Transendothelial Migration

Circulating neutrophils leave the intravascular compartment to enter the tissues in three major steps: margination and rolling on vascular endothelium, attachment to the endothelial cells, and transendothelial migration ( Fig. 56.5 ). In the inflamed tissues, neutrophils are recruited through regional differences in vascular flow and along concentration gradients of chemokines ( Fig. 56.6 , some members in Table 56.1 ), chemotactic tripeptides such as N -formylmethionyl-leucyl-phenylalanine ( N -fMLP), complement fragments (C5a), and leukotrienes (LTB ₄ ). Among chemokines, the CXC subfamily with a glutamate-leucine-arginine tripeptide sequence (such as interleukin-8 [IL-8/CXCL8]) are most important. ^, N -fMLP is an endogenous, chemotactic peptide derived from mitochondria in dying cells that attracts and activates neutrophils and macrophages. It mimics N -formyl oligopeptides released by bacteria and attracts and activates circulating blood leukocytes by binding to specific G-protein–coupled receptors on these cells.

Overview of Neutrophil Functions .

Neutrophils are produced and mature in the bone marrow over a 2-week period. On release from the marrow, neutrophils circulate for 6 to 8 hours before emigrating into tissues. At sites of infection, chemotactic factors enhance neutrophil adhesion to and emigration through the vascular endothelium, and neutrophils migrate in a directed fashion (chemotaxis) toward the pathogens. Phagocytosis of the offending organisms stimulates an increase in production of oxygen metabolites (respiratory burst), which facilitates neutrophil killing of the ingested microbes.

(Reproduced after permission and minor modifications from Benjamin and Maheshwari. Developmental immunology. In: Fanaroff and Martin’s Neonatal-Perinatal Medicine . 47, 752–788.)

Chemokines Are Classified According to the Position of Conserved Cysteine Residues Near the N -Terminus .

In CXC chemokines, two conserved cysteine (C) residues are separated by a variable amino acid (X). CC and C-chemokines have conserved cysteines, whereas the CX3C chemokine, fractalkine, has two cysteines separated by three variable amino acids.

(Reproduced after permission and minor modifications from Benjamin and Maheshwari. Developmental immunology. In: Fanaroff and Martin’s Neonatal-Perinatal Medicine . 47, 752–788.)

In inflamed tissues, alterations in the endothelial surface, with increased leukocyte-binding receptors, and increased gradients of the CXC motif-bearing chemokines (see Fig. 56.5 and Table 56.1 ) promote neutrophil emargination. This process is mediated through a process of repetitive binding and release of selectins (L-selectin on neutrophils, E- and P-selectin on endothelium; Fig. 56.7 ). Rolling neutrophils slow down and attach to endothelium through the binding of β ₂ -integrins to cognate receptors on endothelial cells. The β ₂ -integrins expressed on neutrophils include the leukocyte function-associated antigen 1 (LFA-1) (α _L β ₂ , CD11a/CD18), CR3 (Mac-1, α _M β ₂ , CD11b/CD18), and p150, 95 (α _X β ₂ , CD11c/CD18), which bind endothelial receptors such as the intercellular adhesion molecule 1 and 2 (ICAM-1 and ICAM-2) and vascular cell adhesion molecule 1 (VCAM-1). LFA-1 binds to both ICAM-1 and ICAM-2, whereas Mac-1 and p150, 95 bind exclusively to ICAM-1. These neutrophils undergo activation on the endothelial surface and migrate through the capillary/venular wall, a process that involves the platelet-endothelial cell adhesion molecule (PECAM1, CD31), the integrin-associated protein (CD47), and other junctional molecules ( Fig. 56.8 ).

Adhesion of White Blood Cells to Endothelial Cells Is Mediated by Several Receptor–Ligand Pair Interactions .

A distinct set of endothelial molecules is involved in each stage of leukocyte recruitment (rolling, attachment, and transendothelial migration). In the rolling phase, L-selectin receptors on neutrophils bind to one of several ligands on the endothelial cells. Similarly, in the attachment phase, neutrophil β-integrins bind to the ICAM 1 to 3 or VCAM-1 receptors. ICAM , Intercellular adhesion molecule; VCAM , vascular cell adhesion molecule.

(Reproduced after permission and minor modifications from Benjamin and Maheshwari. Developmental immunology. In: Fanaroff and Martin’s Neonatal-Perinatal Medicine . 47, 752–788.)

Emigration of Neutrophils From the Vascular Space .

Effector molecules are indicated in gray text. Initial capture is mediated by interactions between neutrophil/leukocyte (L)-selectin and endothelial cell (E, P)-selectins and their respective carbohydrate ligands on the opposing cell surface (e.g., P-selectin glycoprotein ligand-1 [PSGL-1]), whereas integrins (e.g., very late antigen 4 [VLA4]) are responsible for firm adherence. Selectin-mediated signaling slows the rolling or tumbling neutrophils, partially countering the shear forces resulting from blood flow. Chemokines diffusing into the bloodstream from sites of microbial invasion in the tissues bind to specific receptors and activate the neutrophil, which is then arrested by integrin-dependent interactions. The activated neutrophil flattens against the endothelium and crawls along the luminal surface of the vessel wall. Platelet-endothelial cell adhesion molecule-1 (PECAM-1), junctional adhesion molecules (JAMs), and endothelial cell-selective adhesion molecule (ESAM) localized to interendothelial cell junctions interact with PECAM-1 and CD99 on the neutrophil surface to permit neutrophil migration between and through endothelial cells. Once in the tissues, the polarized neutrophils move up the mediator concentration gradient to reach the site of microbial invasion. See text for further details. ICAM-1, Intercellular adhesion molecule; LFA-1, leukocyte function–associated antigen; MADCAM1, mucosal addressin cell adhesion molecule 1; PI(3)K, phosphatidylinositol-3-phosphate kinase; SRC, sarcoma; VAV1, vav1 guanine nucleotide exchange factor; VCAM1, vascular cell adhesion molecule 1.

(Reproduced after permission and minor modifications from DeLeo and Nauseef. Scanning electron micrographs of nonadherent and adherent neutrophils. Granulocytic phagocytes. In: Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases . 2020.)

Compared with neutrophils from adults, neonatal neutrophils adhere less avidly to the endothelium. Neonatal neutrophils have lower selectin expression than adults, which might be further reduced by perinatal stress such as in birth asphyxia. In addition, neonatal neutrophils display defective shedding of L-selectin. These properties impair neutrophil rolling on the endothelial surface and thereby the tissue recruitment of these cells. Neutrophil-endothelial adherence and neutrophil transmigration is also limited in neonates due to a developmental deficiency of mac-1 (CD18/CD11b), one of the β ₂ integrins. The transendothelial migration of neutrophils is also limited due to less deformability of neonatal neutrophils.

Chemotaxis

Once outside the blood vessel, neutrophils migrate (see Fig. 56.5 ) along concentration gradients of chemoattractants such as IL-8 and other chemokines, f-MLP, and C5a. These chemotaxins bind high-affinity G-protein–coupled receptors on the leukocyte surface. Bacterial products may be more potent chemoattractants than host chemokines. Neonatal neutrophils migrate slower than do adult neutrophils until 2 to 3 weeks after birth and achieve normal chemotaxis only by 40 to 42 weeks of postconceptional age or later. ^, Although minor infections may enhance chemotaxis in neonates, the migratory responses of neonatal neutrophils may become further depressed during systemic gram-negative sepsis. ^,

Neonatal neutrophils bind various chemoattractants normally, but chemoattractant-induced membrane depolarization, calcium transport, and sugar uptake are less efficient. These chemotactic defects occur because neonatal neutrophils may have a larger, poorly motile neutrophil subpopulation, impaired calcium mobilization, and aberrations in intracellular signaling pathways such as NF-κB activation. ^, Lower Mac-1 expression can also impede chemotaxis due to impaired neutrophil interaction with the extracellular matrix. ^, Inability to effectively direct neutrophils to the bacterial source contributes to the neonatal vulnerability to septicemia.

Phagocytosis

Phagocytosis is a specialized form of endocytosis directed at engulfing solid particles into an internal phagosome, which then “matures” through interactions with the endosomal compartment and eventually fuses with a lysosome for killing of internalized microorganisms and terminal degradation of the cargo (see Fig. 56.5 ). Phagocytosis is more efficient when the target is opsonized by specific immunoglobulin (IG) G. Neutrophils express receptors for IgG (F _cγ receptors I-III, or CD16, CD32, CD64), C3b (CR1), and iC3b (CR3). In some instances, microorganisms may be ingested without opsonization through lectin-carbohydrate (lectins on bacterial fimbriae interact with neutrophil glycoproteins), protein-protein (proteins such as filamentous hemagglutinin that express the arg-gly-asp or RGD amino acid sequence bind to integrins), and hydrophobic-protein (bacterial glycolipids and neutrophil integrins) interactions. ^, The interaction of IgG or complement ( Fig. 56.9 ) receptors on the neutrophil surface with the opsonized particle trigger cytoskeletal rearrangements to enclose the opsonized particle within a phagosome. Phagocytosis is most efficient when organisms are coated with opsonins such as IgG and C ₃ , which interact with cognate receptors (see Fig. 56.5 ). Neutrophils also express integrin receptors for matrix proteins such as fibronectin, laminin, and collagen. ^{, ,}

Complement System Activation Cascade .

Activation of the classic pathway (left) and the alternative pathway (right) causes generation of soluble factors that amplify phagocyte functions and produce a membrane-bound attack complex that damages cell membranes.

(Reproduced after permission and minor modifications from Benjamin and Maheshwari. Developmental immunology. In: Fanaroff and Martin’s Neonatal-Perinatal Medicine . 47, 752–788.)

Preterm neutrophils have impaired phagocytosis, which corrects only in the late third trimester. Preterm neutrophils ingest particles more slowly and ingest fewer bacteria (such as Escherichia coli ). The lack of opsonic activity with a lower concentration of specific antibodies may be a contributing factor. Preterm neutrophils also have lower expression of CD16 (F _cγ RIII) and CD32 (F _cγ RII), which bind IgG. The expression of CD16, but not that of CD32, may improve during the neonatal period.

Intracellular Killing

The phagolysosome exposes the ingested microorganism to toxic substances such as reactive oxygen species (ROS) in an enclosed space (see Fig. 56.5 ). The production of ROS utilizes a “respiratory burst,” where an NADPH-dependent oxidase localized on the phagosome membrane reduces molecular oxygen (O ₂ ) to superoxide anion (O• ₂ ⁻ ) and leads to subsequent generation of peroxide (H ₂ O ₂ ). The hydroxyl radical (OH•, formed in the presence of iron) also contributes to the microbicidal capacity of neutrophils. The oxygen-dependent bactericidal mechanisms can be broadly divided into myeloperoxidase (MPO)-independent (such as H ₂ O ₂ ) and MPO-dependent (MPO catalyzes reactions between H ₂ O ₂ and halides to form highly reactive products). H ₂ O ₂ is a relatively weaker bactericidal agent than the MPO-H ₂ O ₂ -halide system.

Neutrophils also have many nonoxidative killing mechanisms such as low pH (as low as 6.0), broad-spectrum antimicrobial peptides such as defensins, bactericidal/permeability-increasing protein (BPI), lactoferrin, lysozyme, and a variety of cationic proteins. BPI binds lipopolysaccharide and can damage the outer membrane of gram-negative bacteria. Lactoferrin, as an iron chelator, is bacteriostatic and is also involved in neutrophil degranulation and in granulocytopoiesis. Lysozyme hydrolyses bacterial cell wall peptidoglycan. The neutrophil primary granules also contain other cationic antibacterial proteins such as azurocidin, indolicidin, and cathelicidin. ^,

Preterm neutrophils have a less active respiratory burst, which reduces the efficiency of intracellular bacterial killing (in addition to low opsonic activity). ^, The neutrophil respiratory burst in preterm infants improves postnatally as a function of chronologic age in about 2 months but continues to have an overall weaker oxidative burst than adults. The antiviral activity of neonatal neutrophils is also diminished.

Neutrophil extracellular traps (NETs) ( Fig. 56.10 ), which are composed of extracellular strands of decondensed DNA bound to histones, elastase, myeloperoxidase, lactoferrin, and defensins, add to the extracellular antimicrobial defense ability of neutrophils. ^, The formation of NETs, usually referred to as NETosis, usually involves two major pathways: (1) an NADPH oxidase-dependent production of ROS ^, and (2) the complement system. ^, The NET-forming ability of term and preterm neonates is less than that in adults, ^, possibly because of inhibitory mediators such as the NET-inhibitory factor and its related peptides. ^,

Neutrophil extracellular traps (NETs) are blue weblike structures. In this merged image, the circular blue structures are nuclei.

(Reproduced after permission and minor modifications from Baines et al. Biology of neutrophils. In: Middleton’s Allergy: Principles and Practice . 17, 267–277.e1.)

Degranulation

Neutrophil granules ( Fig. 56.11 ) are of at least four types: (1) the “azurophilic” or primary granules (stain with the azure A dye), (2) “specific” or secondary granules, (3) tertiary or gelatinase granules, and (4) secretory vesicles. Azurophilic granules contain MPO (peroxidase-positive granules); proteases such as cathepsins, proteinase-3, and elastase; and antimicrobial proteins such as defensins and the BPI. These granules release their contents into the phagolysosomes for intracellular killing. Specific granules contain antibacterial agents such as lactoferrin, lysozyme, and receptors for complement components. These granules play an important role in extracellular killing and fuse with the cell membrane to release their contents, such as integrins, cytochrome- b 558, receptors for chemotactic agents, and opsonins, to the cell surface by exocytosis.

Neutrophils Contain Many Granules .

The cell here looks trinucleated because the connections between the three lobes are outside the fields of view. The granules are marked by arrows . There is a centrally located centriole (C). The azurophilic granules (white arrows) are electron-dense and contain myeloperoxidase, proteolytic enzymes, antibacterials such as defensins, and bactericidal permeability-increasing protein. Specific granules are relatively electron-lucent and contain lactoferrin, lysozyme, receptors for complement fragments, and f-MLP. The tertiary granules contain gelatinase. The secretory vesicles contain surface membrane-bound receptors.

(Image reproduced after permission and minor modifications from Gartner. Blood and hemopoiesis. Textbook of Histology . 10, 217–248.e2.)

Neutrophils from term neonates contain granules and show degranulation responses similar to adults. However, those from preterm infants release less BPI, elastase, and lactoferrin. ^, Collectively, developmental deficiencies in neutrophil function explain, in part, the susceptibility of neonates to invasive bacterial infections.