Maternal smoking and breast milk interleukin-1α Our objective was to determine whether there is a relationship between IL 1-α levels in milk and smoking habits in nursing mothers, with the consequent implications on the devel- opment of the immune system. Other factors in milk considered for association with infection risk in infancy were the immunomodulators β-endorphin and leptin. β-Endorphin was assessed because it is produced by the epithelial cells (i.e., the primary cells in human milk) (Khachaturian et al. 1958), it exists at detectable levels in human milk (Zanardo et al. 2001), and it inﬂuences the behavior of other cells and tis- sues. Leptin, induced by lipopolysaccharide (LPS) and cytokines, participates in the host response to inﬂammation by modulating the host immune and cytokine responses after LPS (Faggioni et al. 1999). Thus, any effects of a given cytokine could be tested for speciﬁcity by comparison with the other immunologically active components. Materials and Methods Longitudinal and cross-sectional human milk samples were obtained from mothers hospital- ized in regional tertiary maternity care at Padua University (Italy), from June to December 2002. Human milk was collected by mothers via commercial breast pumps, frozen immedi- ately, and stored. Collection date and time were recorded, as were infants’ birth date, gestational age, birth weight, and sex and mothers’ demographic and anthropometrical characteristics [age, education, parity, body mass index (BMI)], diagnoses, pregnancy com- plications, and route of delivery (cesarean sec- tion vs. vaginal). Before the infant’s birth, pregnant women were asked by experienced research midwives to participate in a prospective study. The women received verbal and written informa- tion about the aim and design of the study. Only healthy women ≥ 18 years of age, not taking anti-inflammatory medications at the time of enrollment, with otherwise uncompli- cated gestation, delivery, and puerperium and who planned to obtain care for their new- borns through rooming-in and exclusively breast-feeding their newborns were eligible. Allergy in mothers was not an exclusion crite- rion because of the findings that maternal atopy shows no relationship with cytokine level in milk (Bottcher et al. 2000). After the women accepted participation, their smoking history was recorded. Of the 1,217 eligible participants, 25 of 26 self-identiﬁed as smokers (≥ 5 cigarettes per day through pregnancy until last trimester) were recruited for study participation. One was excluded from the ﬁnal analysis because of maternal fever. Control participants included consecutive women without history of smok- ing and matched a smoking participant on the basis of overall inclusion criteria. A written informed consent was obtained from all participants. Milk specimen collection and processing. Breast milk samples were collected during the third postpartum day (before discharge) and on the 10th postpartum day, close to expression of the colostrum and transitional milk production phases. Collection was standardized to reduce bias and potential diurnal variability in cytokine measurements. Specimens were obtained within 1 hour of the ﬁrst feeding in the morn- ing, deﬁned as 0800 hr to 0900 hr. Mothers were asked not to feed for 3 hr before collec- tion. All mothers were able to provide milk at each sampling point. An aliquot of 3 mL was taken using a manual breast pump. Samples were temporarily stored at –70°C in sterile plas- tic tubes and used for assays. Samples were thawed and centrifuged for 10 min at 1800 rpm at 4°C, after which the lipid layer and cellular elements were removed. The aqueous fraction was filtered (0.45-µm Acrodisc; Gelman Sciences, Ann Arbor, MI, USA) and was used for cytokine, β-endorphin, and leptin determinations, with the lipid layer frozen for other studies. Milk IL1-α, β-endorphin, and leptin assays. Filtered aqueous milk fractions were assayed for IL-1α (by CytElisa Human IL-1α ACCUCYTE; CytImmune Sciences Inc., Rockville, MD, USA), β-endorphin (beta- Endorphin 60 T kit RIA; Nichols Institute Diagnostic, San Juan Capistrano, CA, USA), and leptin (ACTIVE Human Leptin IRMA, DSL-23100; Diagnostic Systems Laboratories Inc., Webster, TX, USA) levels. All fractions were assayed undiluted. The coefficients of variation for intraassay and interassay, respec- tively, are the following: IL-1α, 8.3 and 11.2%; β-endorphin, 4.1 and 7.1%; and lep- tin, 3.7 and 6.6%. Statistical analysis. Unless otherwise speci- fied, results are expressed as mean concen- trations in nanograms per liter or micrograms per liter ± SE. The p = 0.05 signiﬁcance level was used for the statistical analysis. We grouped human milk samples into third postpartum day smoker and nonsmoker mother groups, with 42 and 40 samples per group, respectively, and into 10th postpartum day smoker and non- smoker mother groups, with 42 and 40 samples per group, respectively. Because the concentrations of cytokine IL-1, β-endorphin, and leptin were not normally distributed, we performed a paired analysis with the Mann-Whitney U-test. We used the Student t-test for the analysis of the other data. Ethics. The research protocol was approved by the Hospital Ethical Committee for Human Research of the University of Padua. Results We analyzed 164 milk samples, collected from 82 mothers, 42 smokers, and 40 nonsmokers. Environmental Health Perspectives • VOLUME 113 | NUMBER 10 | October 2005 Measurable immunoreactivity levels were observed in most colostral (third day) and tran- sitional (10th day) milk samples throughout the collection period (> 90%). The smoker mothers of healthy-term infants and control nonsmoker mothers of healthy-term infants who planned to breast-feed their infants were comparable in their anthropometric charac- teristics and those of the respective neonates. The birth weight of the newborn infants of smoker mothers was not significantly lower (Table 1). IL-1α concentrations were significantly reduced in the colostrum of smoker com- pared with the nonsmoker control mothers. β-Endorphin and leptin colostrum concentra- tions were comparable (Table 2). Moreover, β-endorphin concentrations were signiﬁcantly reduced in transitional milk samples compared with colostrum of both smoker and nonsmoker mothers. Also, leptin concentrations were significantly reduced in transitional milk samples compared with colostrum of both smoker and nonsmoker group. IL-1α concentrations were lower in transitional milk than in colostrum samples of smoker and nonsmoker mothers, but without any signiﬁcance (Table 2). We found no significant differences between smoker and nonsmoker lactating mothers in transitional milk concentrations of IL-1α, β-endorphin, and leptin (Table 2). And we found no correlations between reduced IL-1α levels of the colostral milk samples and related immunomodulator β-endorphin and leptin levels. Discussion In this article, we present novel evidence sug- gesting that exposure to tobacco smoke dur- ing pregnancy may affect the development of the immunologic function of the mammary gland, signiﬁcantly inﬂuencing colostral milk provision of the proinflammatory cytokine IL-1α for breast-feeding infants. By contrast, the concentration of the immunomodulatory factors β-endorphin and leptin were unaf- fected. In this context, the possibility that delivery modalities, parity, or BMI affects the levels of cytokines and immunomediators in the breast milk is open to speculation. The identiﬁcation of reduced IL-1α levels in the colostral milk of mothers exposed to tobacco smoke during pregnancy offers sup- port to the hypothesis that some clinical con- sequences of smoking on infancy might be initiated and enhanced by altered levels of inflammatory cytokines (Froen et al. 2000; Sayers and Drucker 1999). Early breast milk may be of particular importance in the development of the mam- malian newborn innate and acquired immu- nity (Goldman et al. 1998). Colostrum has been reported to contain a large amount of 1411