Association Between Serum Macrophage Colony-Stimulating Factor Levels and Monocyte and Thrombocyte Counts in Healthy, Hypoxic, and Septic Term Neonates
Hale Oren
ABBREVIATIONS. G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; M-CSF, macrophage colony-stimulating factor.
Cellular proliferation, maturation, and differentiation of hematopoietic progenitor cells and the regulation of hematopoiesis are dependent, in part, on the supply of highly specific hematopoietic growth factors.[1-3] The dysregulation of hematopoietic growth factor synthesis is an important contributory factor to the complex deficiency of immunologic and hematologic function in the neonate.[4-6] Although term and premature human neonates have high levels of circulating hematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) at birth,[7-12] during stress or states of increased demand for phagocytic immunity the complex dysregulation of neonatal hematopoiesis contributes to the development of peripheral cytopenias, including neutropenia and thrombocytopenia.[6] Although there are several studies that have evaluated the effect and quantity of G-CSF and GM-CSF on this complex dysregulation of neonatal hematopoiesis, there are only a few reports on macrophage colony-stimulating factor (M-CSF) concentrations, which have not directly compared asphyxia versus sepsis in term newborns.[13-16]
M-CSF is a hematopoietic growth factor that stimulates the growth, differentiation, and proliferation of cells of the monocyte-macrophage lineage and induces monocyte production of G-CSF, GM-CSF, interferon-[Gamma] interleukin-1, and tumor necrosis factor.[17-19] M-CSF is produced by a variety of tissue and mesenchymal cells, including marrow stromal cells, fibroblasts, and endothelial cells.[20,21] Placenta is the main source of high serum M-CSF levels in neonates at birth.[14,22,23] It has been demonstrated that the administration of recombinant human M-CSF induces peripheral monocytosis, neutrophilia, and lymphocytopenia in mice, rats, and nonhuman primates.[24-26] M-CSF-induced monocytosis most likely reflects a direct effect of M-CSF on marrow monocyte precursor proliferation, maturation, and release, whereas the neutrophilia and lymphopenia may reflect indirect effects mediated by the known ability of M-CSF to cause the release of the other cytokines.[26] When adverse effects of recombinant human M-CSF were examined in humans and nonhumans, dose-dependent thrombocytopenia has been described.[27-29] Recently, an association between high serum M-CSF levels and thrombocytopenia has been demonstrated in adults.[30-33]
In this study we investigated the serum M-CSF concentrations in healthy, septic, and hypoxic term neonates in the first 24 hours after birth and examined the relationship of serum M-CSF levels and circulating monocyte and thrombocyte counts in these newborn infants.
MATERIALS AND METHODS
Patient Selection
This study was performed prospectively in the Neonatology Unit of the Dokuz Eylul University Hospital, which is a referral center, from September 1997 to August 1999. Seventy-eight infants [is greater than] 37 weeks’ gestational age, without any known prenatal complications, and delivered vaginally were included in the study. The gestational age and birth weight of all neonates were noted.
In patient classification the following 3 groups were defined: group 1, healthy neonates with no risk factors; group 2, neonates who had severe hypoxia, which was defined as a persistently depressed Apgar score with an evidence of severe acidosis after birth[34]; and group 3, neonates who fulfilled the criteria for early-onset sepsis, which was defined as a positive result on 1 or more blood cultures and/or clinical signs or symptoms suggestive of infection within the first few days after birth.[35-37] After obtaining informed consent from each of the mothers, the minimal volume of blood samples was collected for complete blood cell count and serum M-CSF levels by peripheral venipuncture from each infant to prevent inappropriate blood loss. Blood samples from all neonates were obtained within the first 24 hours after birth before any medical therapy.
Assays
Complete blood cell counts were performed on ethylenediaminetetraacetic acid-anticoagulated specimens using a Coulter Counter (Coulter Electronics Inc). A total monocyte cell count was calculated for each complete blood cell count by the percentage of monocytes determined from peripheral blood smears, because manual count may be more accurate than the Coulter Counter result.
For M-CSF measurements, serum was collected after centrifugation of blood samples and stored at -20 [degrees] C until the assays were performed. Serum M-CSF levels were measured with an enzyme-linked immunosorbent assay (Quantikine ELISA Kit, R&D Systems, Minneapolis, MN), which has high specificity, reproducibility, and linearity. A monoclonal antibody specific for M-CSF had been precoated onto a microtiter plate. Standards and samples were pipetted into the wells and any M-CSF present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for M-CSF was added to the wells. After a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells and color developed in proportion to the amount of M-CSF bound in the initial step. The color development was stopped and the intensity of the color was measured. Serum samples were diluted fivefold before the assay, and the minimum detectable dose was typically [is less than] 40 pg/mL. We analyzed single samples from each source because of the small serum volume available from each newborn.
Statistics
Comparison between the 3 groups was investigated with Kruskal-Wallis and Mann-Whitney U tests. The association between serum M-CSF values and monocyte and thrombocyte counts was investigated with Pearson’s correlation test.
RESULTS
A total of 78 infants were enrolled during the study period: 40 in the first group (normal neonates), 20 in the second group (hypoxic neonates), and 18 in the third group (septic neonates). Table 1 gives the minimum, maximum, and mean [+ or -] standard deviation values for gestational age, birth weight, serum M-CSF level, and monocyte and thrombocyte counts of the neonates in groups 1, 2, and 3.
TABLE 1. Gestational Age, Birth Weight, Serum M-CSF Level, and
Monocyte and Thrombocyte Counts of the Neonates in Groups 1, 2, and 3
Group 1 Group 2
(n = 40) (n = 20)
Gestational 39 [+ or -] 1 39 9 [+ or -] 1
age (wk) (37 [+ or -] 40) (37 [+ or -] 40)
Birth 3202 [+ or -] 403 3297 [+ or -] 579
weight (g) (2500 [+ or -] 4200) (2400 [+ or -] 4500)
M-CSF level 1576 [+ or -] 632 2190 [+ or -] 967
(pg/mL) (519 [+ or -] 2930) (817 [+ or -] 4560)
Monocyte count 514 [+ or -] 447 521 [+ or -] 345
([mm.sup.3]) (0-2870) (138-1536)
Thrombocyte count 264 950 [+ or -] 67 779 227 600 [+ or -] 74 415
([mm.sup.3]) (157 000-392 000) (144 000-392 000)
Group 3
(n = 18)
Gestational 39 [+ or -] 1
age (wk) (37-40)
Birth 2962 [+ or -] 526
weight (g) (2000 [+ or -] 4200)
M-CSF level 3548 [+ or -] 2567
(pg/mL) (1134 [+ or -] 10 830)
Monocyte count 523 [+ or -] 299
([mm.sup.3]) (168-1260)
Thrombocyte count 144 722 [+ or -] 42 794
([mm.sup.3]) (7000-217 000)
(*) Results are expressed as mean [+ or -] standard
deviation (minimum-maximum).
The gestational ages and birth weights did not differ significantly between the groups (P = .093; P = .246). Serum M-CSF levels of the septic neonates (group 3) were significantly higher than serum M-CSF levels of both healthy (group 1) and hypoxic (group 2) neonates (P [is less than] .001; P [is less than] .001). Serum M-CSF levels did not differ significantly between the healthy and hypoxic neonates (P = .996). There was no significant correlation between serum M-CSF levels and circulating monocyte counts (r = 0.1400; P = .222). There was a significant inverse correlation between serum M-CSF levels and thrombocyte counts (r = -0.37; P = .001). When this correlation was analyzed according to groups, we determined that this inverse correlation between M-CSF levels and thrombocyte counts was especially significant in the septic neonate group (r = -0.5738; P = .013) but not significant in the healthy and hypoxic neonate groups (r = 0.2972, P = .063; r = -0.1459, P = .539).
DISCUSSION
The high M-CSF levels at birth reported in literature may be attributable to production of M-CSF by the fetal placenta and macrophages in the maternal decidua and may be partly of maternal origin via transplacental transfer.[14,22,38] Because it has been shown that serum levels of M-CSF are significantly higher on day 1 after birth than in cord blood, this growth factor seems to be also actively synthesized after birth in the neonates during this critical developmental period.[14,16] High serum M-CSF levels in neonates gradually decrease after day 3 but are still above adult values during the neonatal period.[14-16,39,40] Because of those alterations in serum M-CSF levels, neonates who were within the first 24 hours after birth were included in our study.
Our data have demonstrated significantly higher concentrations of M-CSF in septic neonates than in normal and hypoxic neonates. Several studies in the literature report high levels of G-CSF and GM-CSF in neonates with infection,[9-11] but we could not find another study reporting high serum M-CSF levels in neonates with sepsis. In a recent report of Ikeno et al,[15] who compared M-CSF levels in normal neonates, no significant difference in M-CSF levels were observed in neonates with infection. Our results may differ from this report because of limited numbers of patients, patient selection, and timing of blood samples. On the other hand, other reports note elevated levels of M-CSF in adult patients with sepsis or severe infection.[40-42] In adult patients with documented infections due to Gram-positive, Gram-negative, or fungal infections, M-CSF levels were higher than in patients without documented infections.[42] It has been also shown that after exposure to cytokines, such as tumor necrosis factor, induced by infection, G-CSF, GM-CSF, and M-CSF are coinduced.[43-45]
In our study, although serum M-CSF levels of hypoxic neonates were slightly higher than M-CSF levels of healthy neonates, there was no statistically significant difference. But Ikeno et al[15] found significantly higher M-CSF levels in neonates with perinatal complications including premature rupture of the membranes, neonatal asphyxia, meconium staining of the amniotic fluid, and maternal anemia.
There was no significant correlation between M-CSF levels and monocyte counts in our study. Roth et al[13] also did not find a correlation between monocyte counts and M-CSF levels in the newborn infants. Possibly the M-CSF levels in the neonate are so high[13-16] that even those infants with lower concentrations already may have attained the threshold necessary for maximum monocyte production. Also, the rise in circulating M-CSF levels observed immediately after birth may lead to increased numbers of macrophages without significantly affecting the numbers of circulating monocytes.[13] It has been shown that the administration of recombinant human M-CSF leads to circulating monocytosis after the first few days of life but not immediately.[25,26,28]
Our data indicate a significant inverse correlation between M-CSF levels and thrombocyte counts. When this correlation was analyzed according to groups, we determined that this inverse correlation was especially significant in neonates with sepsis and not significant in healthy and hypoxic neonate groups. To our knowledge there is no similar reported data demonstrating an inverse correlation between high M-CSF levels and thrombocytopenia in neonates with sepsis. The mechanism of thrombocytopenia is unclear, but several recent studies also supported this negative correlation between high serum M-CSF levels and thrombocytopenia. Vial et al,[27] Munn et al,[28] and Baker et al[29] showed that administration of M-CSF may cause a dose-dependent thrombocytopenia. Baker et al[29] reported that thrombocytopenia produced by M-CSF was not at tributable to suppression of thrombopoiesis, but to increased activity of the monocyte/macrophage system, which causes shortened platelet survival. A study by Francois et al[41] supported this finding; they demonstrated that unexplained thrombocytopenia diagnosed in adult patients with the sepsis syndrome is frequently associated with hemophagocytosis and a high serum M-CSF level. In neonates, thrombocytopenia may occur frequently in sick neonates, and probably more than 1 mechanism leads to thrombocytopenia associated with sepsis.[46-49] According to our results, high M-CSF levels may be one of the factors in the pathogenesis of thrombocytopenia in neonates with sepsis syndrome, probably by causing consumption. Increased M-CSF levels and thrombocytopenia have also been reported in adult patients with immune thrombocytopenic purpura,[30,31] hemophagocytic lymphohistiocytosis,[50] and pregnancy,[32] probably attributable to monocyte/ macrophage-mediated platelet destruction.
Serum M-CSF levels were significantly higher in neonates with sepsis. Although there was no correlation between serum M-CSF levels and circulating monocyte counts, there was an inverse correlation between M-CSF levels and thrombocyte counts, suggesting a possible role for increased M-CSF levels in the pathogenesis of thrombocytopenia in neonates with sepsis.
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Hale Oren, MD; Nuray Duman, MD; Hakan Abacioglu, MD; Hasan Ozkan, MD; and Gulersu Irken, MD
From the From the Departments of Pediatrics and Microbiology, Dokuz Eylul University Faculty of Medicine, Izmir, Turkey. Received for publication Sep 13, 2000; accepted Dec 11, 2000. Address correspondence to Hale Oren, MD, Dokuz Eylul University Faculty of Medicine Department of Pediatrics, 35340 inciralti-Izmir Turkey. E-mail: hale.oren@deu.edu.tr
COPYRIGHT 2001 American Academy of Pediatrics
COPYRIGHT 2001 Gale Group
