Neutrophils (polymorphonuclear leukocytes) and macrophages, the two major types of phagocytic cells in the body, are crucial effector cells for both innate and adaptive immune responses.⁹ Detailed descriptions of these cell types are found in hematology and pathology textbooks (and in Chapter 47). Here, we will summarize their main features related to immune responses. Neutrophils are the most abundant leukocytes in the blood, but are not normally present in any healthy tissue. In responses to signals generated by microbial products and injured cells, neutrophils are rapidly recruited into tissue sites.¹⁰ In contrast, resident macrophages are present in the most healthy tissues of the body at all times, but their numbers increase significantly in response to inflammatory stimuli, due to recruitment of blood monocytes that rapidly differentiate into macrophages once they leave the circulation.¹¹ The resident macrophages in normal tissues are most abundant in the connective tissues underlying epithelial barriers such as the dermis and lamina propria of the gut. Resident macrophages may assume specialized morphologies and functions in particular organs, such as the alveolar macrophages in the lung, Kupffer cells lining hepatic sinusoids, osteoclasts in bone, and microglia cells in the brain. Resident macrophages perform sentinel and first-responder functions, reacting to pathogens or tissue injury by secreting of cytokines and chemokines that promote further recruitment of blood leukocytes, including neutrophils, and blood monocytes.

A major function of both neutrophils and macrophages is the phagocytic ingestion and killing of microbes. Microbial killing is accomplished by generation of reactive oxygen and nitrogen species through the action of phagocyte-oxidase, myeloperoxidase, and inducible nitric oxide synthase, and by proteolysis of the ingested microbes by several different lysosomal enzymes. Neutrophils also produce cytokines and chemokines, which amplify inflammatory responses. Neutrophils are terminally differentiated, cannot divide, and live only a few hours within tissues. They undergo programmed cell death after ingesting microbes. The critical role neutrophils play in innate immunity is reflected by the devastating infectious consequences that are linked to severe reductions in the absolute blood neutrophil counts seen in leukemia and cancer patients. Tissue macrophages may live for years in tissues, and recently recruited macrophages may divide, and live for weeks to months. In addition to microbial killing, macrophages perform the important functions of ingesting dead cells, including neutrophils and tissue debris, and stimulating tissue repair through the production of cytokines and growth factors. Subsets of macrophages have been described that differ in inflammatory and tissue repair functions.^12,13

Classically activated macrophages are induced by interferon-γ (IFNγ) secreting T cells, and perform proinflammatory and microbicidal functions. Alternatively activated macrophages are induced by interleukin (IL)-4- and -5-producing T cells, and produce anti-inflammatory cytokines (e.g., IL-10) and growth factors, which promote angiogenesis and fibrosis.

Natural Killer (NK) cells are bone marrow-derived cells of the innate immune system that arise from the common lymphoid progenitor but do not express highly diverse clonally distributed antigen receptors encoded by somatically rearranged genes, like B and T lymphocytes.^14,15 NK cells make up between 5% and 10% of blood lymphocytes, and are morphologically distinguished as CD16⁺, CD56⁺, and CD3^– large granular lymphocytes. NK cells are activated by contact with target cells that are infected or otherwise stressed, leading to release of perforin and granzymes from cytoplasmic granules, which cause the apoptotic death of the target cells. This killing mechanism is identical to the way CD8⁺ cytotoxic T lymphocytes (CTLs) kill targets, described later. NK cells also secrete interferon γ that promotes inflammation. NK cells distinguish healthy cells from infected or stressed targets by a variety of germline-encoded activating receptors that recognize stress-induced proteins, and inhibitory receptors, such as the killer Ig-like receptors, which recognize class I major histocompatibility complex (MHC) molecules that
are present in most healthy cells. A lack of class I MHC, often termed “missing self,” will result in NK cell activation due to a lack of inhibitory signals.

DCs are ubiquitous bone marrow-derived cells that play a fundamental role in immune surveillance.^16,17,18 They are constitutively present in many tissues but most abundant beneath epithelial barriers (skin, bronchopulmonary, gastrointestinal, genitourinary) and in lymphoid tissues (lymph nodes, spleen). They are named for their morphology in tissues, where they typically display dendrite-like projections that interdigitate between other cells, a feature that maximizes their potential contact with pathogens. DCs perform effector functions of the innate immune system in response to microbial infection, and are essential for initiating adaptive immune responses involving T cells. Two main subsets of DCs, identifiable by cell surface molecules, are conventional DCs and plasmacytoid DCs (pDCs). Conventional DCs are phagocytic cells that take up pathogens or microbial products in infected tissues and transport them into draining lymph nodes for display to T lymphocytes. This function is called antigen presentation, which we will discuss in more detail later in the chapter. Plasmacytoid pDCs are so named because they look like antibody-producing plasma cells but are otherwise unrelated. pDCs respond to viral nucleic acids by secreting abundant type I interferons, which are cytokines that make cells resistant to viral replication.¹⁹ Activation of pDCs by self nucleic acids and resulting abundant type I interferon secretion contributes to the pathogenesis of systemic lupus erythematosus.

Lymphocytes are the principal cells responsible for the defining characteristics of adaptive immunity, namely, extraordinary diversity of antigen specificities, specialization of functions that
adapt to different types of microbial infections, and memory. The majority of lymphocytes are located within lymphoid tissues and epithelial barrier tissue such as skin and gut. There is constant trafficking of between these tissues and the circulation, where they number 1-3 × 10³/mm³. In blood smears, resting lymphocytes appear as relatively homogeneous round cells with a high nuclear:cytoplasmic ratio and a diameter of approximately 7 µM. This uniform morphologic appearance does not reflect the existence of multiple lymphocyte subsets.

There are several subsets of lymphocytes (see Table 6.3). The two major types are B cells and T cells, typically distinguished by flow cytometric or immunohistochemical detection of lineage-specific surface molecules. B cells are the only cells that produce antibodies. Most B cells in blood and lymphoid tissues express membrane Ig, CD19, and CD20. Most T cells express CD3. Developmentally distinct subsets of B cells include follicular B cells, the most abundant and diverse type, and marginal zone B cells, which have a limited range of specificities. T cells have greater developmental and functional heterogeneity than B cells. The two numerically major subsets of T cells are distinguished by the expression of either CD4 or CD8, which correlate with helper or cytotoxic functions, respectively. Together CD4⁺ and CD8⁺ T cells account for approximately 85% to 90% of all T cells, and normally the CD4⁺:CD8⁺ T-cell ratio in blood and lymphoid organs is about 2. Among CD4⁺ T cells, there are several functional subsets, including Th1, Th2, Th17, and regulatory T cells. Other numerically minor subsets of T lymphocytes cell are NKT cells and γδT cells.

Both B and T lymphocytes are also categorized based on their state of functional differentiation, which is related to their prior experience with antigen. B and T lymphocytes that have completed their maturation but have not ever been activated by antigen in the peripheral immune system are called naïve lymphocytes. Naïve B and T cells cannot perform protective functions, but they respond to antigen stimulation in lymphoid organs by clonal expansion and differentiation into effector
lymphocytes, which are relatively short-lived cells that actively contribute to elimination of antigen. After most microbial antigen-specific B- and T-cell responses wane, a population of longlived progeny of the expanded lymphocyte clones persists as memory cells, which mediate rapid and specialized responses to subsequent exposures to the microbial antigens. Memory lymphocytes can survive for many years, and probably undergo a low level of antigen-independent proliferation to maintain their numbers.^20,21,22 In vitro assays for recall responses to antigens after infection or immunization indicate that most memory T cells express the CD45RO surface protein, while naïve T cells, such as found in neonates, are mostly CD45RA⁺. Memory T cells also express the IL-7 receptor. Effector T cells are usually localized within lymphoid organs or in infected tissues, and only rarely are they numerous in the blood, such as during acute EBV infections when they appear as lymphoblasts. Specific details of T-and B-cell antigen recognition, subsets, and effector function will be discussed later in this chapter.

These three cell types all have numerous cytoplasmic granules whose contents are released upon activation, and perform various proinflammatory and microbicidal functions.^23,24,25 Mast cells are bone marrow-derived, stationary tissue cells ubiquitously present adjacent to small blood vessels in most epithelial tissue. Their metachromatically staining granules contain several vasoactive amines such as histamine. Mast cells express highaffinity receptors for IgE antibodies, and cross-linking of the IgE by allergens causes activation of the cells, including granule release, and secretion of newly synthesized cytokines and eicosanoids. Mast cells are major contributors to allergic reactions and defense against parasites. Basophils share most of the properties just described for mast cells, except they are circulating cells of the myeloid lineage, which may be recruited to inflammatory sites. Their role in innate immunity is uncertain, in part because their numbers are low. Eosinophils are circulating granulocytes with eosinophilic granules containing major basic protein and proteolytic enzymes. Eosinophils are recruited to selective inflammatory sites, such as tissue with parasitic worm infections or sites of allergic inflammation. Their granule contents are toxic to helminths and are responsible for tissue damage and remodeling in chronic atopic diseases, such as asthma.

Lymphocyte development, the process by which progenitor stem cells give rise to mature naïve lymphocytes, occurs in the primary or generative lymphoid organs, namely, the bone marrow and thymus. This process is distinct from differentiation of naïve lymphocytes into effector lymphocytes, which occurs in response to antigen recognition in secondary or peripheral lymphoid organs, including lymph nodes, spleen, and mucosal lymphoid tissues.

Microbial invasion and infections can occur anywhere in the body, and the innate immune system deals with this widespread unpredictable threat by the ubiquitous presence of immunocompetent cells in all tissues, and the rapid delivery of additional cells from the circulation. In contrast, the adaptive immune system produces small numbers of millions of different clones of lymphocytes before exposure to microbial antigens, but a particular microbe-specific clone must be activated by specialized APCs before an effective response can be generated. Thus, the initiation of adaptive immune response requires delivery of lymphocytes, APCs, and antigen to common sites within lymphoid tissues. Within lymphoid tissues, subsets of lymphocytes and APCs must be highly motile to maximize the chances of productive physical interactions. Basic features of whole-body and local immune cell trafficking are described below.

Circulating leukocytes, including lymphocytes and myeloid cells, must interact with the endothelial lining of blood vessels in order to migrate into the tissue sites where they perform their effector functions.³⁸ This process is regulated by several factors, including flow characteristics of tissue microvasculature, adhesion molecules on both the leukocytes and endothelial cells, and chemokines produced by endothelial cells and tissue cells, which act on the leukocytes (see Table 6.2). Virtually all leukocyte extravasation occurs in postcapillary venules, which have an average diameter of 10 to 15 µM, and flow characteristics that promote margination of leukocytes to the periphery of the blood stream where they interact physically with endothelial cells. The adhesion molecules involved in the process include integrins on the leukocytes, selectins on the cell surfaces of leukocytes and endothelial cells, selectin ligands on leukocytes and endothelial cells, and integrin ligands on the endothelial cells. Chemokines involved in this process are displayed on the surface of endothelial cells and bind chemokine receptors on the leukocytes (see Table 6.2).³⁹ Extravasation of all types of leukocytes follows a sequential set of steps, referred to as the multistep adhesion cascade, during which leukocytes bind to endothelial cells at appropriate locations, resist the fluid shear stress caused by the blood flow, and exit the vessel. The specific chemokines and adhesion molecules will vary depending on the leukocyte type and tissue site. The sequence of the adhesion cascade begins with low-affinity adhesive interactions and leukocyte rolling on endothelium mediated by P- and E-selectin expressed on activated endothelial cells, which bind to glycoprotein selectin ligands on leukocytes, and L-selectin expressed on leukocytes, which bind ligands on endothelial cells. The next step is activation of leukocyte integrins by chemokines. Chemokines that are produced by phagocytes and endothelial cells in infected or damaged tissues, or are constitutively produced in lymphoid organs, are displayed on the luminal endothelial surface by heparan sulfate glycosaminoglycans, where they can bind to chemokine receptors on rolling leukocytes. Signals generated by the chemokine receptors induce conformational changes in the integrins that enhance their affinity for integrin ligands on the endothelial cells. The next step is stable arrest of leukocytes on endothelium mediated by activated integrins. Leukocyte integrins include leukocyte function-associated antigen (LFA)-1 and Mac-1, which bind to intercellular adhesion molecule (ICAM)-1 on endothelial cells, and very late activation antigen (VLA)-4, which binds to endothelial vascular cell adhesion molecule (VCAM)-1. High-affinity binding of integrins to their endothelial ligands results in cessation of rolling, firm adhesion, and resistance to dislodgement by fluid shear stress. Finally, the leukocytes transmigrate between endothelial cells and out into tissues, a process that requires leukocyte LFA-1 and endothelial ICAM-1, and CD31 expressed on both the leukocyte and the junctional membrane of endothelial cells. VE-cadherin and associated junctional proteins that bridge postcapillary endothelial cells must be temporarily modified to permit passage of the transmigrating leukocytes. Leukocytes may also pass through endothelial cells by a poorly understood process called transcellular migration. Antibody blockade of leukocyte integrins is in clinical use for treatment of inflammatory bowel disease and multiple sclerosis.