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Originally published online as doi:10.1189/jlb.0806521 on April 5, 2007

Published online before print April 5, 2007
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(Journal of Leukocyte Biology. 2007;82:152-160.)
© 2007 by Society for Leukocyte Biology

Circulating CD14–CD36+ peripheral blood mononuclear cells constitutively produce interleukin-10

Lisa Barrett, Chunming Dai, Jane Gamberg, Maureen Gallant and Michael Grant1

Immunology Program, Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada

1 Correspondence: H1809-Immunology, Faculty of Medicine, Memorial University of Newfoundland, 300 Prince Philip Drive, St. John’s, Newfoundland, Canada, A1B 3V6. E-mail: mgrant{at}mun.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The impact of immune regulatory imbalance covers surprising physiological breadth. Although dominance of anti-inflammatory cytokines such as IL-10 is associated with reduced immune responsiveness and susceptibility to persistent infection, conditions such as cardiovascular disease and diabetes are linked to chronic inflammation and lower IL-10 levels. An appropriate threshold for immune activation is critical for optimal protection from infection and conversely, from short- and long-term side-effects of immune effector mechanisms. To assess the possibility that IL-10 plays a role in setting this threshold and that healthy maintenance of immune silence may involve low-level immune suppression, we sought out and characterized human peripheral blood cells constitutively producing the immunosuppressive cytokine IL-10. We determined the surface phenotype of circulating PBMC constitutively producing IL-10 by surface and intracellular flow cytometry and visualized their ultrastructure by electron microscopy. The frequency of IL-10-producing and -secreting cells was estimated by ELISPOT and flow cytometry. Up to 1% of PBMC constitutively produce IL-10. These CD14–CD36+CD61+ nonadherent cells expressed general markers of hematopoietic and progenitor cells (CD45 and CD7) but no stem cell, T cell, B cell, NK cell, monocytes or dendritic cell markers. Inflammation-associated TLRs were also absent. The IL-10-producing cells had prominent nuclei, multiple mitochondria, and abundant rough endoplasmic reticulum. Healthy individuals have PBMC constitutively producing IL-10. Although the lineage of these cells remains unclear, their properties and frequency suggest a potential role in homeostatic or innate immune suppression.

Key Words: immune regulation • hematopoietic cells • cytokines


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of innate and adaptive immunity is essential for combating the constant challenge of invasive viral, bacterial, parasitic, and fungal pathogens. Conversely, preventing inappropriate activation of immune responses and down-regulating appropriate immune responses, once pathogens are cleared or other antigenic stimuli dissipate, are equally essential for avoiding immunopathology. Pathogen clearance by multiple arms of the immune system passively down-regulates immune responses by decreasing antigenic stimuli, but when antigen persists, unresolved immune responses may be more damaging than the infection itself. In such situations or when immune responses arise and propagate in the absence of infection, active immunoregulation by cytokines, regulatory cells, or pharmacological intervention may be appropriate to prevent or limit pathology.

The immunoregulatory cytokine IL-10 plays an essential role in down-modulating adaptive and innate immune responses [1 ]. A variety of hematopoietic cells, including monocytes, mast cells, TH2, regulatory T cells (Tr), dendritic cells (DC), and B cells, produces IL-10, usually in response to particular stimuli [2 ]. IL-10 inhibits TH1 responses indirectly by blocking IL-12 production and MHC Class II up-regulation by APC [3 4 5 ]. IL-10 also suppresses inflammation by inhibiting production of IL-1, TNF, and a variety of chemokines by APC, T cells, and neutrophils [3 , 6 , 7 ]. The physiological relevance of the immunoregulatory effect of IL-10 has been demonstrated clearly in animal models of disease. Too little IL-10 renders mice more susceptible to chronic enterocolitis [8 ] but more resistant to intracellular pathogens such as Listeria and Chlamydia [9 , 10 ], and IL-10 overexpression reduces intravascular inflammation and delays development of atherosclerosis in low density lipoprotein (LDL) receptor-deficient mice [11 ]. In human studies, lower levels of IL-10 production are associated similarly with diseases thought to have an inflammatory component, such as atherosclerosis and diabetes [12 , 13 ]. Administration of exogenous IL-10 reduces symptoms in colitis and inflammatory bowel disease and can moderate disease severity [14 , 15 ]. However, elevated IL-10 levels are associated with chronic bacterial infection [16 17 18 ], decreased DC maturation [19 20 21 22 ], and less effective immune surveillance against tumors [23 ]. Thus, the broad range of IL-10-mediated immunoregulation constitutes a double-edged sword with the beneficial effect of limiting immunopathology, counterbalanced by increased susceptibility to infectious disease and cancer. Although the regulatory role of IL-10 in various pathological states has been well described, there has been little investigation of IL-10 as a potential homeostatic regulator of the immune response. In this light, we report on the frequency, phenotype, and ultrastructure of PBMC which constitutively produce and secrete IL-10 in healthy individuals. Constitutive production of IL-10 by a relatively large number of circulating cells in healthy individuals suggests that these cells could play a role in homeostatic immune regulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Acid citrate dextrose-preserved whole blood was collected by venipuncture from 12 apparently healthy volunteers (six males and six females, ages 26–55 years), recruited from Memorial University Faculty of Medicine personnel (Newfoundland, Canada). Informed consent was obtained from individuals for drawing blood, and ethical approval was obtained from the Memorial University Faculty of Medicine Human Investigation Committee. PBMC were isolated by Ficoll-HyPaque Plus (Amersham Biosciences, Baie d’Urfé, Quebec, Canada) density gradient centrifugation. Cells were washed and suspended at 1 x 106/mL in lymphocyte medium (RPMI supplemented with 10% FCS, 10 mM HEPES, 2 mM L-glutamine, 1% penicillin/streptomycin, and 20 µM 2-ME, all from Invitrogen, Burlington, Ontario, Canada).

Flow cytometry
Surface Staining
The phenotype of fresh PBMC subpopulations was determined by extracellular flow cytometry using antibodies against human myeloid and lymphoid lineage-specific surface markers, as listed in Table 1 . All steps were performed at 4°C, and incubations were performed in the dark to prevent fluorochrome photobleaching. Freshly isolated PBMC (5x105) were washed with cold PBS supplemented with 0.1% BSA (Sigma Chemical Co.), 5 mM EDTA (Sigma Chemical Co.), and 0.02% sodium azide (Sigma Chemical Co.), resuspended in a total volume of 600 µL, and incubated for 20 min with 0.5 µg specific antibody against surface antigens or 0.5 µg appropriate isotype controls (Table 1) . When the primary antibodies were unlabeled, fluorochrome-labeled secondary antibodies were added for 20 min, and cells were washed before fixation. Cells were fixed with 0.5 mL 1% paraformaldehyde (Sigma Chemical Co.) in PBS for 20 min, washed again, and resuspended in 250 µL 1% paraformaldehyde. Fixed, stained cells were stored at 4°C until analysis on a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA).


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  		Table 1. Antibodies for Fresh PBMC Phenotyping

 
Intracellular Staining
Cells were labeled as above for detection of surface antigens with an additional permeabilization step after fixation. Cells were incubated with 0.5 mL DakoCytomation permeabilization reagent together with antibodies against various cytokines or appropriate isotype control antibodies (Table 1) for 20 min. After washing, cells were resuspended in 1% paraformaldehyde in PBS until analysis as above. CellQuest Pro was used for data analysis.

Detection of IL-10 mRNA
The presence of IL-10 mRNA in freshly isolated PBMC subsets was demonstrated by RT-PCR. Aliquots of 1 x 107 PBMC were washed with separation buffer (PBS supplemented with 5 mM EDTA and 0.5% BSA) and incubated for 30 min at 4°C with 5 µg isotype control, anti-CD14, or anti-CD36 antibodies (Table 1) . The cells were washed again and incubated at 3 x 106/ml for 45 min at 4°C in separation buffer with sufficient goat anti-mIgG or rat-anti-mIgM-conjugated magnetic beads (Dynal ASA, Oslo, Norway) for a 10:1 bead:target cell ratio. A magnet was used to separate bead-bound and unbound cells, and flow cytometry indicated depletion of >90% of the targeted subset. Total RNA from bead-bound and unbound cells was extracted with Trizol (Invitrogen) according to the manufacturer’s instructions. cDNA was synthesized using the Amersham cDNA synthesis kit (Amersham Biosciences). Specific primers for PCR were described previously; antisense and sense IL-10 primer sequences were 5'-ACCTGCTCCACGGCCTTGCTCT-3' and 5'-CACCCAGTCTGAACAGCTGC-3', respectively [24 ]. Antisense and sense primers for the ß-actin housekeeping gene were 5'-CAACCGTGAGAAGATGACC-3' and 5'-ATCTCCTGCTCGAAGTCC-3', respectively. PCR conditions for each 50 µL reaction were: 1x PCR buffer, 2.5 mM MgCl2, 200 µM dNTPs, 2.5 U Taq DNA polymerase, and 20 pmol each of the sense and antisense primer (all from Invitrogen). Reactions ran for 35 cycles for IL-10 and 30 cycles for ß-actin with the following cycle times and temperatures: 40 s at 95°C, 30 s at 60°C (IL-10), or 55°C (ß-actin), 1 min at 72°C, with a final extension of 7 min at 72°C. The expected product sizes were ~360 and 339 bp for IL-10 and ß-actin, respectively. Products were separated by electrophoresis on 2% agarose gels with 0.5 µg/mL ethidium bromide (Sigma Chemical Co.) and visualized by UV light. Gels were analyzed using a charged-coupled device camera and ChemiImager (AlphaInnotech, San Leandro, CA, USA) software.

Depletion of CD61+ PBMC
Ten million fresh PBMC were incubated for 20 min with 5 µg-purified anti-CD61 antibody (Table 1) in 0.5 mL flow cytometry buffer at 4°C. Cells were washed and suspended in separation buffer with a 10:1 bead:target cell ratio of goat anti-mIgG-coated magnetic beads (Dynal ASA) for 45 min at 4°C. A magnet was used to remove bead-bound CD61+ cells from the remaining PBMC. Depletion efficiency (98%) was determined by flow cytometric CD61 staining of the remaining PBMC.

Depletion of adherent cells
In some experiments, PBMC were depleted of adherent monocyte cells by plastic adherence. Freshly isolated PBMC at 1 x 106/well were incubated in 24-well, tissue-culture-treated, flat-bottom plates (Corning Costar, Corning, NY, USA) at 37°C, 5% CO2, for 60 min. Nonadherent cells were removed by gently resuspending settled PBMC in 1 mL medium. Efficiency of monocyte removal (97%) was determined by flow cytometry.

ELISPOT for IL-10 production
The number of IL-10-producing cells within intact or CD61-depleted PBMC was determined by ELISPOT. Flat-bottom, polyvinylidene difluoride-coated, 96-well plates (Millipore, Bedford, MA, USA) were prewet with 100 µL 70% ethanol/well (Sigma Chemical Co.), washed with PBS, and coated overnight at 4°C or for 1 h at 37°C with 1.5 µg/well anti-IL-10 antibody (Clone 9D7, Mabtech, Stockholm, Sweden) in PBS. Plates were washed five times with PBS, and 2 x 105 intact PBMC or ~1.9 x 105 CD61-depleted PBMC were added to each well in triplicate. Plates were incubated at 37°C, 5% CO2, for 18 h, washed with PBS, and incubated for 2 h with 0.1 µg/well biotinylated anti-IL-10 antibody (Clone 12G8, Mabtech). Plates were washed, and 100 µL 1:1000 dilution of streptavidin-alkaline phosphatase (Mabtech) was added for 1 h. After washing, 100 µL freshly prepared 3 mg/mL NBT chloride/1.5 mg/mL 5-bromo-4-chloro-3-indolyl phosphate-p-toluidine substrate (BioRad, Hercules, CA, USA) in Tris buffer (pH 9.5) was added to each well for ~20 min. Plates were washed with distilled water to stop the color reaction and air-dried overnight before spot enumeration using the high resolution Zeiss reader system and associated KS software (Carl Zeiss Canada, Ontario). The number of IL-10-producing cells per million PBMC was determined by multiplying the average number of spots/duplicate well x5.

Electron microscopy
The IL-10-producing PBMC population was enriched for visualization by electron microscopy through depletion of T cells, B cells, monocytes, and NK cells. PBMC (1.25x108) were obtained from a healthy donor, divided into 1.0 x 107 aliquots, and incubated with the following antibodies for 25 min at 4°C: Anti-CD2 (7 µg); 6 µg anti-CD3; 6 µg anti-CD14; and 7 µg anti-CD19 (Table 1) . Cells were washed, and goat anti-mIgG-coated magnetic beads (Dynal ASA) were added at a bead:PBMC ratio of 10:1 for 45 min at 4°C. Bead-bound cells were removed magnetically, and red cells lysed from the remaining unlabeled PBMC by 3 min room incubation at room temperature with 0.5 mL erythrocyte lysis buffer (0.15 M ammonium chloride, 10 mM potassium bicarbonate, and 0.1 mM EDTA in distilled water), followed by two PBS washes. The remaining cells (~3.5x106/aliquot) were incubated with 3.5 µg IgM anti-CD36 mAb (Table 1) for 25 min at 4°C, washed in flow buffer, and labeled with 12 nm gold particle-conjugated, goat anti-mIgM beads (Jackson Immunotech Laboratories, West Grove, PA, USA) for 40 min at 4°C. Cells were washed twice, and fixed in 1 mL Karnovsky’s fixative (4 g paraformaldehyde, 5% glutaraldehyde in 0.2 M sodium cacodylate). After 6 h, cells were placed in 1% osmium tetraoxide for 20 min and washed. Cells were dehydrated with increasing alcohol concentrations from 75% to 100% followed by an acetone wash and embedded in epoxide resin overnight at 70°C. Sections (90 nm) were cut, stained with uranite acetate, and examined with a Jeol 1220x electron microscope.

Statistical analyses
All statistical analyses were performed using SPSS Version 9 (SPSS Inc., Chicago, IL, USA). Means were compared using the Mann-Whitney U test or Student’s t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A PBMC subset in healthy individuals constitutively produces and secretes IL-10
To investigate background levels of pro- and anti-inflammatory cytokine production by circulating PBMC, we used intracellular flow cytometry to evaluate constitutive IL-2, IL-10, IL-12, and IFN-{gamma} production by freshly isolated PBMC from healthy individuals. None of the proimmune or proinflammatory cytokines IL-2, IL-12, or IFN-{gamma} were constitutively produced by freshly isolated PBMC from any individual, but CD3-negative PBMC with lymphoid light-scatter characteristics constitutively producing IL-10 were detected in every individual tested (Fig. 1A ). A significant fraction of CD36+ PBMC with lymphoid light-scatter characteristics expressed varying levels of IL-10, and a much smaller fraction of CD36+ monocytoid cells produced low levels of IL-10 (Fig. 1B) . Intracellular flow cytometry indicated ~1% (range 0.37–1.14%) of PBMC from healthy individuals (n=12) constitutively producing IL-10 (Fig. 2 ). Following PBMC isolation, IL-10 production decreased such that after 18 h of cell culture, intracellular IL-10 was detectable in <0.1% of total PBMC (data not shown).


Figure 1
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  		Figure 1. Ex vivo cytokine production by CD3– PBMC from healthy individuals. (A) Constitutive production of IL-2, IFN-{gamma}, IL-12, and IL-10 by CD3+ and CD3– PBMC with lymphoid-scatter characteristics was assessed by intracellular flow cytometry. (B) Production of IL-10 by lymphoid and monocytoid cells was distinguished by side-scatter. Isotype controls are shown below.

 

Figure 2
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  		Figure 2. Comparison of IL-10+ cell frequency in PBMC determined by flow cytometry or ELISPOT in six representative individuals. The picture depicts test wells with PBMC from a representative individual with control wellsunderneath.

 
We used an IL-10 ELISPOT assay to confirm constitutive IL-10 production and to demonstrate IL-10 secretion by freshly isolated PBMC. For the six individuals tested, 318–1171 cells/106 PBMC secreted IL-10 detectable by ELISPOT (Fig. 2) . Negligible numbers of spots developed in the control wells. Although the ELISPOT data confirm spontaneous production of IL-10 by PBMC from healthy individuals, the frequency of IL-10-producing cells detected is approximately one-tenth of that observed by intracellular flow cytometry, reflecting greater sensitivity of flow cytometry or fairly rapid cessation of IL-10 production in vitro. There was a weak but significant correlation between the frequency of IL-10-producing PBMC detected by intracellular flow cytometry and ELISPOT (r=0.88, P<.05). Constitutive production of IL-10 protein was also reflected by detection of IL-10 mRNA in freshly isolated PBMC subsets by RT-PCR (Fig. 3 ). The CD14+ monocyte population and more so, the CD36+ monocyte and lymphoid cell population accounted for IL-10 mRNA in freshly isolated PBMC.


Figure 3
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  		Figure 3. Relative levels of IL-10 mRNA in PBMC subsets. An equal amount of RNA isolated from PBMC (Lane 1), CD14+ PBMC (Lane 2), CD36+ PBMC (Lane 3), PBMC depleted of CD14+ cells (Lane 4), and PBMC depleted of CD36+ cells (Lane 5) was converted to cDNA, subjected to IL-10 and ß-actin-specific PCR, and separated on an agarose gel. The relative intensity of the IL-10 band compared with the ß-actin band is shown in the bar graph below the gel.

 
Detailed phenotype of PBMC constitutively producing IL-10
Initial flow cytometry studies identified the majority of IL-10-producing PBMC as non-T cells with lymphoid light-scatter characteristics. We used the panel of antibodies listed in Table 1 to phenotype these IL-10-producing PBMC more extensively. None of the early precursor and stem cell markers (CD34 and CD117), lymphoid lineage-specific markers (CD2, CD3, and CD19), or myeloid lineage marker CD33 was present on the IL-10-producing cells (Fig. 4 ). The general leukocyte marker CD45 was present together with the nonlineage-specific adhesion molecules CD54 and CD62L. In the absence of other T cell subset makers such as CD4 or CD8, the thymocyte and T cell subset antigen CD7 was expressed on the IL-10-producing cells. None of HLA Class II, CD11c, CD40, CD68, CD80, CD83, or CD86 (molecules found primarily on professional APC) was present on the IL-10-producing cells. Of several activation markers examined (CD25, CD38, and CD69), only CD38, an activation marker increased on T cells and APC after antigen-specific stimulation, was found on the IL-10-producing cells. These data indicate that the lymphoid PBMC constitutively producing IL-10 do not belong to any commonly recognized NK cell, B lymphocyte, T lymphocyte, or DC subset, and they are not professional APC.


Figure 4
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  		Figure 4. Flow cytometric evaluation of relevant protein expression by PBMC constitutively producing IL-10. CD markers shown in enlarged text were expressed on the surface of IL-10+ cells. Myeloperoxidase (MPO) was absent. CD62L, CD62 L-selectin.

 
Two cell surface markers best defined the constitutive IL-10-producing cells and distinguished them clearly from other PBMC with lymphoid-scatter characteristics. All the IL-10-producing cells expressed CD36 (Fig. 5A ) and low levels of CD61 (Fig. 5B) . Cells with high-level CD36 expression (mean channel fluorescence ≥102) were uniformly positive for intracellular IL-10, and 50–80% of the CD36+CD61+ cells expressed IL-10 (Fig. 5B) . To demonstrate constitutive IL-10 production by circulating CD36+CD61+ cells, CD61+ cells were magnetically depleted from freshly isolated PBMC. This removed greater than 98% of the CD36+IL-10+ population in fresh PBMC (Fig. 6A ) as measured by flow cytometry. When PBMC depleted of CD61+ cells were incubated for 18 h in an IL-10 ELISPOT assay, significantly fewer spots were formed (318 IL-10-producing spots/106 cells) than with intact PBMC (733 spots/106 PBMC; Fig. 6B ; t=2.62, P<0.03). This degree of reduction in the ELISPOT assay relative to the 98% reduction in the immediate ex vivo flow cytometry assay indicates partial but incomplete overlap between the PBMC-producing IL-10 ex vivo as detectable by flow cytometry and the PBMC-producing IL-10 in vitro as detected by overnight ELISPOT assay.


Figure 5
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  		Figure 5. Relationship among IL-10 production, CD36 expression, and CD61 expression on PBMC. (a) Cells within the lymphoid-scatter region were analyzed for extracellular CD36 and intracellular IL-10. The lower right panel shows isotype controls. (b) Cells with lymphoid-scatter expressing IL-10 were analyzed for CD36 and CD61 expression. The lower right panel shows isotype controls.

 

Figure 6
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  		Figure 6. Comparison of IL-10-producing cells in PBMC before and after depletion of CD61+ cells. (a) Cells within the lymphoid-scatter region were analyzed for extracellular CD36 and intracellular IL-10, with and without depletion of CD61+ cells. (b) The number of IL-10-secreting cells/106 PBMC (n=6) was enumerated by ELISPOT, before and after depletion of CD61+ cells.

 
Relationship between phenotype and function of IL-10-producing PBMC
As monocytes are the major PBMC population previously reported to express the CD36 and CD61 molecules distinguishing our IL-10-producing PBMC, we examined phenotypic and physical characteristics of the CD36+ IL-10-producing lymphoid cells, which might differentiate them further from monocytes. Lymphocytes and monocytes have different forward (size) and side (granularity) light-scatter characteristics when analyzed by flow cytometry. Most of the PBMC constitutively producing IL-10 have light-scatter characteristics similar to lymphocytes (Fig. 1B) , indicating they are smaller and less granular than monocytes. They also do not express the monocyte marker CD14 (Fig. 7 ). Nonadherence to plastic distinguishes the CD14–CD36+CD61+ IL-10-producing cells further from CD14+CD36+CD61+ monocytes, as there was no depletion of IL-10-producing cells following 2 h PBMC incubation on plastic dishes (data not shown).


Figure 7
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  		Figure 7. Comparison of expression of TLR-2, -4, and -9 and CD14 on CD36+ monocytes and CD36+ cells with lymphoid-scatter characteristics. Results shown are representative of those obtained with 12 individuals.

 
As previously shown, CD36 is present on the IL-10-producing cells, and high CD36 expression appears to be associated with IL-10 production. CD36 is a scavenger receptor for apoptotic bodies and may play an evolutionary role in the innate immune response. Absence of CD14, the LPS receptor that stimulates a monocyte proinflammatory response, phenotypically distinguishes the IL-10-producing cells from CD36+ monocytes. As CD14 is functionally related to the family of TLRs, which recognize pathogen-associate molecular patterns, we investigated expression of several common TLRs on the CD36+ IL-10-producing lymphoid cells. In contrast to the CD14+CD36+CD61+ monocytes, CD14–CD36+CD61+IL10+ cells did not express TLR-2, -4, or -9 (Fig. 7) . The absence of these primarily proinflammatory receptors on the surface of the IL-10-producing cells is consistent with their production of the anti-inflammatory cytokine IL-10 and with a potential immunoregulatory role for these cells.

Ultrastructural characteristics of the IL-10-producing cells
To examine the ultrastructure of the IL-10-producing cells, we enriched for this population by depleting PBMC of T cells, B cells, monocytes, and NK cells with magnetic beads and antibodies against CD2, CD3, CD14, and CD19. Remaining cells were incubated with gold bead-conjugated anti-CD36 antibodies, and the CD36+ cells were visualized by electron microscopy. The CD36+ cells were ~6 µm in diameter, similar to resting lymphocytes, with a high nucleus:cytoplasm ratio and large amount of heterochromatin (Fig. 8 ). Mitochondria were plentiful, and rough endoplasmic reticulum was prominent in the cytoplasm, suggesting these were highly synthetic, metabolically active cells.


Figure 8
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  		Figure 8. Electron micrograph of CD36+CD14– cells. Freshly isolated PBMC were depleted of T cells, B cells, NK cells, and monocytes, labeled with anti-CD36, followed by 12 nm gold particle-conjugated goat anti-mIgM beads, and then visualized by electron microscopy. Arrowheads on the right panels indicate gold beads associated with CD36.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Induction of inflammatory and immune responses must be regulated carefully to allow for effective, protective immunity while avoiding unnecessary inflammation and immunopathology. Stimuli must breach a certain threshold individually and collectively to initiate or sustain inflammation and immunity. Whether an appropriate threshold for immune activation is maintained simply through the requirement for accumulation of positive signals or whether low-level basal immune suppression may be involved is an important issue in relation to susceptibility to chronic infection or chronic inflammation. Consistent with the general view that immune responses do not arise in the absence of stimuli, we found little or no constitutive production of proinflammatory cytokines IL-2, IL-12, or IFN-{gamma} by PBMC from healthy individuals. However, up to 1% of freshly isolated PBMC constitutively produced and secreted IL-10, an important negative regulator of inflammatory and cellular immune responses. Phenotypic and ultrastructural characterization of the cells spontaneously producing IL-10 indicated a PBMC subset previously undescribed, resisting inclusion in any common, recognized lineage.

Constitutive production of IL-10 by freshly isolated, nonstimulated PBMC was readily apparent by ex vivo intracellular flow cytometry or by ELISPOT, following overnight incubation in unsupplemented lymphocyte medium. It is surprising that despite the widespread application of sensitive ELISPOT assays, only one group has reported constitutive IL-10 production previously by PBMC and in this case, only at the mRNA level [25 ]. Counterstaining the IL-10+ PBMC by flow cytometry demonstrated consistent expression of the common leukocyte antigen, CD45, suggesting hematopoietic origin. Although TH2 cells are a common source of IL-10 in response to specific antigens, none of the IL-10+ cells expressed CD2, CD3, CD4, CD8, or CD19, ruling out their assignment within NK, T, or B cell subsets. Immature DC and monocytes also produce IL-10, but the characteristics of the lymphoid IL-10+ cells were inconsistent with either of these PBMC subsets. Unlike immature DC, the IL-10+ cells expressed no HLA-DR or CD1a. In contrast to monocytes, the IL-10+ cells had lymphoid cell light-scatter characteristics and did not express CD14, CD33, CD68, or MPO. The IL-10+ cells are easily distinguished from virtually all other PBMC by their lymphoid light scatter (smaller and less complex than monocytes) and expression of CD36 in the absence of CD14. Expression of other surface markers such as CD5, CD7, CD38, CD54, CD59, and CD61 is interesting and useful for depletion or isolation but offers little obvious insight into the origin or role of these cells. The constitutive IL-10 production is consistent with a role for these cells in low-level basal immunosuppression, and expression of CD62L indicates an ability to exit the bloodstream through high endothelial venules and modulate immune responsiveness within the lymph nodes, where primary immune responses generally originate.

As noted in Results, a small proportion of circulating monocytoid cells was also IL-10+-positive. These cells were easily differentiated from the CD36+CD14– population of lymphoid cells, and removal of CD14+ monocytes through plastic adherence or magnetic beads did not deplete IL-10 protein or mRNA-bearing cells, respectively. Absence of CD14, a primarily proinflammatory molecule related to the TLR, phenotypically distinguishes the CD36+IL-10+ cells from monocytes and also supports their characterization as potentially immunosuppressive, rather than proinflammatory cells. Although signaling through CD14 can also result in late production of IL-10, the phenotypic polarization of these CD36+IL-10+ cells away from immune activation is reflected further by the absence of TLR-2, -4, and -9, additional proinflammatory receptors, which are consistently expressed on CD36+ monocytes and are generally associated with immune activation. In the CD14–CD36+ PBMC subset, we observed a general relationship between IL-10 production and CD36 expression in that all CD36bright cells were producing IL-10 and that the CD36bright cells produced more IL-10 than CD36dim cells. This relationship raises the possibility of a mechanistic link between CD36 expression and the function of these cells. One function of CD36 is in the uptake of apoptotic bodies by macrophages and DC [26 , 27 ] and subsequent modulation of immune responses [28 ]. The relationship between high CD36 expression and high IL-10 production could preferentially deliver apoptotic bodies to IL-10-producing cells, favoring immune suppression over inflammation. Delivery of early apoptotic cells to a macrophage subset producing IL-10 was demonstrated and proposed recently as a mechanism to prevent autoimmunity under steady-state conditions [29 ]. Another CD36 ligand is oxidized LDL (oxLDL), a factor associated with the pathogenesis of atherosclerosis [30 , 31 ]. Selective delivery of oxLDL to IL-10-producing cells through this interaction with CD36 could play a protective role against the putative inflammatory component of atherosclerosis. The malaria-causing parasite Plasmodium falciparum also interacts with CD36 and through this interaction could exploit localized immunosuppression to evade immunity and establish chronic infection [28 ].

Although circulating monocytes express relatively low levels of CD36, few were constitutively producing IL-10; therefore, cell-specific factors other than just expression of CD36 are involved in constitutive IL-10 production. Monocytes exposed in vitro to thiazolidinediones, peroxisomal proliferator-associated receptor-{gamma} agonists commonly used to treat Type II diabetes-associated insulin resistance, express higher levels of CD36 and develop an anti-inflammatory cytokine release profile [32 ]. A rat model of Type II diabetes and hypertension has a dominant CD36 mutation, which precludes expression of CD36 at the transcriptional level [33 ], supporting an immunomodulatory role for CD36 in inflammatory diseases such as diabetes. In humans, the null mutation of a human blood group polymorphism Naka results in loss of CD36 expression [34 ]. This phenotype is prevalent in African and Japanese populations, and it would be interesting to compare circulating IL-10+ PBMC and the rates of various inflammatory diseases between Naka null (with impaired CD36 expression) and appropriate control groups.

Local delivery or production of IL-10 may be the mechanism by which CD36 engagement, in some cases, dampens inflammatory responses [28 , 35 ]. We speculate that the IL-10+CD36+ cells provide a low-level, innate barrier to immune responses and inflammation, suggesting that lower circulating levels of these cells could predispose to development of autoimmune or proinflammatory diseases. Homeostatic regulation of the immune system, similar to what we are suggesting by these IL-10+ cells, is mimicked in the interaction between lung alveolar epithelial cells (AEC) and alveolar macrophages (AM) [36 ]. TGF-ß produced by AEC maintains a homeostatic, suppressive environment in normal lung by inhibiting AM. However, AM are still able to respond to and clear microbial pathogens when necessary by using conformational change to disengage from the AEC inhibition. AM inhibition is restored as the pathogen is cleared, resulting in minimal damage to nearby lung tissue. We suggest that constitutive production of IL-10 by the cells described in this study provides a similar tonic checkpoint on the immune system to down-regulate unnecessary responses in the steady state.

The concept of immune regulatory cells has evolved through various incarnations of suppressor T cells to the relatively concrete CD4+CD25+ Tr cells of today. These cells develop spontaneously and in some cases, mediate their effector functions through nonspecific cytokines such as IL-10 and TGF-ß. However, they are activated through antigen-specific receptors and therefore, constitute an adaptive immunosuppressive response, which can appropriately protect against autoimmunity and inappropriately increase susceptibility to infection and cancer. The existence of circulating antigen-nonspecific cells spontaneously producing the immunosuppressive cytokine IL-10 suggests that like immune reactivity, immune suppression may be organized in innate and adaptive layers with a similar series of interconnections still to be elucidated.


   ACKNOWLEDGEMENTS
 
This research was supported by Canadian Institutes for Health Research (CIHR) Operating grant MOP-41541. L. B. was recipient of a CIHR M.D./Ph.D. fellowship. We thank personnel from the Faculty of Medicine, Memorial University of Newfoundland, who volunteered to donate blood for this study. We also thank the Clinical Immunology Laboratory, Health Care Corporation, St. John’s, for providing some reagents for flow cytometry.

Received August 17, 2006; revised February 13, 2007; accepted March 5, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Moore, K. W., de Waal Malefyt, R., Coffman, R. L., O’Garra, A. (2001) Interleukin-10 and the interleukin-10 receptor Annu. Rev. Immunol. 19,683-765[CrossRef][Medline]
  2. Weiss, E., Mamelak, A. J., La Morgia, S., Wang, B., Feliciani, C., Tulli, A., Sauder, D. N. (2004) The role of interleukin 10 in the pathogenesis and potential treatment of skin diseases J. Am. Acad. Dermatol. 50,657-675[CrossRef][Medline]
  3. De Waal Malefyt, R., Abrams, J., Bennett, B., Figdor, C. G., de Vries, J. E. (1991) Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes J. Exp. Med. 174,1209-1220[Abstract/Free Full Text]
  4. Fiorentino, D. F., Zlotnik, A., Mosmann, T. R., Howard, M., O’Garra, A. (1991) IL-10 inhibits cytokine production by activated macrophages J. Immunol. 147,3815-3822[Abstract]
  5. Koppelman, B., Neefjes, J. J., de Vries, J. E., de Waal Malefyt, R. (1997) Interleukin-10 down-regulates MHC class II {alpha}ß peptide complexes at the plasma membrane of monocytes by affecting arrival and recycling Immunity 7,861-871[CrossRef][Medline]
  6. Nicod, L. P., el Habre, F., Dayer, J. M., Boehringer, N. (1995) Interleukin-10 decreases tumor necrosis factor {alpha} and ß in alloreactions induced by human lung dendritic cells and macrophages Am. J. Respir. Cell Mol. Biol. 13,83-90[Abstract]
  7. D’Andrea, A., Aste-Amezaga, M., Valiante, N. M., Ma, X., Kubin, M., Trinchieri, G. (1993) Interleukin 10 (IL-10) inhibits human lymphocyte interferon {gamma}-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells J. Exp. Med. 178,1041-1048[Abstract/Free Full Text]
  8. Kuhn, R., Lohler, J., Rennick, D., Rajewsky, K., Muller, W. (1993) Interleukin-10-deficient mice develop chronic enterocolitis Cell 75,263-274[CrossRef][Medline]
  9. Dai, W. J., Kohler, G., Brombacher, F. (1997) Both innate and acquired immunity to Listeria monocytogenes infection are increased in IL-10-deficient mice J. Immunol. 158,2259-2267[Abstract]
  10. Yang, X., Gartner, J., Zhu, L., Wang, S., Brunham, R. C. (1999) IL-10 gene knockout mice show enhanced Th1-like protective immunity and absent granuloma formation following Chlamydia trachomatis lung infection J. Immunol. 162,1010-1017[Abstract/Free Full Text]
  11. Pinderski, L. J., Fischbein, M. P., Subbanagounder, G., Fishbein, M. C., Kubo, N., Cheroutre, H., Curtiss, L. K., Berliner, J. A., Boisvert, W. A. (2002) Overexpression of interleukin-10 by activated T lymphocytes inhibits atherosclerosis in LDL receptor-deficient mice by altering lymphocyte and macrophage phenotypes Circ. Res. 90,1064-1071[Abstract/Free Full Text]
  12. Van Exel, E., Gussekloo, J., de Craen, A. J., Frolich, M., Bootsma-Van Der Wiel, A., Westendorp, R. G. (2002) Low production capacity of interleukin-10 associates with the metabolic syndrome and type 2 diabetes: the Leiden 85-Plus Study Diabetes 51,1088-1092[Abstract/Free Full Text]
  13. Pinderski Oslund, L. J., Hedrick, C. C., Olvera, T., Hagenbaugh, A., Territo, M., Berliner, J. A., Fyfe, A. I. (1999) Interleukin-10 blocks atherosclerotic events in vitro and in vivo Arterioscler. Thromb. Vasc. Biol. 19,2847-2853[Abstract/Free Full Text]
  14. Fedorak, R. N., Gangl, A., Elson, C. O., Rutgeerts, P., Schreiber, S., Wild, G., Hanauer, S. B., Kilian, A., Cohard, M., LeBeaut, A., Feagan, B. (2000) Recombinant human interleukin 10 in the treatment of patients with mild to moderately active Crohn’s disease. The Interleukin 10 Inflammatory Bowel Disease Cooperative Study Group Gastroenterology 119,1473-1482[CrossRef][Medline]
  15. Schreiber, S., Fedorak, R. N., Nielsen, O. H., Wild, G., Williams, C. N., Nikolaus, S., Jacyna, M., Lashner, B. A., Gangl, A., Rutgeerts, P., Isaacs, K., van Deventer, S. J., Koningsberger, J. C., Cohard, M., LeBeaut, A., Hanauer, S. B. (2000) Safety and efficacy of recombinant human interleukin 10 in chronic active Crohn’s disease. Crohn’s Disease IL-10 Cooperative Study Group Gastroenterology 119,1461-1472[CrossRef][Medline]
  16. Gomes, N. A., Barreto-de-Souza, V., DosReis, G. A. (2000) Early in vitro priming of distinct T(H) cell subsets determines polarized growth of visceralizing Leishmania in macrophages Int. Immunol. 12,1227-1233[Abstract/Free Full Text]
  17. Yamauchi, P. S., Bleharski, J. R., Uyemura, K., Kim, J., Sieling, P. A., Miller, A., Brightbill, H., Schlienger, K., Rea, T. H., Modlin, R. L. (2000) A role for CD40-CD40 ligand interactions in the generation of type 1 cytokine responses in human leprosy J. Immunol. 165,1506-1512[Abstract/Free Full Text]
  18. Misra, N., Selvakumar, M., Singh, S., Bharadwaj, M., Ramesh, V., Misra, R. S., Nath, I. (1995) Monocyte derived IL 10 and PGE2 are associated with the absence of Th 1 cells and in vitro T cell suppression in lepromatous leprosy Immunol. Lett. 48,123-128[CrossRef][Medline]
  19. Corinti, S., Albanesi, C., la Sala, A., Pastore, S., Girolomoni, G. (2001) Regulatory activity of autocrine IL-10 on dendritic cell functions J. Immunol. 166,4312-4318[Abstract/Free Full Text]
  20. Buelens, C., Verhasselt, V., De Groote, D., Thielemans, K., Goldman, M., Willems, F. (1997) Interleukin-10 prevents the generation of dendritic cells from human peripheral blood mononuclear cells cultured with interleukin-4 and granulocyte/macrophage-colony-stimulating factor Eur. J. Immunol. 27,756-762[Medline]
  21. De Smedt, T., Van Mechelen, M., De Becker, G., Urbain, J., Leo, O., Moser, M. (1997) Effect of interleukin-10 on dendritic cell maturation and function Eur. J. Immunol. 27,1229-1235[Medline]
  22. Morel, A. S., Quaratino, S., Douek, D. C., Londei, M. (1997) Split activity of interleukin-10 on antigen capture and antigen presentation by human dendritic cells: definition of a maturative step Eur. J. Immunol. 27,26-34[Medline]
  23. Brewer, G., Saccani, S., Sarkar, S., Lewis, A., Pestka, S. (2003) Increased interleukin-10 mRNA stability in melanoma cells is associated with decreased levels of A + U-rich element binding factor AUF1 J. Interferon Cytokine Res. 23,553-564[CrossRef][Medline]
  24. Hanna, N., Hanna, I., Hleb, M., Wagner, E., Dougherty, J., Balkundi, D., Padbury, J., Sharma, S. (2000) Gestational age-dependent expression of IL-10 and its receptor in human placental tissues and isolated cytotrophoblasts J. Immunol. 164,5721-5728[Abstract/Free Full Text]
  25. Stordeur, P., Zhou, L., Goldman, M. (2002) Analysis of spontaneous mRNA cytokine production in peripheral blood J. Immunol. Methods 261,195-197[CrossRef][Medline]
  26. Savill, J., Hogg, N., Haslett, C. (1992) Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis J. Clin. Invest. 90,1513-1522[Medline]
  27. Schwartz, B. R., Karsan, A., Bombeli, T., Harlan, J. M. (1999) A novel ß 1 integrin-dependent mechanism of leukocyte adherence to apoptotic cells J. Immunol. 162,4842-4848[Abstract/Free Full Text]
  28. Urban, B. C., Wilcox, N., Roberts, D. J. (2001) A role for CD36 in the regulation of dendritic cell function Proc. Natl. Acad. Sci. USA 98,8750-8755[Abstract/Free Full Text]
  29. Xu, W., Roos, A., Schlagwein, N., Woltman, A. M., Daha, M. R., van Kooten, C. (2006) IL-10 producing macrophages preferentially clear early apoptotic cells Blood 107,4930-4937[Abstract/Free Full Text]
  30. Heinecke, J. W. (1997) Mechanisms of oxidative damage of low density lipoprotein in human atherosclerosis Curr. Opin. Lipidol. 8,268-274[Medline]
  31. Berliner, J. A., Heinecke, J. W. (1996) The role of oxidized lipoproteins in atherogenesis Free Radic. Biol. Med. 20,707-727[CrossRef][Medline]
  32. Asada, K., Sasaki, S., Suda, T., Chida, K., Nakamura, H. (2004) Antiinflammatory roles of peroxisome proliferator-activated receptor {gamma} in human alveolar macrophages Am. J. Respir. Crit. Care Med. 169,195-200[Abstract/Free Full Text]
  33. Aitman, T. J., Glazier, A. M., Wallace, C. A. (1999) Identification of Cd36 (Fat) as an insulin resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats Nat. Genet. 21,76-83[CrossRef][Medline]
  34. Yamamoto, N., Ikeda, H., Tandon, N. N., Herman, J., Tomiyama, Y., Mitani, T., Sekiguchi, S., Lipsky, R., Kralisz, U., Jamieson, G. A. (1990) A platelet membrane glycoprotein deficiency in healthy blood donors: Naka platelets lack detectable GPIV (CD36) Blood 76,1698-1703[Abstract/Free Full Text]
  35. Bzowska, M., Guzik, K., Barczyk, K., Ernst, M., Flad, H. D., Pryjma, J. (2002) Increased IL-10 production during spontaneous apoptosis of monocytes Eur. J. Immunol. 32,2011-2020[CrossRef][Medline]
  36. Takabayshi, K., Corr, M., Hayashi, T., Redecke, V., Beck, L., Guiney, D., Sheppard, D., Raz, E. (2006) Induction of a homeostatic circuit in lung tissue by microbial compounds Immunity 24,475-487[CrossRef][Medline]




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