Nitric oxide Low levels or none Induced following transcriptional activation

of iNOS

Degranulation Major response; induced by

cytoskeletal rearrangement

Not prominent

Cytokine production Low levels per cell Major functional activity, large amounts

per cell, requires transcriptional activation of

cytokine genes

Secretion of

lysosomal enzymes

Prominent Less

Extracellular traps Rapidly induced, by extrusion

of nuclear contents

Little

Fig. 2.8 Distinguishing properties of neutrophils and monocytes. This table lists the major differences

between neutrophils and macrophages. These two cell types share many features, such as phagocytosis,

chemotaxis, and ability to migrate through blood vessels into tissues. HSC, Hematopoietic stem cell; iNOS,

inducible nitric oxide synthase; NET, neutrophil extracellular traps.

34 CHAPTER 2 Innate Immunity

debris. Neutrophils live for only several hours in tissues, so they are the early responders, but they do

not provide prolonged defense.

• Monocytes are less abundant in the blood than neutrophils, numbering 500 to 1000 per µL (see Fig.

2.9B). They also ingest microbes in the blood and in

tissues. During inflammatory reactions, monocytes

enter extravascular tissues and differentiate into

cells called macrophages, which, unlike neutrophils,

survive in these sites for long periods. Thus, blood

monocytes and tissue macrophages are two stages of

the same cell lineage, which often is called the mononuclear phagocyte system (Fig. 2.10). Some macrophages that are resident in different tissues, such as

During early development

In adult homeostasis and inflammatory reactions

Brain:

Microglial

cells Hematopoietic

stem cell

Hematopoietic

stem cell

Hematopoietic

stem cell

Tissue

macrophage

precursor

Monocyte

Differentiation

Monocyte/

dendritic cell

precursor

Macrophage

Activation

Activated

macrophages

Bone marrow

Bone marrow

Blood

Fetal hematopoietic

organs (yolk sac, liver)

Tissue

Blood

Tissue

Liver:

Kupffer cells

Lung:

Alveolar

macrophage

Spleen:

Sinusoidal macrophages

Fig. 2.10 Maturation of mononuclear phagocytes. In the steady state in adults, and during inflammatory

reactions, precursors in the bone marrow give rise to circulating monocytes, which enter peripheral tissues,

mature to form macrophages, and are activated locally. In fetal life, precursors in the yolk sac and liver give

rise to cells that seed tissues to generate specialized tissue-resident macrophages.

A B

Fig. 2.9 Morphology of neutrophils and monocytes. A,

Light micrograph of blood neutrophil shows the multilobed

nucleus, which is why these cells are also called polymorphonuclear leukocytes, and the faint cytoplasmic granules, most of

which are lysosomes. B, Light micrograph of blood monocyte

shows the typical horseshoe-shaped nucleus.

CHAPTER 2 Innate Immunity 35

the brain, liver, and lungs, are derived not from circulating monocytes but from progenitors in the yolk

sac or fetal liver early during the development of the

organism. Macrophages are also found in all connective tissues and organs of the body.

Macrophages serve several important roles in host

defense: they ingest and destroy microbes, they clear

dead tissues and initiate the process of tissue repair,

and they produce cytokines that induce and regulate

inflammation (Fig. 2.11). A number of receptor families

are expressed in macrophages and involved in the activation and functions of these cells. Pattern recognition

receptors discussed earlier, including TLRs and NLRs,

recognize products of microbes and damaged cells and

activate the macrophages. Phagocytosis is mediated

by cell surface receptors, such as mannose receptors

and scavenger receptors, which directly bind microbes

(and other particles), and receptors for antibodies or

products of complement activation that are bound to

microbes. These antibody and complement receptors

are also expressed by neutrophils. Some of these phagocytic receptors activate the microbial killing functions of

macrophages as well. In addition, macrophages respond

to various cytokines.

Macrophages may be activated by two different pathways that serve distinct functions (see Fig. 6.9 in Chapter 6).

These pathways of activation have been called classical

and alternative. Classical macrophage activation is

induced by innate immune signals, such as from TLRs,

and by the cytokine IFN-?, which may be produced in

both innate and adaptive immune responses. Classically

activated macrophages, also called M1, are involved

Toll-like

receptor

Microbe

Complement

receptor

Cytokine

(e.g., IFN-?)

Cytokine

receptor

Cytokines

(TNF, IL-1,

IL-6, IL-12)

Phagocyte

oxidase

Reactive

oxygen

species (ROS)

Nitric

oxide

iNOS Phagocytosis

of microbe into

phagosome

Inflammation,

enhanced

adaptive

immunity

Killing of microbes

Activation

Complement

fragment

Fig. 2.11 Activation and functions of macrophages. In innate immune responses, macrophages are activated by microbial products binding to TLRs and by cytokines, such as NK cell–derived interferon-? (IFN-?),

which lead to the production of proteins that mediate inflammatory and microbicidal functions of these cells.

Cell surface complement receptors promote the phagocytosis of complement-coated microbes as well as

activation of the macrophages. (Macrophage Fc receptors for IgG [not shown] bind antibody-coated microbes

and perform similar functions as the complement receptors.) IL, Interleukin; iNOS, inducible nitric oxide synthase; TNF, tumor necrosis factor.

36 CHAPTER 2 Innate Immunity

in destroying microbes and in triggering inflammation. Alternative macrophage activation occurs in the

absence of strong TLR signals and is induced by the

cytokines IL-4 and IL-13; these macrophages, called

M2, appear to be more important for tissue repair and

to terminate inflammation. The relative abundance of

these two forms of activated macrophages may influence

the outcome of host reactions and contribute to various disorders. We will return to the functions of these

macrophage populations in Chapter 6, when we discuss

cell-mediated immunity.

Although our discussion has been limited to the role

of phagocytes in innate immunity, macrophages are also

important effector cells in both the cell-mediated arm

and the humoral arm of adaptive immunity, as discussed

in Chapters 6 and 8, respectively.

Dendritic Cells

Dendritic cells function as sentinels in tissues that

respond to microbes by producing numerous cytokines,

which serve two main functions: they initiate inflammation and they stimulate adaptive immune responses. They

also capture protein antigens and display fragments of

these antigens to T cells. By sensing microbes and interacting with lymphocytes, especially T cells, dendritic cells

constitute an important bridge between innate and adaptive immunity. We discuss the properties and functions

of dendritic cells further in Chapter 3 in the context of

antigen presentation.

Mast Cells

Mast cells are bone marrow–derived cells with abundant

cytoplasmic granules that are present throughout the

skin and mucosal barriers. Mast cells can be activated by

microbial products binding to TLRs and by components

of the complement system as part of innate immunity

or by an antibody-dependent mechanism in adaptive

immunity. Mast cell granules contain vasoactive amines

such as histamine that cause vasodilation and increased

capillary permeability, as well as proteolytic enzymes

that can kill bacteria or inactivate microbial toxins. Mast

cells also synthesize and secrete lipid mediators (e.g.,

prostaglandins and leukotrienes) and cytokines (e.g.,

tumor necrosis factor [TNF]), which stimulate inflammation. Mast cell products provide defense against

helminths and other pathogens, as well as protection

against snake and insect venoms, and they are responsible for symptoms of allergic diseases (see Chapter 11).

Innate Lymphoid Cells

Innate lymphoid cells (ILCs) are tissue-resident cells

that produce cytokines similar to those secreted by

helper T lymphocytes but do not express T cell antigen receptors (TCRs). ILCs have been divided into

three major groups based on their secreted cytokines;

these groups correspond to the Th1, Th2, and Th17

subsets of CD4+ T cells that we describe in Chapter

6. The responses of ILCs are often stimulated by cytokines produced by damaged epithelial and other cells

at sites of infection. ILCs likely provide early defense

against infections in tissues, but their essential roles in

host defense or immunological diseases, especially in

humans, are not clear.

Natural Killer Cells

NK cells recognize infected and stressed cells and

respond by killing these cells and by secreting the

macrophage-activating cytokine IFN-? (Fig. 2.12). NK

cells are developmentally related to group 1 ILCs and

Virus-infected

cell

NK cell

Macrophage

with

phagocytosed

microbes

IL-12

IFN-?

Killing of

phagocytosed

microbes

Killing of

infected cells

A

B

Fig. 2.12 Functions of natural killer (NK) cells. A, NK cells

kill host cells infected by intracellular microbes, thus eliminating

reservoirs of infection. B, NK cells respond to interleukin-12 (IL12) produced by macrophages and secrete interferon-? (IFN-?),

which activates the macrophages to kill phagocytosed microbes.

CHAPTER 2 Innate Immunity 37

make up approximately 10% of the cells with lymphocyte morphology in the blood and peripheral lymphoid

organs. NK cells contain cytoplasmic granules and

express some unique surface proteins but do not express

immunoglobulins or T cell receptors, the antigen receptors of B and T lymphocytes, respectively.

On activation by infected cells, NK cells empty the

contents of their cytoplasmic granules into the extracellular space at the point of contact with the infected

cell. The granule proteins then enter infected cells and

activate enzymes that induce apoptosis. The cytotoxic

mechanisms of NK cells, which are the same as the

mechanisms used by cytotoxic T lymphocytes (CTLs;

see Chapter 6), result in the death of infected cells. Thus,

as with CTLs, NK cells function to eliminate cellular reservoirs of infection and eradicate infections by obligate

intracellular microbes, such as viruses. In addition, NK

cells may contribute to the destruction of tumors.

Activated NK cells also synthesize and secrete the

cytokine interferon-? (IFN-?), which activates macrophages to become more effective at killing phagocytosed microbes. Cytokines secreted by macrophages and

dendritic cells that have encountered microbes enhance

the ability of NK cells to protect against infections. Three

of these NK cell–activating cytokines are interleukin-15

(IL-15), type I IFNs, and interleukin-12 (IL-12). IL-15

is important for the development and maturation of

NK cells, and type I IFNs and IL-12 enhance the killing

functions of NK cells. Thus, NK cells and macrophages

are examples of two cell types that function cooperatively to eliminate intracellular microbes: macrophages

ingest microbes and produce IL-12, IL-12 activates NK

cells to secrete IFN-?, and IFN-? in turn activates the

macrophages to kill the ingested microbes. As discussed

in Chapter 6, essentially the same sequence of reactions

involving macrophages and T lymphocytes is central to

the cell-mediated arm of adaptive immunity.

The activation of NK cells is determined by a balance between engagement of activating and inhibitory receptors (Fig. 2.13). The activating receptors

recognize cell surface molecules typically expressed

on cells infected with viruses and intracellular bacteria, some cancer cells, and cells stressed by DNA

damage. These receptors enable NK cells to eliminate

A

B

Inhibitory

receptor

Activating

receptor

Activating

ligand for

NK cell

NK cell

not activated;

no cell killing

NK cell

activated;

killing of

infected cell

Self class I

MHC–self

peptide complex

Virus-infected

cell (virus

inhibits class I

MHC expression)

Inhibitory receptor not engaged

NK cell

NK cell

Normal

cell

Inhibitory receptor engaged

Fig. 2.13 Activating and inhibitory receptors of natural killer (NK) cells. A, Healthy host cells express self

class I major histocompatibility complex (MHC) molecules, which are recognized by inhibitory receptors, thus

ensuring that NK cells do not attack normal host cells. Note that healthy cells may express ligands for activating receptors (as shown) or may not express such ligands, but they are not attacked by NK cells because

they engage the inhibitory receptors. B, NK cells are activated by infected cells in which ligands for activating

receptors are expressed (often at high levels) and class I MHC expression is reduced so that the inhibitory

receptors are not engaged. The result is that the infected cells are killed.

38 CHAPTER 2 Innate Immunity

cells infected with intracellular microbes, as well as

irreparably injured cells and tumor cells. One of the

well-defined activating receptors of NK cells is called

NKG2D; it recognizes molecules that resemble class

I major histocompatibility complex (MHC) proteins

and are expressed in response to many types of cellular

stress. Another activating receptor, called CD16, is specific for immunoglobulin G (IgG) antibodies bound to

cells. The recognition of antibody-coated cells results in

killing of these cells, a phenomenon called antibody-dependent cellular cytotoxicity (ADCC). NK cells are the

principal mediators of ADCC. The role of this reaction in antibody-mediated immunity is described in

Chapter 8. Activating receptors on NK cells have

signaling subunits that contain immunoreceptor

tyrosine-based activation motifs (ITAMs) in their cytoplasmic tails. ITAMs, which also are present in subunits

of lymphocyte antigen receptor–associated signaling

molecules, become phosphorylated on tyrosine residues

when the receptors recognize their activating ligands.

The phosphorylated ITAMs bind and promote the activation of cytosolic protein tyrosine kinases, and these

enzymes phosphorylate, and thereby activate, other substrates in several different downstream signal transduction pathways, eventually leading to cytotoxic granule

exocytosis and production of IFN-?.

The inhibitory receptors of NK cells block signaling

by activating receptors and are specific for self class I

MHC molecules, which are expressed on all healthy

nucleated cells. Therefore, class I MHC expression

protects healthy cells from destruction by NK cells. (In

Chapter 3, we describe the important function of MHC

molecules in displaying peptide antigens to T lymphocytes.) Two major families of NK cell inhibitory receptors in humans are the killer cell immunoglobulin-like

receptors (KIRs), so called because they share structural

homology with Ig molecules (see Chapter 4), and receptors consisting of a protein called CD94 and a lectin

subunit called NKG2. Both families of inhibitory receptors contain in their cytoplasmic tails structural motifs

called immunoreceptor tyrosine-based inhibitory motifs

(ITIMs), which become phosphorylated on tyrosine residues when the receptors bind class I MHC molecules. The

phosphorylated ITIMs bind and promote activation of

cytosolic protein tyrosine phosphatases. These enzymes

remove phosphate groups from the tyrosine residues of

various signaling molecules, thereby counteracting the

function of the ITAMs and blocking the activation of

NK cells through activating receptors. Therefore, when

the inhibitory receptors of NK cells encounter self MHC

molecules on normal host cells, the NK cells are shut

off (see Fig. 2.13). Many viruses have developed mechanisms to block expression of class I molecules in infected

cells, which allows them to evade killing by virus-specific

CD8+ CTLs. When this happens, the NK cell inhibitory

receptors are not engaged, and if the virus induces expression of activating ligands at the same time, the NK cells

become activated and eliminate the virus-infected cells.

The role of NK cells and CTLs in defense illustrates

how hosts and microbes are engaged in a constant struggle

for survival. The host uses CTLs to recognize MHC-displayed viral antigens, viruses inhibit MHC expression to

evade killing of the infected cells by CTLs, and NK cells

can compensate for the defective CTL response because

the NK cells are more effective in the absence of MHC

molecules. The winner of this struggle, the host or the

microbe, determines the outcome of the infection. The

same principles may apply to the functions of NK cells

in eradication of tumors, many of which also attempt to

escape from CTL-mediated killing by reducing expression of class I MHC molecules (see Chapter 10).

Lymphocytes with Limited Diversity

Several types of lymphocytes that have some features of

T and B lymphocytes also function in the early defense

against microbes and may be considered part of the

innate immune system. A unifying characteristic of

these lymphocytes is that they express somatically rearranged antigen receptors (as do classical T and B cells),

but the receptors have limited diversity.

• As mentioned earlier, ?d T cells are present in

epithelia.

• NK-T cells express TCRs with limited diversity and

surface molecules typically found on NK cells. They

are present in epithelia and lymphoid organs. They

recognize microbial lipids bound to a class I MHC–

related molecule called CD1.

• Mucosal associated invariant T (MAIT) cells express

TCRs with limited diversity but do not express CD4 or

CD8. They are present in mucosal tissues and are most

abundant in the human liver, accounting for 20% to

40% of all T cells in that organ. Many MAIT cells are

specific for bacterial vitamin B metabolites and likely

contribute to innate defense against intestinal bacteria

that transgress the mucosal barrier and enter the portal

circulation.

CHAPTER 2 Innate Immunity 39

• B-1 cells are a population of B lymphocytes that are

found mostly in the peritoneal cavity and mucosal

tissues, where they produce antibodies in response

to microbes and microbial toxins that pass through

the walls of the intestine. Circulating IgM antibodies

found in the blood of normal individuals, even without specific immunization, are called natural antibodies. They are the products of B-1 cells, and many

of these antibodies are specific for carbohydrates that

are present in the cell walls of many bacteria and for

ABO blood group antigens found on red blood cells

(discussed in Chapter 10).

• Another type of B lymphocyte, marginal-zone B

cells, is present at the edges of lymphoid follicles in

the spleen and other organs and also is involved in

rapid antibody responses to blood-borne polysaccharide-rich microbes.