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Department of Cellular and Physiological Sciences, Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia V6T 1Z3
1To whom requests for reprints should be addressed at Department of Cellular and Physiological Sciences, Faculty of Medicine, The University of British Columbia, 2177 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada. E-mail: joannew{at}interchange.ubc.ca
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Abstract |
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 Top Abstract IntroductionMaternal Alcohol Consumption...Prenatal Alcohol Exposure Alters...Prenatal Ethanol Exposure...References |
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In this review, we focus on the adverse effects of prenatal alcohol exposure on neuroendocrine and immune function, with particular emphasis on the hypothalamic-pituitary-adrenal (HPA) axis and the concept of fetal programming. The HPA axis is highly susceptible to programming during fetal development. Early environmental experiences, including exposure to alcohol, can reprogram the HPA axis such that HPA tone is increased throughout life. We present data that demonstrate that maternal alcohol consumption increases HPA activity in both the maternal female and the offspring. Increased exposure to endogenous glucocorticoids throughout the lifespan can alter behavioral and physiologic responsiveness and increase vulnerability to illnesses or disorders later in life. Alterations in immune function may be one of the long-term consequences of fetal HPA programming. We discuss studies that demonstrate the adverse effects of alcohol on immune competence and the increased vulnerability of ethanol-exposed offspring to the immunosuppressive effects of stress. Fetal programming of HPA activity may underlie some of the long-term behavioral, cognitive, and immune deficits that are observed following prenatal alcohol exposure.
Key Words: prenatal alcohol • fetal programming • corticotropin-releasing hormone • adrenocorticotropic hormone • glucocorticoids • stress • immunity
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Introduction |
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TopAbstractIntroductionMaternal Alcohol Consumption...Prenatal Alcohol Exposure Alters...Prenatal Ethanol Exposure...References |
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Whether alcohol-induced endocrine imbalances actually contribute to the etiology of fetal alcohol syndrome (FAS) is unknown, but it is certainly a possibility (9). The effects of alcohol on the maternal-fetal unit are complex (10–12), and both direct and indirect effects of alcohol on fetal development are known to occur. Alcohol readily crosses the placenta, thus directly affecting developing fetal cells and tissues, including those related to endocrine function. In addition, alcohol-induced changes in maternal endocrine function can disrupt the hormonal interactions between the maternal and fetal systems. Alterations in the normal maternal-fetal hormone balance will indirectly affect the development of fetal metabolic, physiologic, and endocrine functions. Alcohol-induced changes in maternal metabolic and/or endocrine function can also affect the female’s ability to maintain a successful pregnancy, resulting in miscarriage or, if the fetus is carried to term, possible congenital defects.
In this review, we focus on the adverse effects of prenatal alcohol exposure on neuroendocrine and immune function, with particular emphasis on the hypothalamic-pituitary-adrenal (HPA) axis and the concept of fetal programming. Fetal or early programming refers to the concept that early environmental or nongenetic factors can permanently organize or imprint physiological and behavioral systems (13). The HPA axis is highly susceptible to programming during fetal and neonatal development (13). Disturbance of the reciprocal interconnections between maternal and fetal or maternal and neonatal HPA activity may provide a common pathway for fetal programming by which prenatal or perinatal exposure to drugs or other toxic agents produces long-lasting behavioral and physiological consequences (13, 14). We present data that demonstrate that maternal alcohol consumption reprograms the fetal HPA axis such that HPA tone is increased throughout life. Increased exposure to endogenous glucocorticoids throughout the lifespan can alter behavioral and physiological responsiveness and increase vulnerability to illnesses or disorders later in life. Alterations in immune function may be one of the consequences of fetal HPA programming. We discuss studies that demonstrate that alcohol has adverse effects on immune function and that programming of HPA activity may mediate some of the adverse effects of prenatal alcohol exposure on immune competence in later life.
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Maternal Alcohol Consumption Alters HPA Activity and Regulation in the Maternal Female and Her Offspring |
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TopAbstractIntroductionMaternal Alcohol Consumption...Prenatal Alcohol Exposure Alters...Prenatal Ethanol Exposure...References |
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Effects of Alcohol Consumption on HPA Activity of the Pregnant Female.
Data from animal models indicate that maternal alcohol (ethanol) consumption increases maternal adrenal weights, basal corticosterone levels, the corticosterone response to stress, and the corticosterone stress increment, compared with those in pair-fed and control females (7). These changes occur as early as Day 11 of pregnancy, persist throughout gestation, may increase as gestation progresses, and occur even with low concentrations of ethanol in the diet (7, 19). Importantly, maternal nutritional status has no major impact on these effects, indicating that the increased corticoid activity is not due to nutritional factors (7). Furthermore, we have shown that the stimulatory effects of ethanol on HPA activity can extend through parturition, even when alcohol administration is discontinued before parturition (20). These data demonstrate that regular consumption of high doses of ethanol during pregnancy not only raises the set point of HPA function in the mother by increasing both basal and stress levels of corticosterone but may also result in HPA hyperresponsiveness to stressors. The additional finding that binding capacity of corticosterone-binding globulin in ethanol-consuming females is similar to or less than that in control females, both during pregnancy and at parturition, indicates that the increased HPA activity is functionally important and supports the conclusion that maternal ethanol consumption results in both hypersecretion and hyper-responsiveness of the HPA axis (7).
The pregnant female and fetus constitute an interrelated functional unit. Thus, ethanol-induced alterations in maternal HPA activity have significant implications for fetal HPA development. Maternal corticosterone crosses the placenta (21), resulting in suppression of endogenous fetal HPA activity. At the same time, ethanol crosses the placenta and directly activates the fetal HPA axis. Together these direct and indirect effects of ethanol can have permanent organizational effects on neural structures that regulate HPA activity throughout life (22). Whether the increased maternal corticosteroid levels play a role in mediating HPA hyperresponsiveness in fetal ethanol-exposed offspring is as yet unresolved. It has been shown that adrenalectomy of the pregnant dam has no effect on the increased corticoid responses to restraint stress in ethanol-exposed offspring (23). Further, corticosterone treatment of adrenalectomized dams does not mimic the effect of prenatal ethanol on offspring HPA activity (24). These findings suggest that increased maternal corticosteroid levels are not the primary mediator of increased stress responsiveness in ethanol-exposed offspring. In contrast, maternal adrenalectomy reversed the effects of prenatal ethanol on pituitary pro-opiomelanocortin (the precursor of adrenocorticotropic hormone [ACTH]) messenger RNA (mRNA) levels observed in ethanol-exposed offspring (25). Further studies are clearly necessary to elucidate the possible role of maternal glucocorticoids in the HPA hyperresponsiveness of ethanol-exposed offspring. It is likely that a complex interaction between direct and indirect effects of ethanol probably mediates the effects of ethanol on programming of fetal HPA activity.
Effects of Maternal Alcohol Consumption on Offspring HPA Activity.
The Preweaning Period.
The complex interactive effects of ethanol and maternal HPA activity on offspring HPA function are apparent following parturition. Ethanol-exposed fetuses exhibit significantly lower corticosterone levels than control fetuses on Day 19 of gestation (26). In contrast, at birth ethanol-exposed neonates have elevated plasma and brain levels of corticosterone and elevated plasma but reduced pituitary levels of ß-endorphin (20, 27–29). However, throughout the preweaning period ethanol-exposed offspring exhibit blunted HPA and ß-endorphin responses to a wide range of stressors, including ether, novelty, saline injection, and cold stress (20, 27, 29, 30). In addition, ethanol appears to alter the ontogenetic profiles of corticotropin-releasing hormone (CRH) and pro-opiomelanocortin mRNA expression, delaying and exaggerating the rise of CRH expression in female but not male pups and suppressing pro-opiomelanocortin mRNA levels in male but not female pups compared with that in controls throughout the preweaning period (31). These data suggest that sexually dimorphic effects of prenatal ethanol exposure on these two important glucocorticoid-regulated genes contribute to both the immediate and the long-term effects of prenatal ethanol on stress responsiveness.
The significance of the reduced hormonal responsiveness of ethanol-exposed pups during early development remains to be determined. Evidence from our work indicates that in addition to the reduced corticoid responses to stressors, binding capacity of plasma corticosterone-binding globulin is also reduced in ethanol compared with control pups, at least during the first week of life (20). Thus, it is possible that although the corticoid stress response is blunted in ethanol-exposed pups, the ratio of bound to free steroids may not be altered, and the reduction in binding capacity of corticosterone-binding globulin may represent a compensatory response to maintain normal neuroendocrine function. This possibility remains to be tested.
Long-term Effects of Prenatal Alcohol Exposure.
The reduced HPA and ß-endorphin responsiveness observed in ethanol-exposed pups early in life is a transient phenomenon. Following weaning, ethanol-exposed animals are typically hyperresponsive to stressors and to drugs such as ethanol and morphine (32–36). Furthermore, sex differences in response are often observed and may vary, depending on the nature of the stressor and the time course and hormonal end point measured (37–41). For example, in adulthood, ethanol-exposed males and females both exhibit increased corticosterone, ACTH, and/or ß-endorphin responses to stressors such as repeated restraint, foot shock, and immune challenges (33–37, 42, 43). Both males and females also show increased immediate early gene and CRH mRNA levels following stress (33), as well as deficits in habituation to repeated restraint (42). In contrast, in response to prolonged restraint or cold stress, HPA hyperactivity is seen primarily in ethanol-exposed males (41, 44), whereas in response to acute restraint, or acute ethanol or morphine challenge, increased hormone responses occur primarily in ethanol-exposed females (28, 29, 34, 40, 45). Similarly, altered hormone responses in a consummatory task (40) and to predictable and unpredictable restraint stressors (46) suggest deficits in the ability of ethanol-exposed females but not of males to use or respond to environmental cues.
Possible Mechanisms That Mediate HPA Hy-perresponsiveness.
The mechanisms that underlie HPA hyperresponsiveness in ethanol-exposed offspring are just starting to be understood and appear to reflect changes at several levels of the axis. Increased HPA activity could result from increased secretion of secretagogues (e.g., pro-opiomelanocortin, CRH, arginine vasopressin [AVP]), increased pituitary and/or adrenal responsiveness to these secretagogues, increased drive to the hypothalamus, deficits in feedback regulation of HPA activity, and/or alterations in central neurotransmitters regulating the HPA axis. Data suggest that, in fact, multiple mechanisms probably play a role. Furthermore, the finding that prenatal ethanol exposure may differentially alter HPA responsiveness in males and females compared with their control counterparts suggests that the gonadal hormones or altered adrenal-gonadal interactions probably play a role in mediating ethanol effects on HPA activity. In this review, we discuss three of the possible mechanisms shown to be important in mediating HPA hyperresponsiveness in ethanol-exposed animals: alterations in HPA drive, alterations in HPA feedback regulation, and interactions between the adrenal and gonadal axes. Please see several recent reviews (47, 48) for a more complete discussion of mechanisms that mediate fetal alcohol effects on HPA activity and regulation.
Altered HPA Drive and/or Feedback.
Substantial evidence now suggests that enhanced stimulatory inputs or drive to the paraventricular nucleus (PVN) of the hypothalamus are increased by prenatal ethanol exposure. Weanling male and female pups exhibit increased basal levels of CRH mRNA in the PVN (32), and adult ethanol-exposed males exhibit increased basal levels of hypothalamic CRH mRNA and pituitary pro-opiomelanocortin mRNA (25) compared with controls. Preliminary data suggest that ethanol-exposed males also exhibit increased basal CRH mRNA levels following repeated restraint stress (49). However, not all studies have shown these alterations in basal HPA regulation. We found no effect of prenatal ethanol on basal CRH or AVP mRNA levels in adult males and females (44). Similarly, Lee et al. (33) reported no differences in basal CRH heteronuclear RNA (hnRNA) or in basal CRH and AVP median eminence protein levels in ethanol-exposed compared with control animals. Importantly, however, Lee et al. (33) demonstrated that ethanol-exposed animals exhibit enhanced hypothalamic neuronal activity compared with controls in response to both foot shock and an immune challenge. This was reflected in increased mRNA levels of two immediate early genes and increased CRH hnRNA levels. These data provide the first evidence of a selective stress- and cytokine-induced increase in activity of hypo-thalamic CRH neurons in ethanol-exposed animals, indicating a potential hypothalamic mechanism through which alcohol could upregulate the HPA axis.
Recent studies in our laboratory (50–52) were undertaken to explore further the changes in HPA regulation in ethanol-exposed compared with control animals and to try to resolve the conflicting findings on how robust the changes in basal HPA regulation are. Our results support and extend the data (33) that demonstrate that enhanced drive to the hypothalamus is an important mechanism that underlies HPA hyperresponsiveness of ethanol-exposed animals and provide evidence that alterations in sensitivity to corticosterone feedback also play a role. We used adrenalectomy with or without corticosterone replacement as a probe to investigate steady-state HPA function and the role of corticosterone in mediating changes in HPA regulation. Specifically, we assessed hypothalamic CRH and AVP mRNA levels as a measure of hypothalamic activity, CRH Type 1 receptor and pro-opiomelanocortin mRNA levels as an index of pituitary activity, and hippocampal mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mRNA as a measure of feedback regulation. We found that following adrenalectomy, basal plasma ACTH levels were higher in ethanol than in control males, but no significant differences occurred among females from prenatal ethanol and control groups. These data suggest that a corticosterone-independent alteration in HPA regulation is unmasked by removal of the glucocorticoid feedback signal, at least in males. In addition, significant alterations in gene expression were revealed in ethanol-exposed animals. In parallel with changes in plasma ACTH levels, hypothalamic CRH mRNA levels were significantly higher in ethanol-exposed compared with control males following adrenalectomy. A similar increase in CRH mRNA levels was observed in ethanol-exposed females despite the fact that plasma ACTH levels were not differentially elevated compared with controls. Furthermore, corticosterone replacement was less effective in normalizing adrenalectomy-induced alterations in ethanol-exposed animals than in control animals at several levels of the HPA axis, including hippocampal MR mRNA in both males and females, hippocampal GR and hypothalamic AVP mRNA in males, and anterior pituitary CRH receptor mRNA in females. No significant differences occurred among males or females from the three prenatal groups in pituitary pro-opiomelanocortin mRNA levels. The alterations in MR and GR regulation are particularly noteworthy, because our previous studies that used a binding assay found no differences in MR and GR concentrations or binding affinity in the hippocampus and other brain regions between ethanol-exposed and control animals (53, 54). Together these data suggest that ethanol-exposed animals exhibit HPA dysregulation under basal conditions, even in the face of similar basal hormone levels, and differences are further unmasked following perturbations of the system by stress or adrenalectomy. Dysregulation occurs at multiple sites, including the hippocampus, hypothalamus, and pituitary, and appears to reflect a change in the balance between HPA drive and corticosterone feedback regulation. The finding that pro-opiomelanocortin mRNA levels did not differ among groups suggests that at the level of the pituitary there may be compensatory changes in ethanol-exposed animals, such that the enhanced activation seen at the hypothalamus does not translate directly into enhanced pituitary activity. This may be protective for ethanol-exposed animals, minimizing enhanced pituitary activity in the face of enhanced hypothalamic drive. Furthermore, prenatal ethanol exposure appears to have sexually dimorphic effects on HPA regulation, suggesting a role for the gonadal steroids or possibly an alteration in adrenal-gonadal interactions in mediating these effects of ethanol on HPA activity.
Data from an earlier study in our laboratory support this finding that deficits in feedback sensitivity play a role in the HPA hyperactivity of ethanol-exposed animals. In that study, we investigated feedback regulation by injecting dexamethasone, a potent synthetic glucocorticoid, to block endogenous HPA activity and then 3 hrs later subjecting animals to a brief ether stress (55). We found that corticosterone levels were significantly elevated in ethanol-exposed animals compared with control animals following stress. This resistance to dexamethasone suppression suggests a deficit in feedback regulation in the intermediate (2–10 hrs) time domain and may reflect a change in functional activity of the GR, since dexamethasone is a GR agonist. However, these conclusions are made with caution, because increased responsiveness following blockade of endogenous HPA activity could be due, at least in part, to increased HPA drive.
In contrast to these data on intermediate feedback, it is not yet certain whether ethanol-exposed animals also exhibit deficits in the fast (seconds to minutes) feedback time domain. We (56) investigated fast feedback by injecting animals with either corticosterone (to produce a rapid rise in plasma hormone levels, which serves as a negative feedback signal to the hypothalamus) or vehicle, subjecting them immediately to a stressor, and analyzing the plasma hormone response 30 mins later. Administration of corticosterone resulted in a similar blunting of the ACTH response to stress in ethanol as in control animals, suggesting that ethanol-exposed animals do not have a functional deficit in fast feedback regulation. However, Taylor et al. (34) reported elevated plasma ACTH levels in ethanol-exposed compared with control animals at 10 mins after a 1-min intermittent foot shock stress, which is within the fast feedback time domain. Thus, it is possible that a fast feedback deficit exists in ethanol-exposed animals but only during a short period within the fast feedback time domain or only under certain stress conditions.
Recent work in our laboratory also supports the suggestion that the balance between HPA drive and feedback is altered in ethanol-exposed animals (57). We examined the effects of corticosteroid receptor blockade on HPA activity of adult females under both basal and stress conditions. Animals were injected with the MR antagonist spironolactone, the GR antagonist RU38486, or vehicle. Following collection of basal blood samples, rats were subjected to a 1-hr restraint stress, and additional blood samples were collected during and following stress. We found that ethanol-exposed and control females differed in their pattern of ACTH activity following receptor blockade. Both MR and GR blockade significantly increased basal ACTH levels in ethanol but not control animals compared with those in their vehicle-injected counterparts. In addition, MR blockade significantly increased ACTH levels in control females during restraint, whereas GR blockade increased ACTH levels in ethanol-exposed females during restraint and in control females during both restraint and recovery. This differential pattern of responsiveness under basal and stress conditions suggests an alteration in the balance between HPA drive and feedback regulation in ethanol-exposed compared with control females. Furthermore, together with the data from the adrenalectomy study discussed herein, it would appear that ethanol-induced changes in basal HPA regulation may be as important in mediating HPA hyperresponsiveness as those observed following stress. Although ethanol-exposed animals appear able to initiate compensatory mechanisms to maintain normal basal hormone levels under most circumstances, perturbations of the system reveal that tonic or basal HPA tone is, in fact, increased and may play a key role in raising the set point of responsivity following stress.
Altered Adrenal-Gonadal Interactions.
Recently, we began to pursue another line of research focused on investigating the possible role of ethanol-induced alterations in the interactions between the adrenal and gonadal axes in mediating the sexually dimorphic effects of ethanol on male and female offspring. Ongoing studies are examining the effects of gonadectomy with or without hormone replacement on basal and stress levels of the HPA hormones in both males and females. Preliminary data on males indicate that the increased stress-induced ACTH levels seen in ethanol-exposed compared with control males is eliminated by gonadectomy (58). These data suggest that HPA hyperresponsiveness in ethanol-exposed males is mediated, at least in part, by ethanol-induced changes in testosterone regulation or in HPA sensitivity to testosterone. The suppressive effects of testosterone and/or the stimulatory effects of gonadectomy on stress-induced ACTH release are altered in ethanol-exposed compared with control males. In contrast, the role of the gonadal hormones in HPA regulation in ethanol-exposed females appears to be more complex. Preliminary data (59) suggest that overall ethanol-exposed females appear to be less responsive than controls to the effects of estradiol on both reproductive and nonreproductive measures. Furthermore, ethanol-exposed females appear to have decreased tissue responsiveness to estradiol and altered hypothalamic-pituitary-gonadal sensitivity to acute stress. Further studies are needed to elucidate the real impact of prenatal ethanol exposure on interactions between the adrenal and gonadal axes in both males and females.
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Prenatal Alcohol Exposure Alters Development and Function of the Offspring Immune System |
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TopAbstractIntroductionMaternal Alcohol Consumption...Prenatal Alcohol Exposure Alters...Prenatal Ethanol Exposure...References |
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Impaired Immunity in Children with FAS.
Children prenatally exposed to alcohol have an increased incidence of bacterial infections, such as meningitis, pneumonia, otitis media, gastroenteritis, and sepsis, as well as urinary tract and upper respiratory tract infections (60, 61). Early studies reported that these children also had lower cell counts of eosinophils and neutrophils, decreased circulating E-rosette–forming lymphocytes, reduced mitogen-stimulated proliferative responses by peripheral blood leukocytes, and hypogammaglobulinemia (60).
Impaired Immunity in Animal Models of Prenatal Alcohol Exposure.
Research using animal models to investigate immune function of ethanol-exposed offspring has substantiated the clinical evidence of impaired immunity associated with FAS. In addition, it has greatly increased our understanding of these immune deficits in terms of both the spectrum of effects in different organ systems and the mechanisms that mediate the immunoteratogenic effects of alcohol.
Deficits in innate immunity have typically not been observed in animal studies. For example, one large study on nonhuman primates (Macaca nemestrina) found that in utero ethanol exposure did not result in significant differences in total numbers of white blood cells, leukocyte subsets, or monocyte phagocytic activity compared with that in control subjects (62). In contrast, marked deficits in adaptive immunity have been reported in ethanol-exposed animals, involving both cellular and humoral immunity. Furthermore, a marked sexual dimorphism in ethanol effects has been observed, with most deficits occurring in male offspring.
Deficits in Adaptive Immunity.
Inborn errors of immunocompetent cells result in immunodeficiency disorders or increased susceptibility to infections. Recurrent opportunistic infections and infections caused by ubiquitous microorganisms, such as bacteria, viruses, and fungi, typically occur with deficits in cell-mediated immunity. In contrast, deficits in B cells, immunoglobulins, complement, and phagocytes usually lead to infection by encapsulated and pyrogenic bacteria, such as Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus. In children with FAS, both types of recurrent infections have been reported, suggesting that both T-cell– and B-cell–mediated immunity are compromised (60). Work using animal models confirms these findings. For example, the immune response of ethanol-exposed neonates to the intestinal parasite Trichinella spiralis reveals a diminished capacity to respond to the pathogen, demonstrated by an increased intestinal worm count (63, 64). The abnormalities involve depressed T-cell– and B-cell–mediated responses, such as lower serum interleukin (IL)–2 and tumor necrosis factor (TNF) levels and lower immunoglobulin M (IgM) and IgG antibody titers. Interestingly, the detrimental effects of ethanol appear to increase across generations. That is, ethanol pups (second generation) mothered by fetal ethanol-exposed adult offspring (first generation) who themselves consumed ethanol during pregnancy show significantly reduced proliferative responses to T. spiralis antigen and concanavalin A (Con A) stimulation, lower titers of serum IgM and IgG anti–T. spiralis, and lower percentages of T cells and cytotoxic T cells compared with the first-generation ethanol-exposed and pair-fed pups (65). The next two sections will review studies that have described specific deficits in either cell-mediated or humoral immunity.
Deficits in Cell-Mediated Immunity.
Prenatal ethanol exposure alters thymus development in both rats and mice. Delayed ontogeny of the thymus (66), decreased total numbers of thymocytes, and diminished mitogen-induced cell proliferation have been reported in 18- to 19-day-old fetuses (67). Decreased thymus weight, size, and cell counts have also been observed at birth (68). One study in mice found that total thymocyte numbers returned to control levels as early as postnatal Day 6 (69). However, other studies reported that changes in thymus measures persist throughout the preweaning period and even into adolescence (67, 69–72). Similarly, mitogen-induced proliferative responses of thymic cells appears to be suppressed in ethanol-exposed males at weaning (69) but may be increased during the adolescent period (44 days of age) (73, 74). This increase in thymocyte proliferation during adolescence does not appear to be mediated by changes in number of GRs on the thymocytes, and further studies are needed to elucidate the mechanisms that underlie these varying effects of ethanol.
The adverse effects of ethanol on development of the thymus are confirmed by data from in vitro studies that used organ culture to assess the direct effects of ethanol. Total cell numbers and percentages of immature fetal thymocytes (CD4+CD8+) were found to be decreased in a dose responsive manner in ethanol-treated organ cultures (75). This decrease was shown to result from accelerated apoptosis, which then resulted in an increased percentage of more mature thymocytes expressing CD4+CD8– and 
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T-cell receptors (66, 75–77). Similar outcomes were observed in ethanol-exposed animals at 20–40 days of age. That is, thymic cell counts and total numbers of CD4+ and CD8+ cells were decreased throughout this period, and immature CD8+TCR+ and CD8+CD45RC+ thymocytes were reduced at 35 days of age (71). These results suggest that prenatal ethanol treatment alters the later stages of thymocyte maturation (e.g., after double positive [CD4+CD8+] thymocytes acquire T-cell receptor expression).
Prenatal ethanol exposure also appears to have long-term adverse effects on the immune system. Decreased numbers of Thy1.2+, CD4+, CD8+, and IgG+ splenocytes were found in ethanol-exposed compared with control animals (70, 78, 79). Decreased percentages of Thy1.2+ splenocytes were also found in pups born to mothers who consumed ethanol during pregnancy and lactation or lactation alone, indicating direct effects of ethanol on postnatal immune system development (70). Similarly, both rodent and primate studies indicate that splenic lymphocytes from ethanol-exposed males show decreased proliferative responses to mitogens from adolescence through young adulthood (62, 68, 80–83), although the response may normalize by 8 months of age (84, 86). Furthermore, data indicate deficits not only in the response of freshly isolated splenic T cells but also in the response of T blast cells (obtained following culture with Con A) to IL-2 or further Con A stimulation (80–83) in ethanol-exposed offspring. In contrast, ethanol-induced changes in mitogen-induced proliferation of thymocytes appear to normalize in young adulthood (73).
Additional evidence of defective T-cell immune responses in ethanol-exposed animals includes a decreased magnitude of local graft-versus-host response, monitored by lymph node weight, and diminished skin contact hypersensitivity induced by hapten 2,4,6-trinitrochlorobenzene (81).
Deficits in Humoral Immunity.
Humoral immunity appears to be less affected by fetal ethanol exposure than cellular immunity. Data indicate that ethanol-exposed rats do not differ from controls in serum Ig levels after primary and secondary immunization during the prepubertal period (86). Similar results have been observed in nonhuman primates after immunization with tetanus toxoid (62). However, studies of B-cell lineages in murine hematopoietic organs, including bone marrow, spleen, and liver, show abnormal development from fetal life through adolescence. Decreased total numbers of splenic B cells at birth and throughout the preweaning period, as well as decreased proliferative responses of B cells to lipopolysaccharide (LPS; a bacterial cell wall component), were found in ethanol-exposed compared with control animals (86). In addition, a decrease in B220+ hematopoietic cells (a component of the B-cell lineage) on postconception Days 19 and 21 (87) and a delayed maturation of B cells on gestation Day 18 (88) were seen in the liver of ethanol-exposed fetuses. At birth, numbers of both immature (IgM+IgD–) and mature (IgM+IgD+) B cells in spleen and bone marrow were found to be decreased. Most of these recovered to normal levels by 3 to 4 weeks after birth, except for pre-B cells (B220+IgM–) in bone marrow, which remained at lower levels through 5 weeks of life (89). These data demonstrate defects in the B-cell lineage in ethanol-exposed animals, although deficits are not as prominent as those in T-cell development.
Prenatal Ethanol Exposure Increases Vulnerability to Stress-Induced Suppression of Immune Function.
Studies have shown that the challenge to the immune system of chronic stress in adulthood differentially affects animals that have been exposed to ethanol prenatally. That is, similar to the data that demonstrate that basal hormone levels are typically normal in ethanol-exposed animals, specific deficits in the immune system may not be observed under nonstressed conditions but become apparent following exposure to stressors (79, 90). In addition, as seen for endocrine activity, sexually dimorphic effects of prenatal ethanol on immune function are observed.
In ethanol-exposed males but not females, exposure to chronic intermittent stressors selectively downregulated the numbers of thymic and peripheral blood CD43+ cells and peripheral blood CD4+ T cells and marginally decreased the numbers of peripheral blood MHC class II Ia+ (antigen presenting) cells. In contrast, CD43 antigen expression on peripheral blood T cells was selectively upregulated (79). Because these stress-induced immune abnormalities occurred selectively in ethanol-exposed males, despite the finding that stress-induced adrenal hypertrophy was less than that in ethanol-exposed females (79), it is possible that males may be more sensitive to small changes in glucocorticoid levels than females. The possible role of estrogen, as an immunoprotective hormone, and testosterone, as an immunosuppressive hormone, in these sexually dimorphic immune responses remains to be determined.
We have also observed significant interactions between prenatal ethanol and chronic cold stress in female offspring, one of the first demonstrations of fetal ethanol effects on immune function in females (90). After 1 day of cold stress, ethanol-exposed females had significantly increased poke-weed mitogen–induced and Con A–induced lymphocyte proliferation compared with control females. No differences occurred in immune responsiveness among males from the three prenatal groups. However, following 1 or 3 days of cold stress, ethanol-exposed males had significantly increased basal corticosterone levels compared with their counterparts not exposed to cold. These findings are consistent with and extend the data of Halasz et al. (39), who found that the challenge of long-term alcohol exposure in adulthood selectively increased Con A–induced lymphocyte proliferation in ethanol-exposed females but not males. Interestingly, our data are the first to demonstrate an effect of prenatal ethanol and stress on lymphocyte responses to pokeweed mitogen, a T-cell–dependent B-cell mitogen. These data may have important mechanistic implications for understanding the immune deficits seen in ethanol-exposed animals and children prenatally exposed to alcohol, because defective interactions between T and B cells would significantly hinder the development of a normal immune response.
Cytokines and the HPA Axis.
Prenatal ethanol exposure blunts the LPS-induced febrile response in ethanol-exposed compared with control males (91). It has been suggested that a decreased response of central thermoregulatory systems to IL-1ß caused by decreased hypothalamic IL-1ß production after LPS administration may mediate this blunted fever response in ethanol-exposed animals, possibly through an impaired release of endogenous pyrogens (92–94). Interestingly, both maternal adrenalectomy and sham surgery abrogate the effect of ethanol on IL-1ß–induced febrile responses in female offspring, but only adrenalectomy has an effect on male offspring, suggesting that maternal adrenal mediators play an important role in the blunted febrile response of males but that nonadrenal mediators participate in modulation of thermo-regulatory systems in females (95, 96).
During the preweaning period, in parallel with the blunted hormonal responses to stressors, ethanol-exposed animals also exhibit blunted ACTH, ß-endorphin, and TNF-
responses to immune challenges (IL-1ß, LPS), which may persist in males but not females until 35 days of age (37, 94, 97, 98). This altered responsiveness may be mediated by an altered ability of IL-1 to stimulate secretion of ACTH and related peptides (97). Interestingly, ovariectomy before puberty eliminates the difference in the ACTH response between ethanol and control females (37), suggesting that prenatal ethanol exposure and female sex hormones have a common pathway in regulating HPA activity. In contrast, in adulthood, increased ACTH and corticosterone responses to immune signals such as LPS or IL-1ß are observed in ethanol-exposed animals, in parallel with the HPA hyper-responsiveness to stressors that has been observed (94). However, the cytokine response to immune challenges is not similarly increased in ethanol-exposed offspring. Thus, it appears that cytokines probably do not mediate the increased HPA responsiveness to immune signals seen in ethanol-exposed animals (94).
In contrast, we recently found that ethanol-exposed males exhibit increased plasma levels of proinflammatory cytokines following repeated stress (99, 100). Animals were exposed to 9 days of restraint stress, 1 hr per day, and on Day 10 were injected with LPS and terminated at various times after injection. We found that although corticosterone responses to LPS were comparable among groups at the time points measured (60–180 mins), ethanol-exposed animals had greater and more sustained elevations of IL-1ß, TNF-, and IL-6 compared with controls. These data support and extend our previous studies, suggesting that although ethanol-exposed animals may not differ in cytokine responses under basal or nonstressed conditions, they may be more vulnerable to the effects of stress on immune function.
Mechanisms of Ethanol’s Effects on the Developing Immune System.
Direct and Indirect Effects of Ethanol on the Fetus.
Ethanol can directly disrupt the development of the fetal thymus. The highly specified microenvironment of the thymus provided by epithelial and mesenchymal cells is pivotal in attracting immature lymphoid precursors to enable them to be selected and differentiated into mature T cells. Excess ethanol at the time of thymus development inhibits the development of the thymic epithelium and disrupts the microenvironment for maturation of T cells, which leads to impaired T-cell immunity (101). In this regard, it is noteworthy that FAS shares a number of the clinical characteristics of DiGeorge syndrome (102), a congenital immune deficiency syndrome that involves mainly T cells, caused by an abnormality in the development of neural crest–derived components of the thymus and parathyroid glands (103).
Prenatal ethanol exposure also selectively alters thymic gene expression in male fetuses. A significant increase in thymic CRH and decrease in thymic pro-opiomelanocortin gene expression was observed on Day 19 of gestation. These changes appeared unrelated to fetal or maternal corticosterone levels but are possibly induced by the fetal testosterone surge (26). However, the finding that ontogeny of GR sites per thymocyte is different in ethanol-exposed compared with control males in the first 2 months of life suggests that the glucocorticoid hormones play a role in the differential thymic development of ethanol-exposed and control animals (73). Further studies are needed to clarify the role of the glucocorticoids in the altered thymic development of ethanol-exposed offspring.
Data suggest that altered IL-2/IL-2 receptor interactions may play a role in the development of altered immune function in ethanol-exposed animals. For example, despite showing a reduced proliferative response to mitogens, ethanol-exposed animals appear to have normal levels of IL-2 production, IL-2 receptor expression and distribution, calcium influx in T cells, and binding of IL-2 to its receptor. However, the internalization and/or use of IL-2 by lymphoblasts appears to be reduced, and the half-time for dissociation of IL-2 from its receptor is increased in T cells from ethanol-exposed compared with control rats. These findings suggest that impaired intracellular signaling events mediated by IL-2/IL-2R interactions may underlie the immune deficits observed in ethanol-exposed animals (82, 104, 105). In contrast, direct treatment of lymphocytes with acetaldehyde-serum protein, which is formed in vivo as a metabolite of ethanol consumption, was shown to result in decreased IL-2 production but not IL-2 receptor expression (106). This suggests that decreased IL-2 production may also contribute to impaired proliferative responses, at least under some conditions.
Altered neurotransmitter regulation of immune function may also play a role in altered immune function of ethanol-exposed animals. Decreased levels of norepinephrine and ß-adrenoreceptors in the lymphoid organs and diminished synaptic transmission in the spleen and thymus but not the heart of ethanol-exposed compared with control animals has also been reported (81). These changes could result from a higher rate of norepinephrine turnover, which in turn could affect immune capacity through norepinephrine’s role in mediating IL-2 secretion and cytotoxic T-cell responses (81).
Immunity at the Fetal-Placental Interface.
Maternal immunity at the fetal-placental interface is biased toward humoral immunity, and cellular immunity is suppressed to prevent fetal rejection. Cell-mediated immunity is skewed toward T helper cell (Th2) responses and production of cytokines such as IL-3, IL-4, and IL-5 rather than toward Th1 responses and cytokines such as IL-2 and interferon- (107). The observation of elevated cord blood IgE concentrations in ethanol-exposed infants, indicating increased activity of Th2-type responses, supports this notion (108). It has been suggested that progesterone and CD4+ regulatory T cells may be the key factors that enable the fetus to evade immune rejection by mother and thus maintain pregnancy. Progesterone suppresses cytotoxic activity of lymphocytes from pregnant women via progesterone receptors (109). Recent studies show that a timely increase in CD4+CD25+ regulatory T cells, systematically and locally at the maternal-fetal interface, plays an important role in maintaining maternal-fetal tolerance by suppressing an allogeneic response directed against the fetus (110, 111). Maternal ethanol consumption could alter the balance between regulatory T cells and T effector cells and thus contribute to the increased incidence of spontaneous abortions and premature births. Recent preliminary studies in our laboratory (112) suggest that long-term alterations in CD4+ regulatory T cells in ethanol-exposed males may play a role in mediating deficits in T-cell function. We examined dexamethasone-induced apoptosis and rescue by IL-2 of CD4+CD25+ regulatory T cells from ethanol-exposed, pair-fed, and control males. We found that CD4+CD25+ regulatory T cells from ethanol-exposed males are more resistant than those of controls to apoptosis in the presence of IL-2, resulting in increased survival of T regulatory cells. Because T regulatory cells are suppressive in nature, the increased numbers of surviving T regulatory cells could, at least partially, play a role in the immune deficits observed in ethanol-exposed animals.
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Prenatal Ethanol Exposure Reprograms Fetal HPA and Immune Function |
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TopAbstractIntroductionMaternal Alcohol Consumption...Prenatal Alcohol Exposure Alters...Prenatal Ethanol Exposure...References |
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The biological significance of physiological and behavioral programming is not known. However, it has been suggested that environmental factors that act on the mother and fetus can alter the set point or responsiveness of physiological systems and thus prepare the organism for the environment into which it will be born and develop. If, however, this process is initiated by adverse factors during pregnancy (e.g., placental insufficiency) or if the environmental circumstances later in life are different from what was anticipated, then the physiological adaptations that occurred might in themselves result in maladaptive responses and ultimately predispose the organism to disease (13, 113).
The underlying processes that link early growth restriction with these long-term health consequences are not fully understood. However, it is generally accepted that low birth weight per se is unlikely to cause these increased risks for disease. Rather, birth weight likely serves as a marker for the effects of early life events, and a common factor or factors probably underlie both the intrauterine growth retardation and the altered physiological responsiveness (115). The resetting of key hormonal systems by early environmental events may be one mechanism that links early life experiences with long-term health consequences. Studies have specifically identified the HPA axis as one of the key systems likely involved. The HPA axis is highly susceptible to programming during development (13, 114), and recent studies by Phillips et al. (116, 117) demonstrate strong correlations among birth weight, plasma cortisol levels, and the development of hypertension and Type II diabetes. These investigators have suggested that intrauterine programming of the HPA axis may be a mechanism that underlies the observed associations between low birth weight and increased risk of disease.
The routes by which the fetal and early neonatal environment program adult HPA function and behavior are described eloquently in recent papers by Matthews (13) and Welberg and Seckl (113) and illustrated in Figure 1
. As described, structures of the developing limbic system, primarily the hippocampus, hypothalamus, and anterior pituitary, synthesize high levels of corticosteroid receptors and are highly sensitive to glucocorticoids. Exposure to high levels of glucocorticoids during early life can alter the development and subsequent function of both the limbic system and the HPA axis. The limbic system, particularly the hippocampus, regulates HPA activity, and in turn endogenous glucocorticoids modify many aspects of limbic system function. Mechanisms that underlie these mutual effects involve modification of developing neurotransmitter systems and their transporter mechanisms in the brainstem, the development of corticosteroid receptors expression in the hippocampus, and the development and responsiveness of the hypothalamic PVN, which regulates glucocorticoid secretion. Examples of prenatal events that program HPA function include maternal stress, exposure to synthetic glucocorticoids, and nutrient restriction. For example, maternal glucocorticoid treatment programs HPA regulation in adult offspring in a sex-specific manner (118). Examples of postnatal events that program HPA function include early handling, alterations in maternal behavior, exposure to exogenous glucocorticoids, and infection. The overall effect of programming is altered exposure to endogenous glucocorticoids throughout life. This will in turn modify behavior, cognition, learning, memory, and emotion and predispose the individual to cardiovascular and metabolic diseases. The mechanisms that underlie programming of adult HPA function depend on dose, timing, and duration of exposure. Further, although the role of environmental factors in fetal programming is emphasized, it is understood that perinatal environmental and genetic factors mutually influence each other in determining HPA activity and behavior later in life. Moreover, although the effects of programming are often long-lasting, data have shown that postnatal and later environmental events can modulate the effects of prenatal or early programming (13, 113).
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We hypothesize that prenatal ethanol exposure reprograms the fetal HPA axis, resulting in long-term alterations in HPA regulation and responsiveness under both basal and stress conditions and increasing HPA tone throughout life. Given the important role of the HPA hormones in so many aspects of physiological and behavioral function, fetal programming of HPA activity likely underlies some of the long-term behavioral, cognitive, and immune deficits that are observed following prenatal ethanol exposure. Furthermore, it is essential that we begin to explore possible epigenomic mechanisms that underlie the long-term effects of fetal programming in ethanol-exposed offspring. We hypothesize that mechanisms similar to those described by Weaver et al. (119) may be mediating the altered HPA regulation and responsiveness observed following prenatal ethanol exposure. The fact that alcohol itself is known to cause alterations in the methylation state within cells provides support for this hypothesis.
In view of the evidence (116, 117) that fetal programming of the HPA axis underlies, at least in part, the connection between the early environment and adult stress-related and behavioral disorders in humans, further studies are essential to elucidate the mechanisms that underlie prenatal ethanol effects on programming of HPA activity. The data from these investigations will have important implications for the development of therapeutic interventions focused at reversing the long-term adverse effects of prenatal alcohol exposure on behavioral and physiological function of the offspring.
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TopAbstractIntroductionMaternal Alcohol Consumption...Prenatal Alcohol Exposure Alters...Prenatal Ethanol Exposure...References |
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