Endotoxin-induced Chorioamnionitis Modulates Innate Immunity of Monocytes in Preterm Sheep
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美国呼吸和危急护理医学 2005年第1期
University Children's Hospital, Wuerzburg, Germany
Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio
the School of Women's and Infants' Health, The University of Western Australia, Perth, Australia
ABSTRACT
The preterm fetus is immune naive and has immature innate immune function. Although the preterm fetus is frequently exposed to chorioamnionitis, the effects of exposure of the fetal lung to inflammation on innate immune responses are unknown. Using the fetal sheep model of chorioamnionitis, cord blood monocytes were isolated from preterm lambs 1 to 14 days after intra-amniotic endotoxin injection, cultured for approximately 16 hours, and challenged with endotoxin in vitro. Compared with monocytes from adult sheep, the preterm monocytes produced less H2O2 and interleukin-6, and toll-like receptor 4 expression was decreased. Three days after intra-amniotic endotoxin exposure, preterm monocyte responses to in vitro endotoxin challenge demonstrated decreased H2O2 and interleukin-6 production and decreased CD14 and major histocompatibility complex class II expression. Preterm monocyte responses 7 to 14 days after endotoxin tended to exceed those of adults and preterm control animals indicating augmented function. In contrast, a second intra-amniotic endotoxin injection 7 days after the initial endotoxin exposure suppressed monocyte function at 14 days. The fetal monocytes demonstrated patterns of responses consistent with endotoxin tolerance (immune paralysis) as well as maturation of function. Modulation of fetal innate immune responses by exposure to inflammation may alter subsequent immune adaptation after birth.
Key Words: endotoxin tolerance fetal immunity nosocomial infection priming
The fetus is generally recognized as immunologically nave, but chronic indolent chorioamnionitis caused by commensal or low pathogenic organisms occurs in the majority of pregnancies complicated by preterm labor and delivery before 30 weeks gestation (1eC3). Fetal exposure to inflammation/infection or a fetal systemic inflammatory response increases neonatal morbidity and mortality and neurodevelopmental or lung injury (4eC7). Approximately 30% of preterm infants with a very low birth weight also develop nosocomial infections that often have no identifiable source (8, 9). The innate immune system of the preterm is considered to be immature because multiple host defense proteins are low, immune cell numbers are low, and immune cell functions are low relative to cells from older children or adults (10).
Very little information is available about innate immune responses and their modulation in the fetus, although such responses may be central to a number of the injuries associated with preterm birth (7). Fetal exposure to inflammation might either increase or suppress subsequent immune responses (1). Immune responsiveness could be decreased by exposure to inflammatory products resulting in the fetal equivalent of endotoxin tolerance or innate immune paralysis. For example, plasma interleukin (IL)-6 levels are strikingly decreased in fetal sheep after repetitive injections of low dose Escherichia coli endotoxin (11). An example of increased responsiveness is the augmented production of H2O2 and phagocytosis by blood monocytes in fetal sheep 7 days after maternal betamethasone exposure (12).
Chorioamnionitis induced by intra-amniotic endotoxin is primarily a pulmonary exposure for the fetus and results in lung inflammation and striking lung maturation as a result of direct contact of endotoxin or IL-1 with the fetal lung (13eC15). Fetal inflammation occurs mainly in the lung with a modest systemic inflammatory response (lower granulocytes, modest plasma elevations of IL-6) (16, 17). We hypothesized that a result of the modest systemic inflammatory response to intra-amniotic endotoxin-induced lung inflammation would be a modulation of the responses of blood monocytes to a later exposure to endotoxin. Altered monocyte function could affect subsequent immune function in the fetus and newborn. To test this hypothesis, we gave fetal sheep injections of intra-amniotic endotoxin for intervals from 1 to 14 days before preterm delivery. Monocyte function was evaluated by measuring phagocytosis of E. coli, hydrogen peroxide production, and IL-6 production in response to endotoxin stimulation in vitro. Several aspects of endotoxin binding and signal transduction were explored by quantification of endotoxin receptor (CD14) expression, expression of endotoxin signal transducing receptor (toll-like receptor 4 [TLR4]) and major histocompatibility complex (MHC) class II on the monocytes.
METHODS
Animals
The animals were studied in Western Australia with approved protocols by the Cincinnati Children's Hospital (Cincinnati, OH) and the Western Australian Department of Agriculture. Time-mated Merino ewes with singleton fetuses were randomly assigned in groups of five to seven animals to receive a single dose of 10 mg endotoxin (E. coli 055:B5; Sigma, St. Louis, MO) by intra-amniotic injection at 1, 3, 7, or 14 days or at 7 and 14 days before cesarean delivery at 124 days gestational age (18).
Monocyte Isolation and Culture
Fetal cord blood drawn at delivery and blood from healthy pregnant ewes was used to isolate monocytes using Percoll gradients as described before (12, 19). Cells were counted using trypan blue to evaluate viability, and the cells were plated on culture dishes in RPMI 1640 media supplemented by heat-inactivated fetal calf serum. After incubation at 37°C for 2 hours, nonadherent cells were removed by washing. The adherent cell population was 89 ± 4% monocytes for all treatment and control groups. Monocytes in other culture dishes were cultured overnight, and the experiments were done after approximately 16 hours in culture.
Phagocytosis of E. coli
After overnight incubation, monocytes were exposed to fluorescein isothiocyanateeClabeled E. coli (Sigma Chemicals) for 6 hours. Monocytes were suspended, and trypan blue (1.25 mg/ml) was added to one aliquot to measure ingested bacteria only. The total bound and ingested bacteria were measured by flow cytometry (12, 19).
Hydrogen Peroxide Production and IL-6 Concentration
After overnight incubation, monocytes were exposed to 10 and 100 ng/ml E. coli endotoxin for 6 hours (20). Production of hydrogen peroxide by 106 monocytes was measured with an assay kit (OXIS International, Portland, OR). Control samples were exposed to saline instead of endotoxin and were included in all experiments. IL-6 was measured in the cell culture medium of endotoxin-stimulated monocytes with an ovine-specific ELISA (17).
Expression of CD14, TLR4, and MHC II on Monocytes
Sheep-specific CD14 endotoxin receptor and MHC II antibodies (VMRD Inc., Pullman, WA) and a species cross-reacting anti-TLR4 antibody (BD Biosciences, San Jose, CA) were used to measure these surface receptors by fluorescence-activated cell sorter analysis using appropriate secondary antibodies. Control staining was performed with isotype antibodies and with secondary antibody alone to evaluate background fluorescence (17).
Data Analysis
Results are presented as mean ± SEM. Comparisons between endotoxin-treated groups and untreated control animals were by analyses of variance with Student-Newman-Keuls tests used for post hoc analyses. Results were compared with monocytes from healthy adult sheep. Statistical significance was accepted at p < 0.05.
RESULTS
Description of Animals
The birth weights of the animals in the control and intra-amniotic endotoxin-exposed groups were similar (Table 1). Intra-amniotic endotoxin increased total circulating white blood cells for the groups exposed 7 days or 7 and 14 days before delivery but not significantly at 14 days. Neutrophils accounted for the increase in total cell number, whereas blood monocyte numbers did not change with the in vivo endotoxin exposure.
Phagocytosis
Phagocytosis of E. coli by blood monocytes from adult sheep tended to be higher than for fetal sheep at 124 days gestation, but this difference was not statistically significant (Table 1). Intra-amniotic endotoxin had no effect on the phagocytosis of E. coli by cord bloodeCderived monocytes from the preterm lambs.
H2O2 Production
Endotoxin-stimulated blood monocytes from adult sheep produced more H2O2 than did cord blood monocytes from the control preterm lambs after stimulation with 10 or 100 ng/ml endotoxin (Figure 1). Monocytes from adult or fetal sheep produced very little H2O2. Monocytes from lambs exposed to intra-amniotic endotoxin for 1 day and 3 days had suppressed H2O2 production when compared with monocytes from the preterm control animals. However, 14 days after the intra-amniotic endotoxin, the endotoxin-induced H2O2 production exceeded considerably that of both the control preterm and adult monocytes. In striking contrast to the 14-days endotoxin exposed group, H2O2 production was very low after fetal exposure to intra-amniotic endotoxin 7 and 14 days before the in vitro endotoxin challenge.
IL-6 Concentration
Endotoxin concentrations of 10 or 100 ng/ml increased IL-6 concentration in the media of cultured monocytes from adult and preterm control sheep relative to the low IL-6 concentration without endotoxin stimulation (Figure 2). A prior exposure to intra-amniotic endotoxin 3 days before delivery reduced the concentration of IL-6 in culture media after re-exposure to endotoxin in vitro. The IL-6 concentrations in the media of monocytes exposed to 10 or 100 ng/ml endotoxin after the fetal endotoxin exposure were similar to the concentrations of IL-6 from monocytes from adult sheep. The 7- and 14-day repeated exposures of fetuses to intra-amniotic endotoxin had no effects on endotoxin-induced IL-6 concentration compared with preterm control animals.
CD14, TLR4, and MHC II Expression
CD14 expression was not different between monocytes from control preterm lambs and monocytes from adult animals after stimulation with 100 ng/ml endotoxin (Figure 3A). Similar results were obtained with 10 ng/ml endotoxin (data not shown). CD14 expression was decreased qualitatively 1 and 7 days after intra-amniotic endotoxin exposure and significantly after 3 days. The signal-transducing molecule for endotoxin mediated cell activation, TLR4, had a different pattern of expression. TLR4 expression was higher by approximately twofold on monocytes from adult animals than those from preterm control animals (Figure 3B). The expression was increased in cord bloodeCderived monocytes 14 days after intra-amniotic endotoxin. The expression was not different at the other time points. MHC II expression was similar for adult and preterm control monocytes (Figure 3C). Expression was strikingly decreased 3 days after intra-amniotic endotoxin.
DISCUSSION
The temporary suppression of innate immune cell responses by endotoxin has been referred to as "endotoxin tolerance" (21, 22). Endotoxin tolerance is used to describe in vivo models in which a normally lethal endotoxin dose can be tolerated by animals that were previously exposed to sublethal dose of endotoxin (21). Chronically catheterized preterm fetal sheep respond to intravascular endotoxin with a tolerance type response (11). Endotoxin tolerance/immune paralysis is a risk factor for multiorgan failure or sepsis in hospitalized patients (23). Preterm infants are extremely susceptible to nosocomial infections several weeks after preterm birth, at a time when they are often doing well (24). This clinical susceptibility to infection is consistent with immune paralysis, but we are not aware of studies that address this possibility.
The responses of monocytes from fetal sheep to endotoxin are complex. The initial suppression of H2O2 and IL-6 and decreased CD14 and MHC II receptors are typical of endotoxin tolerance. In contrast, the increased production of H2O2 and IL-6 and increased TLR4 expression 14 days after endotoxin exposure indicate an augmented innate host response relative to the control fetal monocytes. However, the repeated exposure at a 7-day interval resulted in profound suppression of H2O2 production. A similar pattern of response occurred with the different endotoxin concentrations tested in vitro. Clinically, chorioamnionitis is often indolent and persists for weeks or months (2). Our results suggest that chronic fetal exposure to inflammation could cause a profound and persistent tolerance-type response and thus put these infants at risk of nosocomial infection (25).
These experiments are a first evaluation of how fetal exposure to inflammation might modulate or affect innate immune responses. The concepts of endotoxin tolerance have been developed primarily in mice and the adult human. In adult mice, endotoxin exposure decreases tumor necrosis factor- and increases IL-1 secretion by monocytes (21). The mechanisms proposed for endotoxin tolerance in humans and mice are controversial but may include downregulation of endotoxin receptors, lipopolysaccharide-binding protein, or CD14 and alterations in the signaling pathways to decrease nuclear factor-B nuclear translocation as well as changes in the cytokine expression profile of the cells (21).
We did not use catheterized animals to avoid the stress of fetal surgery and the potential for infection in catheterized animals. These studies of the fetal innate immune system are unique because the immune system is naive and immature. An example of differences between the preterm and the adult is the observation that the fetal sheep does not respond to ovine tumor necrosis factor- but has systemic and lung inflammatory responses to IL-1 (13, 26). Fetal responses to IL-6 have not been evaluated. The decrease in monocyte surface receptors and suppressed function with single or repetitive fetal endotoxin exposure are consistent with an immune paralysis-type response by immature monocytes (22). The enhanced response 14 days after intra-amniotic endotoxin exposure may represent recovery from immune paralysis together with a maturation of monocyte function. A similar pattern of suppression followed by increased function occurs in response to maternal glucocorticoid exposure (12, 18). Fetal monocytes have inhibited function that resolves to resemble the function of adult monocytes by 7 days after maternal betamethasone exposure (12).
Another unique aspect of these studies is that the intra-amniotic exposure to endotoxin both induces lung maturation and impairs lung development (27eC29). Lung maturation was induced in the fetal sheep by doses of endotoxin from 1 to 100 mg. Similar amounts of intra-amniotic endotoxin were used in murine models of fetal exposure to inflammation (18, 30). In contrast, intravascular endotoxin at a much smaller dose of 5 e/kg causes either fetal death or tolerance to repeated intravascular doses of 1 e/kg in fetal sheep (11). Despite the high amounts of intra-amniotic endotoxin given to fetal sheep, fetal well-being or growth were not impaired (18). The lung inflammation and maturation responses require the direct contact of endotoxin with the fetal lung, presumably by the mixing of fetal lung fluid with amniotic fluid as a result of fetal breathing (15). The intra-amniotic endotoxin caused subtle changes in fetal circulating white blood cells, but the response was not characteristic of a systemic inflammatory response (17). Nevertheless, systemic monocytes were modified to have an immune paralysis-type phenotype by fetal lung inflammation. We previously found that fetal exposure to intra-amniotic endotoxin given 30 days before preterm delivery resulted in increased monocytes and lymphocytes in the alveolar lavages of preterm lambs (31). Therefore, intra-amniotic endotoxin can alter the subsequent immune function of the lung and the systemic monocytes. A cautionary note is that monocytes/macrophages from different tissue locations can respond differently after exposure to inflammation (32). The integrated fetal responses to inflammation remain to be explored.
The long-term implications of alterations in fetal immune responses are just beginning to be explored. Recently, increased cord blood levels of IL-4 and interferon- were associated with less atopy and asthma (33). The indirect evidence that increased maternal antibiotic use during pregnancy increases the risk of asthma in children supports the concept that fetal immune responses may change subsequent immune responses (34, 35). This fetal sheep model will be useful for future studies of long-term effects of lung and systemic innate immune modulation after preterm and term birth.
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Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio
the School of Women's and Infants' Health, The University of Western Australia, Perth, Australia
ABSTRACT
The preterm fetus is immune naive and has immature innate immune function. Although the preterm fetus is frequently exposed to chorioamnionitis, the effects of exposure of the fetal lung to inflammation on innate immune responses are unknown. Using the fetal sheep model of chorioamnionitis, cord blood monocytes were isolated from preterm lambs 1 to 14 days after intra-amniotic endotoxin injection, cultured for approximately 16 hours, and challenged with endotoxin in vitro. Compared with monocytes from adult sheep, the preterm monocytes produced less H2O2 and interleukin-6, and toll-like receptor 4 expression was decreased. Three days after intra-amniotic endotoxin exposure, preterm monocyte responses to in vitro endotoxin challenge demonstrated decreased H2O2 and interleukin-6 production and decreased CD14 and major histocompatibility complex class II expression. Preterm monocyte responses 7 to 14 days after endotoxin tended to exceed those of adults and preterm control animals indicating augmented function. In contrast, a second intra-amniotic endotoxin injection 7 days after the initial endotoxin exposure suppressed monocyte function at 14 days. The fetal monocytes demonstrated patterns of responses consistent with endotoxin tolerance (immune paralysis) as well as maturation of function. Modulation of fetal innate immune responses by exposure to inflammation may alter subsequent immune adaptation after birth.
Key Words: endotoxin tolerance fetal immunity nosocomial infection priming
The fetus is generally recognized as immunologically nave, but chronic indolent chorioamnionitis caused by commensal or low pathogenic organisms occurs in the majority of pregnancies complicated by preterm labor and delivery before 30 weeks gestation (1eC3). Fetal exposure to inflammation/infection or a fetal systemic inflammatory response increases neonatal morbidity and mortality and neurodevelopmental or lung injury (4eC7). Approximately 30% of preterm infants with a very low birth weight also develop nosocomial infections that often have no identifiable source (8, 9). The innate immune system of the preterm is considered to be immature because multiple host defense proteins are low, immune cell numbers are low, and immune cell functions are low relative to cells from older children or adults (10).
Very little information is available about innate immune responses and their modulation in the fetus, although such responses may be central to a number of the injuries associated with preterm birth (7). Fetal exposure to inflammation might either increase or suppress subsequent immune responses (1). Immune responsiveness could be decreased by exposure to inflammatory products resulting in the fetal equivalent of endotoxin tolerance or innate immune paralysis. For example, plasma interleukin (IL)-6 levels are strikingly decreased in fetal sheep after repetitive injections of low dose Escherichia coli endotoxin (11). An example of increased responsiveness is the augmented production of H2O2 and phagocytosis by blood monocytes in fetal sheep 7 days after maternal betamethasone exposure (12).
Chorioamnionitis induced by intra-amniotic endotoxin is primarily a pulmonary exposure for the fetus and results in lung inflammation and striking lung maturation as a result of direct contact of endotoxin or IL-1 with the fetal lung (13eC15). Fetal inflammation occurs mainly in the lung with a modest systemic inflammatory response (lower granulocytes, modest plasma elevations of IL-6) (16, 17). We hypothesized that a result of the modest systemic inflammatory response to intra-amniotic endotoxin-induced lung inflammation would be a modulation of the responses of blood monocytes to a later exposure to endotoxin. Altered monocyte function could affect subsequent immune function in the fetus and newborn. To test this hypothesis, we gave fetal sheep injections of intra-amniotic endotoxin for intervals from 1 to 14 days before preterm delivery. Monocyte function was evaluated by measuring phagocytosis of E. coli, hydrogen peroxide production, and IL-6 production in response to endotoxin stimulation in vitro. Several aspects of endotoxin binding and signal transduction were explored by quantification of endotoxin receptor (CD14) expression, expression of endotoxin signal transducing receptor (toll-like receptor 4 [TLR4]) and major histocompatibility complex (MHC) class II on the monocytes.
METHODS
Animals
The animals were studied in Western Australia with approved protocols by the Cincinnati Children's Hospital (Cincinnati, OH) and the Western Australian Department of Agriculture. Time-mated Merino ewes with singleton fetuses were randomly assigned in groups of five to seven animals to receive a single dose of 10 mg endotoxin (E. coli 055:B5; Sigma, St. Louis, MO) by intra-amniotic injection at 1, 3, 7, or 14 days or at 7 and 14 days before cesarean delivery at 124 days gestational age (18).
Monocyte Isolation and Culture
Fetal cord blood drawn at delivery and blood from healthy pregnant ewes was used to isolate monocytes using Percoll gradients as described before (12, 19). Cells were counted using trypan blue to evaluate viability, and the cells were plated on culture dishes in RPMI 1640 media supplemented by heat-inactivated fetal calf serum. After incubation at 37°C for 2 hours, nonadherent cells were removed by washing. The adherent cell population was 89 ± 4% monocytes for all treatment and control groups. Monocytes in other culture dishes were cultured overnight, and the experiments were done after approximately 16 hours in culture.
Phagocytosis of E. coli
After overnight incubation, monocytes were exposed to fluorescein isothiocyanateeClabeled E. coli (Sigma Chemicals) for 6 hours. Monocytes were suspended, and trypan blue (1.25 mg/ml) was added to one aliquot to measure ingested bacteria only. The total bound and ingested bacteria were measured by flow cytometry (12, 19).
Hydrogen Peroxide Production and IL-6 Concentration
After overnight incubation, monocytes were exposed to 10 and 100 ng/ml E. coli endotoxin for 6 hours (20). Production of hydrogen peroxide by 106 monocytes was measured with an assay kit (OXIS International, Portland, OR). Control samples were exposed to saline instead of endotoxin and were included in all experiments. IL-6 was measured in the cell culture medium of endotoxin-stimulated monocytes with an ovine-specific ELISA (17).
Expression of CD14, TLR4, and MHC II on Monocytes
Sheep-specific CD14 endotoxin receptor and MHC II antibodies (VMRD Inc., Pullman, WA) and a species cross-reacting anti-TLR4 antibody (BD Biosciences, San Jose, CA) were used to measure these surface receptors by fluorescence-activated cell sorter analysis using appropriate secondary antibodies. Control staining was performed with isotype antibodies and with secondary antibody alone to evaluate background fluorescence (17).
Data Analysis
Results are presented as mean ± SEM. Comparisons between endotoxin-treated groups and untreated control animals were by analyses of variance with Student-Newman-Keuls tests used for post hoc analyses. Results were compared with monocytes from healthy adult sheep. Statistical significance was accepted at p < 0.05.
RESULTS
Description of Animals
The birth weights of the animals in the control and intra-amniotic endotoxin-exposed groups were similar (Table 1). Intra-amniotic endotoxin increased total circulating white blood cells for the groups exposed 7 days or 7 and 14 days before delivery but not significantly at 14 days. Neutrophils accounted for the increase in total cell number, whereas blood monocyte numbers did not change with the in vivo endotoxin exposure.
Phagocytosis
Phagocytosis of E. coli by blood monocytes from adult sheep tended to be higher than for fetal sheep at 124 days gestation, but this difference was not statistically significant (Table 1). Intra-amniotic endotoxin had no effect on the phagocytosis of E. coli by cord bloodeCderived monocytes from the preterm lambs.
H2O2 Production
Endotoxin-stimulated blood monocytes from adult sheep produced more H2O2 than did cord blood monocytes from the control preterm lambs after stimulation with 10 or 100 ng/ml endotoxin (Figure 1). Monocytes from adult or fetal sheep produced very little H2O2. Monocytes from lambs exposed to intra-amniotic endotoxin for 1 day and 3 days had suppressed H2O2 production when compared with monocytes from the preterm control animals. However, 14 days after the intra-amniotic endotoxin, the endotoxin-induced H2O2 production exceeded considerably that of both the control preterm and adult monocytes. In striking contrast to the 14-days endotoxin exposed group, H2O2 production was very low after fetal exposure to intra-amniotic endotoxin 7 and 14 days before the in vitro endotoxin challenge.
IL-6 Concentration
Endotoxin concentrations of 10 or 100 ng/ml increased IL-6 concentration in the media of cultured monocytes from adult and preterm control sheep relative to the low IL-6 concentration without endotoxin stimulation (Figure 2). A prior exposure to intra-amniotic endotoxin 3 days before delivery reduced the concentration of IL-6 in culture media after re-exposure to endotoxin in vitro. The IL-6 concentrations in the media of monocytes exposed to 10 or 100 ng/ml endotoxin after the fetal endotoxin exposure were similar to the concentrations of IL-6 from monocytes from adult sheep. The 7- and 14-day repeated exposures of fetuses to intra-amniotic endotoxin had no effects on endotoxin-induced IL-6 concentration compared with preterm control animals.
CD14, TLR4, and MHC II Expression
CD14 expression was not different between monocytes from control preterm lambs and monocytes from adult animals after stimulation with 100 ng/ml endotoxin (Figure 3A). Similar results were obtained with 10 ng/ml endotoxin (data not shown). CD14 expression was decreased qualitatively 1 and 7 days after intra-amniotic endotoxin exposure and significantly after 3 days. The signal-transducing molecule for endotoxin mediated cell activation, TLR4, had a different pattern of expression. TLR4 expression was higher by approximately twofold on monocytes from adult animals than those from preterm control animals (Figure 3B). The expression was increased in cord bloodeCderived monocytes 14 days after intra-amniotic endotoxin. The expression was not different at the other time points. MHC II expression was similar for adult and preterm control monocytes (Figure 3C). Expression was strikingly decreased 3 days after intra-amniotic endotoxin.
DISCUSSION
The temporary suppression of innate immune cell responses by endotoxin has been referred to as "endotoxin tolerance" (21, 22). Endotoxin tolerance is used to describe in vivo models in which a normally lethal endotoxin dose can be tolerated by animals that were previously exposed to sublethal dose of endotoxin (21). Chronically catheterized preterm fetal sheep respond to intravascular endotoxin with a tolerance type response (11). Endotoxin tolerance/immune paralysis is a risk factor for multiorgan failure or sepsis in hospitalized patients (23). Preterm infants are extremely susceptible to nosocomial infections several weeks after preterm birth, at a time when they are often doing well (24). This clinical susceptibility to infection is consistent with immune paralysis, but we are not aware of studies that address this possibility.
The responses of monocytes from fetal sheep to endotoxin are complex. The initial suppression of H2O2 and IL-6 and decreased CD14 and MHC II receptors are typical of endotoxin tolerance. In contrast, the increased production of H2O2 and IL-6 and increased TLR4 expression 14 days after endotoxin exposure indicate an augmented innate host response relative to the control fetal monocytes. However, the repeated exposure at a 7-day interval resulted in profound suppression of H2O2 production. A similar pattern of response occurred with the different endotoxin concentrations tested in vitro. Clinically, chorioamnionitis is often indolent and persists for weeks or months (2). Our results suggest that chronic fetal exposure to inflammation could cause a profound and persistent tolerance-type response and thus put these infants at risk of nosocomial infection (25).
These experiments are a first evaluation of how fetal exposure to inflammation might modulate or affect innate immune responses. The concepts of endotoxin tolerance have been developed primarily in mice and the adult human. In adult mice, endotoxin exposure decreases tumor necrosis factor- and increases IL-1 secretion by monocytes (21). The mechanisms proposed for endotoxin tolerance in humans and mice are controversial but may include downregulation of endotoxin receptors, lipopolysaccharide-binding protein, or CD14 and alterations in the signaling pathways to decrease nuclear factor-B nuclear translocation as well as changes in the cytokine expression profile of the cells (21).
We did not use catheterized animals to avoid the stress of fetal surgery and the potential for infection in catheterized animals. These studies of the fetal innate immune system are unique because the immune system is naive and immature. An example of differences between the preterm and the adult is the observation that the fetal sheep does not respond to ovine tumor necrosis factor- but has systemic and lung inflammatory responses to IL-1 (13, 26). Fetal responses to IL-6 have not been evaluated. The decrease in monocyte surface receptors and suppressed function with single or repetitive fetal endotoxin exposure are consistent with an immune paralysis-type response by immature monocytes (22). The enhanced response 14 days after intra-amniotic endotoxin exposure may represent recovery from immune paralysis together with a maturation of monocyte function. A similar pattern of suppression followed by increased function occurs in response to maternal glucocorticoid exposure (12, 18). Fetal monocytes have inhibited function that resolves to resemble the function of adult monocytes by 7 days after maternal betamethasone exposure (12).
Another unique aspect of these studies is that the intra-amniotic exposure to endotoxin both induces lung maturation and impairs lung development (27eC29). Lung maturation was induced in the fetal sheep by doses of endotoxin from 1 to 100 mg. Similar amounts of intra-amniotic endotoxin were used in murine models of fetal exposure to inflammation (18, 30). In contrast, intravascular endotoxin at a much smaller dose of 5 e/kg causes either fetal death or tolerance to repeated intravascular doses of 1 e/kg in fetal sheep (11). Despite the high amounts of intra-amniotic endotoxin given to fetal sheep, fetal well-being or growth were not impaired (18). The lung inflammation and maturation responses require the direct contact of endotoxin with the fetal lung, presumably by the mixing of fetal lung fluid with amniotic fluid as a result of fetal breathing (15). The intra-amniotic endotoxin caused subtle changes in fetal circulating white blood cells, but the response was not characteristic of a systemic inflammatory response (17). Nevertheless, systemic monocytes were modified to have an immune paralysis-type phenotype by fetal lung inflammation. We previously found that fetal exposure to intra-amniotic endotoxin given 30 days before preterm delivery resulted in increased monocytes and lymphocytes in the alveolar lavages of preterm lambs (31). Therefore, intra-amniotic endotoxin can alter the subsequent immune function of the lung and the systemic monocytes. A cautionary note is that monocytes/macrophages from different tissue locations can respond differently after exposure to inflammation (32). The integrated fetal responses to inflammation remain to be explored.
The long-term implications of alterations in fetal immune responses are just beginning to be explored. Recently, increased cord blood levels of IL-4 and interferon- were associated with less atopy and asthma (33). The indirect evidence that increased maternal antibiotic use during pregnancy increases the risk of asthma in children supports the concept that fetal immune responses may change subsequent immune responses (34, 35). This fetal sheep model will be useful for future studies of long-term effects of lung and systemic innate immune modulation after preterm and term birth.
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