THE EGYPTIAN JOURNAL OF IMMUNOLOGY Vol. 19 (2), 2012 Page: 01-07 Interleukin 1β Level in Human Colostrum in Relation to Neonatal Hyperbilirubinemia 1 Nabil Gamil Mohamed, 1 Gehan Lotfy Abdel Hakeem, 1 Mahmoud Shabaan Ali, 2 Ashraf Osman Mohamed Departments of 1 Pediatric and 2 Clinical Pathology, Faculty of Medicine, El Menia University, El Menia, Egypt. Breast-fed infants have higher bilirubin level than formula-fed infants which is of undetermined etiology. The cholestatic effect of cytokines (e.g. IL-1β, IL-6) is believed to result from the repression of genes that normally mediated the hepatic uptake, metabolism, and biliary excretion of bile salts and bilirubin. The present study aimed to assess the level of interleukin 1β (IL1β) in early milk of nursing mothers and its relation to neonatal jaundice. Sixty full term neonates and their mothers were included. They were classified into two groups; group I included Forty neonates patients with physiological jaundice and group 11 included twenty age and sex matched controls neonates without jaundice. Milk samples were collected from mothers for estimation of serum IL-1β level by ELISA, and blood samples were collected from the neonates for measuring total serum bilirubin (TSB) level. A significant difference (P< 0.01) in the level (pg/dl) of IL-1β was found in early milk between group I (10.25 ± 4.23) and group II (3.75 ± 2.07). The total serum bilirubin level was higher in group I than group II (10.91 ± 3.25 mg/dl versus 3.88 ± 0.78 mg/dl) (P < 0.01). A significant positive correlation was found between of IL-1β in breast milk and total serum bilirubin levels (r = 0.494, P < 0.001). It is concluded that Elevated levels of IL-1β in the colostrum provide additional data to understand one of path physiologic mechanisms of breast milk jaundice. Breast-fed infants compared with formula fed infants have higher bilirubin levels of unexplained etiology (Zanardo et al., 2007). Theories to explain the involved mechanisms include insufficient milk transfer to the neonate, (Bertini et al., 2001), lack or no hepatic excretion of bilirubin, and increased intestinal absorption of bilirubin. In addition, suppression of hepatic excretion of bilirubin may explain the jaundice associated with breastfeeding. (Zanardo et al., 2007) The exposure to acquired cholestatic causes, such as drugs, hormones, proinflammatory cytokines or biliary obstruction results in altered expression and function of hepatic uptake and excretory system changes contributing to the occurrence of jaundice (Trauner et al., 2005). In particular, increased production of cytokines including IL-8 and IL-10 has been proved in patients with clinical jaundice (Kimura et al., 2001, Honsawek et al., 2005). It is believed that the cholestatic effect of cytokines (e.g. IL-1β, IL-6) may result from the alteration of genes mediating the hepatic uptake, metabolism, and biliary excretion of bilirubin and bile salts (Bolder et al., 1997; Moseley et al., 1997). Patients and Methods This study included sixty full term neonates and their mothers who had cesarean section or vaginal delivery at El Menia University Hospital for children and Matai Reproductive Health Hospital during the period from March to July 2010. The neonates and their mothers were classified according to presence of neonatal jaundice into cases and control groups. Group I (case) Included 40 neonates with jaundice and their Mothers. A) Neonates Forty full term neonates (37-41weeks) were included. Their gestational age, sex, weight were recorded. B) Mothers Their age and antenatal history were recorded.
2 IL1β Level in Human Colostrum in Relation to Neonatal Hyperbilirubinemia Group II (Control) Included 20 full term neonates without jaundice and their mothers. A) Neonates Twenty full term neonates (37-41weeks) were included. Their gestational age, sex, weight were recorded. B) Mothers Their age and antenatal history were recorded. Inclusion Criteria A) Mothers 1-18 years old or more. 2- No history of inflammatory diseases or medications prior to and at the time of enrollment. 3- Uncomplicated pregnancy, delivery and purperium. 4- Exclusively breast-feeding of their newborns was eligible. B) Neonates 1- Healthy full term neonates, their gestational age range (37-41 weeks). 2- Not having any signs of birth injuries, congenital infections, or respiratory distress. Exclusion Criteria A) Mothers 1- Medical diseases during pregnancy as Diabetes Mellitus or Hypertension. 2- Taking anti-inflammatory Medications at the time of enrollment and in the previous one month. 3- Complicated delivery and purperium. 4- Previous neonates with a history of neonatal jaundice. 4- Not exclusively breast-feeding to their newborns. B) Neonates 1- Preterm infants. 2- Neonates having any signs of birth injuries, congenital infections, poor suckling, respiratory distress or signs of neonatal sepsis. Data Management and Analysis The collected data was revised, coded, tabulated and introduced to a PC using Statistical package for Social Science (SPSS 15.0. for windows; SPSS Inc, Chicago, IL, 2001). Numerical data were expressed as Mean, Standard deviation (SD), Minimum and maximum values (range). Non-numerical data were expressed as Frequency and percentage. Analytical statistics including Independent-Samples T, Correlation analysis (using Pearson's method) the correlation coefficient denoted symbolically "r", Chi-Square test. P< 0.05: Significant Results The data in this study are formulated as tables and figures. In the present study, mean total serum bilirubin in group I was 10.9+3.25mg/dl while was 3.88+0.78mg/dl in group II (figure 1). Mean Interleukin-1β level in group I was 10.35+4.23pg/dl and was 3.75+2.1pg/dl in group II (figure 2). Comparing the studied and control groups, significant differences were found regarding Interleukin-1β level (P=0.013) and total serum bilirubin (P=0.001) while no significant differences between the two groups regarding gestational age (P=0.357), neonatal weight (P=0.707), direct bilirubin (P=0.513), hemoglobin (P=0.27), RBCs (P=0.22), WBCs (P=0.87) and platelets (P=0.517) (table 1). Significant positive correlations between IL-1β level in colostrum and both total serum bilirubin (P=0.0001, r=0.722) (figure 3, table 2) and direct bilirubin (P=0.001, r=0.588) levels in group I (table 2).
THE EGYPTIAN JOURNAL OF IMMUNOLOGY 3 Table 1. Comparison of Laboratory findings between neonates with jaundice and controls Parameter Group I (n = 40) Group II (n = 20) P Value Gestational age (weeks) 38.725 38.65 NS Interleukin-1β in Colostrums (pg/ml) 10.35 3.75 0.013 Weight of neonate(kg) 3.1 2.97 NS Total bilirubin (mg/dl) 10.91 3.88 0.0001 Direct bilirubin (mg/dl) 0.62 0.4 NS Hemoglobin (gm/dl) 15.09 14.9 NS Red blood cells ( 10 12 /l) 4.71 4.75 NS White blood cells ( 10 9 /l) 10.26 10.88 NS Platelets ( 10 9 /l) 236.13 256.75 NS *P 0.0l: is significant. Ns: not significant Table 2. Correlation study results between IL-1β (pg/ml) and the studied variables in group I Parameter Interleukin-1β in colostrums (pg/ml) Value r *P Value Gestational age (wks) 38.7 0.081 NS Weight of neonate (kg) 3.008 0.158 NS Total bilirubin (mg/dl) 10.91 0.494 0.001 Direct bilirubin (mg/dl) 0.623 0.588 0.0001 Hemoglobin (gm/dl) 15.09 0.060 NS Red blood cells ( 10 12 /l) 4.709 0.176 NS White blood cells ( 10 9 /l) 10.26 0.007 NS Platelets ( 10 9 /l) 236.13 0.056 NS *P 0.0l: is significant. Ns: not significant
4 IL1β Level in Human Colostrum in Relation to Neonatal Hyperbilirubinemia IL-1β (pg/ml) 16 14 12 10 8 6 4 2 0 Mean ± SD IL-1β in Colostrum in both groups Group I (n = 40) Group II (n = 20) Figure 1. Mean serum IL1β levels in both studied groups. Group I= neonates with physiological jaundice. Group II= controls. 16 Mean ± SD total bilirubin in both groups 14 T otal bilirubin (mg/dl) 12 10 8 6 4 2 0 Group I (n = 40) Group II (n = 20) Figure 2. Mean total serum bilirubin levels in both studied groups. Group I= neonates with physiological jaundice. Group II= controls.
THE EGYPTIAN JOURNAL OF IMMUNOLOGY 5 20 18 16 14 IL-1β (pg/ml) 12 10 8 6 4 2 0 P = 0.0001 r = 0.722 0 5 10 15 20 25 T otal bilirubin (mg/dl) Figure 3. Correlation between total serum bilirubin and IL-1β level in group I. Discussion Many factors are associated with the occurrence of neonatal jaundice (NJ). Regarding gender, Seidman et al., 1999 reported that after controlling for race, birth weight, gestational age (GA), sepsis, and in a multiple regression model, the males had significantly higher bilirubin than females agreeing with the study proved significant increase in IL-1β in the colostrum of mothers of jaundiced breast-fed neonates compared with breast-fed neonates without neonatal jaundice (Zanardo et al., 2007). Ghadeer et al., 2009 found that there is a highly statistical significance difference between jaundiced breast fed neonates and those breast fed infants without neonatal jaundice regarding level of IL-1β in breast milk. Another study suggesting that the mammary gland immunologic function through altering cytokine levels may affect jaundice development in neonates. The identification of elevated IL-1β concentration in colostrum of mothers with jaundiced neonates and the clinical consequences of breast-feeding initiated through this immunological bas (Zanardo et al, 2007). Also, may be explained by sequestration and protection of cytokines and chemokines until they reach the intestine (Calhoun et al, 1999) which also supported by the hypothesis that interleukin-1 receptor antagonism improves gastric emptying.
6 IL1β Level in Human Colostrum in Relation to Neonatal Hyperbilirubinemia Although cytokines are known to play a critical role in the intestinal absorption, the molecular basis remains unclear (Michael et al., 1999). Constitutively enterocytes can express pro-inflammatory cytokines and this response is up-regulated by inflammatory stimuli such as endotoxin and IL-1β (Zanardo et al., 2007). With excessive proinflammatory cytokine production (IL-8) an inflammatory stimulation after interaction between immature human enterocytes and excessively released cytokines (IL-1β, lipopolysaccharides), causing destruction of enterocyte barrier (Defranco et al., 1998; Nanthakumar et al., 2000). Additionally, inflammatory cytokines such as TNF-α, interferon (IFN)-γ, and IL-1β, produced by epithelial cells and cells of the innate and adaptive immune systems contribute to dysfunction of epithelial barrier enterocytes (Van Haver et al., 2009). Another theory of relative distal expression of cytokines (IL-1β and ll-6) in the small intestine and different bacterial species increasing the expression of proinflammatory cytokines. IL-1β and growth factors and altering vascular smooth muscle differentiation changing from a contractile to a proliferative phenotype, which in turn leads to a change in the expression levels of contractile proteins such as actin and myosin heavy chains, causing force generation reduction (Shirkey et al., 2006) supported by the suggestion that IL-1β inhibits the initial Ca 2+ dependent contraction of intestinal smooth muscle (Hsieh et al., 2000; Wenhui et al., 2009). Also, diminished intestinal motility enables more bilirubin deconjugation by the luminal enzyme β-glucuronidase, allowing more reabsorption of unconjugated bilirubin (James et al., 2007). Studying the correlation between level of ll-1β in colostrum and level of neonatal TSB, Ghadeer et al., 2009 reported that there is a highly statistical significant positive correlation between level of ll-1β in colostrum and level of neonatal TSB, but this was in contrast with the opinion that neither IL-1β concentrations in colostrum nor other studied cytokines (IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α) were correlated to serum bilirubin concentrations of neonates with NJ(1). Factors that may contribute to the development of neonatal jaundice include exposure to various breast milk components such as 3-alpha-20-beta pregnanediol, nonesterified fatty acids, and glucuronidase, or injurious agents (e.g. drugs, hormones, proinflammatory cytokines,) results in alteration in bilirubin pathway, changes that may maintain and contribute to appearance of jaundice. In addition, the suppression of genes that normally mediates this bilirubin pathway and excretion (Zanardo et al., 2007). Regulatory mechanisms that affect bile acid transporter, expression and function will help to develop novel rational options to treat cholestasis and chronic cholestatic liver diseases (Sertac et al., 2004). Depending on IL-1β assay only in colostral milk without putting in consideration that concentrations of IL-1β in the early, transitional and mature milk are different may make a limitation of our study. Milk composition is affected with several factors including delivery weather normal or instrumental, multiple parturition, and with day time, at each feeding, also milk proteins produced and secreted in the mammary gland are constitute the major percentage of human milk proteins and have more postpartum variation compared with other serum proteins that are passively transferred into milk (Londerdal et al., 1995). We conclude that elevated levels of IL-1β in the colostrum provide additional data to understand one of path physiologic mechanisms of breast milk jaundice and immunologic role of mammary gland in the occurrence of neonatal jaundice.
THE EGYPTIAN JOURNAL OF IMMUNOLOGY 7 References 1. Bertini G, Dani C, Tronchin M, Rubaltelli FF. (2001). Is breast feeding really favoring early neonatal jaundice? Pediatrics; 107: e41. 2. Bolder U, Ton-Nu HT, Schteingart CD, Frick E, Hofmann AF. (1997). Hepatocyte transport of bile acids and organic anions in endotoxemic rats, impaired uptake and secretion. Gastroenterology; 112: 214-225. 3. Calhoun, DO, Darlene A, Mathilde Lunøe, Yan Du, Robert D. Christensen. (1999). Concentrations of granulocyte colony-stimulating factor in human milk after in vitro simulations of digestion. Pediatr Res; 46: 767-771. 4. DeFranco AL, Crowley MT, Finn A, Hambleton J, Weinstein SL. (1998). The role of tyrosine kinases and map kinases in LPS-induced signaling. Prog Clin Biol Res; 397: 119-136. 5. Ghadeer A, Lerine B, Safaa S. (2009). Interleukin 1β in human colostrum in relation to neonatal jaundice: a thesis. Faculty of Medicine Ain-Shams University, 89-104. 6. Honsawek S, Chongsrisawat V, Vejchapipat P, Thawornsuk N, Tangkijvanich P, Poovorawan Y. (2005). Serum interleukin-8 in children with biliary atresia: relationship with disease stage and biochemical parameters. Pediatr Surg Int; 21: 73-77. 7. Hsieh JT, Farley JM. (2002). Characterization of contractile function and expression of muscarine receptors, G proteins and adenyl cyclase in culture muscle of swine. J Biomed Sci; 9(4): 339-47. 8. James CJ, Kivlahan C. (2007). Emergency medicine approach to neonatal hyperbilirubinemia. Emerg Med Clin N Am; 25(4) 1117-1135. 9. Kimura F, Miyazaki M, Suwa T, Sugiura T, Shinoda T, Itoh H, Nagakawa K, Ambiru S, Shimizu H, Yoshitome H. (2001). Antiinflammatory response in patients with obstructive jaundice caused by biliary malignancy J Gastroenterol Hepatol; 16: 467-472. 10. Londerdal B, Atkinson S. (1995). Human milk proteins. In Handbook of Milk Composition. Jensen R (ed), San Diego: Academic Press, 351-368. 11. Michael D. Josephs, Guozhang Cheng, Riadh Ksontini, Lyle L. Moldawer, Michael P. Hocking. (1999). Products of cyclooxygenase 2 catalysis regulates postoperative bowel motility. J Surg Res; 86(1): 50-54. 12. Moseley RH. (1997). Sepsis-associated cholestasis. Gastroenterology; 112: 302-306. 13. Nanthakumar NN, Fusunyan RD, Sanderson I, Walker WA. (2000). Inflammation in the developing human intestine: a possible pathophysiologic contribution to necrotizing enterocolitis. Proc Natl Acad Sci USA; 97: 6043-6048. 14. Seidman DS, Ergaz ZP. (l999). Predicting the risk of jaundice in full term healthy newborn. J Perinatal; 19: 564-567. 15. Sertac Kip. N, Konstantinos N. Lazaridis, Anatoliy I. Masyuk, Patrick L. Splinter, Robert C. Huebert, Nicholas F. LaRusso. (2004). Differential expression of cholangiocyte and ileal bile acid transporters following bile acid supplementation and depletion. World J Gastroentrol; 10(10): 1440-1446. 16. Shirkey TW, Siggers RH, Goldade BG, Marshall JK, Drew MD, Laarveld B, Van Kessel AG. (2006). The effect of commensal bacteria on intestinal morphology and expression of proinflammatory cytokines in the gonotobiotic pig. Exp Biol Med; 231: 1333-1345. 17. Trauner M, Wagner M, Fickert P, Zollner G. (2005). Molecular regulation of hepato- biliary transport systems: clinical implications for understanding and treating cholestasis. J Clin Gastroenterol; 39: 111-124. 18. Van Haver ER, Sangild PT, Oste M, Siggers JL, Weyns AL, Van Ginneken CJ. (2009). Dietdependent mucosal colonization and interleukin- 1beta responses in preterm pigs susceptible to necrotizing enterocolitis. J Pediatr Gastroenterol Nutr; 49 (1): 90-98. 19. Wenhui HU, Fangl Sunila M, Karnuaus M. (2009). Muscle cell biology and cell motility. Am J Physiol; 296: 1310-1320. 20. Zanardo Vincenzo, Rosanna Golin, Maurizio Amato, Daniele Trevisanuto, Flaviano Favaro, Diego Faggian, Mario Plebani (2007). Cytokines in human colostrum and neonatal jaundice. Pediatric Res; 62(2): 191-194.