cd14' cells in granulocyte colony-stimulating factor (g-csf

CD14' Cells in Granulocyte Colony-Stimulating Factor (G-CSF)-Mobilized Peripheral Blood Mononuclear Cells Induce Secretion of Interleukin-6 and

G-CSF by Marrow Stroma By Marco Mielcarek, Bryan A. Roecklein, and Beverly Torok-Storb

The ability of granulocyte colony-stimulating factor (G- CSFI-mobilized peripheral blood mononuclear cells (G- PBMCs) to induce secretion of cytokines in primary long- term marrow cultures (LTC) or in the human marrow stromal cell line HS23 was compared with that of marrow mononuclear cells. Equal numbers of G-PBMCs or marrow mononuclear cells were added to stromal cultures, supernatants were harvested at day 4 and levels of interleukin-la (IL-la), IL-lp, IL-2, IL-6, G-CSF, and tumor necrosis factor a (TNFa) were determined. G-PBMCs induced 21.4-fold higher levels of IL-6 and 12.5-fold higher levels of G-CSF in LTC cocultures compared with marrow mononuclear cells and induced 20.6- fold more IL-6 and 6.3-fold more G-CSF when added to HS23

G RANULOCYTE COLONY-stimulating factor (G- CSF)-mobilized peripheral blood mononuclear cells

(G-PBMCs) have been used increasingly for hematopoietic reconstitution after myeloablative therapy."' With few ex- ceptions, patients receiving G-PBMCs have experienced earlier platelet recovery compared with historical control groups of marrow In contrast, the time course of neu- trophil recovery has not been consistently improved.

The rapidity of platelet recovery seen in patients receiving G-PBMCs was initially explained by the increased number of CD34' cells provided in the G-PBMCs product compared with a marrow harvest. However, an analysis of the correlation between 'the number of CD34' cells and days to engraftment showed no benefit above a dose of 5 X lo6 cells/ kg.'" This would suggest that differences in the number of CD34' cells between marrow and G-PBMCs are not entirely responsible for the better engraftment kinetics of G-PBMCs. Alternative explanations would include qualitative differences in CD34' cells or quantitative/qualitative differences in accessory cell populations.

The possibility that accessory cells, eg, T cells and monocytes, may be functionally different is supported by the observation that G-PBMCs, which contain 10-fold more T cells than does marrow," do not cause more frequent or severe

From the Program in Transplantation Biology, Clinical Research Division, Fred Hutchinson Cancer Research Center, and the Depart- ment of Medicine, University of Washington, Seattle, WA.

Submitted May 24, 1995; accepted September l , 1995. Supported by Grants No. DK34431, CA18221, CA18029, and

HL.36444 from the National tnstitutes of Health, Department of Health and Human Services (DHHS), Bethesda, MD. M.M. is supported by a "Dr-Mildred-Scheel-Fellowship' ' of Deutsche Krebs- hilfe.

Address reprint requests to Marco Mielcarek, MD, Fred Hutchin- son Cancer Research Center, 1124 Columbia St, "318, 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. 0 1996 by The American Society of Hematology. 0006-4971/96/8702-0026$3.00/0

574

cells. Experiments using sorted populations of CDZO', CD3+, and CD14' cells showed that CD14' cells within G-PBMCs were responsible for triggering the production of IL-6 and G-CSF. The effect did not require cell-cell contact and was inhibited when neutralizing antibodies to IL-la and IL-1p were used in combination. In these experiments, the greater stimulating ability of G-PBMCs is most likely attributable to the greater number of CD14+ cells in G-PBMCs (26.1% -c 2.3%) compared with marrow (2.5% + 0.8%). because equal numbers of CD14+ cells sorted from marrow and G-PBMCs showed comparable ability to induce IL-6 and G-CSF when placed directly on stromal cells. 0 1996 by The American Society of Hematology.

acute graft-versus-host disease (GVHD).'.'' This is unex- pected given the observation that donor T cells are responsible for acute GVHD, and reducing their numbers in marrow harvests is associated with a reduced incidence and severity of GVHD.

Precisely how T-cell and monocyte function may differ between marrow and G-PBMCs has not been defined and whether such differences, if they do exist, can modulate stromal cell function in the hematopoietic microenvironment is unknown.

The marrow microenvironment plays a critical role in controlling the maintenance, proliferation, and differentiation of hematopoietic cells.".'4 It consists of stromal elements such as fibroblasts, reticular adventitial cells, endothelial cells, adipocytes, and accessory cells such as T lymphocytes and macrophages.15,'6 These stromal or accessory cells secrete hematopoietic growth factors and cytokines that are presented to hematopoietic cells in a soluble form or bound to extracellular matrix molecules. After myeloablative therapy, this complex microenvironment is disrupted. To reconstitute this system, accessory cells, such as T cells and monocytes, must be provided by the graft to interact with stromal cells of the host." Therefore, it is reasonable to speculate that both the number and quality of these donor-derived cells could influence the function of the microenvironment.

In the present study, we used an immortalized human marrow stromal cell line, HS23,I8 and primary stromal cell cultures to investigate how accessory cells derived from G- PBMCs or marrow modulate stromal function. The data suggest that CD14' cells isolated from either source can induce interleukin-6 (IL-6) and G-CSF secretion by marrow stromal cells. Unmanipulated G-PBMCs contain more CD14' cells than marrow and therefore induce more cytokine expression. However, CD34-selected G-PBMCs contain a lower number of CD14' cells than marrow and still engraft more rapidly, suggesting that, if CD14' cells in G-PBMCs play a role in rapid engraftment it may be due to as yet undefined qualitative rather than quantitative differences.

MATERIALS AND METHODS

Donors, marrow, und G-PBMC processing. Marrow and G- PBMCs were obtained from normal donors after informed consent

Blood, Vol 87, No 2 (January 15), 1996: pp 574-580

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CD14' CELLS IN MOBILIZED PERIPHERAL BLOOD 575

as defined by the Internal Review Board at the Fred Hutchinson Cancer Research Center (FHCRC; Seattle, WA). G-PBMC donors were mobilized with recombinant human G-CSF (rhG-CSF) at 16 pg/kg/d (Amgen, Inc. Thousand Oaks, CA) by administering two subcutaneous injections per day for 5 days, 4 days before G-PBMC collection and once after the first collection. Leukapheresis was performed using a continuous flow blood cell separator ( C o b Labora- tories, Lakewood, CO) on 2 consecutive days beginning on day 5 of rhG-CSF administration. Samples for experiments were taken either from the first or second day of collection. Cells were suspended in Hank's Balanced Salt Solution (HBSS)/l% bovine serum albumin (BSA) and centrifuged at 200g for 10 minutes to remove platelets before hemolysis in hemolysis buffer (150 mmol/L ammo- niumchloride and 12 mmol/L sodium bicarbonate).

Marrow was obtained from allogeneic donors by aspiration from the iliac crest under general anesthesia. Samples were collected into 10% Normosol R (Abbott Laboratories, North Chicago, IL) supplemented with 10 IUlmL preservative-free heparin. Buffy coats or Ficoll (Accu-Prep; Accurate Chemicals, Westbury, NY; density, 1.077 g/mL)-separated cells were hemolysed, washed once in HBSS containing 1% BSA, and resuspended in a Minimal Essential Me- dium (a MEM) containing 10% fetal calf serum (FCS). Cells were used for experiments without further processing or labeled for cell sorting.

Long-term cultures (LTCs) and marrow stromal cell lines. LTCs were established as described by Gartner and Kaplan." Briefly, buffy coat cells from marrow aspirates were plated in T-75 flasks (Costar, Cambridge, MA) at 1 to 2 X 106/mL. Adherent cells were grown in LTC medium containing Iscove's modified Dulbecco's medium (IMDM), 12.5% horse serum, 12.5% fetal calf serum, L-glutamine (0.4 mg/mL), sodium pyruvate (1 mmoVL), penicillin (100 U/mL), streptomycin sulfate (100 pg/mL), hydrocortisone sodium succinate (lo-' mol/L), and P-mercaptoethanol mom) and fed weekly by demidepletion. Stromal layers were maintained at 37°C in an atmosphere of 5% CO2. After reaching confluency, the adherent layers were trypsinized once and grown to confluency again in a T- 75 flask to deplete hematopoietic cells before transfer to 24-well plates (Costar). Experiments were performed with 3-week-old LTCs after cells were confluent.

The marrow stromal line HS23" was grown in RPMI 1640 medium supplemented with 10% FCS, L-glutamine (0.4 mg/mL), sodium pyruvate ( l m o a ) , penicillin (100 U/mL), and streptomycin sulfate (100 pg/mL) (complete RPMI medium). After reaching confluency, cells were trypsinized and transferred to 24-well plates (Costar). Cells were allowed to reach confluency again before the coculture experiments were initiated.

Staining and sorting for B cells, T cells, and monocytes. To sort for B cells, T cells, and monocytes, approximately 100 X lo6 marrow cells or G-PBMCs were stained with Leu16 (anti-CD20 phycoer- ythrin [PE] direct conjugate; Becton Dickinson, San Jose, CA), IOT3b (anti-CD3 fluorescein isothiocyanate [FITC] direct conjugate; Amac, Westbrook, ME), or LeuM3 (anti-CD14 PE direct conjugate; Becton Dickinson), respectively. Staining was performed for 20 minutes on ice. Cells were washed twice in HBSS/I% BSA and sorted on the FACSTAR-PLUS (Becton Dickinson). B and T lymphocytes were sorted by setting the first gate on the population with low orthogonal and intermediate forward light scatter and a second gate on the cells positively stained with the monoclonal antibody to either CD20 or CD3. Monocytes were sorted by setting one gate on cells with slightly higher orthogonal light scatter compared with the lym- phocyte population and with relatively high forward scatter. The second gate was based on high expression of the CD14 antigen. Sort windows for CD14' cells from bone marrow and G-PBMCs are described in Fig 1. Purity was always greater than 98% as assessed by routine fluorometric purity check after each individual sort. Cells

were counted and resuspended in a MEM supplemented with 10% FCS, penicillin (100 U/mL), and streptomycin sulfate ( 1 0 0 &mL) (complete a MEM). Viability always exceeded 95% as determined by trypan blue exclusion.

Coculture of marrow stroma and hematopoietic cells. To assay for the release of cytokines into coculture supernatants, G-PBMCs, marrow cells, or purified cell fractions were suspended in defined numbers in 1 mL complete CY MEM and plated on LTCs or HS23 that had been grown to confluency on 24-well plates (Costar). Cocul- tures were incubated at 37°C in an atmosphere of 5 % CO,. After 4 days, conditioned media (CM) were harvested, centrifuged briefly to remove cells and debris, and stored at 4°C for not longer than 1 week before cytokine levels were determined.

To study contact requirements for cytokine secretion, G-PBMCs or marrow cells were separated from stromal cells by 0.4-pm Transwell polycarbonate inserts (Costar) and coculture experiments were performed as described above.

Inhibition of IL-6 and G-CSF release by marrow stroma. To investigate if IL-l was the humoral mediator of IL-6 and G-CSF secretion by marrow stroma, HS23 was grown to confluency on 24- well plates and incubated in the presence of complete a MEM only, CM from CD14' cells, or recombinant IL-I, in the presence and absence of neutralizing antibodies to IL-la, IL-Ip (R&D Systems, Minneapolis, MN; 3.3 pgmL and 33.0 pg/mL, respectively). Super- natants were harvested after 48 hours and assayed for IL-6 and G- CSF levels.

IL-6 and G-CSF enzyme-linked immunosorbent assays (ELISAS). Analysis of cytokine levels was provided by Allan Farrand of the shared resources of the FHCRC. Briefly, cytokine levels were determined using 96-well plates (Costar) coated overnight at 4°C with 1 pg/mL of antihuman IL-6 (Boehringer Mannheim, Indianapolis, IN) or antihuman G-CSF (Oncogene Science, San Diego, CA) in 50 mmol/L Na carbonate, pH 9.5. The plates were washed three times and nonspecific activity was blocked by incubating the coated plates with 5% Blotto/phosphate-buffered saline (PBS) (Carnation Non- Fat Dry Milk in PBS; Nestle Foods, Glendale, CA) at room tempera- ture (RT) for 30 minutes. After additional washes, diluted samples were incubated overnight at 4°C in high salt assay buffer (1 % mouse serum/PBS-t + 0.5 m o m NaCI). Detection of captured IL-6 was accomplished by the addition of a 1 :750 dilution of sheep antihuman IL-6-biotin conjugate and detection of captured G-CSF by the addition of a 1:1500 dilution of sheep antihuman G-CSF-biotin conjugate (The Binding Site, San Diego, CA) in 1% mouse serum/PBS- T at RT for 2 hours. After washing, the detection of captured IL-6 and G-CSF sandwich was determined by using peroxidase VEC- TASTAIN ABC Reagent (Vector Labs, Burlingame, CA) in PBS- T at RT for 30 minutes. Finally, the plates were washed again and substrate (0.1 mg/mL TMB; Sigma, St Louis, MO) with H202 was added. The reaction was stopped with 1 mol/L H2S04. Optical density was determined at 450 nm using a microplate reader (Emax; Molecular Devices, Sunnyvale, CA). Unknown IL-6 values were calculated from a standardized curve using rhIL-6 (R&D Systems). Interassay and intraassay CVs were determined to be less than IO%, with an assay sensitivity of less than 4 pg/mL. All samples, stan- dards, and controls were run in duplicate.

RESULTS

Cytokine levels in culture supernatants of G-PBMCs or BM-MNC and marrow stroma. The supernatants generated in 4-day cocultures of stroma plus marrow or G-PBMCs were assayed for IL-la, IL-IP, IL-2, IL-6, G-CSF, and tumor necrosis factor a (TNFcr). As shown in Table 1, IL-6 levels detected in cocultures of G-PBMCs and primary stroma were, on average, 21.4-fold and G-CSF levels 12.5-




576 MIELCAREK, ROECKLEIN, AND TOROK-STORB

L FSC-Height

Table 1. IL-6 and G-CSF Levels (in Nanograms per Milliliter) in Culture Supernatants From Stroma Alone (Control) and After

the Addition of Marrow or G-PBMCs

Control c Marrow + G-PBMCs Experiment

No. Stroma 11-6 G-CSF IL-6 G-CSF IL-6 G-CSF

1 LTC 15.6 0.25 24.1 0.29 46.5 0.48 2 LTC 0.7 ND 15.2 0.23 25.3 0.82 3 LTC 0.2 ND 33.3 1.73 149.7 18.58 4 LTC 0.2 ND 1.8 ND 58.8 2.31 5 LTC 37.6 0.40 38.6 0.87 122.3 2.84 6 LTC 5.6 0.07 14.9 0.66 54.0 4.86 7 LTC 0.1 ND 2.7 ND 294.8 17.44 1 HS23* 0.8 0.03 1.9 0.04 112.0 >5.00 2 HS23 1.7 ND 48.3 8.96 134.8 36.60 3 HS23 2.0 ND 40.7 6.67 955.8 31.84 4 HS23 1.8 0.20 12.1 1.59 140.0 49.85 7 HS23 3.5 0.24 41.7 10.83 476.1 53.68

Stroma was cultured alone (control) or with either 5 x IO5 marrow cells (+ Marrow) or G-PBMCs (+ G-PBMCs). G-PBMCs induced significantly more IL-6 and G-CSF secretion by LTCs ( P = .018) or HS23 ( P = ,043) than did marrow cells (Wilcoxon's signed rank test). Data represent the mean (in nanograms per milliliter) of replicate determi- nations made in different experiments.

Abbreviation: ND, not detectable. * HS23 cultured in the presence of 1 ng/mL IL-la secretes 830 -t

109 ng/mL IL-6 and 49.7 ? 4.0 ng/mL G-CSF; IL-1p induces secretion of 501 2 54 ng/mL IL-6 and 49.8 f 1.8 ng/mL G-CSF.

Fig 1. The forward (FSC) and orthogonal (SSC) light scattering properties of G-PBMCs (A) and marrow (B) were used to es- tablish a gate as indicated by the box. The expression of CD14 by cells within this FSClSSC gated population is shown for G- PBMCs (C) and marrow (D). The box indicates the sort window used for collecting CD14' cells.

fold higher than those detected in cocultures of marrow and primary stroma ( P = .018; Wilcoxon's signed rank test). When cells were cultured on HS23, a similar pattern of L- 6 and G-CSF production could be shown (20.6-fold higher IL-6 and 6.3-fold higher G-CSF levels of G-PBMCs versus marrow; P = .043). The levels of L-la, IL-lP, L-2, and TNFa did not differ significantly.

CD14' cells within G-PBMCs are responsible for IL-6 and G-CSF release. To identify the cell populations among G-PBMCs responsible for stimulating IL-6 and G-CSF production, B cells, T cells, and monocytes were isolated by flow cytometry based on their expression of CD20, CD3, and CD14, respectively, and defined numbers of sorted cells were added to LTC or HS23. Data indicated that only CD14+ cells were able to trigger the IL-6 and G-CSF release in the same order of magnitude as unfractionated G-PBMCs (Fig 2). CD3' cells, when plated directly on LTC but not on HS23, induced some L - 6 and G-CSF secretion. However, because the CD3+ cells only functioned in direct contact with primary cultures, it is likely that their effect is mediated through macrophages present in the LTC. Separating CD14+ cells from stroma by 0.4-pm porous policarbonate mem- branes did not reduce their ability to stimulate L - 6 and G- CSF production by stroma. Stromal cells or G-PBMCs alone generated only relatively low levels of L - 6 and G-CSF. IL-la and IL-lP released by CDJ4' cells are humoral

mediators of IL-6 and G-CSF release by marrow stroma. To determine whether L - 6 and G-CSF are secreted by stroma or CD14+ cells, conditioned medium generated from sorted CD14+ cells was harvested after 4 days and added to the stromal line HS23. Conditioned media from 1 X lo5




CD14' CELLS IN MOBILIZED PERIPHERAL BLOOD 577

5 Fig 2. Differential induction and contact require-

C

ments for G-PBMC stimulation of IL-6 and G-CSF se- E cretion by stroma. Unfractionated G-PBMCs or sorted populations (CD3'. CD20+, and CD14+ cells) .g were plated directly on stroma or separated by a 0.4- p m porous polycarbonate membrane (+membrane). The experiments were performed with primary stroma (LTC) or HS23 as indicated. CM was harvested on day 4 and assayed for IL-6 ( W ) or G-CSF (B). Shown is one of three experiments with each determination performed in duplicates and (a) through (f) indicating cell populations added to the cultures: (a) 5 x l o 5 unsorted G-PBMCs, (b) 1.5 x l o 5 CD3' cells, (c) 1 x lo5 CD14+ cells, (dl 3.8 x 104CD20+ cells, (e) 5 x l o 5 unsorted G-PBMCs without stroma, and (f) stroma only.

11-6 (ng/rnl), 48 hrs

0 100 200 300

LTC

-1

a b c d e f

LTC + membrane '0 m,

a b c d e f

HS 23

yal

HS 23 + membrane

400 I I L

control F

anti-IL-l a+p 1 I . , . , . , . , . , . , . , 0 10 20 30 40 50 60 70

G-CSF (nglrnl), 48 hrs

Fig 3. Neutralizing antibodies to IL- la and IL-1p used in combination inhibit IL-6 and G-CSF induction by CD14 conditioned medium (CDlCCM). HS23 stromal cells were cultured alone (control) or in the presence of CD14-CM. The addition of neutralizing antibody to IL- lo (anti-IL-la) at 3.3 pg/rnL or antibody t o IL-10 (anti-IL-lp) at 33 pgl mL had no effect on IL-6 (W) and G-CSF (0) production. However, the addition of both antibodies effectively prevented IL-6 and G-CSF secretion. The CD14-CM added t o the cultures contained only 3.9 ng l mL IL-6 and 0.44 ng/mL G-CSF.

CD14+ cells obtained from G-PBMCs induced secretion of IL-6 (260.5 2 14.3 ng/mL [mean t SEMI) and G-CSF (51.8

9.2 ng/mL). HS23 cultured alone had 4.4 t 0.26 ng/mL L - 6 and 0.09 5 0.05 ng/mL G-CSF. Conditioned media from LTC or HS23 did not induce significant IL-6 or G- CSF production from G-PBMCs (results not shown). The data suggest that the marrow stromal cells were induced to secrete IL-6 and G-CSF by a humoral factor released by CD14+ cells.

ELISA analysis of conditioned media from CD14+ cells indicated that IL-la and IL-1p were present at 569.5 5 98.4 and 2,128.8 t 425.6 pg/mL, respectively. Furthermore, the addition of either IL-la or IL-lp (1 ng/mL) to LTCs or HS23 induced L - 6 and G-CSF secretion (data not shown). Therefore, it was reasonable to test if neutralizing antibodies to IL-la, IL-lp, or both were capable of inhibiting IL-6 and G-CSF production by marrow stroma induced with CD14- CM (Fig 3). The combination of neutralizing antibodies to IL-la and L- lp almost completely inhibited L 6 and G- CSF secretion by HS23 stimulated with CD14-CM. Neu- tralizing antibodies to only one of those two cytokines were not sufficient to inhibit IL-6 and G-CSF secretion, indicating that each cytokine may account for the effect.

CD14+ cells obtained from marrow and G-CSF-mobi- lizedperipheral blood harvests have equal abilities to induce IL-6 release by marrow stroma. To investigate if CD14' cells from G-PBMCs and marrow have different abilities to induce IL-6 release in marrow stroma, CD14+ cells were purified from both sources by cell sorting and plated in equal numbers on LTC or HS23. The sort windows chosen for G- PBMCs and marrow are shown in Fig 1. The first gate was set based on the typical forward and side light scattering properties of monocytes. The second gate was based on relatively high expression of the CD14 antigen.





500 1

300 -

200 -

100 -

HS23

0 4 . , . , , , . \ ~

50 25 10 Stroma only

LTC 400

I

300 -

200 -

G-PBMC m a w

l o l l

only

Number of CD14+ cells (x 103)

Fig 4. The ability of equal numbers of CD14' cells obtained from G-PEMCs and marrow to induce IL-6 secretion by marrow stroma. Equal numbers of CD14+ cells (50 x IO', 25 x lo3, and 10 x IO') from the two sources were plated on the marrow stromal line HS23 or LTCs as indicated. Supernatants were harvested on day 4 and assayed for IL-6. Values are means and SEM in one of three representa- tive experiments.

As shown in Fig 4, IL-6 levels in CMs were correlated to the number CD14' cells obtained from either G-PBMCs or marrow and there was no significant difference in their potential to induce the IL-6 response.

Quantitative differences in CD14+ cells between standard marrow and peripheral blood harvests. The percentage of CD14' cells in fresh unselected samples of G-PBMCs from healthy donors was 26.1% ? 2.3% (n = 8). After CD34 selection using the CEPRATE (Cell Pro Inc, Bothell, WA),2'' the proportion of CD14' cells is reduced to 4.0% ? 1.1 % (n = 14). This is comparable to both the 4.0% t 0.9% seen in Ficoll separated marrow (n = 7) and the 2.55% ? 0.83% seen in marrow buffy coat preparations (n = 4).

DISCUSSION

G-PBMCs have been widely used for autologous hernato- poietic reconstitution after myeloablative therapy. The rela- tive ease of harvest without the necessity of general anesthesia, the potential depletion of tumor cells, and the favorable

engraftment patterns seen after G-CSF mobilization have made G-PBMCs an attractive alternative to autologous marrow. The use of G-PBMCs in allogeneic transplantation has followed a slower pace due to concerns regarding G-CSF treatment of healthy donors, insufficient data on long-term engraftment potential, and the possibility of more severe GVHD. However, recent reports suggest that acute GVHD incidence and severity are not increased and that engraftment is stable for more than 1 ear.'.^.^' In addition, platelet recovery in patients receiving allogeneic G-PBMCs is as rapid as that reported for autologous patient^.^.^

The rapid platelet recovery seen in both the autologous and allogeneic setting i s unlikely to be entirely explained by the greater number of CD34' cells in G-PBMCs compared with marrow. Recent studies have shown that, after reaching a threshold of about 5 X IO6 cellslkg, there is no further acceleration in engraftment kinetics,'" suggesting other factors besides increased numbers of CD34' cells may contrib- ute to rapid platelet reconstitution. One alternative hypothesis is that factors extrinsic to the CD34' cells, including a greater number or functionally altered accessory cells may facilitate engraftment.

To investigate mechanisms not related to differences in- trinsic to the CD34+ cells, we initiated studies to compare the effects of equal numbers of marrow cells and G-PBMCs on stromal function. Our data indicated that significantly higher levels of IL-6 and G-CSF could be detected when G- PBMCs were plated on stroma compared with marrow. This finding was consistent in both allogeneic and autologous systems, showing that the effect was not major histocompat- ability complex (MHC) dependent (data not shown). We showed that IL-6 and G-CSF were released by marrow stromal cells and that signalling was mediated by IL-la and IL- lp secreted by CD14' cells. Neutralizing antibodies to IL-la and IL-10, when used in combination, could almost completely eliminate the response, whereas neutralizing antibodies to either IL-la or IL-1p used alone did not have significant inhibitory effects. This indicates that both cytokines were independent stimuli for IL-6 and G-CSF secretion.

IL-6 is a pleiotropic cytokine that has been shown to enhance maturation and polyploidization of megakaryocytes in vitro.22 This leads to accelerated reconstitution of platelets after chemotherapy or radiation in dogs:' primates?',24 and

We, therefore, hypothesize that high levels of IL-6 produced and presented in the hematopoietic microenvironment after G-PBMC transplantation could accelerate platelet recovery. It has been reported earlier that the percentage of monocytes in leukapheresis products after intensive chemotherapy with or without G-CSF administration can reach up to 50% and that monocytes in the recovery phase after chemotherapy produce significantly more IL-6 and IL-lp in vitro than do steady-state peripheral blood mono- cy te~ .~ ' However, we detected IL-6 concentrations in supernatants from CD14' cells cultured with stroma 10- to 100- fold higher than supernatants from CD14' cells cultured alone and showed that marrow stromal cells rather than the monocytes were the major source for IL-6.

Liu et a128 reported a correlation between the absolute




CD14’ CELLS IN MOBILIZED PERIPHERAL BLOOD 579

number of monocytes and IL-6 serum levels in patients being mobilized with chemotherapy and G-CSF for autologous transplantation. However, no significant correlation was found between serum levels of IL-6 and the time course of hematopoietic recovery.29 Several other groups have moni- tored IL-6 serum levels after marrow transplantation. Ho et a13’ reported a peak in IL-6 serum levels (21.1 ? 4.0 pg/ mL) within the first 7 days after autologous transplantation of granulocyte-macrophage colony-stimulating factor (GM- CSF)-mobilized peripheral blood mononuclear cells. A direct comparison to patients receiving marrow was not provided in this study, but Schwaighofer et a13’ reported IL-6 serum levels peaking by day 11 after allogeneic bone marrow transplantation. In several studies a correlation between ele- vated IL-6 serum levels and transplant-related infectious complication^^"^^ or GVHD34 was observed. A direct comparison of IL-6 serum levels between patients undergoing G-PBMC and marrow transplantation would be of interest. However, we propose that cytokines such as IL-6 that are secreted and presented by stromal cells may have more rele- vant concentrations in the hematopoietic microenvironment than in serum.

Interestingly, CD34-selected G-PBMCs seem to retain the early platelet engraftment ~apab i l i t y~*-~~ of unselected G- PBMCs despite a greater than 2 log depletion for CD14’ cells. Based on our data, the total number of CD14’ cells in a CD34-selected product is lower than in a typical unselected marrow graft. Therefore, if CD14+ cells do play a role in facilitating early hematopoietic reconstitution, we can propose at least two hypothesis. First, it is possible that G- PBMCs contain more monocyte precursors. This would make it possible to generate a larger number of CD14+ cells quickly, providing G-PBMCs with a quantitative advantage in accessory cell function. This theory is supported in part by the observation that a much higher percentage of CD34 cells in G-PBMCs coexpress the differentiation markers CD33, CD13, and CD1 IC than do CD34 cells in m a r r ~ w . ~ * . ~ ~ Because CD14 is expressed on relatively late stages of monocyte differentiation,40 it would not be useful for identifying the premonocyte population.

A second hypothesis is that CD14+ cells in G-PBMCs may be qualitatively different from those in marrow. Although we did not detect differences in their ability to induce IL-6 secretion by marrow stroma, it is important to consider that our experimental design does not control for possible functional differences that may exist in regard to their in vivo ability to home to the marrow. Preliminary results did not show any significant differences in the expression of adhesion molecules L-Selectin, VLA-4, LFA-1, Mac-l, or PECAM-1 (results not shown) between the two populations. However, it is well accepted that integrins such as VLA-4 or Mac- l can be activated and that the overall level of surface expression does not change with their functional s ta t~s .4’ .~~ Whether functional differences do exist between these two sources of CD14’ cells remains unclear. Whether such differences, if they do exist, may impact on homing remains to be determined.

In summary, we present evidence that CD14+ cells induce IL-6 and G-CSF secretion by stroma. If this phenomenon

occurs in vivo, it is reasonable to hypothesize that relatively high concentrations of IL-6 and G-CSF produced in the marrow microenvironment could play a critical role in hematopoietic recovery. It is possible that the expression of other cytokines, in addition to IL-6 and G-CSF, may also be modu- lated by accessory cells. Clearly, a better understanding of accessory cell-stromal cell interactions may lead to the design of more efficacious strategies for controlling hematopoietic reconstitution.

ACKNOWLEDGMENT

We thank Ludmila Golubev for her technical assistance and Har- riet Childs and Paul Mullin for typing the manuscript.

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1996 87: 574-580

M Mielcarek, BA Roecklein and B Torok-Storb secretion of interleukin-6 and G-CSF by marrow stroma(G-CSF)-mobilized peripheral blood mononuclear cells induce CD14+ cells in granulocyte colony-stimulating factor

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cd14' cells in granulocyte colony-stimulating factor (g-csf

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