PEDIATRICS Vol. 121 No. 3 March 2008, pp. e496-e505 (doi:10.1542/10.1542/peds.2007-0878)
ARTICLE |
Role of Complement and CD14 in Meconium-Induced Cytokine Formation
a Institute of Immunology
b Department of Pediatric Research, University of Oslo and Rikshospitalet University Hospital, Oslo, Norway
c Tanox Inc, Houston, Texas
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ABSTRACT |
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OBJECTIVE. Meconium aspiration syndrome has a complex, poorly defined pathophysiology. Meconium is a potent activator of complement in vitro and in vivo; the latter is associated with a systemic inflammatory response. The complement system and Toll-like receptors are 2 important upstream components of the innate immune system that act partly independently in the inflammatory network. The aim of this study was to investigate the relative role of complement and CD14 in meconium-induced cytokine production.
METHODS. Human adult (n = 6) and cord whole blood (n = 6) anticoagulated with lepirudin was collected and distributed into tubes that contained inhibitory antibodies (anti-CD14, anti-C2, anti–factor D, or combinations thereof). The tubes were preincubated for 5 minutes before addition of meconium or buffer and then incubated for 4 hours at 37°C. Complement activation was measured by quantification of the terminal sC5b-9 complement complex by enzyme-linked immunosorbent assay. A panel of 27 inflammatory mediators (cytokines, chemokines, and growth factors) was measured by using multiplex technology.
RESULTS. Fourteen of the 27 mediators measured were induced by meconium both in cord and adult blood. In cord blood, 2 additional chemokines were induced and the inflammatory response was, in general, more potent. Blocking of complement or CD14 differentially reduced the formation of most mediators, anti-CD14 being more effective. Notably, the combined inhibition of complement and CD14 almost completely abolished meconium-induced formation of the cytokines and the chemokines and markedly reduced the formation of growth factors. The endogenous lipopolysaccharide content of meconium could not explain the CD14-mediated response.
CONCLUSIONS. Meconium-induced triggering of the cytokine network is differentially mediated by complement and CD14. A combined inhibition of these effector mechanisms may be an alternative approach to reduce the inflammatory reaction in meconium aspiration syndrome.
Key Words: meconium • complement system • lipopolysaccharide • CD14 • Toll-like receptor • cord blood
Abbreviations: MAS—meconium aspiration syndrome • TNF-
—tumor necrosis factor • IL—interleukin • TLR—Toll-like receptor • PBS—phosphate-buffered saline • IgG1—immunoglobulin G1 • TCC—terminal sC5b-9 complement complex • ELISA—enzyme-linked immunosorbent assay • AU—arbitrary units • IFN-
—interferon • MIP—macrophage inflammatory protein • G-CSF—granulocyte colony-stimulating factor • GM-CSF—granulocyte macrophage colony-stimulating factor • FGF—fibroblast growth factor • VEGF—vascular endothelial growth factor • IP-10—interferon-inducible protein
Meconium aspiration syndrome (MAS), an important cause of respiratory distress in the term newborn, is a serious condition with high morbidity and mortality.1,2 The pathophysiology is complex and not well defined, including airway obstruction, pulmonary hypertension, epithelial injury, surfactant inactivation, and inflammation.1,3 Fetal asphyxia4 and infection are suggested to be main causative agents.5,6
Meconium produces inflammatory responses in both animal models and newborns with MAS.7 After intratracheal instillation of meconium in animals, there is an intense pulmonary inflammatory reaction with influx of polymorphonuclear leukocytes, monocytes/macrophages, and T cells within a few hours. The production of proinflammatory cytokines further propagates parenchymal lung cell injury,8,9 and apoptotic epithelial cells are present in meconium-containing lungs.10,11
We previously showed that meconium is a potent activator of the complement system in vitro as well as in vivo in a newborn pig model of MAS, the latter being associated with a systemic inflammatory response reflected by cytokine production and changes in neutrophil function.12,13 Complement activation preceded and was correlated with increased production of tumor necrosis factor (TNF-) and interleukin 1β (IL-1β). Furthermore, the activation of complement was correlated with lung dysfunction and mortality.14
The complement system and Toll-like receptors (TLRs), including the CD14-associated TLR4/MD2 complex, are 2 important components of the innate immune system that both act upstream and partly independently.15 Meconium with its complex composition may activate both pathways, leading to secondary cytokine production. Because MAS may be associated with chorioamnionitis16,17 and meconium used for in vitro experiments contains lipopolysaccharide, CD14-dependent signaling18 may contribute to meconium-induced cytokine production. Meconium may contain agents other than lipopolysaccharide, which could interact with CD14 under sterile in vivo conditions. A possible role for the lipopolysaccharide content in meconium to induce cytokines via the CD14/TLR4/MD2 complex in experimental models in vitro and in vivo is not known.
The aim of this study was to investigate whether meconium, in addition to activating complement, could induce CD14-mediated cytokine production. Specific and combined inhibition of the 2 systems was used to define their relative roles in meconium-induced cytokine production. Endogenous lipopolysaccharide was quantified in meconium, and exogenously lipopolysaccharide was added to meconium to evaluate a possible role for lipopolysaccharide in the CD14-mediated response.
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METHODS |
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Reagents and Equipment
All materials and solutions in this study were endotoxin-free according to the manufacturers. Polypropylene tubes were NUNC cryotubes (Nalgene NUNC, Roskilde, Denmark). Sterile phosphate-buffered saline (PBS) was from Dulbecco (Paisley, United Kingdom), and lepirudin (Refludan) was from Hoechst (Frankfurt am Main, Germany).
Antibodies
Mouse monoclonal antibodies blocking C2 (clone 175–26, immunoglobulin G1 [IgG1]) and factor D (clone 166–32, IgG1) and an isotype-matched control (clone G3-519, IgG1) were produced and purified under identical conditions.19,20 The combination of anti-C2 and anti–factor D efficiently blocks all 3 initial pathways of complement. Mouse anti-human CD14 (clone 18D11) was purchased from Diatec (Oslo, Norway).
Meconium
Meconium was collected with a wooden spatula from diapers of 48 healthy newborns and stored in 50-mL propylene centrifuge tubes (Corning Inc, Acton, MA) at –20°C. The meconium was then thawed, pooled, and processed in PBS and then freeze-dried (HETO-FD3; Heto, Holten, Denmark). The meconium was reconstituted with PBS to a final concentration 100 mg/mL and then frozen in aliquots at –20°C until the day of the experiment. No bacteria were detected after cultivation. The lipopolysaccharide content, measured using a standard Limulus Amebocyte Lysate Assay (LAL QCL-1000; Cambrex Bio Science, Walkersville, MD), was found to be 20 pg/mg meconium.
Lipopolysaccharide
Ultrapure Escherichia coli lipopolysaccharide (0111:B4 strain-TLR4 ligand; InvivoGen, San Diego, CA) was purchased for lipopolysaccharide investigations.
Experimental Set-up
Human whole blood from 6 adult healthy donors and cord blood from 6 placentas (normal deliveries of healthy newborns), anticoagulated with lepirudin at 50 µg/mL,21 were collected and distributed into tubes that contained PBS, inhibitory antibodies (anti-CD14 100 µg/mL blood, anti-C2 100 µg/mL blood, anti–factor D 50 µg/mL blood, or control antibody). Informed consent was obtained from all donors. The samples were preincubated for 5 minutes before addition of either PBS or meconium. The meconium concentration was 1 mg/mL blood. Baseline sample (T0), containing only blood and PBS, was processed immediately, whereas the other samples were incubated for 4 hours at 37°C. Negative control sample (T4) contained blood and PBS. At the end of the incubation, ethylenediaminetetraacetic acid was added to all samples to a final concentration of 20 mM to avoid additional complement activation. Then the tubes were centrifuged for 15 minutes at 1400g (4°C). The plasma samples were stored at –70°C.
Adult whole-blood samples from 3 donors, anticoagulated with lepirudin, were incubated with lipopolysaccharide at 10, 100, 1000, or 10000 pg/mL blood for 4 hours at 37°C. In parallel experiments, the same amounts of lipopolysaccharide as mentioned previously were added to meconium at 1 mg/mL blood, providing an increasing concentration of lipopolysaccharide in a fixed concentration of meconium. Baseline sample (T0) containing blood and PBS was processed immediately. At the end of the incubation, ethylenediaminetetraacetic acid was added to all samples as described, then the tubes were centrifuged and the plasma samples were stored at –70°C.
Complement Activation
Complement activation was measured by quantification of the terminal sC5b-9 complex (TCC) using an enzyme-linked immunosorbent assay (ELISA) as previously described and later modified.22,23 The assay is based on a monoclonal antibody (aE11) highly specific for a neoepitope exposed in C9 when incorporated into TCC. Values are given in arbitrary units (AU) defined by a serum standard activated with zymosan and defined to contain 1000 AU/mL.
Cytokine Analysis
The following cytokines, chemokines, and growth factors were measured on a Bioplex Array Reader (Luminex 100; Bio-Rad Laboratories, Hercules, CA) using Bio-Plex Human Cytokine 27-plex panel (Bio-Rad Laboratories): IL-1Ra; IL-1β; IL-2; IL-4; IL-5; IL-6; IL-7; IL-8; IL-9; IL-10; IL-12p70; IL-13; IL-15; IL-17; TNF-; interferon (IFN-); macrophage inflammatory protein 1 (MIP-1); MIP-1β; eotaxin; MCP-1; granulocyte colony-stimulating factor (G-CSF); granulocyte macrophage colony-stimulating factor (GM-CSF); basic fibroblast growth factor (FGF); vascular endothelial growth factor (VEGF); interferon-inducible protein (IP-10); regulated upon activation, normal T cell expressed and secreted; and platelet-derived growth factor bb. IL-8 was repeated in an IL-8 single-plex panel (Bio-Rad Laboratories) with 3 different donors of adult blood and cord blood, respectively, because the values were beyond the standard curve in the 27-plex panel and needed to be diluted and analyzed separately.
Data Presentation and Statistics
Data are medians and interquartile ranges of 6 experiments for all parameters except for IL-8 and lipopolysaccharide, for which data are medians and ranges of 3 experiments. Because of the large numbers of readouts and experiments with different inhibitors and their combination, statistical calculation was limited to the primary study aim, namely to compare the meconium-induced increase of a mediator with the effect of combined inhibition with complement and anti-CD14. Nonparametric Wilcoxon's test was used for all experiments with 6 observations and Student's t test for IL-8 with 3 experiments. A 2-tailed P value of <.05 was considered significant.
Ethics
This study was approved by the Norwegian Regional Ethical Committee.
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RESULTS |
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Spontaneous Activation and Controls
The mediators induced by meconium are shown in Figs 1 through 7. For all of them, only a very modest spontaneous activation of complement and formation of cytokines, chemokines, and growth factors in blood samples without meconium were observed from start (T0) to 4 hours of incubation (T4), giving a low background control compared with the values obtained after incubation with meconium. The isotype-matched antibody control consistently showed no inhibitory effect on meconium-induced mediator induction throughout the study, in contrast to the combined inhibition of complement and CD14, as detailed next. Unless otherwise stated, data are medians and interquartile ranges of 6 experiments.
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Complement Activation
Adult Blood
TCC in the baseline sample (T0) increased from 3 AU/mL (1–4 AU/mL) (median and interquartile range) to 19 AU/mL (8–38 AU/mL) after 4 hours of incubation (T4). Addition of meconium increased TCC formation to 190 AU/mL (146–198 AU/mL). Anti-C2 and anti–factor D antibodies completely blocked meconium-induced complement activation, giving TCC values of 2 AU/mL (1–4 AU/mL). As expected, anti-CD14 and the isotype-matched control antibody had no effect on meconium-induced TCC formation (Fig 1, left).
Cord Blood
TCC in T0 increased from 0 AU/mL (0–1 AU/mL) to 5 AU/mL (4–6 AU/mL) in T4. Addition of meconium increased TCC formation to 48 AU/mL (44–61 AU/mL). Anti-C2 and anti–factor D antibodies completely blocked complement activation, giving TCC values of 0 AU/mL (0–5 AU/mL). As for adult blood, anti-CD14 and the isotype-matched control antibody had no effect on meconium-induced TCC formation (Fig 1, right).
Whole-Blood Inflammatory Mediators
Using multiplex technology, we measured 27 different cytokines, chemokines, and growth factors. Fourteen and 16 of these were induced by meconium in adult and cord blood, respectively. The following mediators were not induced by meconium in adult blood: IL-2; IL-4; IL-5; IL-7; IL-9; IL-12p70; IL-13; IL-17; eotaxin; MCP-1; IP-10; regulated upon activation, normal T cell expressed and secreted; and platelet-derived growth factor bb. The same mediators were negative in cord blood, except for eotaxin and IP-10, which were induced in cord blood but not in adult blood (see chemokines).
Proinflammatory Cytokines
TNF-: Adult Blood and Cord Blood
The substantial meconium-induced formation of TNF- in both adult and cord blood was reduced by anti-CD14 and anti-C2/D, respectively. The combined inhibition of CD14 and C2/D almost completely abolished the formation of TNF- in both adult (P = .002) and cord blood (P = .002; Fig 2, top).
IL-1β: Adult Blood and Cord Blood
The substantial meconium-induced formation of IL-1β in both adult and cord blood was reduced by anti-CD14 and anti-C2/D, respectively. The combined inhibition of CD14 and C2/D almost completely abolished the formation of IL-1β in both adult (P = .002) and cord blood (P = .026; Fig 2, middle).
IL-6: Adult Blood and Cord Blood
The substantial meconium-induced formation of IL-6 in both adult and cord blood was reduced by anti-CD14 but not by anti-C2/D. The combined inhibition of CD14 and C2/D, however, almost completely abolished the formation of IL-6 in both adult (P = .002) and cord blood (P = .002; Fig 2, bottom).
IFN-: Adult Blood
Meconium increased IFN- formation to 576 pg/mL (565–777 pg/mL), which was reduced to 415 pg/mL (320–550 pg/mL) by anti-CD14 but not by anti-C2/D. The combined inhibition of CD14 and C2/D almost completely abolished the formation of IFN- to 212 pg/mL (179–269 pg/mL; P = .008; data not shown).
IFN-: Cord Blood
Meconium increased IFN- formation to 1624 pg/mL (1326–2183 pg/mL), which was reduced to 489 pg/mL (348–723 pg/mL) by anti-CD14 and to 1174 pg/mL (607–1479 pg/mL) by anti-C2/D. The combined inhibition of CD14 and C2/D almost completely abolished the formation of IFN- to 365 pg/mL (290–386 pg/mL; P = .002; data not shown).
IL-15: Adult Blood
Meconium increased IL-15 formation to 504 pg/mL (430–690 pg/mL), which was reduced to 294 pg/mL (251–354 pg/mL) by anti-CD14 and to 291 pg/mL (253–365 pg/mL) by anti-C2/D. The combined inhibition of CD14 and C2/D markedly reduced the formation of IL-15 to 267 pg/mL (232–375 pg/mL; P = .004; data not shown).
IL-15: Cord Blood
Meconium increased IL-15 formation to 167 pg/mL (123–244 pg/mL), which was reduced to 58 pg/mL (44–72 pg/mL) by anti-CD14 and to 82 pg/mL (59–149 pg/mL) by anti-C2/D. The combined inhibition of both CD14 and C2/D markedly reduced the formation of IL-15 to 45 pg/mL (23–51 pg/mL; P = .002; data not shown).
Chemokines
IL-8: Adult Blood and Cord Blood
The substantial meconium-induced formation of IL-8 in both adult (n = 3) and cord blood (n = 3) was reduced by anti-CD14 and anti-C2/D, respectively. The combined inhibition of CD14 and C2/D almost completely abolished the formation of IL-8 in both adult (P = .01) and cord blood (P = .008; Fig 3, top).
MIP-1: Adult Blood and Cord Blood
The substantial meconium-induced formation of MIP-1 in both adult and cord blood was reduced by anti-CD14 and anti-C2/D in adult blood but only by anti-CD14 in cord blood. The combined inhibition of CD14 and C2/D, however, almost completely abolished the formation of MIP-1 in both adult (P = .002) and cord blood (P = .002; Fig 3, middle).
MIP-1β: Adult Blood and Cord Blood
The substantial meconium-induced formation of MIP-1β in both adult and cord blood was reduced by anti-CD14 and anti-C2/D in adult blood but only by anti-CD14 in cord blood. The combined inhibition of CD14 and C2/D, however, almost completely abolished the formation of MIP-1β in both adult (P = .009) and cord blood (P = .016; Fig 3, bottom).
IP-10 and Eotaxin: Adult Blood
IP-10 and eotaxin were not induced by meconium in adult blood in contrast to cord blood.
IP-10: Cord Blood
The substantial meconium-induced formation of IP-10 in cord blood was reduced by anti-CD14 and anti-C2/D, respectively. The combined inhibition of CD14 and C2/D almost completely abolished the formation of IP-10 (P = .002; Fig 4, top).
Eotaxin: Cord Blood
The substantial meconium-induced formation of eotaxin in cord blood was reduced by anti-CD14 and anti-C2/D, respectively. The combined inhibition of CD14 and C2/D almost completely abolished the formation of eotaxin (P = .002; Fig 4, bottom).
Growth Factors
G-CSF: Adult Blood and Cord Blood
The substantial meconium-induced formation of G-CSF in both adult and cord blood was reduced by anti-CD14 and anti-C2/D, respectively. The combined inhibition of CD14 and C2/D almost completely abolished the formation of G-CSF in both adult (P = .002) and cord blood (P = .002; Fig 5, top).
GM-CSF: Adult Blood and Cord Blood
The substantial meconium-induced formation of GM-CSF in both adult and cord blood was reduced by anti-CD14 and anti-C2/D, respectively. The combined inhibition of CD14 and C2/D markedly reduced the formation of GM-CSF in both adult (P = .031) and cord blood (P = .002; Fig 5, middle).
Basic FGF: Adult Blood and Cord Blood
The substantial meconium-induced formation of basic FGF in both adult and cord blood was reduced by anti-CD14 and anti-C2/D, respectively. The combined inhibition of CD14 and C2/D almost completely abolished the formation of basic FGF in both adult (P = .002) and cord blood (P = .041; Fig 5, bottom).
Vascular Endothelial Growth Factor
Meconium increased VEGF formation modestly in both adult blood and cord blood, but the increase was not reduced by CD14 or complement inhibitors (data not shown).
Antagonists
IL-1Ra: Adult Blood and Cord Blood
The substantial meconium-induced formation of IL-Ra in both adult and cord blood was not reduced by either anti-CD14 or anti-C2/D in adult blood but was reduced by anti-CD14 in cord blood. The combined inhibition of CD14 and C2/D markedly reduced the formation of IL-1Ra in both adult (P = .0002) and cord blood (P = .031; Fig 6, left).
Effect of Lipopolysaccharide on Cytokine Formation
A possible role for lipopolysaccharide in meconium-induced cytokine formation was then investigated. Four proinflammatory cytokines that are known to be induced by lipopolysaccharide were measured.
Tumor Necrosis Factor
Increasing concentrations of lipopolysaccharide from 10 to 10000 pg/mL blood increased the formation of TNF- dose-dependently. Incubation with meconium alone, containing 20 pg endogenous lipopolysaccharide/mg meconium, increased the formation of TNF- substantially, but no additional increase in TNF- formation was seen after adding up to 10000 pg/mL lipopolysaccharide together with meconium before incubation (Fig 7, top left).
IL-1β, IL-6, and IL-8
The results observed for IL-1β (Fig 7, top right), IL-6 (Fig 7, bottom left), and IL-8 (Fig 7, bottom right) were virtually identical to that described for TNF-.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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In this study, we demonstrated for the first time that meconium induced a potent CD14-mediated inflammatory reaction reflected by synthesis of a broad panel of cytokines, chemokines, and growth factors in human adult and cord whole blood. Inhibition of CD14 markedly reduced the production of a number of the inflammatory mediators and complement inhibition reduced some of the mediators to a certain extent, whereas a combined inhibition of CD14 and complement showed a remarkable effect on virtually all of the mediators induced. This combined effect was more pronounced than could be predicted from the effect of separate inhibition of CD14 and complement. These data indicate a synergistic effect of blocking these 2 main branches of innate immunity on the inflammatory response induced by meconium.
Complement is the main fluid-phase branch of innate immunity. Meconium was shown to be a potent activator of the complement system,12 and the potent complement anaphylatoxin C5a was shown to be essential in meconium-induced granulocyte activation.13 MAS is associated with an inflammatory response closely correlated with lung dysfunction,13 and lung inflammation is reflected by local production of proinflammatory cytokines in experimental MAS.8,24 Thus, complement is 1 potential candidate to induce lung and systemic inflammation in newborns with MAS. This study supports this assumption but highlights the other main innate immunity branch to be at least as important for activation of the cytokine network. The degree of meconium-induced complement activation obtained in cord blood was markedly lower than that in adult blood, as expected because cord blood is known to contain approximately half the amount of complement components compared with adult blood.25,26 This may partly explain the relatively lower power of complement compared with CD14 when comparing these branches in cord blood.
The second main branch of innate immunity is the cellular response, composed mainly of the TLR family, of which the CD14/TLR4/MD2 complex is a main constituent. This receptor recognizes lipopolysaccharide among a number of other ligands. Tentatively, meconium is sterile when located in the normal fetal bowel but may contain variable amounts of lipopolysaccharide under certain conditions. First, meconium from normal deliveries may be contaminated by lipopolysaccharide during collection and preparation, influencing the results from in vitro and in vivo experimental studies using normal meconium. Second, fetal membranes, even from deliveries by cesarean section at term, had microbial organisms present (70%), although only <25% showed inflammatory cells present.27 Third, intrauterine infections may increase bacterial and lipopolysaccharide load. Fetal membranes obtained after preterm labor or after prolonged premature rupture of membranes are mainly double positive for bacteria deep within the membranes and for inflammatory cells in amnion or chorion.27 The lipopolysaccharide content in the meconium used in this study was as low as 20 pg/mg meconium. Whether this very low lipopolysaccharide content is a normal physiologic concentration of newborn meconium or is attributable to contamination during collection and storage, despite attempts to avoid this, is not known. Importantly, it is known that a lipopolysaccharide content 100-fold higher (in the range of µg/mL) is needed to induce complement activation.28 Thus, the complement activation potency of meconium is definitely not related to lipopolysaccharide, although a potential for cytokine production could not be excluded at this level. Incubation of adult whole blood with increasing concentrations of lipopolysaccharide up to 10000 pg/mL increased the formation of TNF-, IL-1β, IL-6, and IL-8 dosage dependently to values expected; however, meconium alone, containing only 20 pg lipopolysaccharide/mg meconium, induced a markedly higher formation of these cytokines. Adding lipopolysaccharide up to 10000 pg/mL to the meconium did not increase the cytokine formation further. These data suggest that the CD14-mediated meconium-induced cytokine production is caused mainly by factors other than lipopolysaccharide. Although lipopolysaccharide is the main exogenous ligand for TLR4, a number of endogenous candidates (eg, heparan sulfate, hyaluron oligomers, fibrinogen) have been identified.29 Normal, sterile meconium is entirely endogenous and contains innumerable constituents, which could contribute to the CD14-mediated cytokine synthesis. It is therefore not surprising that meconium, containing such a broad spectrum of substances with various biological potential, is able to induce an innate immune response when it meets whole blood.
Because MAS is a disease of the newborn, the study was performed in both adult and cord whole blood, the latter to emphasize the relevance of the disease and the former to pinpoint the difference between immature and mature immunity. It is interesting that the meconium-induced cytokine formation was generally more potent in cord blood than in adult blood. Sixteen of the 27 mediators studied were increased in cord blood, in contrast to 14 in the adult blood. Thus, meconium induced formation of the chemotaxins IP-10 and eotaxin in cord blood but not in adult blood. Furthermore, meconium was much more potent in inducing formation of 7 mediators (IL-1β, IFN-, IL-8, MIP-1, MIP-1β, G-CSF, and IL-1Ra) in cord blood than in adult blood, whereas the meconium-induced formation of only 3 mediators (TNF-, GM-CSF, and basic FGF) was higher in adult blood. Bessler et al30 found fewer CD14-positive mononuclear cells and a lower CD14 density on each cell when comparing cells that were isolated from cord blood versus adult blood. Despite these observations, cord blood seemed in our study to be generally more sensitive to activation by meconium than adult blood. Lipopolysaccharide exposure to cord blood and adult blood measured in flow cytometry gating monocytes showed a significantly reduced expression of TNF- in adult blood compared with cord blood in contrast to a greater percentage of fetal cells expressing IL-8, whereas the expression of IL-1β and IL-6 were the same for both cord and adult blood.31 Lipopolysaccharide-induced production of IL-6 was higher in cord blood than in adult blood, but the increase in TNF- production was higher in adult blood.32 Altogether, these data indicate that there are differences in the response of adult and cord blood and that the response may differ between stimuli. In general, cord blood seems to be more sensitive to activation by meconium than adult blood, probably because of different activities of intracellular responses to CD14 stimulation or other receptor-mediated differences between adult and cord blood.
Because cord blood seems to be more sensitive to meconium than adult blood by producing more cytokines, it is of interest that the effect of inhibition of CD14 and complement is also more efficient in cord blood. Anti-CD14 inhibited the formation of 5 mediators (TNF-, IL-1β, IL-6, MIP-1, and MIP-1β) in adult whole blood by >50%, whereas in cord whole blood, anti-CD14 inhibited the formation of 11 mediators (TNF-, IL-1β, IL-6, IFN-, IL-15, IL-8, MIP-1, MIP-1β, G-CSF, GM-CSF, and eotaxin) >60%. Similarly, complement inhibition reduced the formation of 5 mediators (TNF-, IL-15, MIP-1β, GM-CSF, and basic FGF) in adult whole blood by >40%, whereas in cord whole blood, complement inhibition reduced the formation of 6 mediators (TNF-, IL-1β, IL-15, GM-CSF, IP-10, and eotaxin) by >45%. Most interesting is that the combined inhibition of both complement and CD14 almost completely abolished the formation of most cytokines both in adult blood and in cord blood. Indeed, >85% of the TNF-, IL-1β, IL-6, IL-8, MIP-1, and MIP-1β formation was inhibited by the combined inhibition of CD14 and complement.
This study is an in vitro investigation, and we emphasize that the data should be interpreted with caution with respect to extrapolation to in vivo experimental and clinical MAS. Other cell types are present in the lungs and participate in lung injury in vivo. The incubation period was limited to 4 hours. This is the period in which in our hands the physiologic conditions in the blood was relatively stable. Some cytokines, such as IL-10, need longer time to be synthesized in vitro; therefore, it cannot be excluded that meconium in vivo may induce the formation of more cytokines than observed here. Despite the limitations of the model, we suggest that principally an analogous situation would most likely occur when meconium is aspirated into the lungs of the newborn and there meets both local cells and blood cells from the circulation. Local and subsequent triggering of the 2 main branches of innate immunity would then be suspected. Altogether, it is reasonable to suggest that meconium in vivo is a potent inductor of a broad panel of inflammatory mediators that may contribute to the pathophysiology of MAS.
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CONCLUSIONS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Meconium-induced cytokine production is mediated by complement and CD14. Their relative roles differ among the cytokines, and the CD14 effect cannot be explained by the lipopolysaccharide content of meconium. A combined inhibition of CD14 and complement may be an alternative approach to reduce the inflammatory reaction in MAS.
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ACKNOWLEDGMENTS |
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This project was supported by the Research Council of Norway, the Research Council of Rikshospitalet-Radiumhospitalet, and the Family Blix Foundation.
We thank Anne Pharo and Merethe Sanna Borgen for excellent advice during laboratory analysis.
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FOOTNOTES |
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Accepted Jul 19, 2007.
Address correspondence to Bodil Salvesen, MD, Department of Pediatric Research, Rikshospitalet, N-0027 Oslo, Norway. E-mail: bodil.salvesen{at}medisin.uio.no
Financial Disclosure: Dr Fung is employed by Tanox Inc; the other authors have indicated they have no financial relationships relevant to this article to disclose.
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PEDIATRICS (ISSN 1098-4275). ©2008 by the American Academy of Pediatrics
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