Vol. 58, No. 9
INFECTION AND IMMUNITY, Sept. 1990, p. 3002-3008 0019-9567/90/093002-07$02.00/0 Copyright C) 1990, American Society for Microbiology
Alteration of the Functional Effects of Granulocyte-Macrophage Colony-Stimulating Factor on Polymorphonuclear Leukocytes by Membrane-Fluidizing Agents E. STEPHEN
BUESCHER,'*
SARAH M.
AND
McILHERAN,l STEVEN M. BANKS,2
SAROJ VADHAN-RAJ3
Department of Pediatrics, University of Texas Medical School at Houston,' and Department of Clinical Immunology and Biological Therapy, University of Texas M. D. Anderson Cancer Center,3 Houston, Texas 77030, and Office of the Director of the Intramural Research Program, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 208922 Received 21 March 1990/Accepted 22 June 1990
Locomotion and oxidative metabolism of polymorphonuclear leukocytes from 15 patients receiving recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) were examined in vitro. At the end of each GM-CSF treatment course, polymorphonuclear leukocyte (PMN) chemotactic responses were suppressed and no enhancement of formyl-peptide-stimulated superoxide production was observed. The priming of PMN superoxide production normally seen after in vitro GM-CSF exposure was also blunted in these cells. By using control donor PMN, two membrane-fluidizing agents, pentoxifylline and butanol, were shown to normalize suppressed PMN chemotaxis caused by in vitro GM-CSF (1 nM) exposure. Pentoxifylline, but not butanol, also reversed the effects of in vitro GM-CSF on PMN superoxide production. When PMN obtained from six patients at the end of GM-CSF therapy were exposed to pentoxifylline in vitro, the chemotactic suppression typically observed was significantly improved. The data suggest that GM-CSF may affect PMN function via mechanisms involving membrane fluidity or cell deformability or both.
The recent cloning of human colony-stimulating factors has rapidly led to their application as therapeutic agents in conditions of bone marrow failure or suppression. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used successfully to reverse bone marrow suppression in acquired immune deficiency syndrome (11), myelodysplastic syndrome (34), aplastic anemia (33), autologous bone marrow transplant (4), and cancer chemotherapy (1). It has been assumed that phagocytic cells produced during therapy with GM-CSF are functionally normal or, perhaps, functionally enhanced. We and others (5, 14, 33) have previously examined selected in vitro functions of phagocytic cells from patients undergoing phase I therapy with GM-CSF. As a group, these patients either had been heavily pretreated for their underlying diseases or had inherently abnormal bone marrows. Not surprisingly, they also had depressed chemotactic responses "off' of GM-CSF therapy, compared with responses of normal controls. In this group of patients, GM-CSF exposure in vivo did not significantly alter their abnormal chemotaxis, although a significant suppressive effect of in vitro GM-CSF exposure could be shown with control cells (5). We were concerned that the base-line functional abnormalities of the patients in our previous study might have obscured the true effects of GM-CSF therapy on in vitro phagocytic cell functions. Additionally, we were interested in examining whether GM-CSF-associated functional alterations could be reversed. Thus, the following studies were performed.
GM-CSF treatment were examined. These patients had received minimal chemotherapy before entry into the GMCSF study and were not expected to show either chemotherapy-related or disease-related suppression of neutrophil function. A minimum of 21 days after their most recent chemotherapy, each patient was treated with a continuous infusion of recombinant human GM-CSF (Immunex Corp., Seattle, Wash.) for 14 days, rested for 7 days, received chemotherapy for 5 days (Cytoxan, Adriamycin, and dacarbazine), rested for 2 days, and then again received a continuous 14-day GM-CSF infusion (Fig. 1). In later patients, the protocol was modified to administer chemotherapy over 3 days, followed immediately by the second course of GMCSF. The doses of GM-CSF used (in micrograms per square meter per day) were as follows: 120, four patients; 250, six patients; 500, four patients. Samples for polymorphonuclear leukocyte (PMN) functional assays were obtained from patients on five occasions, as shown in Fig. 1: (i) before the first course of GM-CSF (referred to as PRE#1), (ii) on day 13 or 14 of the first GM-CSF infusion (referred to as END#1), (iii) immediately prechemotherapy (referred to as BETWEEN), (iv) immediately before the second GM-CSF infusion (referred to as PRE#2), and (v) on day 13 or 14 of the second GM-CSF infusion (referred to as END#2). Each patient was paired with a control volunteer donor whose cells were collected and studied in parallel on the same days the cells of the patient were studied. Cell preparations. PMN were purified from heparinized (1 U/ml) peripheral blood by Hypaque-Ficoll separation, dextran sedimentation, and hypotonic lysis (3). PMN locomotion. PMN locomotive responses were examined by using an underagarose method. Quadruplicate 60- by 15-mm petri dishes were loaded with ME agarose (FMC Corp., Rockland, Maine) (1.2% agarose in minimum essential medium, 5% autologous serum), and pairs of parallel
MATERIALS AND METHODS Patient selection. Fourteen patients with recently diagnosed soft-tissue sarcomas entering a phase I-II trial of *
Corresponding author. 3002
VOL. 58, 1990
EFFECTS OF GM-CSF ON PMN FUNCTION
CHEAIORx
_~L~GM-CSF-2
GGM-CSF - I
PRE *1
END*I 14
BETWEEN PRE*2
21
28
I END
42
DAY OF STUDY FIG. 1. Time line showing the relationship of each patient specimen to the administration of two courses of GM-CSF treatment. CHEMORx, Chemotherapy.
troughs were cut into each plate (trough dimensions, 2 by 5 mm; 4 mm between troughs). The troughs were positioned perpendicular to the radius at the 12, 3, 6, and 9 o'clock positions in each dish. Each outer trough was filled with 20 ,ul of stimulant (Hanks balanced salt solution with Ca2+ and Mg2+ [HBSS], zymosan-activated serum [AcS], 5 x 10-7 M formylmethionylleucylphenylalanine [fMLP], or fMLP in AcS [A+f]), and the inner trough was filled with 20 RI of purified cells (2.5 x 107/ml). The plates were incubated in 5% CO2 at 37°C for 4 h, fixed with Formalin, stained by modified Wright-Giemsa (Diff-Quik) stain, and projected with a histology slide projector. The distance (in centimeters) from the trough edge to the leading front of cells was measured, and the quadruplicate values were averaged. Leading front distances were expressed in arbitrary units equal to the distances (in centimeters) measured on the projected images. PMN superoxide production. Superoxide production was quantitated by measuring superoxide dismutase-inhibitable reduction of ferricytochrome c by PMN (17). In the first eight patients, both patient and control cells were tested in three different conditions: condition 1, freshly purified cells; condition 2, purified cells incubated in HBSS plus 10% autologous, heat-inactivated serum for 2 h at 37°C; condition 3, purified cells incubated in HBSS plus 10% autologous, heat-inactivated serum plus 100 pM GM-CSF (Immunex) for 2 h at 37°C. Condition 1 was used to examine whether in vivo GM-CSF exposure enhanced PMN superoxide production. Conditions 2 and 3 were used to examine whether the enhancement of fMLP-stimulated superoxide production observed after in vitro GM-CSF exposure (7, 15) persisted during GM-CSF treatment. The 10% autologous serum present in all assays was used to prevent clumping of cells during the 2-h, 37°C incubation. Each assay was run in triplicate: 400 RI of cells (2.5 x 106/ml) was combined with HBSS and cytochrome c (65 ,uM final), with or without fMLP (10-7 M final), with or without 113 U of superoxide dismutase to a total volume of 1.5 ml. Samples were tumbled at 37°C for 30 min, and after incubation, the triplicate assays were pelleted at 18,000 x g for 15 s. The supernatants were then collected, and their A550S were determined. Superoxide production was calculated with an extinction coefficient of 21 and is expressed as nanomoles per 106 cells per 30 min. When cell yields from patient blood samples were low, chemotaxis assays were performed first and any remaining cells were used in the superoxide assay. When this occurred, fresh-cell experiments were performed in preference to the 2-h incubation experiments. In patients 9, 10, 11, 13, and 14, only conditions 2 and 3 (see above) were examined with and without fluidizer exposures. Exposure + GM-CSF was for 60 min rather than 120 min, and the cells were stimulated with 106 M fMLP. Fluidizer exposures. (i) Chemotaxis. Agarose chemotaxis assay plates were made up as described above, but with
3003
100 ,ug of pentoxifylline (PTXF) per ml, 0.25% n-butanol (BUTNL), 1 nM GM-CSF, 100 jig of PTXF per ml plus 1 nM GM-CSF, or 0.25% BUTNL plus 1 nM GM-CSF incorporated into the agarose. Purified PMN from adult volunteer donors were used in these assays as described above. Cells from patients 9 to 14 were used to examine how in vitro PTXF modified the effects of in vivo GM-CSF exposure on chemotaxis. For these studies, only PTXF was incorporated into the agarose of the chemotaxis plates. (ii) Superoxide production. Purified PMN from adult volunteer donors were resuspended in HBSS without Ca2+ or Mg2+ containing 5% autologous, heat-inactivated serum either with or without 100 pM GM-CSF. Cells were incubated at 37°C for a total of 60 min. After the first 45 min of incubation, two samples of each mixture (with and without GM-CSF) were taken and BUTNL (0.25% final) or PTXF (100 ,ug/ml final) was added. Incubation was continued for 15 more min, and superoxide production was then assayed as described above with 10-6 M fMLP for stimulation. Cells from patients 9 to 14 were also used to examine how in vitro PTXF exposure modified the effects of in vivo GM-CSF exposure on PMN superoxide production. These experiments were performed exactly as described above. Unless otherwise noted, all reagents were purchased from Sigma Chemical Co., St. Louis, Mo. Statistical analysis. The effects of in vivo GM-CSF treatment were examined by comparing the differences within patient-volunteer control pairs at the PRE and END points of each GM-CSF treatment course by an analysis of variance (25). The rationale for this approach was that across multiple patient-control pairs, these patient-minus-control differences would be expected to sum to zero in the absence of a treatment effect. An average GM-CSF effect on chemotaxis was estimated for each patient by calculating the change in the patientcontrol differences between PRE#1 and END#1 and between PRE#2 and END#2. This estimate of the average GM-CSF effect for each patient was introduced into a t test, with residual error from the analysis of variance used as the reference source of variability. In instances when data at a given time point were missing, data collected at the remaining time point of the pair were discarded so that unbiased estimates could be obtained. Clinical data were reported in terms of the mean + standard error (SE), and statistical significance was declared at P < 0.05. The membrane fluidizer experiments were analyzed by examining the PTXF or BUTNL effects separately. The analyses used a three-way ANOVA controlling for the donor, absence or presence of GM-CSF, and absence or presence of the fluidizer. RESULTS Patients. Fourteen patients were examined on 57 occasions. Eight of the fourteen patients provided fewer than five specimens. The first GM-CSF infusion was evaluated in 12 patients; the second GM-CSF infusion was evaluated in 9
patients. Purity of cell preparations. The percentages of PMN present in cell preparations from control donors and patients at each of the study time points are given in Table 1. Eosinophils composed the majority of contaminating cells in patient preparations made at time points END#1 (45% ± 7%), but eosinophils composed only 16% ± 6% at END#2. Effects of in vivo GM-CSF therapy on PMN locomotion.
3004
INFECT. IMMUN.
BUESCHER ET AL.
significant effect on unstimulated locomotion. In vitro 100 pLg of PTXF per ml enhanced unstimulated locomotion (P = 0.007). Chemotaxis to AcS (P = 0.003), fMLP (P = 0.001), and A+f (P = 0.02) were also slightly enhanced, consistent with a previous report (29). Exposure of cells to 0.25% BUTNL had no significant effect on unstimulated locomotion or chemotaxis to AcS or fMLP but slightly enhanced chemotaxis to A+f (P = 0.01). Exposure of cells to the combination of GM-CSF and PTXF or GM-CSF and BUTNL resulted in locomotive responses (both unstimulated locomotion and chemotaxis to all three stimuli) not significantly different statistically from those of unexposed control cells. Stimulation of PMN by 10-6 M fMLP resulted in production of 6.8 + 1.3 nmol of superoxide per 106 cells per 30 min (Fig. 3). Incubation of these same cells with 100 pM GMCSF for 60 min prior to stimulation resulted in approximately 2.3-fold enhancement of superoxide production, (15.5 1.9 nmol/106 cells per 30 min, P < 0.005). Exposure of cells to either 100 ,ug PTXF per ml or 0.25% BUTNL alone prior to fMLP stimulation resulted in suppression of superoxide production (2.9 0.9 and 4.3 1.4 nmol/106 cells per 30 min, respectively), consistent with previous reports (10, 30, 35). Exposure to the combinations GM-CSF plus PTXF and GM-CSF plus BUTNL resulted in different effects. Exposure to GM-CSF plus PTXF resulted in superoxide production no different from that of unexposed cells, the effects of one agent balancing the effects of the other. GM-CSF plus BUTNL resulted in superoxide production significantly greater than that of unexposed cells (P = 0.001) and no different from that observed with cells exposed only to GM-CSF. The sequence of exposure to GM-CSF-PTXF or GM-CSF-BUTNL did not affect the levels of superoxide produced (data not shown). Effects of PTXF on in vivo GM-CSF-treated cells. The effects of in vitro exposure to PTXF were examined with cells from six patients (Fig. 4). When patients were off GM-CSF treatment (time points PRE#1, BETWEEN, and PRE#2), PTXF exerted a slight enhancing effect on unstimulated locomotion. When patients were on GM-CSF treatment (END#1 and END#2), in vitro PTXF exposure significantly improved chemotactic responses to AcS (P = 0.02), fMLP (P = 0.004) and A+f (P = 0.01). This effect decreased the suppression of chemotaxis observed at these times by 52% for AcS, by 31% for fMLP, and by 32% for A+f (Fig. 4). The effects of in vitro PTXF exposure on superoxide production by patient cells are shown in Fig. 5. Without any in vitro exposures, superoxide production by patient cells off GM-CSF was not different from superoxide production by cells on GM-CSF. In vitro PTXF exposure significantly suppressed superoxide production by cells off GM-CSF (P 0.001) but did not significantly affect cells on GM-CSF (P >
TABLE 1. Purity of PMN preparations obtained from patients and controls at each study time point
no
exposure to
Mean % ± SE of PMN in preparation at: PMN source
Patients Controls
PRE#1
END#1
(n=9)
(n=12)
95 94
53 ± 6 94 ± 1
1 1
a For patients, n
=
BETWEEN"
92 ± 2 94 ± 1
END#2
PRE#2
(n=11)
(n=9)
88 94
80 96
7 1
7 1
11; for controls, n = 10.
Patient and paired control chemotaxis results at the five time points studied are shown in Table 2. Mean unstimulated locomotion and chemotaxis responses of control cells were stable over time. Patient cell unstimulated locomotion responses were equal to those of the controls at all time points examined, showing no GM-CSF treatment effect. Patient cell chemotaxis responses were not different from control cell responses at time points PRE#1 and PRE#2, demonstrating that patient cells were capable of normal chemotaxis. At time points END#1 and END#2, patient cells showed suppressed chemotactic responses to all three chemoattractant stimuli. At the BETWEEN time point, chemotactic responses were midway between the low END#1 and the normal PRE#2 values. The calculated average GM-CSF effects for these results are -1.05 cm for AcS (P = 0.008), -1.87 cm for fMLP (P = 0.001), and -1.94 cm for A+f (P = 0.0002), with the negative values representing suppression of stimulated chemotaxis. Effects of in vivo GM-CSF therapy on formyl-peptidestimulated superoxide production. Patient and paired control cell superoxide production at the five time points studied are shown in Table 3. When freshly purified cells were examined, patient and control cells were not different at any time point, demonstrating that in vivo exposure to GM-CSF (END#1 and END#2) did not enhance superoxide production by patient cells. When control cells were exposed to 100 pM GM-CSF in vitro for 2 h before stimulation, the resulting superoxide production was increased an average of threefold over that of fresh cells (Table 3). When patient cells were exposed to this same condition, superoxide production was enhanced an average of 2.3-fold at times PRE#1, BETWEEN, and PRE#2. At times END#1 and END#2, average superoxide production was not enhanced by in vitro exposure to GMCSF, demonstrating that in vivo GM-CSF treatment blunted this in vitro effect of GM-CSF. Effects of membrane fluidizers on in vitro GM-CSF-treated cells. In vitro exposure of normal PMN to 1 nM GM-CSF resulted in suppression of chemotaxis (Fig. 2) to AcS, fMLP, and A+f by 32, 32, and 31% (P < 0.0001, P = 0.0006, and P = 0.0002, respectively). This concentration of GM-CSF had
±
±
±
TABLE 2. Chemotaxis responses by PMN from patients receiving GM-CSF treatment and from their paired controls Leading front distance (mean ± Stimulus
PRE#1 in (n
Patients
HBSS AcS fMLP A+f
1.5 3.2 3.7 4.3
± + + +
0.1 0.4 0.3 0.3
=
13):
Controls
1.8 3.5 4.2 4.8
± ± ± ±
0.2 0.3 0.3 0.3
END#1 in (n Patients
1.4 2.4 2.2 2.8
± 0.1 ± 0.2 ± 0.2
± 0.3
=
13):
Controls
1.5 3.7 4.6 5.1
± 0.2 ± 0.3 ± 0.3
± 0.3
BETWEEN in (n Patients
1.5 3.4 3.7 4.5
± ± ± ±
0.2 0.3 0.5 0.4
aFor each pair of underlined values, P < 0.05 by analysis of variance (see text).
=
SE)" at:
12):
PRE#2 in (n = 10):
Controls
Patients
± ± ± ±
2.1 ± 0.3 4.0 ± 0.4 4.9 ± 0.3 5.5 ± 0.3
1.6 3.6 4.6 5.1
0.2 0.3 0.3 0.2
Controls 1.9 4.1 4.8 5.3
± ± + +
0.2 0.3
0.3 0.4
END#2 in (n = 9): Patients 1.5 2.6 2.3 2.9
± 0.2 + 0.2 + 0.3 + 0.3
Controls 2.0 3.9 4.4 5.2
± ± ± ±
0.2 0.3 0.5 0.4
EFFECTS OF GM-CSF ON PMN FUNCTION
VOL. 58, 1990
3005
TABLE 3. Superoxide production by PMN from patients receiving GM-CSF treatment and from their paired controls Superoxide production (mean nmol ± SE) at: Treatment
PRE#1 in (n = 5)a:
Patients
END#1 in (n = 8):
Controls
Patients
Controls
BETWEEN in (n = 6)b: Patients
PRE#2 in (n = 5): Patients
Controls
Controls
END#2 in (n = 3): Patients
Controls
None (fresh cells) 2.0 ± 0.5 1.77 0.4 5.9 ± 2.0 3.7 ± 1.5 3.7 ± 1.9 3.4 ± 1.2 4.5 ± 1.4 4.4 ± 2.6 4.0 ± 1.9 2.7 ± 1.5 No GM-CSF' 2.8 ± 0.9"d 2.8 ± 1.11 4.3 ± 1.8 5.2 ± 1.41 4.0 ± 0.71 6.3 ± 1.21 6.9 ± 1.11 6.7 ± 2.8 3.9 ± 1.7 2.4 ± 1.5 100 pM GM-CSFc 4.9 + 1.0 1 5.8+ 1.71 4.2 ± 1.9 9.0 ± 2.21 7.0+ 1.2 1 9.6 ± 1.91 11.3 +2.51 10.2 ± 3.21 5.1 ± 2.0 11.3 ± 3.41 For the fresh cells, n = 6. For the fresh cells, n = 5. ' For 120 min at 37°C. d For braced values, P < 0.05 by analysis of variance (see text). b
0.25). In vitro exposure of cells off GM-CSF to 100 pM GM-CSF resulted in significantly enhanced superoxide production (P = 0.002), but this exposure did not affect cells on GM-CSF. Combined in vitro exposure to GM-CSF and PTXF resulted in patient cell superoxide production no different from that of unexposed cells when patients were both on and off GM-CSF treatment (P > 0.25 and P = 0.081, respectively). DISCUSSION GM-CSF, one of a number of recently cloned colony stimulating factors, has the demonstrated capacity to support granulocyte progenitor proliferation both in vitro and in vivo (1, 4, 11, 33, 34). GM-CSF exerts functional effects on PMN after in vitro exposure; these effects include enhanced formyl-peptide-stimulated superoxide production (7, 15, 31) and, at high doses, suppression of chemotaxis (5, 10). In studies using cells from patients undergoing treatment with GM-CSF, it has been reported that superoxide production is enhanced when cells are examined very early in the course of treatment (12 h) (30), which may correlate with the in vitro observation. Additionally, a single report of depressed in vivo migration of PMN during continuous-infusion GM-CSF therapy has been published (23), whereas others, including
6-
HBSS
ourselves, have previously reported no suppression of in vitro chemotactic responses by GM-CSF treatment. Because in vitro GM-CSF exposure suppresses the chemotactic responses of normal control PMN, it was unexpected that such a suppressive effect was not observed in our initial studies of in vivo-exposed PMN. We were concerned that the inherent locomotive abnormalities in the patients that we initially examined might obscure real GM-CSF effects, and so we sought a patient group in which we could document normal chemotactic responsiveness before treatment with GM-CSF had begun. To date, no studies of the in vivo effects of GM-CSF therapy on in vitro chemotaxis have documented normal pretreatment locomotive responses in the patients examined. The studies we performed were intended to carefully examine whether the PMN functional effects present after in vitro GM-CSF exposure were also present at the end of 14 days of continuous infusions of GM-CSF in vivo and whether the effects of GM-CSF on PMN function could be modified or reversed. Using a patient group in which pre-GM-CSF treatment chemotactic responses were normal, we found significant suppression of PMN chemotaxis at the end of each 14-daylong continuous infusion of GM-CSF. Between courses of treatment, chemotactic responses returned to normal levels.
PMLP
Ac S
A+
5Q
4-
2
0
(a
Z
3-
Q
O CONTROL (13)
2I
* PTXF (1O)
A BUTNL. (9)
I_NO GM-CSF
GM-CSF
NO
GM-CSF
NO
GM-CSF
NO
GM-CSF
GM-CSF GM-CSF GM-CSF FIG. 2. Effects of in vitro exposure to 1 nM GM-CSF and membrane fluidizers (100 jig of PTXF per ml or 0.25% BUTNL) on control PMN locomotive responses. The data shown are the mean ± SE leading front distances observed with the locomotive stimuli shown at the top of the figure.
INFECT. IMMUN.
BUESCHER ET AL.
3006
15-
20 -
O NO IN VITRO EXPOSURE * IN VITRO PTXF EXPOSURE IN VITRO GM-CSF EXPOSURE * IN VITRO PTXF + GM-CSF
Q
Q
15-
INFECTION AND IMMUNITY, Sept. 1990, p. 3002-3008 0019-9567/90/093002-07$02.00/0 Copyright C) 1990, American Society for Microbiology
Alteration of the Functional Effects of Granulocyte-Macrophage Colony-Stimulating Factor on Polymorphonuclear Leukocytes by Membrane-Fluidizing Agents E. STEPHEN
BUESCHER,'*
SARAH M.
AND
McILHERAN,l STEVEN M. BANKS,2
SAROJ VADHAN-RAJ3
Department of Pediatrics, University of Texas Medical School at Houston,' and Department of Clinical Immunology and Biological Therapy, University of Texas M. D. Anderson Cancer Center,3 Houston, Texas 77030, and Office of the Director of the Intramural Research Program, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 208922 Received 21 March 1990/Accepted 22 June 1990
Locomotion and oxidative metabolism of polymorphonuclear leukocytes from 15 patients receiving recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) were examined in vitro. At the end of each GM-CSF treatment course, polymorphonuclear leukocyte (PMN) chemotactic responses were suppressed and no enhancement of formyl-peptide-stimulated superoxide production was observed. The priming of PMN superoxide production normally seen after in vitro GM-CSF exposure was also blunted in these cells. By using control donor PMN, two membrane-fluidizing agents, pentoxifylline and butanol, were shown to normalize suppressed PMN chemotaxis caused by in vitro GM-CSF (1 nM) exposure. Pentoxifylline, but not butanol, also reversed the effects of in vitro GM-CSF on PMN superoxide production. When PMN obtained from six patients at the end of GM-CSF therapy were exposed to pentoxifylline in vitro, the chemotactic suppression typically observed was significantly improved. The data suggest that GM-CSF may affect PMN function via mechanisms involving membrane fluidity or cell deformability or both.
The recent cloning of human colony-stimulating factors has rapidly led to their application as therapeutic agents in conditions of bone marrow failure or suppression. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used successfully to reverse bone marrow suppression in acquired immune deficiency syndrome (11), myelodysplastic syndrome (34), aplastic anemia (33), autologous bone marrow transplant (4), and cancer chemotherapy (1). It has been assumed that phagocytic cells produced during therapy with GM-CSF are functionally normal or, perhaps, functionally enhanced. We and others (5, 14, 33) have previously examined selected in vitro functions of phagocytic cells from patients undergoing phase I therapy with GM-CSF. As a group, these patients either had been heavily pretreated for their underlying diseases or had inherently abnormal bone marrows. Not surprisingly, they also had depressed chemotactic responses "off' of GM-CSF therapy, compared with responses of normal controls. In this group of patients, GM-CSF exposure in vivo did not significantly alter their abnormal chemotaxis, although a significant suppressive effect of in vitro GM-CSF exposure could be shown with control cells (5). We were concerned that the base-line functional abnormalities of the patients in our previous study might have obscured the true effects of GM-CSF therapy on in vitro phagocytic cell functions. Additionally, we were interested in examining whether GM-CSF-associated functional alterations could be reversed. Thus, the following studies were performed.
GM-CSF treatment were examined. These patients had received minimal chemotherapy before entry into the GMCSF study and were not expected to show either chemotherapy-related or disease-related suppression of neutrophil function. A minimum of 21 days after their most recent chemotherapy, each patient was treated with a continuous infusion of recombinant human GM-CSF (Immunex Corp., Seattle, Wash.) for 14 days, rested for 7 days, received chemotherapy for 5 days (Cytoxan, Adriamycin, and dacarbazine), rested for 2 days, and then again received a continuous 14-day GM-CSF infusion (Fig. 1). In later patients, the protocol was modified to administer chemotherapy over 3 days, followed immediately by the second course of GMCSF. The doses of GM-CSF used (in micrograms per square meter per day) were as follows: 120, four patients; 250, six patients; 500, four patients. Samples for polymorphonuclear leukocyte (PMN) functional assays were obtained from patients on five occasions, as shown in Fig. 1: (i) before the first course of GM-CSF (referred to as PRE#1), (ii) on day 13 or 14 of the first GM-CSF infusion (referred to as END#1), (iii) immediately prechemotherapy (referred to as BETWEEN), (iv) immediately before the second GM-CSF infusion (referred to as PRE#2), and (v) on day 13 or 14 of the second GM-CSF infusion (referred to as END#2). Each patient was paired with a control volunteer donor whose cells were collected and studied in parallel on the same days the cells of the patient were studied. Cell preparations. PMN were purified from heparinized (1 U/ml) peripheral blood by Hypaque-Ficoll separation, dextran sedimentation, and hypotonic lysis (3). PMN locomotion. PMN locomotive responses were examined by using an underagarose method. Quadruplicate 60- by 15-mm petri dishes were loaded with ME agarose (FMC Corp., Rockland, Maine) (1.2% agarose in minimum essential medium, 5% autologous serum), and pairs of parallel
MATERIALS AND METHODS Patient selection. Fourteen patients with recently diagnosed soft-tissue sarcomas entering a phase I-II trial of *
Corresponding author. 3002
VOL. 58, 1990
EFFECTS OF GM-CSF ON PMN FUNCTION
CHEAIORx
_~L~GM-CSF-2
GGM-CSF - I
PRE *1
END*I 14
BETWEEN PRE*2
21
28
I END
42
DAY OF STUDY FIG. 1. Time line showing the relationship of each patient specimen to the administration of two courses of GM-CSF treatment. CHEMORx, Chemotherapy.
troughs were cut into each plate (trough dimensions, 2 by 5 mm; 4 mm between troughs). The troughs were positioned perpendicular to the radius at the 12, 3, 6, and 9 o'clock positions in each dish. Each outer trough was filled with 20 ,ul of stimulant (Hanks balanced salt solution with Ca2+ and Mg2+ [HBSS], zymosan-activated serum [AcS], 5 x 10-7 M formylmethionylleucylphenylalanine [fMLP], or fMLP in AcS [A+f]), and the inner trough was filled with 20 RI of purified cells (2.5 x 107/ml). The plates were incubated in 5% CO2 at 37°C for 4 h, fixed with Formalin, stained by modified Wright-Giemsa (Diff-Quik) stain, and projected with a histology slide projector. The distance (in centimeters) from the trough edge to the leading front of cells was measured, and the quadruplicate values were averaged. Leading front distances were expressed in arbitrary units equal to the distances (in centimeters) measured on the projected images. PMN superoxide production. Superoxide production was quantitated by measuring superoxide dismutase-inhibitable reduction of ferricytochrome c by PMN (17). In the first eight patients, both patient and control cells were tested in three different conditions: condition 1, freshly purified cells; condition 2, purified cells incubated in HBSS plus 10% autologous, heat-inactivated serum for 2 h at 37°C; condition 3, purified cells incubated in HBSS plus 10% autologous, heat-inactivated serum plus 100 pM GM-CSF (Immunex) for 2 h at 37°C. Condition 1 was used to examine whether in vivo GM-CSF exposure enhanced PMN superoxide production. Conditions 2 and 3 were used to examine whether the enhancement of fMLP-stimulated superoxide production observed after in vitro GM-CSF exposure (7, 15) persisted during GM-CSF treatment. The 10% autologous serum present in all assays was used to prevent clumping of cells during the 2-h, 37°C incubation. Each assay was run in triplicate: 400 RI of cells (2.5 x 106/ml) was combined with HBSS and cytochrome c (65 ,uM final), with or without fMLP (10-7 M final), with or without 113 U of superoxide dismutase to a total volume of 1.5 ml. Samples were tumbled at 37°C for 30 min, and after incubation, the triplicate assays were pelleted at 18,000 x g for 15 s. The supernatants were then collected, and their A550S were determined. Superoxide production was calculated with an extinction coefficient of 21 and is expressed as nanomoles per 106 cells per 30 min. When cell yields from patient blood samples were low, chemotaxis assays were performed first and any remaining cells were used in the superoxide assay. When this occurred, fresh-cell experiments were performed in preference to the 2-h incubation experiments. In patients 9, 10, 11, 13, and 14, only conditions 2 and 3 (see above) were examined with and without fluidizer exposures. Exposure + GM-CSF was for 60 min rather than 120 min, and the cells were stimulated with 106 M fMLP. Fluidizer exposures. (i) Chemotaxis. Agarose chemotaxis assay plates were made up as described above, but with
3003
100 ,ug of pentoxifylline (PTXF) per ml, 0.25% n-butanol (BUTNL), 1 nM GM-CSF, 100 jig of PTXF per ml plus 1 nM GM-CSF, or 0.25% BUTNL plus 1 nM GM-CSF incorporated into the agarose. Purified PMN from adult volunteer donors were used in these assays as described above. Cells from patients 9 to 14 were used to examine how in vitro PTXF modified the effects of in vivo GM-CSF exposure on chemotaxis. For these studies, only PTXF was incorporated into the agarose of the chemotaxis plates. (ii) Superoxide production. Purified PMN from adult volunteer donors were resuspended in HBSS without Ca2+ or Mg2+ containing 5% autologous, heat-inactivated serum either with or without 100 pM GM-CSF. Cells were incubated at 37°C for a total of 60 min. After the first 45 min of incubation, two samples of each mixture (with and without GM-CSF) were taken and BUTNL (0.25% final) or PTXF (100 ,ug/ml final) was added. Incubation was continued for 15 more min, and superoxide production was then assayed as described above with 10-6 M fMLP for stimulation. Cells from patients 9 to 14 were also used to examine how in vitro PTXF exposure modified the effects of in vivo GM-CSF exposure on PMN superoxide production. These experiments were performed exactly as described above. Unless otherwise noted, all reagents were purchased from Sigma Chemical Co., St. Louis, Mo. Statistical analysis. The effects of in vivo GM-CSF treatment were examined by comparing the differences within patient-volunteer control pairs at the PRE and END points of each GM-CSF treatment course by an analysis of variance (25). The rationale for this approach was that across multiple patient-control pairs, these patient-minus-control differences would be expected to sum to zero in the absence of a treatment effect. An average GM-CSF effect on chemotaxis was estimated for each patient by calculating the change in the patientcontrol differences between PRE#1 and END#1 and between PRE#2 and END#2. This estimate of the average GM-CSF effect for each patient was introduced into a t test, with residual error from the analysis of variance used as the reference source of variability. In instances when data at a given time point were missing, data collected at the remaining time point of the pair were discarded so that unbiased estimates could be obtained. Clinical data were reported in terms of the mean + standard error (SE), and statistical significance was declared at P < 0.05. The membrane fluidizer experiments were analyzed by examining the PTXF or BUTNL effects separately. The analyses used a three-way ANOVA controlling for the donor, absence or presence of GM-CSF, and absence or presence of the fluidizer. RESULTS Patients. Fourteen patients were examined on 57 occasions. Eight of the fourteen patients provided fewer than five specimens. The first GM-CSF infusion was evaluated in 12 patients; the second GM-CSF infusion was evaluated in 9
patients. Purity of cell preparations. The percentages of PMN present in cell preparations from control donors and patients at each of the study time points are given in Table 1. Eosinophils composed the majority of contaminating cells in patient preparations made at time points END#1 (45% ± 7%), but eosinophils composed only 16% ± 6% at END#2. Effects of in vivo GM-CSF therapy on PMN locomotion.
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significant effect on unstimulated locomotion. In vitro 100 pLg of PTXF per ml enhanced unstimulated locomotion (P = 0.007). Chemotaxis to AcS (P = 0.003), fMLP (P = 0.001), and A+f (P = 0.02) were also slightly enhanced, consistent with a previous report (29). Exposure of cells to 0.25% BUTNL had no significant effect on unstimulated locomotion or chemotaxis to AcS or fMLP but slightly enhanced chemotaxis to A+f (P = 0.01). Exposure of cells to the combination of GM-CSF and PTXF or GM-CSF and BUTNL resulted in locomotive responses (both unstimulated locomotion and chemotaxis to all three stimuli) not significantly different statistically from those of unexposed control cells. Stimulation of PMN by 10-6 M fMLP resulted in production of 6.8 + 1.3 nmol of superoxide per 106 cells per 30 min (Fig. 3). Incubation of these same cells with 100 pM GMCSF for 60 min prior to stimulation resulted in approximately 2.3-fold enhancement of superoxide production, (15.5 1.9 nmol/106 cells per 30 min, P < 0.005). Exposure of cells to either 100 ,ug PTXF per ml or 0.25% BUTNL alone prior to fMLP stimulation resulted in suppression of superoxide production (2.9 0.9 and 4.3 1.4 nmol/106 cells per 30 min, respectively), consistent with previous reports (10, 30, 35). Exposure to the combinations GM-CSF plus PTXF and GM-CSF plus BUTNL resulted in different effects. Exposure to GM-CSF plus PTXF resulted in superoxide production no different from that of unexposed cells, the effects of one agent balancing the effects of the other. GM-CSF plus BUTNL resulted in superoxide production significantly greater than that of unexposed cells (P = 0.001) and no different from that observed with cells exposed only to GM-CSF. The sequence of exposure to GM-CSF-PTXF or GM-CSF-BUTNL did not affect the levels of superoxide produced (data not shown). Effects of PTXF on in vivo GM-CSF-treated cells. The effects of in vitro exposure to PTXF were examined with cells from six patients (Fig. 4). When patients were off GM-CSF treatment (time points PRE#1, BETWEEN, and PRE#2), PTXF exerted a slight enhancing effect on unstimulated locomotion. When patients were on GM-CSF treatment (END#1 and END#2), in vitro PTXF exposure significantly improved chemotactic responses to AcS (P = 0.02), fMLP (P = 0.004) and A+f (P = 0.01). This effect decreased the suppression of chemotaxis observed at these times by 52% for AcS, by 31% for fMLP, and by 32% for A+f (Fig. 4). The effects of in vitro PTXF exposure on superoxide production by patient cells are shown in Fig. 5. Without any in vitro exposures, superoxide production by patient cells off GM-CSF was not different from superoxide production by cells on GM-CSF. In vitro PTXF exposure significantly suppressed superoxide production by cells off GM-CSF (P 0.001) but did not significantly affect cells on GM-CSF (P >
TABLE 1. Purity of PMN preparations obtained from patients and controls at each study time point
no
exposure to
Mean % ± SE of PMN in preparation at: PMN source
Patients Controls
PRE#1
END#1
(n=9)
(n=12)
95 94
53 ± 6 94 ± 1
1 1
a For patients, n
=
BETWEEN"
92 ± 2 94 ± 1
END#2
PRE#2
(n=11)
(n=9)
88 94
80 96
7 1
7 1
11; for controls, n = 10.
Patient and paired control chemotaxis results at the five time points studied are shown in Table 2. Mean unstimulated locomotion and chemotaxis responses of control cells were stable over time. Patient cell unstimulated locomotion responses were equal to those of the controls at all time points examined, showing no GM-CSF treatment effect. Patient cell chemotaxis responses were not different from control cell responses at time points PRE#1 and PRE#2, demonstrating that patient cells were capable of normal chemotaxis. At time points END#1 and END#2, patient cells showed suppressed chemotactic responses to all three chemoattractant stimuli. At the BETWEEN time point, chemotactic responses were midway between the low END#1 and the normal PRE#2 values. The calculated average GM-CSF effects for these results are -1.05 cm for AcS (P = 0.008), -1.87 cm for fMLP (P = 0.001), and -1.94 cm for A+f (P = 0.0002), with the negative values representing suppression of stimulated chemotaxis. Effects of in vivo GM-CSF therapy on formyl-peptidestimulated superoxide production. Patient and paired control cell superoxide production at the five time points studied are shown in Table 3. When freshly purified cells were examined, patient and control cells were not different at any time point, demonstrating that in vivo exposure to GM-CSF (END#1 and END#2) did not enhance superoxide production by patient cells. When control cells were exposed to 100 pM GM-CSF in vitro for 2 h before stimulation, the resulting superoxide production was increased an average of threefold over that of fresh cells (Table 3). When patient cells were exposed to this same condition, superoxide production was enhanced an average of 2.3-fold at times PRE#1, BETWEEN, and PRE#2. At times END#1 and END#2, average superoxide production was not enhanced by in vitro exposure to GMCSF, demonstrating that in vivo GM-CSF treatment blunted this in vitro effect of GM-CSF. Effects of membrane fluidizers on in vitro GM-CSF-treated cells. In vitro exposure of normal PMN to 1 nM GM-CSF resulted in suppression of chemotaxis (Fig. 2) to AcS, fMLP, and A+f by 32, 32, and 31% (P < 0.0001, P = 0.0006, and P = 0.0002, respectively). This concentration of GM-CSF had
±
±
±
TABLE 2. Chemotaxis responses by PMN from patients receiving GM-CSF treatment and from their paired controls Leading front distance (mean ± Stimulus
PRE#1 in (n
Patients
HBSS AcS fMLP A+f
1.5 3.2 3.7 4.3
± + + +
0.1 0.4 0.3 0.3
=
13):
Controls
1.8 3.5 4.2 4.8
± ± ± ±
0.2 0.3 0.3 0.3
END#1 in (n Patients
1.4 2.4 2.2 2.8
± 0.1 ± 0.2 ± 0.2
± 0.3
=
13):
Controls
1.5 3.7 4.6 5.1
± 0.2 ± 0.3 ± 0.3
± 0.3
BETWEEN in (n Patients
1.5 3.4 3.7 4.5
± ± ± ±
0.2 0.3 0.5 0.4
aFor each pair of underlined values, P < 0.05 by analysis of variance (see text).
=
SE)" at:
12):
PRE#2 in (n = 10):
Controls
Patients
± ± ± ±
2.1 ± 0.3 4.0 ± 0.4 4.9 ± 0.3 5.5 ± 0.3
1.6 3.6 4.6 5.1
0.2 0.3 0.3 0.2
Controls 1.9 4.1 4.8 5.3
± ± + +
0.2 0.3
0.3 0.4
END#2 in (n = 9): Patients 1.5 2.6 2.3 2.9
± 0.2 + 0.2 + 0.3 + 0.3
Controls 2.0 3.9 4.4 5.2
± ± ± ±
0.2 0.3 0.5 0.4
EFFECTS OF GM-CSF ON PMN FUNCTION
VOL. 58, 1990
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TABLE 3. Superoxide production by PMN from patients receiving GM-CSF treatment and from their paired controls Superoxide production (mean nmol ± SE) at: Treatment
PRE#1 in (n = 5)a:
Patients
END#1 in (n = 8):
Controls
Patients
Controls
BETWEEN in (n = 6)b: Patients
PRE#2 in (n = 5): Patients
Controls
Controls
END#2 in (n = 3): Patients
Controls
None (fresh cells) 2.0 ± 0.5 1.77 0.4 5.9 ± 2.0 3.7 ± 1.5 3.7 ± 1.9 3.4 ± 1.2 4.5 ± 1.4 4.4 ± 2.6 4.0 ± 1.9 2.7 ± 1.5 No GM-CSF' 2.8 ± 0.9"d 2.8 ± 1.11 4.3 ± 1.8 5.2 ± 1.41 4.0 ± 0.71 6.3 ± 1.21 6.9 ± 1.11 6.7 ± 2.8 3.9 ± 1.7 2.4 ± 1.5 100 pM GM-CSFc 4.9 + 1.0 1 5.8+ 1.71 4.2 ± 1.9 9.0 ± 2.21 7.0+ 1.2 1 9.6 ± 1.91 11.3 +2.51 10.2 ± 3.21 5.1 ± 2.0 11.3 ± 3.41 For the fresh cells, n = 6. For the fresh cells, n = 5. ' For 120 min at 37°C. d For braced values, P < 0.05 by analysis of variance (see text). b
0.25). In vitro exposure of cells off GM-CSF to 100 pM GM-CSF resulted in significantly enhanced superoxide production (P = 0.002), but this exposure did not affect cells on GM-CSF. Combined in vitro exposure to GM-CSF and PTXF resulted in patient cell superoxide production no different from that of unexposed cells when patients were both on and off GM-CSF treatment (P > 0.25 and P = 0.081, respectively). DISCUSSION GM-CSF, one of a number of recently cloned colony stimulating factors, has the demonstrated capacity to support granulocyte progenitor proliferation both in vitro and in vivo (1, 4, 11, 33, 34). GM-CSF exerts functional effects on PMN after in vitro exposure; these effects include enhanced formyl-peptide-stimulated superoxide production (7, 15, 31) and, at high doses, suppression of chemotaxis (5, 10). In studies using cells from patients undergoing treatment with GM-CSF, it has been reported that superoxide production is enhanced when cells are examined very early in the course of treatment (12 h) (30), which may correlate with the in vitro observation. Additionally, a single report of depressed in vivo migration of PMN during continuous-infusion GM-CSF therapy has been published (23), whereas others, including
6-
HBSS
ourselves, have previously reported no suppression of in vitro chemotactic responses by GM-CSF treatment. Because in vitro GM-CSF exposure suppresses the chemotactic responses of normal control PMN, it was unexpected that such a suppressive effect was not observed in our initial studies of in vivo-exposed PMN. We were concerned that the inherent locomotive abnormalities in the patients that we initially examined might obscure real GM-CSF effects, and so we sought a patient group in which we could document normal chemotactic responsiveness before treatment with GM-CSF had begun. To date, no studies of the in vivo effects of GM-CSF therapy on in vitro chemotaxis have documented normal pretreatment locomotive responses in the patients examined. The studies we performed were intended to carefully examine whether the PMN functional effects present after in vitro GM-CSF exposure were also present at the end of 14 days of continuous infusions of GM-CSF in vivo and whether the effects of GM-CSF on PMN function could be modified or reversed. Using a patient group in which pre-GM-CSF treatment chemotactic responses were normal, we found significant suppression of PMN chemotaxis at the end of each 14-daylong continuous infusion of GM-CSF. Between courses of treatment, chemotactic responses returned to normal levels.
PMLP
Ac S
A+
5Q
4-
2
0
(a
Z
3-
Q
O CONTROL (13)
2I
* PTXF (1O)
A BUTNL. (9)
I_NO GM-CSF
GM-CSF
NO
GM-CSF
NO
GM-CSF
NO
GM-CSF
GM-CSF GM-CSF GM-CSF FIG. 2. Effects of in vitro exposure to 1 nM GM-CSF and membrane fluidizers (100 jig of PTXF per ml or 0.25% BUTNL) on control PMN locomotive responses. The data shown are the mean ± SE leading front distances observed with the locomotive stimuli shown at the top of the figure.
INFECT. IMMUN.
BUESCHER ET AL.
3006
15-
20 -
O NO IN VITRO EXPOSURE * IN VITRO PTXF EXPOSURE IN VITRO GM-CSF EXPOSURE * IN VITRO PTXF + GM-CSF
Q
Q
15-
