Zanardo et al. cytokines, but little is known as to their concentrations and associations within breast milk (Laiho et al. 2003). IL-1 is probably the first cytokine that has been quantified in human milk (Garofalo and Goldman 1998; Munoz et al. 1990). The synthesis, processing, secretion, and activity of IL-1 are tightly regu- lated events. For instance, the presence of an IL-1 receptor antagonist is an important mechanism through which IL-1 signaling is down-regulated, and therefore its activity is reduced (Dinarello 1997). Of interest, IL-1β receptor antagonists have been measured in human breast milk (Buescher and Malinowska 1996). To what extent the biologic effect of the cytokine in milk is modulated by the pres- ence of soluble receptors and other cytokine antagonist is open to speculation. Smoking is a major risk factor for both infantile infections and SIDS. Such infections, both viral and bacterial, also increase the SIDS risk (Gordon et al. 2002; Guntheroth 1989). In experimental animals, nicotine has been shown to depress both the primary and sec- ondary immune response of the lungs, lymph nodes, and spleen (Sayers and Drucker 1999). Interestingly, most studies have shown a decrease in proinflammatory cytokine pro- duction (IL-1α, IL-6, and tumor necrosis factor-α) by lung macrophages in smokers, accounting for the greater concentration of tobacco in the lungs (Froen et al. 2000; Sayers and Drucker 1999). These cells have proven Characteristic Maternal data Maternal age (years) Maternal smoking (cigarettes/day) Maternal education (years) Parity [primiparous (%)] BMI (kg/m2) Vaginosis (%) Labor induction (%) Duration of labor (hr) Mode of delivery [vaginal (%)] Birth data Gestational age (weeks) Birth weight (kg) Sex (%male) Apgar score 1st min 5th min Table 1. Demographics, pregnancy, and birth outcomes characteristics of the study groups. very sensitive to both smoking history and the duration of the smoke-free period (Zeidel et al. 2002). Similarly, the greater concentration of tobacco components reported in the breast milk of smoker mothers could imply a differ- ent magnitude of immune impairment of the mammary gland macrophages (Klonoff-Cohen et al. 1995). The possibility that altered levels of cytokines and other biologic factors in breast milk modify the immune function in the nursed infants is a very attractive hypothesis (Jones and Warner 2000). Physiologic delays or imbalance in the production of immune fac- tors can occur in mammals, and such delays increase the risk of infection and SIDS (Laiho et al. 2003). Human milk is an important source of other bioactive substances, including hormones, growth factors, and immunologic factors such as cytokines, but the functional consequences of an overexpression or of a down-regulation of most milk immunomodulatory constituents in neonates are unknown. In our study, the levels of β-endorphin and leptin in breast milk were not related to smoking habits of nursing mothers. The lack of additive effects and the dissimilarity in response to tobacco smoke might indicate that these immunomodulatory molecules are involved in different pathways. In this context, it is interesting that nicotine can cause a release of β-endorphin in various brain regions, and that cytokines and endotoxin can cause a release of β-endorphin in different Smokers (n = 42) Nonsmokers (n = 40) 31.1 ± 0.9 3.2 ± 0.7 10.5 ± 2 35.1 25.3 ± 4.0 2.3 7.1 8.9 ± 4.2 88 39.0 ± 0.3 3.23 ± 0.14 44 8.8 ± 0.4 9.5 ± 0.1 31.2 ± 1.1 10.6 ± 3 41.4 25.7 ± 4.2 2 7.2 8.6 ± 4.3 92 40.0 ± 0.3 3.48 ± 0.09 42 9.2 ± 0.2 9.8 ± 0.1 tissues. Moreover, in mice, leptin, induced by LPS and cytokines, participates in the host response to inﬂammation by modulating the host immune and cytokine responses (Faggioni et al. 1999). There are certain limitations in our work. First, only colostral and transitional milk sam- ples were collected. It is well established that milk composition differs with delivery modali- ties, increasing parturition, throughout the day, at each feeding, and with time. Generally, milk proteins that are produced and secreted in the mammary gland are expected to con- stitute the largest proportion of protein in human milk and to have more postpartum variation than do serum proteins that are pas- sively transferred into milk (Londerdal and Atkinson 1995). Having multiple cytokine and immunomediators measures taken over time from breast-feeding mothers with a dose–response relationship between smoking levels and levels of agents would permit veriﬁ- cation of the assumption underlying this analysis. In addition, the selection of IL-1, of leptin, and of β-endorphin, among the wide range of immunomodulatory factors pre- sent in breast milk and interconnecting the neuroendocrine and immune systems, has been made in the context of the present study mainly for convenience and not according to solid scientiﬁc reasons. However, the study has several strengths. Our measure of cytokines in colostrum has been used in previous studies and is valid. Furthermore, most of the colostral and tran- sitional milk samples (> 90%) in the study contained IL-1α, so this is a large enough group to be confident of the results and of measurable presence of IL-1α in the early milk. Nevertheless, it is premature to know how the findings will pertain to later infec- tions, because most children of smoking mothers do not develop infections in the early perinatal time. Conclusion Our data support the suggestion that some clinical consequences of smoking might be initiated and enhanced by the production of inflammatory cytokines from colostrum. Reduced colostral IL-1α concentrations pro- vide additional data to the delineation of the pathophysiologic mechanism that includes mammary gland immunologic dysfunction in the cascade of events that can lead to infec- tions and SIDS. To our knowledge, this is the first study on the effects of smoking on the content of cytokines and immunomedia- tors in human milk. Prospective studies are required to thoroughly assess the differences in the composition of breast milk from moth- ers with different smoking histories and the immunologic consequences of this for the offspring. VOLUME 113 | NUMBER 10 | October 2005 • Environmental Health Perspectives Data are presented as mean ± SD unless otherwise indicated. Table 2. IL-1α, β-endorphin, and leptin concentrations in the colostrum and transitional milk of smoker compared with nonsmoker mothers. IL-1α (ng/L) β-Endorphin (ng/L) Leptin (µg/L) 1412 Data are presented as mean ± SE, or p-value from Mann-Whitney U-test. *p < 0.05; **p < 0.01. Colostrum Smokera 17.2 ± 4.0 353.5 ± 29.1 1.6 ± 0.3 Nonsmokerb 38.4 ± 7.4 317.5 ± 27.6 1.5 ± 0.2 Transitional milk Smokerc Nonsmokerd 14.4 ± 5.2 152.8 ± 26.5 0.7 ± 0.1 21.7 ± 12.5 127.7 ± 2.3 0.5 ± 0.1 p-Value a vs. b* a vs. c** b vs. d** a vs. c* b vs. d**