Neutrophil Production and Function in Newborn Infants

18 q 2000 Blackwell Science Ltd

Review

NEUTROPHIL PRODUCTION AND FUNCTION IN NEWBORN INFANTS

Bacterial infection is a major cause of death and long-termmorbidity in preterm neonates. Infection rates amongneonates undergoing intensive care range from 25% to50% (Stoll et al, 1996; Cooke et al, 1997; Fanaroff et al,1998) and, in spite of effective antibiotic therapy, sepsis-related mortality has remained constant at 15±20% fornearly two decades (Gladstone et al, 1990).

The reason for such high sepsis rates and attendantmortality are largely due to the immaturity of bactericidalmechanisms. Clinical evidence of the preterm neonate'simmune incompetence is the pattern of bacterial infectionswhich closely parallels that seen in patients with profoundneutropenia (Stoll et al, 1996) and the frequent develop-ment of neutropenia in response to bacterial sepsis (Engleet al, 1984; Gessler et al, 1995). These two phenomena canbe directly related to immaturity of neutrophil function andproduction.

NEUTROPHIL PRODUCTION

Newborn infants, whether born at term (36±40 weeksgestation) or very preterm (24±31 weeks gestation) haveperipheral blood neutrophil counts very similar to olderchildren and adults (Manroe et al, 1979; Mouzinho et al,1994). However, they differ from adults in their response tobacterial sepsis. Although adults develop a sustainedneutrophil leukocytosis by releasing preformed neutrophilsfrom the marrow storage pool into the circulation andincreasing proliferation by recruiting more committedgranulocyte progenitors (granulocyte±macrophage colony-forming units; GM±CFU) into the cell cycle, pretermneonates frequently become neutropenic (Christensen,1989).

Our understanding of the kinetics of neutrophil produc-tion in immature newborn infants largely comes fromstudies in rodents. These have shown that newborn ratshave a total pool of GM-CFU that is less than 10% of the GM-CFU per gram body weight of adults and unlike adults, whohave a large pool of quiescent progenitors to recruit into thecell cycle in the face of sepsis, over 75% of GM-CFU in non-infected newborn rats are in active cell cycle (Christensenet al, 1984; Christensen, 1988). Circumstantial evidence fora similar immaturity of granulopoiesis in human neonatescomes from studies in infants born at or near term which

show a similar pattern of continuous near maximal GM-CFUproliferation (Christensen et al, 1986) compared with thelarge reserve of non-proliferating GM-CFU in human adults(Fauser & Messner, 1979).

The immediate fall in neutrophil count that accompaniesneonatal sepsis may be due to more than an inability toincrease the proliferation of early progenitors. Profounddepletion of marrow neutrophils, as assessed histologically,has frequently been demonstrated in septic neutropenicinfants and is associated with a high mortality (Christensenet al, 1980; Wheeler et al, 1984). From this, one mightconclude that the most important determinant of criticalneutrophil depletion, susceptibility to overwhelming sepsisand resulting death may be the available total mass ofpreformed neutrophils in the marrow at the time ofinfection.

Studies in newborn rats have demonstrated that theirabsolute neutrophil cell mass per gram of body weight isonly one-quarter of that of adult animals and is even less inpreterm rat pups. After birth, the neutrophil cell mass pergram of body weight expands to reach adult levels afterabout 4 weeks (Erdman et al, 1982).

Recently, evidence has been presented that newbornhuman infants born before 32 weeks gestation likewisehave a total neutrophil cell mass which is < 20% of adultvalues, using the plasma concentration of soluble FcRIII(sFcRIII) as a surrogate marker (Carr & Huizinga, 2000).sFcRIII is the plasma form of the neutrophil membranereceptor FcgRIII. Recent work has demonstrated thatsFcRIII is derived from apoptotic neutrophils and itsconcentration in plasma reflects the total body neutrophilmass as well as overall production of neutrophils in thebone marrow (Huizinga et al, 1990a; 1994; Homberg et al,1995). Preterm neonates have very low concentrations ofsFcRIII. In contrast, term neonates have plasma sFcRIIIconcentrations and, by implication, neutrophil storeswithin the normal adult range. Furthermore, in neonatesborn prematurely, the very low sFcRIII levels present atbirth increase postnatally to reach adult values by thefourth postnatal week (Carr et al, 1992a). These dataindicate that preterm neonates have a neutrophil pool sizeat birth and a timescale of postnatal expansion almostidentical to those established by direct measurement innewborn rats.

In summary, immaturity of granulopoiesis in pretermneonates is manifest by a low neutrophil cell mass, a limitedspare capacity for increasing progenitor proliferation and, asa consequence, the frequent occurrence of neutropenia inresponse to sepsis.

British Journal of Haematology 2000, 110, 18±28

Correspondence: Dr Robert Carr, Department of Haematology, StThomas' Hospital, Lambeth Palace Road, London SE1 7EH, UK.

E-mail: [email protected]

NEUTROPHIL FUNCTION

ChemotaxisThe most consistently observed functional abnormality ofneonate neutrophils is reduced chemotaxis. In most assays,neutrophils from newborn infants migrate at about half thespeed travelled by adult cells (Miller, 1971; Klein et al, 1977;Pahwa et al, 1977; Anderson et al, 1981; Boner et al, 1982;Sacchi et al, 1982; Krause et al, 1986a; Carr et al, 1992b;Wolach et al, 1998). Chemotaxis, or cell movement towardsan inflammatory stimulus, involves the cell's ability todetect an inflammatory signal, adhere to tissue matrixthrough adhesion receptors and redistribute those receptorsover the cell membrane in order to propel the cell forwardwhile letting go of the surface it is leaving behind. Thisprocess is measured in the laboratory by stimulating cellmovement under a layer of agar, through a micropore filteror, in more recent studies, through cultured monolayers ofvascular endothelium.

Three observations of neutrophil behaviour illustrate thedifference between neonatal and adult cells. (i) A proportionof neutrophils in the blood stream roll along the vessel wallby loose attachment to the vascular endothelium. As theypass an inflammatory focus, they come into contact withactivated endothelium and are arrested by firm adherenceto the endothelial cells, between which they then migrate toenter the extravascular matrix. Neonatal neutrophils dis-play less interaction with endothelial monolayers inconditions of flow than adult cells. Rolling adhesion isdiminished, fewer cells attach to activated endothelium andfewer cells migrate to the subendothelial tissue (Andersonet al, 1991). (ii) Adult neutrophils adhere to protein-coatedglass after activation by a chemotactic stimulus. If the cellsare given a second stimulus and the glass inverted, thecells hang from the surface and ultimately detach. Fewer cellsfrom neonates will attach to glass after the first stimulusand they fail to detach with the second (Anderson et al,1981). (iii) Albumin-coated latex beads bind randomly tothe surface of adult neutrophils after exposure to achemotactic factor in low dose. With a second and largerstimulus, the cells take up a bipolar shape with ruffledcytoplasmic membrane directed towards the stimulus (thelamellipod) and a tail-like extension (the uropod) away fromthe stimulus. As this happens, the latex beads congregate onthe uropod. With a third stimulus, the beads on the uropodare released and new binding sites appear on the lamellipod(Smith & Hollers 1980). Neonatal neutrophils fail to take upa complete bipolar shape and fail to redistribute beadbinding sites to the uropod/tail of the cell (Anderson et al,1981).

The explanation for these abnormal patterns of behaviourlies in the abnormal expression and dynamics of twofamilies of cell membrane adhesion molecules, the b2

integrins and the selectins, together with abnormalities ofthe neonate neutrophil cytoskeleton.

L-Selectin and vascular rolling. Neutrophil rolling alongendothelium under conditions of flow is mediated by theselectins (Kansas, 1996). These molecules initiate the earliestevents in leucocyte±endothelial adhesion by capturing

leucocytes from the blood stream and so mediate a looseadherence that allows rolling along the vessel wall. Theprincipal selectin on neutrophils is L-selectin, which islocated on the tips of surface folds of unstimulatedneutrophils where contact is easily made with the vascularendothelium. The process is aided by E-selectin, expressedon activated endothelial cells, and P-selectin on bothendothelium and activated platelets aggregating at sites ofvascular injury. Several studies have shown neutrophilsfrom term neonates to express less than half the number ofL-selectin receptors on their surface compared with adultneutrophils (Anderson et al, 1991; ToÈroÈk et al, 1993;Rebuck et al, 1995; Koenig et al, 1996; Mariscalco et al,1998). Confirmation that this underlies the reduced rollingattachment of neonatal cells to endothelium comes from astudy in which adult neutrophils had their L-selectinreceptors blocked by antibody, which reduced their inter-action with endothelium to the level of neonatal cells(Mariscalco et al, 1998).

Mac-1, adhesion and chemotaxis. L-selectin itself is insuffi-cient to arrest neutrophil rolling; this requires activationand increased expression of the b2 integrins Mac-1 and LFA-1to bind the neutrophil firmly before chemotactic crawlingcan begin (Lawrence & Springer, 1991; Hughes et al, 1992;Carlos & Harlan, 1994; Simon et al, 1995; Smith et al,1989; Von Andrian et al, 1991). The critical importance ofthese adhesion molecules is demonstrated by observation ofchildren with an inherited deficiency of the b chaincommon to these two receptors and consequent absent orlow expression of both Mac-1 and LFA-1. In this syndrome,known as leucocyte adhesion deficiency (LAD), the neutro-phils do not adhere, aggregate or migrate in vitro or in vivoand the affected children suffer recurrent and often life-threatening bacterial infections (Todd & Fryer, 1988). Invitro experiments blocking the individual receptors withantibodies show Mac-1 to be the more important of thetwo receptors for neutrophil adhesion and chemotaxis(Anderson et al, 1986).

In resting neutrophils, low levels of Mac-1 are distributedevenly over the cell surface (Smith & Hollers 1980). Duringcell locomotion, Mac-1 receptors are translocated to the tailof the cell while the new receptor is translocated from storesin peroxidase-negative cytoplasmic granules (Bainton et al,1987) to the leading edge of the migrating cells (Franciset al, 1989). When neonatal neutrophils are stimulated invitro they do not up-regulate the number of Mac-1molecules on the cell surface to the same extent as adultcells (Anderson et al, 1987; Carr & Davies, 1990; Carr et al,1992b; ToÈroÈk et al, 1993). This is due to both a reducedtotal cell content of preformed Mac-1, which in termneonates is about 60% of adult cell Mac-1 (Abughali et al,1994; McEvoy et al, 1996), and a reduced translocation ofpreformed receptor from cytoplasmic granules to the cellsurface (Todd et al, 1984; Anderson et al, 1987; Jones et al,1990). The secondary translocation of receptors to theuropod is also impaired (Anderson et al, 1981). Theseabnormalities of Mac-1 correlate closely with the reducedchemotaxis of neonatal neutrophils observed in in vitroassays (Anderson et al, 1987).

q 2000 Blackwell Science Ltd, British Journal of Haematology 110: 18±28

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Studies of neutrophil adhesion and transmigration throughendothelial monolayers have shown that term neonate cellsdisplay about 50% transmigration compared with adult cells,and antibody blocking has shown that the neonatal defectcan likewise be explained almost entirely by their reducedMac-1 expression (Anderson et al, 1990). In the termneonate, LFA-1 expression and LFA-1-dependent adhesionto endothelium appears intact (Anderson et al, 1990, 1987).

Immaturity of the vascular endothelium may alsocontribute to reduced neutrophil transmigration in pretermneonates. In a newborn rodent model, neutrophils fromadult animals had delayed transendothelial migration dueto reduced expression of P-selectin on the newbornendothelium. When human fetal endothelium was exam-ined, the level of P-selectin varied with gestational age,being absent or only weakly expressed before 27 weeks butexpressed normally at term (Lorant et al, 1999).

Although reduced up-regulation of Mac-1 on neonatalneutrophils is a consistent finding, and the close correlationbetween this and reduced adhesion to glass coverslips instatic assay has been clearly demonstrated (Anderson et al,1987), there remains doubt as to whether this quantitativeabnormality is responsible for all the in vitro abnormalities ofchemotaxis and transendothelial migration or for the in vivosusceptibility to infection. Heterozygotes for leucocyteadhesion deficiency have a similar reduction in stimulatedMac-1 expression, yet they display minimal neutrophilchemotactic functional defects and no more infection thannormal individuals (Anderson et al, 1985). Of greaterimportance may be altered receptor dynamics. Neonateneutrophils show impaired shedding of L-selectin afterstimulation (Koenig et al, 1996), and Mac-1 up-regulation,flow over the cell and shedding are all abnormal. Theimportance of receptor shedding is demonstrated by thesequential stimulation of adult cells which ultimately leadsto a reduction in overall adhesiveness associated with loss ofadhesion proteins from the uropod and thus, by `letting go',forward movement is achieved. Neonatal neutrophils, whichfail to redistribute the adhesion sites to the uropod, increasetheir adhesiveness with sequential stimulation (Andersonet al, 1981), thus forward motion is impeded (Smith et al,1979).

The neutrophil cytoskeleton. The altered receptor dynamics,described above, may be linked to abnormalities of theneutrophil cytoskeleton and a lack of cell fluidity. Earlystudies using cell elastimetry demonstrated neonatalneutrophils to be more rigid than adult cells (Miller,1979). More specific evidence for abnormalities of theneutrophil cytoskeleton is provided by the observation thatneonatal neutrophils have increased basal levels of filamen-tous F-actin (Hilmo & Howard, 1987). After stimulation,neonatal cells with defective chemotaxis fail to polymeraserapidly monomeric G-actin to increase the F-actin cellcontent to the peak level observed in normally migratingcells (Sacchi et al, 1987; Harris et al, 1993). In addition,others have found reduced microtubule formation, whichcorrelates with the inability of neonate cells to achieve afully bipolar shape change on stimulation (Anderson et al,1984).

Effects of prematurity and stress on chemotaxis. Thediscussion so far has been based on data from termneonates in whom neutrophils were tested either at, orwithin a few days of, birth. Do preterm neonates, who havea higher incidence of sepsis, display greater defects inneutrophil migration and associated receptor expression?

The neutrophils of clinically stable preterm neonates asimmature as 24 weeks gestation when tested within a fewdays of birth appeared functionally very similar to terminfants. In vitro chemotaxis of neutrophils from clinicallystable preterm neonates born between 24 and 32 weeksgestation was identical to that of neutrophils from terminfants in our studies (Carr et al, 1992b) and those of others(Krause et al, 1986a; Bektas et al, 1990). This is associatedwith similar abnormalities of adhesion to endothelialmonolayers in a static assay (Carr et al, 1992b).

We found L-selectin expression on unstimulated neutro-phils from neonates , 32 weeks gestation to be < 40% ofthat on adult cells (unpublished observations), which is verysimilar to that reported for term neonate neutrophils(Rebuck et al, 1995). Likewise, preterm neutrophils displaya reduced up-regulation of Mac-1 expression after stimula-tion, that is similar to term cells (Carr et al, 1992b).However, this phenotypic similarity may hide a moresignificant difference between immature preterm and terminfants. A study of preterm infants which measured the totalcell content of Mac-1 both in cytoplasmic stores and on thecell membrane showed the Mac-1 content to range from10% of adult levels at 27 weeks to 48% at 36 weekscompared with 57 ^ 4% in six term infants. There was ahighly significant linear correlation between Mac-1 cellcontent and gestational age (McEvoy et al, 1996).

The clinical instability of preterm infants makes it difficultto separate the effects of clinical stress (either cardiorespira-tory or septic) from the effect of prematurity on neutrophilfunction. We found that uninfected neonates ventilated forsevere respiratory distress syndrome had increased chemo-taxis compared with stable preterm and term infant cells(Carr et al, 1992b). In contrast, a study of septic preterminfants showed that Gram-negative septicaemia depressedchemotactic responses whereas superficial infection hasbeen associated with enhanced chemotaxis (Laurenti et al,1980). Another study of clinically stressed preterm neonatesshowed them to have reduced chemotaxis, as a group,compared with healthy infants, although infants with sepsisand respiratory distress were not analysed separately(Krause et al, 1986a).

Postnatal maturation of chemotaxis. If, in utero, theimmature chemotactic response of neutrophils remainsconstant between 24 weeks and term, what is the influenceof birth? What is the time-course of postnatal maturationand does it differ between infants born prematurely and atterm?

The earliest study of postnatal neutrophil maturationcompared preterm neonates born before 34 weeks gestationwith those born after 34 weeks and with full term deliveries(Sacchi et al, 1982). Term infants had achieved adultchemotactic performance by 2 weeks postnatal age. Pre-terms born between 34 and 36 weeks had immature

20 Review


chemotaxis 2 weeks after birth, but normal adult chemo-taxis by 40±42 weeks post-conceptional age. More imma-ture preterms born before 34 weeks, when tested at42 weeks, showed some improvement in chemotacticperformance, but it remained significantly impaired com-pared with adults. Three other studies have extended andconfirmed these findings. Eisenfeld et al, (1990) followed 37healthy term infants for 2´5 years from birth. Chemotaxiswas 40% of adult values at birth, unchanged at 5 d butnormal by 10±32 d and thereafter. We (Carr et al, 1992b)followed 23 preterms aged 24±32 weeks (median27 weeks) over 2 months. Chemotaxis was unchangedfrom birth during the second and third postnatal week,but showed significant improvement during the secondmonth. However, at final testing, it remained abnormalcompared with adults, even though all assays were under-taken when the infants were free of sepsis or other clinicalstress. This observation has been confirmed by others(Usmani et al, 1991). The link between chemotaxis andMac-1 expression is extended by our observation thatneutrophil Mac-1 receptor expression on stimulated cellsincreased in parallel with improving chemotaxis (Carr et al,1992b).

In summary, neutrophil migration, assessed by in vitroassays, is abnormal at birth, although similar in both termand preterm neonates. Term infants rapidly establishnormal chemotactic function. In immature preterm infants,the process of postnatal maturation begins 2±3 weeks afterpreterm birth but proceeds very slowly. Why this should beso and how much the process is delayed by sepsis and otherimmunological stimuli in the postnatal period has not, atpresent, been established.

PhagocytosisHaving left the vascular compartment, the cell follows aconcentration gradient of chemotactic factors until itreaches the site of microbial invasion. Here, it encountershigh concentrations of inflammatory mediators and cyto-kines, forward movement ceases and it up-regulatesreceptors involved in phagocytosis and activation of thecell's bactericidal machinery. Reactive oxygen species aregenerated through the respiratory burst and bactericidalsubstances released from cytoplasmic granules into thephagolysosome.

The process of phagocytosis and killing by neutrophils ismediated through receptors for both complement and the Fcdomain of immunoglobulin and therefore operates moreefficiently when the organism is opsonized by specificantibody and appropriate complement fragments (Yanget al, 1989).

Complement receptors and phagocytosis. The opsonicactivity of complement resides in the surface-boundfragments of C3:C3b, which is recognized by the comple-ment receptor 1 (CR1), and the more stable iC3b, which isrecognized by complement receptor 3 (CR3). CR1 expres-sion has been found to be normal on neonate neutrophilsby most investigators (Anderson et al 1987; Bruce et al,1987).

The most important receptor for phagocytosis may be

CR3, which is the same receptor as Mac-1, discussed above.This versatile integrin has a number of different bindingdomains: for ICAM-1 on endothelium (discussed aboveunder Mac-1), for iC3b and for a lectin-like binding site whichcan bind bacteria independent of opsonization (Smith, C.L.et al, 1990). The importance of the CR3/Mac-1 receptor forneutrophil bactericidal function is again demonstrated by itsabsence in patients with leucocyte adhesion deficiency, inwhom cells not only have chemotactic defects but also showdefective cytocidal activity in vitro (Todd & Freyer, 1988).CR3 may have particular importance for neonatal neutro-phil phagocytosis in the setting of low type-specific antibodyand opsonin deficiencies in term neonates (Geelen et al,1990; Droussou et al, 1995) and severe hypogammaglobu-linaemia (Ballow et al, 1986) and deficient complementactivity (Kovar et al, 1983; Notarangelo et al, 1984) inpreterm infants. Its ability to bind to bacteria throughlectins on their surface may compensate for lack ofopsonization. A detailed study of the role of complementreceptors for phagocytosis of group B streptococci (GBS)(Smith, C.L. et al, 1990) showed that in hypogammaglobu-linaemic serum CR3 can achieve significant binding of GBStype III in the absence of opsonization through the lectinbinding site. Furthermore, whereas CR1 blocking byantibodies had only a minor effect on the function ofneonatal neutrophils, CR3 1 CR1 blockage almost comple-tely prevented type III GBS uptake. It has likewise beenshown that CR3 can bind Escherichia coli in the absence ofopsonization by direct reaction with lipopolysaccharide(Wright et al, 1989).

The ability of CR3 to mediate phagocytosis in the settingof low immunoglobulin and complement levels suggeststhat this receptor's reduced expression on the neutrophils ofpreterm neonates is an important factor in their difficulty indealing with bacterial sepsis.

Fc gamma receptors and phagocytosis. Neutrophils expresstwo classes of Fcg receptor that together mediate thebinding, ingestion and killing of bacteria. FcgRII is atransmembrane structure that mediates IgG-induced phago-cytosis (Tosi & Berger, 1988; Huizinga et al, 1989; Indik et al,1995) and initiates superoxide generation and the respira-tory burst (Looney et al, 1986; Huizinga et al, 1989).Neutrophil FcgRIII is a phosphoinositol (PI)-linked structurewhich lacks transmembrane and cytoplasmic domains(Huizinga et al, 1988; Selvaraj et al, 1988). In spite of itslack of direct contact with cytoplasmic second messengersystems, it is able to activate neutrophil granule exocytosis(Huizinga et al, 1990b) and phagocytosis (Salmon et al,1987). The mechanism by which FcgRIII activates the cellsremains uncertain, however it has been suggested that theextra mobility of FcgRIII within the cell membrane, aconsequence of its PI linkage, may allow it to bring IgG-opsonized particles into close proximity with FcgRII or CR3receptors which, in turn, trigger the bactericidal mechan-isms (Indik et al, 1995; McKenzie & Schreiber, 1998). Thehypothesis that FcgRIII can concentrate opsonized particlesin the vicinity of transmembrane receptors, together withevidence that it can trigger opsonin-independent phagocy-tosis (Salmon et al, 1987), suggests that FcgRIII, like CR3,


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may have particular importance in neonates with lowopsonic capacity.

In term neonates, both FcgRII and FcgRIII receptors arepresent in normal numbers on cord blood neutrophils (Carr& Davies, 1990; Payne et al, 1993). On preterm neonateneutrophils, FcgRII is also near normal, being expressed at80±88% of adult levels. In contrast, the expression of theFcgRIII receptor is significantly less on preterm neonatecells, being < 50% of adult levels in infants born before32 weeks. Postnatally, we found membrane-bound FcgRIIIto increase rapidly so that by 2 weeks after birth receptornumbers were normal. Whether low receptor number atbirth reflects reduced synthesis (Edwards et al, 1990) orincreased shedding (Tosi & Zakem, 1992) remainsunknown.

Phagocytosis in term neonates. The majority of studiesexamining the ability of neonatal neutrophils to phago-cytose bacteria in vitro have used cells separated from theirnative plasma and bacterial targets opsonized with adultimmunoglobulin and complement. Under these circum-stances of full opsonization, ingestion of both Gram-positiveand Gram-negative bacteria by neutrophils from termneonates has been normal (Dossett et al, 1969; Forman &Steihm, 1969; McCracken & Eichenwald, 1971; Mills et al,1979). In contrast, phagocytosis of Candida has beenabnormal compared with adults (Miller, 1969; Al-Hadithyet al, 1981; Bektas et al, 1990). The explanation for thisdifference may rest with the size of the particle. The greatersize of the candida cells suggests that the mechanism ofingestion may have a closer relationship to chemotaxis thanbacterial phagocytosis. This view is supported by thepostnatal improvement in phagocytosis of candida, whichreaches normal adult levels after 2 postnatal weeks, atimescale similar to chemotaxis maturation (Al-Hadithyet al, 1981).

Effect of prematurity and stress on phagocytosis. Two studieshave examined neutrophils from clinically stable pretermneonates (28±36 weeks gestation) using separated neutro-phils and adult plasma for opsonization. Phagocytosis ofStaphylococcus aureus was found to be equal to adult andterm infant cells (Gahr et al, 1985; Bektas et al, 1990). Weadopted a different approach and examined phagocytosis ofE. coli in a whole blood assay, a system which moreaccurately reflects normal physiology and the effect of thereduced opsonins especially in preterm infants. Termneonate neutrophils performed as well as adult cells.However, preterm neonates (23±33 weeks gestation), whowere neither septic nor had respiratory disease, showedsignificantly impaired phagocytosis in terms of the numberof cells taking up bacteria, the number of bacteria ingestedby active cells and the time-course of uptake (Falconer et al,1995). Furthermore, when the neonates were retested 1±2 months later, phagocytosis remained depressed with noevidence of postnatal maturation.

The explanation is almost certainly the critical influenceof low opsonization in preterm neonate blood. This view issupported by three studies that together demonstrated (i)that phagocytosis by adult neutrophils could be reduced toneonate levels by suspending adult cells in preterm neonate

serum, when S. aureus (Forman & Stiehm, 1969) or GBS III(KaÈllman et al, 1998) were used as targets; (ii) pretermneutrophils could perform as well as adult cells when adultserum or therapeutic immunoglobulin was added to thetarget staphylococci (Forman & Stiehm, 1969; Fujiwara et al,1997); (iii) after intravenous immunoglobulin infusions intofour preterm neonates, which raised their plasma IgGconcentration to normal adult levels, the ex vivo neutrophilphagocytosis significantly improved to overlap with fourterm neonates tested simultaneously (Fujiwara et al, 1997).Thus, term and preterm neonate neutrophils can phagocy-tose bacteria normally, but only if the targets are adequatelyopsonized.

In the face of infection or non-infective clinical stress,such as respiratory distress syndrome, the ability of neonateneutrophils to ingest fully opsonized Gram-positive bacteria,including GBS, appears to be maintained (Wright et al,1975; Shigeoka et al, 1979, 1981; Harris et al, 1983), butphagocytosis of E. coli becomes impaired (Wright et al,1975).

The respiratory burstThe respiratory burst is the major mechanism by whichneutrophils kill engulfed bacteria (Klebanoff, 1992; Hamp-ton et al, 1998; Babior, 1999). The importance of theoxidative burst is demonstrated by the susceptibility toinfection of individuals with chronic granulomatous disease,in which the NADPH oxidase is inactive (Segal, 1988; Smith& Curnutte, 1991). Similarly, Staphylococcus epidermidiscannot be killed in experimental conditions of oxygendepletion which virtually abolishes the respiratory burst(Weiss et al, 1982), an observation which may be ofrelevance to neonatal practice where S. epidermidis infectionis common.

Respiratory burst in neonates. The respiratory burst activityof neonate neutrophils is best summarized by a recent studyin which the cells were activated by the clinically relevantstimulus of GBS III and fully opsonized by serotype-specificIgG and complement (KaÈllman et al, 1998). Term neonateneutrophils had a chemiluminescence (CL) response iden-tical to adults, but preterm (28±34 weeks) neutrophilsresponded with a very much smaller CL peak. Asphagocytosis is normal in preterm cells under suchconditions of optimal opsonization, it would appear thatpreterm neonates have less capacity to generate bactericidaloxygen metabolites. The normal CL response of full-termneonates in response to opsonized GBS or zymosan has beenconfirmed by others (Shigeoka et al 1981; Peden et al, 1987;Bektas et al, 1990). Similarly, the reduced respiratory burstactivity of preterm neonates has been confirmed using thesame bacterial stimuli (Peden et al, 1987; Bektas et al,1990).

The relevance of the reduced respiratory burst activity toincreased sepsis in preterm infants is supported by in vitrostudies of bacterial killing. Intracellular killing of S. aureus orE. coli by neutrophils from term neonates has consistentlybeen found to be normal (Dossett et al, 1969; Forman &Stiehm, 1969; Mills et al, 1979), whereas, and in line withthe respiratory burst findings, killing of staphylococci was

22 Review


impaired in preterm neonates with birthweight , 2000 g(Gahr et al, 1985).

When challenged by sepsis or other clinical or experi-mental `stress', the respiratory burst of term neonateneutrophils becomes less active (Droussou et al, 1997), incontrast to adults who tend to increase respiratory burstmetabolism in response to sepsis (Barbour et al, 1980;Babior, 1984). Term neonates with GBS III sepsis showedneutrophil CL responses of only 50±75% compared withuninfected infants, and this was associated with a 10-foldreduction in their ability to kill fully opsonized GBS in vitro(Shigeoka et al 1979). A similar, though less marked,reduction in CL was found in uninfected infants withrespiratory distress (Shigeoka et al 1981). In vitro, challeng-ing neonate neutrophils with larger numbers of E. coli or S.aureus per cell also leads to reduced chemiluminescence andbacteria killing, whereas under the same conditions adultcell bactericidal function is maintained (Mills et al, 1979).

Postnatal maturation of the respiratory burst in pretermneonates has been examined in two studies. Driscoll et al(1990) studied 57 preterm infants sequentially over a 2-month period. During the first postnatal week, the peakchemiluminescence in response to opsonized zymosan wasnormal for the group as a whole, although the moreimmature infants (24±28 weeks gestation) had significantlylower peak CL than infants born at 29±35 weeks. Duringsubsequent testing over 2 months, mean CL for the cohortwas lower than adult controls. During this period, the lowerCL did not correlate with gestational age, but was associatedwith more clinical interventions. Although serious infec-tions developed more frequently in infants with low CL inthe first week, the subsequent and persistent suppression ofthe respiratory burst seemed more likely to be a conse-quence of clinical stress and prolonged intensive care.Indeed, an increasing proportion of infants had a low CLresponse as their time in intensive care progressed.Healthier infants, who would perhaps have been more likelyto show maturation of the respiratory burst function, werenot studied once they were discharged. Usmani et al (1991)likewise showed CL to remain suppressed, with only minorimprovement over 21 d after preterm birth.

Superoxide generation. There is an additional and intri-guing aspect of the neonate respiratory burst. Although thegeneration of bactericidal hydroxyl radicals is reduced(Ambruso et al, 1979; Strauss & Snyder, 1983), the initialphase of the respiratory burst, as represented by superoxide(O2

2) generation, is increased in neonate neutrophilscompared with adults (Yamazaki et al, 1998). Thisphenomenon was investigated by Ambruso et al (1984,1987), who found that the kinetics of the neonates' NADPHoxidase system, which generates O2

2, was qualitativelydifferent from adult oxidase enzymes. In addition, there wasan increase in O2

2 production by cord blood neutrophilscollected after labour, in contrast to caesarean section,which suggested priming for increased activity duringparturition (Ambruso et al, 1987).

Lactoferrin and myeloperoxidase. There is a close correlationbetween the reduced generation of hydroxyl radicals byneonatal neutrophils and a reduced cell content of

lactoferrin (Ambruso & Johnston, 1981). Lactoferrin is abactericidal component of neutrophil-specific granules. Ithas direct bacteriostatic properties (Lehrer & Ganz, 1990)and, when saturated with iron, enhances the production ofhydroxide ions (Ambruso & Johnston, 1981; Klebanoff &Waltersdorph, 1990). Others have confirmed that termneonate cells contain only half the amount of lactoferrinfound in adult cells (Anderson et al, 1987; Kjeldsen et al,1996) and that levels are lower still in preterm infants(Anderson et al, 1987). An alternative and importantcatalyst for the generation of toxic oxygen radicals ismyeloperoxidase, which is involved in the generation ofhypochlorous acid from hydrogen peroxide. It is present innormal concentrations in term neonate azurophil granules(Ambruso et al, 1984; Jones et al, 1990; Kjeldsen et al,1996), although it too is significantly reduced in infantsborn prematurely (Rider et al, 1988). Thus, the explanationfor the paradox of reduced bactericidal oxidants, as detectedby CL, when the generation of superoxide and hydroxygenperoxide is increased appears to rest with inadequate storesof lactoferrin and myeloperoxidase to catalyse the laterstages of the respiratory burst.

CONCLUSIONS AND THERAPEUTIC PROSPECTS

Over the past 20 years, the functional differences betweenneonate and adult neutrophils have been well described,and since the review by Hill (1987) the differences betweenpreterm and term infants have been further identified.However, there remains no unifying theory to explain thevarious alterations in function that have been so carefullydocumented.

A theory in vogue during the 1980s was that of increasedneutrophil heterogeneity (Gallin, 1984). That is to say,neonates have a larger population of inactive phagocyticcells which can be detected through functional studies butwhich are not related to morphological immaturity (Krauseet al, 1990). This was supported by studies which showedneonates to have a larger population of non-motile cells inchemotaxis assays (Krause et al, 1986b, 1989) and a higherpercentage of cells that do not produce an oxidativerespiratory burst when stimulated (Gessler et al, 1996;Drossou et al, 1997). However, this does not explain why theinactive cells are different.

Most investigators invoke `immaturity of function' withthe implication that there is a process of functionaldifferentiation within individual cells that is incomplete.There is some support for this concept in that there isevidence for reduced content of some proteins critical forfunction, e.g. Mac-1 and LFA-1 (Abughali et al, 1994;McEvoy et al, 1996), and lactoferrin and gelatinase(Ambruso et al, 1984; Kjeldsen et al, 1996). These findingsimply that the biosynthetic capacity of the neutrophilprecursors is reduced. However, many proteins and enzymesystems appear normal, therefore altered protein synthesisdoes not appear to provide the complete picture.

An alternative explanation is that the cells' membranereceptor phenotype and performance in functional assaysresults from an altered activation state, perhaps as a


Review 23

consequence of the birth process itself or postnatal contactwith bacteria and other stimuli to the immune system. Thishypothesis is supported by the observation that membranereceptors for FcgRIII and L-selectin, which are markedlyreduced after birth, are expressed normally on neutrophilsobtained from fetal blood samples (Smith et al, 1990; Koeniget al, 1992; Smith & Tabsh, 1993). These particularreceptors are shed by activated cells. Other receptors,known to be up-regulated by cell activation, are present inincreased numbers on the surface of `unstimulated' neonatecells, e.g. Mac-1 (Carr et al, 1992b; Rebuck et al, 1995) andCD14 (unpublished observations). Increased superoxidegeneration in newborn infants also appears to be relatedto priming of the NADPH oxidase (Ambruso et al, 1987),whereas superoxide generation by fetal neutrophils appearsnot to be increased (Newburger, 1982). Studies of neonateneutrophils over the next decade should be aimed atelucidating the external influences operating within theimmune system which underlie these observed differences.

Armed with this knowledge, are we able to devisestrategies to enhance neonate neutrophil production andfunction? Correcting the deficient neutrophil storage pool inpreterm infants is, perhaps, the most immediately achiev-able goal, through the availability of granulocyte colony-stimulating factor (G-CSF) and granulocyte±macrophagecolony-stimulating factor (GM-CSF) (for a review, see Carr &Modi, 1997). Pilot studies in human neonates show bothCSFs can increase peripheral blood neutrophils and expandthe marrow reserves (Gillan et al, 1994; Cairo et al, 1995).Our own studies have demonstrated that prophylactic GM-CSF can prevent postnatal neutropenia (Carr et al, 1999).This and other preliminary studies suggest that CSFs aremore effective at improving clinical outcomes in pretermneonates when used prophylactically rather than oncesepsis is established (for a review, see Modi & Carr, 2000),but more, well designed clinical studies need to be carriedout.

Phagocytosis depends critically on adequate opsonization.Thus, intravenous immunoglobulin should improve thepreterm neonate's ability to defend itself against bacterialpathogens. Recent systematic reviews of available data(Jenson & Pollock, 1997; Ohlsson & Lacy, 1999a, b) suggestthat intravenous immunoglobulin therapy in infants withestablished sepsis may improve outcome and this warrantsfurther investigation. However, the clinical benefit achievedby the prophylactic use of immunoglobulin has beendisappointing. The effect of supplementing complementhas not been adequately explored.

The major functional deficiencies of neonatal neutrophils,namely cell movement and bacterial killing, seem at presentless amenable to therapy. One line of investigation suggeststhat gamma interferon (IFN-g) may have potential fortherapeutic benefit (Hill et al, 1999). In a comparative study,this cytokine was the most effective at correcting abnorm-alities of cell movement in vitro (Hill et al, 1991). It has alsobeen demonstrated that IFN-g production by neonatalmononuclear cells is reduced, in response to mitogens orgroup B streptococci (Wilson et al, 1986; Joyner et al, 1999).These observations raise the possibility that increasing

IFN-g levels in vivo might be beneficial. Given that IFN-g is aTH1 cytokine which tends to be increased by GM-CSF andsuppressed by G-CSF, these data would tend to favour theuse of GM-CSF for the prophylactic enhancement of bothneutrophil number and function.

Infection in preterm neonates remains a significantclinical problem. Finding effective strategies to enhancethe neonatal immune system is a pressing goal for the nextdecade.

ROBERT CA RRDepartment of Haematology,King's College,St Thomas' Hospital,London, UK

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Keywords: newborn infant, preterm, neutrophil function,granulopoiesis.

28 Review


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Neutrophil Production and Function in Newborn Infants