The effect of GM-CSF and G-CSF on human neutrophil function

hnmunopharmaco ELSEVIER

Immunopharmacology 29 (1995) 111-l 19

The effect of GM-CSF and G-CSF on human neutrophil function Loretta A. Bober*, Michael J. Grace, Catherine Pugliese-Sivo, Albert0 Rojas-Triana, Tracey Waters, Lee M. Sullivan, Satwant K. Narula Schering-Plough Research Institute, Department of Immunology, 2015 Galloping Hill Road, Kenilworth. NJ 07033, USA (Received 13 April 1994; accepted 7 September 1994)

Abstract A direct comparison of GM-CSF and G-CSF in a panel of in vitro neutrophil-function assays was performed to investigate any differences in activity profiles. In our modified chemotactic assay, GM-CSF rapidly increased the migratory capacity of polymorphonuclear cells (PMNs) to move toward fMLP and LTB,. In contrast, G-CSF only stimulated PMN migration towards fMLP. GM-CSF, but not G-CSF, increased PMN cytotoxic killing of C. n&cans blastospores. The expression of PMN surface antigens associated with Fc- and complement-mediated cell-binding (FcyRl, CR-I and CR-3j, and adhesion signalling (ICAM-I), was increased after the exposure of GM-CSF, but not to G-CSF. In contrast these CSFs demonstrated relative equipotency in their ability to induce PMN anti-bacterial phagocytosis, and to restore the Staphylococcus aureus killing capacity of dexamethasone-suppressed neutrophils. The phagocytic activity of PMNs for opsonized yeast, as well as hexose-monophosphate shunt activity, was equivalent following GM-CSF or G-CSF treatment. We discuss the significance of the difference in activity profiles in this article.) Keywords: GM-CSF;

G-CSF; Neutrophil;

Cytokine

1. Introduction The hematopoietic growth factors are integral members of the regulatory network controlling the production of the major classes of blood cells and * Corresponding author. Tel.: (I-908) 298-3085; Fax: (l-908) 298-3084. Abbreviations: CR-l, CD35; CR3, CD1 lb; Dex, dexamethasone: FcyR1, CD64; fMLP, N-formyl-met-leu-phe; G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte-macrophage colony stimulating factor; ICAM, CD54; LTB,, (S’S7 12R]-dihydroxy-[6z-Se-lOe-014z]-eicosatetranoic acid); M-CSF, macrophage colony stimulating factor; MFI, mean fluorescent intensity; MI-FITC, Micrococcus Iy.Todeikticus-fluorescein conjugate; NBT, nitroblue tetrazolium; PMN, polymorphonuclear leukocyte 0162-3109/95/$9.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0162-3109(94)00050-6

the stimulation of mature cell function (Bach et al., 1973). In particular, GM-CSF and G-CSF have been the focus of much attention due to their promising therapeutic potential. A number of review papers delineating the stimulatory properties of these CSFs have appeared recently (Herrman et al., 1992; Rose, 1992; Roilides and Pizzo, 1992). Despite the fact that GM-CSF and G-CSF appear to share a number of activities, there are few reports in which the CSFs have been compared to each other. In the present study, we compared GM-CSF and G-CSF directly in a series of experiments designed to cover a wide-range of mature neutrophil functions, particularly emphasizing assays that elucidate activity on bacterial and fungal cells. These data support the proposed clinical use for

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L.A. Bober et al. / Immur~opharmacol~g~ 29 (199.5) I I I -I 19

the CSFs in the treatment of immunosuppressed patients who are otherwise at high risk of fungal infection (Metcalf, 1987). We report that GM-CSF demonstrated a stronger potential for stimulating a C. albicans anti-fungal defense as compared to G-CSF. However, GM-CSF and G-CSF were equally effective in our anti-bacterial defense assays, including helping steroid-impaired PMNs to maintain a cytotoxic response to bacteria.

2. Materials

and methods

2. I. PMN isolation Venous blood was drawn by venipuncture from normal healthy non-fasting volunteers. Granulocytes (neutrophils) were purified under sterile conditions by gravity-sedimentation through 4”/, dextran/saline, washed and separated using a two-step Percoll density gradient adapted from the method of Pertoft et al. (1978). The recovered granulocyte band was free of monocyte contamination (< 1%) by a Wright-Giemsa blood stain and a non-specific esterase stain for monocytes. The purity of the neutrophil band ranged between 90-95% with eosinophils being the major contaminating cell type. 2.2. Cytokirzes Recombinant human GM-CSF had a specific activity of 1.25 x lo8 IU/mg. Recombinant human G-CSF (Neupogen) (spec. act. 1 + 0.6 x lo8 Ujmg) was obtained from Amgen (Thousand Oaks, CA, USA). M-CSF was the kind gift of the Genetics Institute (Cambridge, MA, USA). The cytokines were compared in all assay systems on a proteinweight basis. Protein concentrations were determined by HPLC analysis. Endotoxin contamination of all cytokines was below detectable levels as measured by Limulus amebocyte assay. 2.3. Chemotaxis A standard chemotaxis procedure using individual Boyden chambers was modified by shortening the cell incubation time with the CSFs to only 15 min (1 x lo6 cells/chamber) followed by a 15min migra-

tion through 3.0~pm PVP-free nucleopore filters (Weisbart et al., 1987). The chemoattractants used were N-formyl-met-leu-phe (fMLP) at a concentration of 10e8 M or LTB, ((5S7 12R)-dihydroxy-(6z8e-lOe-014z)-eicosatetranoic acid) at a concentration of 3 x lo-’ M. Both were puchased from Sigma (St. Louis, MO, USA). Following migration, filters were removed, stained with Wright-Giemsa, scraped to remove unmigrated cells, and mounted bottom-up to allow for enumeration of cells that had migrated through the pores. A microscopic evaluation of 8 fields per group was used to generate the mean number of cells per field per donor. Statistical analysis using Student’s z-test was done on individual data and across the six donor panel. Control experiments using media alone as the chemoattractant confirmed that the observed migration of PMNs was not chemokinetic (data not shown). Preliminary experiments had shown that the mobilization responses of GM-CSF treated PMNs were extremely rapid in some donors, occurring as soon as 5 min after placement in the chambers. We used an average 15min migration time as the most representative time of migration across the 10 donor panel. 2.4. Fungalphugocytosis, NBT and cytotoxicity assays The phagocytic capacity of neutrophils (2 x lo6 cells/ml) was evaluated by measuring the ingestion of opsonized heat-killed yeast after an 18-h incubation with the CSFs (Patterson-Deltield and Lehrer, 1977). CSF-treated PMNs were incubated for 45 min with heat-killed baker’s yeast after which a mixture of Trypan blue and eosin Y was added to the cells to facilitate the microscopic evaluation of yeast ingestion, and the number of phagocytic cells. The avidity (mean number of yeast ingested per 30 cells) and percent phagocytosis (200 cells) measurements for each experiment were multiplied to calculate the phagocytic index. The data were calculated for statistical significance based on the mean phagocytic index across a panel of 7 donors using Student’s t-test. The determination of hexose-monophosphate shunt activity was determined using an NBTreduction assay (Baechner and Nathan, 1968). PMNs were obtained from duplicate samples of the cell suspensions used for phagocytic capacity. These

cells were incubated for 45 min at 37°C in RPM1 1640 containing 2% FCS, 2 mM L-glutamine with 0.06 PM phorbol myristate acid and 0.5 mg/ml NBT. NBT-reduced positive cells, i.e., those containing a black formazan deposit, were evaluated by microscopy from alcohol-fixed Wright-Giemsa stained cellsmears. Cells were counted in triplicate at 100 cells per open field. Statistical significance was calculated across the 6 donor panel using Student’s t-test. Neutrophil cytotoxicity against C. albicans blastospores was analyzed using a germination assay (Cutler and Thomas, 1984). Effector-target (E/T) ratios were chosen at which normal untreated neutrophils have a minimal capacity to kill C. albicuns. This allowed for a maximal demonstration of a cytokine effect in our assay. Following an 18-h incubation, PMNs were recovered from culture, washed and diluted to the appropriate E/T ratios. PMNs were incubated with C. albicuns blastospores (C43 strain) in the presence of human serum and the target yeast (3000 organisms) for 45 min. After incubation, the cells were transferred to 24-well dishes precoated with 1:;i, bovine serum albumin (B SA) in deionized water and incubated for 30 min. The plates were centrifuged, the supernatant was removed and 0.5 ml of a 2% corn meal agar suspension was added (Sigma). These plates were incubated for 30 min to ensure full germination of any surviving yeast blastospores. Control-yeast preparations (4 wells per plate), were processed as described, and were used as the baseline for survival. The number of germ tubes (gt) per high power field was counted for each well, e.g., 8 separate fields at 200 x magnification. The endpoint number reflected viability because preliminary experiments had demonstrated that the same number of micro-colonies per high power field would develop if incubation were continued for 24 h. The percent surviving yeast was calculated by the formula: mean number of gt (media or cytokine treated) x 100 %= mean of gt (no treatment) 2.5. Bacterial phugocytosis and killing Bacterial phagocytosis was measured using a quantitative flow-cytometric assay in which 2 x lo6 PMNs were exposed to opsonized fluoresceinlabeled bacteria, Micrococcus (vsodeikticus (MI-

FITC) (Oben and Foreman, 198X). After incubation with MI-FITC for 1.5min at 37 ‘C, the PMNs were recovered and washed, and then were exposed to lysozyme (0.03 mg/ml) for 30 min, rewashed, and fixed with 10% paraformaldehyde. 20000 events were collected using an open gate and analyzed for percent of fluorescent positive PMNs using a Becton-Dickinson FACScanTM. The percentage of total positive cells was derived by analysing FL1 parameters and removing the autofluorescence and the negative control events (paraformaldehyde-fixed cells exposed to MI-FITC). The data were analyzed for statistical validity using the Wilcoxon signedrank test. PMNs were assayed for bacterial killing using a S. uureu~ fluorochrome assay (Bellavati-Pires et al., 1989). Prior to assay, cell suspensions (2 x lo6 cells) were incubated for 18 h at 37 oC and 5 y0 CO2 with CSFs, CSFs and dexamethasone (10 ’ M), dexamethasone alone, or media. On the day of assay, PMNs were recovered, washed, and 2 x lo4 preopsonized S. aweus (6538P: SPRI assay units) organisms were added per tube and incubated in a shaking water bath for 30 min at 37°C. The suspensions were washed in Hanks’-buffered saline with 1y0 gelatin, stained with acridine orange (1:30 of a 1P; stock), and washed four times before counting. Enumeration was accomplished using open-field epifluorescent microscopy. The percentage of cells containing intracellular red (dead) bacteria were calculated from a field count of 600 cells per sample. Statistical analysis was performed across the panel of 6 donors using Student’s t-test. 2.6. FACS evaluation of cell-surface markers For analysis of cell-surface antigen expression (Macey et al., 1992), PMNs ((0.5-l) x lo6 cells/ml) were incubated in a 50:50 mixture of RPM1 1640 and DMEM/Fl2 (high glucose) containing 107~ FCS, 6 mM L-glutamine, 1“/A non-essential amino acids and 1y0 penicillin-streptomycin for 18 h in the presence or absence of cytokines. PMNs were harvested from culture, washed and incubated with monoclonal antibodies specific for the markers by adding the monoclonal antibody to the cell pellet and leaving on ice for 30 min. All antibodies were FITC direct-conjugates purchased

L.A. Bober et al. / Immunopharmaroiogy 29

114

from Becton-Dickinson Immunocytometry Systems (San Jose, CA, USA). Markers used were CD64 (FcyRl); CD54 (ICAM-1); CD35 (CR-l); CDllb (CR3) and CD 1lc (~150). Isotype controls of FITCconjugated mouse IgG,, IgG,,, or IgG,, were included in all experiments. After staining, cells were washed in Dulbecco’s modified phosphate-buffered saline without Ca*+ /Mg*+ containing 1y0 BSA and 0.01% sodium azide. Propidium iodide was added prior to fluorometric analysis for viability discrimination. PMNs were collected and analyzed using a Becton Dickinson FACScanTM flow cytometer. For each tube, 15000 separate events were collected in list mode for 4 parameters (FSC, SSC, FL1 and FL2) using open statistical gates in the FL1 parameter. Data were analyzed using LYSIS II.

(19951

I1 I-l 19

Table 1). PMN migration towards fMLP was also enhanced in the 0.5 rig/ml treatment GM-CSF group. G-CSF treated PMNs were assayed in parallel experiments, and only trended for migration towards fMLP at the 5 rig/ml level of cytokine (Table 1). GM-CSF pretreatment of PMNs significantly affected cellular migration toward LTB, (Table 1). Over the panel of 10 donors tested, PMN migration was enhanced 2-fold in the 5 rig/ml GMCSF treatment group (60 k 11 mcf) vs. untreated (P< 0.05). The pre-exposure of PMNs to a 0.5 rig/ml concentration of GM-CSF also stimulated cellular migration towards LTB4. G-CSF at all levels tested had no effect on PMN migration towards LTB, (Table 1). 3.2. Efsects of GM-CSF on neutrophilphagocytosis and killing of fungal targets

3. Results 3.1. Effects of GM-CSF and G-CSF on neutrophil chemotaxis As shown in Table 1, the pre-exposure of PMNs to GM-CSF for 15 min stimulated the chemotactic response of the cells toward fMLP (a model for bacterial cell-wall constituents). In samples taken from 10 donors, the mean cells per field (mcf) in the 5 rig/ml GM-CSF treated groups (9Ok 22) was 2-fold higher than in untreated cells (45 & 8;

Table 1 Pretreatment with GM-CSF-induced PMN migration fMLP and LTB, to a greater extent than G-CSF GM-CSF

toward

Concentration (n&4

fMLP

LTB,

fMLP

LTB,

fMLP

LTB,

5.00 0.50 0.05

90222 69+10 56_+9

60+11” 49&7” 42_+6

81+22 51*11 43 2 9

29&4 28*5 25+4

34+5 37klO 235

23 k 3 21k4 16_+2

G-CSF

M-CSF

PMNs were pre-incubated with CSFs for 15 min, washed, then allowed to migrate for 15 min toward the indicated chemoattractam. Data are expressed as mean cells per field k S.E.M. of 10 donors, 8 fields per individual determination. M-CSF was used as a negative control, data equivalent to untreated, control cells from these donors. Untreated: fMLP 45 f 8; LTB, 29 k 7. ’ PsO.05 by Student’s t-test.

Phagocytic function was measured by exposing cyotkine-treated PMNs to opsonized heat-killed yeast targets. Hexose-monophosphate function (NBT reduction) for the treated PMNs was determined in parallel cultures. Preliminary experiments demonstrated that donor variability in the untreated group was highest when the cells were tested for phagocytic function shortly after isolation from whole blood. However, by 18 h post-isolation, the untreated PMNs regained a quiescent state when tested for phagocytic activation. Pretreatment of PMNs with either GM-CSF or G-CSF (at concentrations from 5 rig/ml to 0.005 rig/ml) enhanced the phagocytic response, and there was no statistical difference between the two cytokines in the degree of phagocytic activation (Table 2). There was a pronounced dichotomy between the activity profile between the two cytokines when the PMNs were presented with live C. albicans as a target. We used effector-target ratios which were titrated to be below those maximally effective for untreated PMNs (i.e., at a 25O:l E/T ratio 257; of the yeast inoculum germinated). As the E/T ratio was lowered, an increasing percentage (627;) of the yeast targets germinated (Table 3). GM-CSF significantly reduced the percentage of surviving C. albicans to at both the 5 and 0.5 rig/ml concentrations. Even at the lowest GM-CSF concentration (0.05 ngiml), there was a marked reduction in the percentage of C. al-

115

L.A. Boher et al. i Immunopharmacology 29 (1995) 1 I l-l 19 Table 2 Phagocytic function for yeast particles and NBT reduction is enhanced in PMNs by both CSFs Concentration hdd)

GM-CSF

5.000 0.500 0.050 0.005

G-CSF

PI

%NBT

PI

;;NBT

369% 19" 299k28" 164_+29 136225

74+3" 70+4" 52_+5 3222

264t22" 226+27" 185_+28 116k20

75+6" 57*5" 41+3" 38k5

PMNs were preincubated with CSFs for 18 h prior to assay. Data represent mean + S.E.M. of 7 donors. M-CSF used as a negative control with data equivalent to that obtained from untreated donor cells. PI, phagocytic index = avidity x “; phagocytic cells. Untreated: (PI), 78 + 10; %NBT-positive 20 2 4. a PSO.05 Student’s t-test.

bicans blastospores able to germinate from the cytokine-treated PMNs (Table 3). In marked contrast, PMNs treated with G-CSF demonstrated no change in the percentage of yeast germination compared to normal untreated cells (Table 3).

Table 3 Pretreatment with GM-CSF, but not with G-CSF, primed freshly isolated human PMNs to kill Candida ulbicans blastospores Treatment

None GM-CSF 5.00 0.50 0.05

Percent surviving C. albicans effecter/target 15O:l

5O:l

2S:l

25&l

3319

5029

62+_7

11*5" 10_+4" 15*4

1624" 24_t5" 27+7b

2426" 3426" 41 -+ 7"

25 k 8 26k8 3229

35&8 36*1 44* 18

51+9 45&9 565 10

G-CSF (ngiml) 5.00 2157 0.50 2629 0.05 27+7

on phugocytosis

We utilized a FACS-based analysis system to determine whether there were concentration-dependent potency differences between the CSFs. Based on an analysis of 20000 cells per donor (6 donors), over a concentration range of 5.0 rig/ml to 0.005 ngj ml, both cytokines were equally effective in stimulating PMNs to phagocytize FITC-labeled bacteria (Table 4). Both cytokines were also extremely potent in this assay. The exposure of PMNs to either CSF at concentrations as low as 0.05 pg/ml triggered a 2-fold increase in the percentage of phagocytic cells (individual donor data not shown). This threshold of CSF activation was found to be donor-dependent. The data in Table 4 represent the plateau region of the dose response for direct comparison of the two CSFs. A comparison of the two CSFs for their capacity to induce PMNs to ingest and kill S. aUreUSdemonstrated that neither was able to enhance bacterial killing above that of normal untreated PMNs except for a slight effect at the 5 rig/ml concentration of GM-CSF (Table 5). To more clearly demonstrate the activation potential of these cytokines, the phagocytic response of the PMNs was impaired by the addition of dexamethasone to the cultures. The dexamethasone addition reduced by half the number

ratio

25O:l

(@ml) 8*3” 1024" 13*5

3.3. Effect of GM-CSF and G-CSF and killing of bacterial cells

PMNs were pre-incubated with CSFs for 18 h prior to assay. Data represent mean + S.E.M. of 6 donors. Effector cell numbers were adjusted after CSF incubations so that E/T ratios were achieved using a constant target number of 3000 yeast. M-CSF was used as a negative control with no activation of PMNs measurable for Candida killing. a PsO.05; Student’s t-test (media vs. CSF-treated): b PCO.10; Student’s t-test (media vs. CSF-treated).

Table 4 Pretreatment of PMNs with either GM-CSF their capacity for bacterial phagocytosis Concentration (ngiml)

5.000 0.500 0.050 0.005

or G-CSF enhanced

Percent total cells with MI-FITC GM-CSF

G-CSF

81 26” 8Oi6" 7826" 85+6"

86k4" 82?5" 7725" 8025"

Neutrophils were pre-incubated with cytokine for 18 h prior to assay. Data represent mean f S.E.M. of 6 donors. Percent cells = 20000 gated events analyzed by FACScan. M-CSF was used as a negative control with no activation of PMN for bacterial phagocytosis above control level. Untreated = 63 k 7. MI-FITC, Micrococcus lysodiekticus-fluorescein conjugated. a P
116

LA. Bober et al. : Immunophavmacology 29 (19951 11 l-l 19

Table 5 Co-incubation of neutrophils with hematopoietic factors and dexamethasone prevented a steriod-induced suppression of bacterial killing capacity Treatment Media 66 2 4 GM-CSF (rig/ml) 5.0 0.5 G-CSF (ngiml) 5.0 0.5 Dexamethasone Dex + GM-CSF 5.0 0.5 Dex + G-CSF 5.0 0.5

Mean :j0 cells dead bacteria

78 i: 7 66~4 68 + 9 6024 37 + 3”

(Cdllb), ICAM(CD54) and to some extent CD1 lc. On the other hand, G-CSF did not enhance the basal-level expression of these cell-surface markers to any consistent degree across the panel of donors tested (Table 6). The basal-levels of expression for the surface antigens analyzed were obviously sufficient for effective bacterial phagocytosis and killing, since there was no significant difference observed between GM-CSF and G-CSF in the functional assays. However, the increased antiCandidial activity of GM-CSF treated PMNs may be possibly explained by the effect of GM-CSF on the enhancement of these pertinent PMN surface antigens.

69 + 5b 70 + 3b

4. Discussion 63_+6b 61+3b

PMNs were pre-incubated with media, CSF or CSF+ dex (10e5 M) or dex (10e5 M) for 18 h before assay. Cells were incubated with live S. aureu~ for 30 min before termination of assay. M-CSF was used as a negative control in parallel cultures with no activation ofPMNs detectable. Data represent mean + S.E.M. of 3 donors. a PCO.05; Student’s f-test (Dexamcthasone alone vs. media alone); h P-CO.05; Student’s l-test (Dexamethasone + CSF vs. dexamethasone alone).

While there are numerous review articles summarizing the results of studies using GM-CSF and G-CSF (Metcalf, 1987; Groopman et al., 1989; Weisbart and Golde, 1989; Rose, 1992; Roilides and Pizzo, 1992; Crosier and Clark, 1992; Neumanaitis, 1993), few of these studies have addressed the question of the relative efficacy of these two CSFs by direct comparative study. We studied both CSFs in

Table 6

of PMNs containing dead bacteria (37 _+3) vs. that of dexamethasone-untreated cells (66 5 4; Table 5). Co-addition of either GM-CSF or G-CSF protected PMNs from the steroid-induced suppression of bacterial killing (Table 5). The percentage of PMNs containing dead bacteria in the CSF-treated dexamethasone cultures was equivalent to that of cultures without steroid treatment (Table 5).

Cell-surface marker analysis on PMNs after pre-incubation either GM-CSF or G-CSF

3.4. Effect of GM-CSF and G-CSF on cell-surface markers

G-CSF (rig/ml) 20.0 5.0 0.5 Untreated

We measured changes in PMN surface antigens that were relevant to the process of opsonized cellular adhesion and ingestion. We chose the 18 h post-CSF exposure time-point to be consistent with the phagocytic assay. GM-CSF enhanced the PMN surface basal-level of expression for FcqRI (CD64), the complement receptors CR-l (CD3.5) and CR3

Mean fluorescent intensityb

Cytokine treatment” GM-CSF

(ngiml)

20.0 5.0 0.5

with

FcgRl CD64 38(3) 3X4) 33(4)

ICAM CR-1 CD54 CD35 114(7)’ 18(l)’ 106(11) 16(l) lOl(11) 15(l)

CR-3 CDllb 530(99)” 464(100) 464(100)

p150 CDllc 47(8) 43(7) 44(7)

23(4)

84(10) 95(4) 77(7)

13(l) 12(l) 11(l)

348(78) 359(78) 337(72)

41(8) 39(6) 38(6)

23(J)

74(S)

II(l)

350(64)

37(6)

300)

260)

a MNs were pre-incubated with media or CSFs for 18 h prior to assay. M-CSF was included as a negative control with no activation potential for these cell-surface markers. b Data represent the mean (SEM) of the MFI from three separate donors. ’ PcO.05, Student’s t-test; untreated vs. CSF.

L.A. Bober et al. i Ii?~mtmopharmacolo~~ 29 (1995) 11 I -I 19

parallel at equal protein concentrations to determine their efficacy in a series of in vitro assays targeting PMN phagocytosis and killing, as well as changes in cell-surface markers relevant to PMN function. We observed that GM-CSF demonstrated some unique qualitative and quantitative differences from G-CSF in the panel of neutrophil function assays. In particular, GM-CSF stimulated PMN chemotaxis toward LTB,, whereas G-CSF did not; GM-CSF, but not G-CSF, enhanced PMN killing of C. albicans blastospores; and, GM-CSF enhanced the PMN surface expression for Fc- and complement-mediated antigens. However, GM-CSF and G-CSF were equipotent in all anti-bacterial functions tested. The interpretation of the effects of these CSFs on PMN chemotaxis is complex. GM-CSF and G-CSF have been reported to be a chemotactic factor for PMNs (Wang et al., 1987; 1988). For GM-CSF, there are also some reports that the CSF is a negative signal for chemotaxis (Buescher et al., 1988; Kownatzki et al., 1990). We found, as did Weisbart and co-workers (1987) that the timing of the PMN exposure to the CSF, as well as the use of a short migration time across the filter, is critical for the demonstration of enhanced movement of PMNs. Both CSFs at 5 rig/ml stimulated PMN movement toward fMLP when the incubation time (< 30 min) and migration time across the filter were kept short (15 min). GM-CSF was more potent than G-CSF, retaining its activity at the lower concentrations. Under these conditions, GM-CSF-treated PMNs also were able to migrate toward LTB,. In contrast, G-CSF-treated PMNs were unresponsive to this signal. Weisbart et al. (1989) attributed the rapid movement of GM-CSF-treated PMNs to the lateral mobilization of adhesion-related molecules on the cell surface of the neutrophil. We observed a dramatic increase in PMN surface expression for ICAMfollowing an incubation for 18 h with GM-CSF. ICAM- 1 expression was also observed to be elevated as quickly as 2 h post-exposure to GM-CSF (data not shown). The necessity for GM-CSF to be studied at short incubation and migration times may also be due to the continuation of events that occur after exposure to this CSF. Weisbart et al. (1987) found that as exposure to GM-CSF continued past 30 min, the PMN lost mobility while its capacity to generate

117

superoxide increased, and immobilization factors necessary for retaining the cell at the site of inflammation also increased. Gasson et al. (1984) have reported that rhGM-CSF is identical to neutrophil inhibition factor (NIF-T). The differences present in the literature suggest the importance of choosing the correct timing for the measurement of chemotaxis. The mobilization of GM-CSF treated PMNs toward LTB4 might be interpreted as harmful to the host since this could result in an exacerbation of inflammation (Haworth et al., 1991). However, for a wound healing application this property could be of benefit because the rapid arrival of PMN would suppress bacterial outgrowth and initiate the healing response. In fact, GM-CSF has been proposed as having potential for wound healing (Kaplan et al., 1992). Our data do not indicate that G-CSF-treated PMNs would respond in an equivalent fashion. These findings support previous suggestions that G-CSF is erratic in its ability to stimulate PMN migration toward chemoattractants (Bronchud et al., 1988; Sartorelli et al., 1991) Neutrophil surface antigens which previously have been implicated in supporting competent phagocytic function were measured after incubation with either CSF. GM-CSF treatment of PMNs was observed to enhance the basal-level surface expression of FcyRl, C-3bi and CR-l. This supports the observation that GM-CSF upregulates the complement receptors CR-l and CR-3 in vivo (Moore et al., 1992). On the other hand, G-CSF treatment of PMNs had little or no effect on enhancing basallevel expression for these surface antigens. Both CSFs were equipotent in stimulating phagocytosis of opsonized yeast and in increasing the activation state of the PMNs as judged by the percent increase in foramazan-positive staining cells. As well, both CSFs were equipotent in preventing a dexamethasone-induced depression in PMN phagocytosis and killing. Bacterial phagocytosis and killing capacity of PMNs has been reported to be significantly enhanced by either of these cytokines in several different studies (Mayer et al., 1987; Roilides et al., 1990; 1991). Thus, it appears that the basal-level of expression for FcyRl and the complement receptors was sufficient to provide functional anti-bacterial activity in vitro. Despite the efficacy of GM-CSF and G-CSF on

118

L.A. Bober et al. / Immunopharmacolog~~ 29 (I 995) 111 -I 19

phagocytosis and bacterial killing, we found a dichotomy of CSF activity when we tested the ability of CSF-activated PMNs to kill C. albicans in vitro. GM-CSF was markedly superior to G-CSF in enhancing PMN killing. Roilides et al. (199 1) have previously reported that G-CSF treatment enhanced neither phagocytosis nor fungicidal activity of normal PMN against C. albicans blastoconidia. G-CSF however, has been found to enhance the PMN oxidative burst in response to both the blastoconidia and pseudohyphae of C. albicans (Roilides et al., 1993). Other investigators have shown that when incubation with the CSF is prolonged during the challenge period with Cundidu, PMNs will become capable of killing the blastoconidia (Yamamoto et al., 1993). In the short challenge period that we and others have used (approx. 1 h), it has been speculated that there is a difference between the CSFs in mediating phagolysosome formation or closure in the cell (Cech and Lehrer, 1984). Our results indicate that GM-CSF may have superior potential clinical utility for the treatment of anti-fungal, and in particular anti-candidial infections arising from immunosuppressive chemotherapy (Jones, 1981; Elis et al., 1988). When this advantage is combined with equivalent anti-bacterial activity, as compared with G-CSF, and the ability to stimulate monocyte-directed cellular immunity, GMCSF is potentially better suited as an anti-infective adjunct therapy, resulting in patients having less days on antibiotic therapy and experiencing shorter hospitalization periods (Metcalf, 1989).

Acknowledgements The authors would like to thank Drs. Jay Fine and Paul Trotta for their helpful discussions and critique. We would also like to thank Ms. Luminita Justice for her assistance in the NBT experiments and Ms. Maria Carter for her preparation of this manuscript.

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