Premed · Premed · Immunology
Lecture 8: Phagocytes and Natural Killer Cells
Immunology
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the process of phagocytosis and the mechanisms used to kill ingested microorganisms
- Compare and contrast the roles of neutrophils, macrophages, and dendritic cells in innate immunity
- Explain the mechanisms by which NK cells recognize and kill target cells
- Describe antibody-dependent cellular cytotoxicity (ADCC)
- Discuss macrophage polarization (M1 vs. M2) and its functional significance
Lecture Content
I. Professional Phagocytes
Three main types of professional phagocytes carry out the innate immune system's cellular defense. Neutrophils are the most abundant and serve as the first responders, but they are short-lived. Macrophages are tissue-resident and recruited cells that are longer-lived and multifunctional. Dendritic cells primarily function as antigen-presenting cells and are less microbicidal than the other two. Non-professional phagocytes such as epithelial cells and fibroblasts have limited phagocytic capacity.
II. The Process of Phagocytosis
Phagocytosis proceeds through four steps. In recognition and attachment, phagocytes can recognize pathogens directly through PRRs such as the mannose receptor, scavenger receptors, and Dectin-1, but opsonin-mediated recognition is far more efficient. IgG opsonins are recognized by Fcγ receptors (FcγRI/CD64, FcγRII/CD32, FcγRIII/CD16), C3b and iC3b opsonins are recognized by complement receptors (CR1 and CR3), and collectins such as MBL and SP-A bind their respective receptors. During engulfment, pseudopods extend around the particle through a zipper mechanism driven by actin polymerization, enclosing it in a membrane-bound vesicle called a phagosome. Phagosome maturation follows as the phagosome fuses sequentially with early endosomes, late endosomes, and lysosomes, forming a phagolysosome that is acidified to pH 4.5-5.0 and contains hydrolytic enzymes. This maturation process involves Rab GTPases, specifically the transition from Rab5 to Rab7.
Killing and degradation occurs through both oxygen-dependent and oxygen-independent mechanisms. The oxygen-dependent mechanisms constitute the respiratory burst: NADPH oxidase (phagocyte oxidase) assembles on the phagosomal membrane and produces superoxide anion (O2-), which is converted to H2O2 by superoxide dismutase. Myeloperoxidase (MPO) then combines H2O2 with chloride to generate hypochlorous acid (HOCl, essentially bleach), the most potent microbicidal agent in the system. Additionally, iNOS produces nitric oxide, which combines with superoxide to form peroxynitrite (ONOO-), a reactive nitrogen species. Oxygen-independent mechanisms include defensins (which disrupt microbial membranes), lysozyme (which degrades peptidoglycan), lactoferrin (which sequesters iron), cathepsins and other acid hydrolases, elastase and collagenase, and BPI (bactericidal permeability-increasing protein, which neutralizes LPS and kills Gram-negative bacteria).
Chronic granulomatous disease (CGD) illustrates the importance of the respiratory burst. This condition results from deficiency in NADPH oxidase components (most commonly gp91phox, which is X-linked). Patients cannot generate a respiratory burst and suffer severe recurrent infections with catalase-positive organisms (Staphylococcus aureus, Aspergillus, Burkholderia, Serratia), which destroy their own H2O2 and thereby eliminate the substrate for the MPO system.
<image>A step-by-step diagram of phagocytosis. Panel A: Recognition -- a macrophage with surface receptors (Fc receptors binding IgG-opsonized bacteria, CR3 binding C3b-opsonized bacteria, and mannose receptor binding bacterial surface carbohydrates directly). Panel B: Engulfment -- pseudopods extending around the bacterium via the zipper mechanism, with actin filaments highlighted along the extending pseudopods. Panel C: Phagosome formation and maturation -- the bacterium enclosed in a phagosome that fuses with lysosomes to form a phagolysosome (pH 4.5). Panel D: Killing mechanisms -- inside the phagolysosome, NADPH oxidase on the membrane generating superoxide; myeloperoxidase producing HOCl; defensins and lysozyme attacking the bacterial wall; reactive nitrogen species (NO, peroxynitrite) shown. Bacterial debris at the final stage indicates degradation.</image>
III. Neutrophils
Neutrophils are the most abundant white blood cell, constituting 60-70% of circulating leukocytes. They have a short half-life of 6-8 hours in blood and 1-2 days in tissues. They are highly efficient phagocytes equipped with pre-formed granules organized into three categories: primary (azurophilic) granules containing myeloperoxidase, defensins, lysozyme, elastase, and BPI; secondary (specific) granules containing lactoferrin, collagenase, lysozyme, and NADPH oxidase components; and tertiary granules containing gelatinase and MMPs. Degranulation releases granule contents into phagosomes or into the extracellular space.
A distinctive neutrophil capability is the formation of neutrophil extracellular traps (NETs). During NETosis -- a form of cell death distinct from both apoptosis and necrosis -- neutrophils release decondensed chromatin (DNA) studded with histones and granule proteins. These NETs trap and kill extracellular bacteria. However, excessive NET formation contributes to autoimmunity (particularly SLE) and thrombosis.
IV. Macrophages
Macrophages derive from either circulating monocytes or embryonic precursors (in the case of tissue-resident macrophages) and are long-lived, surviving weeks to months in tissues. Their functions encompass phagocytosis and pathogen killing, antigen processing and presentation via MHC class II, production of cytokines and chemokines (TNF-α, IL-1, IL-6, IL-12, IL-10), tissue remodeling and repair, and clearance of apoptotic cells through efferocytosis.
Macrophage polarization describes the spectrum of functional states macrophages can adopt. M1 (classically activated) macrophages are induced by IFN-γ from Th1 cells and NK cells along with TLR ligands such as LPS. They exhibit enhanced microbicidal activity, produce pro-inflammatory cytokines (TNF-α, IL-1, IL-12, IL-23), and generate ROS and NO. M1 macrophages support Th1-type responses and defense against intracellular pathogens and tumors. M2 (alternatively activated) macrophages are induced by IL-4 and IL-13 from Th2 cells and ILC2s. They produce anti-inflammatory mediators (IL-10, TGF-β) and promote wound healing, fibrosis, and tissue remodeling, supporting Th2-type responses, parasite defense, and allergy. M2 macrophages can be further subdivided into subtypes: M2a (induced by IL-4/IL-13), M2b (induced by immune complexes plus TLR stimulation), and M2c (induced by IL-10 and glucocorticoids). Importantly, M1/M2 polarization represents a spectrum rather than a strict binary, and macrophages can shift their phenotype in response to changing signals. Macrophage activation syndrome (MAS) represents the pathological extreme of excessive macrophage activation, resulting in hemophagocytosis and cytokine storm.
V. Natural Killer (NK) Cells
NK cells are large granular lymphocytes that belong to the innate immune system as part of innate lymphoid cell group 1. They do not require prior sensitization or MHC-restricted antigen recognition to kill target cells, and they constitute approximately 5-15% of peripheral blood lymphocytes. Key markers are CD56, CD16 (FcγRIIIA), and NKp46. Two functional subsets exist: CD56bright CD16dim cells are primarily cytokine producers (IFN-γ, TNF-α) found in lymph nodes and tissues, while CD56dim CD16bright cells are the predominant cytotoxic effectors found in blood.
VI. NK Cell Recognition: Activating and Inhibitory Receptors
NK cell activation is controlled by a balance of signals from activating and inhibitory receptors. Inhibitory receptors recognize MHC class I on normal healthy cells and include KIRs (killer immunoglobulin-like receptors), which bind specific HLA class I alleles (HLA-A, HLA-B, HLA-C), and the NKG2A/CD94 heterodimer, which binds HLA-E (a non-classical MHC I molecule). When inhibitory signals dominate, the NK cell is restrained from killing. Activating receptors recognize stress-induced ligands and include NKG2D, which binds MICA, MICB, and ULBPs (upregulated on infected, transformed, or stressed cells); natural cytotoxicity receptors (NCRs) such as NKp30, NKp44, and NKp46, which bind viral hemagglutinins and heparan sulfate proteoglycans; CD16 (FcγRIIIA), which binds the IgG Fc region and mediates ADCC; and NKG2C/CD94, which is the activating counterpart to NKG2A.
The missing-self hypothesis explains a key aspect of NK function: virus-infected cells and tumor cells often downregulate MHC class I to escape detection by CTLs. This loss of MHC I removes the inhibitory signal to NK cells, leading to their activation and killing of the target. NK cells thus fill the gap left by CTLs when MHC I is absent. Induced-self recognition provides a complementary mechanism: stress, infection, or transformation induces expression of activating ligands such as MICA and MICB, and strong activating signals can override inhibition even when MHC I is present.
<image>A two-panel diagram showing NK cell recognition. Panel A (No killing -- normal cell): A healthy host cell displaying MHC class I molecules on its surface. NK cell inhibitory receptors (KIRs) engage MHC I, sending a dominant inhibitory signal (red). Activating receptors (NKG2D) find no stress ligands. The balance favors inhibition -- NK cell does not kill. Panel B (Killing -- target cell): A virus-infected or tumor cell with downregulated MHC I (missing-self) and upregulated stress ligands (MICA/MICB). Inhibitory KIRs have no ligand (no MHC I). Activating receptors NKG2D engage MICA/MICB, and NKp46 engages viral ligands. The balance favors activation -- NK cell releases perforin and granzymes toward the target cell, and the target cell undergoes apoptosis. A balance/scale icon shows the concept of integration of activating vs. inhibitory signals.</image>
VII. NK Cell Killing Mechanisms
NK cells kill through several mechanisms. The perforin-granzyme pathway involves formation of an immunological synapse with the target cell, polarization of lytic granules toward the synapse, release of perforin (which forms pores in the target cell membrane), and entry of granzymes (serine proteases, especially granzyme B) through the perforin pores, activating the caspase cascade and triggering apoptosis. The death receptor pathway involves NK cell expression of FasL (CD95L) and TRAIL (TNF-related apoptosis-inducing ligand), which bind Fas (CD95) and DR4/DR5 on target cells, respectively, activating the extrinsic apoptosis pathway. Antibody-dependent cellular cytotoxicity (ADCC) occurs when CD16 (FcγRIIIA) on NK cells binds IgG coating target cells, triggering the release of perforin and granzymes. This mechanism is important for therapeutic monoclonal antibodies such as rituximab and trastuzumab. NK cells also produce cytokines, including IFN-γ (which activates macrophages and promotes Th1 responses) and TNF-α (which is pro-inflammatory and has direct antitumor effects), within hours of activation.
VIII. NK Cell Activation by Cytokines
Several cytokines activate NK cells. IL-12 from macrophages and DCs activates NK cells and enhances IFN-γ production. IL-15 is critical for NK cell development, survival, and homeostasis. IL-18 synergizes with IL-12 for IFN-γ production. Type I interferons (IFN-α/β), produced early in viral infections, enhance NK cytotoxicity. IL-2 promotes NK cell proliferation and activation, which has been the basis for IL-2 therapy in cancer.
