Ontogeny
of Fetal Sheep Polymorphonuclear
ByRussell W.
Jennings,
N. Scott Adzick,
Michael
T. Longaker,
and Thomas
Leukocyte Brian W. Duncan,
Phagocytosis
Heinz Scheuenstuhl,
K. Hunt
San Francisco, California l Premature infants and neonates are vulnerable to bacterial sepsis. This susceptibility may be due to the relative immaturity of their immune systems. To determine if neonates and, in particular, premature infants have decreased polymorphonuclear leukocyte (PMN) phagocytosis, wa tested PMN phagocytosis of Staphylococcus aureus as a function of gestational age in the fetal lamb model. Because phagocytosis is made more efficient by the presence of opsonins in plasma, fetal and postnatal PMN phagocytosis were also measured after exposure to fetal and adult plasma. PMNs were isolated from fetal lambs at 104,114,124, and 141 days’ gestation (term gestation for the fetal lamb is 145 days), as well as from IO-day-old neonatal sheep and adult sheep. Labeled S ~ureus were opsonized by incubation in either fetal or adult plasma, or left unopsonized for baseline values. Phagocytosis was measured as a percent of adult PMN phagocytosis after adult plasma opsonization. It was found that fetal PMN function is limited by two factors during the early third trimester: a primary defect in the ability of the PMN to phagocytose S aureus despite adequate opsonization, and the diminished ability of autologous fetal plasma to opsonire bacteria. The defect in PMN phagocytosis disappears late in the third trimester, but the inability of the fetal plasma to opsonize effectively continues until after birth. Copyright o 1991 by W.B. Saunders Company INDEX WORDS: Polymorphonuclear sis; fetal immunity; bacterial sepsis.
leukocyte,
phagocyto-
I
premature infants, NFANTS, PARTICULARLY are at increased risk for sepsis. This susceptibility to infection may be due to the relative immaturity of the neonatal immune system. Previous studies have shown a decreased number of polymorphonuclear leukocytes (PMNs) in blood from the umbilical cord of premature infants, but studies of PMN function in these babies have been controversial. To determine when fetal PMNs are capable of phagocytosis, and to determine if fetal PMN phagocytosis is augmented by plasma opsonins, we tested PMN phagocytosis of Staphylococcus aureus without opsonization as a function of gestational age in fetal, term, and adult sheep. Because phagocytosis is made more efficient by opsonins in plasma, fetal and postnatal PMN phagocytosis were also measured after opsonization with fetal and adult sheep plasma. MATERIALS
AND
METHODS
Time-dated pregnant ewes of gestational ages 90 to 141 days (term is 145 days) were obtained from Torrel Farms (Ukiah, CA). Maternal adult sheep blood was obtained during general halothane/ oxygen anesthesia by jugular vein cannulation and collected into sodium EDTA and heparin. Fetal sheep blood was collected by direct umbilical arterial cannulation during fetal surgery using JournaiofPediatric
Surgery,
Vol26, No
7
(July), 1991: pp 853-855
techniques described previously.’ A portion of each sample was evaluated for complete blood count. All blood samples were processed in parallel.
PMN Isolation To isolate the PMNs, the plasma was first separated from the cellular components by centrifugation at 2,500 rotations/min (rpm) for 10 minutes. The PMNs were maintained at 4°C at all times. The plasma was saved for the opsonization step. The red cells were removed by shock hypotonic lysis three times. After each lysis the solution was returned to normal tonicity with cold 10X phosphate buffer saline. The cells were then isolated by centrifugation at 2,500 rpm. To separate the PMNs from the other leukocytes and remaining red cells, a two-step discontinuous density gradient of 80% and 50% Percoll (Pharmacia, Piscataway, NJ) was used (densities of 1.092 and 1.055 gimL). After isolation of the PMNs, their viability and concentration were determined by ethidium bromide and acridine orange dye using a fluoresence microscope and a hemocytometer. The PMNs were resuspended to 1 X 10’ to 1 x 10” per milliliter. The purity and maturity of the PMNs was evaluated by light microscopy using Wright’s stain.
Bacterial Preparation Laboratory-grade pansensitive S aureus (ATCC 29213) was grown in culture with ‘H-glycine (Amersham, Arlington Heights, IL). The bacteria were then washed three times with phosphatebuffered saline to remove all unincorporated radiolabeled glycine. The bacteria were then incubated in 50% maternal plasma or fetal plasma diluted with phosphate-buffered saline for 1 hour at 37°C to allow opsonization of the bacteria, or in phosphate-buffered saline alone as a control. Concentrations of the bacteria were determined using a turbidity curve and plate counts.
Phagocytosis Assay The isolated PMNs and either opsonized or unopsonized S aurem were then incubated together for 1 hour at 37°C to allow phagocytosis at a 1OO:lratio of bacteria to PMNs. A 10011ratio was chosen to provide a guaranteed excess of bacteria. Preliminary studies showed phagocytosis of enough colony forming units per PMN to significantly alter the ratio of bacteria to PMNs when lower counts of bacteria were used. The extracellular bacteria that had not been phagocytosed were dissolved with 10 U/mL of lysostaphin (Sigma Chemical Co, St Louis, MO) by incubation for 45 minutes at 37%X.*The PMNs were removed by centrifugation
From The Fetal Treatment Program and The Wound Healing Laboratory, Department of Surgery, University of California, San Francisco, CA. Presented at the 23rd Annual Meeting of the Pacific Association of Pediatric Surgeons, Kona, Hawaii, June 3-6, 1990. Address reprint requests to N. Scott Adzick, MD, 585 HSE, UCSF Medical Center, Third & Pamassus Aves, San Francisco, CA 941430570. Copyright o 1991 by W.B. Saunders Company 0022-3468l91J2607-0025$03.00/O
853
854
JENNINGS ET AL
and washed twice with phosphate-buffered saline to remove all extracellular radioactivity. A representative sample was smeared and stained to check for intracellular bacteria. To measure the phagocytosis activity, the PMNs containing the bacteria were dissolved in 0.1% SDS (sodium dodecyl sulfate) and the radiolabeled bacteria were counted in 5 mL of Ecolyte liquid scintillation fluid on a LKB 1209 Rackbeta scintillation counter (LKB Products, Piscataway, NJ).
To assess the ability of the PMNs to phagocytose S aweus without the aid of opsonization, the phagocytosis assay was performed using phosphate-buffered saline. Fetal PMNs gradually develop phagocytic capabilities during the third trimester, but fetal PMNs always showed diminished phagocytosis compared with adult sheep PMNs (Fig 1). Fetal sheep phagocytosis was uniformly low ranging from 24% + 9% of adult levels at 103 days, to 33% k 6% of adult levels at 141 days. The neonatal sheep PMN phagocytosis was 36% k 6% of adult levels. Adult sheep PMN phagocytosis of unopsonized bacteria was 44% + 14% of the phagocytosis value with opsonization. The use of adult plasma opsonization of the bacteria resulted in adult levels of phagocytosis in both the neonatal sheep PMNs and in the fetal sheep PMNs of gestational ages 124 and 141 days. In contrast, the 106day and the 114-day fetal sheep PMNs demonstrated much less phagocytosis (35% f 8% and 63% + 11% of adult levels, respectively). Finally, autologous fetal sheep or neonatal plasma opsonization of the bacteria prior to phagocytosis decreased phagocytosis in all groups of PMNs compared with adult plasma opsonizafion. In particular, autologous fetal sheep plasma opsonization did not improve fetal sheep PMN phagocytosis over that obtained without opsonization at any of the fetal sheep gestational age&
DataAnalysis Alldata were processed using Excel version 2.2 (Microsoft Corp, Redmond, WA). Statistical analysis was performed with Statview 512+ (Brainpower Inc, Calabasas, CA). All data are reported as mean 1?1SD. Differences between values were tested for significance using the Student-Newman Keul’s test and two-way unbalanced analysis of variance (ANOVA) at P < .05. RESULTS
PMNs were obtained from fetal sheep ranging in gestational age from 90 days to term (term is 145 days), as well as neonatal (10 days old), and adult sheep. The blood from fetal sheep less than 104 days gestational age contained too few PMNs to accurately assay for phagocytosis (n = 3). Complete blood counts on the adult sheep showed normal PMN counts (1,008 to 4,218 PMNs/kL). Similarly, neonatal (n = 6), 141-day (n = 12), and 124-day (n = 12) fetal blood had normal PMN counts for gestational age (294 to 1,000 PMNs/pL). One hundred fourteen-day (n = 9) fetal blood contained 5% PMNs (125 to 525 PMNs/ pL), and 104-day (n = 4) fetal blood contained 4% PMNs (24 to 52 PMN&L). Younger fetal blood showed a normal predominance of lymphocytes.3 The viability of the isolated PMNs prior to the phagocytosis assay was always greater than 95% by ethydium bromide and acridine orange stain. Gram stains of representative smears after phagocytosis showed intracellular gram-positive cocci, but no extracellular bacteria. 1.4 q 1.2
No
DISCUSSION
Premature infants are vulnerable to bacterial sepsis. Investigations of neonatal PMN function have shown varying degrees of functional impairment compared with adult PMNs.~ The gram-positive bacteria, such as S aweus, are controlled by the body predominantly by PMN phagocytosis. This phagocytosis can occur via relatively nonspecific recognition, but is
Opsonizafion
H
Fetal Op~nization
q
Adult Opsonization
* l-
:*
T
**
T
1 1.0
Proportion of Adult Phagocytosis
0.8 0.6
124d
Gestational
141d
Age
Fig 1. Fetal, neonatal, and adult sheep PMN phagocytoris of S 8ureus. All values are compared with adult PMN phagocy tosis of adult plasma opsonized S aureus. Error bars are standard deviation. l%
of Fetal Sheep Polymorphonuclear
ByRussell W.
Jennings,
N. Scott Adzick,
Michael
T. Longaker,
and Thomas
Leukocyte Brian W. Duncan,
Phagocytosis
Heinz Scheuenstuhl,
K. Hunt
San Francisco, California l Premature infants and neonates are vulnerable to bacterial sepsis. This susceptibility may be due to the relative immaturity of their immune systems. To determine if neonates and, in particular, premature infants have decreased polymorphonuclear leukocyte (PMN) phagocytosis, wa tested PMN phagocytosis of Staphylococcus aureus as a function of gestational age in the fetal lamb model. Because phagocytosis is made more efficient by the presence of opsonins in plasma, fetal and postnatal PMN phagocytosis were also measured after exposure to fetal and adult plasma. PMNs were isolated from fetal lambs at 104,114,124, and 141 days’ gestation (term gestation for the fetal lamb is 145 days), as well as from IO-day-old neonatal sheep and adult sheep. Labeled S ~ureus were opsonized by incubation in either fetal or adult plasma, or left unopsonized for baseline values. Phagocytosis was measured as a percent of adult PMN phagocytosis after adult plasma opsonization. It was found that fetal PMN function is limited by two factors during the early third trimester: a primary defect in the ability of the PMN to phagocytose S aureus despite adequate opsonization, and the diminished ability of autologous fetal plasma to opsonire bacteria. The defect in PMN phagocytosis disappears late in the third trimester, but the inability of the fetal plasma to opsonize effectively continues until after birth. Copyright o 1991 by W.B. Saunders Company INDEX WORDS: Polymorphonuclear sis; fetal immunity; bacterial sepsis.
leukocyte,
phagocyto-
I
premature infants, NFANTS, PARTICULARLY are at increased risk for sepsis. This susceptibility to infection may be due to the relative immaturity of the neonatal immune system. Previous studies have shown a decreased number of polymorphonuclear leukocytes (PMNs) in blood from the umbilical cord of premature infants, but studies of PMN function in these babies have been controversial. To determine when fetal PMNs are capable of phagocytosis, and to determine if fetal PMN phagocytosis is augmented by plasma opsonins, we tested PMN phagocytosis of Staphylococcus aureus without opsonization as a function of gestational age in fetal, term, and adult sheep. Because phagocytosis is made more efficient by opsonins in plasma, fetal and postnatal PMN phagocytosis were also measured after opsonization with fetal and adult sheep plasma. MATERIALS
AND
METHODS
Time-dated pregnant ewes of gestational ages 90 to 141 days (term is 145 days) were obtained from Torrel Farms (Ukiah, CA). Maternal adult sheep blood was obtained during general halothane/ oxygen anesthesia by jugular vein cannulation and collected into sodium EDTA and heparin. Fetal sheep blood was collected by direct umbilical arterial cannulation during fetal surgery using JournaiofPediatric
Surgery,
Vol26, No
7
(July), 1991: pp 853-855
techniques described previously.’ A portion of each sample was evaluated for complete blood count. All blood samples were processed in parallel.
PMN Isolation To isolate the PMNs, the plasma was first separated from the cellular components by centrifugation at 2,500 rotations/min (rpm) for 10 minutes. The PMNs were maintained at 4°C at all times. The plasma was saved for the opsonization step. The red cells were removed by shock hypotonic lysis three times. After each lysis the solution was returned to normal tonicity with cold 10X phosphate buffer saline. The cells were then isolated by centrifugation at 2,500 rpm. To separate the PMNs from the other leukocytes and remaining red cells, a two-step discontinuous density gradient of 80% and 50% Percoll (Pharmacia, Piscataway, NJ) was used (densities of 1.092 and 1.055 gimL). After isolation of the PMNs, their viability and concentration were determined by ethidium bromide and acridine orange dye using a fluoresence microscope and a hemocytometer. The PMNs were resuspended to 1 X 10’ to 1 x 10” per milliliter. The purity and maturity of the PMNs was evaluated by light microscopy using Wright’s stain.
Bacterial Preparation Laboratory-grade pansensitive S aureus (ATCC 29213) was grown in culture with ‘H-glycine (Amersham, Arlington Heights, IL). The bacteria were then washed three times with phosphatebuffered saline to remove all unincorporated radiolabeled glycine. The bacteria were then incubated in 50% maternal plasma or fetal plasma diluted with phosphate-buffered saline for 1 hour at 37°C to allow opsonization of the bacteria, or in phosphate-buffered saline alone as a control. Concentrations of the bacteria were determined using a turbidity curve and plate counts.
Phagocytosis Assay The isolated PMNs and either opsonized or unopsonized S aurem were then incubated together for 1 hour at 37°C to allow phagocytosis at a 1OO:lratio of bacteria to PMNs. A 10011ratio was chosen to provide a guaranteed excess of bacteria. Preliminary studies showed phagocytosis of enough colony forming units per PMN to significantly alter the ratio of bacteria to PMNs when lower counts of bacteria were used. The extracellular bacteria that had not been phagocytosed were dissolved with 10 U/mL of lysostaphin (Sigma Chemical Co, St Louis, MO) by incubation for 45 minutes at 37%X.*The PMNs were removed by centrifugation
From The Fetal Treatment Program and The Wound Healing Laboratory, Department of Surgery, University of California, San Francisco, CA. Presented at the 23rd Annual Meeting of the Pacific Association of Pediatric Surgeons, Kona, Hawaii, June 3-6, 1990. Address reprint requests to N. Scott Adzick, MD, 585 HSE, UCSF Medical Center, Third & Pamassus Aves, San Francisco, CA 941430570. Copyright o 1991 by W.B. Saunders Company 0022-3468l91J2607-0025$03.00/O
853
854
JENNINGS ET AL
and washed twice with phosphate-buffered saline to remove all extracellular radioactivity. A representative sample was smeared and stained to check for intracellular bacteria. To measure the phagocytosis activity, the PMNs containing the bacteria were dissolved in 0.1% SDS (sodium dodecyl sulfate) and the radiolabeled bacteria were counted in 5 mL of Ecolyte liquid scintillation fluid on a LKB 1209 Rackbeta scintillation counter (LKB Products, Piscataway, NJ).
To assess the ability of the PMNs to phagocytose S aweus without the aid of opsonization, the phagocytosis assay was performed using phosphate-buffered saline. Fetal PMNs gradually develop phagocytic capabilities during the third trimester, but fetal PMNs always showed diminished phagocytosis compared with adult sheep PMNs (Fig 1). Fetal sheep phagocytosis was uniformly low ranging from 24% + 9% of adult levels at 103 days, to 33% k 6% of adult levels at 141 days. The neonatal sheep PMN phagocytosis was 36% k 6% of adult levels. Adult sheep PMN phagocytosis of unopsonized bacteria was 44% + 14% of the phagocytosis value with opsonization. The use of adult plasma opsonization of the bacteria resulted in adult levels of phagocytosis in both the neonatal sheep PMNs and in the fetal sheep PMNs of gestational ages 124 and 141 days. In contrast, the 106day and the 114-day fetal sheep PMNs demonstrated much less phagocytosis (35% f 8% and 63% + 11% of adult levels, respectively). Finally, autologous fetal sheep or neonatal plasma opsonization of the bacteria prior to phagocytosis decreased phagocytosis in all groups of PMNs compared with adult plasma opsonizafion. In particular, autologous fetal sheep plasma opsonization did not improve fetal sheep PMN phagocytosis over that obtained without opsonization at any of the fetal sheep gestational age&
DataAnalysis Alldata were processed using Excel version 2.2 (Microsoft Corp, Redmond, WA). Statistical analysis was performed with Statview 512+ (Brainpower Inc, Calabasas, CA). All data are reported as mean 1?1SD. Differences between values were tested for significance using the Student-Newman Keul’s test and two-way unbalanced analysis of variance (ANOVA) at P < .05. RESULTS
PMNs were obtained from fetal sheep ranging in gestational age from 90 days to term (term is 145 days), as well as neonatal (10 days old), and adult sheep. The blood from fetal sheep less than 104 days gestational age contained too few PMNs to accurately assay for phagocytosis (n = 3). Complete blood counts on the adult sheep showed normal PMN counts (1,008 to 4,218 PMNs/kL). Similarly, neonatal (n = 6), 141-day (n = 12), and 124-day (n = 12) fetal blood had normal PMN counts for gestational age (294 to 1,000 PMNs/pL). One hundred fourteen-day (n = 9) fetal blood contained 5% PMNs (125 to 525 PMNs/ pL), and 104-day (n = 4) fetal blood contained 4% PMNs (24 to 52 PMN&L). Younger fetal blood showed a normal predominance of lymphocytes.3 The viability of the isolated PMNs prior to the phagocytosis assay was always greater than 95% by ethydium bromide and acridine orange stain. Gram stains of representative smears after phagocytosis showed intracellular gram-positive cocci, but no extracellular bacteria. 1.4 q 1.2
No
DISCUSSION
Premature infants are vulnerable to bacterial sepsis. Investigations of neonatal PMN function have shown varying degrees of functional impairment compared with adult PMNs.~ The gram-positive bacteria, such as S aweus, are controlled by the body predominantly by PMN phagocytosis. This phagocytosis can occur via relatively nonspecific recognition, but is
Opsonizafion
H
Fetal Op~nization
q
Adult Opsonization
* l-
:*
T
**
T
1 1.0
Proportion of Adult Phagocytosis
0.8 0.6
124d
Gestational
141d
Age
Fig 1. Fetal, neonatal, and adult sheep PMN phagocytoris of S 8ureus. All values are compared with adult PMN phagocy tosis of adult plasma opsonized S aureus. Error bars are standard deviation. l%
