Tumor necrosis factor-a and interleukin-l ~ production by human fetal Kupffer cells William H. Kutteh, MD, PhD: William E. Rainey, PhD: Bruce Beutler, MD: and Bruce R. Carr, MD" Dallas, Texas This study describes the isolation and characterization of human fetal Kupffer cells. We demonstrated that these cells have the potential to respond to cS'tokines and lipopolysaccharide with an increased production of tumor necrosis factor-a and interleukin-113. Kupffer cells were characterized by: (1) morphologic characteristics after adherence to plastic, (2) staining for a-naphthyl acetate esterase, (3) immunofluorescence with monoclonal antibodies, and (4) phagocytosis of latex beads. More than 90% of the adherent cells were identified as macrophages. Kupffer cells cultured with lipopolysaccharide were able to produce interleukin-113 and tumor necrosis factor-a in a time- and dose-dependent fashion and maximal secretion was observed with the use of 10 /-1g of lipopolysaccharide per milliliter within 8 hours of treatment. We have demonstrated mature functional activity of human fetal Kupffer cells at an early gestational age (13 to 19 weeks) and discussed the roles that these cells may play in development and protection of the fetus. (AM J OasTET GVNECOL 1991 ;165:112-20.)
Key words: Kupffer cells, interleukin-1f3, tumor necrosis factor-a, human fetus
Kupffer cells, which represent the largest population of resident tissue macrophages in the adult, I play major roles in antigen processing and elimination, detoxification of endotoxin, secretion of cytokines, mediation of multiple immune reactions, maintenance of hepatocyte stability and function, and lipid and cholesterol homeostasis.2.3 In humans the fetal liver, as a fraction of body mass, is large compared with that of the adult. The liver represents approximately 10% of the total fetal weight at 10 weeks' gestation! The human fetal liver has a diverse cellular composition with about 60% parenchymal cells (hepatocytes), 20% non parenchymal cells (including Kupffer cells and fat storing cells), and 20% hematopoietic cells. 5 Although the presence of Kupffer cells at early stages of human development is 'well documented,6.lo the functional capabilities of these cells have not been clearly determined. Kupffer cells have been identified in sinusoid-like structures at 5 weeks' gestation and were present in fully developed sinusoids between 6 to From the Division of Reproductive Endocrinology and the Cecil H. and Ida Green Center for Reproductive Biology Sciences, Department of Obstetrics and Gynecology," and the Department of Internal Medicine/ University of Texas Southwestern Medical Center. This work was supported in part by National Institutes of Health grant HD07190. W.H.K. is the recipient of the American Fertility Society-Ortho Distinguished Fellowship in Reproduction. Presented at the Seventy-second Annual Meeting of the endocrine Society, Atlanta, Georgia, June 20-23, 1990. Received for publication September 14, 1990; revised December 28, 1990; accepted January 18,1991. Reprint requests: William H. Kutteh, MD, PhD, Department ofObstetrics and Gynecology-J6.114, 5323 Harry Hines Blvd., University of Texas Southwestern Medical Center, Dallas, TX 75235-9032.
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8 weeks. 6 , 7 Macrophage precursors, originating from the yolk sac, have been identified in fetal liver at 9 to 10 weeks postconception." It has been suggested that these cells function in removal of debris as one maturing embryonic tissue replaces another. 9 Indeed, yolk sac macro phages from 7-week gestations were noted to contain primitive erythroblasts. 1O In the adult, Kupffer cell functions are well defined, and they play important roles in the clearance of biologically harmful materials, including bacterial lipopolysaccharide, microorganisms, and clotting factors. II Furthermore, endotoxin, derived from enteric bacterial flora, is a normal component of portal venous blood but is efficiently removed during passage through the liver by Kupffer cells." II In addition, it is clear that Kupffer cells secrete a wide variety of cytokines that may modify immune functions, including interleukin1/3 (IL-1/3), tumor necrosis factor-a (TNF-a), hepatocyte stimulating factor, and interferons. 12·15 Important roles for fetal Kupffer cells in immune homeostasis have been proposed. Production of the important immunoregulary peptide TNF-a can be induced with endotoxin in fetal and newborn rat hepatic macrophages, demonstrating functional activity of fetal rodent Kupffer cells. 16 TNF-a exhibits diverse activities that can range from local mediation of cellular or tissue homeostasis to systemic events resulting in alterations of physiologic processes. These diverse activities depend on the concentration of TNF-a at the cellular, tissue, or systemic level. 17 Isolated monocytes from cord blood of preterm neonates produced significantly less TNF-a when stimulated with lipopolysaccharide than
TNF-ex and IL-1~ production by Kupffer cells
Volume 165 Number I
cells x 10 7
% viable
%exNAE+
- 250
98.1
0.9
137
94.2
1.4
8
91.7
21.4
1
2.4
94.3
49.3
ADHERENT CELLS
1.3
96.8
90.2
WHOLE TISSUE
1 :. . . . . . . . . . . . . 1~~~:a~ ~~ ...
113
Nylon dissociation 0.05% collagenase
SINGLE CELLS
3min
Pellet SUPERNATANT (Hepatocytes) 1375 x g for 10min :"......................... Resuspend pellet F/H 1.090 glcc :
...
Pellet INTERFACE (Red blood cells) Complete media :"......................... Overnight culture
...
Nonadherent (Discard)
(Kupffer cells)
Fig. 1. Kupffer cell isolation procedure. Kupffer cells were purified from specimens of whole human fetal liver as outlined in text. Mechanical and enzymatic dissociation into single cell suspensions, followed by several centrifugation steps, preceded placing cells into overnight culture. Columns refer to total cells in a representative preparation, their viability as determined by try pan blue dye exclusion, and percentage of cells that were positive with ex-naphthyl acetate esterase (NAE) staining. See Material and methods for details.
monocytes from term neonates, suggesting that this decreased responsiveness may contribute to the increased susceptibility of the preterm neonate to infection. ls However, in pregnant women with preterm intraamniotic infection, increased levels ofTNF-a in amniotic fluids may result in preterm rupture of the membranes. 19• 2o We hypothesized that Kupffer cells that are obtained from fetuses at 13 to 19 weeks' gestation might possess functional activities that could be important in the development and protection of the fetus. The purposes of this investigation were (1) to develop isolation and purification techniques, (2) to establish short-term culture, (3) to characterize the functional activities, and (4) to investigate potential activators of human fetal Kupffer cells. Purified Kupffer cells could be stimulated to produce IL-113 and TNF-a. We describe a number of activators of fetal Kupffer cells and discuss the potential local (paracrine and autocrine) and systemic (endocrine) effects of these secreted mediators.
Material and methods Human fetal tissues. Fetal tissues were obtained at the time of therapeutic termination at 13 to 19 weeks' gestation, as determined by fetal foot length. Tissues were obtained in accordance with the Donors Anatomical Gift Act of the state of Texas. A consent form and protocol approved by the Institutional Review Board of the University of Texas Southwestern Medical Center at Dallas were used. Kupffer cell isolation (Fig. 1). Methods used for the isolation of Kupffer cells from human fetal liver were
based on published principles!!. 22 Fresh tissue was rinsed in calcium-free phosphate-buffered saline solution, pH 7.4, with penicillin (0.1 mg/ml), streptomycin (100 flom/ml), and amphotericin (2.5 flog/ml). The liver was puncture perfused in calcium-free buffer to disrupt the desmosomal attachments of parenchymal cells,23 then minced into 1 em' pieces in 25 mmol/L HEPES with 5 mmol/L calcium buffer (pH 7.5) containing 0.05% collagenase type IV (Sigma, St. Louis) for 10 minutes at 25° C. The pieces were dissociated between layers of nylon mesh in RPMI 1640 (Gibco, Grand Island, N.Y.) with 2% heat-inactivated calf serum (Gibco), filtered through nylon, and centrifuged at 100 g for 3 minutes. (This pellet, depleted of Kupffer cells and cell fragments, was used as the source of hepatocytes, as described below). The supernatant, which was enriched for Kupffer cells, was centrifuged at 375 g for 10 minutes; then the pellet was resuspended in media, layered onto Ficoll Hypaque 1.090 gm/ml (Whitaker Bioproducts, Walkersville, Md.), and centrifuged at 350 g for 40 minutes at 20° C. The buffy interface was removed, rinsed three times, resuspended in RPMI 1640 with 10% fetal calf serum and antibiotics, and then placed into culture plates as described below. Cytocentrifuge preparations of various cell fractions were stained for a-naphthyl acetate esterase (Sigma), one of the most reliable markers for identification of macrophages. 11 Hepatocytes. The pellet containing hepatocytes was treated with 0.5 mmol/L ammonium chloride buffer at 0° C for 5 minutes to lyse red blood cells. Hepatocytes were rinsed three times, then resuspended into equal
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Kutteh et al.
volumes of Dulbecco's modified Eagle medium and Ham's nutrient mixture F-12 with 2 mmol/L Lglutamine (DME/FI2, Gibco), 5% calf serum, and antibiotics. An enzyme-linked immunosorbent assay for ufetoprotein (AFP), a product of fetal hepatocytes, was performed on cell supernatants with commercially obtained assay kits (Pharmacia, Piscataway, N.J.). Interassay coefficient of variation was 90% were judged to be macrophages on the basis of the ability to ingest 0.8 ].Lm latex beads, a-naphthyl acetate esterase staining, and immunofluorescence with monoclonal antibody 3C1O (Fig. 2, a, b, and c). Fig. 2, a, demonstrates an adherent Kupffer cell after incubation with 0.8 ].Lm latex beads and extensive washing to remove noningested beads. Phagocytosis of multiple beads in the cytoplasm was characteristic of adherent cells. Fig. 2, b, is representative of a-naphthyl acetate used as substrate to reveal the dark-stained cytoplasm identifying esterase activity in Kupffer cells. This photomicrograph is from a population of cells just before placement into culture, where typically 50% of the cells were positive. Fig. 2, c, is a photomicrograph of adherent Kupffer cells visualized with indirect immunofluorescence staining techniques with mouse monoclonal antibody 3CIO. Typically, >90% of adherent cells were positive with this method. Similar immunofluorescence staining patterns were obtained with the use of mouse monoclonal antibodies EMB-II and anti-Leu-M5. Less than 1% of the adherent cells were positive when stained with monoclonal antibodies anti-Leu-2a, anti-Leu-3a + 3b, or anti-Leu-4 + 5b, indicating that our populations contained very few human T cells (data not shown). Effect of lipopolysaccharide treatment on TNF-«, IL-IJ3, and AFP production by human fetal liver cells. TNF -a, IL-I~, and AFP were quantified in the culture media of I X 10 6 adherent human fetal Kupffer cells incubated with 10 ].Lg lipopolysaccharide per milliliter
of complete culture media for 24 hours. In addition, we assayed the same products in the culture supernatants of purified human fetal hepatocytes. As shown in Fig. 3, IL-I~ and Tl\'F-a were produced by human fetal Kupffer cells in significant quantities after stimulation with lipopolysaccharide. Baseline, unstimulated production was 20 pg/mlllOh cells for IL-I~ and 30 pg/mlllO li cells for TNF-a. Hepatocyte culture supernatants contained very low levels of TN'F -a or IL-I ~ with and without lipopolysaccharide stimulation, indicating minimal contamination with Kupffer cells. As shown in Fig. 3, 0.1 fJ.-m has been described as an important criterion to distinguish Kupffer cells from other sinusoidal cells of the liver.'o In agreement with our findings of active phagocytosis, cells from midgestation human fetal liver that stained positive with OKM-I, a mouse monoclonal antibody to human macrophages, apparently had phagocytosed maternal immunoglobulins or immune complexes, since they contained both immunoglobulin iight chain markers, K
118
Kutteh et a/. Am
Julv 1991
J Obstet Gynecol
1200 .IL-1 ~
!!2 Qi 1000 U
Key words: Kupffer cells, interleukin-1f3, tumor necrosis factor-a, human fetus
Kupffer cells, which represent the largest population of resident tissue macrophages in the adult, I play major roles in antigen processing and elimination, detoxification of endotoxin, secretion of cytokines, mediation of multiple immune reactions, maintenance of hepatocyte stability and function, and lipid and cholesterol homeostasis.2.3 In humans the fetal liver, as a fraction of body mass, is large compared with that of the adult. The liver represents approximately 10% of the total fetal weight at 10 weeks' gestation! The human fetal liver has a diverse cellular composition with about 60% parenchymal cells (hepatocytes), 20% non parenchymal cells (including Kupffer cells and fat storing cells), and 20% hematopoietic cells. 5 Although the presence of Kupffer cells at early stages of human development is 'well documented,6.lo the functional capabilities of these cells have not been clearly determined. Kupffer cells have been identified in sinusoid-like structures at 5 weeks' gestation and were present in fully developed sinusoids between 6 to From the Division of Reproductive Endocrinology and the Cecil H. and Ida Green Center for Reproductive Biology Sciences, Department of Obstetrics and Gynecology," and the Department of Internal Medicine/ University of Texas Southwestern Medical Center. This work was supported in part by National Institutes of Health grant HD07190. W.H.K. is the recipient of the American Fertility Society-Ortho Distinguished Fellowship in Reproduction. Presented at the Seventy-second Annual Meeting of the endocrine Society, Atlanta, Georgia, June 20-23, 1990. Received for publication September 14, 1990; revised December 28, 1990; accepted January 18,1991. Reprint requests: William H. Kutteh, MD, PhD, Department ofObstetrics and Gynecology-J6.114, 5323 Harry Hines Blvd., University of Texas Southwestern Medical Center, Dallas, TX 75235-9032.
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8 weeks. 6 , 7 Macrophage precursors, originating from the yolk sac, have been identified in fetal liver at 9 to 10 weeks postconception." It has been suggested that these cells function in removal of debris as one maturing embryonic tissue replaces another. 9 Indeed, yolk sac macro phages from 7-week gestations were noted to contain primitive erythroblasts. 1O In the adult, Kupffer cell functions are well defined, and they play important roles in the clearance of biologically harmful materials, including bacterial lipopolysaccharide, microorganisms, and clotting factors. II Furthermore, endotoxin, derived from enteric bacterial flora, is a normal component of portal venous blood but is efficiently removed during passage through the liver by Kupffer cells." II In addition, it is clear that Kupffer cells secrete a wide variety of cytokines that may modify immune functions, including interleukin1/3 (IL-1/3), tumor necrosis factor-a (TNF-a), hepatocyte stimulating factor, and interferons. 12·15 Important roles for fetal Kupffer cells in immune homeostasis have been proposed. Production of the important immunoregulary peptide TNF-a can be induced with endotoxin in fetal and newborn rat hepatic macrophages, demonstrating functional activity of fetal rodent Kupffer cells. 16 TNF-a exhibits diverse activities that can range from local mediation of cellular or tissue homeostasis to systemic events resulting in alterations of physiologic processes. These diverse activities depend on the concentration of TNF-a at the cellular, tissue, or systemic level. 17 Isolated monocytes from cord blood of preterm neonates produced significantly less TNF-a when stimulated with lipopolysaccharide than
TNF-ex and IL-1~ production by Kupffer cells
Volume 165 Number I
cells x 10 7
% viable
%exNAE+
- 250
98.1
0.9
137
94.2
1.4
8
91.7
21.4
1
2.4
94.3
49.3
ADHERENT CELLS
1.3
96.8
90.2
WHOLE TISSUE
1 :. . . . . . . . . . . . . 1~~~:a~ ~~ ...
113
Nylon dissociation 0.05% collagenase
SINGLE CELLS
3min
Pellet SUPERNATANT (Hepatocytes) 1375 x g for 10min :"......................... Resuspend pellet F/H 1.090 glcc :
...
Pellet INTERFACE (Red blood cells) Complete media :"......................... Overnight culture
...
Nonadherent (Discard)
(Kupffer cells)
Fig. 1. Kupffer cell isolation procedure. Kupffer cells were purified from specimens of whole human fetal liver as outlined in text. Mechanical and enzymatic dissociation into single cell suspensions, followed by several centrifugation steps, preceded placing cells into overnight culture. Columns refer to total cells in a representative preparation, their viability as determined by try pan blue dye exclusion, and percentage of cells that were positive with ex-naphthyl acetate esterase (NAE) staining. See Material and methods for details.
monocytes from term neonates, suggesting that this decreased responsiveness may contribute to the increased susceptibility of the preterm neonate to infection. ls However, in pregnant women with preterm intraamniotic infection, increased levels ofTNF-a in amniotic fluids may result in preterm rupture of the membranes. 19• 2o We hypothesized that Kupffer cells that are obtained from fetuses at 13 to 19 weeks' gestation might possess functional activities that could be important in the development and protection of the fetus. The purposes of this investigation were (1) to develop isolation and purification techniques, (2) to establish short-term culture, (3) to characterize the functional activities, and (4) to investigate potential activators of human fetal Kupffer cells. Purified Kupffer cells could be stimulated to produce IL-113 and TNF-a. We describe a number of activators of fetal Kupffer cells and discuss the potential local (paracrine and autocrine) and systemic (endocrine) effects of these secreted mediators.
Material and methods Human fetal tissues. Fetal tissues were obtained at the time of therapeutic termination at 13 to 19 weeks' gestation, as determined by fetal foot length. Tissues were obtained in accordance with the Donors Anatomical Gift Act of the state of Texas. A consent form and protocol approved by the Institutional Review Board of the University of Texas Southwestern Medical Center at Dallas were used. Kupffer cell isolation (Fig. 1). Methods used for the isolation of Kupffer cells from human fetal liver were
based on published principles!!. 22 Fresh tissue was rinsed in calcium-free phosphate-buffered saline solution, pH 7.4, with penicillin (0.1 mg/ml), streptomycin (100 flom/ml), and amphotericin (2.5 flog/ml). The liver was puncture perfused in calcium-free buffer to disrupt the desmosomal attachments of parenchymal cells,23 then minced into 1 em' pieces in 25 mmol/L HEPES with 5 mmol/L calcium buffer (pH 7.5) containing 0.05% collagenase type IV (Sigma, St. Louis) for 10 minutes at 25° C. The pieces were dissociated between layers of nylon mesh in RPMI 1640 (Gibco, Grand Island, N.Y.) with 2% heat-inactivated calf serum (Gibco), filtered through nylon, and centrifuged at 100 g for 3 minutes. (This pellet, depleted of Kupffer cells and cell fragments, was used as the source of hepatocytes, as described below). The supernatant, which was enriched for Kupffer cells, was centrifuged at 375 g for 10 minutes; then the pellet was resuspended in media, layered onto Ficoll Hypaque 1.090 gm/ml (Whitaker Bioproducts, Walkersville, Md.), and centrifuged at 350 g for 40 minutes at 20° C. The buffy interface was removed, rinsed three times, resuspended in RPMI 1640 with 10% fetal calf serum and antibiotics, and then placed into culture plates as described below. Cytocentrifuge preparations of various cell fractions were stained for a-naphthyl acetate esterase (Sigma), one of the most reliable markers for identification of macrophages. 11 Hepatocytes. The pellet containing hepatocytes was treated with 0.5 mmol/L ammonium chloride buffer at 0° C for 5 minutes to lyse red blood cells. Hepatocytes were rinsed three times, then resuspended into equal
114
Kutteh et al.
volumes of Dulbecco's modified Eagle medium and Ham's nutrient mixture F-12 with 2 mmol/L Lglutamine (DME/FI2, Gibco), 5% calf serum, and antibiotics. An enzyme-linked immunosorbent assay for ufetoprotein (AFP), a product of fetal hepatocytes, was performed on cell supernatants with commercially obtained assay kits (Pharmacia, Piscataway, N.J.). Interassay coefficient of variation was 90% were judged to be macrophages on the basis of the ability to ingest 0.8 ].Lm latex beads, a-naphthyl acetate esterase staining, and immunofluorescence with monoclonal antibody 3C1O (Fig. 2, a, b, and c). Fig. 2, a, demonstrates an adherent Kupffer cell after incubation with 0.8 ].Lm latex beads and extensive washing to remove noningested beads. Phagocytosis of multiple beads in the cytoplasm was characteristic of adherent cells. Fig. 2, b, is representative of a-naphthyl acetate used as substrate to reveal the dark-stained cytoplasm identifying esterase activity in Kupffer cells. This photomicrograph is from a population of cells just before placement into culture, where typically 50% of the cells were positive. Fig. 2, c, is a photomicrograph of adherent Kupffer cells visualized with indirect immunofluorescence staining techniques with mouse monoclonal antibody 3CIO. Typically, >90% of adherent cells were positive with this method. Similar immunofluorescence staining patterns were obtained with the use of mouse monoclonal antibodies EMB-II and anti-Leu-M5. Less than 1% of the adherent cells were positive when stained with monoclonal antibodies anti-Leu-2a, anti-Leu-3a + 3b, or anti-Leu-4 + 5b, indicating that our populations contained very few human T cells (data not shown). Effect of lipopolysaccharide treatment on TNF-«, IL-IJ3, and AFP production by human fetal liver cells. TNF -a, IL-I~, and AFP were quantified in the culture media of I X 10 6 adherent human fetal Kupffer cells incubated with 10 ].Lg lipopolysaccharide per milliliter
of complete culture media for 24 hours. In addition, we assayed the same products in the culture supernatants of purified human fetal hepatocytes. As shown in Fig. 3, IL-I~ and Tl\'F-a were produced by human fetal Kupffer cells in significant quantities after stimulation with lipopolysaccharide. Baseline, unstimulated production was 20 pg/mlllOh cells for IL-I~ and 30 pg/mlllO li cells for TNF-a. Hepatocyte culture supernatants contained very low levels of TN'F -a or IL-I ~ with and without lipopolysaccharide stimulation, indicating minimal contamination with Kupffer cells. As shown in Fig. 3, 0.1 fJ.-m has been described as an important criterion to distinguish Kupffer cells from other sinusoidal cells of the liver.'o In agreement with our findings of active phagocytosis, cells from midgestation human fetal liver that stained positive with OKM-I, a mouse monoclonal antibody to human macrophages, apparently had phagocytosed maternal immunoglobulins or immune complexes, since they contained both immunoglobulin iight chain markers, K
118
Kutteh et a/. Am
Julv 1991
J Obstet Gynecol
1200 .IL-1 ~
!!2 Qi 1000 U
