of anti-inflammatory mediators IL-4, IL-10, IL-13, and transforming growth factor beta (TGF-β). This decrease in IL-12 and resultant increase in TH2 cells correlates with adverse outcomes. A listing of pro- and anti-inflammatory mediators may be found in Tables 1 and 2 . Table 3 groups the effectors of the innate and acquired pathways with their functions.

TABLE 1

Proinflammatory Mediators

Mediator	Action
IL-1	IL-1 is pleiotropic. Locally, it stimulates cytokine and cytokine receptor production by T cells as well as stimulating B-cell proliferation. Systemically, IL-1 modulates endocrine responses and induces the acute phase response.
IL-6	Il-6 induces acute phase reactants in hepatocytes and plays an essential role in the final differentiation of B cells into Ig-secreting cells. Additionally, IL-6 has anti-inflammatory properties.
IL-8	IL-8 is one of the major mediators of the inflammatory response. It functions as a chemoattractant and is also a potent angiogenic factor.
IL-12	IL-12 regulates the differentiation of naïve T cells into TH1 cells. It stimulates the growth and function of T cells and alters the normal cycle of apoptotic cell death.
TNF-α	TNF-α is pleiotropic. TNF-α and IL-1 act alone or together to induce systemic inflammation as mentioned previously. TNF-α is also chemotactic for neutrophils and monocytes, as well as increasing neutrophil activity.
MIF	MIF forms a crucial link between the immune and neuroendocrine systems. It acts systemically to enhance the secretion of IL-1 and TNF-α.

IL, Interleukin; Ig, immunoglobulin; MIF, migration inhibitory factor; TH1, T-helper lymphocyte; TNF, tumor necrosis factor.

TABLE 2

Anti-Inflammatory Mediators

Mediator	Action
IL-4	IL-4, IL-3, IL-5, IL-13, and CSF2 form a cytokine gene cluster on chromosome 5q, with this gene particularly close to IL-13.
IL-10	IL-10 has pleiotropic effects in immunoregulation and inflammation. It downregulates the expression of TH1 cytokines, MHC class II antigens, and costimulatory molecules on macrophages. It also enhances B-cell survival, proliferation, and antibody production. In addition, it can block NF-κB activity and is involved in the regulation of the JAK-STAT signaling pathway.
IL-11	IL-11 stimulates the T-cell-dependent development of immunoglobulin-producing B cells. It is also found to support the proliferation of hematopoietic stem cells and megakaryocyte progenitor cells.
IL-13	IL-13 is involved in several stages of B-cell maturation and differentiation. It upregulates CD23 and MHC class II expression, and promotes IgE isotype switching of B cells. It downregulates macrophage activity, thereby inhibiting the production of proinflammatory cytokines and chemokines.
IFN-α	IFN-α enhances and modifies the immune response.
TGF-β	TGF-β regulates the proliferation and differentiation of cells, wound healing, and angiogenesis.
α-MSH	α-MSH modulates inflammation by way of three mechanisms: (1) direct action on peripheral inflammatory cells; (2) actions on brain inflammatory cells to modulate local reactions; and (3) indirect activation of descending neural anti-inflammatory pathways that control peripheral tissue inflammation.

CSF, Colony-stimulating factor; IFN, interferon; Ig, immunoglobulin; IL, interleukin; JAK-STAT, Janus kinase/signal transducers and activators of transcription; MHC, major histocompatibility complex; MSH, melanocyte stimulating hormone; NF-κB, nuclear factor κB; TGF, transforming growth factor; TH, T helper.

TABLE 3

Innate and Acquired Effector Pathways and Functions

Effector	Functions
Innate
Complement, proteins, and polypeptides	Disrupt defensins, properdin, pathogen membranes and facilitate
Tuftsin, lysozyme	Phagocyte activation and phagocytosis
Fibrin and coagulation proteins	Isolate pathogens, promote phagocytosis, chemokine release, TH1 differentiation
Neutrophils	Bacterial phagocytosis, entrapment with neutrophil extracellular traps (NETs)
Basophils	Allergic reactions for mold, dust, and other foreign substances, releasing histamine and heparin
Eosinophils	Phagocytosis and allergic reactions against bacteria and parasites
Mast cells	Effect allergic and anaphylactic responses, releasing histamine and heparin
Monocytes, macrophages, and dendritic cells	Phagocytosis, professional antigen presentation, lymphocyte activation and differentiation
Natural killer cells	Cell-mediated killing of some virally infected cells or tumor cells
NKT cells (CD1d-restricted T cells)	Lymphocyte and APC activation
γδ T cells	APC activation, phagocytosis
Acquired
αβ T cells	CD8 restricted cell-mediated killing
	CD4 restricted lymphocyte activation and regulation, cell-mediated killing
B cells, B1 B cells, plasmacytes	Immunoglobulin production
	IgM: polymeric, complement fixing, membrane bound for B-cell activation, or free
	IgD : often membrane bound IgG: complement fixing
	IgE : allergen response
	IgA : serum or mucosal immunity, often polymeric

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 (DR isotype) receptor expression is decreased, leading to a loss of antigen-presenting capacity and decreased TNF-α production. PGE2, IL-10, and TGF-β all contribute to this “immunoparalysis.”

T-helper cells differentiate into either into one of several subsets that may promote proinflammatory responses, promote humoral responses, or perform regulatory functions to limit immune responses. Subsets include proinflammatory TH1 cells, TH2 cells that promote humoral responses, TH9 cells that promote antiparasitic and allergic responses as well as antitumor responses and autoimmunity in some cases, TH17 cells that promote antifungal responses, regulatory T cells that are required to maintain self-tolerance, and follicular helper T cells that facilitate B-cell humoral responses. TH1 and TH2 cells predominate early in the setting of trauma. TH1 cells promote the proinflammatory cascade through the release of IL-2, interferon-γ, and TNF-β, and TH2 cells produce anti-inflammatory mediators. Monocytes/macrophages, through the release of IL-12, stimulate the differentiation of T-helper cells into TH1 cells. Because IL-12 production can be depressed following major trauma, there can be a shift toward TH2 cells, 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 PMNLs “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 PMNLs are mediated by chemoattractants such as chemokines and complement anaphylatoxins. Colony-stimulating factors (CSFs) likewise stimulate monocytopoiesis or granulocytopoiesis and reduce apoptosis of PMNLs 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 PMNLs. 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 shear-resistant 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 immunoglobulin-superfamily members. The arrested leukocytes cross the endothelial layer via bicellular and tricellular endothelial junctions in a process coined diapedesis. This is mediated by platelet-endothelial cell adhesion molecules (PECAM), proteins expressed on both the leukocytes and intercellular junctions of endothelial cells.

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