Developmental Immunology

Normal PMN Concentrations and Neutropenia.

Circulating concentrations of PMNs increase dramatically at birth, peak at 12-24 hours, and then decline slowly by 72 hours to remain stable during the rest of the neonatal period. Blood PMN concentrations can be interpreted using one of several available reference ranges. The absolute neutrophil count can be calculated from a routine complete blood count (CBC) by multiplying the white cell count with the sum of segmented and band neutrophil percentages on the differential count. Manroe et al. were the first to compile reference ranges for blood neutrophil concentrations in neonates using data from a cohort of 434 neonates born at 38.9 ± 2.4 weeks’ gestation. They showed that the neutrophil counts peaked at 12-24 hours with 95% confidence limits of 7,800-14,500/µL and then stabilized at a lower value of 1,750 by 72 hours of life. A stable upper limit was achieved at 6.6 days of age. Although these ranges were useful for term and late preterm neonates, these did not include many preterm infants. To address this deficiency, Mouzinho et al. compiled ANC values from 1,788 CBCs drawn from 63 neonates born at 29.9 ± 2.3 weeks’ gestation. Their revised charts were comparable to those from Manroe et al. near the upper limits of blood neutrophil concentrations but showed greater variation at the lower limit. In a more recent study, Schmutz et al. compiled a new set of reference ranges for ANCs using data from 30,354 CBCs from neonates born at 23-42 weeks’ gestation. Besides the large sample size, a major strength of this study was the use of automated blood counting instrumentation, which allowed greater consistency in identification of neutrophils. In the interval between 72 and 240 hours after birth, the ANC ranged between 2700-13,000/µL (5 ^th -95th percentile) for infants >36 weeks’ gestation, between 1000-12,500/µL at 28-36 weeks, and 1300-15,300/µL at <28 weeks’ gestation. The upper limits of ANC in this data set were higher than ranges reported by both Manroe and Mouzinho, which may have been due to the high altitude at which the participating centers were located.

When searching for the cause of neutropenia (<1500 PMNs/mm ⁹ ) in infants, a strong suspicion of infection is warranted, although maternal preeclampsia, premature birth, birth depression, intraventricular hemorrhage, and hemolytic disease may result in low peripheral PMN counts. Persistent neutropenia is one feature that is frequently seen in patients who succumb to overwhelming sepsis. Neutropenia in these infants may be due to increased margination of activated circulating cells or to rapid depletion of circulating and bone marrow storage pool PMNs. Failure to provide adequate numbers of PMNs to the sites of microbial invasion may contribute to the risk of overwhelming sepsis in neonates.

Rolling and Adhesion.

PMN adhesion to endothelial cells is a crucial step in the recruitment of these leukocytes to inflammatory sites. Although PMNs can adhere to normal or activated vascular endothelium, inflammation is associated with hemodynamic and biochemical changes in blood vessels that facilitate leukocyte adhesion to the vessel endothelial lining. PMNs flowing through an inflamed venule may transiently adhere to the endothelium to “brake” their flow, causing the neutrophils to “tumble” or “roll” on the vascular surface. Leukocyte rolling is mediated via the interaction of L-selectin molecules on neutrophils with endothelial glycoproteins called addressins ( Fig. 47.2 ): glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1), CD34/sgp90, endomucin, podocalyxin, endoglycan, and the mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1). Endothelial cells also express two selectin molecules, P-selectin/CD62P and E-selectin/CD62E, which interact with the P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin on leukocytes. Engagement of these selectins activates signaling pathways in neutrophils that initiate neutrophil rolling and activation.

Circulating neutrophils usually roll along vascular endothelial cells with transient interactions between neutrophil selectins on the cell surface and counter-receptors on the endothelium. When endothelial cells are activated during inflammation, more selectin counter-receptors are expressed, and additional proteins (intercellular adhesion molecule [ICAM]-1 and ICAM-2) of the immunoglobulin supergene family, which serve as ligands for the neutrophil integrin family, are activated. Specific receptor-ligand interactions at the cell surface membrane facilitating neutrophil adhesion to vascular endothelium are shown in Fig. 47.2 .

After a few rolling events, leukocytes slow down and rest on the endothelium. During this phase, the interaction of leukocyte integrins with endothelial receptors initiates signaling events in the endothelium leading to transendothelial migration of leukocytes. Integrins involved in neutrophil trafficking include three heterodimeric proteins, each of which consists of one immunologically distinct “α” subunit (leukocyte functional antigen-1; CD11a, Mac-1; CD11b, p150, 95; CD11c), along with a common “β” subunit (CD18). Monoclonal antibodies directed against CD11b/CD18 inhibit PMN aggregation, spread, chemotaxis, and adhesion to endothelial cells. CD11b/CD18 and CD11c/CD18 serve as complement receptors and mediate complement-coated (fragment iC3b) particle ingestion by PMNs. CD11a/CD18 has been shown to be important in PMN killing of target cells through antibody-dependent mechanisms. Patients with a heritable deficiency of these leukocyte cell surface glycoproteins have recurrent infections characterized by failure to accumulate granulocytes at sites of infection.

Increased interactions of neutrophil cell surface integrins with endothelial ICAM-1 and ICAM-2 results in firm neutrophil-endothelial adhesive interactions. After tight adhesion, the PMN begins a process of diapedesis through adjacent endothelial cells and the intact underlying basement membrane. Once the PMN has penetrated the basement membrane, the cell migrates from the blood vessel into the area of inflammation. In the tissues, initial random migration (chemokinesis) becomes progressively directed (chemotaxis) toward the nidus of microorganisms along concentration gradients of bacterial products or chemotactic factors.

Adhesion of PMNs to artificial surfaces in vitro is comparable for unstimulated PMNs isolated from neonatal or adult blood. When PMNs from term neonates are stimulated with chemotactic factors, adhesion is greatly diminished, however, compared with cells isolated from adults. More profound deficits are displayed by PMNs from preterm infants. Neonatal PMNs show lower selectin expression, defective shedding of L-selectin, and less stimulated mobilization and overall expression of CD11b/CD18 glycoproteins on the plasma membrane. Other causes of diminished stimulated adherence include impaired capacity to upregulate cell surface chemotactic receptors, lower granular content of lactoferrin, less fibronectin binding to the cell surface, and less shape change (failure to increase significantly overall cell surface area on chemotactic factor stimulation). Downregulation of L-selectin expression on term newborn cord blood granulocytes and monocytes during acute inflammation has been shown, although the pattern and level of shedding vary from neutrophils isolated from adult subjects.

Compared to neutrophils from adults, neutrophils from both term and preterm neonates adhere poorly to endothelium. Neonatal neutrophils have lower selectin and β-integrin expression. In neonates, L-selectin expression is lower at birth than in adults and decreases further during the first 24-72 hours. Neonatal “stress,” as in perinatal asphyxia, may further reduce L-selectin expression on neutrophils. In addition, neonatal neutrophils display defective shedding of L-selectin. The combination of lower expression and impaired shedding of L-selectin reduces the frequency of neutrophil rolling events, which are a rate-limiting step in the tissue recruitment of neutrophils. Characteristics of preterm vascular endothelium such as lower P -selectin expression further contribute to these defects in neutrophil recruitment.

Neonatal neutrophils have lower expression of Mac-1(CD11b/CD18), which correlates with lower neutrophil–endothelial adherence and transmigration. In addition, neutrophils from both preterm and term infants are unable to upregulate Mac-1 expression following stimulation by bacterial products.

Transendothelial migration of neutrophils is affected to some degree by deformability of neutrophils. Although initial studies reported conflicting data, deformability of mature resting neutrophils appears to be similar in healthy preterm neonates to their full-term counterparts and adults. However, the release of immature neutrophils from the NSP during sepsis is associated with an overall reduction in neutrophil deformability.

Interventions such as administration of recombinant G-CSF increase the expression of β ₂ -integrins but lower L-selectin expression on neonatal neutrophils (both term and preterm). In contrast, early dexamethasone administration decreases β ₂ integrin expression on neutrophils.

Chemotaxis.

Chemotaxis is defined as the directed cellular migration along the concentration gradient of a chemoattracting substance. This movement involves a series of orchestrated events, including the binding of the chemoattractant to cell surface receptors, generation of an intracellular second messenger that is coupled to the receptor-ligand binding, and remodeling of the plasma membrane and cytoskeleton to produce shape changes and proper orientation of the cellular contents toward the highest concentration of the chemoattractant. Morphologically, the PMN orients toward the chemoattractant with the formation of a lamellipodium along the leading edge. Most of the intracellular organelles remain at the posterior pole of the cell (uropod). As the cell moves, the leading edge adheres to available surfaces, and contraction of cytoskeletal microfilaments (actin and myosin) pulls along the rest of the cell. Many aspects of this process are poorly developed in PMNs isolated from neonatal blood. Some of the chemotactic defects of PMNs present during the neonatal period persist throughout early childhood.

Neutrophils from both term and preterm neonates have an impaired chemotactic response and migrate slowly compared to adult cells. Although neutrophils from term infants achieve normal chemotactic function by 2 weeks after birth, such postnatal neutrophil maturation begins 2-3 weeks after birth in immature preterm infants and proceeds very slowly. Neutrophils from preterm infants born at 34-36 weeks’ gestation achieve normal chemotaxis by 40-42 weeks’ postconceptional age (PCA). In more immature preterm infants (<34 weeks), neutrophil chemotaxis improves with time but remains impaired in comparison to adults, even at 42 weeks’ PCA. The presence of various clinical confounders makes it difficult to separate the effects of clinical stress from the effects of prematurity on neutrophil function. Gram-negative sepsis may depress neutrophil chemotaxis whereas superficial infections are associated with enhanced chemotaxis.

Neonatal neutrophils bind various chemoattractants normally. However, chemoattractant-induced membrane depolarization, calcium transport, and sugar uptake are relatively less efficient. Neonatal neutrophils show an incremental chemotactic response to increasing chemokine concentrations, but these responses remain lower than adult neutrophils. The chemotactic defect in neonatal neutrophils may be multifactorial, affected by factors such as a larger, poorly motile neutrophil subpopulation; impaired calcium mobilization; and aberrations in intracellular signaling pathways such as NF-B activation. Lower Mac-1 expression can also impede chemotaxis due to impaired neutrophil interaction with the extracellular matrix.

In preterm infants, intrapartum exposure to magnesium sulfate reduces both neutrophil chemotaxis and random motility. Theophylline concentrations in the high therapeutic range (84 µmol/L or 15 µg/mL) cause dose-dependent reductions in neutrophil chemotaxis. Cells from preterm infants are particularly sensitive to this effect. In contrast, theophylline concentrations in the low therapeutic range (28 µmol/L or 5 µg/mL) increase neutrophil activity. Indomethacin, too, has an adverse effect on neutrophil chemotaxis, which is more pronounced in preterm infants. Both G-CSF and GM-CSF increase neutrophil chemotactic responsiveness to other chemoattractants.

Phagocytosis.

Phagocytosis is a process of particle ingestion. Most particulate matter must be opsonized (coated) with IgG, complement fragments C3b or iC3b, fibronectin, or other proteins before being recognized and engulfed by PMNs ( Fig. 47.3 ). Neutrophils express IgG receptors such as Fcγ receptors I-III (or CD16, CD32, CD64), C3b (CR1), and iC3b (CR3). After binding of the opsonized microbe by an appropriate cell surface receptor, the PMN extends pseudopods to surround the particle and form a phagocytic vacuole. Microbes may also be ingested without opsonization through several interactions like lectin-carbohydrates on bacterial fimbriae that interact with neutrophil glycoproteins, protein–protein (such as filamentous hemagglutinin that express the argly-asp), and hydrophobic-protein (bacterial glycolipids and neutrophil integrins) interactions.

Neutrophils from preterm neonates show impaired phagocytosis, which corrects by late third trimester or term gestation to become comparable to adults. Preterm neutrophils ingest particles more slowly and ingest fewer bacteria. The lack of opsonic activity is an important consideration, as preterm infants often have lower concentrations of specific antibodies. Adult neutrophils lose their phagocytic efficiency if suspended in serum of preterm infants. Similarly, neutrophils of preterm neonates can increase their phagocytic function following exposure to adult serum or therapeutic immunoglobulin preparations.

Compared to term neonates and adults, preterm neutrophils have a lower expression of CD16 (F _c γRIII) and CD32 (F _c γRII), the two most abundant neutrophil IgG receptors. In “stressed” preterm neonates with severe RDS or sepsis, CD16 expression may be even lower. Whereas CD16 expression normally increases to adult levels over the first 3 weeks of life, CD32 deficiency does not correct with time. CD32 is the high-affinity receptor for IgG ₂ (important against encapsulated bacteria), and hence CD32 deficiency may represent an important immune defect in preterm neonates. Unlike CD16 and CD32, CD64 expression on neonatal neutrophils (both preterm and term) may be higher than neutrophils from adult subjects. CD64 is not affected by neonatal “stress” as in respiratory distress and/or prolonged rupture of membranes, and emerging data suggest that CD64 might be a useful early marker for bacterial infections.

Recombinant G-CSF and GM-CSF both activate neutrophil phagocytosis. The benefits of intravenous immunoglobulin (IVIG) as a source of opsonic activity remain uncertain. A major limitation may be in the formulation of current IVIG preparations, which may not have adequate concentrations of antibodies against neonatal pathogens.

Microbicidal Activity.

The phagolysosome provides an enclosed space in which an ingested microbe is exposed to high concentrations of toxic substances while limiting the exposure of the phagocyte and other cells to these potentially injurious agents. The major killing mechanism in neutrophils involves the generation of highly reactive free oxygen radicals in a “respiratory burst.” An NADPH-dependent respiratory burst oxidase localized on the cell membrane (and therefore, the phagosome membrane) reduces molecular oxygen (O ₂ ) to superoxide anion (O• ₂ ^- ). Subsequent generation of hydrogen peroxide (H ₂ O ₂ ) and the hydroxyl radical (OH•, formed in the presence of iron) also contributes to the microbicidal capacity of neutrophils.

These oxygen-dependent bactericidal mechanisms can be broadly divided into myeloperoxidase (MPO)-independent (such as hydrogen peroxide) and MPO-dependent (MPO catalyzes reactions between H ₂ O ₂ and halides to form highly reactive products). H ₂ O ₂ is a weak bactericidal agent per se, but the MPO-H ₂ O ₂ -halide system increases its efficacy by nearly 50-fold. The bactericidal effects of free oxygen radicals are due to oxidizing effects on various components of the bacterial cell wall.

Although preterm neutrophils have a higher oxygen consumption and normal/elevated release of superoxide and H ₂ O ₂ , the respiratory burst is, overall, depressed. This deficiency may be more marked in preterm infants with a high severity of sickness. Neutrophil oxidative burst remains impaired in preterm infants despite opsonization with IgG and complement.

Perinatal events can influence the respiratory burst in neonatal neutrophils. Labor and vaginal delivery activates the generation of free oxygen radicals in neonatal neutrophils. In contrast, perinatal distress can suppress the neutrophil respiratory burst. Both LPS and cytokines such as interferons and TNF can prime neutrophils for an accelerated respiratory burst in vitro, but prolonged exposure to these agents (during sepsis) can dampen their effect. Reduced respiratory burst activity in preterm infants correlates with impaired intracellular killing of S. aureus or E. coli. Whereas bacterial killing by neutrophils from term neonates has consistently been found to be normal, killing of staphylococci was impaired in preterm neonates.

The postnatal maturation of respiratory burst response varies according to gestational age. During the first week, infants born at 24–28 weeks have a lower respiratory burst than those born at 29-35 weeks. The differences between preterm infants born at different gestational ages disappear in about 2 months, but overall, neutrophils from preterm infants continue to have a weaker oxidative burst than adults. The postnatal maturation of the respiratory burst may not be seen at all in sick preterm infants receiving intensive care.

In “stressed” preterm infants receiving intensive care, recombinant GM-CSF can boost the neutrophil respiratory burst to levels seen in term neonates. Similarly, G-CSF can enhance neutrophil respiratory burst response in septic preterm infants. In adults, hypertonic saline may enhance host response to bacterial challenge by augmenting superoxide formation in neutrophils. Intracellular killing may also be augmented by fluoroquinolones such as ciprofloxacin, which have a potent intraphagosomal bactericidal activity against both gram-positive and gram-negative bacteria.

Neutrophils also contain additional elaborate nonoxidative killing mechanisms, including cationic proteins like defensins, bactericidal/permeability-increasing protein (BPI), and LL-37; proteolytic enzymes such as lysozyme, proteinase 3, and neutrophil elastase; and metal chelator proteins such as lactoferrin. Defensins are broad-spectrum antimicrobial peptides with activity against gram-positive and gram-negative bacteria, fungi, and enveloped viruses. BPI binds lipopolysaccharide (LPS) and blocks its effects, can damage the outer membrane of gram-negative bacteria, and has some opsonic activity. Lactoferrin, an iron chelator, is bacteriostatic as it deprives bacteria of the iron required for growth. Lactoferrin is also involved in neutrophil degranulation, in oxygen radical production, and in granulocytopoiesis. Lysozyme hydrolyzes a glycoside bond in the bacterial cell wall peptidoglycan. Primary granules also contain other cationic antibacterial proteins such as azuricidin, indolicin, and cathelicidins.

Another elegant antimicrobial defense system demonstrated by neutrophils is their ability to form neutrophil extracellular traps (NETs). Upon activation, neutrophils can undergo a type of cell death in which chromatin is released from the cell in the form of NETs. These NETs also contain histones and other antimicrobial proteins that function to trap and kill microbes in the extracellular space. However, neonatal neutrophils are deficient in their ability to form NETs. The mechanisms leading to NET formation and its ability to fight infectious agents is still not well understood and is an active area of investigation.

Degranulation.

Neutrophils contain three major types of granules: (1) the “azurophilic” granules (stain positive with the azure A dye), (2) “specific” granules (do not stain with azure A), and (3) “gelatinase” granules. Azurophilic granules contain myeloperoxidase (MPO); proteolytic enzymes such as cathepsin G, proteinase-3, and neutrophil elastase; and antimicrobial proteins such as defensins and the bactericidal permeability-increasing protein (BPI). These granules release their contents into the phagolysosomes and are involved in intracellular killing. The specific granules contain antibacterial agents such as lactoferrin and lysozyme, receptors for complement components, and bacterial products such as f-MLP. Specific granules fuse with the cell membrane to release their contents by exocytosis and also bring functionally important membrane proteins such as integrins, cytochrome- b ₅₅₈ , and receptors for chemotactic agents and opsonins to the cell surface. Specific granules play an important role in extracellular killing. Gelatinase granules contain fewer antimicrobial cargo but are storage sites for gelatinases and other metalloproteases.

Neutrophils from term neonates have granule content and degranulation responses similar to adults. However, neutrophils from preterm infants have a lower capacity to release BPI, elastase, and lactoferrin than in adults and term infants. G-CSF activates mature neutrophils without degranulation of primary granules, whereas GM-CSF induces degranulation and exocytosis of granule contents. However, anti-inflammatory agents such as corticosteroids and indomethacin inhibit the degranulation of secondary granules.

Cytokine Production.

Resident macrophages are often the first phagocytic cells of the innate immune system to encounter invading pathogens. These cells serve important host defense functions through phagocytosis and also as sentinel cells that regulate local inflammatory responses by producing various cytokines and chemokines. Most studies on monocyte/macrophage cell function have been done on cord blood, and fetal cells have not been studied to the same extent so far. Term cord-blood monocytes produce IL-1, IFN-α, and TNF in concentrations that are comparable to adults, but the levels of IFN-γ, IL-8, IL-10, and GM-CSF are lower. These cells also produce lower concentrations of extracellular proteins like fibronectin and bioreactive lipids like leukotriene B ₄ . Impaired monocyte function in neonates may be partially responsible for poorer cytokine responses of neonatal T cells.

Generally, mononuclear phagocyte recruitment and accumulation lag behind the brisk PMN influx by 6-12 hours, but the former process persists for several days. In addition to the host defense functions, mononuclear phagocytes also help regulate the process of tissue debridement and initiation of wound repair. During resolution of inflammation, the macrophage populations switch from a pro-inflammatory to an anti-inflammatory phenotype. Emerging evidence indicates that macrophages are dynamic and heterogeneous cells, which are polarized into the classically activated M1 macrophages that express various inflammatory signals and the M2 macrophages that display an anti-inflammatory profile. The effect of development on macrophage polarization is unclear.