Aust. Pnediatr.
J.
1977). 1 3 : 287-289
Neutrophil Phagocytic and Bactericidal Dysfunction Induced by Bilirubin Y. H. THONG,' A. FERRANTE3 and D. NESS3 Department of Paediatrics, University of Adelaide, T h e Adelaide Children's Hospital, North Adelaide, South Australia Thong, Y. H., Ferrante, A., and Ness, D. (1977). Aust. Paediatr. J., 13, 287-289. Nentrophil phagocytic and bactericidal dysfunction induced hy bilirubin. The effect of unconjugated bilirubin on neutrophil polymorphs was studied in-vitro by standard phagocytic and bactericidal assays. At concentrations attainable clinically, bilirubin was shown to inhibit both phagocytosis and killing of Staphylococcus aureus. These results suggest that jaundiced newborns may have an increased susceptibility to infection.
INTRODUCTION
We have previously shown that bilirubin has suppressive effects on the hexose-monophosphate Neonatal jaundice is a common problem in paediatrics. At high levels of serum bilirubin, IHMP) shunt of human polymorphonuclear kernicterus is a well-known complication. Less leucocytes (Thong and Rencis, 1977). In the known is the fact that unconjugated bilirubin present studies, we have investigated the is toxic to tissues other than the brain. In 1847, in-vitro effects of bilirubin on neutrophil Virchow noted visceral pigmentation in infants phagocytosis and bactericidal capacity. with kernicterus. Necrotic lesions in many organs were also seen in jaundiced infants MATERIALS AND METHODS without kernicterus (Bernstein and Landing, Blood was obtained from healthy donors and 1962). Of particular interest were the lesions in neutrophils isolated by centrifugation on n spleen and bone marrow (Bernstein and hyuaque-ficoll gradient (Boyum, 1968). The Landing, 1962), and peripheral leucocytes cells were washed twice and resuspended in (Vietti, 1959). That the immune system may medium 199 for the experiments. Preliminary be involved in neonatal hyperbilirubinaemia is studies were performed to establish that substantiated by findings of lower levels of unconjugated bilirubin is not toxic (as assessed immunoglobulins (Ansaldi et al., 1968) , and by trypan-blue dye exclusion) to neutrophils specific antibodies (Nejedla, 1970; Diaz del at the concentrations used in these experiments, Castillo, 1973) in infants experiencing jaundice The bactericidal assay was performed by a in the newborn period. Lymphocyte responsivestandard technique (Holmes et al., 1966). ness to phytohaemogglutinin (DeSanctis, et al., Briefly, 5 x 10' neutrophils were mixed with 1968; Rola-Pleszczynski et al., 1975) and 5 x 10' S. aureus phage-type 96 in 10 ml mixed lymphocyte culture (Rola-Pleszczynski screw-capped plastic tubes, each tube containet al., 1975) was also found to be impaired. ing 2.57, pooled human serum (albumin concentration 75 mg/dl or approximately Senior Lecturer. 1.3 x 105M). Experiments were performed in ' Scientific Officer. Laboratory Technician. 0.5 ml volumes. Bilirubin-treated tubes Received June 14, 1977. contained the following concentrations of
288
unconjugated bilirubin solubilized in 10 4hl sodium carbonate: 1 >: 10 'M, 2 x 10--'M. and 4 x 10-4M (6 mg, 12 mg, and 24- mg dl respectively). Control tubes contained the solvent only. At the end of two hours incubation with the tubes rotated end over end, a t 37"C, 0.1 ml aliquots were removed from each tube and diluted in sterile distilled water for disrupting the neutrophils, and serial dilutions plated on nutrient agar for the quantitation of viable colonies.
I00
00
PERCENT 60
40
KILLED IN
a
No. bacteria at zero time - No. killed by lysostaphin + Number of bacteria at zero time x 100
RESULTS The results of the bactericidal experiments are presented in Figure 1. There was significant impairment of bacterial killing in tubes containing 2 x 10-4M and 1. x 10 4M bilirubin, but not 1 x 10-'M bilirubin, as compared to control i P < 0.001, P < 0.001 and P > 0.02 respectively). At bilirubin concentrations of 4 x 10-4M, percentage of bacteria phagocytosed was 62.2 rt 7.1 (mean % SEM of 5 experiments) compared to 96.9 i- 1.2 in control. This difference was statistically significant ( t = 4.48, P < 0.001).
DISCUSSION
S. AUREUS
2 HOURS
nutrient agar for the quantitation of viable colonies. The per cent of bacteria phagocytosed M. as calculate,d as follows :
L
0 MO-' 4;10-~ M BILIRUBIN CONCl NTR ATION
FIGURE1 : Effect of unconjugated bilirubin on neutrophil bactericidal activity. Results represent mean 2 SEM of 5 experiments.
Phagocytosis experiments were performed by the method of Tan et al. (1971). The procedure was similar to the bactericidal assay described above, except that at the end of 2 hours incubation, lysostaphin was introduced to the mixture at a final concentration of 5 pg/ml for the killing of extracellular S. aureus. After 10 minutes incubation a t 37"C, 2.5% trypsin was added to terminate the reaction. Serial dilutions in distilled water were plated on
Neutrophils play an important role in host resistance to infection. Fundamental to their role in defence is the ability to ingest and kill microorganisms (Bellanti and Dayton, 1975). The results of the present studies indicate that neutrophil bactericidal capacity is impaired in the presence of bilirubin, and that concomitant inhibition of phagocytosis is partially responsible for this finding. The mechanisms by which bilirubin inhibits nautrophil function have not been identified. They may be related to the known ability of bilirubin to suppress a number of cellular enzyme activities. Bilirubin has been shown to uncouple oxidative phosphorylation (Zetterstrom and Ernster, 1956), inhibit electron transfer iCowger et aZ., 1965), and induce mitochondria1 swelling (Noir et al., 1972). In particular, bilirubin has been shown to inhibit HMP shunt activity of phagocytosing neutrophils (Thong and Rencis, 1977). Increased production of hydrogen peroxide and superoxide constitutes an important microbicidal mechanism of these cells (Bellanti and Dayton, 1975). In patients with chronic granulomatous disease, both HMP shunt activity and bactericidal capacity are defective iHolmes et al., 1966).
I n the clinical situation, binding of bilirubin
by albumin reduces its bioavailability and toxicity. But even at serum bilirubin concentrations within the binding capacity of serum albumin, toxic damage to cells has been observed after longer periods of exposure (Thaler, 1971). presumably as a result of transfer t o the cells of small amounts of unbound, unconjugated bilirubin (Odell et al., 1969; Schiff et al., 1972). Though our studies suggest that jaundiced newborns may be more susceptible to infection, whether or not they are actually at risk is still to be determined. Newborns with moderately severe hyperbilirubinaemia, undergoing treatment with phototherapy (Lucey, 1972), may be a suitable group for study, due to their prolonged exposure to fairly high levels of bilirubin. Of some pertinence to this question is the clinical observation that patients with congenital atresia of the bile ducts, in whom a substantial fraction of their sera bilirubin may be unconjugated, have an increased susceptibility to intercurrent infection (Sherlock, 1958). Also, jaundice is the second commonest clinical finding in neonate5 with sepsis (Gluck et al., 1966) ; although hyperbilirubinaemia is generally regarded as a consequence rather than a predisposing cause of sepsis, the reverse may be operative in some cases.
REFERENCES ANSALDI,N., FIANDINO, G., and CIRIOOTI,G. (1968), Changes in the immunoglohulin level in hyperbilirubinaemic premature infants, Minerva Pediatr.,
20: 1982.1984.
BELLANTI,J. A., and DAYTON,D. H. (1975). Th e Dhagocytic cell in host resistance. New York, Raven Press. BERNSTEIN,J., and LANDING,B. H. (1962), Extraneural lesions associated with hyperhiliruhinaemia and kernicterus, Am. J. Pathol., 40: 371-384. BOYUM, A. (1968). Isolation of mononuclear rells and granulocytes from human blood, Scand. J. Clin. Lab. Invest. (suppl. 9 7 ) , 21: 77-89. COWGER,M. L., Ico, R. P., and LABEE,R. F. (1965). T he mechanisms of bilirubin toxicity studied with purified respiratory enzyme and tissue culture systems, Biochemistry, 4: 2763-2770.
DESANCTIS.C., MALANDRA, C., ZANETTI,P., FABRIS, C., and PONZONE,A. (1968), Neonatal hyperbilirubinaemia and response of lymphocytes to phytohaemagglutinin, Minerva Pediatr., 20: 2011-
2013.
Llr~iz
uEt.
CcmLLo, E. (19731,
rubinarinia.
Its
ticarniia of the 13: 1131-1148.
relation
IlCWkJOL.Ii,
X u I m t a l h!prrlJilito thc ininiunological nt.wl)clrn infant. Guc.
Pediatr. (.‘/in..Vorth Anr.,
HOLMES,B., QOIE, 1’. C,., W I K I ) I I ( I R ~I). T . 13.. COol), K. A. ( 1 9 6 6 1 , Fatal granuhniatous di
anti
of childhoud: Inlmrn abnormalit! of pliagov) tic function, Luncet, 1 : 1225.1227. LUCKY,J . F. (1972) , Nronalal jaundice and pliot(ntherapy, Pediutr. Clin. North A m . , 19: 827-839. NEJEDLA,Z. 11970) , TI]? d
J.
1977). 1 3 : 287-289
Neutrophil Phagocytic and Bactericidal Dysfunction Induced by Bilirubin Y. H. THONG,' A. FERRANTE3 and D. NESS3 Department of Paediatrics, University of Adelaide, T h e Adelaide Children's Hospital, North Adelaide, South Australia Thong, Y. H., Ferrante, A., and Ness, D. (1977). Aust. Paediatr. J., 13, 287-289. Nentrophil phagocytic and bactericidal dysfunction induced hy bilirubin. The effect of unconjugated bilirubin on neutrophil polymorphs was studied in-vitro by standard phagocytic and bactericidal assays. At concentrations attainable clinically, bilirubin was shown to inhibit both phagocytosis and killing of Staphylococcus aureus. These results suggest that jaundiced newborns may have an increased susceptibility to infection.
INTRODUCTION
We have previously shown that bilirubin has suppressive effects on the hexose-monophosphate Neonatal jaundice is a common problem in paediatrics. At high levels of serum bilirubin, IHMP) shunt of human polymorphonuclear kernicterus is a well-known complication. Less leucocytes (Thong and Rencis, 1977). In the known is the fact that unconjugated bilirubin present studies, we have investigated the is toxic to tissues other than the brain. In 1847, in-vitro effects of bilirubin on neutrophil Virchow noted visceral pigmentation in infants phagocytosis and bactericidal capacity. with kernicterus. Necrotic lesions in many organs were also seen in jaundiced infants MATERIALS AND METHODS without kernicterus (Bernstein and Landing, Blood was obtained from healthy donors and 1962). Of particular interest were the lesions in neutrophils isolated by centrifugation on n spleen and bone marrow (Bernstein and hyuaque-ficoll gradient (Boyum, 1968). The Landing, 1962), and peripheral leucocytes cells were washed twice and resuspended in (Vietti, 1959). That the immune system may medium 199 for the experiments. Preliminary be involved in neonatal hyperbilirubinaemia is studies were performed to establish that substantiated by findings of lower levels of unconjugated bilirubin is not toxic (as assessed immunoglobulins (Ansaldi et al., 1968) , and by trypan-blue dye exclusion) to neutrophils specific antibodies (Nejedla, 1970; Diaz del at the concentrations used in these experiments, Castillo, 1973) in infants experiencing jaundice The bactericidal assay was performed by a in the newborn period. Lymphocyte responsivestandard technique (Holmes et al., 1966). ness to phytohaemogglutinin (DeSanctis, et al., Briefly, 5 x 10' neutrophils were mixed with 1968; Rola-Pleszczynski et al., 1975) and 5 x 10' S. aureus phage-type 96 in 10 ml mixed lymphocyte culture (Rola-Pleszczynski screw-capped plastic tubes, each tube containet al., 1975) was also found to be impaired. ing 2.57, pooled human serum (albumin concentration 75 mg/dl or approximately Senior Lecturer. 1.3 x 105M). Experiments were performed in ' Scientific Officer. Laboratory Technician. 0.5 ml volumes. Bilirubin-treated tubes Received June 14, 1977. contained the following concentrations of
288
unconjugated bilirubin solubilized in 10 4hl sodium carbonate: 1 >: 10 'M, 2 x 10--'M. and 4 x 10-4M (6 mg, 12 mg, and 24- mg dl respectively). Control tubes contained the solvent only. At the end of two hours incubation with the tubes rotated end over end, a t 37"C, 0.1 ml aliquots were removed from each tube and diluted in sterile distilled water for disrupting the neutrophils, and serial dilutions plated on nutrient agar for the quantitation of viable colonies.
I00
00
PERCENT 60
40
KILLED IN
a
No. bacteria at zero time - No. killed by lysostaphin + Number of bacteria at zero time x 100
RESULTS The results of the bactericidal experiments are presented in Figure 1. There was significant impairment of bacterial killing in tubes containing 2 x 10-4M and 1. x 10 4M bilirubin, but not 1 x 10-'M bilirubin, as compared to control i P < 0.001, P < 0.001 and P > 0.02 respectively). At bilirubin concentrations of 4 x 10-4M, percentage of bacteria phagocytosed was 62.2 rt 7.1 (mean % SEM of 5 experiments) compared to 96.9 i- 1.2 in control. This difference was statistically significant ( t = 4.48, P < 0.001).
DISCUSSION
S. AUREUS
2 HOURS
nutrient agar for the quantitation of viable colonies. The per cent of bacteria phagocytosed M. as calculate,d as follows :
L
0 MO-' 4;10-~ M BILIRUBIN CONCl NTR ATION
FIGURE1 : Effect of unconjugated bilirubin on neutrophil bactericidal activity. Results represent mean 2 SEM of 5 experiments.
Phagocytosis experiments were performed by the method of Tan et al. (1971). The procedure was similar to the bactericidal assay described above, except that at the end of 2 hours incubation, lysostaphin was introduced to the mixture at a final concentration of 5 pg/ml for the killing of extracellular S. aureus. After 10 minutes incubation a t 37"C, 2.5% trypsin was added to terminate the reaction. Serial dilutions in distilled water were plated on
Neutrophils play an important role in host resistance to infection. Fundamental to their role in defence is the ability to ingest and kill microorganisms (Bellanti and Dayton, 1975). The results of the present studies indicate that neutrophil bactericidal capacity is impaired in the presence of bilirubin, and that concomitant inhibition of phagocytosis is partially responsible for this finding. The mechanisms by which bilirubin inhibits nautrophil function have not been identified. They may be related to the known ability of bilirubin to suppress a number of cellular enzyme activities. Bilirubin has been shown to uncouple oxidative phosphorylation (Zetterstrom and Ernster, 1956), inhibit electron transfer iCowger et aZ., 1965), and induce mitochondria1 swelling (Noir et al., 1972). In particular, bilirubin has been shown to inhibit HMP shunt activity of phagocytosing neutrophils (Thong and Rencis, 1977). Increased production of hydrogen peroxide and superoxide constitutes an important microbicidal mechanism of these cells (Bellanti and Dayton, 1975). In patients with chronic granulomatous disease, both HMP shunt activity and bactericidal capacity are defective iHolmes et al., 1966).
I n the clinical situation, binding of bilirubin
by albumin reduces its bioavailability and toxicity. But even at serum bilirubin concentrations within the binding capacity of serum albumin, toxic damage to cells has been observed after longer periods of exposure (Thaler, 1971). presumably as a result of transfer t o the cells of small amounts of unbound, unconjugated bilirubin (Odell et al., 1969; Schiff et al., 1972). Though our studies suggest that jaundiced newborns may be more susceptible to infection, whether or not they are actually at risk is still to be determined. Newborns with moderately severe hyperbilirubinaemia, undergoing treatment with phototherapy (Lucey, 1972), may be a suitable group for study, due to their prolonged exposure to fairly high levels of bilirubin. Of some pertinence to this question is the clinical observation that patients with congenital atresia of the bile ducts, in whom a substantial fraction of their sera bilirubin may be unconjugated, have an increased susceptibility to intercurrent infection (Sherlock, 1958). Also, jaundice is the second commonest clinical finding in neonate5 with sepsis (Gluck et al., 1966) ; although hyperbilirubinaemia is generally regarded as a consequence rather than a predisposing cause of sepsis, the reverse may be operative in some cases.
REFERENCES ANSALDI,N., FIANDINO, G., and CIRIOOTI,G. (1968), Changes in the immunoglohulin level in hyperbilirubinaemic premature infants, Minerva Pediatr.,
20: 1982.1984.
BELLANTI,J. A., and DAYTON,D. H. (1975). Th e Dhagocytic cell in host resistance. New York, Raven Press. BERNSTEIN,J., and LANDING,B. H. (1962), Extraneural lesions associated with hyperhiliruhinaemia and kernicterus, Am. J. Pathol., 40: 371-384. BOYUM, A. (1968). Isolation of mononuclear rells and granulocytes from human blood, Scand. J. Clin. Lab. Invest. (suppl. 9 7 ) , 21: 77-89. COWGER,M. L., Ico, R. P., and LABEE,R. F. (1965). T he mechanisms of bilirubin toxicity studied with purified respiratory enzyme and tissue culture systems, Biochemistry, 4: 2763-2770.
DESANCTIS.C., MALANDRA, C., ZANETTI,P., FABRIS, C., and PONZONE,A. (1968), Neonatal hyperbilirubinaemia and response of lymphocytes to phytohaemagglutinin, Minerva Pediatr., 20: 2011-
2013.
Llr~iz
uEt.
CcmLLo, E. (19731,
rubinarinia.
Its
ticarniia of the 13: 1131-1148.
relation
IlCWkJOL.Ii,
X u I m t a l h!prrlJilito thc ininiunological nt.wl)clrn infant. Guc.
Pediatr. (.‘/in..Vorth Anr.,
HOLMES,B., QOIE, 1’. C,., W I K I ) I I ( I R ~I). T . 13.. COol), K. A. ( 1 9 6 6 1 , Fatal granuhniatous di
anti
of childhoud: Inlmrn abnormalit! of pliagov) tic function, Luncet, 1 : 1225.1227. LUCKY,J . F. (1972) , Nronalal jaundice and pliot(ntherapy, Pediutr. Clin. North A m . , 19: 827-839. NEJEDLA,Z. 11970) , TI]? d
