Published online before print May 21, 2008
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,1
* Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio, USA;
Department of Biology, John Carroll University, Cleveland, Ohio, USA; and
Section of Leukocyte Biology, Baylor College of Medicine, Houston, Texas, USA
1 Correspondence: Department of Pediatrics, Mount Sinai School of Medicine, Annenberg Bldg., Rm. 14-08, 1 Gustave L. Levy Place, New York, NY 10029, USA. E-mail: michael.tosi{at}mssm.edu
ABSTRACT
Markedly deficient expression of membrane-activated complex 1 (Mac-1; CD11b/CD18) by polymorphonuclear neutrophils (PMN) of human neonates compared with adults is well documented. To define postnatal maturation of Mac-1 expression of PMN, lysates of PMN from 21 infants, aged 1–14 months, and concurrent adult controls were assayed by ELISA for total cell content of Mac-1 and LFA-1 (CD11a/CD18), and LFA-1 content was within the normal adult range at all ages tested. Mac-1 content was 
50% of adult levels for infants 1–2 months of age and steadily increased to reach normal adult levels by 11–12 months of age. For a separate group of 25 infants, aged 0.5–11 months, measurement of surface expression of Mac-1 and LFA-1 on activated PMN by immunofluorescence flow cytometry yielded results that were similar to those obtained by ELISA.
Key Words: β-2 integrins • children • development
Well-recognized deficiencies in adhesion and migration of neonatal polymorphonuclear neutrophils (PMN) [1 , 2 ], highly relevant to antibacterial defenses, may be accounted for by one or more of several biochemical deficiencies described previously [3 4 5 6 7 8 ]. One well-studied example is the reduced expression of the important leukocyte β-2 integrin adhesion molecule membrane-activated complex 1 (Mac-1) or CD11b/CD18. Normally, Mac-1 surface expression can be increased as much as eight- to tenfold upon optimal stimulation by chemoattractants, as a result of translocation of a large intracellular storage pool of Mac-1 to the plasma membrane [9 ]. In PMN of term neonates, this up-regulation of Mac-1 is deficient, and we have reported previously that this deficiency could be accounted for by a reduction in the total PMN content of Mac-1, 50–60% of normal adult levels [10 ]. PMN Mac-1 content was found to be reduced even further in infants born prematurely and to be related directly to gestational age, and infants born between 26 and 28 weeks gestation had PMN that contained 20% or less of adult levels of Mac-1 [11 ]. LFA-1 (CD11a/CD18) expression and cell content in PMN of term infants are equivalent to adult controls, although it is reduced in infants born at less than 35 weeks gestation [11 ]. The current study extends our previous findings, characterizing the postnatal maturation of PMN Mac-1 expression, using measurements of total cell content and up-regulated surface expression.
Generally, healthy infants from 1 to 14 months of age who had been born at term (
37 weeks gestation) and who attended outpatient clinics at Rainbow Babies and Children’s Hospital of University Hospitals of Cleveland (Cleveland, OH, USA) were recruited after verbal permission to do so was obtained from each infant’s primary physician. Venous blood (4.0–8.0 ml) was obtained for the study only if an infant was already scheduled to undergo venipuncture for clinical indications, usually for follow-up of a questionable neonatal screening test or iron deficiency anemia. Written, informed consent to obtain the additional blood volume was obtained from the accompanying parent(s) or guardian(s). The study included healthy adults as controls and was approved by the Institutional Review Board of Rainbow Babies and Children’s Hospital of University Hospitals of Cleveland.
Data from a separate group of 25 infants were examined retrospectively for Mac-1 and LFA-1 surface expression on stimulated PMN. These infants ranged in age from 0.5 to 11 months and were referred over a 4-year period to the clinical laboratory of the Section of Leukocyte Biology, Department of Pediatrics (Baylor College of Medicine, Houston, TX, USA), for evaluation for possible defects of leukocyte function. To avoid confounding effects of any clinical abnormalities on the results of interest, data from any infant who was diagnosed with such a defect were excluded in advance from the current study. Extraction of these retrospective clinical data, stripped of all personal identifiers, was governed by a limited dataset agreement and approved by the Institutional Review Board of Baylor College of Medicine and Affiliated Hospitals.
For measurements of total cell content of Mac-1 and LFA-1, PMN were isolated from anticoagulated venous blood of each infant and a concurrent adult control, as described previously [10
], resulting in PMN suspensions with purity and viability of >95%. PMN were pelleted and lysed for 60 min in 1% Nonidet P-40 with protease inhibitors as described previously. After centrifugation at 10,000 g for 60 s, the supernatant containing solubilized proteins was recovered and stored at –80°C until assayed. For each infant studied, PMN from a healthy adult control concurrently were isolated, lysed, and stored as described. The total cell content of Mac-1 and LFA-1 in stored PMN lysates (5x105 cell equivalents) was measured using a sandwich ELISA assay described previously [10
, 11
]. Preliminary assays for LFA-1 and Mac-1 in serial dilutions of normal adult PMN lysates confirmed in advance a linear relationship between absorbance by ELISA and the number of PMN equivalents between 105 and 106 PMN (a range of 20–200% of control input). Samples from 21 infants from 1 to 13 months of age and concurrent adult controls were studied prospectively. Figure 1
shows a plot of the PMN content of Mac-1 and LFA-1 for each infant, expressed as a percentage of its adult control against the infant’s postnatal age in months to the nearest half-month. The Mac-1 content of PMN in infants in the current study was 45–50% of adult controls during the first 2 months of life, similar to previous findings for PMN from cord blood of infants born at term [10
, 11
]. PMN Mac-1 content rose progressively with increasing postnatal age and was consistently within the adult range by 11–12 months of age. When the data for Mac-1 were analyzed by simple linear regression (R=0.905), the line derived from these data reached 100% of control at 12 months of age. Consistent with our previous findings for term neonates [10
], the LFA-1 content of infant PMN throughout the age range studied was equivalent to that for adult controls. Whether the presence of iron-deficiency anemia in a number of infants in this specific study group might have influenced results for Mac-1 PMN content is unknown, although we are aware of no data to suggest such an effect.
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Figure 1. Deficient PMN content of Mac-1 in early infancy increases gradually to normal adult levels by 11–12 months of postnatal age. The Mac-1 ( ) and LFA-1 ( ) content of PMN from 21 infants ranging in age from 1 to 13 months, as measured by a sandwich ELISA, is expressed as a percentage of the corresponding value for PMN from each infant’s concurrent adult control. Values for LFA-1 content in infant PMN are equivalent to adult controls over the entire age range studied. Mac-1 content of infant PMN is markedly reduced in early infancy, as shown, but rises to levels that are consistently in the adult control range between 11 and 12 months of age. A line derived from the data for Mac-1 by simple linear regression (R=0.905) is at 43% at 0 months and reaches 100% at 12 months.
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55% of adult levels at birth and rising to 100% of adult controls by 11 months of age, similar to the data for total cell content of Mac-1 above. Figure 2B
shows the corresponding data for LFA-1 expression from the same infants, equivalent to adult levels at birth and throughout the age range studied. These studies of Mac-1 and LFA-1 surface expression on stimulated PMN are consistent with the above results for total cell content of these adhesion molecules. It is of note that these results for up-regulated Mac-1 surface expression exhibit a much greater degree of variability than do the above results for total cell Mac-1 content, especially during the first few months of life. This greater variability might be predicted, as the magnitude of up-regulated surface expression depends on several variables, including available cell content, responsiveness to the stimulus applied, the efficiency or completeness of translocation of stored Mac-1 to the cell surface, and possibly, gestational age, which could not be ascertained for this anonymous, retrospective sample. Despite this greater variability, the results in aggregate are in accord with those above for total cell content of Mac-1 and LFA-1 measured by ELISA.
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Figure 2. Deficient up-regulated surface expression of Mac-1 in early infancy increases to normal adult levels by 11 months of age. Surface expression of Mac-1 (A) and LFA-1 (B) on stimulated PMN (10–7 M fMLPx20 min, 37°C) from 25 infants ranging in age from 0.5 to 11 months as measured by immunofluorescence flow cytometry is expressed as a percentage of the corresponding value for PMN from each infant’s concurrent adult control. A line derived from the data for Mac-1 by simple linear regression (R=0.433) is at 56% at age 0 months and reaches 100% at 11 months. Values for LFA-1 expression are equivalent to normal adult levels throughout the age range studied.
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Received March 11, 2008; revised April 15, 2008; accepted April 24, 2008.
REFERENCES
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