Polymorphonuclear leukocyte function in newborn infants

862

Brief clinical and laboratory observations

The Journal of Pediatrics November 1978

Brief clinical and laboratory observations Polymorphonuclear leukocyte function in newborn infants Joan W. Stoerner, M.D., Larry K. Pickering, M.D.,* Eugene W. Adeoek III, M.D., and Frank H. Morriss, Jr., M.D., Houston, Texas

PHAGOCVTOSIS and killing of bacteria by human polymorphonuclear leukocytes is accompanied by a number of oxidative alterations, including increases in hexose monophosphate shunt activity, oxygen consumption, and hydrogen peroxide production. I The purposes of this study were (1) to investigate H MPS activity in resting and phagocytizing PMNL obtained from infected and healthy infants, (2) to determine the bactericidal activity of PMNL obtained from healthy control infants and adults in the presence of either adult or infant serum, using Staphylococcus aureus and Escherichia coli as the test organisms, and (3) to compare bactericidal activity with HMPS activity in healthy control infants and adults. MATERIALS

AND METHODS

The study population consisted of infants in Hermann Hospital and healthy adults at The University of Texas Medical School at Houston. Informed consent for participation in this study was obtained from the parents of infants and from the adult participants. Eight preterm infants with bacteremia and/or meningitis due to E. coli, S. aureus, or group B streptococcus were studied during antibiotic therapy. Eleven healthy newborn infants greater than 35 weeks' gestation and 21 healthy adults served as controls. Heparinized blood (15 units/ml blood) was obtained at the time of delivery from the umbilical vein of the 11 control neonates, from an indwelling arterial catheter in the infected neonates, or by venipuncture from adults. Plastic syringes and siliconized test tubes were used to collect the blood. Total and differential WBC counts were From the Department of Pediatrics and the Program in Infectious Diseases and Clinical Microbiology, The University of Texas Medical School at Houston. Presented in part at the annual meeting of the Southern Society for Pediatric Research, New Orleans, January, 1978. *Reprint address: The University of Texas Medical School. 228 Freeman Bldg.. 6431 Fannin St., Houston, TX 77030.

performed on all samples. HMPS activity of resting and phagocytizing PMNL in whole blood from infected and control infants and adults was measured according to the method of Keusch et al. ~ Results are reported as counts/ 106 PMNL/30 minutes' incubation and represent duplicate determinations. Bactericidal activity of separated PMNL from eight control neonates and 21 control adults was determined using S. aureus (502A) and E. coli (H-10407) as the test organisms. PMNL were separated by the dextran sedimentation method of Skoog and Beck? The bactericidal activity of PMNL were measured by a modification of the pour-plate method of Quie et al.' Only sterile, siliconized glassware and sterile reagents were used. All tests were initiated within one hour of blood collection and were performed in duplicate. The bacteria to PMNL ratio utilized was 4:1, To assess the opsonophagocytic effect of serum factors, control adult and infant cells were tested with fresh pooled human sera from three adult donors and with autologous infant sera at a 10% concentration. Abbreviations used HMPS: hexose monophosphate shunt PMNL: polymorphonuclear leukocyte(s) P/R: phagocytizing/resting HMPS activity ratio WBC: white blood cell R: resting P: phagocytizing RESULTS

By unpaired t test analysis, neither the WBC nor the absolute PMNL count of infected infants differed significantly from that of control infants. Mean R-HMPS activity of infected infants was not significantly different from those of the control infants and adults. However, the P-HMPS activity of PMNL from infected infants was significantly (P < 0.05) less than that of control infants (989 ± 181 vs 1,385 _+ 119). Although the increase in activity between resting and phagocytizing cells for the infected infants was significantly less than the increase for

0022-3476/78/110862+ 03500.30/0 © 1978 The C. V. Mosby Co.

Volume 93 Number 5

Brief clinical and laboratory observations

863

2400¸

3$

20oo,

a

e-

~.~

~

~

,

,,

o

1600' ..~,....,.

1200'

D

",~,,,y t i z i n og

oO

.o a

-:o

o

•

•

Z

" 0

0

2

"

. ~"~..~,, •

"

• "

4

6

~ 8

y = 712- 0.54 x

~ "•

p < .005

10

12

Absolute Polymorphonuclear 103 cells/ram 3

14

16

18

Leukocytes

Fig. l. The relationship of the absolute polymorphonuclear leukocyte count to resting (closed circles) and phagocytizing (open squares) HMPS activities of PMNL for all infants studied.

control infants, the P/R ratio of infected infants did not differ significantly from that of either control adults or control infants when these ratios were compared by the appropriate nonparametric test. By linear regression analysis, PMNL from all infants (control and infected) had resting and phagocytizing HMPS activities which were inversely related to the absolute PMNL count. This relationship was not found for PMNL from control adults. Fig. 1 illustrates the relationship of HMPS activity to the absolute PMNL count for all infants. The PMNL from normal, healthy infants incubated with either adult or infant sera-killed S. aureus as well as did PMNL from adults inadult sera. Fig. 2 illustrates the mean bactericidal activity using E. coli as the test organism for the celt and serum combinations after 120 minutes' incubation. By unpaired t test analysis, PMNL from adults incubated with pooled adult sera-killed E. coli significantly better than did PMNL from infants incubated with either infant sera (P < 0.005) or pooled adult sera (P < 0.05) after 120 minutes. By paired t test analysis, PMNL from adults incubated with adult sera had greater bactericidal activity after 30 and 120 minutes' incubation than when incubated with infant sera (P < 0.01). Bactericidal activity of PMNL from infants using E. coli as the test organism was greater at 30 and 120 minutes when incubated with adult sera than with infant sera (P < 0.05 and P < 0.01, respectively). For control infants and adults the simultaneous determinations of whole blood HMPS activity and bactericidal activity of separated PMNL against S. aureus and E. coli were performed. Resting and phagocytizing HMPS activities of PMNL in whole blood were compared with

100 9O 8O

÷

ro ;7 8o

m

10 C

Adult Cells and Adult Serum

Adult Cells and Infant Serum

Infant Cells

Infant Cells

and Infant Serum

end Adult Serum

Fig. 2. The mean ( _ SEM) bactericidal activity using E. coil as the test organism for PMNL from infants and adults with either infant sera or pooled adult sera after 120 minutes' incubation. bactericidal activity of adult PMNL incubated with pooled adult sera and infant PMNL incubated with infant sera. There was no significant relationship by linear regression analysis between HMPS activity, whether resting or phagocytizing, and killing of S. aureus or E. coli by PMNL from either adults or infants. DISCUSSION Both R- and P-HMPS activities of PMNL from newborn infants were inversely correlated with the absolute PMNL count. This relationship suggests that PMNL from a newborn infant with a low absolute PMNL count have greater metabolic activity per cell. Two possible explanations are suggested for this inverse correlation: (1) a quantitative feedback mechanism for regulating meta-

864

Brief clinical and laboratory observations

bolic activity according to the PMNL population may exist or (2) the number of PMNL which are metabolically immature may be greater in newborn infants who have released marrow or marginated PMNL into the circulation to increase the number of circulating PMNL. Neither the total WBC count nor the absolute PMNL count of infected infants differed from that of control infants, as has been demonstrated previously? In control adults, Rand P-HMPS activities of PMNL were not related to the absolute PMNL count; however, the range of absolute PMNL values was narrow. Both normal 6.7 and decreased 8 bactericidal activity of PMNL from term newborn infants using S. aureus as the test organism in the presence of adult serum have been reported. Low-birth-weight infants have been shown to have decreased bactericidal activity of PMNL incubated with autologous serum? In our study, no significant difference among the populations studied was found for bactericidal activity of PMNL against S. aureus. Both infant and adult PMNL exhibited greater killing of E. coli when incubated with adult sera compared to incubation with infant sera. However, infant PMNL incubated with adult sera did not kill E. coli as effectively as did adult cells incubated in adult sera. These findings indicate deficient factors or the presence of inhibitors in the serum of newborn infants and also suggest the presence of a cellular defect in the ability of PMNL from newborn infants to kill E. coli. Simultaneous determinations of R- and P-HMPS activities and bactericidal activity failed to demonstrate a relationship between this index of PMNL metabolism and the killing ability of either adult or infant PMNL. In summary, this study confirms the existence of two defects in the neonatal host defense mechanism: (1) P-PMNL from infected infants have less than normal enhancement of metabolic activity, and (2) normal newborn infants lack serum factor(s) necessary for (or

The Journal of Pediatrics November 1978

possess inhibitors of) normal bactericidal activity against E. coli. Furthermore, three new findings of our investiga-

tion contribute to the understanding o f the neonatal defense mechanism: (1) the HMPS activity of PMNL from infants is inversely proportional to the absolute PMNL count, (2) bactericidal activity is not correlated with the HMPS activity under the conditions studied, and (3) the decreased bactericidal activity exhibited by PMNL from normal infants is not fully corrected by adult sera. The authors gratefully acknowledge the competent technical assistance of Betty Jo Webster and the expert help of Alice L. Johnston and Bettie Casey in preparing the manuscript. REFERENCES

1. Karnovsky ML: The metabolism of leukocytes, Semin Hematol 5:156, 1968. 2. Keusch GT, Douglas SD, and Mildvan D: 14C-glucose oxidation in whole blood: a clinical assay for phagocytic dysfunction, Infect Immun 5:414, 1972. 3. Skoog WA, and Beck WS: Studies on the fibrinogen, dextran, and phytohemagglutinin methods of isolating leuckocytes, Blood 11:436, 1956. 4. Quie PG, White JG, Holmes B, et al: In vitro bactericidal capacity of human polymorphonuclear leukocytes: Diminished activity in chronic granulomatous disease of childhood, J Clin Invest 46:668, 1967. 5. Anderson DC, Pickering LK, and Feigin RD: Leukocyte function in normal and infected neonates, J PEDIATR85:420, 1974. 6. Park BH, Holmes B, and Good RA: Metabolic activities in leukocytes in newborn infants, J PEDIATR76:237, 1970. 7. Wright WC, Ank BJ, Herbert J, et al: Decreased bactericidal activity of leukocytes of stressed newborn infants, Pediatrics 56:879, 1975. 8. Coen R, Grush O, and Kauder E: Studies on the bactericidal activity and metabolism of the leukocyte in full-term neonates, J PEDIATR75:400, 1969. 9. Forman ML, and Stiehm ER: Impaired opsonic activity but normal phagocytosis in low birth weight infants, N Engl J Med 281:926, 1969.

Central parenteral alimentation in newborn infants: A new technique for catheter placement Nestor E. Vain, M.D.,* Keith E. Georgeson, M.D., Chul C. Cha, M.D., and O. Ward Swarner, M.D., Loma Linda, Calif.

From the Departments of Pediatrics and Surgery, Loma Linda University Medical Center. *Reprint address: Department of Pediatrics, Loma Linda University Medical Center, Loma Linda, CA 92350.

PARENTERAL NUTRITION is frequently used for premature infants as a supplement to milk feedings: Many neonatologists have suggested total parenteral nutrition as the first choice for very low-birth-weight