Changes with Development and During Bacterial Infection

0031-3998/85/1912-1278$02.00/0 PEDIATRIC RESEARCH Vol. 19, No. 12, 1985 Copyright O 1985 International Pediatric Research Foundation, Inc. Printed in U.S.A.

Neutrophil Myeloperoxidase Concentration: Changes with Development and during Bacterial Infection

ROBERT D. CHRISTENSEN AND GERALD ROTHSTEIN Departments of Pediatrics and Internal Medicine, University of Utah School ofMedicine, Salt Lake City, Utah 84132

ABSTRACT. In experimental animals, the quantity of neutrophil MPO during recovery from infection is the result of myeloperoxidase (MPO) in a volume of whole blood, and deleting one myelocyte division. the neutrophil concentration in that same volume, were determined and the results expressed as units of MPO MATERIALS AND METHODS (lO-')/neutro~hil- Two situations are reported in which the MPO quantiJiCation.Our standard method for the measure- MPo/neutrOphil was found change; ment of myeloperoxidase has been published previously (3, 4). during the growth and and during Briefly, leukocytes, separated from whole blood on discontinuous bacterial infection. Premature and newborn rats had only gradients, are washed and suspended in M~c~~'~5A medium, 25% of the MPO/neutrophil found in adults. One to three after which contaminating erythrocytes are subjected to hypo- wk O1ds had the enzyme During tonic lysis. Final cell concentrations are determined by electronic fatal bacterial infection, MPO/neutrophil fell rapidly, often counting (Coulter Electronics, Hialeah, FL), and cell differential to undetectable levels, but during sublethal infections, fol- counting. Myeloperoxidase is extracted from cells by suspending lowing a 24-h lag period in and a 48-h lag in them in 0.5% hexadecyltrimethylammonium bromide (Sigma neonates, the concentration increased to twice normal. Chemical co., st. ~~~i~,MO) in 50 m~ potassium phosphate (Pediatr Res 19: 1278-1282,1985) buffer, pH 6.0. Specimens are subjected to sonication in an ice bath for 10 s (Heat Systems-Ultrasonics, Plainview, NY), fol- Abbreviations lowed by freeze-thawing and repeat sonication. MPO is assayed spectrophotometrically; 0.1 ml of the material to be measured is MPO, myeloperoxidase CFU, colony forming unit mixed with 2.9 ml of 50 mM phosphate buffer, pH 6.0, containing 0.167 mg/ml o-dianisidine dihydrochloride (Sigma Chemical CFUc, colony forming unit in culture Co.) and 0.0005% hydrogen peroxide (Mallinckrodt, Paris, KY). The change in absorbance at 460 nm is measured with a Beck- man DU spectrophotometer (Beckman Instruments, Fullerton, CA). One unit of MPO activity is defined as that amount MPO is an antimicrobial enzyme located within the primary degrading one micromole of peroxide per minute at 25" C (5). granules of mammalian neutrophils (1, 2). In this study we The micromethod differs from the standard method in that employed a method for quantification of MPO using only 50 pl neutrophils are not separated from the blood. Instead, 50 PI of of blood, thus enabling its determination in very small subjects. blood are drawn into a heparinized microcapillary tube (Dade We assessed two situations in which we suspected that the Diagnostics, Inc., Miami, FL) and transferred to a test tube concentration of MPO in neutrophils might vary; during devel- containing 2 ml Hanks' balanced salt solution. Hypotonic lysis opment and during bacterial infection. First, blood samples from of the erythrocytes is accomplished by adding 6 ml distilled water rats at various stages of pre- and postnatal development were and mixing for 2 min, after which 2 ml of 3.5% NaCl is added. tested. In premature and newborn animals, the quantity of MPO The mixture is then centrifuged at 1200 x g for 5 min. The per neutrophil was only 25% of that found in adults, while supernatant is removed and 0.5 ml hexadecyltrimethylammon- neutrophils from 1 to 3 wk olds contained 50% of the adult ium bromide buffer (50 mM phosphate with 0.5% hexadecyltri- enzyme concentration. Next, the effect of bacterial infection on methylammonium bromide) added. The sample is then frozen neutrophil MPO content was measured. During fatal infections, at -70" C and assayed for MPO as described above. In both the concentration of MPO fell significantly, often to undetectable methods, the mean neutrophil MPO value was calculated by levels. In contrast, during sublethal infections mean neutrophil dividing the units of MPO from a known quantity of blood by myeloperoxidase did not fall, but rather, increased to twice the number of neutrophils measured in that same quantity of normal concentrations following a 24-h lag period in adults and blood. Values are expressed as units MPO (10-')/neutrophil. a 48 hour lag in neonates. We speculate that: 1) the low MPO Animals. Sprague-Dawley rats (Simonson Laboratories, Gil- concentration in the neutrophils of neonates is a result of addi- roy, CA) of various ages were studied. Groups of 10 or more tional myelocyte divisions, 2) the fall in neutrophil MPO during animals were taken prematurely at 19 and at 20 days gestation lethal infection is due to MPO exocytosis, and 3) the increase in (term is 21 days) by performing an hysterotomy on timed- pregnant females. Other animals were studied late on the first Received December 7, 1984; accepted July 23, 1985. day of life (6- 18 h old), on the 2nd day, between the 7th and 9th Address reprint requests to Dr. R. D. Christensen, Division of Hematology, day, at 3, 4, 6, 9, and 12 wk of age. Animals were weaned from University of Utah School of Medicine, 50 North Medical Drive, Salt Lake City, UT 84132. their mothers when were between 3 and wk old. At that Supported by Public Health Service Grant HD-14419 and a grant from the time they were caged in groups of five to eight and allowed water Thrasher Research Fund. and rat food ad libitum. 1278 NEUTROPHIL MPO DURING DEVELOPMENT AND INFECTION Blood samples were obtained in a manner previously described (6). Briefly, the animals were first subjected to inhalation anes- thesia (Methoxyflurane, Abbott Laboratories, N. Chicago, IL) and then, in those 2 days old or younger, the external jugular vein was incised with scissors. Blood was collected directly into heparinized capillary tubes for MPO determination and electronic cell counting. In older animals, blood was drawn by venipuncture from the inferior vena cava into a syringe containing heparin (Panheparin, Abbott Laboratories) in a concentration of 10-20 U/ml blood. A 50-p1 aliquot was taken for the micromethod determination using a heparinized capillary tube. In all animals, a blood smear was prepared, stained with Wright stain, and a 100-200 cell differential performed. The accuracy and 3.0 reproducibility of neutrophil enumeration by this method have previously been established (7). 2 3.0 4.0 5.0 8 6.08 8 7.03 8 8.0o I 9.08 I 10.01 Bacteria. Type I11 group B streptococci, isolated from a human 4'1..MACRO METHOD neonate, were used to produce either lethal or sublethal infec- MPO UNITS (10-VNEUT tions. The organism was identified by the precipitin method Fig. 1. This study compares MPO/neutrophil values performed by using rabbit antisera (8) and grown overnight at 37" C in Todd- two differentmethods. Seventeen blood samples, each represented by a Hewett broth. After washing with phosphate-buffered saline, dot, were tested by the standard (gradient sedimentation) method and aliquots were frozen at -70" C. Before administering bacteria to plotted on the abscissa, and also by the micromethod, plotted on the animals, the aliquots were thawed and grown overnight in fresh ordinate. The correlation coefficient for the two methods was 0.9430. Todd-Hewitt broth. Bacteria were then sedimented by centrifu- gation and the concentrated organisms were washed three times in phosphate-buffered saline. The organisms were then diluted in phosphate-buffered saline to an optical density (Spectronic 20, Bausch and Lomb, Inc, Rochester, NY) corresponding to a final concentration of either lo", lo9, or lo7 CFU/ml. One microliter of bacterial suspension per gram of body weight was then used for animal inoculation. In 4-wk-old animals, a lethal infection was produced by an intraperitoneal inoculation of lo8 CFU/g body weight. Sublethal infection was produced in 4 wk olds by inoculation of lo6CFU/g. Lethal infection was produced in newborn animals by an intraperitoneal inoculation of lo6 CFU/g body weight, while sublethal infection was produced by inoculation of 1O4 CFW/g. Statistical analysis. Single variable linear regression analysis was used to compare the two myeloperoxidase determinations. All other data were analyzed by means of Student's t test. RESULTS 1/2 4 6 8 10 12 14 16 18 1st SAMPLE Testing the micromethod. To test the reliability of the micro- MPO UNITS (IO-')/NEUT method for MPO measurement, the MPO of neutrophils pre- Fig. 2. The intratest variation of the micromethod for measurement pared by gradient sedimentation was compared with values on of MPO/neutrophil was assessed by performing duplicate tests from blood from the same samples, using the micromethod. Blood for single blood samples. Each sample is represented by a dot. Values for the testing was obtained from 12 adult humans and five adult rats. first micromethod determination are plotted on the abscissa and from MPO by the standard method ranged from 3.6 to 8.5 x U/ the second on the ordinate. The correlation coefficient for the duplicate neutrophil (mean = 5.9), and by the micromethod from 4.0 to determinations was 0.978. 8.8 x U/neutrophil (mean = 6.1) (Fig. 1). The correlation coefficient for the two methods was 0.9430. Intratest variation of the micromethod was assessed by obtain- Mean neutrophil MPO during development. Blood neutrophil ing duplicate 50-p1 samples of venous blood, and performing concentration and MPO/neutrophil determinations were ob- MPO analysis on each (Fig. 2). The correlation coefficient for tained at 10 different age groups; each composed of 10 or more the duplicate samples was 0.978. animals (Fig. 3). (Both the blood neutrophil concentration and During preparation of cells for the micromethod assay, al- the MPO/neutrophil value are shown in each of the remaining though the red blood cell lysate supernatant is removed, it is figures. This is done in order to illustrate situations in which the likely that small amounts of hemoglobin contaminate the sam- two are discordant, as during the neutrophilia associated with ple. In order to determine whether contaminating hemoglobin birth, or concordant, as during the profound neutropenia of fatal might interfere with the accuracy of the micromethod assay, sepsis in neonates.) The blood neutrophil concentration in- various amounts of red blood cell lysate (10, 50, 100, and 250 creased just before birth, from 820 * 110/mm3 at 19 days of pl), prepared by hypotonic lysis of whole blood, were added to gestation to 2780 + 110/mm3 at 20 days gestation (mean + 2.5-ml suspensions of neutrophils (3 x 106/ml) obtained from a SEM, p = <0.001). By 6-18 h following delivery the blood 4-wk-old rat and separated by gradient separation. Micromethod neutrophil cocentration had diminished to 1000 + 160/mm3, MPO quantification was then camed out. When extremely large but by 7-9 days of age, it had again increased; 17 10 + 260/mm3 quantities of lysate (100 p1 or more) were added, some diminu- (p= 0.036). No further changes of significance were observed tion in the observed MPO did indeed occur (from 3.3-2.9 U). through 12 wk of age. However, at hemoglobin levels which approximate those found Mean neutrophil myeloperoxidase did not change over the in our samples (5-10 p1 lysate/ml) or when up to five times those first four periods of observation (19 days gestation, 20 days levels (50 j~1lysate/ml) were added, no artifactual diminution in gestation, 6-18 h and 2 days), but increased from 1.5 + 0.2 U MPO quantification occurred. (10-7)/neutrophil at 2 days of age to 3.0 + 0.4 U (10-7)/neutro- CHRISTENSEN AND ROTHSTEIN

r p < 0.05 vs 2 days 4 -6.0 a:0 I- 5.0 2 z\ -4.0 -b -3.0 g *Blood Neutrophils I $" r P < 0.005 vs 2 days OMean Neutrophil MPO V) I Mean f SEM -2.0 Ir: 2 3

;..$.6.b~*6:18~1=+s HRS DAYS DAYS Fig. 3. Blood neutrophil concentration (solid circles) and units of MPO (10-7)/neutrophil(open circles) in developing rats. Groups of 10 or more animals were tested at various pre- and postnatal ages, from 19 days gestation to 12 wk of age. phi1 at 7-9 days (p= 0.003). By 6 wk of age the mean neutrophil 4 WEEK-OLD RATS MPO had increased further to 5.5 + 0.7 (p < 0.005). At 9 wk of age, neutrophil MPO was 5.8 + 0.7 and 5.8 f 0.6 U neutrophil was observed at 12 wk. 2000 Mean Neutrophil MPO during Bacterial Infection. Lethal infection. When twenty-five 4-wk-old rats were inoculated with lo8 type 111 group B streptococci/g body weight, death occurred in all within 8-1 2 h. Sixty other animals received the same inoculum and then were sacrificed at various intervals for determinations of blood neutrophil count and neutrophil MPO concentration (Fig. 4A). After 6 h the blood neutrophil concentration had fallen to 330 +- 70/mm3 (p < 0.0025 versus preinoculation value); this was preceded by a fall in MPO/neutrophil(2.3 + 0.7 at 3 h, p < 0.05 versus preinoculation value). The mean neutrophil MPO level remained low in the lethally infected animals (0.6 + 0.1 at 4 h, 2.7 + 0.9 at 5 h, 3.2 + 0.6 at 6 h, p < 0.05 versus preinoculation). Newborn rats were also given a lethal inoculum of type 111 group B streptococci. However, unlike 4-wk-old animals, a much smaller bacterial inoculum (lo6 CFU/g) was needed to produce death by 8-12 h. Following a very brief period of neutrophilia (Fig. 4B) the neutrophil count fell significantly. Six hours after the inoculation profound neutropenia was present (150 + 701 mm3, p < 0.001). As in the adults, the mean neutrophil myeloperoxidase content fell. Four hours after the inoculation the value was 0.79 + 0.14 (p = 0.001) and after 6 h no MPO was detectable. Sublethal infection. In 4-wk-old rats, when the bacterial inoculum was decreased from 10' to lo6 CFlJ/g, no deaths occurred. Early in the course of infection, the blood neutrophil count increased from 1 I00 _+ 80/mm3 to 2250 + 350/mm3 (p = 0.05), but no further changes of significance were detected during the 96 h of observation (Fig. 5A). In contrast to the 4-wk-old animals Blood Neutrophils o Meon Neutrophil MPO with lethal infections, the mean neutrophil myeloperoxidase in Meant SEM I * this group did not decrease. In fact, an increase was observed by ,,I? 24 h 13.9 _+ 0.5 U(10-7)/neutrophil at 6 h versus 6.5 +- 0.8 at 24 123456 h, p < 0.021. HOURS AFTER LETHAL INOCULATION A sublethal infection was produced in neonatal animals by Fig. 4. Blood neutrophil concentration (solid circles) and units of reducing the bacterial inoculum from lo6 to lo4 CFU/g. As is MPO (10-7)/neutrophil (open circles) in (A) 4-wk-old rats and (B) shown in Figure 5B, like the sublethally infected 4 wk olds, their newborn rats, during a lethal infection. mean neutrophil MPO increased. The increase occurred between 24 h (1.2 -f- 0.3) and 48 h (4.0 -t 0.6) (p = 0.002) following the DISCUSSION inoculation. The MPO/neutrophil value at 48 h in these animals MPO is an enzyme constituent of the primary or azurophilic is also over twice the value observed in noninoculated control granules of neutrophils (1). It is also found in the primary animals at 48 h of life (shown in Fig. 3). granules of eosinophils, basophils, and in far lower concentra- NEUTROPHIL MPO DURING DEVELOPMENT AND INFECTION 1281 their technology did not permit actual quantification of MPO I 1 4 WEEK-OLD RATS and therefore, they could not determine whether the neonate's MPO/neutrophil was reduced. Ambruso et al. (14), using spec- trophotometric methods similar to those employed in our present study, recently reported that MPO/neutrophil values in the cord blood of term neonates was similar to that of adults. In contrast, MPOIneutro~hilconcentrations in memature neonates have not been reported. However, using this micromethod, we have tested six noninfected preterm infants (27-29 wk gestation) within their first 2 days of life, and have found their ~~~/neutro~hilvalues, analogous to the situation in rat pups at term, to be only about 25% of the adult value (Christensen RD, unpublished observation)... - -. 1;-addition to the marked age-related changes in neutqophil MPO content in rats, we observed significant changes &ring bacterial infection. Both neonatal and adult animals experienced a marked, rapid reduction in MPO/neutrophil during lethal Blood Neulrophils NEWBORN RATS 0 Mean Neutrophil MPO bacterial infections. In fact, even before neutropenia occurred, the mean neutrophil MPO decreased. In contrast, during the I IMeantSEM *I course of sublethal bacterial infections, neutrophil MPO concentration did not decrease, but rather increased to about double the previous values. Decreases in neutrophil MPO concentration, observed both in very young rats and also in those with lethal infections, might be brought about by one of three mechanisms. First, less enzyme might be synthesized during the promyelocyte stage. Second, the number of cell divisions for individual myelocytes might increase, thus further dividing a fixed quantity of MPO among additional progeny. Third, neutrophils in the circulation might lose the enzyme. Previous experiments support the concept that in uninfected neonates the mechanism probably involves the I addition of more myelocyte divisions. Evidence in favor of this 123456 24 48 72 96 can be found in the studies of Grould and Helpap (15), who HOURS AFTER SUBLETHAL INOCULATION injected rats with tridiated thymidine and based on labeling Fig. 5. Blood neutrophil concentration (solid circles) and units of index, concluded that neonates experience one additional mye- MPO (10-')/neutrophil (open circles) in (A) 4-wk-old rats and (B) locyte division. Further support for an increased number of newborn rats during a sublethal infection. divisions in the mitotic pool of neonates is derived from experiments in which we measured 2500 mature, stored neutrophils/ tions, in the granules of monocytes and macrophages (9). In CFUc in prematurely delivered rats, 1200 mature cells/CFUc in neutrophils the enzyme is produced only during the promyelo- 1-day-old animals and a further decrease to 500 neutrophils/ cyte stage, during which the primary granules bud from the CFUc in adults (16). Two additional myelocyte divisions in the proximal or concave face of the Golgi apparatus (10). In the premature animals, and one additional division in the 1-day-old myelocyte stage, the distal or convex surface of the Golgi begins group could explain the larger number of daughter cells to stem to produce specific granules, but no further production of MPO cells observed in that study. Excess loss of enzyme in circulating occurs (10). Thus, with each myelocyte division a fixed quantity neutrophils is an unlikely cause of the diminished neutrophil of MPO is divided between daughter cells. Generally three to MPO in uninfected neonates, and diminished production of five myelocyte divisions take place (1 1) giving each mature MPO by the promyelocytes of young animals remains an un- neutrophil '/8 to l/32 the MPO concentration of the late promye- tested possible alternative explanation. locyte. During a lethal bacterial infection, a different mechanism Methods that are currently available for measurement of MPO appears to be responsible for the reduced neutrophil MPO con- in blood require the separation of neutrophils from erythrocytes. centration. The rapidity with which the MPO dropped in that Because several milliters of blood are needed, MPO quantifica- setting strongly argues against decreased synthesis of MPO by tion has not been possible in very small subjects. In this study promyelocytes or increased myelocyte divisions as mechanisms, we developed a micromethod with which 50 p1 of blood could since the promyelocyte to PMN transit time is undoubtedly be assayed. MPO activity by this micromethod correlated well several days (17), yet the MPO fell in only 4 to 6 h. We maintain with that measured by standard methods and intratest variability that the speed with which the MPO fell during lethal infections appeared acceptable. Utilizing the micromethod for MPO meas- argues strongly that it was due to exocytosis of MPO by circulat- urement in developing rats, we observed marked age-related ing neutrophils. We have previously demonstrated that neutro- differences. the neutrophils of young animals, whether prema- phils in suspension export MPO extracellularly when bacteria ture, term-newborns or 2 days of age, had only 25% of the MPO are added (3), thereby rapidly diminishing the MPO/neutrophil concentration observed in adults. Animals 1-3 wk old had 50% concentration. Similarly, Lash et al. (18) have shown in vivo of the adult concentration. release of secondary granule constituents by circulating neutro- Human neonates born at term probably do not have the phils. In addition, Pebmer and Kinkade (19) have shown that marked diminution in neutrophil MPO concentration which we differences in MPO activity could be related to selective exocy- observed in neonatal rats. Corberand et al. (12), using a cyto- tosis of the enzyme. Therefore, both kinetic arguments and in chemical scoring method, reported that neutrophils of term vitro studies suggest that exocytosis of MPO is the most likely human neonates had MPO concentrations about 10%lower than mechanism for the observed rapid decrease in neutrophil MPO those observed in adults. Moms et al. (13) used electronmicros- concentration during fatal bacterial infection. copy to examine neutrophils obtained from human cord blood The increase in MPOIneutro~hil.which we observed in two and found no differences in the ratio of primary to secondary conditions-during growth to adulthood and following sublethal granules compared to adults. However, they emphasized that infections-might occur by one of three mechanisms: 1) increas- 1282 CHRISTENSEN A ND ROTHSTEIN ing MPO synthesis by promyelocytes, 2) deleting myelocyte Nancy Taylor for technical assistance and Dr. Hany R. Hill for divisions, or 3) decreasing the rate of exocytosis of MPO by supplying the bacteria. circulating neutrophils. In animals with sublethal infections, the REFERENCES fact that the increase in MPO/neutrophil occurred within only 1. Klebanoff SJ 1968 Myeloperoxidase-halide-hydrogen perooxide antibacterial 24-48 h, suggests that increased MPO synthesis is not the mech- system. J Bacterial 95:2 13 1-2 138 anism. Fliedner et al. (17) have shown that the emergence time 2. Babior BM 1978 Oxygen-dependent microbial killing by phagocytes. N Engl J (labeled myelocyte to blood transit time) is about 48 h in rats. Med 298:659-668 Therefore, in order to produce twice the usual amount of MPO 3. ~radle~PP, Christensen RD, Rothstein G 1982 Cellular and extracellular myeloperoxidase in pyogenic inflammation. Blood 60:6 18-622 at the promyelocyte stage, and then to generate mature blood 4. 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