Effect of Recombinant Granulocyte Colony-Stimulating Factor on Neutrophil Kinetics in Normal Young and Elderly Humans

By Thomas H. Price, Gurkamal S. Chatta, and David C. Dale

Recombinant granulocyte colony-stimulating factor (G-CSF) was administered to healthy young (n = 32) and elderly (n = 19) volunteers (0 pg/d, 30 pg/d, or 300 pg/d) to determine its effect on neutrophil production, blood kinetics, and tissue migration. Measurements included blood counts (daily), marrow neutrophil pool sizes and neutrophil tissue migra- tion (baseline and day 5). blood kinetics (day 6), and marrow transit time while on drug (days 6 to 14). G-CSF markedly expanded the marrow neutrophil mitotic pool and shortened the transit time of the postmitotic pool (control, mean = 6.4 days; 300 pgld, mean = 2.9 d). G-CSF increased neutrophil

ECOMBINANT granulocyte colony-stimulating factor R (G-CSF) is now widely used for the treatment of acute and chronic ne~tropenia.'.~ As its name implies, this cytokine was originally defined on the basis of its capacity to stimulate the proliferation of granulocyte progenitor cells in vitro." Subsequent studies demonstrated its ability to stimulate mar- row myeloid proliferation, to induce neutrophilia, to acceler- ate neutrophil recovery after chemotherapy or bone marrow transplantation (BMT), and to enhance the functional capa- bilities of mature neutrophil^."^ There is increasing evidence that G-CSF plays an important role as an endogenous regula- tor of granulocyt~poiesis.~~~ The kinetic mechanisms respon- sible for its effects are less well understood, and systematic studies to determine the quantitative effect of G-CSF admin- istration on marrow, blood, and tissue kinetics of neutrophils in healthy subjects have not been reported.

Advanced age has been shown in several studies to be associated with increased morbidity and mortality because of infection.1°-12 Aging may be associated with a decrease in neutrophil p rodu~t ion '~ , '~ and increased toxicity from cancer ~hemotherapy.'~ One possible explanation for this apparent blunting of the neutrophil response in the elderly is a reduced or altered response to endogenous G-CSF. It is also possible that it is predominantly the humoral, rather than the cellular response, which is blunted with aging and that this deficiency can be overcome with endogenous stimulators such as G- CSF. From a clinical perspective it is particularly important to define the effects of cytokines such as G-CSF in the elderly, given the higher frequency of cancer, greater prob- lems with its treatment, and potentially greater benefits to the use of these agents in this population. For these reasons we have examined the effects of G-CSF on neutrophil pro- genitor colony forming unit-granulocyte macrophage (CFU- GM) numbers, marrow neutrophil pool sizes and transit time, blood neutrophil kinetics, and the tissue inflammatory re- sponse in healthy young subjects and have compared them with those of healthy elderly subjects.

MATERIALS AND METHODS

Subjects were young (age 20 to 30) or elderly (age 70 to 80) normal volunteers. Some data on these subjects and their response to G-CSF has been reported previously.16 All subjects were nonsmokers who, at entry, had normal blood counts and no history of hematologic abnormality, recent infectious disease, use of medica- tion, or significant medical illness. All subjects gave informed con-

Subjects.

production without significantly altering blood neutrophil half-life or margination. Compared to control, neutrophil ac- cumulation in skin chambers decreased by about 50% in the 300 pg/d group in both young and elderly subjects. G-CSF induced neutrophilia by stimulating proliferation of marrow neutrophil precursors and accelerating neutrophil entry into the blood. Decreased neutrophil inflammatory responses measured with the skin chamber technique may be because of the relative immaturity of the circulating cells or to alter- ations in neutrophil phenotype induced by G-CSF. 0 1996 by The American Society of Hematology.

sent for this study, which was approved by the University of Wash- ington Human Subjects Review Committee.

Each subject had baseline measurements including a medical history, physical examination, complete blood count, BM aspiration for determination of marrow pool sizes. and skin chamber test for measurement of the in vivo neutrophil inflam- matory response. In addition, buccal neutrophils were determined from an oral wash with saline. Nineteen young and 19 elderly sub- jects were separately randomized to receive 30 pg/d G-CSF (14 subjects), 30 pg/d G-CSF (14 subjects), or no G-CSF (10 subjects). G-CSF was administered subcutaneously at 8 AM on study days 1 through 14. Complete blood counts were determined daily just before G-CSF administration. On day 5 a repeat marrow aspiration for marrow neutrophil quantitation was performed as were repeat skin chamber and oral wash studies. Blood neutrophil kinetic studies using tritiated diisopropylfluorophosphate (3H-DFF') labeled autolo- gous neutrophils were performed on day 6. On day 7 subjects were given an injection of tritiated thymidine and samples drawn over the next 8 days for determination of marrow postmitotic pool transit time. Blood neutrophil kinetics studies were performed in an addi- tional eight young subjects who had not received G-CSF. To examine further the effects of G-CSF on the in vivo neutrophil inflammatory response, skin chamber studies were performed on an additional 13 young subjects receiving 300 pg/d G-CSF. Marrow culture studies (CFU-GM assays) were performed on these same additional subjects. G-CSF (300 pg/mL) was supplied by Amgen Corp (Thousand Oaks, CA).

Marrow differentials were determined by count- ing 500 nucleated cells on Wright's stained smears using standard techniques. Results for each marrow neutrophil compartment were expressed as a percent of the total nucleated cell population. Results were also normalized to the total marrow erythroblast population and expressed as neutrophiYerythrocyte ratios.

Study design-overview.

EM pool sizes.

From the Department of Medicine, University of Washington,

Submitted June 30, 1995; accepted February 27, 1996. Supported by a fellowship from the Brookdale National Founda-

tion (G.S.C.); a Geriatric Academic Physician (GAP) Award (AG00503) from the National Institutes of Health (G.S.C.): and a research contract from Amgen. Inc (Thousand Oaks, CA).

Address reprint requests to Thomas H. Price, MD, Puget Sound Blood Center, 921 Terry Ave, Seattle, WA 98104.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

Puget Sound Blood Center, Seattle.

0 1996 by The American Society of Hematology. 0006-4971/96/8801-0018$3.oO/0

Blood, Vol 88, No 1 (July l), 1996: pp 335-340 335

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336 PRICE, CHATTA, AND DALE

BM culture studies. Heparinized BM aspirates were obtained from the posterior iliac crest under local anesthesia. The cell suspen- sion was diluted 1:l with NCTC 109 tissue culture medium, and the mononuclear cell population was isolated by Ficoll-Hypaque (Pharmacia, Piscataway, NJ) sedimentation (400 g) and washed three times with TC199 or Hanks' balanced salt solution (HBSS). In vitro colony formation was determined in a serum-containing system as previously described.14 Adherent cells were removed by two I-hour incubations in 75 cm2 plastic flasks (Coming, Coming NY) con- taining 20 X lo6 cells in 20 mL TC199 supplemented with 10% (voUvol) fetal calf serum (FCS) at 37" in 5% CO,. The nonadherent cells (2 X lo5 cells/mL) were cultured in 0.3% agar containing 20% FCS with or without the addition of growth factors. Maximum growth was stimulated by a mixture of three recombinant hematopoi- etic growth factors: interleukin-3 (IL-3) (40 pmoVL), granulocyte macrophage-CSF (GM-CSF) (50 pmoVL), and G-CSF (282 pmol/ L) (all provided by Amgen) or by phytohemagglutinin-stimulated leukocyte conditioned medium. Colony growth was determined on day 14.

The transit time through the marrow postmitotic pool was measured following a study day 7 injection of 10 pCiikg 3H-thymidine (50 to 90 Ci/mmol, New England Nuclear, Boston, MA) as previously described." Twenty milliliter blood sam- ples for determination of neutrophil specific activity were obtained daily for 2 days, then q12 hours for a total of 8 days. Neutrophils were isolated from each blood sample by Ficoll-Hypaque sedimenta- tion (Pharmacia), dextran sedimentation, and NH,CI lysis of contam- inating red cells. The radioactivity of a measured number of cells was determined by liquid scintillation counting. Cell specific activity was plotted as a function of time after injection of the isotope, the resulting curve representing the sum of influx of labeled cells into the circulation from the marrow and the efflux of labeled cells into the tissues as indicated by the formula:

Neutrophil transit time.

g(t) = cH(t) + H'(t)

in which g (t) equals the rate of entry of labeled cells from the marrow at time t; c is a constant determined by dividing In 2 by the t (hours) of the blood neutrophils; H(t) is the neutrophil specific activity at time t; and H'(t) is the slope of the curve at time t. The transit time of the postmitotic pool was obtained by subtracting from the maximum g (t) the average time required for 50% of the last myelocyte generation to enter the postmitotic pool. The latter figure was assumed to be 8 hours based on the data of Cronkite et al.'*

The blood recovery and sur- vival of autologous neutrophils was measured on day 5. Approxi- mately 500 mL of blood was withdrawn into a plastic bag containing acid-citrate dextrose, formula A (ACD-A) or citrate-phosphate-dex- trose-adenine (CPDA1) anticoagulant (Fenwal PL130; Baxter Cor- poration, Deerlield, IL). The neutrophils were labeled in vitro with 100 pCi of 'H-DFP as previously described." The isotope (3H-DFP, 6 to I O Ci/mmol) was obtained from the manufacturer (New England Nuclear) as a radiochemical and was sterilized and diluted in propyl- ene glycol to a concentration of 1 mCi/mL; 0.1 mL of this mixture was diluted in 10 mL saline immediately before use. After removing an aliquot of the labeled blood for determination of neutrophil spe- cific activity (previously described), the labeled blood was reinfused into the subject over a 5 to 10 minute period. Twenty milliliter blood samples were obtained at 10 minutes and at 1 , 2, 3, 4, 6, 8, 11, and 24 hours after infusion for determination of neutrophil specific activity. Blood neutrophil half-time was determined by the method of least squares through the most linear portion of a semilogarithmic plot of these data points. Neutrophil recovery was defined as the fraction of infused cells circulating at time zero, determined by the value of the y-intercept of the extrapolated survival curve. The frac- tion of infused cells not recovered in the circulation was considered

Blood neutrophil kinetic studies.

to represent the marginal pool. Neutrophil tumover was calculated as outlined by Athens et al."

For measure- ment of neutrophil accumulation in skin chambers, a 2 cm2 area of the volar forearm was abraded by scraping with a surgical scalpel blade and was covered with a glass chamber, which was fastened in place with adhesive compound and tape. The chamber was filled with a mixture of 10% autologous serum and saline to which had been added 100 p/mL streptokinaselstreptodomase (Behringwerks AG; MarburgLahm, Germany) to prevent neutrophil clumping as previously described.*' The chamber fluid was replaced at 4 and 8 hours and removed at 24 hours. The number of white cells in each fluid sample was determined by an electronic particle counter (Coulter, Hialeah, FL). Skin chamber studies were performed on the first 38 subjects at baseline and at day 5. Studies to determine the acute effects of G-CSF in 13 additional subjects were done on day 1, beginning immediately after a dose of 300 p g G-CSF.

Neutrophil accumulation in the oral cavity was assessed by the method of Wright et al." The subject was asked to swish hisher mouth with 25 mL of saline for 30 seconds, return the specimen to a sputum cup, and to immediately repeat the procedure to obtain a duplicate sample. Specimens were centrifuged at 200g for 10 min- utes within one hour of collection. The pellet was resuspended in 1 mL HBSS containing 2 pg/mL acridine orange and incubated 15 minutes in a 37" shaking water bath. The cells were then thoroughly resuspended and the number of neutrophils determined by a hemocy- tometer count using fluorescence microscopy.

Results are expressed as mean 2 1 SEM unless otherwise specified. Student's t-test or paired t-test was used to determine the significance of differences between groups.

In vivo measurement of inflammatory response.

Statistical analysis.

RESULTS

The effect of G-CSF on routine hematologic values in these subjects has been reported pre- viously.'6 In summary, there was a dose and time dependent increase in blood neutrophil counts, the 8 AM (Pre-G-CSF dose) count averaging 20.5 X 109/1 and 28.6 X 10'fl after 5 and 15 days of 300 &kg/d G-CSF, respectively. Bands and segmented neutrophils both increased over this time period, but the relative immaturity of the neutrophils, represented by the fraction of the cells that were bands, increased only with higher dose of the drug. After G-CSF administration, neutrophils on the peripheral blood smear appeared larger than normal with very blue cytoplasm, as reported pre- viously.*' No consistent or significant changes were seen in blood lymphocyte, monocyte, basophil, or eosinophil counts. Hematocrit changes were only seen at day 15, undoubtedly because of the fact that 500 to 750 mL of blood had been withdrawn between day 5 and day 15 for laboratory measure- ments, and there were no differences between subjects given different doses of the drug. Platelet counts were not affected by 15 days of G-CSF. There were no substantive differences between the values for the young and elderly subjects.

The effect of 5 days of G-CSF therapy (300 pg/d) on marrow differential counts in these subjects has been previously reportedI6 and indicated an expansion of the mitotic compartment with a relative depletion of the segmented neutrophils. When these values are expressed in relation to the erythroid compartment (Fig I), a relative increase in all morphologic types of neutrophils is apparent with significant increases in promyelocytes, my- elocytes, and metamyelocytes. Because the absolute size of

Hematologic values.

Marrow neutrophil pool sizes.

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EFFECT OF G-CSF ON NEUTROPHIL KINETICS 337

1.2

1 0

tu .- - [I: 0.8

[1: 5 0.6

g 0.4 3 z

x .- 0 a, -

L 5 0.2

0 Blasts Pro

*

+

E b

1 Myelo Meta Band

ooay 0

+

Fig 1. BM neutrophil pool sizes at day 0 and after 5 days of 300 p g G-CSF. Values normalized to constant marrow nucleated ery- throid cell population. (*I Different from baseline value ( P < .05). Error bars represent 1 SEM.

the marrow erythroid compartment would not have been expected to change appreciably over the first 5 days of G- CSF therapy, this normalization of the myeloid values to a constant erythron size provides a more absolute measure of changes in the size of the neutrophil pools for this time point. The fact that a smaller effect is seen in the maturational compartments than in the proliferative compartment is attrib- utable, at least in part, to an acceleration of mature cell movement through the marrow and into the blood, as indi- cated by the results for the marrow transit times (discussed later). There were no differences in marrow pool size mea- surements for young and elderly subjects (data not shown).

Marrow myeloid progenitor cell assays were performed on 1 1 young normal subjects receiv- ing 300 pg/d G-CSF. With maximal growth stimulation (IL- 3, GM-CSF, G-CSF), CFU-GM values after 5 days of G- CSF (18 2 5/10' cells plated) were slightly reduced from baseline (26 2 8/10' cells plated), although the difference was not statistically significant. However, the mononuclear cell preparations used for these cultures contained all nucle- ated cells up to and including the metamyelocyte stage. Be- cause CFU-GM are expressed as colonies per nucleated cells plated, the expansion of the myeloblast-metamyelocyte pools seen with G-CSF administration will serve to dilute the pro- genitor cells plated and may result in an underestimation of the CFU-GM pool size.

The transit time of the marrow postmitotic pool, measured by the pattern of emer- gence of labeled neutrophils in the blood after intravenous injection of tritiated thymidine, is shown in Fig 2. In normal subjects not treated with G-CSF the mean transit time is 6.4 t 0.3 days, a value similar to that obtained by Dancey et al.24 The value is shortened substantially on G-CSF treatment, reaching means of 4.3 t 0.2 days and 2.9 2 0.1 days after treatment with 30 pg/d G-CSF and 300 pg/d G-CSF, respec- tively. The differences between the values at all three drug levels are statistically significant (P < .01).

Results of blood kinetic mea- surements are shown in Table l. There was no significant

Marrow culture studies.

Marrow neutrophil transit time.

Neutrophil blood kinetics.

I I I I I I I I

0 1 2 3 4 5 6 7 6 Days after 3H-TdR

Fig 2. Marrow postmitotic pool transit time: The data points through which the curves are drawn represent average relative neu- trophil specific activities for subjects receiving 300 p g G-CSF (n = 121, 30 p g G-CSF (n = 10). and no G-CSF (n = 6). Actual values indicated for transit time represent mean -t 1 SEM of calculated values for individual subjects (see text).

effect of G-CSF treatment on the fraction of labeled neutro- phils accounted for in the circulation (% recovery), an indica- tor of the distribution of blood neutrophils between the circulating and marginal pools. Mean blood neutrophil half- disappearance time was slightly prolonged in subjects receiv- ing G-CSF, but the values were not statistically significantly different from those in the controls in this relatively small group of subjects. Calculation of the neutrophil turnover, the daily production of neutrophils by the marrow, suggests that the kinetic mechanism responsible for the neutrophilia is principally that of increased neutrophil production by the marrow. The effect of G-CSF to increase neutrophil produc- tion was the same in young and elderly subjects (data not shown).

The neutrophil inflammatory re- sponse was assessed by measuring neutrophil accumulation in a skin chamber and in the oral mucosa (Table 2). Skin chamber neutrophils after 5 days of G-CSF decreased in proportion to the dose, reaching less than half the baseline value in both young and elderly subjects receiving 300 pg/ day. There was no apparent acute effect of G-CSF on neutro- phil localization to the skin chamber because results obtained in skin chambers placed at the time of the first 300 pg G- CSF dose (125.9 t 35.0 X IO6, n = I I) were not different from baseline values. Buccal neutrophils were more plentiful in elderly subjects who were not edentulous, presumably

Inflammatory response.

Table 1. Effect of G-CSF on Blood PMN Kinetics. ~~

Recovery PMN Turnover Dose n PMNIuL (%) t% (hr) (X io7nc~1d)

0 12 3.5 2 0.4 71 2 9 10.4 2 1.5 64 -t 11 30pg 11 8 . 2 2 0 . 6 t 8 7 2 1 0 15 .922 .1 1 0 3 r 2 3

3 0 0 p g 9 23.7 2 1.9tS 62 2 10 13.5 t 1.7 389 2 47t*

Abbreviation: PMN, polymorphonuclear leukocyte. Mean 2 SEM.

t Different from control ( P < . O I L * Different from 30 pg G-CSF ( P < .OIL

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338 PRICE, CHAlTA, AND DALE

Table 2. In Vivo Neutrophil Migration*

5 Days

Baseline 0 30 pg 300 pg

Young Old Young Old Young Old Young Old

Blood PMN (x106/L) n 19 19 5 5 7 7 7 7

3.04 t 0.16 3.29 2 0.19 3.25 t 0.46 4.15 t 0.27 7.61 5 0.79 7.63 2 0.77 17.31 t 2.35 23.69 2 2.14 Skin Chamber

PMN ( ~ 1 0 ~ ) n 19 14 5 3 7 5 7 6 129.7 t 22.7 117.7 2 19.1 129.7 t 29.8 118.5 t 51.7 100.1 t 18.7 102.6 f 44.1 46.0 5 16.6t 49.9 2 18.0t

Buccal PMN (Xl06) n 19 18 5 5 7 7 6 7

Abbreviation: PMN, polymorphonuclear leukocyte. * Mean -c SEM. t Different from baseline (P < .05).

0.52 ? 0.10 2.27 t 0.70 1.09 2 0.39 5.54 5 2.67 0.26 t 0.03 1.28 t 0.84 0.66 t 0.23 1.39 2 0.55

related to gingival inflammation. There was neither a sig- nificant increase or decrease in buccal neutrophils with G- CSF treatment. AS shown in Table 2, there was a marked contrast between the elevated blood neutrophil counts, the simultaneous diminution of skin chamber neutrophil local- ization, and the constant or slight reduction in buccal neutro- phil localization in the young and elderly subjects, respec- tively.

Mild bone aching was reported by sev- eral subjects, both elderly and young, during the period of G-CSF administration. It was not necessary to discontinue treatment in any subject. No other side effects attributable to the drug were seen.

Adverse effects.

DISCUSSION

G-CSF is a potent stimulator of in vivo granulocytopoiesis and is an essential factor for maintenance of normal blood neutrophil count^.^^^^',^ Serum levels of G-CSF, for example, are elevated in infection,’’ and animals lacking G-CSF are ne~tropenic.~.~ Previous studies have shown that G-CSF stimulates proliferation of marrow neutrophil precursor^^^^^^ concomitant with increases in the blood neutrophil Neutrophil transit time through the marrow postmitotic pool has been reported to be shortened by G-CSF, but the magni- tude of the effect has been controversial. Studies in two patients with metastatic breast cancer suggested that the tran- sit time was shortened to one day, -20% of the normal value,30 but more recent studies in mice have suggested a more modest 55% reduction,’’ similar to the findings in this report. G-CSF is not thought to affect neutrophil blood kinet- ics, but direct measurements of neutrophil margination and survival have not been reported previously. Limited studies have suggested that G-CSF does not affect neutrophil local- ization to an inflammatory site.”,” Few of these studies have been performed on normals, thus raising the possibility that some of the findings were influenced by other abnormalities in the subjects. Although it is known that G-CSF stimulated mobilization of marrow neutrophils is no different for elderly than for young no other information is available on a possible influence of age on G-CSF effects.

As has been previously reported, in the present study there was a dose dependent increase in blood neutrophil count seen with G-CSF administration, an effect that was identical for young and elderly subjects. A relative increase in imma- ture neutrophils was seen only at the higher doses of the drug. There was no consistent effect of G-CSF on blood levels of other white blood cells or platelets. The decrease in hematocrit seen after 15 days of G-CSF administration undoubtedly reflected blood loss caused by phlebotomy and not an effect of G-CSF.

Marrow neutrophil pool sizes were measurably increased after 5 days of 300 pg/d G-CSF. This increase was most apparent in the mitotic pool compartments where absolute pool sizes were increased approximately 150%. The apparent lesser effect on the postmitotic neutrophil pool sizes was most likely caused by the accelerated delivery of these near- mature neutrophils into the circulation, shown by the dose dependent shortening of the marrow transit time seen in Fig 2. This shortened maturation time seen with G-CSF is similar to that seen in other situations where neutrophils are mobi- lized from the marrow storage pool such as infection or inflammation or administration of substances such as cortico- steroids or end~toxin.~’

Blood kinetic studies indicate that neutrophil margination is not appreciably affected by G-CSF in the doses studied. There is at most a modest prolongation of blood neutrophil survival, although the values are not significantly different from normal. The data suggest that the increased blood neu- trophil count seen with G-CSF administration is principally attributable to increased delivery of neutrophils from the marrow to the blood, whether the result of early mobilization or increased granulocytopoiesis. Although not shown, there are no differences seen between young and elderly subjects in these blood kinetic measurements.

Previous studies have suggested that tissue neutrophil lo- calization is impaired after the administration of GM-CSF but is unaltered by the administration of G-CSF.32,33,36,’7 In the present study, it is apparent that the tissue inflammatory response, as measured by neutrophil localization to skin chambers, was significantly reduced if measured after 5 days

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EFFECT OF G-CSF ON NEUTROPHIL KINETICS 339

of high-dose G-CSF (300 pg/d) in both young and elderly subjects, despite marked blood neutrophilia. Similarly, buc- cal neutrophils were not increased in proportion to the dramatic increase in circulating neutrophil counts seen with G-CSF stimulation. The apparent decrease in buccal neutro- phils was not statistically significant, although the failure to detect a real difference may be because of the variability of the measurements and the relatively small sample size. Skin chamber accumulation was not different from normal when studied immediately after the first 300 pg dose of G-CSF, suggesting that reduced localization is not caused by G-CSF per se; rather it appears more likely to reflect the fact that neutrophils released from the marrow after several days of G-CSF stimulation have different levels of surface adhesion proteins than do neutrophils released from the marrow with- out ~ t i m u l a t i o n , ~ ~ , ~ ~ differences that may affect the neutro- phil's ability to migrate from the blood into the tissues. The reduced neutrophil response seen in the skin chamber studies does not necessarily imply that these neutrophils would be unavailable to a patient with an infection. These cells made under the influence of G-CSF have been shown to be capable of upregulation of cell surface markers on activation by in- flammatory mediators"; an in vivo infectious challenge may well provide the same activation of the circulating neutro- phils. That G-CSF stimulated neutrophils are capable of re- sponding to an infection normally is suggested by the various clinical studies performed in both animals and humans sug- gesting that G-CSF treatment may have a positive effect on survival and resolution of i n f e ~ t i o n . ~ ~ The failure of neutrophilia per se to affect the quantity of neutrophils accu- mulating at the inflammatory sites suggests that when neutro- phils are adequate in number, the number of cells localized is primarily dependent upon the inflammatory stimulus, not simply the number of available neutrophils. The observation that tissue infiltration by neutrophils is minimal in transgenic mice with marked G-CSF induced neutrophilia is consistent with this formulation."

The effect of G-CSF was no different in young and elderly subjects in any of the results reported here. The only ob- served difference between the different age groups was that elderly subjects had higher numbers of oral neutrophils. This was true whether or not G-CSF had been administered and was easily attributable to differences in gingival health.

These studies have shown that G-CSF stimulates marrow neutrophil delivery into the blood by stimulating prolifera- tion of both progenitor cells and the identifiable marrow mitotic pool and by accelerating the transit time through the postmitotic pool. Blood kinetics, as reflected by margination or survival of neutrophils, is unaffected by G-CSF therapy, and the neutrophilia observed in these subjects was entirely accounted for by increased marrow neutrophil production. The neutrophils circulating after several days of G-CSF stim- ulation are different from those produced in the absence of G-CSF. They are larger, their complement of surface molecules is different, and their ability to localize to an inflammatory site, at least in the absence of a major inflam- matory stimulus, is reduced. In view of clinical studies sug- gesting a salutary effect of G-CSF administration during infection, it is plausible that under a more severe inflamma-

tory stimulus, neutrophil delivery may be normalized or even enhanced.

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27. Begley CG, Nicola NA, Metcalf D: Proliferation of normal human promyelocytes and myelocytes after a single pulse stimula- tion by purified GM-CSF or G-CSF. Blood 7 1 :640, 1988

28. Morstyn G, Souza LM, Keech 3, Sberidan W, Campbell L, Alton NK, Green M, Metcalf D, Fox R: Effect of granulocyte colony stimulating factor on neutropenia induced by cytotoxic chemother- apy. Lancet 1:667, 1988

29. Bronchud MH, Scarffe JH, Thatcher N, Crowther D, Souza LM, Alton NK, Testa NG, Dexter TM: Phase VI1 study of recombi- nant human granulocyte colony-stimulating factor in patients receiv- ing intensive chemotherapy for small cell lung cancer. Br J Cancer 56:809, 1987

30. Lord BI, Bronchud MH, Owens S, Chang J, Howell A, Souza L, Dexter TM: The kinetics of human granulopoiesis following treat- ment with granulocyte colony-stimulating factor in vivo. Proc Natl Acad Sci USA 86:9499, 1989

3 I , Uchida T, Yamagiwa A: Kinetics of rG-CSF-induced neutro- philia in mice. Exp Hematol 20:152, 1992

32. Peters WP, Kurtzberg J, Atwater S, Borowitz M, Gilbert C, Rao M, Cume M, Shogan J, Jones RB, Shpall El, Souza L: Compar- ative effects of rHuG-CSF and rHuGM-CSF on hematopoietic recon- stitution and granulocyte function following high dose chemotherapy and autologous bone marrow transplantation (ABMT). Blood 72:130a, I988 (abstr, suppl 1)

33. Hammond WP, Price TH, Souza LM, Dale DC: Treatment of cyclic neutropenia with granulocyte colony-stimulating factor. N Engl J Med 320:1306, 1989

34. Chatta GS, Price TH, Stratton JR, Dale DC: Aging and mar- row neutrophil reserves. J Am Geriatr Soc 42:77, 1994

35. Dale DC, Fauci AS, Guerry D, Wolff SM: Comparison of agents producing a neutrophilic leukocytosis in man. J Clin Invest 56:808, 1975

36. Peters WP, Stuart A, Affronti ML, Kim CS, Coleman RE: Neutrophil migration is defective during recombinant human granu- locyte-macrophage colony-stimulating factor infusion after autolo- gous bone marrow transplantation in humans. Blood 72:1310, 1988

37. Addison IE, Johnson B, Devereux S, Goldstone AH, Linch DC: Granulocyte-macrophage colony-stimulating factor may inhibit neutrophil migration in vivo. Clin Exp Immunol 76: 149, 1989

38. Liles WC, Rodger ER, Dale DC: Differential regulation of human neutrophil surface expression of CD14, CDI Ib, CD18 and L-selectin following the administration of G-CSF in vivo and in vitro. Clin Res 42:304A, 1994 (abstr)

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40. Dale DC: Potential role of colony-stimulating factors in the prevention and treatment of infectious diseases. Clin Infect Dis 18:S180, 1994 (suppl 2)

41. Silver GM, Gamelli RL, O’Reilly M: The beneficial effect of granulocyte colony-stimulating factor (G-CSF) on survival after Pseudomonas burn wound infection. Surgery 106:452, 1989

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44. Chang JM, Metcalf D. Gonda TJ, Johnson GR: Long-term exposure to retrovirally expressed granulocyte-colony-stimulating factor induces a nonneoplastic granulocytic and progenitor cell hy- perplasia without tissue damage in mice. J Clin Invest 84: 1488, 1989

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