PEDIATRICS Vol. 108 No. 2 August 2001, pp. 329-332

Association Between Serum Macrophage Colony-Stimulating Factor Levels and Monocyte and Thrombocyte Counts in Healthy, Hypoxic, and Septic Term Neonates

Hale Ören, MD, Nuray Duman, MD, Hakan Abacioglu, MD, Hasan Özkan, MD, and Gülersu Irken, MD

From the From the Departments of Pediatrics and Microbiology, Dokuz Eylül University Faculty of Medicine, Izmir, Turkey.

	ABSTRACT

Top Abstract MaterialsMethods Results Discussion References

Objective.  Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that mainly stimulates the growth, differentiation, and proliferation of cells of the monocyte-macrophage lineage. There are only limited numbers of studies about M-CSF levels in neonates, but high levels of serum M-CSF have been reported in septic and some thrombocytopenic adult patients. In this study, we investigated the serum M-CSF levels in healthy, septic, and hypoxic term neonates on the first day of life and examined the relationship of serum M-CSF levels and circulating monocyte and thrombocyte counts in these newborn infants.

Study Design.  Three groups were defined in this prospective study: group 1, healthy neonates with no risk factors (n = 40); group 2, neonates who had severe hypoxia (n = 20); and group 3, neonates who fulfilled the criteria for early-onset sepsis (n = 18). Blood samples were collected for complete blood cell count and serum M-CSF levels by peripheral venipuncture from each infant in the first 24 hours after birth before any medical therapy.

Results.  The gestational ages and birth weights did not differ significantly between the groups. Serum M-CSF levels of the septic neonates were significantly higher than of both healthy and hypoxic neonates, but did not differ significantly between the healthy and hypoxic neonates. There was no significant correlation between serum M-CSF levels and circulating monocyte counts, but there was a significant inverse correlation between serum M-CSF levels and thrombocyte counts. 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, but not significant in the healthy and hypoxic neonate groups.

Conclusions.  Serum M-CSF levels are significantly higher in neonates with sepsis. High serum M-CSF levels may have a possible role in the pathogenesis of thrombocytopenia in neonates with sepsis.  Key words:  macrophagecolony-stimulating factor, thrombocytopenia, monocyte, sepsis, neonate.

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-, 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

Top Abstract MaterialsMethods Results Discussion References

Patient Selection

This study was performed prospectively in the Neonatology Unit of the Dokuz Eylül University Hospital, which is a referral center, from September 1997 to August 1999. Seventy-eight infants >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 birth³⁴; 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°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 <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

Top Abstract MaterialsMethods Results Discussion References

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 ± 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. 

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 < .001; P < .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

Top Abstract MaterialsMethods Results Discussion References

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 al¹⁵ 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 al¹³ 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 al²⁹ showed that administration of M-CSF may cause a dose-dependent thrombocytopenia. Baker et al²⁹ reported that thrombocytopenia produced by M-CSF was not attributable to suppression of thrombopoiesis, but to increased activity of the monocyte/macrophage system, which causes shortened platelet survival. A study by Francois et al⁴¹ 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.

	FOOTNOTES

Received for publication Sep 13, 2000; accepted Dec 11, 2000.

Address correspondence to Hale Ören, MD, Dokuz Eylül University Faculty of Medicine Department of Pediatrics, 35340 nciralt-zmir Turkey. E-mail: hale.oren{at}deu.edu.tr

	ABBREVIATIONS

G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; M-CSF, macrophage colony-stimulating factor.

	REFERENCES

Top Abstract MaterialsMethods Results Discussion References

1. Sieff C Hematopoietic growth factors. J Clin Invest 1987; 79:1549-1557
2. Clark S, Kamen R The human hematopoietic colony-stimulating factors. Science 1987; 236:1229-1237 [Abstract/Free Full Text]
3. Nathan D Regulation of hematopoiesis. Pediatr Res 1990; 27:423-431 [Medline]
4. Cairo MS Therapeutic implications of dysregulated colony-stimulating factor expression in neonates. Blood 1993; 82:2269-2272 [Free Full Text]
5. Rondini G, Chirico G Hematopoietic growth factor levels in term and preterm infants. Curr Opin Hematol 1999; 6:192-197 [CrossRef][Medline]
6. Christensen R Hematopoiesis in the fetus and neonate. Pediatr Res 1989; 26:531-535 [Medline]
7. Laver J, Duncan E, Abboud M, High levels of granulocyte and granulocyte-macrophage colony-stimulating factors in cord blood of normal full-term neonates. J Pediatr 1990; 116:627-632 [CrossRef][Medline]
8. Russell ARB, Davies EG, Scopes GJD, Daly S, Gordon-Smith EC Plasma granulocyte-colony stimulating factor concentrations (G-CSF) in the early neonatal period. Br J Haematol 1993; 86:642-644 [CrossRef]
9. Gessler P, Kirchmann N, Kientsch-Engel R, Haas N, Lasch P, Kachel W Serum concentrations of granulocyte colony-stimulating factor in healthy term and preterm neonates and in those with various diseases including bacterial infections. Blood 1993; 82:3177-182 [Abstract/Free Full Text]
10. Bailie KEM, Irvine AE, Bridges JM, McClure BG Granulocyte and granulocyte-macrophage colony-stimulating factors in cord and maternal serum at delivery. Pediatr Res 1994; 35:164-168 [Medline]
11. Weimann E, Rutkowski S, Reisbach G G-CSF, GM-CSF and IL-6 levels in cord blood: diminished increase of G-CSF and IL-6 in preterm with perinatal infection compared to term neonates. J Perinat Med 1998; 26:211-218 [Medline]
12. Cokun M, Kardelen F, Oygür N, Ündal L, Sava A, Yein O Granulocyte and granulocyte-macrophage colony-stimulating factors, their receptors and interleukin-3 levels in newborns. Turk J Pediatr 1997; 39:295-301 [Medline]
13. Roth P Colony-stimulating factor 1 levels in the human newborn infant. J Pediatr 1991; 119:113-116 [CrossRef][Medline]
14. Ishii E, Masuyama T, Yamaguchi H, Production and expression of granulocyte- and macrophage-colony-stimulating factors in newborns: their roles in leukocytosis at birth. Acta Haemotol 1995; 94:23-31
15. Ikeno K, Koike K, Fukuromoto T, Shimizu T, Nagatomo M, Komiyama A Increased macrophage-colony stimulating factor levels in neonates with perinatal complications. Early Hum Dev 1996; 46:229-237 [CrossRef][Medline]
16. Roth P, Stanley ER Colony-stimulating factor-1 expression in the human fetus and newborn. J Leukoc Biol 1995; 58:432-437 [Abstract]
17. Stanley ER, Guilbert LJ, Tushinski RJ, Baltelmez SH A mononuclear phagocyte lineage-specific hemopoietic growth factor. J Cell Biochem 1983; 21:151-159 [CrossRef][Medline]
18. Warren MK, Ralph P Macrophage growth factor CSF-1 stimulates human monocyte production of interferon, tumour necrosis factor, and colony stimulating activity. J Immunol 1986; 137:2281-2285 [Abstract]
19. Moore RN, Oppenheim J, Farrar J, Carter C, Waheed A, Shadduck R Production of lymphocyte-activating factor (interleukin 1) by macrophages activated with colony-stimulating factors. J Immunol 1980; 125:1302-1305 [Abstract]
20. Gimble JM, Pietrangeli C, Henley A, Characterization of murine bone marrow and spleen derived stromal cells. Analysis of leukocyte marker and growth factor mRNA transcript levels. Blood 1989; 74:303-311 [Abstract/Free Full Text]
21. Sieff CA, Niemeyer CM, Mentzer SJ, Faller DV Interleukin-1, tumor necrosis factor, and the production of colony-stimulating factors by cultured mesenchymal cells. Bood 1988; 72:1316-1323
22. Daiter E, Pampfer S, Yeung Y, Barad D, Stanley E, Pollard J Expression of colony-stimulating factor-1 in the human uterus and placenta. J Clin Endocrinol Metab 1992; 74:850-858 [Abstract]
23. Wegmann TG, Athanassakis I, Guilbert L, The role of M-CSF and GM-CSF in fostering placental growth, fetal growth and fetal survival. Transplant Proc 1989; 21:566-568 [Medline]
24. Koren S, Klimpel GR, Fleishman WR Treatment of mice with macrophage-colony stimulating factor (CSF-1) prevents the in vivo myelosuppression induced by murine alpha, beta and gamma interferons. J Biol Resp Modifiers 1986; 5:481-489
25. Hume DA, Parli P, Donahue RE, Fidler IJ The effect of human recombinant macrophage colony-stimulating factor (CSF-1) on the murine mononuclear phagocyte system in vivo. J Immunol 1988; 141:3405-3409 [Abstract]
26. Ulich TR, Castillo J, Watson LR, Yin S, Garnick MB In vivo hematologic effects of recombinant human macrophage colony-stimulating factor. Blood 1990; 75:846-856 [Abstract/Free Full Text]
27. Vial T, Descotes J Clinical toxicity of cytokines used as haemopoietic growth factors. Drug Saf 1995; 13:371-406 [Medline]
28. Munn DH, Garnick MB, Cheung NV Effects of parenteral recombinant human macrophage colony-stimulating factor on monocyte number, phenotype, and antitumor cytotoxicity in nonhuman primates. Blood 1990; 75:2042-2048 [Abstract/Free Full Text]
29. Baker GR, Levin J Transient thrombocytopenia produced by administration of macrophage colony-stimulating factor: investigations of the mechanism. Blood 1998; 91:89-99 [Abstract/Free Full Text]
30. Zeigler ZR, Rosenfeld CS, Nemunaitis JJ, Besa EC, Shadduck RK Increased macrophage colony-stimulating factor levels in immune thrombocytopenic purpura. Blood 1993; 81:1251-1254 [Abstract/Free Full Text]
31. Nomura S, Yasunaga K, Fujimara K, Kuramato A, Okuma M, Nomura T High dose intravenous gamma globulin reduces macrophage colony-stimulating factor levels in idiopathic thrombocytopenic purpura. Int J Hematol 1996; 63:227-234 [CrossRef][Medline]
32. Paloran V, Coupey L, Donnard M, Berrada L, Naud MF Elevation of serum M-CSF concentrations during pregnancy and ovarian hyperstimulation. Br J Haematol 1994; 86:675-677 [Medline]
33. Yong K, Salooja N, Donahue RE, Hedge U, Linch DC Human macrophage colony stimulating factor levels are elevated in pregnancy and in immune thrombocytopenia. Blood 1992; 80:2897-2902 [Abstract/Free Full Text]
34. Carter BS, Haverkamp AD, Merenstein GB The definition of acute perinatal asphyxia. Clin Perinatol 1993; 20:287-304 [Medline]
35. Bone RC, Balk RA, Cerra FB, American College of Chest Physicians Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure for the use of innovative therapies in sepsis. Chest 1992; 101:1481-1483 [Free Full Text]
36. Uzuner N, lekel H, Özkan H, en A, Yenice S, Çevik N Urinary nitrite excretion in low birth weight neonates with systemic inflammatory response syndrome. Biol Neonate 1997; 71:362-366 [Medline]
37. Placzek MM, Whitelaw A Early and late neonatal septicemia. Arch Dis Child 1983; 58:728-731 [Abstract/Free Full Text]
38. Saito S, Motoyoshi K, Ichijo M, Saito M, Takaku F High serum human macrophage colony stimulating factor level during pregnancy. Intern J Hematol 1992; 55:219-225
39. Aihara T, Misago M, Hanamura T, Within-day and day-to-day variations of serum M-CSF levels in healthy volunteers. Rinsho Byori 1993; 41:268-272 [Medline]
40. Hanamura T, Motoyoshi K, Yoshida K, Quantitation and identification of human monocytic colony-stimulating factor in human serum by enzyme-linked immunosorbent assay. Blood 1988; 72:886-892 [Abstract/Free Full Text]
41. Francois B, Trimoreau F, Vignon P, Fixe P, Praloran V, Gastinne H Thrombocytopenia in the sepsis syndrome: Role of hemophagocytosis and macrophage colony stimulating factor. Am J Med 1997; 103:114-120 [CrossRef][Medline]
42. Cebon J, Layton JE, Maher D, Morstyn G Endogenous haemopoietic growth factors in neutropenia and infection. Br J Haematol 1994; 86:265-274 [Medline]
43. Oster W, Lindemann A, Mertelsmann R, Herrmann F Production of macrophage-, granulocyte-, granulocyte-macrophage- and multi-colony-stimulating factor by peripheral blood cells. Eur J Immunol 1989; 19:543-547 [Medline]
44. Weiss M, Moldower LL, Schneider EM Granulocyte colony-stimulating factor to prevent the progression of systemic nonrespondiveness in systemic inflammatory response syndrome and sepsis. Blood 1999; 93:425-439 [Free Full Text]
45. Koeffler HP, Gasson J, Tabler A Transcriptional and posttranscriptional modulation of myeloid colony-stimulating factor expression by tumor necrosis factor and other agents. Mol Cell Biol. 1988; 8:3432-3436 [Abstract/Free Full Text]
46. Ören H, rken G, Ören B, Olgun N, Özkan H Assessment of clinical impact and predisposing factors for neonatal thrombocytopenia. Ind J Pediatr. 1994; 61:551-558 [Medline]
47. Homans A Thrombocytopenia in the neonate. Pediatr Clin N Am. 1996; 43:737-756 [CrossRef][Medline]
48. Blanchette VS, Rand ML Platelet disorders in newborn infants: diagnosis and management. Semin Perinatol. 1997; 21:53-62 [CrossRef][Medline]
49. Roberts IA, Murray NA Review: management of thrombocytopenia in neonates. Br J Hematol 1999; 105:864-870 [CrossRef][Medline]
50. Akashi K, Hayashi S, Gondo H, Involvement of interferon- and macrophage colony-stimulating factor in pathogenesis of hemophagocytic lymphohistiocytosis in adults. Br J Hematol. 1994; 87:243-250 [Medline]