The effects of colony stimulating factors on human monocyte cell function

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Copyright

J. Immunopharmoc., 0 1995 International Printed

Vol. 17, No.

5, pp. 385-392, 1995 Elsevier Science Ltd Society for Immunopharmacology in Great Britain. All rights reserved Ol92-0561/95 $9.50 + .oo

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THE EFFECTS

OF COLONY STIMULATING FACTORS ON HUMAN MONOCYTE CELL FUNCTION

LORETTA A. BOBER, MICHAEL J. GRACE, CATHERINE PUGLIESE-SIVO, ALBERT0 ROJAS-TRIANA, LEE M. SULLIVAN and SATWANT K. NARULA Schering-Plough Research Institute, Department of Immunology, 2015 Galloping Hill Road, Kenilworth, NJ 07033, U.S.A. (Received

3 1 October 1994 and in final form 10 January 1995)

Abstract - We used a panel of fUnctiona assays to compare directly the pattern and potency of GM-CSF and M-CSF on monocyte activity associated with cell-mediated immune defense. GM-CSF and M-CSF were found to be equivalent both in their capacity to stimulate human monocyte functionsin vitro and in their pattern of monocyte activation. The two CSFs were effective in inducing monocyte chemotaxis towards either fMLP or LTB, at equivalent concentrations across a panel of donors. GM-CSF and M-CSF demonstrated equipotency in the induction of monocyte phagocytosis of heat-killed baker’s yeast and in the regulation of the hexose-monophosphate shunt (NBT reduction). Both were also found to be equivalent in preventing steroid (dexamethasone)-induced suppression of monocyte anti-bacterial (Cundida albicuns) and anti-fungal (Stap/~yZococcusoureus) phagocytic capacities. GM-CSF was somewhat more effective than M-CSF in stimulating monocyte C. albicuns killing at a lower E:T ratio. Keywords

: CM-CSF, G-CSF, monocyte.

The monocyte-macrophage is a pivotal cell in the immune defense network, capable of functioning as an effector cell through its phagocytic capacity, and also as a transducer cell through its cytokine production capacity. The biological activities that have been elucidated for GM-CSF and M-CSF position them as important soluble mediators of host cellular defense. Both granulocyte-macrophage stimulating factor (GMCSF) and macrophage stimulating factor (M-CSF) have been shown to be important in the regulation of monocyte cell number and in the modulation of the mature monocyte functional capacity (Crosier & Clark, 1992; Metcalf, 1990; Roilides & Pizzo, 1992; Rowe & Rapoport, 1992; Munn & Chung, 1992). Colony-stimulator factor (CSF)-producing cells are found in large numbers in most organs but, in particular, they are concentrated in epithelial and endothelial tissues where most initial contacts with infectious organisms occur (Groopman et al., 1989). The production of CSFs by these cells is enhanced rapidly upon contact with invasive pathogens (Bickel et al., 1987). In contrast to other CSFs, M-CSF is unique in that it is a normal constituent of human sernm (Hanamura et al., 1988). Because of these properties, GM-CSF and M-CSF have been intensively studied as promising therapeutic modalities for treating infectious disease crises that frequently arise in

immunocompromised patients (Nemunaitis et al., 1991; Demetri & Antman, 1992). Although a substantial body of the scientific literature already exists describing the effects of GMCSF and M-CSF on mature monocyte functions, few studies directly compare the potency and spectrum of activities of these two molecules. The focus of the present study was to compare directly these two CSFs in a panel of monocyte fUnctiona assays.

EXPERIMENTAL PROCEDURES

Cell isolation

Venous blood was drawn by venipuncture from normal human non-fasting volunteers and the mononuclear cell layer was separated by density centrifugation using a Hypaque-Ficoll gradient. These cell preparations were determined to be about 30% monocytic by non-specific esterase staining. We decided to conduct the studies with this preparation rather than seek further purification of the monocytes for two reasons. Most methods of purifying monocytes to 80% or greater purity also tend to activate the cells for 38.5

L. A.

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BOBER et al

biological responsiveness and thus potentially obviate our ability to observe significant differences between M-CSF and GM-CSF. In vivo, monocytes exist with contextual relationship to lymphocytes, and are known to interact both by soluble mediators and receptorligand interaction. In addition, the presence of lymphocytes in bacterial phagocytosis and killing assays has been shown not to significantly affect monocyte responsiveness (Peterson et OZ., 1977). The viability of the cells in our preparations, as determined by trypan blue exclusion, was always greater than 95% at the end of the CSF incubation period (18 h). For all culture conditions following purification, except where otherwise indicated, MNCs were suspended at 2 x 10” cells/ml in DMEM/F12 media containing 10% FBS, 2 mM t.-glutamine and penicillin-streptomycin, and cultured for 18 h at 5% CO, and 37°C in a humid incubator.

Chemotaxis The migration responses of CSF-treated monocytes toward standard chemoattractants were measured in a Boyden chamber system (Wang et al., 1987). Following a 30 min incubation with either GM-CSF or M-CSF, mononuclear cell (MNC) preparations were washed free of the CSF and the cells were resuspended at a concentration calculated to deliver 1 x 10’ cells on to a 5 pm PVP-free filter in the Boyden chamber. The chemoattractants (300 pl), either N-formyl-Met-Leu-Phe (fMLP; 1 x 10 8 M) or LTB, ([5s, 12R]-dihydroxy-[6Z, 8E, lOE, 14Z]-eicosatetranoic acid; 3 x 10 ’M), had been loaded into the bottom of the Boyden chambers. The experimental time for cellular migration (65 min) was determined to be optimum for both cytokines. Upon termination of the migration time, the filters were removed, stained with Wright-Giemsa, scraped to remove all unmigrated cells, and then reverse-mounted to allow for enumeration of cells that had migrated through the filter pores. Microscopic evaluation of eight fields per group at 400 x magnification was used to generate the count of mean cells per field per donor. Statistical evaluation using the Student’s t-test was performed on individual data and across the six donor panel. Control experiments demonstrated that cellular migration was not due to random migration (data not shown).

Fungal phugocytosis, cytotoxicity assays

monophosphate

shunt,

and

The phagocytic capacity of monocytes was measured by the ingestion of opsonized heat-killed baker’s yeast. MNC cultures were incubated with either GM-CSF or M-CSF for 18 h prior to phagocytic assay to stimulate

monocyte response in the assay. The 18 h incubation time-point was chosen because it allowed time for untreated monocytes to return to a quiescent state after activation from the Ficoll-Hypaque separation. After incubation, MNCs were washed to remove the CSFs, and were then co-incubated for 45 min at 37°C with heat-killed yeast. A mixture of trypan blue and eosin Y dyes was added to the MNC and yeast mixture following the co-incubation time to facilitate microscopic evaluation of ingested yeast and to differentiate the number of phagocytic cells (Patterson-Delatield & Lehrer, 1977). The monocyte avidity for yeast (mean number of yeast cells ingested per 30 phagocytic cells) and percentage of phagocytic cells (out of 200 MNCs counted) were multiplied to calculate the phagocytic index. The data were analyzed using the Student’s t-test for statistical significance using the mean phagocytic index as determined from the individual data, and also across the panel of six donors. In parallel with the yeast-phagocytic assay, MNC cultures were examined for hexose-monophosphate shunt activity using an NBT-reduction assay (Baechner & Nathan, 1968). MNC cultures were incubated in RPMI- 1640 medium containing 2% fetal-calf serum, 2 mM L-glutamine, 2.5 pM phorbol myristate acid and 0.5 mg/ml NBT for 45 min at 37°C in 5% CO,. NBTreduction, i.e. cultures containing a black formazan deposit, was evaluated from alcohol-fixed WrightGiemsa stained cell smears using standard light microscopy. Counts were made using triplicate fields of at least 100 cells per field. Statistical significance was calculated for each individual donor and across the six donor panel using the Student’s t-test. Monocyte cytotoxicity against Candida albicans blastospores was analyzed using a germination assay (Cutler & Thompson, 1984). Effector : target (E : T) ratios were determined at which normal untreated monocytes had a minimal capacity to kill C. albicans. This allowed for the maximal demonstration of a CSF enhancement of cell function. MNC cultures were incubated with either GM-CSF or M-CSF for 48 h prior to assay. Preliminary experiments had demonstrated that there was less variability across the donor panel when MNC cultures were incubated for at least 48 h prior to assay. After CSF incubation, rthe MNCs were washed to remove the CSF and diluted to the desired E : T ratios with the C. afbicans blastospores (C43, SPRI Microbiology Dept) added at a standardized 3000 organisms. The MNC-blastospore co-cultures were incubated in medium containing fresh human serum for 60 min at 37°C in a shaking water bath. At termination, the cellular mixture was transferred to 24-well dishes that had been pre-coated with 1% bovine serum albumin (BSA). The plates were incubated for 30 min at 37’C. If any cells

Colony Stimulating Table 1. Monocyte

Concentration+

387

migration toward fMLP and LTB, is enhanced by prior exposure to GM-CSF or M-CSF

(ng/ml)

5.00 0.50 0.05

Factors

GM-CSF fMLP LTB, 75(7.8)* 70(10.6)* 58(12.8)*

57(4.4)* 55(9.9)* 56(8.9)*

M-CSF tMLP

LTB,

88(2.1)* 75(12.5)* 64(9.2)

51(1 l.l)* 53(12.2)* 46(8.3)

Untreated cells BvlLP = 4 1 (4.8) LTB, = 26 (3.1) ‘Unseparated monocytes were exposed to CSFs 30 min before exposure to chemoattractant. Cells were allowed to migrate across the filter for 65 min. Data are expressed as mean cells per field (S.E.M.) of ten donors; eight fields per individual determination. G-CSF was assayed as a negative control for monocyte activation. *P
were still intact at this point, the lysis step with water/ BSA was repeated. The plates were centrifuged, the resolved supematant was removed, and 0.05 ml of a 2% corn-meal agar (Sigma Chemical Co., St Louis, MO, U.S.A.) was added. The plates were then incubated for a further 30 min to ensure full germination of surviving yeast blastospores. Control yeast preparations were processed by the same procedure and used as the baseline for the determination of germination after exposure. The number of germ tubes (gt) per high-power field (200x magnification with an inverted microscope) was enumerated for each well for eight separate fields. Preliminary experiments demonstrated that essentially the same number of microcolonies per high power field would develop if incubation was continued for 24 h. The percentage of surviving yeast was calculated using the formula: % Viable = [mean # gt (media or cytokine) x lOO]/mean # gt (Can&& alone).

Bacterial phagocytosis

and killing

Bacterial phagocytosis was measured using a quantitative flow cytometric assay in which 2 x lo6 MNCs were mixed with fluorescein-labeled Micrococcus lysodeikticus (MI-FITC) (Oben & Foreman, 1988). After co-incubation of the MNC cultures with MI-FITC for 5 min at 37’C, the cells were immediately fixed with 10% paraformaldehyde, washed, and then exposed to lysozyme (0.03 mg/ml) for 30 min to remove adherent extracellular bacteria. Unstimulated MNC cultures from normal human donors were observed to ingest a maximum level of MI-FITC after only 5 min co-incubation time. This short incubation time limited our ability to discern any CSFinduced enhancement of bacterial phagocytosis (Leijh

et al., 1981). Therefore, 10” M dexamethasone was coadded with either GM-CSF or M-CSF to the MNC cultures at the onset of the CSF treatment, 18 h prior to the bacterial phagocytosis assay. The percentage of total phagocytic cells containing MI-FITC was compared between dexamethasone-treated and normal-untreated cells to the respective CSF treatments. The percentage of total ingested cells was calculated on the basis of 5000 events that had been collected through an FSC vs SSC monocyte live-gate using a Becton-Dickinson FACScanTM. Autofluorescence and negative control (paraformaldehyde fixed cells exposed to MI-FITC) events were subtracted prior to derivation of the per cent total events. The data were analyzed for statistical significance using the Student’s t-test. The determination of cytokine enhancement of Staphylococcus aureus killing by MNCs was also evaluated with dexamethasone-induced suppression. Again, MNC cultures were co-treated with dexamethasone and either GM-CSF or M-CSF for 18 h prior to assay. MNCs were washed to remove the steroid and CSF, 2 x lo4 pre-opsonizeds. aureus organisms (6538P; obtained from the SPRI Bioassay Unit) were added per tube (E : T ratio = 100 bacteria per cell), and the mixture was incubated in a shaking water bath for 30 min at 37°C. The suspensions were washed extensively in Hank’s buffered saline containing 1% gelatin, stained with acridine orange (1 : 30 dilution of a 1% stock), and washed exhaustively prior to counting. Preparations were kept on ice until wet mounts were examined for the percentage of cells containing intracellular red (dead) bacteria. The percentage kill (cells with dead bacteria) was calculated from an open-field microscopic count of 600 cells per sample examined at 400x magnification using a Leitz Ortholux fluorescent

L. A. BOBER et al.

388 Table 2. Phagocytosis

Concentration+

of yeast particles and NBT reduction is enhanced GM-CSF or M-CSF

(&ml)

Phagocytic:

GM-CSF %NBT+b index

199(21)* 177(26)* 127(19)

5.00

0.500 0.050 0.005

95(9)

49.8(0.9)* 50.9(4.5)’ 50.7(4.5)* 24.5(2.6)

in monocytes

after incubation

with

M-CSF Phagocyticl 178(24)* 168(23)* 120(17) 85( 14)

index

%NBT+Q 54.9(3.8)* 41.1(2.9)+ 33.4(3.4)* 26.4(4.0)

Untreated cells Phagocytic index = 72 (10) Per cent NBT positive = 15.8 (1.6) +Unseparated monocytes were exposed to CSFs for 18 h prior to assay. Data represent mean (S.E.M.) of results from a panel of seven donors except for the 0.005 group where n = 5 donors. G-CSF was used as a negative control in parallel cultures. IPhagocytic index; avidity x per cent phagocytic cells 6% NBT+ = no. Fotmazan positive/Total no. cells. *P
microscope. Acridine orange has a green fluorescence when reacted with living cells and a red fluorescence when reacted with dead cells (Szkaradkiewicz, 1992). Statistical analysis was performed using the Student’s t-test.

Materials Recombinant human M-CSF (2.5 mg/ml; specific activity 2 x lo6 U/mg) was a generous gift from Genetics Institute (Boston, MA, U.S.A.). Recombinant human GM-CSF (specific activity of 1.OO x 1O81 U/mg) was obtained from Schering-Plough Research Institute. For our studies, the cytokines were compared on a protein-weight basis rather than on units of activity. Protein content was determined by HPLC analysis. G-CSF (Neupogen/Filgrastim; Amgen, Thousand Oaks, CA, U.S.A.) was used as a specificity control throughout these experiments. Endotoxin levels were below detectable limits by Limulus amebocyte lysate assay.

RESULTS

Efl^ect of GM-CSF and M-CSF on monocyte chemotaxis One of the main functions of the mononuclear phagocyte system is the clearance of infectious organisms and immune complexes from the host circulation. Extravasation of these cells in response to infectious or inflammatory stimuli is among the first cellular responses to occur. Any disruption of the ability of monocytes to migrate toward a site of infection such as that which occurs in patients treated with high-dose steroids or in AIDS patients leads to a collapse in the necessary initial steps towards an efficient immune response.

In the present study, the pre-exposure of monocytes to either GM-CSF or M-CSF for 30 min prior to transfer to chemotaxis chambers enhanced the chemotactic response of the monocytes towards fMLP, a model for bacterial cell wall constituents, at least two-fold over media (Table 1). GM-CSF retained a significant activity on the monocytes in culture even at the lowest (0.05 ng/ml) concentration tested (Table 1). Both CSFs were equally potent in enhancing the migration of monocytes in the MNC cultures toward LTB,, a potent chemoattractant produced at inflammatory sites. Over the panel of ten donors tested, the migration of monocytes was enhanced at least two-fold towards LTB, by both GM-CSF and M-CSF. With GM-CSF, the enhanced migration was present at the lowest concentration tested (Table 1).

Effect of GM-CSF and M-CSF on monocyte phagocytosis and killing of fungal targets Phagocytic function was measured by exposing either GM-CSF or M-CSF treated monocytes in MNC cultures to opsonized heat-killed yeast targets. Hexosemonophosphate shunt activity (NBT reduction) was measured in parallel as a corroborative measurement of CSF-mediated cell activation. MNC cultures exposed to 5.0 or 0.5 ng/ml of either GM-CSF or M-CSF were significantly enhanced compared to medium control cultures for both the phagocytic index and percentage of NBT-reduced cells (Table 2). The per cent of NBTreduced cells was observed to still be enhanced in MNC cultures treated with 0.05 ngiml of either CSF (Table 2). However, at this level the phagocytic index of the cytokine-treated MNC cultures was diminished (Table 2).

Colony Stimulating Factors Table 3. Pretreatment of human monocytes with GM-CSF or M-CSF enhanced killing capacity for C. albicans blastospores Per cent surviving C. albicuns Effector : Target ratio Treatment’ of cells

150: 1

50: I

25 : 1

10: 1

None

48(6)

54(8)

65(6)

60(12)

20(3)** 27(4)** 35(g)*

27(3)** 34(5)** 41(7)**

28(5)** 41(6)** 49(7)’

GM-CSF (@ml) 5.00 18(5)** 0.50 26(5)** 31(7)* 0.05 M-CSF (rig/ml) 5.00 19(3)** 0.50 23(4)** 27(6)** 0.05

19(l)** 26(5)** 36(5)*

24(l)** 38(7)** 46(4)*

35(4)** 56(5) 61(7)

‘Unseparated monocytes were exposed for 48 h to CSFs before cells were recovered from culture. Effector cell numbers were adjusted so that these ratios were achieved when using a constant target number of 3000 yeast. G-CSF was used as a negative control without effect in this assay. *P
The CSF-treated monocytes were also tested for their ability to kill C. albicans blastospores. The effectortarget ratios were chosen to be below those maximally effective for untreated monocytes, i.e. at 150 : 1: 48% of the yeast targets germinated after exposure to untreated monocytes and as the effector cell number was lowered an increasing percentage (-60-65%) of yeast blastospores germinated. The incubation of MNC cultures with GM-CSF or M-CSF for 48 h was effective in enhancing the killing capacity of the cultures for ingested C. albicuns blastospores. The killing efftciency of the CSF-treated MNCs was diminished only at the very lowest concentration tested for both CSFs (0.05 @ml). The loss of CSF efficacy was found to be variable within the donor population tested. MNC cultures from a few of the donors were effective at killing even at a 10 : 1 ratio after CSF treatment (Table 3). Neither CSF was capable of directly affecting fungal growth by inhibiting germination (data not shown). We did not observe any effect of either CSF on the viability or the percentage of monocytes in culture. Effects of GM-CSF and M-CSF on monocyte phugocytosis and killing of bacteria The ingestion of opsonized bacterial particles was an extremely efficient process in untreated MNC

389

Table 4. Both GM-CSF and M-CSF prevented a dexamethasoneinduced suppression of monocyte phagocytosis of bacterial particles Treatment’ of cells

Per cent total cells with MI-FITC’ GM-CSF M-CSF None 5 ng/ml 5 ng/ml

None

78(3)

80(l)

81(2)

Dexamethasone 105M 106M 10-l M

53(4)* 59(6)* 62(5)

74(7)** 78(4)** 82(2)**

75(6)** 75(3)** 78(4)**

‘Monocytes were pre-incubated with CSFs for 18 h. Data represent mean (S.E.M.) of six donors. *Per cent cells = 5000 gated events analyzed by flow cytometry. G-CSF was used as a negative control. MI-FITC, Fluorescinated A4icrococcus lysodeikticus; monocytes exposed to MI-FITC for 5 min. *WO.OS; Student’s t-test (dex alone vs none). **P
cultures. As a result, it was difficult to superimpose any CSF-mediated effect. To demonstrate more clearly any potential effect of the CSFs for bacterial ingestion, dexamethasone was added to the MNC cultures. We observed that the addition of either GM-CSF or M-CSF to the dexamethasone-containing MNC cultures prevented the steroid effect. The ingestion of the FITClabeled bacteria was equivalent to that obtained from normal non-steroid treated cells (Table 4). Interestingly, GM-CSF treated MNC cultures retained slightly better killing efficacy even at the lowest E : T ratio tested. The effect of the CSFs to prevent steroid suppression of the MNC cultures was also observed in a bacterial killing assay. We observed that the addition of dexamethasone to MNC cultures induced a two-fold reduction in the percentage of monocytes containings. uureus after a 30 min incubation period. This reduction in bacterial killing capacity in the steroid-treated cultures was abolished by the co-addition of either GM-CSF or M-CSF with dexamethasone. Both CSFs were completely effective in abolishing the dexamethasoneinduced suppression even at the 0.5 ng/ml concentration (Table 5).

DISCUSSION

A defective monocyte-mediated cellular defense system increases patient risk of recurrent and severe bacterial or fungal infections that are often life-

L. A. BORER

390

Table 5. Co-incubation of monocytes steroid-induced suppression Mean % cells dead bacteria

Treatment+ of cells None +GM-CSF 5.0 0.5

t M-CSF (@ml) 5.0 0.5

39(2) 3Y3)

with GM-CSF or M-CSF prevented of bacterial killing capacity Treatment+ of cells Dexamethasone

35(2) (ng/ml)

etal.

Dex+GM-CSF 5.0 0.5

33(2) 34(2)

a

Mean % cells dead bacteria 14(l)*

(ng/ml)

Dex+M-CSF (ng/ml) 5.0 0.5

33(2)** 33(2)**

31(2)** 32(2)**

+Monocytes were pre-incubated with CSF and/or dexamethasone (10 5 M) for 18 h. Cells were exposed to opsonized S. aureus bacteria for 30 min before staining with acridine orange. Data represent a mean (S.E.M.) of six donors; for each donor six fields of 100 cells were evaluated for calculation of individual percentage of cells containing dead (red) bacteria. *P
**PcO.O5; Student’s t-test (dex+CSF vs dex alone).

threatening. Such immunocompromised patients usually have either a clinical monopenia or an impairment of monocyte phagocyte function. Abnormal monocyte chemotaxis and phagocytosis have been identified as contributing factors in the immune collapse documented to occur in AIDS patients (Smith et al., 1984; Lane & Fauci, 1985). Cancer patients have also been documented to have failures in monocyte chemotaxis, phagocytosis, and in cytokine production (Dammacco et al., 1982; Eccles & Alexander, 1974; Kjeldsberg & Ray, 1978; Tan et al., 1986). In the present study, the exposure of monocytes in MNC cultures to either M-CSF or GM-CSF, at concentrations that are therapeutically achievable in vivo (Munn & Cheung, 1992), resulted in monocytes that were mobilized very quickly to migrate toward chemoattractants representative of those found in bacterial lesions or trauma sites. As with the neutrophil (Weisbart et al., 1986), any demonstration of enhanced chemotaxis is highly dependent on the length of cellular exposure to CSF and length of migration time through the filter. Cells cannot maintain readiness for migration indetinitely and as exposure to the activating agent continues, other functions of these cells (i.e. superoxide production and cytokine release) that prevent cell migration become paramount (Weisbart et al., 1986); Hugli, 1989). Even though these CSFs are themselves chemoattractants (Wang et al., 1987, 1988), a bolus injection to a patient of the CSF would most likely result in a rapid deployment of mature cells to the sites of infection, trauma, cancer, etc., where local activation responses would be dominant. Both CSFs were found to be equally effective in

stimulating MNC culture phagocytosis and killing of yeast targets (Wang et al., 1989; Smith et al., 1990). In our assay systems, we have not been able to demonstrate that either of the CSFs could further enhance MNC culture killing activity at high E : T ratios (>150 : 1). However, at lower E : T ratios the killing efficiency of CSF-treated MNC cultures was significantly better than that of control untreated MNC cultures. These in vitro results indicate that the CSFs may have their greatest utility in patients where monocyte cell numbers and/or function are lowered or compromised by disease, chemotherapy, stress, or trauma (Dammacco et al., 1992; Bach et al., 1973; Klein et al., 1984). Use of the CSFs in these situations may simultaneously bolster cell numbers and maximize the activation capacity of the existing cell population. Future experiments to study the effect of IL-3 in comparison or in combination with either M-CSF or GM-CSF may be useful in helping to elucidate further the clinical implications (Ganser et al., 1993). The potential for either GM-CSF or M-CSF to support immunocompromised patients through a crisis is further strengthened by our demonstration that the addition of these CSFs will prevent the suppression of cell function induced by concomitant steroid addition. Drugs commonly used as therapy for cancer, transplant rejection crises, etc., often place the patient at risk for increased nosocomial infection (Szkaradkiewicz, 1992; Bach et al., 1973; Klein et al., 1984; Athlin & Domellof, 1987; Dammacco, 1992). The CSFs may actually work to counterbalance the effects of steroids by supporting mature cell function and number. The addition of GMCSF to monocyte cultures has been reported to prevent

Colony Stimulating

the dexamethasone-induced reduction of class II antigen (Sadeghi et al., 1992). Children receiving methylprednisolone as an immunosuppressive therapy have been reported to have higher concentrations of serum GM-CSF (Tuncer et al., 1992). GM-CSF support therapy has been demonstrated to be effective and well tolerated in AIDS patients with Kaposi’s sarcoma (Scadden, 1992). M-CSF therapy has been reported to enhance survival ofpatients with severe fungal infection for at least 100 days (Nemunaitis et al., 1991). GM-CSF and M-CSF were compared at equal protein concentrations and were observed to have equivalent

Factors

391

activation potentials in most of the assays used in this study. The only exception observed was the maintenance of the anti-candidal killing potential by GM-CSF-treated PMN cultures at low E : T ratio. GM-CSF, with its ability to activate monocytes, neutrophils, and eosinophils, may have a slightly better therapeutic potential than a lineage-restricted cytokine such as M-CSF. Our data suggest that CSF support therapy may have greatest potential clinical application and benefit in disease states where cell number and mature cell t%nction have been impaired.

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