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Originally published online as doi:10.1189/jlb.0605297 on August 8, 2006

Published online before print August 8, 2006
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(Journal of Leukocyte Biology. 2006;80:862-869.)
© 2006 by Society for Leukocyte Biology

Immature monocytes from G-CSF-mobilized peripheral blood stem cell collections carry surface-bound IL-10 and have the potential to modulate alloreactivity

A. R. Fraser*,1, G. Cook*,2, I. M. Franklin*,{dagger}, J. G. Templeton*, M. Campbell{dagger}, T. L. Holyoake*,{dagger} and J. D. M. Campbell*

* Academic Transfusion Medicine Unit, Section of Experimental Haematology, Division of Cancer Sciences and Molecular Pathology, University of Glasgow, Glasgow, United Kingdom; and
{dagger} Bone Marrow Transplant Unit, Glasgow Royal Infirmary, Glasgow, United Kingdom

1 Correspondence: Division of Immunity, Infection and Inflammation, Glasgow Biomedical Research Centre, 120 University Avenue, Glasgow G12 8TA, UK. E-mail: af82y{at}clinmed.gla.ac.uk


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ABSTRACT
 
Production of the anti-inflammatory cytokine IL-10 by monocytes has been implicated as a probable negative regulator of graft-versus-host disease (GvHD) in patients undergoing allogeneic stem cell transplants (SCT). Monocytes from G-CSF-mobilized peripheral blood stem cell (gmPBSC) collections have been reported to produce more IL-10 than unmobilized monocytes in response to proinflammatory factors such as LPS. Why this should occur is unclear. In this study, monocyte phenotype and IL-10 localization and release were investigated in PB mononuclear cells (MNC) from 27 healthy donors mobilized for allogeneic SCT and from 13 patients with hematological malignancies mobilized for autologous SCT. All isolates contained elevated total percentages of monocytes in comparison with unmobilized PB, a high proportion of which displayed an immature phenotype. Stimulation of gmPB MNC with an inflammatory stimulus [fixed Staphylococcus aureus cells (SAC)] induced rapid up-regulation of CD14, indicating conversion to mature status. Localization studies indicated that IL-10 was predominantly present, bound on the surface of CD64+/CD14low/neg immature monocytes. Inflammatory stimuli (LPS, polyinosinic:polycytidylic acid, or SAC) induced release of variable quantities of IL-10 from the cell surface. MNC, separated into surface IL-10-positive or -negative fractions, differed in their ability to stimulate alloreactivity in MLR, and IL-10+ MNC induced significantly lower levels of proliferation than IL-10– MNC. Thus, the subset of immature monocytes carrying surface-bound IL-10 in gmPB has the potential to modulate alloreactivity and GvHD after allogeneic SCT through cell-to-cell contact and released IL-10.

Key Words: GvHD • transplantation


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INTRODUCTION
 
Allogeneic stem cell transplantation (SCT) is currently the only therapy that offers the potential of a cure for hematological malignancies or bone marrow (BM) failure. Stem cells can be collected from BM harvested from HLA-matched donors. An alternative is to use stem cells isolated from donor peripheral blood (PB). G-CSF is used to mobilize stem cells from the BM into the periphery, and these G-CSF-mobilized PB stem cell (gmPBSC) collections from matched donors are now increasingly used to reconstitute BM function [1 2 3 ] with improved engraftment kinetics over standard BM transplant [4 , 5 ].

Initial concerns regarding a possible increase in the incidence of acute graft-versus-host disease (aGvHD) with the use of gmPB SCT have not been realized, despite a 2–3 log increase in the number of CD3+ cells present in gmPB SCT when compared with BM harvests [6 , 7 ]. However, a number of studies have indicated a comparative increase in the incidence and severity of chronic GvHD (cGvHD) associated with the use of gmPB SCT [8 , 9 ]. This possible suppression of aGvHD, followed by a "bounce-back" increase in cGvHD, has led to the suggestion that factors present at the time of transplant may suppress initial GvHD, but their effects decrease over time [10 ].

A standard gmPB SCT collection contains large numbers of monocytes, as a result of the direct effects of G-CSF (through differentiation of precursors) and enrichment during the leukapheresis procedure, which together, can produce up to 50 times more monocytes than would be collected during a typical BM harvest [11 ]. Monocytes isolated from gmPB have been shown to suppress alloreactivity and proliferation of activated T cells [12 ]. This monocyte-induced in vitro suppression has been attributed to increased IL-10 production [13 ].

Monocytes from gmPB are likely to encounter elevated levels of proinflammatory stimuli in the recipient as a result of release of microbial products, as a consequence of tissue damage to the digestive tract from conditioning therapy prior to transplant. These pathogen-derived molecules are potent activators of monocytes and other APC and are likely to have a profound influence on monocyte maturity, phenotype, and cytokine release. Monocytes from gmPB have been shown to require lower levels of stimulation with proinflammatory factors such as LPS to produce IL-10 rather than nonmobilized monocytes [13 , 14 ]. There is some evidence that gmPB monocytes may "spontaneously" produce IL-10, although the mechanisms underlying this observation are unclear [15 ]. Thus, the relatively high proportion of monocytes primed to make IL-10 may represent a significant population in the SCT, which could influence early induction of GvHD. Understanding the mechanisms governing IL-10 effects in this context is crucial to develop strategies to control GvHD.

This study was designed to elucidate the mechanism underlying the preferential production of IL-10 by monocytes from gmPB. We examined gmPB from healthy donors (for allogeneic transplant) and from patients with hematological malignancies (for autologous transplant). Relative percentages of monocytes were assessed in each group and compared against numbers from steady-state (nonmobilized) PB and phenotypes analyzed using flow cytometry. Monocytes were categorized into mature and immature populations and examined for surface-bound IL-10. The effect of proinflammatory factors on IL-10 release was investigated using flow cytometry and quantified by ELISA. Monocyte maturity and presence of surface-bound IL-10 were found to correlate with inhibitory effect in MLR. The data obtained represent a novel mechanism of IL-10 regulation, which may play a role in the modulation of GvHD.


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MATERIALS AND METHODS
 
Donors and G-CSF mobilization of PBSC
Samples of gmPB from 27 normal, healthy donors and 13 patients with hematological malignancies [multiple myeloma (n=5), non-Hodgkin’s lymphoma (n=5), Hodgkin’s lymphoma (n=2), and chronic myeloid leukaemia (n=1)] were collected following the Local Research Ethics Committee project approval and informed consent. Donors received recombinant human G-CSF (Lenograstim®, Chugai Pharma, UK) at 10 µg/kg/day, starting 4 days prior to leukapheresis, which was performed on Days 5 and 6 post-G-CSF commencement, using a continuous flow cell separator (Cobe, UK) processing 10–15 L PB. After collection, mononuclear cells (MNC) were isolated over ficoll (Lymphoprep, Nycomed, Norway), cryopreserved in 10% DMSO/90% human AB serum, and stored in liquid nitrogen until required. Control, nonmobilized (steady-state) PB (ssPB) was collected from normal, healthy volunteers as above. Magnetic cell sorting of PB MNC into IL-10-positive and -negative fractions was done using fresh samples after removal of granulocytes by ficoll separation.

Reagents and antibodies
Fluorochrome-conjugated mAb against human CD3, CD14, CD19, CD54, CD58, CD64, CD80, CD86, HLA-DR, IL-10, IL-12, and suitable isotype controls plus the intracellular cytokine staining kit were obtained from PharMingen/Becton Dickinson Ltd. (Oxford, UK). Mitomycin C, LPS, polyinosinic:polycytidylic acid [poly (I:C)], and DNase I were obtained from Sigma (Poole, UK). "Pansorbin"-fixed Staphylococcus aureus cells (SAC) were supplied by Calbiochem (Nottingham, UK). Anti-PE and CD14 magnetic microbeads and separation columns (LS) were supplied by Miltenyi Biotec (Surrey, UK).

Cell preparation
Cryopreserved samples were recovered from liquid nitrogen based on a method described previously [16 ]. Briefly, cells were thawed and resuspended in 400 Kunitz units DNase I in PBS and incubated for 5 min at 37°C. The cells were then added slowly into 10 ml warm RPMI-1640 medium (Sigma) and pelleted at 200 g for 5 min. The pellet was resuspended in further 400 Kunitz units DNase I for 5 min at 37°C, and the cells were then washed three times to remove residual DMSO and DNase. Dead cells and residual granulocytes were then removed, and MNC were isolated by centrifugation over ficoll, followed by three washes in warm RPMI-1640. Fresh PB MNC were purified over ficoll, red cell-lysed using 0.83% ammonium chloride, and washed as before.

Analysis of IL-10 production
The anti-inflammatory cytokine IL-10 was detected on ssPB and gmPB MNC using an intracellular cytokine staining kit (PharMingen/Becton Dickinson Ltd.). Briefly, cells were cultured overnight with or without stimulation (1 µg/ml SAC) plus "Golgi-Stop" (monensin) to inhibit secretion of cytokines, then permeabilized using kit reagents as per instructions, and probed with IL-10-PE antibody. The cells were compared against isotype control by flow cytometry. Monocyte-specific IL-10 was quantified by gating on CD64+ cells. Localization of IL-10 was identified by surface versus intracellular labeling. Briefly, resting, unstimulated gmPB MNC were incubated with anti-IL-10-PE antibody, with or without cell permeabilization (as per the PharMingen kit) and analyzed against isotype controls by flow cytometry. Again, gating ensured only CD64+ monocytes were assessed for IL-10. Monocytes were also labeled with anti-IL-12-PE antibody to confirm that cytokine-specific binding occurred.

Inflammatory stimulation of MNC
The effects of proinflammatory stimulators on MNC were assessed using flow cytometry and quantitative ELISA (see below). MNC from gmPB and ssPB (1x107/well) were cultured in complete medium (CM; RPMI 1640 plus 5% human AB serum plus 1% penicillin/streptomycin) and challenged with proinflammatory stimulators overnight [1 µl/ml SAC or 12.5 ng/ml poly (I:C) or 1 µg/ml LPS]. Cells were then collected, washed, and labeled for flow cytometry (see below). Culture supernatants were stored frozen at –70°C until use and tested in a quantitative IL-10 capture ELISA (Biosource, UK) as per kit instructions. Specific IL-10 release was quantified against standard kit controls.

Flow cytometry analysis
MNC (5x105/sample) were stained by direct immunofluorescence for multiparameter flow cytometry versus isotype-matched controls. Cells were visualized on a FACSCaliber flow cytometer (PharMingen/Becton Dickinson Ltd.) and analyzed using "Cell Quest" software. For each sample, 10,000 events were analyzed.

Cell sorting
Populations of cells enriched for mature or immature monocytes were isolated by sorting using CD14 microbeads and LS magnetic separation columns, according to the manufacturer’s instructions (Miltenyi Biotec). This produced isolates containing predominantly CD64+/CD14high or CD64+/CD14low/neg cells. Monocytes carrying surface-bound IL-10 were separated by staining the CD14low/neg fraction with anti-IL-10-PE, followed by anti-PE microbeads and then isolated using LS columns as per the manufacturer’s instructions (Miltenyi Biotec).

MLR
MLR were performed using freshly prepared ssPB MNC as responders at 106 cells/ml in CM. Stimulator MNC were preincubated with 25 µg/ml mitomycin C (Sigma) for 30 min at 37°C to inhibit proliferation and then washed three times with CM. The CD14high, CD14low/neg/IL-10–, and CD14low/neg/IL-10+ MNC fractions were used as stimulators. The MLR assays were performed at a ratio of 1:1 responders:stimulators, incubated for 5 days in 5% CO2 in air at 37°C. Proliferation was assessed by the addition of 0.5 µCi tritiated thymidine (Amersham Biosciences, UK) per well, 18 h prior to analysis. Incorporation of radiolabel was quantified using a Packard Matrix 96 counter.

Statistical analysis
Data were summarized by means plus standard deviation. Statistical comparisons were performed using Student’s t-test for parametric data and the Mann Whitney U-test for comparison of nonparametric data. Statistical significance was set at P < 0.05 with a 95% confidence interval.


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RESULTS
 
Monocyte phenotype in gmPB and ssPB
The MNC from ssPB contained a mean of 15.7 ± 2.0% CD64+ cells and a mean fluorescence intensity (MFI) for CD14 of 883 ± 123 (Fig. 1A ). In contrast, gmPB MNC contained a much greater percentage of monocytes. Healthy donor gmPB MNC contained a mean of 55.2 ± 4.0% CD64+ cells, and malignancy patient gmPB MNC contained 72.1 ± 4.0% CD64+ cells (Fig. 1A) . Mean CD14 expression by CD64+ cells in healthy donor (MFI 275±60) or malignancy patient gmPB (MFI 327±34) was significantly lower than in the unmobilized ssPB (P<0.001; Fig. 1A ).


Figure 1
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  		Figure 1. CD14 expression by monocytes from ssPB and gmPB. (A) The mean percentage of CD64+ monocytes (bars) and MFI of CD14 expression (line) by CD64+ monocytes from ssPB samples (n=12), healthy donor (HD) gmPB (n=27), and malignancy patient (M) gmPB (n=13) cells. The CD14 MFI was significantly lower in HD and M gmPB monocytes than in ssPB (P<0.001). (B) Comparative flow cytometric analysis of monocytes from representative ssPB and healthy donor gmPB MNC, demonstrating the presence of two distinct populations of monocytes in gmPB (CD64+/CD14high and CD64+/CD14low/neg). (C) Differential phenotype of monocyte populations in gmPB taken from a representative patient, CD64+/CD14high [Region 1 (R1)] and CD64+/CD14low/neg (Region 2), shown with percentage cells in each quadrant. Both monocyte populations were CD54+ (>95%), but CD14high cells were CD58high (>99%), HLA-DR+, and CD86low/neg, and CD14low/neg cells were CD58low/neg and negative for HLA-DR and CD86 (<3% positive). Neither population stained for CD80. Thus, CD14high cells had a mature monocyte phenotype, and the CD14low/neg cells had a more immature phenotype. SSC, Side-scatter; FSC, forward-scatter.

However, MNC from gmPB demonstrated heterogeneity in CD14 expression of CD64+ cells and a mixture of CD14high cells and CD14low/neg cells (Fig. 1B) . CD14high cells expressed surface molecules characteristic of mature monocytes, HLA-DR, CD86, CD54, and CD58 (Region 1, Fig. 1C ). The CD14low/neg cells expressed lower levels of CD54 than CD14high cells and were low or negative for CD86 or HLA-DR markers (Region 2, Fig. 1C ). This suggests that the CD14low/neg monocytes (the majority of monocytes in gmPB isolates) express an immature phenotype in comparison with monocytes from ssPB.

Monocyte responses to inflammatory stimuli
Transplantation-conditioning regimes induce a proinflammatory environment with the generation of cytokines and release large quantities of bacterial products into the circulation following damage to the gastrointestinal blood barrier. To mimic this in vitro, gmPB (n=6) and ssPB (n=6) MNC were stimulated with proinflammatory molecules, LPS (bacterial, signaling through CD14 and TLR4), SAC (bacterial, signaling though multiple TLR but not CD14-dependent), and poly (I:C) (synthetic viral dsRNA, signaling through TLR3) [17 , 18 ]. Changes in CD14 expression were assessed by flow cytometry. Stimulation of ssPB monocytes with SAC did not produce a significant change in expression of CD14. However, SAC stimulation of normal donor or malignancy patient gmPB MNC resulted in a significant up-regulation in surface expression of CD14 on CD64+ cells compared with unstimulated gmPB (P<0.0001; Fig. 2 ). LPS and poly (I:C) also increased CD14 expression on CD64+ cells (data not shown).


Figure 2
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  		Figure 2. CD14 modulation in response to SAC stimulation. Comparative expression of CD14 in monocytes from cohorts of ssPB or gmPB isolates (n=6 each group), prior to and after overnight stimulation with SAC. Monocytes from ssPB demonstrated a minor reduction in mean CD14 MFI after stimulation, whereas monocytes from gmPB significantly up-regulated CD14 expression after culture with SAC (*, P<0.0001).

IL-10 production by monocytes
IL-10 expression and localization in the ssPB and gmPB monocyte populations were investigated using flow cytometry. As expected, monocytes from ssPB isolates demonstrated low, intracellular IL-10 staining, and only 7.3 ± 1.9% of CD64+ MNC stained positively after 24 h culture without stimulus. The percentage of IL-10-positive cells increased significantly to 37.2 ± 10.3% after overnight stimulation with SAC (P=0.02; Fig. 3A ). Nonpermeabilized ssPB cells did not stain for IL-10 or IL-12, indicating no surface-bound cytokine.


Figure 3
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  		Figure 3. IL-10 expression and localization by monocytes. (A) ssPB (n=12) and gmPB (combined healthy donor and malignancy isolates, n=22) MNC cultured in vitro overnight with medium only (unstim) or with SAC stimulation, permeabilized and labeled with anti-IL-10 PE antibody. Stimulation of ssPB with SAC overnight significantly increased the number of CD64+ monocytes expressing IL-10 (*, P=0.02), whereas gmPB monocytes demonstrated high initial levels of IL-10, which dropped slightly after SAC stimulation. (B) Unstimulated gmPB CD64+ cells from 27 healthy donors and 13 malignancy patients assayed for surface or intracellular staining for IL-10. Approximately 20% of unpermeabilized (surface-bound) and 25% of permeabilized (total) CD64+ cells stained IL-10-positive, confirming that the vast majority of IL-10 detected was localized on the cell surface.

In contrast, monocytes from gmPB isolates demonstrated a different pattern of IL-10 expression, and 32.7 ± 5.5% of unstimulated CD64+ monocytes were taken from healthy donor and malignancy gmPB MNC, staining positively for intracellular and surface IL-10. After SAC stimulation, the proportion of IL-10+ cells in gmPB MNC dropped to 27.8 ± 5.8%. FACS analysis of permeabilized versus nonpermeabilized gmPB monocytes labeled with anti-IL-10 antibody demonstrated that ~80% of the IL-10 present was surface-bound rather than intracellular (19.9% IL-10+ by surface staining and 24.5% IL-10+ for intracellular plus surface staining; Fig. 3B ). No surface-bound IL-12 was detected, indicating that the IL-10 results were not a phenomenon of nonspecific, anticytokine antibody binding.

Monocytes from gmPB MNC were separated based on CD14 and surface IL-10 expression. The sorted cells were then assessed by flow cytometry, and the vast majority of IL-10+ monocytes demonstrated an immature CD14low/CD64+ phenotype (Fig. 4B ).


Figure 4
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  		Figure 4. Determination of maturation status of IL-10+ monocytes, which were isolated by sequential fractionation using magnetic bead sorting for CD14 and IL-10. (A) FACS plots of a representative gmPB sample. Monocytes were isolated from gmPB MNC using MACS CD14 microbeads, producing highly enriched CD64+/CD14high and CD64+/CD14low/neg populations with purities >95%. The CD64+/CD14low/neg population was then stained with anti-IL-10-PE antibody and sorted into IL-10-positive and -negative fractions using MACS anti-PE beads. The IL-10+ fraction was highly enriched for cells bearing surface IL-10 (>91%). (B) To confirm that surface IL-10 staining was largely confined to CD64+/CD14low/neg monocytes, gmPB MNC were sorted into IL-10-positive and -negative fractions as above but without prior CD14 fractionation. Cells from the IL-10+ fraction stained for expression of CD14 and CD64 as shown by histogram. Solid histogram, Isotype control; lines indicate CD64 or CD14 staining. Isolated IL-10+ cells expressed the characteristic CD64+/CD14low/neg phenotype of immature monocytes.

To investigate whether the surface-bound IL-10 could be released from monocytes, healthy donor (n=13) and malignancy patient (n=12) gmPB MNC were cultured overnight without stimulation or with LPS, SAC, or poly (I:C). The cells were assayed by flow cytometry before and after stimulation (Fig. 5 ), and IL-10 released into culture supernatants was quantified using a capture ELISA (Fig. 6 ). MNC not exposed to inflammatory stimuli overnight retained surface IL-10 and no corresponding IL-10 release into the supernatant (5.71±10.9 pg/ml). In contrast, SAC-, LPS-, or poly (I:C)-stimulated gmPB monocytes became largely IL-10-negative on a flow cytometric plot (Fig. 5 , SAC data only), and significant quantities of IL-10 were released into culture supernatants (Fig. 6) . Increases in supernatant IL-10 levels were detected after all stimuli [SAC: 410.1±1024.1 pg/ml, P=0.033; LPS: 232.5±563.8 pg/ml, P=0.030; poly (I:C): 127.0±287.8 pg/ml, P=0.024]. Variation in IL-10 release was observed between individual isolates in response to inflammatory stimuli, which may reflect heterogeneous receptor distribution or an inherent variation in IL-10 production and localization.


Figure 5
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  		Figure 5. Change in IL-10 expression on gmPB MNC after stimulation. Flow cytometry plots of gmPB MNC taken from a representative healthy donor at collection and after stimulation with SAC overnight. FACS analysis confirmed that unstimulated CD64+ monocytes stained strongly for surface IL-10, which was largely removed after overnight stimulation with SAC.


Figure 6
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  		Figure 6. ELISA quantification of IL-10 released after stimulation. Supernatants from overnight cultures of gmPB MNC from 12 healthy donor and 12 malignancy patient isolates, with or without inflammatory stimuli, were tested by quantitative capture ELISA. Unstimulated cells showed little or no IL-10 release. SAC treatment induced greatest mean IL-10 release from gmPB MNC, although a degree of variation was observed. All treatments resulted in significantly increased IL-10 release in comparison with unstimulated controls [LPS, P=0.030; SAC, P=0.033; poly (I:C), P=0.024]. Graph shows individual patients ({diamondsuit}) and mean IL-10 release for each treatment (–).

Influence of gmPB monocytes on alloreactivity in vitro
MLR studies were conducted to test whether the monocyte populations identified here had different immunomodulatory capacities. Monocytes were isolated from gmPB MNC using CD14 beads and then further separated on the basis of surface IL-10 expression using anti-IL-10-PE- and anti-PE-labeled magnetic beads. The sorted cells were then assessed by flow cytometry, and the vast majority of IL-10+ cells showed a CD14low/CD64+ immature monocyte phenotype (Fig. 4B) . The CD14high, CD14low/neg/IL-10–, and CD14low/neg/IL-10+ fractions isolated were used as stimulators in a one-way MLR (five gmPB MNC vs. five ssPB MNC). The CD14high monocytes were found to be allostimulatory, as expected, but there was a marked difference in the ability of the IL-10+ and IL-10– populations to induce alloreactivity (Fig. 7 ). The CD14low/neg/IL-10+ cells induced far less proliferation than the CD14high cells and significantly lower proliferation than the CD14low/neg/IL-10– cells (P=0.02). Thus, the IL-10+ monocytes were the poorest allostimulators, and the IL-10– monocytes were highly immunostimulatory. This confirms a biological significance for the surface-bound IL-10 carried by the immature monocytes.


Figure 7
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  		Figure 7. Effect of monocyte maturity and surface IL-10 load on MLR, and MLR data showed proliferation of ssPB MNC in response to stimulus with monocytes isolated from gmPB MNC, expressed as a stimulation index (induced proliferation over appropriate unstimulated control). Although CD14high monocytes induced strong proliferation, CD14low/neg/IL-10– proved the most allostimulatory. In contrast, CD14low/neg/IL-10+ cells were poorly allostimulatory and resulted in significantly lower proliferation than CD14low/neg/IL-10– cells (*, P<0.02).


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DISCUSSION
 
GvHD continues to be a major complication of allogeneic gmPB SCT, despite the administration of potent, post-graft immunosuppression [19 ]. However, aGvHD is less common with gmPB SCT than conventional BM transplant. Evidence has shown that MNC from normal individuals who receive G-CSF can suppress the activation of T cells to alloantigen and CD3 stimulation through the action of IL-10 [20 ]. This has been suggested as a possible explanation for the lack of increased aGvHD incidence after gmPB SCT, despite a huge increase in T cells administered. Several groups have suggested that the increased monocyte number in stem cell grafts may influence this outcome, and the results presented here strengthen this argument.

In this study, the phenotype of monocytes isolated from gmPB was investigated, and it was shown for the first time that there are clear differences in monocyte quantity and maturation status between mobilized and nonmobilized PB MNC. It was demonstrated that monocyte phenotypes were altered in gmPB in comparison with ssPB, and CD64+ monocytes expressed significantly lower levels of CD14 on their surface. It has been shown previously that CD14low monocytes are present in normal PB and account for ~10% of the monocyte population [21 ]. In this study, a similar figure was observed, and less than 15% of ssPB expressed a CD64+/CD14low/neg phenotype. Higher percentages of CD14low/neg monocytes have been detected in the PB of patients with diseases, including malignancy and chronic infections [22 , 23 ]. In each case, the presence of this monocyte population was attributed to an increase in hematopoietic growth factor release in the circulation. However, all these studies reported a significantly lower frequency of CD14low/neg monocytes than was found in our study of allogeneic donors. The artificial situation of donor mobilization is likely to provide a more extreme induction of immature precursors, hence, the large numbers of CD64+/CD14low/neg/HLA-DRlow cells found here. This distinct population was confirmed as immature, as it demonstrated low expression of costimulatory molecules such as CD58 and CD86.

The reduced expression of CD14 on monocytes from gmPB observed in this study is also functionally important, as it correlates with immature status and indicates the ability of these monocytes to respond to LPS. The LPS receptor (CD14) is essential in the control of monocyte function, as it initiates many signaling cascades inside the cell [24 ]. As CD14 is not highly expressed on a large proportion of gmPB monocytes, they may respond poorly to LPS stimulation, and thus, LPS may not be the most effective factor for use in activation studies [25 ]. Recently, synergistic LPS signaling has been described through CD14 interaction with TLR4 [26 ]. Although relative levels of TLR4 were not specifically investigated on monocytes in this study, SAC was used for stimulation, as it does not rely on CD14 signaling for activation but functions by binding to multiple TLR [18 ].

Monocyte-derived cytokines and in particular, IL-10 have been implicated as key mediators of acute GvHD suppression after gmPB SCT [13 , 20 ]. Although monocytes from gmPB can suppress T cell alloreactivity in vitro, previous studies have not clearly elucidated the mechanisms of suppression of aGvHD. A previous study showed that gmPB monocytes contained higher levels of IL-10 mRNA transcripts than ssPB monocytes [13 ]. However, comparable levels of IL-10 secretion from ssPB and gmPB monocytes were observed in response to LPS stimulation. Therefore, increased transcription of IL-10 was not necessarily a factor in suppression of alloreactivity by gmPB monocytes. We have shown in this study that there are significantly increased numbers of monocytes in gmPB collections and that this major cell population carries IL-10 and has the potential to participate in controlling the inflammatory response.

In our study, flow cytometry of whole or permeabilized monocytes demonstrated that a large percentage of resting, immature gmPB monocytes carries IL-10 bound to their surface. After exposure to stimuli, there appears to be a rise in intracellular IL-10 but a concomitant loss of IL-10 from the surface, which resulted in a small net loss of total IL-10. As well as the potential to suppress proliferation of activated T cells, monocytes carrying surface-bound IL-10 have been shown to have significantly reduced bactericidal function and oxidative burst capacity [27 , 28 ]. Surface-bound or free IL-10 can inhibit IFN-{gamma}-induced NO generation, which suggests that it can act via cell-to-cell contact and specific receptor binding [29 ]. Both mechanisms are inhibitory through identical signaling pathways [30 , 31 ]. The IL-10 was stably bound on the monocyte surface and was not released during culture unless in response to proinflammatory factors. The variation in quantities of IL-10 released suggests that IL-10 may be bound to the surface through mechanisms other than attachment to its cognate receptor alone. IL-10 release was triggered by all inflammatory factors used here, regardless of receptor target [CD14/TLR4 for LPS, TLR3 for poly (I:C), or multiple targets including TLR2/4 for SAC], and coincided with the development of a mature CD14+ monocyte phenotype. This confirms that immature monocytes carrying IL-10 on the surface can act as a reservoir of IL-10 released in response to inflammatory triggers. The released IL-10 could then act to suppress immune responses in a variety of ways, including suppression of T cell responses and inhibition of dendritic cell function. By these mechanisms, gmPB monocytes carrying IL-10 could suppress aGvHD reactions in patients given SCT.

Direct action of surface-bound IL-10 upon immune reactions was demonstrated using MLR and confirmed that CD14low/neg monocytes carrying surface-bound IL-10 were poor allostimulators in comparison with CD14high monocytes or the comparable IL-10neg immature monocyte population. Thus, this novel CD64+/CD14low/neg/IL-10+ monocyte population has the ability to modulate alloreactivity in transplant patients and may provide the link among gmPB SCT, IL-10 production, and suppression of alloreactivity.

In this study, we have identified a novel mechanism for gmPB monocyte mediation of aGvHD suppression via surface-bound IL-10. Contact with proinflammatory agents induces mass release of this surface-bound IL-10. Therefore, large numbers of monocytes entering the inflammatory environment of the post-transplant conditioning patient have the potential to release a wave of anti-inflammatory IL-10. We also determined that IL-10+ monocytes are poorly allostimulatory compared with IL-10– monocytes, providing a direct correlation between surface IL-10+ cells and anti-inflammatory effects. Induction and maintenance of GvHD are complex, and additional factors such as donor and patient cytokine gene polymorphisms and the presence of regulatory CD4+/CD25+ cells may affect GvHD progression [32 , 33 ]. As monocytes are relatively long-lived within gmPB SCT [34 , 35 ], it is tempting to suggest that the CD64+/CD14low/neg/IL-10+ population identified in this study may augment or prolong a comparatively GvHD-free state in the transplant recipient, and as the monocyte numbers decline over the months, control over this state wanes, and cGvHD is the ultimate outcome for many patients. Certainly, a "burst" of IL-10 from the cells identified here is likely to have an anti-aGvHD effect, and further clinical studies are required to correlate the presence of these cells with GvHD outcome.


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ACKNOWLEDGEMENTS
 
Research was supported by the University of Glasgow and the Scottish National Blood Transfusion Service. T. L. H. was funded by the Leukaemia Research Fund, and A. R. F. was funded by an unrestricted research grant from Chugai-Pharma (UK).


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FOOTNOTES
 
2 Current address: Transplant Immunology Group, Academic Department of Haematology and Oncology, University of Leeds, LS7 9TF, UK. Back

Received June 3, 2005; revised May 2, 2006; accepted June 2, 2006.


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