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Neonatal Leukocyte Physiology and Disorders


Key Points

  • The neonate and the young infant depend primarily on the innate immune system for host defense. With limited prior exposure to infectious and environmental antigens, the adaptive immune arm is still in a phase of structural and functional development.

  • Neutropenia is frequently encountered during the neonatal period.

  • Neonatal neutrophils show a wide range of functional deficiencies in movement, phagocytosis, and microbial killing.

  • With the exception of a few deficiencies, neonatal monocytes and macrophages are functionally comparable to their counterparts in adults.

  • Neonatal T-cell and B-cell populations are still developing. Several adaptive mechanisms, such as the presence of B1 cells that can function without assistance from T cells and the production of immunoglobulins with polyspecific antigen binding, are unique to the neonate and partially mitigate the deficiencies in adaptive immunity.

  • The innate lymphoid cells are a recently described exciting new subset in innate immunity that are likely to play a major role during the neonatal period and early infancy.

This chapter presents an overview of neonatal leukocyte physiology and quantitative and qualitative disorders of leukocytes. Topics include the normal physiology and defects associated with neonatal hematopoiesis, neutrophils, monocytes, lymphocytes, dendritic cells (DCs), and innate lymphoid cells (ILCs) ( Fig. 71.1 ). Novel therapeutic approaches are also discussed.

Fig. 71.1
Leukocyte Populations in the Neonate and the Young Infant.
Hematopoietic stem cells differentiate along the myeloid and lymphoid lineages to ultimately give rise to leukocyte populations that participate in the innate (blue background) or adaptive (orange background) immune responses. DC , Dendritic cell; ILC , innate lymphoid cell; NK , natural killer.

Neutrophil Physiology and Function

Ontogeny

Neutrophils are an important line of defense in the cellular innate response (see Fig. 71.1 ). The life cycle of a neutrophil can conceptually be divided into three phases, representing time spent in (1) marrow, (2) blood, and (3) tissues. The earliest neutrophilic precursors, myeloblasts, promyelocytes, and myelocytes, are capable of cell division and thus are referred to as the neutrophil proliferative pool (NPP). In later stages of maturation, neutrophils lose their ability for cell division. These metamyelocytes, bands, and segmented neutrophils continue to differentiate in situ and constitute the neutrophil storage pool (NSP). In adults, the NSP is a sizeable reservoir of neutrophils that can be rapidly mobilized into the bloodstream when needed. However, the NSP is relatively much smaller in the midgestation fetus and preterm infant and can be readily exhausted during sepsis. The NSP contains about 6 × 10 9 cells per kilogram in adult rats. In contrast, the rat NSP at 19 days’ gestation contains only about 0.9 × 10 9 cells per kilogram, which expands marginally to 1.2 × 10 9 cells per kilogram at term (21 days). Unlike in fetal and newborn rodents, in whom the liver and spleen house a significant fraction of the NPP and NSP, neutrophil production and storage in human neonates occur primarily in the bone marrow.

Circulating and Marginated Blood Neutrophil Pools

Circulating neutrophils are distributed into two compartments of approximately equal size, the circulating and marginated pools. As the name suggests, neutrophils in the circulating compartment freely circulate in the bloodstream, whereas those in the marginated compartment are transiently attached to the endothelium. In healthy adults, the circulating and marginated pools both contain approximately 0.4 × 10 7 cells per kilogram. The marginated cells can move into the circulation for short periods of 30 to 45 minutes after strenuous crying or exercise or following administration of epinephrine or corticosteroids.

Neutrophils remain in circulation for a few hours (half-life of about 6.3 hours) and then migrate into the tissues. During infection, neutrophil trafficking into the tissues is increased. In fetal sheep, intra-amniotic exposure to endotoxin caused an initial drop in circulating neutrophil counts due to tissue emigration, which was followed by a gradual increase over the next 6 days. The length of time that neutrophils spend in the tissues and their subsequent fate is not well understood.

Neutrophil Heterogeneity

There is now evidence for the existence of different neutrophil subsets with distinct molecular markers. Details of differences in function are still emerging. Three molecular markers have been evaluated most frequently:

  • Olfactomedin 4 (OLFM4) is a glycoprotein that has been suggested to act as a tumor suppressor and has recently been identified in specific granules of approximately 25% of circulating human neutrophils. The expression of OLFM4 could negatively regulate the efficiency of bacterial killing in a subset of neutrophils.

  • The surface glycoprotein CD177 (NB1) is a 55-kDa glycosylphosphatidylinositol-anchored receptor that is expressed at various levels on circulating neutrophils. Several distinct functions have recently been attributed to CD177, including high-affinity binding to platelet–endothelial cell adhesion molecule–1 and the ability to associate with the serine protease PR3. During infection, CD177+ neutrophil subsets may show increased tissue infiltration as aided by the associated cell surface PR3.

  • Intercellular adhesion molecule (ICAM)-1–positive neutrophils are believed to undergo reverse transendothelial migration from the tissues to enter the bloodstream and may be involved in the systemic dissemination of inflammation.

Recent studies demonstrate additional heterogeneity in neutrophil phenotypes depending on age, microenvironment, priming by microbiota, and exposure to inflammation. These distinct phenotypes result in differences in cytokine production, phagocytosis, and neutrophil extracellular traps (NETs) production. For example, while neutrophils in the spleen are CD26L low , CD11b hi , ICAM-1 hi and produce NETs, neutrophils in lymph nodes express CCR7, LFA-1, and CXCR4 and interact with T lymphocytes and mediate T cell activation. Similarly, exposure to antibiotic-resistant Staphylococcus aureus results in two distinct subsets of murine neutrophils based on TLR expression, cytokine production, and macrophage activation potential. PMN-1 neutrophils express TLR2/4/5/8, produce IL-12, and classically activate macrophages; PMN-2 neutrophils express TLR2/4/7/9, produce IL-10, and alternatively activate macrophages. Thus, neutrophils are complex inflammatory cells that can mediate distinct immune and non-immune functions both during homeostasis and in various disease states.

Neonatal Neutropenia

Statistically, neutropenia is defined as an absolute neutrophil count (ANC) less than two standard deviations below the mean value, or below the fifth percentile, for postnatal age. Manroe et al. established reference values for ANCs in term and preterm infants during the first 28 days of life for both healthy infants and those with perinatal complications. Mouzinho et al. studied serial white blood cell counts in healthy preterm very low-birthweight (VLBW) infants to investigate whether this patient cohort had neutrophil counts different from those found in previous studies in which cohorts consisted mostly of term infants. They detected a wider range of the ANC, mostly resulting from a downward shift of the lower boundary, especially during the first 60 hours of life. However, there was no difference in absolute total immature neutrophil counts or in the ratio of immature neutrophil counts to total neutrophil counts. Schmutz et al. showed that the ANC peaked at 6 to 8 hours for neonates born at 28 weeks’ gestation or later but at 24 hours for those born before 28 weeks’ gestation. Table 71.1 provides the 5th and 95th percentiles for ANCs at 72 to 240 hours among neonates in that study. In that study, ANCs were higher in neonates born after a prolonged period of labor than in those born by elective cesarean delivery. Female infants also had higher ANCs, averaging about 2000/μL more than their male counterparts.

Table 71.1
Absolute Neutrophil Count in Neonates at 72–240 Hours of Age
From Schmutz N, Henry E, Jopling J, Christensen RD. Expected ranges for blood neutrophil concentrations of neonates: the Manroe and Mouzinho charts revisited. J Perinatol . 2008;28(4):275–281.
Gestational Age 5th Percentile 95th Percentile
>36 weeks 2700/μL 13,000/μL
28–36 weeks 1000/μL 12,500/μL
<28 weeks 1300/μL 15,300/μL

In neutropenic infants, the pathophysiologic mechanisms include exhaustion of myeloid progenitors, inadequate response of the progenitor cells to proliferative or maturational signals, and increased usage and destruction. Fig. 71.2 highlights the causes of neutropenia in the newborn period. Some of the more frequently encountered causes of neonatal neutropenia are discussed in the following sections.

Fig. 71.2, Causes of Neonatal Neutropenia.

Sepsis-Induced Neutropenia

Neonates with overwhelming sepsis often develop neutropenia, which illustrates some of the differences between adult and neonatal neutrophils. Neonates have fewer neutrophil progenitors and a diminished precursor storage pool, so neutrophils are easily depleted in stress conditions. Following experimental sepsis with group B streptococci, adult rats respond with a transient decrease in circulating neutrophil counts, followed by significant neutrophilia associated with a twofold to threefold increase in the progenitor pool (colony-forming unit–megakaryocyte) and an increase in the proliferative rate to 75% of the maximal capacity. In contrast, neonatal rats under the same conditions had a decrease of 50% of their progenitor pool and failed to increase their myeloid proliferative rate, which, as discussed previously, was already at near maximal levels. Most important, during experimental sepsis, neonatal rats had further depletion of their already reduced NSP reserves by almost 80%, compared with a decline of 33% in adult rats.

Immune-Mediated Neonatal Neutropenia

Immune-mediated neutropenia is important to consider as a diagnostic possibility in infants with persistent neutropenia. Alloimmune neonatal neutropenia occurs as a result of maternal sensitization to neutrophil antigens present on the infant’s neutrophils (paternally acquired) that are not present on the maternal neutrophils, with subsequent production of immunoglobulin G (IgG). Neutrophil-specific antibodies are found in the maternal and infant sera, but the mother has a normal neutrophil count. Alloimmune neonatal neutropenia is estimated to occur at a frequency of 3% of live births. The antigens most commonly involved in the United States are human neutrophil antigen (HNA)-1a, HNA-1b, and HNA-2a. Because the antibodies are IgG, which crosses the placenta, peripheral blood counts show profound neutropenia. The condition is self-limiting and typically resolves within 6 to 7 weeks, during which time the neonate is susceptible to infections, mostly cutaneous in nature. Most infections are mild, although infants with profound neutropenia are at risk of life-threatening infections and should be monitored closely. Neonatal autoimmune neutropenia occurs when mothers have antineutrophil antibodies that cross transplacentally and bind fetal neutrophils. This form of neutropenia is generally milder in severity than alloimmune neutropenia.

Maternal Hypertension–Associated Neutropenia

One of the most common and well-described causes of transient neonatal neutropenia is maternal hypertension. Neonatal neutropenia is inversely related to birthweight and gestational age and directly related to the severity of hypertension. Infants of hypertensive mothers seem to have decreased production of neutrophils, but the cause is uncertain. Several studies have demonstrated a decrease in the numbers of neutrophil progenitor cells, decreased cycling of these cells, a relatively normal NPP and NSP, and the absence of a “left shift.” Studies show conflicting evidence for the risk of infection in these infants, although the risk is probably low because neutropenia resolves in most cases within 72 hours, and almost always in 5 to 7 days.

Idiopathic Neutropenia of Prematurity

This is generally a benign form of neutropenia that presents after the early neonatal period (weeks 4 to 10). Neutropenia is usually transient and recovers spontaneously in a majority of patients. Differential leukocyte counts show few immature neutrophils (in the presence of neutropenia), suggesting a transient suppression of neutrophil regeneration in these patients. In some premature infants, idiopathic neutropenia can be severe (sometimes ANC <500/μL) and prolonged, but even in these infants neutropenia resolves spontaneously.

Treatment of Neonatal Neutropenia

Recombinant granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are often used to treat neonatal neutropenia. G-CSF stimulates neutrophil production, maturation, and release from the marrow and also reduces neutrophil apoptosis. GM-CSF generates both granulocyte and macrophage colonies from precursor cells. G-CSF is the primary systemic regulator of the circulating neutrophil concentrations. GM-CSF may not play a major role in the steady state but is induced in inflamed tissues.

Recombinant G-CSF or GM-CSF has been used in neonatal sepsis with conflicting results. In a meta-analysis of five studies, G-CSF was shown to reduce mortality (odds ratio 0.17, 95% confidence interval [CI] 0.03 to 0.70). However, this protective effect was no longer significant when nonrandomized studies were excluded. The role of GM-CSF in the prevention or treatment of neonatal sepsis is also unclear. Carr et al. reviewed the efficacy and safety of G-CSF/GM-CSF in the treatment or prophylaxis of neonatal sepsis. In preterm infants with suspected sepsis, G-CSF or GM-CSF did not reduce mortality at 14 days after the start of therapy (relative risk [RR] of death 0.71, 95% CI 0.38 to 1.33). However, in a subgroup of 97 infants with sepsis and neutropenia (ANC <1700/μL), there was a significant reduction in mortality (RR 0.34, 95% CI 0.12 to 0.92; number needed to treat 6, 95% CI 3 to 33).

Recombinant G-CSF is highly effective in correcting immune-mediated neutropenia. Along with its effects on neutrophil production, G-CSF may decrease antigen expression on neutrophils, rendering them less vulnerable to circulating antibodies, and also improve neutrophil function. Similarly, G-CSF is also effective in many patients with congenital neutropenias. Kostmann syndrome presents in early infancy with low ANCs, often less than 200/μL, and recurrent bacterial infections. These patients have mutations in the neutrophil elastase gene ELANE . G-CSF can reduce the need for antibiotics and hospitalization and can improve survival in these patients. Cyclic neutropenia is another rare hematologic disorder that may be modified by the use of G-CSF. Cyclic neutropenia is characterized by regular drops in ANCs to levels as low as 250/μL at 3-week intervals. Although the marrow may look normal during periods of higher ANCs, it shows a characteristic “maturational arrest” of myeloid cells during or just before the onset of severe neutropenia. G-CSF treatment can be used to raise neutrophil counts during periods of severe neutropenia. These patients may not respond to GM-CSF. While neutropenia resolves spontaneously in most patients with idiopathic neutropenia of prematurity, in one study, G-CSF treatment induced a rapid rise in ANC, indicating an adequate marrow neutrophil reserve in these patients.

G-CSF is administered intravenously or subcutaneously at a dosage of 5 to 10 μg/kg/day. The response to G-CSF therapy and a rise in ANC usually occurs within 24 to 48 hours. In an occasional patient, G-CSF therapy will not raise blood neutrophil counts. In such a patient, G-CSF doses can be increased in increments of 10 μg/kg at 7- to 14-day intervals if the ANC remains below 1000/μL. Doses can be reduced or withheld once the ANC exceeds 5000/μL. G-CSF treatment is usually tolerated in neonates without adverse effects. Long-term G-CSF therapy in congenital neutropenias has been associated with splenomegaly, thrombocytopenia, osteoporosis, myelodysplastic syndrome/leukemia, and the development of anti–G-CSF antibodies.

Intravenous immunoglobulin (IVIG) is effective in about 50% of infants with alloimmune and autoimmune neutropenia. IVIG can mobilize neutrophils from the NSP, although repeated doses may be needed for a sustained effect. While steroids are generally not effective in alloimmune neutropenia, may raise the ANC for short periods in autoimmune neutropenia of infancy. Exchange transfusions are generally not effective in immune-mediated neutropenia.

Monocyte Physiology and Dysfunction

Ontogeny

Embryonic macrophages are found among hematopoietic cells in the yolk sac at 3 to 6 weeks’ gestation. The fetal liver becomes the primary site of hematopoiesis from 6 weeks until midgestation, and the bone marrow then becomes the lifelong center of blood cell production. Monocytes are present in high proportions in the early hematopoietic tissues, with approximately 70% of hematopoietic cells at 4.5 weeks’ gestation morphologically identifiable as monocytes. This proportion falls from 1% to 2% during the next 6 weeks as erythroid cells become predominant. The precursors of monocytes, monoblasts, and promonocytes continue to be present in the fetal liver; however, intravascular monocytes are not observed until the fifth month of gestation. Circulating monocytes do not appear with regularity until hematopoiesis is first established in the bone marrow after the 10th week of gestation.

During ontogeny, macrophages in the fetal liver express CD11b as early as 12 weeks’ gestation. The classic monocyte marker, CD14, does not appear until about 15 to 21 weeks’ gestation. CD14 expression on circulating mononuclear cells is equivalent in cord blood and adult peripheral blood. CD11a, CD11b, and CD11c are expressed in lower densities on cord blood monocytes than on adult cells. There is also a lower expression of class II major histocompatibility complex (MHC) antigens HLA-DR, HLA-DP, and HLA-DQ on neonatal monocytes compared with adult monocytes. The density of these class II MHC antigens has been correlated with the antigen-presenting capacity of monocytes in vitro, although the effect of this deficiency on neonatal host defense is not clear. Other important monocyte markers are the receptors for the Fc moiety of IgG (FcγR) and the Toll-like receptors (TLRs). FcγR receptors are important in the process of monocyte and macrophage phagocytosis of microbes and antibody-dependent cytotoxicity. Monocytes constitutively express the high-affinity receptor FcγRI (CD64) and FcγRII (CD32). TLRs play a critical role in recognition of microbial pathogens. Term neonatal monocytes express normal basal levels of TLR2 and TLR4 but show reduced tumor necrosis factor (TNF) release in response to stimulation with a range of TLR agonists. Cord blood monocytes of preterm neonates, however, have lower TLR4 expression than adult peripheral blood monocytes.

Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that regulates the proliferation, differentiation, and functional activation of monocytes. Normally detected in human serum, M-CSF plays an important role in enhancing the effector functions of monocytes and macrophages. Serum M-CSF levels are increased in cord blood and rise further during the neonatal period.

Circulating Monocytes

Term infants show a relative monocytosis that persists through the neonatal period. Although there is some disagreement about normal blood monocyte counts in neonates, we have described normal ranges of absolute monocyte counts (AMCs) using data from more than 62,000 blood counts. In this cohort, blood monocyte concentrations increased almost linearly between 22 and 42 weeks’ gestation. Monocyte concentrations also increased during the first 2 weeks postnatally. These data are consistent with previous kinetic studies in human fetuses that show a similar maturational increase in the concentrations of monocyte precursors. In neonates, monocytosis has been associated with prematurity, blood transfusions, albumin infusions, and theophylline therapy. Monocytosis has also been described in infants with congenital infections such as candidiasis and syphilis. and in association with immune-mediated neutropenia. In contrast, monocytopenia is not seen frequently in neonates, except in growth-restricted preterm infants who may have low monocyte counts as part of an overall suppression of all leukocyte lineages. Recently, we showed that a fall in AMCs can be a useful diagnostic marker of necrotizing enterocolitis (NEC) in VLBW infants. In this study, we compared blood counts obtained at the onset of feeding intolerance with the last available counts obtained before the onset of symptoms. In an infant with feeding intolerance, a drop in AMC of more than 20% indicated NEC with a sensitivity of 0.70 (95% CI 0.57 to 0.81) and specificity of 0.71 (95% CI 0.64 to 0.77). The negative predictive value was 88%, indicating that the test may be valuable for exclusion of the diagnosis of NEC in infants with feeding intolerance due to other causes. Despite modest diagnostic accuracy, AMC is a convenient tool because the information is already available at no extra cost in complete blood counts from automated hematology analyzers.

Monocyte Subsets

Increasing evidence indicates that peripheral blood monocytes are a heterogeneous population comprised of two major subpopulations. The predominant subtype is “classic” CD14 + CD16 - monocytes, which express C-C chemokine receptor (CCR)2, CD64, and CD62L and represent nearly 80% to 90% of all blood monocytes. The remaining fraction comprises the “nonclassic” CD14 low CD16 + monocytes that lack CCR2. Both subsets express the receptor for fractalkine, CX3C chemokine receptor (CX 3 CR)1, but CD14 low CD16 + monocytes characteristically express higher levels. CD16 + monocytes are composed of at least two populations with distinct functions. Monocytes that express CD16 and CD14 (CD14 + CD16 + ) also express the Fcγ receptors CD64 and CD32, have phagocytic activity and produce TNF and interleukin (IL)-1β in response to lipopolysaccharide (LPS). In contrast, monocytes that express CD16 but very low levels of CD14 (CD14 dim CD16 + ) lack the expression of other Fc receptors, are poorly phagocytic and do not produce TNF or IL-1β in response to LPS. This subset of “resident” monocytes patrol blood vessels in the steady state and extravasate during infection with Listeria monocytogenes or during tissue healing.

Monocyte Function

Monocytes are capable of directed movement (chemotaxis) in response to chemoattractants produced by bacteria or by host cells at the site of injury or invasion. The chemotactic capabilities of neonatal and adult peripheral blood monocytes have been compared, and chemotaxis was found to be less pronounced in neonates than in adults ( Table 71.2 ).

Table 71.2
Function and Phenotype of Adult Versus Neonatal Mononuclear Cells
Cells Function/Phenotype Adult Neonate
Monocytes Chemotaxis ↑ ↓
Phagocytosis ↑ ↓
Adhesion ≈ ≈
Respiratory burst ≈ ≈
Dendritic cells Expression of CD83, CD86 ↑ ↓
Mixed lymphocyte reaction ↑ ↓
IL-12 (p40) production ↑ ↓
IL-12 production ↑ ↓
IL-10 production ↓ ↑
Natural killer cells Expression of CD8, CD57 ↑ ↓
Expression of ICAM-1, CD161 ↑ ↓
Cytolytic activity ↑ ↓
ICAM-1 , Intercellular adhesion molecule1; IL , interleukin.

During an acute infection, circulating monocytes become activated and migrate into the tissues. During this process, monocytes adhere to the endothelium through the interaction of the integrins (CD11a, CD11b, CD11c, and CD18) expressed on the monocyte cell membrane, with ICAM-1 or ICAM-2 on the endothelial surface. Finally, the activated monocyte moves through the endothelium to the site of inflammation or infection. Preliminary studies demonstrate that the levels of monocyte adhesion molecule expression are comparable in neonate and adult peripheral blood. The CD11b–CD18 complex (macrophage one antigen/complement receptor 3) promotes monocyte trafficking to the sites of infection by binding ICAM-1 and is also involved in the recognition of opsonized microbial pathogens.

Antimicrobial activity of monocytes includes oxygen-dependent mechanisms such as the respiratory burst, which through a complex series of reactions forms highly reactive hydroxyl radicals that damage host and microbial membranes. The ability of fetal and neonatal monocytes to kill pathogens ( Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli , and Candida albicans ) is generally comparable to that of the monocytes in adult peripheral blood (see Table 71.2 ), although isolated preterm monocytes may show weaker superoxide production and degranulation in vitro than monocytes from term neonates.

On exposure to microbial antigens, monocytes and macrophages become activated by collaborative actions of soluble recognition proteins, including CD14 and TLRs, and produce several cytokines and chemokines contributing to the inflammatory process. IL-1β, interferon (IFN)-α, and TNF are synthesized at similar levels in adults and neonates. Kaufman et al. detected lower TNF production in LPS-stimulated adherent monocytes from preterm infants compared with monocytes from term infants; however, no difference was seen in the production of IL-1β or IL-6. They also found lower expression of CD11b and CD18 adhesion receptor subunits in preterm monocytes. Although TLR expression of term neonatal monocytes is similar to that of adult monocytes, there are important functional differences. Levy et al. reported that cord blood monocytes are less sensitive to TLR ligands than adult monocytes for TNF induction. The innate immune responses of neonatal monocytes to TLR agonists may be biased toward high IL-6 levels but low TNF levels in vitro because of distinct neonatal cellular (monocyte) and humoral (serum) factors, and such a pattern was also evident in vivo.

Developmental Defects in the Phagocytic Immune System in Neonates

The immaturity of the phagocytic immune system predisposes neonates to increased morbidity and mortality during bacterial sepsis. This impairment is attributed to developmental deficiencies in both the innate immune system and the adaptive immune systems (see Fig. 71.1 ). In the following section, we use the term phagocyte to refer mainly to neutrophils and the monocyte/macrophage lineage. Although immature DCs also show phagocytic activity, there is limited information about these cells in the neonate. The phagocyte system depends on the presence of adequate numbers of phagocytes in circulation to function efficiently, the ability to respond to signals from the sites of inflammation, the ability of phagocytes to migrate to these sites, and the capability of phagocytes to ingest and kill invading microorganisms.

Several studies show important differences in TLR expression on neonatal versus adult leukocytes. The basal expression of TLR2 and TLR4 in neutrophils and monocytes from healthy adults and term neonates is similar. During sepsis, neonates showed a sustained upregulation of TLR2 on monocytes but not on neutrophils, and there was no change in TLR4 expression on monocytes or neutrophils. In VLBW infants, monocytes express TLR4 at much lower levels than in mature infants and adults and show significantly lower LPS-stimulated IL-1β, IL-6, and TNF release in vitro. Further, CD14 and MD-2, co-receptors for gram-negative and gram-positive cell wall constituents alongside TLR2 and TLR4, respectively, are also expressed at lower levels on preterm infant monocytes. Stimulation of newborn monocytes with LPS produced a significant decrease in the expression of MyD88, supporting the premise of impaired TLR4-mediated signaling Belderbos et al. compared TLR-induced cytokine responses of whole blood leukocytes from cord blood from healthy term neonates, neonatal venous blood at the age of 1 month, and adult venous blood. On TLR4 activation, neonatal leukocytes (both at birth and at 1 month of age) showed a skewed pattern of cytokine expression, with low levels of T-helper (T h ) type 1–polarizing cytokines such as IL12p70 and IFN-α and more of the anti-inflammatory cytokine IL-10. In contrast, cytokine responses to TLR3, TLR7, and TLR9 matured by 1 month of age.

Recent studies indicate that epigenetic changes during development may underlie the observed maturation in monocyte function with advancing gestational age. Histone modifications H3K4me3 and H3K4me1 affect promoter sites of various immune genes (IL1B, IL6, TNF) in monocytes. Increased H3K4me3 activation at these promoter sites (compared to H3K4me1 activity leading to increased H3K4me3/H3K4me1 ratio) is observed in monocytes with advancing gestational age and thought to be important in modifying the epigenetic landscape of monocytes to a more mature and functional state with age.

The immaturity of neonatal host defense is also characterized by profound deficiencies in quantitative and qualitative phagocytic effector cell function, particularly during stress or bacterial sepsis. The initial step in mounting a host defense response is directed migration or chemotaxis ( chemo refers to a chemical substance; taxis refers to rearrangement) of activated phagocytes toward the site of microbial invasion. Such movement occurs along concentration gradients of chemoattractants, which may include bacterial products such as N -formyl- l -methionyl- l -leucylphenylalanine (f-MLP), leukotriene B 4 , complement products such as C5a, or chemokines. At the site of inflammation, the phagocytes ingest and kill the pathogens by oxygen-dependent and oxygen-independent mechanisms. Neutrophils from both preterm and term neonates show numerous qualitative abnormalities, including decreased deformability and impaired functions, including chemotaxis, phagocytosis, adherence, bacterial killing, aggregation, and oxidative metabolism. In contrast to neutrophils, neonatal monocytes are more comparable in function to the monocytes of adults.

GM-CSF increases adult neutrophil oxidative metabolism by augmenting superoxide anion production. Additionally, GM-CSF increases chemotaxis, promotes phagocytosis of Staphylococcus aureus and augments neutrophil aggregation by the increased expression of surface adhesion molecules. GM-CSF is not a direct stimulant of neonatal neutrophil function, but cord blood leukocytes show increased superoxide production if primed with GM-CSF before exposure to f-MLP or opsonized zymosan particles.

The diminished inflammatory response of neonatal neutrophils results in a high incidence of microbial invasion. Significant defects in the upregulation of surface-active glycoprotein receptors (C3bi) and reduced aggregation also predispose the neonate to impaired response to bacterial infection. C3bi expression has been compared in adult and neonatal neutrophils and was found to be significantly less in neonatal neutrophils when stimulated by f-MLP– or zymosan-activated serum. However, cord blood neutrophils incubated with GM-CSF demonstrated a significant induction of C3bi expression. Also, a significant increase in C3bi expression was seen when cord blood neutrophils were pretreated with GM-CSF and subsequently stimulated with the calcium ionophore A23187. The upregulation of the C3bi receptor by GM-CSF also appears to correlate with an enhancement of neutrophil aggregation. GM-CSF also primes neutrophils for increased neutrophil aggregation following agonist (f-MLP) stimulation.

G-CSF and TNF have also been reported to modulate the function of adult and neonatal neutrophils in a manner similar to that of GM-CSF. Priming cord blood neutrophils with G-CSF or TNF and subsequent stimulation with f-MLP induces the expression of C3bi receptors and enhances the bacterial and phagocytic activity and superoxide generation.

Lymphocyte Contributing to Acquired Immunity

Lymphocytes form critical components of the acquired immune system and constitute 20% of blood leukocytes. There are two broad categories: (1) T lymphocytes, which mature in the thymus and subsequently seed peripheral lymphatic organs, including the spleen and lymph nodes; and (2) B lymphocytes, which are produced in the bone marrow, mature in secondary lymphoid organs, and subsequently differentiate into antibody-secreting plasma cells. The following sections outline the development and function of T and B lymphocytes in the neonate.

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