Vol. 22, No. 1
INFECTION AND IMMUNITY, Oct. 1978, p. 57-61 0019-9567/78/0022-0057$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Age-Dependent Variations in Polymorphonuclear Leukocyte Chemiluminescence DENNIS E. VAN EPPS,* JAMES S. GOODWIN, AND SHIRLEY MURPHY Departments of Medicine, Microbiology, and Pediatrics, University of New Mexico School of Medicine, Bernalillo County Medical Center, Albuquerque, New Mexico 87131 Received for publication 1 August 1978
Polymorphonuclear leukocytes from 46 adults (age 18 to 35), 19 adults (age 70 to 91), 10 children (age 1 to 3), and 22 neonates (cord blood samples) were tested for their chemiluminescence response to opsonized zymosan. Results indicated that both cord blood leukocytes and those from individuals over 70 were significantly lower (P < 0.05) in their chemiluminescence response. Furthermore, when the latter group was divided into two subsets, one containing subjects over 80 years of age and the other containing subjects between 70 and 80 years of age, those over 80 showed a chemiluminescence response significantly lower (P < 0.05) than those between 70 and 80. The kinetics of the chemiluminescence response was similar with all samples except the neonatal cells, where the response appeared to peak and subside more slowly. These data demonstrate that polymorphonuclear leukocyte chemiluminescence is depressed in the very young and the very old. chemiluminescence was observed with neonatal PMNs as compared to those of normal adults. This decrease was not observed in children 1 to 3 years of age, but many adults over 70 did show decreased chemiluminescence activity. Chemiluminescence by neonatal PMNs also showed kinetic differences, when compared with adult PMNs, which were not seen in those people over 70 with decreased chemiluminescence.
The functional capabilities of human neonatal polymorphonuclear leukocytes (PMNs) have been frequently studied with conflicting results. Some studies indicate that PMN phagocytosis may be depressed in neonates (8, 9), while other studies show no difference between phagocytosis by neonatal PMNs and adult PMNs (4). Other workers have shown that hexose monophosphate shunt activity may be depressed in neonatal cells undergoing phagocytosis (2, 3), while various other reports indicate increased resting levels of Nitro Blue Tetrazolium dye reduction with a slightly elevated stimulated Nitro Blue Tetrazolium test (13) or decreased (J. A. Bellanti, B. E. Cantz, D. A. Maybee, and R. J. Schlegel, Pediatr. Res. 3:376, no. 114, 1969) or normal Nitro Blue Tetrazolium dye reduction (7). In still other studies, Miller has also shown that neonatal neutrophil chemotaxis is depressed as well as PMN membrane deformabil-
MATERIALS AND METHODS
ity (10, 11).
In this study, neonatal (cord blood) PMN function was evaluated by the chemiluminescence technique (6) and compared among children 1 to 3 years of age, adults 18 to 35 years of age, and adults greater than 70 years of age. This technique measures the liberation of light from PMNs stimulated with opsonized zymosan and is believed to be dependent upon a variety of PMN properties including surface contact with zymosan, hexose monophosphate shunt activity, the myeloperoxidase system, and the generation of singlet oxygen (1). A marked depression in 57
Cell sources. All subjects were selected randomly from a healthy population. Neonatal PMNs were prepared from heparinized cord blood samples within 3 h of delivery. Mothers were not receiving general anesthesia for delivery. Fifty percent of mothers received a short-acting narcotic (alpha prodine hydrochloride), 90% received a local anesthetic (xylocaine), and 10% received nothing. Samples from children aged 1 to 3 were obtained from normal children free from infection and medication at the time of study. Blood samples from 18- to 35-year-old adults were obtained from nonhospitalized volunteers, and adults over 70 years of age (70 to 91 years) were obtained from three sources: seven subjects had been admitted to the General Medical Ward of the County Hospital for nursing home placement; seven subjects were inmates of a local nursing home; and five subjects were members of a local senior citizens group who volunteered to donate blood. All subjects were ambulatory and none had a diagnosed infection or systemic disease or was on medications other than occasional analgesics, laxatives, or sedatives. Cell preparations. PMNs were prepared from heparinized peripheral blood by Plasmagel (HTI, Buf-
58
INFECT. IMMUN.
VAN EPPS, GOODWIN, AND MURPHY
falo, N.Y.) sedimentation of erythrocytes (RBCs) followed by Ficoll-Hypaque centrifugation to remove mononuclear cells (14). Final cell preparations contained greater than 90% PMNs, which were adjusted to 107 PMNs per ml in phosphate-buffered saline (pH 7.2). In some instances, contaminating RBCs were lysed using a 40C, 30-s distilled water lysis. Cells were then washed and resuspended in phosphate-buffered saline. Generally, RBC lysis was not performed, since a high percentage of fetal cord blood leukocytes (WBCs) were sensitive to this treatment. Therefore, comparisons between groups of subjects were made with samples which had not undergone the lysis treatment, to make comparisons valid. In cord blood preparations, slightly more eosinophils, bands, and mononuclear cells were noted (mean, 3% bands, 6% eosinophils, and 7% mononuclear cells) than in adult preparations (mean, 1.5% bands, 3% eosinophils, and 1% mononuclear cells). Other cell preparations were similar to normal adult cell preparations in morphology. Chemiluminescence assay. Chemiluminescence was performed as previously described (6). Zymosan particles which had been opsonized with fresh normal serum were used as a stimulant (6). Light emission was measured in a nonrefrigerated Nuclear Chicago /?-scintillation counter used out of coincidence with a window setting of 0, 10. A mixture of 0.5 ml of PMNs (5 X 106) and 0.2 ml of opsonized zymosan (10 mg/ml) was suspended in phosphate-buffered saline to a final volume of 1 ml in an 18-h dark-adapted Beckman Poly Q vial. Tubes were counted for 1 min at 5- to 7-min time intervals.
RESULTS Chemiluminescence profiles on PMNs from normal adults and fetal cord blood. Cumulative results of PMN chemiluminescence profiles on three adults and three neonates showing depressed chemiluminescence are shown in Fig. 1. A profound depression in the peak chemiluminescence response was observed with these neonatal PMNs as compared with adult PMNs. In addition, the kinetics of chemiluminescence differed between adult and these neonatal PMNs, with neonatal PMNs showing a peak response between 11 and 15 min and adults showing a peak response between 6 and 9 min. Furthermore, the response by neonatal PMNs subsided more slowly than the adult chemiluminescence response. Comparison of peak PMN chemiluminescence responses in neonates, children (ages 1 to 3), adults (ages 18 to 35), and adults over 70 years of age. Figure 2 shows a summary of the peak chemiluminescence response in 46 adults (18 to 35 years of age), 10 children (1 to 3 years of age), 22 neonates, and 19 adults over 70 years of age (range 70 to 91). The kinetics of the chemiluminescence response of both the 1- to 3-year-old children and the greater-than70-year-old adults were similar to those obtained with the 18- to 35-year-old adults. Only neonatal
35r 3*
°Q2 x
CL 0
20
Is5 A.-
-
-
-4"-
-
-
-
5
12 4 6 8 10 12 14 16 18 20 22426 28 30
Minutes FIG. 1. PMN chemiluminescence profiles on three normal adults ( ) and three fetal cord blood samples (-----) in response to opsonized zymosan. The data shown are a composite of three normal adults and three fetal cord blood samples. 44 42 38 36 tE Q 34
32 K 30 28 26 24 22
I
.
9
to 01
I'
0:-
I
*-
.
IsX 0:
I
14K1
*
00
.
' 12 K) 8
t.
61 8-3ym I -3ysws
>7Oyeas Fea COrd Blood
FIG. 2. Comparison of peak PMN chemiluminescence responses offetal cord blood cells and subjects aged I to 3, 18 to 35, and over 70. Groups were statistically compared using the Dunnett test (5).
PMNs with a suppressed peak chemiluminescence response showed a difference in chemiluminescence kinetics (Fig. 1). No association between the mother's anesthetic and neonatal cell
VARIATIONS IN CHEMILUMINESCENCE
VOL. 22, 1978
chemiluminescence was observed. The mean peak chemiluminescence response for 18- to 35year-old adults was 30,936 + 6,845; for 1- to 3year-old children it was 26,053 ± 4,965; for adults over 70 years it was 24,727 ± 9,571; and for neonates it was 16,033 ± 8,138. In tests with neonatal cells, 15 of 22 gave a chemiluminescence response of less than 20,000 cpm. It should be noted that cell preparations from cord blood samples contained slightly more band forms, eosinophils, and mononuclear cells (mean, 3% bands, 6% eosinophils, and 7% mononuclear cells) than adult controls (mean, 1.5% bands, 3% eosinophils, and 1% mononuclear cells). This difference was minor and could not account for the marked reduction in chemiluminescence. This was confirmed by titration experiments on normal cells, which indicated that a 20% reduction in the number of PMNs resulted in only a 14% reduction in chemiluminescence. Thus, the number of bands, eosinophils, and mononuclear cells found in cord blood samples was not a probable explanation for the decreased chemiluminescence observed with neonatal cells. Al though no significant difference was observed between 18- to 35-year-old adult PMNs and those of 1- to 3-year-old children (Fig. 2), PMNs both of adults over 70 years of age (P < 0.05) and of neonates (P < 0.01) showed an overall significant depression in their chemilumines-
59
tions. When RBC-to-WBC ratios
were determined on five adult PMN preparations and six neonatal PMN preparations (three with depressed chemiluminescence and three with normal chemiluminescence), it was apparent (Table 1) that RBC contamination could not totally account for depressed neonatal PMN chemiluminescence. Evidence for this is shown with cord blood samples 1 and 4, with reduced chemiluminescence and RBC-to-WBC ratios of 4 and 5, as compared to cord blood samples 2 and 3 and adult samples 1 and 5, with normal chemiluminescence values and comparable RBC-to-WBC ratios. Attempts were made to lyse RBCs and compare adult and neonatal PMNs (Table 1). As shown, lysis treatment resulted in a substantial loss of cord blood PMNs with no apparent effect on adult PMNs. In each case, chemiluminescence was measured before and after RBC lysis. Distilled-water lysis of RBCs was an efficient way to remove contaminating RBCs from adult PMN preparations, as shown. However, when cord blood PMN preparations were lysed with water, a significant number of PMNs were lost, and a substantial number of RBCs remained (Table 1). The overall effect of RBC lysis was an increased chemiluminescence response with both neonatal and adult cells, although the data are difficult to interpret because a mean loss of cence responses. 42% of PMNs was observed with neonatal cells Another possible reason for depressed chemi- after lysis. These data indicate that at least a luminescence with neonatal PMNs may be the portion of neonatal PMNs differ from normal suppressive effects of contaminating RBCs in adult PMNs in their susceptibility to distilledPMN preparations. Ficoll-Hypaque purification water lysis. of PMNs resulted in a substantial number of Although there was a significant difference RBCs in cord blood and adult PMN prepara- between the overall response of adults over 70
TABLE 1. Comparison of neonatal and adult PMN chemiluminescence before and after lysis of RBCs Before lysis After lysis
SampleRB/RC RBC/ RBC/ Chemiluminescence Chemiluminescence
%osf
%WIBCof
Fetal cord blood 1 2 3 4 5 6 Mean
12,354 26,083 31,073 12,372 24,487 10,474 19,474 ± 8,780
4 4 6 5 3 8 5
27,397 28,635 29,831 31,362 36,211
4 2 2 6 2
64 54 31 44 16
30,687 ± 3,418
3
42
32,566 35,995 27,124 23,859 27,357 29,380
5 2 2 3 4 3
34,597 37,901 48,402 39,456 40,340 40,139 ± 5,111
3 0.5 0 0 0 1
2 0 0 0 0 1
Adult blood 1 2 3 4 5 Mean
4,837
60
VAN EPPS, GOODWIN, AND MURPHY
INFECT. IMMUN.
and that of the 18- to 35-year-old adult population (Fig. 2), the majority of these individuals showed normal levels of chemiluminescence, with 12 of 19 giving a response of greater than 20,000 cpm. It is of interest that the mean age of those individuals over 70 showing less than 20,000 cpm chemiluminescence was 85 ± 5 years, whereas those showing greater than 20,000 cpm had a mean age of 77 ± 5. This difference in mean age was significant at the P < 0.02 level. When these individuals were divided between those over 80 (10 of 19) and those between 70 and 80 (9 of 19), the mean peak chemiluminescence was significantly lower (P < 0.05) for those individuals over 80 (20,443 ± 8,371) than for those between 70 and 80 (30,015 ± 8,252). Only one of the over-70-year-old subjects was receiving sedatives (phenothiazine), and this 82-yearold patient showed a chemiluminescence response of 30,240 cpm. The remaining subjects were healthy individuals. The situation was quite different with neonate PMNs, where the majority were consistently decreased in chemiluminescence (15 of 22 individuals below 20,000
increased number of RBCs contaminating neonatal PMN preparations. Experiments designed to remove RBCs by lysis indicated that both neonate and adult PMN chemiluminescence could be enhanced by RBC lysis, although RBC removal from neonatal cell preparations could never be totally achieved. As a consequence of RBC lysis, a large portion of neonatal PMNs was also lost. This loss was not observed with adult PMNs and indicates that neonatal PMNs may differ from adult PMNs in fragility. This may be an important consideration in all studies dealing with neonatal PMNs where RBC lysis is a standard procedure. Our data indicate that, after lysis, neonatal PMN chemiluminescence approaches normal adult levels, although these values may be difficult to interpret since a mean loss of 42% of PMNs was observed after lysis. The remaining cells resemble adult PMNs in their resistance to lysis and their chemiluminescence activity, but are probably not a true representation of the total neonatal PMN population. The PMNs that were susceptible to lysis may represent a subpopulation of cells, possibly less mature, less active in chemiluminescence, or more rigid. The latter has been demonstrated previously by others (11). Neonatal PMN lysis makes it difficult to determine if RBC contamination is the cause of the decreased chemiluminescence response in neonatal PMN, although comparison of RBC-to-WBC ratios in adult and neonatal PMN preparations shows that neonatal PMN preparations with comparable RBC contamination may still show a greatly reduced chemiluminescence response and implies that these cells differ from adult PMNs. It is apparent that not all neonatal PMN preparations show a reduced chemiluminescence response (Table 1, Fig. 2). The reason for decreased neonatal PMN chemiluminescence in some samples and not others is not understood at this time, but may be related to several other previously defined defects in PMN function. These previously described defects include depressed phagocytosis (8, 9) and hexose monophosphate shunt activity (Bellanti et al., Pediatr. Res. 3:376, no. 114, 1969), both of which may be involved in chemiluminescence, although the former is not essential for chemiluminescence (R. D. Nelson, M. Herron, R. L. Simmons, and P. G. Quie, Fed. Proc. 36:4214A, 1977). This depression in phagocytic and hexose monophosphate shunt activity has been controversial, since some investigators have shown no difference between neonatal and adult cells with respect to these functions (4, 7). The discrepancies in these results may be due to one of several things: to the method of cell preparation, to differences in analysis of Nitro
cpm). DISCUSSION This study demonstrates the tremendous variation in PMN chemiluminescence responses from individual to individual. Although the chemiluminescence response by different individuals covers a broad range, a significantly decreased response was observed with the majority of fetal cord blood cells (15 of 22) and some subjects over age 70 (7 of 19). In the older subjects, subdividing this age group into two categories revealed that individuals between 70 and 80 years of age showed a much higher mean chemiluminescence response than subjects over 80 years of age. This implies that the PMN chemiluminescence response may deteriorate beyond a certain age and may possibly be associated with a general depression of host defense mechanisms previously observed with age (12). Although chemiluminescence responses in children aged 1 to 3 years were comparable to adult levels, a decreased responsiveness in neonatal PMNs was observed. This was apparent in the majority of fetal cord blood PMNs tested. In addition to a decreased peak chemiluminescence response in most cases, these same neonatal PMNs differed from adult PMNs in the kinetics of the chemiluminescence response. Neonatal PMN chemiluminescence peaked more slowly and tapered off more slowly than adult PMN chemiluminescence. One possible explanation for the differences in neonatal and adult PMN chemiluminescence could be the
VOL. 22, 1978
Blue Tetrazolium dye reduction by either optical density (13; Bellanti et al., Pediatr. Res. 3:376, no. 114, 1969) or microscopic observation for the presence of intracellular precipitate (7), or to differences in the expression of data either as absolute values (13) or as a function of the resting levels of Nitro Blue Tetrazolium dye reduction (Bellanti et al., Pediatr. Res. 3:376, no. 114, 1969). In some cases the latter may affect results, since resting dye reduction levels for neonatal cells may be elevated. Thus, this particular defect is still controversial. In addition, both membrane deformability and chemotaxis have been shown to be decreased with neonatal cells (11) and may be related to chemiluminescence defects. The chemiluminescence studies we have performed would support the contention that neonatal PMNs generally differ functionally from adult PMNs, although the degree of difference may vary with individuals. These studies demonstrate that PMN chemiluminescence may be depressed in the very young and the very old. Since PMN chemiluminescence may be intimately involved in bactericidal activity, such variations in this response may be extremely important in host defense against bacterial organisms. ACKNOWLEDGMENTS We thank Mary Lynn Garcia for her excellent technical assistance and the Bernalillo County Medical Center Obstetrics staff for their aid in obtaining cord blood samples essential for this project. This work was supported by Public Health Service grants CA-20819 from the National Cancer Institute and AI-1343302 from the National Institute of Allergy and Infectious Dis-
VARIATIONS IN CHEMILUMINESCENCE
2.
3. 4.
5. 6.
7.
8. 9. 10. 11.
12. 13.
eases.
LITERATURE CITED 1. Allen, R. C., S. J. Yevich, R. A. Orth, and R. H. Steele. 1974. The superoxide anion and singlet molecular oxy-
14.
61
gen: their role in the microbial activity of the polymorphonuclear leukocyte. Biochem. Biophys. Res. Commun. 60:909-917. Coen, R., 0. Grush, and E. Kauder. 1969. Studies of bactericidal activity and metabolism of leukocytes in full-term neonates. J. Pediatr. 75:400-406. Donnell, G. N., W. G. Ng, J. E. Hodgman, and W. R. Bergren. 1967. Galactose metabolism in the newborn infant. Pediatrics 39:829-837. Dooset, J. H., R. C. Williams, Jr., and P. G. Quie. 1969. Studies on interaction of bacteria, serum factors and polymorphonuclear leukocytes in mothers and newborns. Pediatrics 44:49-57. Dunnett, C. W. 1955. A multiple comparison procedure for comparing several treatments with a control. Am. Stat. Assoc. J. 50:1096-1121. Hill, H. RK, N. A. Hogan, J. F. Bale, and V. G. Hemming. 1977. Evaluation of non-specific (alternative pathway) opsonic activity by neutrophil chemiluminescence. Int. Arch. Allergy Appl. Immunol. 53:490-497. McCraken, G. H., and H. F. Eichenwald. 1971. Leukocyte function and the development of opsonic and complement activity in the neonate. Am. J. Dis. Child. 121:120-126. Matoth, Y. 1952. Phagocytic and ameboid activities of the leukocytes in the newborn infant. Pediatrics 9:748-754. Miller, M. E. 1969. Phagocytosis in the newborn infant-humoral and cellular factors. J. Pediatr. 74:255-259. Miller, M. E. 1971. Chemotactic function in the human neonate-humoral and cellular aspects. Pediatr. Res. 5:487-492. Miller, M. E. 1975. Developmental maturation of human neutrophil motility and its relationship to membrane deformability, p. 295-307. In J. A. Bellanti and D. H. Dayton (ed.), The phagocytic cell in host resistance. Raven Press, New York. Palmer, D. L, and W. P. Reed. 1974. Delayed hypersensitivity skin testing. II. Clinical correlates and anergy. J. Infect. Dis. 130:138-143. Park, B. H., B. Holmes, and R. A. Good. 1970. Metabolic activities in leukocytes of newborn infants. J. Pediatr. 76:237-241. Van Epps, D. E., and R. C. Williams, Jr. 1976. Suppression of leukocyte chemotaxis by human IgA myeloma components. J. Exp. Med. 144:1227-1242.
INFECTION AND IMMUNITY, Oct. 1978, p. 57-61 0019-9567/78/0022-0057$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Age-Dependent Variations in Polymorphonuclear Leukocyte Chemiluminescence DENNIS E. VAN EPPS,* JAMES S. GOODWIN, AND SHIRLEY MURPHY Departments of Medicine, Microbiology, and Pediatrics, University of New Mexico School of Medicine, Bernalillo County Medical Center, Albuquerque, New Mexico 87131 Received for publication 1 August 1978
Polymorphonuclear leukocytes from 46 adults (age 18 to 35), 19 adults (age 70 to 91), 10 children (age 1 to 3), and 22 neonates (cord blood samples) were tested for their chemiluminescence response to opsonized zymosan. Results indicated that both cord blood leukocytes and those from individuals over 70 were significantly lower (P < 0.05) in their chemiluminescence response. Furthermore, when the latter group was divided into two subsets, one containing subjects over 80 years of age and the other containing subjects between 70 and 80 years of age, those over 80 showed a chemiluminescence response significantly lower (P < 0.05) than those between 70 and 80. The kinetics of the chemiluminescence response was similar with all samples except the neonatal cells, where the response appeared to peak and subside more slowly. These data demonstrate that polymorphonuclear leukocyte chemiluminescence is depressed in the very young and the very old. chemiluminescence was observed with neonatal PMNs as compared to those of normal adults. This decrease was not observed in children 1 to 3 years of age, but many adults over 70 did show decreased chemiluminescence activity. Chemiluminescence by neonatal PMNs also showed kinetic differences, when compared with adult PMNs, which were not seen in those people over 70 with decreased chemiluminescence.
The functional capabilities of human neonatal polymorphonuclear leukocytes (PMNs) have been frequently studied with conflicting results. Some studies indicate that PMN phagocytosis may be depressed in neonates (8, 9), while other studies show no difference between phagocytosis by neonatal PMNs and adult PMNs (4). Other workers have shown that hexose monophosphate shunt activity may be depressed in neonatal cells undergoing phagocytosis (2, 3), while various other reports indicate increased resting levels of Nitro Blue Tetrazolium dye reduction with a slightly elevated stimulated Nitro Blue Tetrazolium test (13) or decreased (J. A. Bellanti, B. E. Cantz, D. A. Maybee, and R. J. Schlegel, Pediatr. Res. 3:376, no. 114, 1969) or normal Nitro Blue Tetrazolium dye reduction (7). In still other studies, Miller has also shown that neonatal neutrophil chemotaxis is depressed as well as PMN membrane deformabil-
MATERIALS AND METHODS
ity (10, 11).
In this study, neonatal (cord blood) PMN function was evaluated by the chemiluminescence technique (6) and compared among children 1 to 3 years of age, adults 18 to 35 years of age, and adults greater than 70 years of age. This technique measures the liberation of light from PMNs stimulated with opsonized zymosan and is believed to be dependent upon a variety of PMN properties including surface contact with zymosan, hexose monophosphate shunt activity, the myeloperoxidase system, and the generation of singlet oxygen (1). A marked depression in 57
Cell sources. All subjects were selected randomly from a healthy population. Neonatal PMNs were prepared from heparinized cord blood samples within 3 h of delivery. Mothers were not receiving general anesthesia for delivery. Fifty percent of mothers received a short-acting narcotic (alpha prodine hydrochloride), 90% received a local anesthetic (xylocaine), and 10% received nothing. Samples from children aged 1 to 3 were obtained from normal children free from infection and medication at the time of study. Blood samples from 18- to 35-year-old adults were obtained from nonhospitalized volunteers, and adults over 70 years of age (70 to 91 years) were obtained from three sources: seven subjects had been admitted to the General Medical Ward of the County Hospital for nursing home placement; seven subjects were inmates of a local nursing home; and five subjects were members of a local senior citizens group who volunteered to donate blood. All subjects were ambulatory and none had a diagnosed infection or systemic disease or was on medications other than occasional analgesics, laxatives, or sedatives. Cell preparations. PMNs were prepared from heparinized peripheral blood by Plasmagel (HTI, Buf-
58
INFECT. IMMUN.
VAN EPPS, GOODWIN, AND MURPHY
falo, N.Y.) sedimentation of erythrocytes (RBCs) followed by Ficoll-Hypaque centrifugation to remove mononuclear cells (14). Final cell preparations contained greater than 90% PMNs, which were adjusted to 107 PMNs per ml in phosphate-buffered saline (pH 7.2). In some instances, contaminating RBCs were lysed using a 40C, 30-s distilled water lysis. Cells were then washed and resuspended in phosphate-buffered saline. Generally, RBC lysis was not performed, since a high percentage of fetal cord blood leukocytes (WBCs) were sensitive to this treatment. Therefore, comparisons between groups of subjects were made with samples which had not undergone the lysis treatment, to make comparisons valid. In cord blood preparations, slightly more eosinophils, bands, and mononuclear cells were noted (mean, 3% bands, 6% eosinophils, and 7% mononuclear cells) than in adult preparations (mean, 1.5% bands, 3% eosinophils, and 1% mononuclear cells). Other cell preparations were similar to normal adult cell preparations in morphology. Chemiluminescence assay. Chemiluminescence was performed as previously described (6). Zymosan particles which had been opsonized with fresh normal serum were used as a stimulant (6). Light emission was measured in a nonrefrigerated Nuclear Chicago /?-scintillation counter used out of coincidence with a window setting of 0, 10. A mixture of 0.5 ml of PMNs (5 X 106) and 0.2 ml of opsonized zymosan (10 mg/ml) was suspended in phosphate-buffered saline to a final volume of 1 ml in an 18-h dark-adapted Beckman Poly Q vial. Tubes were counted for 1 min at 5- to 7-min time intervals.
RESULTS Chemiluminescence profiles on PMNs from normal adults and fetal cord blood. Cumulative results of PMN chemiluminescence profiles on three adults and three neonates showing depressed chemiluminescence are shown in Fig. 1. A profound depression in the peak chemiluminescence response was observed with these neonatal PMNs as compared with adult PMNs. In addition, the kinetics of chemiluminescence differed between adult and these neonatal PMNs, with neonatal PMNs showing a peak response between 11 and 15 min and adults showing a peak response between 6 and 9 min. Furthermore, the response by neonatal PMNs subsided more slowly than the adult chemiluminescence response. Comparison of peak PMN chemiluminescence responses in neonates, children (ages 1 to 3), adults (ages 18 to 35), and adults over 70 years of age. Figure 2 shows a summary of the peak chemiluminescence response in 46 adults (18 to 35 years of age), 10 children (1 to 3 years of age), 22 neonates, and 19 adults over 70 years of age (range 70 to 91). The kinetics of the chemiluminescence response of both the 1- to 3-year-old children and the greater-than70-year-old adults were similar to those obtained with the 18- to 35-year-old adults. Only neonatal
35r 3*
°Q2 x
CL 0
20
Is5 A.-
-
-
-4"-
-
-
-
5
12 4 6 8 10 12 14 16 18 20 22426 28 30
Minutes FIG. 1. PMN chemiluminescence profiles on three normal adults ( ) and three fetal cord blood samples (-----) in response to opsonized zymosan. The data shown are a composite of three normal adults and three fetal cord blood samples. 44 42 38 36 tE Q 34
32 K 30 28 26 24 22
I
.
9
to 01
I'
0:-
I
*-
.
IsX 0:
I
14K1
*
00
.
' 12 K) 8
t.
61 8-3ym I -3ysws
>7Oyeas Fea COrd Blood
FIG. 2. Comparison of peak PMN chemiluminescence responses offetal cord blood cells and subjects aged I to 3, 18 to 35, and over 70. Groups were statistically compared using the Dunnett test (5).
PMNs with a suppressed peak chemiluminescence response showed a difference in chemiluminescence kinetics (Fig. 1). No association between the mother's anesthetic and neonatal cell
VARIATIONS IN CHEMILUMINESCENCE
VOL. 22, 1978
chemiluminescence was observed. The mean peak chemiluminescence response for 18- to 35year-old adults was 30,936 + 6,845; for 1- to 3year-old children it was 26,053 ± 4,965; for adults over 70 years it was 24,727 ± 9,571; and for neonates it was 16,033 ± 8,138. In tests with neonatal cells, 15 of 22 gave a chemiluminescence response of less than 20,000 cpm. It should be noted that cell preparations from cord blood samples contained slightly more band forms, eosinophils, and mononuclear cells (mean, 3% bands, 6% eosinophils, and 7% mononuclear cells) than adult controls (mean, 1.5% bands, 3% eosinophils, and 1% mononuclear cells). This difference was minor and could not account for the marked reduction in chemiluminescence. This was confirmed by titration experiments on normal cells, which indicated that a 20% reduction in the number of PMNs resulted in only a 14% reduction in chemiluminescence. Thus, the number of bands, eosinophils, and mononuclear cells found in cord blood samples was not a probable explanation for the decreased chemiluminescence observed with neonatal cells. Al though no significant difference was observed between 18- to 35-year-old adult PMNs and those of 1- to 3-year-old children (Fig. 2), PMNs both of adults over 70 years of age (P < 0.05) and of neonates (P < 0.01) showed an overall significant depression in their chemilumines-
59
tions. When RBC-to-WBC ratios
were determined on five adult PMN preparations and six neonatal PMN preparations (three with depressed chemiluminescence and three with normal chemiluminescence), it was apparent (Table 1) that RBC contamination could not totally account for depressed neonatal PMN chemiluminescence. Evidence for this is shown with cord blood samples 1 and 4, with reduced chemiluminescence and RBC-to-WBC ratios of 4 and 5, as compared to cord blood samples 2 and 3 and adult samples 1 and 5, with normal chemiluminescence values and comparable RBC-to-WBC ratios. Attempts were made to lyse RBCs and compare adult and neonatal PMNs (Table 1). As shown, lysis treatment resulted in a substantial loss of cord blood PMNs with no apparent effect on adult PMNs. In each case, chemiluminescence was measured before and after RBC lysis. Distilled-water lysis of RBCs was an efficient way to remove contaminating RBCs from adult PMN preparations, as shown. However, when cord blood PMN preparations were lysed with water, a significant number of PMNs were lost, and a substantial number of RBCs remained (Table 1). The overall effect of RBC lysis was an increased chemiluminescence response with both neonatal and adult cells, although the data are difficult to interpret because a mean loss of cence responses. 42% of PMNs was observed with neonatal cells Another possible reason for depressed chemi- after lysis. These data indicate that at least a luminescence with neonatal PMNs may be the portion of neonatal PMNs differ from normal suppressive effects of contaminating RBCs in adult PMNs in their susceptibility to distilledPMN preparations. Ficoll-Hypaque purification water lysis. of PMNs resulted in a substantial number of Although there was a significant difference RBCs in cord blood and adult PMN prepara- between the overall response of adults over 70
TABLE 1. Comparison of neonatal and adult PMN chemiluminescence before and after lysis of RBCs Before lysis After lysis
SampleRB/RC RBC/ RBC/ Chemiluminescence Chemiluminescence
%osf
%WIBCof
Fetal cord blood 1 2 3 4 5 6 Mean
12,354 26,083 31,073 12,372 24,487 10,474 19,474 ± 8,780
4 4 6 5 3 8 5
27,397 28,635 29,831 31,362 36,211
4 2 2 6 2
64 54 31 44 16
30,687 ± 3,418
3
42
32,566 35,995 27,124 23,859 27,357 29,380
5 2 2 3 4 3
34,597 37,901 48,402 39,456 40,340 40,139 ± 5,111
3 0.5 0 0 0 1
2 0 0 0 0 1
Adult blood 1 2 3 4 5 Mean
4,837
60
VAN EPPS, GOODWIN, AND MURPHY
INFECT. IMMUN.
and that of the 18- to 35-year-old adult population (Fig. 2), the majority of these individuals showed normal levels of chemiluminescence, with 12 of 19 giving a response of greater than 20,000 cpm. It is of interest that the mean age of those individuals over 70 showing less than 20,000 cpm chemiluminescence was 85 ± 5 years, whereas those showing greater than 20,000 cpm had a mean age of 77 ± 5. This difference in mean age was significant at the P < 0.02 level. When these individuals were divided between those over 80 (10 of 19) and those between 70 and 80 (9 of 19), the mean peak chemiluminescence was significantly lower (P < 0.05) for those individuals over 80 (20,443 ± 8,371) than for those between 70 and 80 (30,015 ± 8,252). Only one of the over-70-year-old subjects was receiving sedatives (phenothiazine), and this 82-yearold patient showed a chemiluminescence response of 30,240 cpm. The remaining subjects were healthy individuals. The situation was quite different with neonate PMNs, where the majority were consistently decreased in chemiluminescence (15 of 22 individuals below 20,000
increased number of RBCs contaminating neonatal PMN preparations. Experiments designed to remove RBCs by lysis indicated that both neonate and adult PMN chemiluminescence could be enhanced by RBC lysis, although RBC removal from neonatal cell preparations could never be totally achieved. As a consequence of RBC lysis, a large portion of neonatal PMNs was also lost. This loss was not observed with adult PMNs and indicates that neonatal PMNs may differ from adult PMNs in fragility. This may be an important consideration in all studies dealing with neonatal PMNs where RBC lysis is a standard procedure. Our data indicate that, after lysis, neonatal PMN chemiluminescence approaches normal adult levels, although these values may be difficult to interpret since a mean loss of 42% of PMNs was observed after lysis. The remaining cells resemble adult PMNs in their resistance to lysis and their chemiluminescence activity, but are probably not a true representation of the total neonatal PMN population. The PMNs that were susceptible to lysis may represent a subpopulation of cells, possibly less mature, less active in chemiluminescence, or more rigid. The latter has been demonstrated previously by others (11). Neonatal PMN lysis makes it difficult to determine if RBC contamination is the cause of the decreased chemiluminescence response in neonatal PMN, although comparison of RBC-to-WBC ratios in adult and neonatal PMN preparations shows that neonatal PMN preparations with comparable RBC contamination may still show a greatly reduced chemiluminescence response and implies that these cells differ from adult PMNs. It is apparent that not all neonatal PMN preparations show a reduced chemiluminescence response (Table 1, Fig. 2). The reason for decreased neonatal PMN chemiluminescence in some samples and not others is not understood at this time, but may be related to several other previously defined defects in PMN function. These previously described defects include depressed phagocytosis (8, 9) and hexose monophosphate shunt activity (Bellanti et al., Pediatr. Res. 3:376, no. 114, 1969), both of which may be involved in chemiluminescence, although the former is not essential for chemiluminescence (R. D. Nelson, M. Herron, R. L. Simmons, and P. G. Quie, Fed. Proc. 36:4214A, 1977). This depression in phagocytic and hexose monophosphate shunt activity has been controversial, since some investigators have shown no difference between neonatal and adult cells with respect to these functions (4, 7). The discrepancies in these results may be due to one of several things: to the method of cell preparation, to differences in analysis of Nitro
cpm). DISCUSSION This study demonstrates the tremendous variation in PMN chemiluminescence responses from individual to individual. Although the chemiluminescence response by different individuals covers a broad range, a significantly decreased response was observed with the majority of fetal cord blood cells (15 of 22) and some subjects over age 70 (7 of 19). In the older subjects, subdividing this age group into two categories revealed that individuals between 70 and 80 years of age showed a much higher mean chemiluminescence response than subjects over 80 years of age. This implies that the PMN chemiluminescence response may deteriorate beyond a certain age and may possibly be associated with a general depression of host defense mechanisms previously observed with age (12). Although chemiluminescence responses in children aged 1 to 3 years were comparable to adult levels, a decreased responsiveness in neonatal PMNs was observed. This was apparent in the majority of fetal cord blood PMNs tested. In addition to a decreased peak chemiluminescence response in most cases, these same neonatal PMNs differed from adult PMNs in the kinetics of the chemiluminescence response. Neonatal PMN chemiluminescence peaked more slowly and tapered off more slowly than adult PMN chemiluminescence. One possible explanation for the differences in neonatal and adult PMN chemiluminescence could be the
VOL. 22, 1978
Blue Tetrazolium dye reduction by either optical density (13; Bellanti et al., Pediatr. Res. 3:376, no. 114, 1969) or microscopic observation for the presence of intracellular precipitate (7), or to differences in the expression of data either as absolute values (13) or as a function of the resting levels of Nitro Blue Tetrazolium dye reduction (Bellanti et al., Pediatr. Res. 3:376, no. 114, 1969). In some cases the latter may affect results, since resting dye reduction levels for neonatal cells may be elevated. Thus, this particular defect is still controversial. In addition, both membrane deformability and chemotaxis have been shown to be decreased with neonatal cells (11) and may be related to chemiluminescence defects. The chemiluminescence studies we have performed would support the contention that neonatal PMNs generally differ functionally from adult PMNs, although the degree of difference may vary with individuals. These studies demonstrate that PMN chemiluminescence may be depressed in the very young and the very old. Since PMN chemiluminescence may be intimately involved in bactericidal activity, such variations in this response may be extremely important in host defense against bacterial organisms. ACKNOWLEDGMENTS We thank Mary Lynn Garcia for her excellent technical assistance and the Bernalillo County Medical Center Obstetrics staff for their aid in obtaining cord blood samples essential for this project. This work was supported by Public Health Service grants CA-20819 from the National Cancer Institute and AI-1343302 from the National Institute of Allergy and Infectious Dis-
VARIATIONS IN CHEMILUMINESCENCE
2.
3. 4.
5. 6.
7.
8. 9. 10. 11.
12. 13.
eases.
LITERATURE CITED 1. Allen, R. C., S. J. Yevich, R. A. Orth, and R. H. Steele. 1974. The superoxide anion and singlet molecular oxy-
14.
61
gen: their role in the microbial activity of the polymorphonuclear leukocyte. Biochem. Biophys. Res. Commun. 60:909-917. Coen, R., 0. Grush, and E. Kauder. 1969. Studies of bactericidal activity and metabolism of leukocytes in full-term neonates. J. Pediatr. 75:400-406. Donnell, G. N., W. G. Ng, J. E. Hodgman, and W. R. Bergren. 1967. Galactose metabolism in the newborn infant. Pediatrics 39:829-837. Dooset, J. H., R. C. Williams, Jr., and P. G. Quie. 1969. Studies on interaction of bacteria, serum factors and polymorphonuclear leukocytes in mothers and newborns. Pediatrics 44:49-57. Dunnett, C. W. 1955. A multiple comparison procedure for comparing several treatments with a control. Am. Stat. Assoc. J. 50:1096-1121. Hill, H. RK, N. A. Hogan, J. F. Bale, and V. G. Hemming. 1977. Evaluation of non-specific (alternative pathway) opsonic activity by neutrophil chemiluminescence. Int. Arch. Allergy Appl. Immunol. 53:490-497. McCraken, G. H., and H. F. Eichenwald. 1971. Leukocyte function and the development of opsonic and complement activity in the neonate. Am. J. Dis. Child. 121:120-126. Matoth, Y. 1952. Phagocytic and ameboid activities of the leukocytes in the newborn infant. Pediatrics 9:748-754. Miller, M. E. 1969. Phagocytosis in the newborn infant-humoral and cellular factors. J. Pediatr. 74:255-259. Miller, M. E. 1971. Chemotactic function in the human neonate-humoral and cellular aspects. Pediatr. Res. 5:487-492. Miller, M. E. 1975. Developmental maturation of human neutrophil motility and its relationship to membrane deformability, p. 295-307. In J. A. Bellanti and D. H. Dayton (ed.), The phagocytic cell in host resistance. Raven Press, New York. Palmer, D. L, and W. P. Reed. 1974. Delayed hypersensitivity skin testing. II. Clinical correlates and anergy. J. Infect. Dis. 130:138-143. Park, B. H., B. Holmes, and R. A. Good. 1970. Metabolic activities in leukocytes of newborn infants. J. Pediatr. 76:237-241. Van Epps, D. E., and R. C. Williams, Jr. 1976. Suppression of leukocyte chemotaxis by human IgA myeloma components. J. Exp. Med. 144:1227-1242.
