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Normal and Abnormal Neutrophil Physiology in the Newborn
Introduction
The response of the immune-competent host to invasive pathogens includes a variety of local and systemic mechanisms. Among these are humoral elements, such as complement or immunoglobulins (Igs), as well as cellular defenses that involve both innate (nonspecific but immediate) and adaptive (antigen-specific, programmed) immune responses. Neutrophils, or polymorphonuclear cells, comprise a critical arm of the innate immune system (reviewed by Nauseef and colleagues and Nemeth and colleagues ) and are the most abundant cell type in the circulating pool of immune cells. Inadequate numbers of circulating neutrophils can compromise host defense, which increases the risk of infection-associated morbidity and mortality. However, in addition to adequate circulating numbers, proper functioning of neutrophils is required to mount an effective antimicrobial defense. Individuals with dysfunctional neutrophils are at risk for severe local and systemic bacterial and fungal infections despite having normal numbers of circulating neutrophils or even neutrophilia in some patients. ,
During ontogeny, the fetus develops in a protected environment. Accordingly, aggressive innate immune defense mechanisms are unnecessary and would be maladaptive for proper fetal growth and development. Conversely, neonates and particularly preterm neonates are at considerable risk for bacterial and fungal infections after birth, necessitating a transitional period that promotes the adaptation of immune function to life outside the protective uterus. , This chapter discusses the role of neutrophils in host defense and inflammation during fetal life and in the neonate, with a specific emphasis on the functions modulated by host immaturity.
Neutrophil Homeostasis
Neutrophils, the major class of leukocytes in the blood, are the host’s “first responders” against invading pathogens. Neonates with abnormally low neutrophil numbers (neutropenia) are at increased risk for infection, and a failure to rapidly replenish neutrophils during sepsis contributes to mortality. Neutrophil homeostasis is a finely tuned process that orchestrates the balance between neutrophils lost by attrition (apoptosis, programmed cell death) and the production of adequate numbers of new cells to protect the health of the host.
After birth, the bone marrow is the primary site of neutrophil production and storage. (See Chapter 108 for a detailed discussion of this process.) Under stable, noninfected conditions, neutrophil progenitors and their developing progeny are retained in bone marrow niches, in part through interactions between the chemokine receptor CXCR4 and its ligand CXCL12 ( Fig. 119.1 ). Under homeostatic conditions, neutrophils undergo a differentiation pathway in the bone marrow. Along this pathway, several neutrophil subsets with distinct molecular signatures and proliferation rates have been defined, which eventually lead to the formation of mature neutrophils that gradually egress from the bone marrow into the circulation to maintain enough circulating neutrophils for immune surveillance. The migration of aging neutrophils back to the bone marrow appears to be a critical factor in neutrophil homeostasis. , Recent evidence also suggests an important regulatory role of the intestinal microbiome and homeostatic neutrophil trafficking into peripheral tissues in this process.
In contrast, under conditions of stress, pro-inflammatory signaling releases cytokines and chemokines into the bloodstream. This process is driven in part by T-cell-mediated release of interleukin (IL)-17, which increases circulating levels of granulocyte-colony stimulating factor (G-CSF). G-CSF is a cytokine important to neutrophil production as well as their mobilization from the bone marrow into the vasculature, in part through inhibitory actions on CXCL12 (C-X-C motif chemokine 12, or stromal cell-derived factor-1, SDF-1). Under normal circumstances, resolution of inflammatory processes involves macrophage-mediated phagocytosis (efferocytosis) and clearance of apoptotic tissue neutrophils. Efferocytosis of senescent neutrophils promotes antiinflammatory mechanisms in macrophages, including a decrease in IL-23 release, which leads to a reduction in IL-17 secretion and hence G-CSF release.
Neutrophil Activation Mechanisms
Overview
Under homeostatic conditions, mature neutrophils released by the bone marrow traverse the circulatory system in an inactivated, quiescent form. Alterations in the environment, such as those induced by microbial invasion of tissues, exposure to circulating microbial products, or tissue damage (sterile inflammation) , can initiate the activation of neutrophils and their recruitment to the site of injury. , As a result, neutrophils migrate through the vascular wall into inflamed tissues and enter a state of readiness to initiate their immune functions. However, neutrophil-mediated inflammation is a double-edged sword: dysregulation of the activating mechanisms so critical to host protection can incidentally lead to exaggerated or persistent inflammatory responses that contribute to tissue injury and chronic diseases.
A variety of inflammatory mediators, alone or in combination, can engage surface receptors on neutrophils and induce their activation, either directly or through priming ( Fig. 119.2 ). These include bacterial products such as lipopolysaccharide (LPS, an endotoxin) and the bacterial peptide formyl- methionyl-leucyl-phenylalanine ( f MLP), complement factors (C5a), inflammatory cytokines, leukotriene B 4 , and platelet-activating factor (PAF). Here, we will provide a short overview on how these factors interact with receptor systems on the neutrophil surface. In addition, we will also describe signal transduction processes through activation of these receptor systems, which are instrumental for mounting an effective immune response. Finally, we will discuss how these receptor systems work in the fetus and neonate, although much remains unknown in this regard.
G Protein-Coupled Receptors
Contact of neutrophils with bacterial peptides and various inflammatory molecules such as leukotriene LTB4, f MLP, complement fragment C5a, and chemokines including CXCL8 (IL-8) or CCL2 induce the engagement of inhibitory G (G αi )-protein-coupled receptors (GPCRs). According to the stimulus, the following G αi -protein-coupled receptors are involved (respective ligands are in parenthesis): leukotriene receptor BLT1 (LTB4), formyl peptide receptors FPR1 and 2 ( f MLP), complement receptor C5aR (C5a), and chemokine receptors CXCR1 and 2 (CXCL8). Upon ligand binding, G αi -protein-coupled receptors trigger intracellular signaling processes leading to respective effector responses by the activated neutrophil. Signaling through G αi -coupled receptors follows characteristic cell signaling pathways including activation of neutrophil-specific Src tyrosine kinase family members Hck, Fgr, and Lyn, nonreceptor tyrosine kinase Syk, phospholipase Cβ (triggering an increase in intracellular Ca 2+ ), and phosphatidylinositol 3-kinases (PI3K) (see Fig. 119.2 ). Depending on the stimulus, engagement of respective G αi -coupled receptors can induce all aspects of neutrophil effector functions including integrin activation and adhesion, chemotaxis, phagocytosis, granule release, and the production of reactive oxygen species (ROS).
Type I and Type II Cytokine Receptors
Several type I and type II cytokine receptors expressed on the neutrophil surface (see Fig. 119.2 ) bind to a diverse group of cytokines. Type I cytokine receptors on neutrophils include granulocyte colony-stimulating factor receptor (G-CSFR), which binds to G-CSF, and granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR), which binds to GM-CSF. These receptors (particularly G-CSFR) are primary mediators of neutrophil production, differentiation, and function. Type II cytokine receptors on neutrophils include those for type I interferons, IFN-α and IFN-β, which bind to (IFN)-α/β receptor; the type II interferon, IFN-γ, which binds to the IFN-γ receptor; and the type III interferon, IFN- λ, which binds to the IFN- λ receptor. Type I and II interferon signaling are critical for acute antiviral and inflammatory responses. The balance between type I and type II interferon signaling in neutrophils is a subject of recent interest in the cancer field. While type I interferon signaling shifts the balance to an antitumor (neutrophil N1) phenotype with increased killing capacity of tumor cells, activation of type II interferon signaling favors tumor supporting (neutrophil N2) functions including production of proangiogenic factors (vascular endothelial growth factor [VEGF], matrix metalloproteinase-9 [MMP9]). Recently, type III interferon signaling through the IFN- λ receptor in neutrophils has been reported to be crucial during infection with Aspergillus fumigatus . Interestingly, engagement of both type I and type II cytokine receptors primarily activate signaling through members of the JAK and STAT family, although other signaling pathways involving MAPK or PI3K can also be involved (see Fig. 119.2 ).
Pattern Recognition Receptors in Neutrophils
Functioning as an integral part of the innate immune system, pattern-recognition receptors (PRRs) on neutrophils interact with ligands expressed by microbial pathogens or generated as endogenous danger signals. All these ligands carry common structural motifs termed pathogen-associated molecular patterns (PAMPs) for microbial-derived factors (e.g., LPS, bacterial DNA, or double-stranded viral RNA) and damage-associated molecular patterns (DAMPs) for endogenous danger signals (e.g., HMGB1, S100A8/A9). , Among PRRs, Toll-like receptors (TLRs) play a predominant role in neutrophils (see Fig. 119.2 ). Nearly all TLRs (with the exception of TLR3 and possibly TLR7) are expressed at basal levels in neutrophils and interact with microbial DNA and RNA, as well as lipids, carbohydrates, and proteins. Engagement of TLRs primes neutrophils for enhanced activation in response to secondary stimuli. In addition, recent work demonstrated that TLR2 and TLR4 stimulation can also induce rapid induction of neutrophil effector functions through MyD88-dependent processes, which do not require transcriptional activity. , Specific TLRs are responsible for recognizing different sources of PAMPs or DAMPs. In general, engagement of TLRs activates downstream signaling pathways, particularly those involving NFκB (nuclear factor kappa B), p38 MAPK, and JNK, all of which respond to cellular stress. Besides TLRs, several other groups of pattern recognition receptors on neutrophils have been described. These include retinoic acid inducible gene I-like receptors (RLRs), nucleotide-binding oligomerization domain-like receptors (NLRs), AIM2-like receptors, and DNA sensors such as the recently described transcription factor Sox2. In addition, some members of the C-type lectin receptor family including Dectin-1 and TREM-1 (Triggering Receptor Expressed on Myeloid cells 1) also belong to the PRR family. Through its binding to β-glucans, commonly found in various fungi, Dectin-1 is involved in antifungal defense mechanisms and signals via an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic tail. The other C-type lectin, TREM-1, synergizes with additional activating receptors on neutrophils during inflammatory responses and has recently been reported to influence neutrophil chemotaxis, ROS production, and transepithelial migration.
Neutrophil Fc Receptors
Neutrophils express several receptors that recognize the constant region (Fc) of IgG or IgA (see Fig. 119.2 ). This enables neutrophils to bind IgG- and IgA-opsonized particles and pathogens reacting against it, resulting in various proinflammatory responses including phagocytosis and degranulation. The following Fc receptors are expressed in neutrophils (see Fig. 119.2 ): FcαRI (CD89), which binds to IgA; and FcγRIA (CD64A), FcγRIIA (CD32A), and FcγRIIIB (CD16B), all of which bind to IgG. FcαRI, FcγRIA, and FcγRIIA are classical activation receptors. These carry an ITAM at the cytoplasmic tail, as in the case of FcγRIIA, or they are associated with the ITAM-bearing signaling unit Fc receptor γ chain in the case of FcαRI and FcγRIA. Upon ligand binding, ITAMs are phosphorylated by Src family kinases, triggering a signaling cascade involving the nonreceptor tyrosine kinase Syk, PI3K, and phospholipase Cγ (PLCγ) (see Fig. 119.2 ). Similar to stimulation of FcγRIA and FcγRIIA by IgG, FCαRI stimulation of neutrophils by multimeric IgA triggers a variety of proinflammatory functions including phagocytosis, degranulation, ROS production, and neutrophil extracellular trap (NET) formation. Multimeric IgA also induces the release of leukotriene LTB4, which attracts additional neutrophils. In contrast, the binding of FcαRI to monomeric serum IgA exerts antiinflammatory responses.
Neutrophil Activation Mechanisms in Neonates
In contrast to the extensive body of work defining activation mechanisms in adult neutrophils, the influence of developmental stage on neutrophil activation processes and associated pathology in neonates is less understood. Neonatal neutrophils, even in preterm infants, have the capacity for activation under conditions of stress, such as respiratory distress or sepsis. , However, neonatal inflammatory responses may be excessive relative to those of adults. Neonatal animals exhibit exaggerated inflammatory reactions in response to injury, such as intestinal ischemia-reperfusion, , which are findings that recapitulate the exaggerated inflammatory responses observed in human neonates.
In vitro studies also suggest that neonatal neutrophils may be intrinsically “primed for action” even under basal conditions. Neonatal neutrophils exhibit constitutive activation of the transcription factor NF-κB, which is associated with increased generation of proinflammatory mediators such as IL-8 or expression of anti-apoptotic proteins such as FLICE. , Pertinently, NF-κB activation is a critical component of inflammatory injury in neonates. , Neutrophil activation of NF-κB in neonates is also linked to basal activity of signaling mechanisms that promote cell survival, such as Akt, a gatekeeper for DNA damage response/repair.
While the aforementioned studies indicate that neonatal neutrophils are hyperreactive and constitutively activated, others suggest a relative resistance of neonatal neutrophils to certain activation pathways, such as those involving TLR4 or the Src kinase, Lyn. , These findings are supported by in vivo studies investigating neutrophil recruitment in the mouse fetus, which showed a strong fetal age-dependent inability of neutrophils to extravasate into inflamed tissue. Similar results were found in an in vitro flow chamber study where an ontogenetically regulated reduction in rolling and adhesion of cord blood neutrophils was observed. How can we explain these presumably conflicting reports on hyper- and hypoactive neutrophils? Potential reasons for the observed differences in studies of ex vivo neutrophils might be related to mode of delivery (cesarean section vs. vaginal delivery), blood source (placental cord blood vs. postnatal peripheral blood sampling), and processing techniques (isolation and treatments). In a recent exciting study by the Viemann group, it became evident that the innate immune system, including its pattern recognition receptors, are well prepared for the transition from intrauterine life to the challenges of postnatal life. This transition brings about rapid changes in innate immune cell function and programming. Future work in this area is urgently warranted to more completely define neonatal neutrophil inflammatory responses and to distinguish these from inflammatory responses by fetal neutrophils. Clarifying these issues will certainly also help in identifying new potential age-adjusted therapeutic strategies for premature and mature infants suffering from severe infections or from other inflammatory conditions.
Neutrophil Recruitment
Overview: The Neutrophil Recruitment Cascade
Neutrophil recruitment is an important immunologic process that enables neutrophils to leave the circulating pool of blood-resident cells and extravasate into inflamed tissue in order to fulfill their task of host defense. Neutrophil recruitment follows a well-defined cascade of adhesion and activation events (see Fig. 119.1 ), consisting of initial neutrophil capture (tethering) and rolling along the inflamed endothelium, followed by firm arrest. Subsequently, neutrophils strengthen their adhesive bonds and spread over the underlying endothelial surface (to better withstand shear forces exerted by the flowing blood) before they start “crawling” along the endothelial lining to find an appropriate spot for transmigration into inflamed tissues. , Neutrophil transmigration, that is passage through the endothelial lining, occurs mostly in a paracellular fashion and is followed by penetration of the underlying vascular basement membrane before the neutrophil reaches the inflamed tissue. ,
Neutrophil recruitment is primarily mediated by groups of adhesion molecules that include the selectins and selectin ligands, integrins, and the Ig gene superfamily. Each serves a specific purpose related to the recruitment process.
Selectins and Selectin Ligands
The selectin (CD62) family constitutes a group of C-type lectins that bind to specific carbohydrate moieties on selectin ligands in a calcium-dependent manner. , Members of this family include leukocyte-expressed L-selectin (CD62L); E-selectin (CD62E), expressed on inflamed endothelial cells; and P-selectin (CD62P), found on activated endothelial cells and activated platelets. Selectin ligand activity depends on critical posttranslational glycosylation steps during selectin ligand synthesis including α2,3 sialylation and α1,3 fucosylation leading to the generation of the tetrasaccharide sialyl Lewis X (sLe X ) found at the terminus of core2-decorated O-glycans. ,
The most prominent selectin ligand, the sialomucin P-selectin glycoprotein ligand-1 (PSGL-1), is expressed on the neutrophil surface and binds to all three selectins. Besides PSGL-1, L-selectin can function as a selectin ligand by interacting with E-selectin. Notably, this interaction has been observed in the human system but not in the mouse. , Initially considered as part of a braking system to slow down circulating neutrophils when interacting with the inflamed endothelium, more recent work has identified important signaling functions of selectin/selectin ligand interactions. Signaling through PSGL-1 relies on the adapter molecules DAP12 and FcRγ and induces the activation of β 2 integrins. Interestingly, the engagement of PSGL-1 (or L-selectin) by E-selectin mediates the release of the DAMP molecule S100A8/A9 from neutrophils. Released S100A8/A9 in turn binds to TLR 4 in an autocrine fashion and stimulates β 2 integrin activation, suggesting that an additional extracellular activation loop is necessary for β 2 integrin activation during the recruitment process. ,
Integrins
Neutrophil-expressed integrins including the β2 integrins LFA-1 (αLβ2) and Mac-1 (αMβ2) are crucial players in neutrophil recruitment and involved in all steps of the recruitment cascade (see Figs. 119.1 and 119.2 ). Integrins not only provide an adhesive platform critical for firm neutrophil arrest on the endothelium in a microenvironment, where pulling forces are exerted constantly on the attached neutrophil, but neutrophil integrins also exhibit critical signaling functions during the recruitment process.
In general, β2 integrins on circulating neutrophils are kept in a bent, inactive conformation, which strongly impairs interactions of integrins with their ligands in trans , although ligand binding, as recently reported, is possible in cis, leading to inhibition of leukocyte adhesion. During the recruitment process neutrophils make intimate contact with endothelial cells, providing neutrophils the opportunity to interact with adhesion and activation molecules bound to the endothelial surface (see Fig. 119.1 ). There are a number of surface receptors on neutrophils, which upon ligation with their specific ligands (mostly found on the luminal surface of endothelial cells) mediate activation of β2 integrins, a process called inside out signaling . Surface receptors inducing β2 integrin activation with relevance during the recruitment phase include GPCR such as chemokine receptors, formyl peptide receptors, receptors for LTB4, PAF, and C5a. In addition, and as mentioned above, selectin ligands such as PSGL-1 have been shown to activate β2 integrins in neutrophils , Recently, several inhibitors of integrin activation have been reported, including developmental endothelial locus-1 (Del-1), fibroblast growth factor 23 (FGF23), and growth-differentiation factor-15 (GDF-15), which add to the complexity of how neutrophil recruitment is regulated.
In contrast to inside-out signaling, outside-in signaling refers to signaling events following the engagement of integrins with their ligands and associated integrin clustering. As outside-in signaling during the recruitment process often occurs at the same time as inside-out signaling events, and because it uses at least in part the same kinases and adaptor molecules (including Src kinases, Syk, Talin, and Kindlin3), these two processes are often difficult to differentiate. In the context of neutrophil recruitment, adhesion strengthening, spreading, and crawling are typical events related to outside-in signaling. These processes require extensive rearrangement of the actin cytoskeleton and are regulated by a complex network of signaling events aiming to prepare the attached neutrophil for its migratory journey into tissue. , Besides β2 integrins, recent work has elucidated that the β1 integrins VLA3 and VLA6, which are laminin-binding integrins, are involved in the penetration of neutrophils through the vascular basement membrane. For this step, neutrophil VLA3 and VLA6 are mobilized from intracellular storage granules to the cell surface, where they become available for binding to laminins, key components of the vascular basement membrane. ,
Immunoglobulin Gene Superfamily
Similar to integrins, members of the Ig gene superfamily are involved in all steps along the recruitment cascade. Endothelial members include ICAM-1 (CD54), ICAM-2 (CD102), VCAM-1 (CD106), PECAM-1 (CD31), MAdCAM-1, and junctional adhesion molecule (JAM) family members. Endothelial ICAM-1 binds to LFA-1 and Mac-1 on neutrophils and firmly anchors neutrophils to the inflamed endothelium. ICAM-1 is upregulated on the endothelium following stimulation by inflammatory cytokines, including tumor necrosis factor-α (TNF-α), IL-1, and IFN-γ. Interestingly, engagement of endothelial ICAM-1 by β2 integrins also leads to signaling events inducing the phosphorylation of VE-cadherin, an important molecule at endothelial junctions. PECAM-1, present on neutrophils and other hematopoietic cells as well as on platelets and the endothelium, is critical for neutrophil extravasation. Structurally, PECAM-1 is a member of the Ig immunoreceptor tyrosine-based inhibitory motif (Ig-ITIM) family. Phosphorylation of its ITIM domain can promote cell proliferation and activation as well as inhibit signaling events related to adhesion and migration. JAM-A, -B, and -C contribute to cell-cell contact integrity; their expression at tight junctions of endothelial and epithelial cells influences barrier permeability during inflammation. JAM molecules are involved in neutrophil transendothelial migration. In addition, cleavage of JAM-C by neutrophil elastase has been demonstrated to be critical in triggering reverse neutrophil transmigration , a process describing the return of extravasated neutrophils into the blood circulation. Although the function of reverse neutrophil transmigration is not completely clear, it may potentially expand inflammatory activity to other tissues.
Ontogenetic Regulation of Neutrophil Recruitment
General Considerations
Comparative studies between neutrophils of adults and those of fetuses and neonates have shown developmental impairments of neutrophil recruitment that can adversely affect their capacity to protect the host against invasive pathogens. These impairments affect all steps along the inflammatory cascade. Studies in fetal and neonatal animal models as well as in human neonates have shown that diminished neutrophil capacity for rolling and firm adherence under flow conditions is directly related to gestational age. ,
Specific Impairments of Neutrophil Adhesion Molecules in Neonates
Studies in fetal and neonatal animal models have shown that neutrophil capacity for rolling and firm adherence under flow conditions is influenced by ontogenetic alterations. , Ex vivo studies of human preterm and term neonates have confirmed these findings. Neutrophil adherence defects are related to low intrinsic levels or functional capacities of neutrophil and/or endothelial adhesion molecules, as described below. These impairments appear to be developmentally programmed because the “recruitability” of neutrophils isolated from the placental cord blood of preterm infants matched those of their postconception age-matched already born counterparts. ,
Selectin Impairments in Neonates
Selectin-dependent rolling of neutrophils has been studied in flow chamber assays using neutrophils isolated from human preterm and term neonates. To test L-selectin dependent rolling, flow chambers were coated with human umbilical vein endothelial cells stimulated with IL-1. Treatment with neutralizing antibody to L-selectin inhibited the tethering and rolling of adult neutrophils to the activated endothelium while this treatment had a minimal effect on neonatal neutrophils, consistent with diminished L-selectin function. Additional in vitro studies revealed that neutrophils from mature neonates showed reduced levels of L-selectin while fetal neutrophils express surface levels of L-selectin similar to those of adults. , P- and E-selectin-dependent neutrophil rolling has been investigated in flow chambers coated with P- or E-selectin. For both selectins, neutrophil rolling was diminished in preterm infants but increased with advancing gestational age. This can be explained, at least in part, by developmentally regulated PSGL-1 expression on neutrophils and E-selectin expression on the endothelium. , As mentioned above, selectin/selectin-ligand interactions together with chemokine-dependent activation mediate the transition of neutrophils from rolling to β 2 integrin-mediated arrest/firm adhesion on endothelial cells. Using flow chambers and cord blood neutrophils from premature and mature infants, a strong reduction in the induction of firm neutrophil arrest and adhesion under flow was observed in flow chambers coated with E-selectin, ICAM-1, and CXCL1, suggesting that the induction of adhesion is impaired in neutrophils from term and preterm infants.
β2 Integrin Impairments in Neonates
Stimulated cord blood neutrophils exhibit variable defects in integrin-dependent adhesion, chemotaxis, and transmigration compared to adult neutrophils. , , This can be explained in part by diminished surface and total Mac-1 expression in neutrophils of term and preterm infants. In contrast, expression of LFA-1, the other prominent β 2 integrin on neutrophils, does not change significantly during fetal ontogeny.
Neonatal neutrophils are also impaired in their capacity to localize to inflammatory sites. , In immature animals, neutrophil accumulation is delayed in response to experimentally inflamed peritoneum or following lung injury. Decreased functional expression of Mac-1 appears to contribute to deficits in neonatal recruitment and accumulation, while other functions such as phagocytosis remain intact.
Specific Impairments of Endothelial Adhesion in Neonates
The influence of ontogeny on endothelial adhesion molecule expression and function remains incompletely understood. Much of our current understanding has been based on extensive studies utilizing in vitro models of cultured human endothelial cells from umbilical veins (HUVECs) or foreskin, or in vivo animal models. LPS-stimulated HUVEC showed reduced upregulation of E-selectin and ICAM-1 surface expression, indicating that the endothelial compartment also contributes to impaired neutrophil recruitment during ontogeny. Neonatal neutrophils exhibit impaired transendothelial migration as a result of abnormal CD11b-ICAM-1 interactions, , findings confirmed by intravital microscopy of fMLP-stimulated yolk sac vessels in the mouse. Deficient P- and E-selectin expression in fetal and neonatal animals has also been linked to impaired leukocyte accumulation and localization. Similarly, human preterm neonates exhibit decreased endothelial P-selectin surface expression.
Specific Impairments in Transendothelial Migration and Chemotaxis in Neonates
Previous studies in neonatal humans and in animal models indicate an age-dependent response of neutrophil localization to inflammatory stimuli. , Compared to adults, neonates exhibit decreased neutrophil accumulation in various organs, such as the lungs, in response to inflammatory stimuli.
Neonatal leukocyte functional impairments are related to both structural differences as well as to alterations in adhesive capacity. Preterm and term neutrophils exhibit inadequate migration responses to chemoattractants in a gestational age-dependent manner. , Neonatal neutrophils exhibit decreased random (chemokinesis) and directed (chemotaxis) migration. In the first weeks of life, neutrophil chemotactic function is only 50% of that observed in adult neutrophils. This impairment is related to adhesive or activation defects, as suggested by lower intracellular free calcium levels and decreased generation of filamentous (F)-actin. , Extrinsic factors such as sepsis, antenatal steroids, maternal magnesium therapy, and delivery by cesarean section may further reduce chemotaxis. While these functional impediments increase the neonatal risk for infection, particularly in preterm infants, they may also represent a teleologic mechanism to limit excessive inflammatory responses in utero. In fact, intravital observations of leukocyte extravasation following local stimulation with fMLP has been performed in murine yolk sac vessels of the living fetus still connected to the mother. These experiments showed an ontogenetically regulated increase in myeloid cell extravasation, supporting the concept that age-limited inflammatory responses might be beneficial for proper fetal growth and development.
Antimicrobial Functions of Neutrophils
Inflammatory neutrophils recruited to tissue sites of microbial invasion engage a variety of attack mechanisms. These processes include phagocytosis, oxidative metabolism, degranulation, and the formation of NETs.
Phagocytosis
Overview
The process of phagocytosis , or leukocyte ingestion of foreign particles, has been recognized since the 19th century. The earliest descriptions of phagocytosis were published by Joseph Richardson and William Osler in 1869 and 1875, respectively ; although credit for the term and its discovery are commonly attributed to Élie Metchnikoff, who shared the 1908 Nobel Prize in Physiology or Medicine with Paul Ehrlich. Phagocytosis is so crucial to host defense that two categories of leukocytes are classified as “professional phagocytes” and neutrophils lead the charge in that regard.
A preparatory step for phagocytosis is opsonization, the process of “tagging” the targeted pathogens by coating them with opsonins. An opsonin may be an Ig (especially IgG), fibronectin, and complement-derived cleavage products (C3b and C3bi). This coating prepares pathogens for ingestion by phagocytes, including neutrophils. Opsonized pathogens bind to neutrophils through specific surface receptors such as CD11b. Adhesive contact between opsonized particles and inflammatory neutrophils initiates both phagocytosis and the neutrophil respiratory burst , a process that generates reactive oxygen intermediates (ROIs) that mediate intracellular killing and degradation of ingested pathogens.
Neutrophil receptors for Ig opsonins include Fc receptors that recognize and bind the Fc portion of the IgG molecule. Neutrophils possess three distinct Fc receptors: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRI is the high-affinity receptor for IgG. It is not expressed at high levels on the cell surface until the neutrophil undergoes activation, and it lacks a direct role in promoting phagocytosis. However, it may potentiate phagocytosis mediated by other receptors. FcγRII and FcγRIII are constitutively expressed lower-affinity Fc receptors that are important to the efficiency of neutrophil phagocytosis.
Perhaps the most important receptor for phagocytosis is complement receptor 3 (CR3, also known as Mac-1 ). CR3 is a β 2 -integrin, a heterodimer of CD18 (common to all members of this integrin family) and CD11b, which contains the C3bi binding site and has a recognition domain for other matrix proteins including fibrinogen. CR3 also contains a lectin-like binding site that can bind bacteria independent of opsonization. When C3bi binds to CR3, it initiates two pathogen-killing mechanisms: phagocytosis and the respiratory burst (described below).
Binding of an opsonized target to the neutrophil cell surface through any of the aforementioned mechanisms activates intracellular microfilaments that enable pseudopods to extend and surround the target, capturing it inside the cell. This membrane-bound vacuole contains the target, or “phagosome,” and fuses with intracellular granules. Granule release of toxic contents into the vacuole facilitates pathogen killing and removal.
Neonatal Impairments in Phagocytosis
Phagocytosis is a complex process that is impacted by multiple factors including the involved surface receptors, the opsonization status of the target, and the properties of the target itself. As such, phagocytosis of neonatal neutrophils is impacted by the developmental stage and setting of the infant. Neonatal neutrophils have reduced CR3 receptor pools relative to adults, and neutrophils from preterm infants have reduced FcγRII and FcγRIII. Phagocytic activity of neonatal neutrophils can also be impaired by reduced availability of opsonins. Maternal Ig is actively transported across the placenta, but the majority of such transfer occurs in the third trimester, leaving preterm infants with considerably less than term infants. A number of studies have investigated neonatal neutrophil phagocytosis covering a range of targets including bacteria and fungi. Some suggest defects, particularly in preterm infants, where others did not, likely reflecting variability in assay technique and conditions, as well as the numerous variables that impact the process. ,
Oxidative Microbial Killing
Overview
Neutrophil activation is accompanied by a marked increase in oxygen consumption and glucose utilization known as the respiratory burst , a reaction that is crucial in enabling phagocytes to degrade internalized pathogens. This process, catalyzed by the membrane-bound phagocyte oxidase complex, generates an array of ROIs that are toxic to surrounding microbial targets. Among these ROIs are hydrogen peroxide (H 2 O 2 ) and superoxide anion (O 2 − ). The phagocyte oxidase complex comprises a 91-kDa flavoprotein and a 22-kDa heme protein, collectively referred to as flavocytochrome b 558 . In resting states, flavocytochrome b 558 is mostly sequestered in the membranes of secretory vesicles and specific granules, where it can be rapidly activated through degranulation.
NADPH is required for generating O 2 − , and the binding sites of flavocytochrome b 558 provide the cognate ligand. In a carefully orchestrated, highly regulated process, soluble cytoplasmic components including p47 phox , p67 phox , p40 phox , and the GTPase, Rac1 help “build” the cluster of proteins called the NADPH oxidase complex and catalyze electron donation from NADPH to O 2 to produce superoxide. Chronic granulomatous disease (CGD), manifested by an increased susceptibility to infection secondary to an inability to mount the respiratory burst, results from deficiencies in flavocytochrome b 558 , p47 phox , or p67 phox .
After the phagocytic oxidase generates O 2 − , superoxide dismutase rapidly converts it to H 2 O 2 . Further reactions involving O 2 − and H 2 O 2 in the presence of iron produce a hydroxide ion and a hydroxyl radical. Because oxidative killing carries a high threat for damage to healthy tissue, processes are in place to control and limit the reaction. Other neutrophil products including catalase and glutathione peroxidase help to neutralize these reactive molecules and prevent potential injury to healthy tissue.
Neutrophil azurophilic granules utilize two additional mechanisms of oxidative killing. Myeloperoxidase (MPO) from the granules can catalyze the formation of chloramines and hypochlorous acid (the active component in bleach) from H 2 O 2 and chloride. Inducible nitric oxide synthase (iNOS) is also present in azurophilic granules. When induced, the synthase produces the highly reactive molecule nitric oxide (NO). NO production generates reactive nitrogen intermediates that complement the antimicrobial activity of ROS.
Neonatal Respiratory Burst
The respiratory burst in neonatal neutrophils has been studied extensively since the 1970s. Most evidence suggests that, at birth, neutrophils of full-term infants elicit a respiratory burst equivalent to that in adult neutrophils. In contrast, preterm infants are deficient in this response. This holds true for both spontaneous and fMLP-stimulated neutrophil respiratory burst in neonates with or without signs of infection. Additionally, “stressed” preterm infants with sepsis or respiratory distress can exhibit severe impairments in neutrophil-mediated antibacterial function and delayed maturation of the respiratory burst. Other perinatal variables also affect the respiratory burst. Cord blood neutrophils collected after labor and vaginal delivery generate more ROS in response to stimulation with Escherichia coli and have higher CD11b expression than those collected after cesarean section.
In addition to quantifiable differences between neonatal and adult respiratory burst responses, qualitative characteristics differ as well. Compared to adult cells, neonatal neutrophils generate more O 2 − from the initial stage of the respiratory burst both at baseline and in response to stimulation. This may be related to the altered content of membrane-associated flavocytochrome b 558 relative to cytoplasmic phox components. However, bactericidal oxidants generated later in the pathway, such as hydroxyl radicals, are reduced in neonates. The reduced neonatal content of MPO, which generates chloramines and hypochlorous acid from H 2 O 2 , and lactoferrin, which enhances hydroxide ion production when saturated with iron, are thought to contribute to the deficiencies in generating these bactericidal constituents.
Finally, there is evidence that the oxidative burst is sensitive to therapy with dexamethasone used in the treatment of bronchopulmonary dysplasia (BPD) in premature infants. Although respiratory burst activity increases with postnatal age in very low-birth-weight infants, exposure to dexamethasone diminishes responses relative to age-matched controls. Lower levels of antioxidant enzymes, such as glutathione, in neonates compared with adults, may also contribute to neutrophil-mediated toxicity in the local tissue environment.
Degranulation
Neutrophils are categorized as granulocytes based on the diverse array of densely packed granules and secretory vesicles highly visible within these cells. All of these components are key players in the antimicrobial defenses mediated by neutrophils. Additionally, these granules contain receptors, adhesion molecules, and inflammatory mediators that play vital roles in nearly every aspect of neutrophil function.
Following neutrophil activation, intracellular granules and secretory vesicles translocate to the neutrophil surface. , Precise control of granule release is essential not only in transforming these cells from innocuous patrollers to deadly effector cells, but also in limiting their potential for causing collateral damage to healthy tissues.
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