The Inflammatory Response

Neutrophils

The neutrophil, a type of polymorphonuclear (PMN) leukocyte, is a potent mediator of acute inflammation and often the first cell type recruited in response to injury and infection. As a circulating PMN leukocyte, neutrophils have a short half-life of approximately 8 hours; the longevity of the neutrophil is increased in response to inflammatory signals, although the exact duration is a topic of debate. Neutrophils are continuously produced in the bone marrow in response to granulocyte colony-stimulating factor (G-CSF), and their production is regulated by interleukin (IL)-17 from T-cells and IL-23 from macrophages. The neutrophil undergoes a process of tethering, rolling, adhesion, crawling, and transmigration to move from the bloodstream to the tissue ( Fig. 3.1 ). Neutrophils contain three types of proinflammatory granules – azurophilic (primary) granules, specific (secondary) granules, and gelatinase (tertiary) granules. Proteolytic contents of these granules can be released extracellularly or into the intracellular phagosome to aid elimination of invading microbes. Neutrophils also release fiber meshwork to which histones, proteins, and enzymes adhere; this is the neutrophil extracellular trap (NET). Extracellular pathogens are trapped within the NET to prevent spread of the pathogen and aid phagocytosis.

Although classically considered a key mediator of the initial inflammatory response, the functions of the neutrophil have been shown to extend beyond the acute inflammatory period. The neutrophil granules contain a number of proteases that are essential for tissue remodeling and wound healing. They directly stimulate angiogenesis via release of vascular endothelial growth factors (VEGFs). In addition, neutrophils display plasticity, and, although typically proinflammatory, antiinflammatory subsets of neutrophils have been identified in certain pathologic states.

Macrophages

Named for its ability to consume and degrade extracellular debris, the macrophage is a key player in innate immunity. Monocytes, the precursor to the macrophage, differentiate into macrophages in response to infection and tissue injury. Not displayed on immature monocytes, the macrophage expresses a large array of pattern recognition receptors (PRRs) – receptors that recognize a variety of intracellular and extracellular danger signals. In response to PRR stimulation, macrophages neutralize, invading pathogens via phagocytosis and lysosomal degradation; they additionally secrete proinflammatory mediators, including IL-1β and tumor necrosis factor-α (TNF-α) that recruit other immune cells to the damaged tissue. Macrophages also process antigenic substances and present them on their surface to help stimulate the differentiation of helper T cells; thus, macrophages are professional antigen-presenting cells (APCs).

Similar to neutrophils, once thought to be a single cell type, the macrophage demonstrates plasticity and phenotypic variance depending upon its environment. M1 macrophages express proinflammatory cytokines and proteolytic substances; they are predominant in viral and bacterial infection. M1 macrophages stimulate proinflammatory helper T cells. While M1 macrophage products facilitate a beneficial inflammatory response against invading microbes, they can result in a dangerous inflammatory state for the human host. High concentrations of M1-type cytokines correlate with mortality in sepsis models. M2 macrophages are essential for tissue remodeling and wound healing; they express a variety of antiinflammatory markers, including IL-10. Macrophages are abundant throughout the body. Their functions vary depending on the tissue in which they reside. For example, Kupffer cells of the liver and microglia of the central nervous system are macrophages.

Dendritic Cells

Dendritic cells bridge the innate and adaptive immune response as the major professional APC. Upon encountering foreign material, the dendritic cell will engulf and degrade pathogen-derived proteins. These antigenic proteins are loaded onto a major histocompatibility (MHC) complex class I or class II molecule. The antigen-MHC complex is transported to the surface of the dendritic cell, and the dendritic cell travels from the tissue to the lymphoid organs, primarily the lymph nodes, and the spleen. Within the lymphoid organs, it stimulates naïve, resting T cells to differentiate into either cytotoxic T cells or helper T cells. Extracellular proteins are processed within the dendritic cell lysosome, and they are presented in conjunction with the MHC class II molecule to activate CD4+ helper T cells. In contrast, intracellular proteins are processed within the cytosol by the proteasome, and they are presented via the MHC class I molecule to CD8+ cytotoxic T cells. Certain subsets of dendritic cells, however, process extracellular proteins through a process called cross-presentation and allow for presentation of these molecules via MHC class I. Through the process of MHC-antigen presentation, the adaptive immune response begins.

Dendritic cells additionally stimulate T cell activity via surface ligands, such as CD80 and CD86, and via production of proinflammatory cytokines, such as IL-12. As a result of its many costimulatory mechanisms, dendritic cells are highly efficient at provoking the adaptive immune response. While macrophages and B cells are also considered APCs, they do not function at this level of efficiency for adaptive immune stimulation.

Dendritic cells also process self-antigens and nonpathogenic antigens. Presentation of this antigen type to a naïve T cell induces the regulatory T cell – an immunosuppressive type cell essential for tolerance and immune homeostasis. Disorders of this pathway result in autoimmunity to self-antigens and allergy response against nonpathogenic environmental material. The fact that dendritic cells use similar machinery both to induce an active immune response to foreign pathogens and to induce a tolerant response toward self-antigens is an interesting paradox and an area of interest in cancer immunobiology. Tumor cells can be considered master evaders of the immune system. One of their many and only partially understood mechanisms of immune evasion is via inhibition of dendritic cell function.

T Cell

T and B lymphocytes are the primary effector cells of the adaptive immune system; T cells are the primary effector cell of the cellular immune response, while B cells primarily mediate the humoral immune response. T and B cells are unique in their ability to recognize specific antigens and rapidly respond through clonal expansion. T and B cells are essential for the development of immune memory.

T cell activation is a complex, multifaceted process. It can be simplified to three key steps. While keeping in mind that activation of the immune system is not a linear process (multiple events involving multiple cell types take place simultaneously) there are many branch points within the pathway that influence the ultimate outcome. Once transported to the lymphoid organs, mature dendritic cells present antigen-MHC complexes to naïve T cells. Antigens derived from cytosolic proteins are presented via the MHC class I molecule; the antigen-MHC class I complex activates CD8+ cytotoxic T cells. Antigens derived from extracellular proteins are presented via the MHC class II molecule; the antigen-MHC class II complex activates CD4+ helper T cells. Whereas MHC class I molecules can be found on all nucleated cells, MHC class II is confined to APCs. Although consistent with classic teaching, emerging research indicates that the formation of antigen-MHC complexes is not so straight forward. Recent studies have shown that activation of certain PRRs can alter whether a protein is loaded onto an MHC class I or MHC class II receptor following uptake. For example, toll-like receptor 4 (TLR4) is a PRR most famous for its role in recognizing lipopolysaccharide, a key component of the cell wall of extracellular gram-negative bacteria. Activation of TLR4 at the cell surface transiently results in an increase in cross-presentation and thus an increase in loading of antigenic peptides onto MHC class I molecules with activation of CD8+ cytotoxic T cells. However, once engulfed within the endosome, TLR4 switches to promote loading of antigenic peptides onto MHC class II molecules; this ultimately promotes a CD4+ helper T cell predominant immune response.

As self-antigens and benign environmental antigens are able to be loaded on to MHC molecules, presentation of the antigen-MHC complex alone is not sufficient to activate the adaptive immune pathway. Costimulatory molecules are additionally necessary for full T cell activation, most notably, CD80 and CD86, located on the activated dendritic cell and its interaction with CD28 upon T cells ( Fig. 3.2 ). Stimulation of CD28 pathways results in a lower threshold for T cell activation and production of IL-2.

Cytokines are also essential for full T cell activation, and the innate cytokine milieu varies based upon the type of PRR that has been stimulated. IL-12, IL-6, and TNF-α potentiate acute inflammation and influence T cell differentiation. IL-1 is essential for upregulating the acute-phase response. Interferon (IFN) type 1 drives an antiviral predominant response and drives activation of CD8+ cytotoxic T cells. In the context of CD4+ helper T cells, IL-12 promotes differentiation of helper T cell type 1 (Th1) cells. IL-4 promotes differentiation to Th2 cells. IL-6 and transforming growth factor-β (TGF-β) promote differentiation of Th17 cells. TGF-β can also promote differentiation to regulatory-type T cells in the absence of infection. In summary, T cell activation is achieved by three key steps: presentation of an antigen-MHC complex to a naïve T cell by a mature dendritic cell, costimulation of the T cell by surface molecules located on the dendritic cell, and the presence of cytokines produced by cells of the innate immune system.

Each activated T cell produces a unique profile of cytokines to elicit a variety of downstream effects. Of the CD4+ helper T cell lineage, the best-characterized cells are Th1, Th2, and Th17 cells. In regard to infection, Th1 cells primarily fight intracellular pathogens and do so via upregulation of IFN-γ and propagation of the inflammatory response. Th2 cells function to clear extracellular pathogens and mediate the allergic response through production of IL-4, IL-5, and IL-13. A growing body of research indicates that a healthy immune response is heavily influenced by the proportional response of Th1 and Th2 cells.

Th17 cells differentiate in response to extracellular pathogens and fungi; they are frequently implicated in autoimmune disorders, and Th17 cells can acquire the characteristic of Th1 cells in chronic inflammatory states. Th17 cells drive production of IL-17. Regulatory T cells, another class of CD4+ helper T cells, are essential for the development of memory and tolerance to self-antigens; they produce potent antiinflammatory cytokines such as IL-10 and TGF-β. CD8+ cytotoxic T cells target cells that have been infected with a virus for destruction, and they produce the potent proinflammatory cytokine IFN-γ.

In general, studies have shown that the adaptive T cell–dependent inflammatory response is dampened following general anesthesia, surgical stress, blood transfusion, hypothermia, hyperglycemia, and postoperative pain; this occurs with a simultaneous increase in adrenocorticotropic hormone (ACTH) and glucocorticoids. As T cells play a role in the destruction of circulating tumor cells and the prevention of micrometastasis, this observation has particular importance within the realm of surgical oncology. A recent study compared the postoperative T cell profile of patients undergoing surgery for invasive breast cancer and for benign fibroadenomas. Postoperatively, no change in the T cell profile was exhibited in patients within the fibroadenoma group, whereas patients within the invasive breast cancer group exhibited an increase in regulatory T cells. The regulatory T cells increase at 72 hours postoperatively correlated with a larger tumor size, human epidermal growth factor receptor-2 (HER2) positivity, and decrease in the length of disease-free survival. A lower burden of Th1 cells was correlated with a greater tumor burden and HER2 positivity. This suggests that postoperative immunosuppression may leave patients vulnerable to metastases and invites opportunity for research into immunomodulation in the postoperative immunosuppressed state.

B Cell

B cells, the primary effector cell of the humoral immune response, produce antibodies or immunoglobulins (Ig) and function as professional APCs. B cells initially develop in the bone marrow, where their cellular maturation can be correlated to the structural rearrangement of the immunoglobulin gene segments. B cells undergo a process termed V(D)J recombination in which a number of genetic recombinant events among gene segments V, D, and J of the immunoglobulin light and heavy chains ultimately allow for the production of different immunoglobulins; immunoglobulins have the capacity to recognize more than 5×10 ¹³ different antibodies. During the process of V(D)J recombination, the B cell progresses through the pro-B and pre-B cell phases. Following V(D)J recombination, surface-bound IgM marks the entrance of the B cell into the immature B cell state; it is at this point in its life cycle that it leaves the bone marrow and migrates to the spleen. Within the spleen, immature B cells will become naïve follicular or marginal zone B cells.

Marginal zone B cells function in the spleen as the first line of defense against blood borne invaders. Independent of T cells, these B cells can rapidly produce soluble IgM during the early stages of infection. Naïve follicular B cells can be found within the lymph nodes or as circulating B cells. Their activation is T cell–dependent. Activation of follicular B cells results in a process termed class switching in which B cells transition from the production of IgM antibodies to the production of other classes of immunoglobulin, primarily IgG, IgA, and IgE, during times of infection. During the transition from IgM to other types of immunoglobulins, further genetic rearrangements occur that result in immunoglobulins with a higher affinity for the antigen recognized by the B cell. Memory B cells are B cells that are maintained following an immune response. These memory cells retain the capacity to produce high-affinity immunoglobulins toward a certain antigen and, should that antigen ever be introduced again, these B cells can rapidly mount a robust immunologic response.

Toll-Like Receptors

DAMPs are recognized by cellular receptors, broadly termed PRR, that are found on the cell surface or intracellularly. PRRs are evolutionarily conserved receptors that respond to specific PAMPs. These PAMPs are essential for survival from invading microbes and are not easily altered; microbes are typically unable to alter PAMPs in an attempt to evade the immune system. The best-characterized class of PRR involved in the inflammatory response is the toll-like receptor (TLR) family. The Toll signaling pathway was initially characterized in Drosophila melanogaster . The Toll protein had a known nuclear factor-κB (NF-κB)–dependent role in activation of B cells in response to lipopolysaccharide, a component of the gram-negative cell wall and a classic PAMP. IL-1, an important mediator of fever, T-cell activation, and the acute phase response had also previously demonstrated NF-κB–dependent signaling. The discovery that the IL-1 receptor (IL-1R) shared a homologous motif with the Drosophila protein, Toll, marked a key advancement in the understanding of intracellular signaling pathways of the innate immune system.

The TLR is a transmembrane protein with an extracellular ligand-binding domain and an intracellular signaling domain. TLRs are expressed on the cell surface or within the endosome. Binding of a DAMP prompts dimerization of the TLR and subsequent intracellular activation of multiple signaling pathways. Ten human TLRs have been identified, each recognizing various PAMPs and triggering a variety of downstream cellular responses. TLR4 plays a key role in recognition of bacterial LPS, while TLR1, TLR2, and TLR6 recognize other common bacterial lipoproteins. TLR4 additionally plays a role in recognition of high-mobility group box protein 1 (HMGB1) and heat shock protein 70, two common alarmins, as well as mediation of sterile inflammation in the setting of ischemia-reperfusion injury. TLR3 recognizes double-stranded ribonucleic acid (RNA), and TLR7 and TLR8 recognize single-stranded RNA specific to viral invaders.

TLRs function through NF-κB and mitogen-activated protein kinase intracellular pathways to upregulate a number of proinflammatory cytokines, including IL-1 and TNF-α. This allows for activation of neighboring innate immune cells, and for activation of cell lines involved in adaptive immunity, including helper T cells, cytotoxic T cells, regulatory T cells, and B cells.

Inflammasome

Another well-characterized family of PRR is the nucleotide-binding and oligomerization domain (NOD)–like receptor (NLR) family. NLRs are assembled in the cytoplasm to form a key intracellular structure known as the inflammasome – an essential intracellular PRR. NLRs complex with apoptosis-associated speck-like protein containing a caspase recruitment domain to form the inflammasome. The inflammasome plays an essential role in regulating sterile inflammation via recognition of endogenous alarmins, as well as activating the innate immune response via recognition of foreign PAMPs.

The best studied NLR is NLRP3. Once an NLRP3 inflammasome has been primed, it activates protease caspase 1. Caspase 1 is essential in the cleaving and subsequent secretion of proinflammatory cytokines IL-1β and IL-18 by macrophages in addition to the proinflammatory alarmin HMGB1. Endogenous factors capable of priming the NLRP3 inflammasome include hypoxia, complement, reactive oxygen species, oxidized low-density lipoproteins, amyloids, and misfolded proteins. The inflammasome plays a key role in the sterile inflammatory process that accompanies metabolic diseases, atherosclerosis, and neuroinflammatory disorders. Emerging evidence suggests that the NLRP3 inflammasome also plays a role in cardiomyopathy associated with sepsis.