Mature Neutrophil Function Research Articles

PRAM-1 Is Required for Optimal Integrin-Dependent Neutrophil Function

PML-retinoic acid receptor alpha (RARalpha) regulated adaptor molecule 1 (PRAM-1) is an intracellular adaptor molecule that is upregulated during the induced granulocytic differentiation of promyelocytic leukemic cells and during normal human myelopoiesis. This report describes the generation of PRAM-1-deficient mice and an analysis of the function of this adaptor in neutrophil differentiation and mature neutrophil function. We demonstrate here that neutrophil differentiation is not impaired in PRAM-1-deficient mice and that PRAM-1-deficient neutrophils function normally following engagement of Fcgamma receptors. In contrast, mature PRAM-1-null neutrophils exhibit significant defects in adhesion-dependent reactive oxygen intermediate production and degranulation. Surprisingly, other integrin-dependent responses, such as cell spreading and activation of several signaling pathways, are normal. Together, these findings demonstrate the uncoupling of key integrin-dependent responses in the absence of PRAM-1 and show this adaptor to be critical for select integrin functions in neutrophils.

Stat3 Is Required for In Vivo G-CSF-Responsiveness and Neutrophil Function.

Granulocyte-colony stimulating factor (G-CSF) and its receptor (G-CSFR) are essential for neutrophil development. The G-CSF/G-CSFR pathway supplies normal circulating neutrophil levels and supports neutrophil terminal maturation. These activities have been exploited clinically, where G-CSF is used frequently to treat congenital or therapeutically induced neutropenia, or to mobilize hematopoietic stem cells for bone marrow transplant. Previous work from our laboratory and others has shown a role for the major G-CSF-responsive signaling molecule STAT3 in granulopoiesis. It is well established that STAT3 has an important negative function in controlling neutrophil production. Importantly, our studies have elucidated a critical positive regulatory role for STAT3 in G-CSF-responsiveness and development of functional neutrophils in vivo. Mice lacking STAT3 in the bone marrow (TIE2cre/Stat3f/Δ) fail to upregulate circulating neutrophil levels in response to a single dose of G-CSF administered subcutaneously, while their wild type littermates demonstrate a two-fold increase in circulating neutrophil levels (e.g., Gr-1+/CD11b+ cells) under these conditions. Inspection of bone marrow responses revealed an increase in the Gr-1lo/CD11bhi population and a corresponding decrease in the Gr-1hi/CD11bhi population of littermate control animals following G-CSF administration. Strikingly, mice that lack functional STAT3 in the bone marrow fail to respond to G-CSF similarly; these animals do not demonstrate an induction in the Gr-1lo/CD11bhi population, or a decrease in the Gr-1hi/CD11bhi population within the bone marrow following G-CSF treatment. These results indicate that in vivo G-CSF administration recruits early progenitors (e.g., Gr-1lo/CD11bhi cells) into the granulocytic lineage while concomitantly decreasing levels of more mature granulocytes (e.g., Gr-1hi/CD11bhi) within the marrow, which are likely mobilized to the peripheral circulation, and suggest an essential role for STAT3 in these responses. Furthermore, neutrophils isolated from TIE2cre/Stat3f/Δ mice demonstrate defective chemotaxis in response to MIP-2. This deficiency appears to be cell autonomous since granulocytes derived from an ex vivo differentiation system show a similar phenotype. Defective chemotaxis may be due in part to improper actin rearrangement dynamics, since STAT3 deficient neutrophils show enhanced actin polymerization in response to MIP-2 compared to wild type controls. To examine potential pathways by which STAT3 may function, we investigated the expression of the PU.1 transcription factor in TIE2cre/Stat3f/Δ mice. PU.1 is known to control expression of genes that are essential for mature neutrophil functions. Our results demonstrate that PU.1 is expressed at high levels in the Gr-1lo/CD11bhi population relative to the Gr-1hi/CD11bhi population isolated from bone marrow of wild type mice. Ex vivo G-CSF treatment of wild type bone marrow stimulates an increase in the proportion of Gr-1lo/CD11bhi cells within the culture, as well as the total PU.1 protein level of the population. By contrast, induction of Gr-1lo/CD11bhi cells and total PU.1 expression by G-CSF treatment ex vivo is abrogated in bone marrow from TIE2cre/Stat3f/Δ mice. Importantly, these effects are not due to significant changes in cell survival or selection in the ex vivo culture system. Collectively, our results indicate that STAT3 is essential for G-CSF-mediated neutrophil responses and function in vivo, and suggest that STAT3-dependent regulation of PU.1 may be an important intermediate in this pathway.

The murine myeloid cell line 32Dcl3 as a model system for studying neutrophil functions

The murine myeloid cell line 32Dcl3 is one of the few cell lines that can terminally differentiate into neutrophils. Granulocyte colony-stimulating factor (G-CSF) drives the differentiation of these cells; therefore, G-CSF receptor signaling for neutrophil proliferation and differentiation has been studied extensively using this cell line as a model. Differentiated 32Dcl3 cells exhibit a striking morphologic similarity to normal neutrophils; however, the degree to which differentiated 32Dcl3 cells are functionally similar to normal neutrophils remains unknown. In this study, we compared the function of differentiated 32Dcl3 cells with mouse neutrophils. Our results demonstrate that a subclone of differentiated 32Dcl3 cells (32Dcl3C) exhibits normal neutrophil functions of phagocytosis, degranulation, adhesion and shape change in response to appropriate stimuli. These observations suggest that this cell line can serve as an effective model system to study similar mature neutrophil functions. However, 32Dcl3C cells fail to produce superoxide in response proper stimuli.

The relationship between endogenous cortisol, blood micronutrients, and neutrophil function in postparturient Holstein cows.

Neutrophil function, blood micronutrients, and cortisol concentrations were measured in 43 clinically healthy postparturient Holstein cows. Estimated 305-day mature equivalent milk production and neutrophil function were related to results of the blood micronutrient concentrations and neutrophil function tests. Cattle had low to normal zinc concentrations; normal to high selenium, vitamin E, and cortisol concentrations; and normal copper concentrations. Blood selenium (P = .03) and zinc (P = .027) concentrations were both significant predictors of neutrophil adhesion, and selenium (P < .001) was a significant predictor of neutrophil cytochrome C reduction (superoxide production). Fourteen of 20 (70%) cattle with blood selenium concentrations > 300 ng/mL had neutrophil adhesion, and 15 of 20 (75%) had cytochrome C reduction above the mean value for this group. There was also a significant correlation (r = 0.331; P = .037) between cytochrome C reduction and estimated milk production. These findings suggest that neutrophils from postparturient dairy cows with higher blood concentrations of selenium have greater potential to kill microbes, and that cattle with greater superoxide production may have higher milk production.

Convergent alteration of granulopoiesis, chemotactic activity, and neutrophil apoptosis during mouse selection for high acute inflammatory response

Neutrophil homeostasis was investigated in two mouse lines, AIRmax and AIRmin, genetically selected for high or low acute inflammatory response (AIR) and compared with unselected BALB/c mice. Mature neutrophil phenotype and functions appeared similar in the three mouse lines. However, an unprecedented phenotype was revealed in AIRmax animals characterized by a high neutrophil production in bone marrow (BM), a high number of neutrophils in blood, a high concentration of chemotactic agents in acrylamide-induced inflammatory exudates, and an increased resistance of locally infiltrated neutrophils to spontaneous apoptosis. In vitro, BM production of neutrophils and eosinophils was accompanied by an unusual high up-regulation of cytokine receptors as assessed by antibodies to CD131, which bind the common beta chain of receptors to interleukin (IL)-3, IL-5, and granulocyte macrophage-colony stimulating factor. An accelerated neutrophil maturation was also observed in response to all-trans retinoic acid. Several candidate genes can be proposed to explain this phenotype. Yet, more importantly, the results underline that genetic selection, based on the degree of AIR and starting from a founding population resulting from the intercross of eight inbred mouse lines, which display a continuous range of inflammatory responses, can lead to the convergent selection of alleles affecting neutrophil homeostasis. Similar gene combinations may occur in the human with important consequences in the susceptibility to inflammatory or infectious diseases and cancer.

The Relationship between Endogenous Cortisol, Blood Micronutrients, and Neutrophil Function in Postparturient Holstein Cows

Neutrophil function, blood micronutrients, and cortisol concentrations were measured in 43 clinically healthy postparturient Holstein cows. Estimated 305-day mature equivalent milk production and neutrophil function were related to results of the blood micro-nutrient concentrations and neutrophil function tests. Cattle had low to normal zinc concentrations; normal to high selenium, vitamin E, and cortisol concentrations; and normal copper concentrations. Blood selenium (P= .03) and zinc (P= .027) concentrations were both significant predictors of neutrophil adhesion, and selenium (P < .001) was a significant predictor of neutrophil cytochrome C reduction (superoxide production). Fourteen of 20 (70%) cattle with blood selenium concentrations >300 ng/mL had neutrophil adhesion, and 15 of 20 (75%) had cytochrome C reduction above the mean value for this group. There was also a significant correlation (r= 0.331; P= .037) between cytochrome C reduction and estimated milk production. These findings suggest that neutrophils from postparturient dairy cows with higher blood concentrations of selenium have greater potential to kill microbes, and that cattle with greater superoxide production may have higher milk production.

Granulocyte colony-stimulating factor promotes adhesion of neutrophils.

Granulocyte colony stimulating factor (G-CSF) is well known for its ability to drive the maturation and mobilization of neutrophils. G-CSF also appears to have the potential to activate functions of mature neutrophils, influencing recruitment at sites of inflammation and tissue injury. We investigated the ability of G-CSF to stimulate adhesion of isolated blood neutrophils. G-CSF induced significant adherence to intercellular adhesion molecule (ICAM)-1 that was both macrophage antigen-1 (Mac-1) and leukocyte function-associated antigen-1 dependent. The kinetics of G-CSF-stimulated adhesion to ICAM-1 peaked at 11 min without detectable surface upregulation of Mac-1. This was in marked contrast to chemokines, in which peak activation of adhesion is seen within 1 min of stimulation. In contrast to chemokine-induced adhesion, G-CSF stimulation was not inhibited by pertussis toxin. G-CSF also augmented the attachment of neutrophils to activated human umbilical vein endothelial cells (HUVEC) through specific effects on neutrophils, because HUVEC appear to lack functional G-CSF receptors.

Molecular aspects of anti-inflammatory action of G-CSF.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine which stimulates the production of neutrophils in the bone marrow and modulates cellular functions of mature neutrophils. Besides neutrophils and their precursors, monocytes are direct target cells of G-CSF action. G-CSF influences monocyte functions in an anti-inflammatory way: The stimulation of monocytes with G-CSF results in an attenuation of LPS-induced release of IL-1beta, TNF-alpha, IL-12 and IL-18. G-CSF exerts its biological functions on neutrophils and monocytes via membrane-bound receptors. Seven different human G-CSF receptor isoforms have been described which are generated by alternative splicing. The physiologic roles of these isoforms and the expression pattern on various cell types are still unknown. The signal transduction pathway of G-CSF receptors involves the activation of JAK tyrosine kinases and STAT transcription factors and the ras/mitogen-activated protein kinase pathway.

Myeloid hematopoietic growth factors and their role in prevention and/or treatment of neonatal sepsis

Sepsis continues to be an important cause of morbidity and mortality among both full-term and preterm infants, secondary to an immaturity in neonatal host defense. The incidence of neonatal sepsis ranges from 30% in very low birth weight infants to 0.4% in preterm neonates and 0.1% in term neonates. The dysregulation of the expression and production of hematopoietic cytokines in the neonate contributes to quantitative and qualitative deficiencies in neonatal myeloid progenitor activity and decreased availability and function of mature effector neutrophils. These abnormalities contribute in large part to the increased incidence and mortality associated with neonatal sepsis. In this review, we have summarized and analyzed the studies investigating the dysregulation, expression and production of myelopoietic growth factors in neonates, the preclinical in vivo effects of myelopoietic growth factors in neonatal animals, the preclinical in vivo effects of myelopoietic growth factors during experimental sepsis in neonatal animals, the in vitro effects of growth factors on human neonatal phagocytic immunity, and clinical results of myelopoietic growth factors in human neonates. Future studies should be focused on investigating other abnormalities of neonatal host defense and multiple and simultaneous approaches to circumvent identified defects to attempt to reduce both the incidence and severity of neonatal host defense.

A new hypothalamic polypeptide is a regulator of myelopoiesis.

The effect of hypothalamic proline-rich polypeptide (PRP) against Pseudomonas aeruginosa infection in BALB/c mice with leukopenia was investigated. Mice were treated with cyclophosphamide (CPA) and were then injected with PRP 24 h after CPA treatment. The lethal doses of P. aeruginosa were injected to mice when the number of peripheral blood leukocytes reached a nadir on day 5 after CPA administration. The administration of PRP significantly increased the survival of infected mice, and had a pronounced protective effect during the period of development of the infection. The number of bacteria in internal organs of PRP-treated mice was significantly lower than that in control mice. In PRP-treated mice, the neutrophil levels in peripheral blood started to increase 7 days after CPA administration and were consistently higher, and they were more mature than those in controls. Our results may indicate the ability of PRP to stimulate recovery of myelopoiesis and enhance mature neutrophil function.

Granulocyte colony-stimulating factor receptor expression on neutrophils of term and preterm neonates with and without signs of infection.

Neutrophils are an essential component of the human host defence system against infection. Recombinant human granulocyte colony-stimulating factor induces neutrophilia and enhances effector functions of mature neutrophils. Since the biological effects of granulocyte colony-stimulating factor (G-CSF) are mediated by its receptor, we investigated the expression of G-CSF receptor on the surface of neutrophils of term and preterm neonates (n = 22) with and without signs of infection and of healthy adults (n = 13) by flow cytometry. In healthy adults, the percentage of neutrophils expressing G-CSF receptor was higher compared to cord blood of term and preterm neonates (87% vs 53%, P < 0.05). Between 2 and 32 h of life, neonates with signs of infection showed lower values of G-CSF receptor expression compared to neonates without signs of infection (32% vs 54%, P < 0.05). No correlation was detectable between expression of G-CSF receptor and gestational age. Expression of granulocyte colony-stimulating factor receptor on neutrophils is lower than in adults. This may adversely affect granulopoiesis and neutrophil function during the neonatal period. Moreover, granulocyte colony-stimulating factor receptor expression seems to be down-regulated during neonatal infection.

In vitro effects of GM-CSF on mature peripheral blood neutrophils.

GM-CSF can play a crucial role in regulating the neutrophil-mediated inflammatory response. This growth factor is a proliferative stimulus for bone marrow neutrophil stem cell precursors and has at least 3 important roles in regulating neutrophil-mediated immunity: a) a direct effect on the proliferation and development of neutrophil progenitors; b) synergistic activity with other haemopoietic growth factors; c) stimulation of the functional activity of mature neutrophils. The production of GM-CSF may be triggered directly by exogenous factors such as antigens and endotoxins, or indirectly through the release of cytokines by a variety of cells including lymphocytes, activated macrophages and endothelial cells exposed to products of mononuclear phagocytes. Such production of GM-CSF may serve to quickly release mature neutrophils from the bone marrow in response to infections. Moreover, enhancement of the function of mature neutrophils may also augment their ability to migrate to infective sites and then phagocytose and kill pathogens. Increased expression of CD11b/CD18 may play a fundamental part in this mechanism because this receptor is essential for the adhesion of neutrophils to the endothelium. Both phagocytosis and oxidative burst activity increase as a result of the action of GM-CSF and the increased expression of complement- and Fc-receptors can augment opsono-phagocytosis. A further level of neutrophil up-regulation occurs by increasing the functional life span of neutrophils by GM-CSF. Thus, by delaying neutrophil apoptosis, GM-CSF greatly extends the time over which neutrophils may function at inflammatory sites. GM-CSF can thus exert a variety of important regulatory controls of neutrophil function during bacterial infections. Both the number and the functional status of neutrophils is highly regulated by GM-CSF. It is also possible that GM-CSF produced within localised sites of acute inflammation or infection may attract, trap and then activate neutrophils within this site.

G-CSF receptor expression on neutrophils of healthy term and preterm neonates and in those with infection

Background: Granulocyte colony-stimulating factor (G-CSF) has been shown to induce neutrophilia and to enhance mature effector neutrophil function. Whereas serum concentrations of G-CSF are high during infection, nothing is known about G-CSF receptor (G-CSF-R) expression on neutrophils of neonates.

The cytoplasmic domain of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha subunit is essential for both GM-CSF-mediated growth and differentiation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates differentiation, survival, and proliferation of colony-forming unit-granulocyte-macrophage progenitor cells. The biologic actions of GM-CSF are mediated by binding to a specific receptor consisting of two chains designated as alpha and beta subunits. We have demonstrated that the murine FDC-P1-derived cell line WT-19 transfected with the human GM-CSF receptor alpha and beta subunits (GM-CSFRalpha and beta) can be induced to differentiate by the addition of human GM-CSF (hGM-CSF). By expressing a series of GM-CSFRalpha mutants in WT19 cells, we have determined the amino acid domains of the GM-CSFRalpha cytoplasmic domain that regulate cell differentiation, proliferation, and survival. We found that the membrane proximal proline-rich domain and adjacent 16 residues are essential for both hGM-CSF-dependent cell proliferation and differentiation. In contrast, the C-terminal region of the GM-CSFRalpha cytoplasmic domain was not necessary for cell differentiation mediated by hGM-CSF, but the removal of this region severely impaired the ability of hGM-CSF to support cell survival. While the activation of JAK2, Shc, Erk, and STAT5 proteins correlated with hGM-CSF-mediated cell growth, cellular differentiation occurred in the absence of activation of these signal transduction pathways.

Distinguishing features of basophil and neutrophil activation by major basic protein

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Immunolocalization of granulocyte-colony-stimulating factor in human glial and primitive neuroectodermal tumors.

Granulocyte-colony-stimulating factor (G-CSF) is a hematopoietic cytokine that regulates the differentiation of myeloid progenitors and the function of mature neutrophils. It is produced in vitro by monocytes/macrophages, mesothelial cells, fibroblasts and endothelial cells after appropriate induction by inflammatory mediators like IL-1 and TNF. Normal as well as tumorous glial cells can also be induced to produce CSFs in vitro. However, little is yet known about the in vivo expression of G-CSF as a mediator in inflammation and malignancy within the human central nervous system. The aim of the present study was to investigate by immunostaining the expression of the G-CSF protein within non-tumorous and tumorous glial tissues, and primitive neuroectodermal tumors. Using the murine monoclonal anti-G-CSF TM 82/60 antibody, we found high G-CSF expression in astrocytoma WHO grades I and II and reactive brain tissue, low expression in astrocytoma WHO grade III, and none in glioblastoma, oligodendroglioma WHO grades II and III, and medulloblastoma. In consecutive sections of the tissue samples, G-CSF protein was localized in GFAP-positive glial cells, but not in macrophages/microglial cells, which expressed HLA-DR, detected by the antibody CR3/43. Computer-assisted microdensitometric evaluation of the intensity of immunostaining for G-CSF and statistic analysis of the data revealed significant differences between the diagnostic entities studied (p < 0.0001). We conclude that in vivo expression of G-CSF is a characteristic of reactive as well as tumorous astrocytes, with the latter losing this feature at higher degrees of dedifferentiation.

The role of G-CSF in mature neutrophil function is not related to GM-CSF-type cell priming.

Because of uncertainties regarding the comparability of granulocyte-macrophage and granulocyte colony-stimulating factors with regard to their effects on mature neutrophils (PMNs), we compared the actions of the two cytokines on reactive oxidant production and granular secretion by these cells. We found that chemiluminescence (CL) stimulated by formylmethionyl-leucyl-phenylalanine (fMLP) was not influenced by G-CSF (0.1-100 ng/ml), whereas GM-CSF priming (10 ng/ml) caused a nearly twofold increase in this PMN response. Moreover, the reactivity of PMNs treated with GM-CSF and G-CSF in combination was not different from that of PMNs treated with GM-CSF alone. GM-CSF (10 ng/ml) increased the rate of O2- production by 79%, caused a fivefold increase in fMLP-induced myeloperoxidase (MPO) secretion, and strongly enhanced CD11b expression. In contrast, G-CSF (50 ng/ml) only slightly increased O2- production (by 15%), and MPO secretion and CD11b expression remained unchanged. Both cytokines together gave results similar to those obtained with GM-CSF alone. In the presence of platelets (which by themselves enhanced PMN reactivity), the differences in the effects of the two cytokines persisted. We conclude that the priming effect of G-CSF on mature PMNs is negligible compared with that of GM-CSF. Our results are in conflict with previous reports of much more pronounced G-CSF effects but in accord with recent work showing the failure of this cytokine to induce a range of effects produced by GM-CSF. We therefore suggest that the primary role of G-CSF in mature PMN function is still unclear but may be related to the control of PMN distribution in view of the mobilizing and marginating effects of the cytokine in vivo.

Serum Concentrations of Granulocyte Colony-Stimulating Factor in Healthy Term and Preterm Neonates and in Those With Various Diseases Including Bacterial Infections

The neonate is uniquely susceptible to severe and overwhelming bacterial infections. One of the most important deficits in the neonatal host defense system seems to be a quantitative and qualitative deficiency of the myeloid and the phagocytic system. Future optimal therapy of neonatal sepsis may include the use of adjuvant immunologic therapy. Granulocyte colony-stimulating factor (G-CSF) has been shown to induce neutrophilia and to enhance mature effector neutrophil function. To evaluate the role of G-CSF with respect to infection, we examined serum levels of G-CSF in term and preterm neonates, using an enzyme-linked immunosorbent assay method. G-CSF levels in healthy neonates showed peak levels up to 7 hours after birth, followed by an increase in total neutrophil cell (TNC) counts. Both G-CSF levels determined between 4 and 7 hours after birth and peak TNC counts correlated with the gestational age of the neonates. The state of nutrition, maternal treatment with glucocorticoids, maternal infection and hypertension, and the mode of delivery influenced peak G-CSF levels. Neonates with signs of infection between 4 and 7 hours after birth had higher levels of G-CSF than did healthy neonates (1,312 +/- 396 pg/mL v 176 +/- 19 pg/mL). In conclusion, the presented results of serum concentrations of G-CSF in relation to TNC counts and various diseases suggests an important role of G-CSF in the regulation of granulopoiesis during the neonatal period.

Serum concentrations of granulocyte colony-stimulating factor in healthy term and preterm neonates and in those with various diseases including bacterial infections

The neonate is uniquely susceptible to severe and overwhelming bacterial infections. One of the most important deficits in the neonatal host defense system seems to be a quantitative and qualitative deficiency of the myeloid and the phagocytic system. Future optimal therapy of neonatal sepsis may include the use of adjuvant immunologic therapy. Granulocyte colony-stimulating factor (G-CSF) has been shown to induce neutrophilia and to enhance mature effector neutrophil function. To evaluate the role of G-CSF with respect to infection, we examined serum levels of G-CSF in term and preterm neonates, using an enzyme-linked immunosorbent assay method. G-CSF levels in healthy neonates showed peak levels up to 7 hours after birth, followed by an increase in total neutrophil cell (TNC) counts. Both G-CSF levels determined between 4 and 7 hours after birth and peak TNC counts correlated with the gestational age of the neonates. The state of nutrition, maternal treatment with glucocorticoids, maternal infection and hypertension, and the mode of delivery influenced peak G-CSF levels. Neonates with signs of infection between 4 and 7 hours after birth had higher levels of G-CSF than did healthy neonates (1,312 +/- 396 pg/mL v 176 +/- 19 pg/mL). In conclusion, the presented results of serum concentrations of G-CSF in relation to TNC counts and various diseases suggests an important role of G-CSF in the regulation of granulopoiesis during the neonatal period.

Effects of perioperative granulocyte colony-stimulating factor on horses with ascending colonic ischemia.

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein that regulates the proliferation and maturation of hematopoietic progenitor cells and modulates the function of mature neutrophils. The responses to administration of G-CSF alone, and in combination with antimicrobials, were studied in an equine model of ascending colon ischemia. Complete segmental colonic ischemia (3.75 hours) with pelvic flexure enterotomy was created in four treatment groups. Group 1 horses received recombinant canine G-CSF (10 micrograms/kg, every 24 hours, intramuscularly), gentamicin sulfate (2.2 mg/kg, every 8 hours, intravenously), and potassium penicillin G (40,000 IU/kg, every 6 hours, intravenously). Group 2 horses were treated with the G-CSF vehicle and antimicrobials as for group 1. Group 3 horses received G-CSF and the antimicrobial drug vehicles, and group 4 horses served as the untreated control receiving G-CSF vehicle and antimicrobial vehicles. The results for 20 horses, five horses in each group, were compared. Treatment with G-CSF was associated with an increased concentration of white blood cells, band neutrophils, neutrophils, lymphocytes, and monocytes in the peripheral blood after surgery. Antimicrobial administration had no detectable effect on cell concentrations after surgery. Administration of G-CSF was associated with an increased concentration of nucleated cells in the peritoneal fluid including neutrophils, small mononuclear cells and large mononuclear cells. Horses that developed incisional infections had lower neutrophil concentrations in the peripheral blood on postoperative day 2 than horses without infected incisions. These results suggested that the prophylactic administration of G-CSF may be useful in the treatment of patients at risk for developing neutropenia after surgery.