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Original Research |

Circulating Myeloid-Derived Suppressor Cells Are Increased and Activated in Pulmonary HypertensionMyeloid Suppressor Cells in Pulmonary Hypertension FREE TO VIEW

Michael E. Yeager, PhD; Cecilia M. Nguyen, BS; Dmitry D. Belchenko, BA; Kelley L. Colvin, MS; Shinichi Takatsuki, MD; D. Dunbar Ivy, MD, FCCP; Kurt R. Stenmark, MD
Author and Funding Information

From the Department of Pediatrics, Division of Pulmonary and Critical Care Medicine (Drs Yeager and Stenmark, Ms Nguyen, and Mr Belchenko), the Department of Pediatrics, Pulmonary Hypertension Program and Pediatric Heart Lung Center, Children’s Hospital (Dr Ivy and Ms Colvin), and the Gates Center for Regenerative Medicine and Stem Cell Biology (Dr Stenmark), University of Colorado, Denver, Aurora, Colorado; and the Department of Pediatrics, Omori Medical Center (Dr Takatsuki), Toho University, Tokyo, Japan.

Correspondence to: Michael E. Yeager, PhD, University of Colorado Denver Health Sciences, Pediatrics Critical Care, 12700 E 19th Ave, Box C218, Aurora, CO 80045; e-mail: michael.yeager@ucdenver.edu


Funding/Support: This study was funded by an Actelion Entelligence Young Investigator Award to Dr Yeager; the National Institutes of Health, Specialized Centers of Clinically Oriented Research [Grant HL-084923-020]; the National Institutes of Health, Program Project [Grant HL-014985-35]; the Jayden DeLuca Foundation; the National Institutes of Health, General Clinical Research Center National Center for Research Resources [Grant M01-RR00069]; and the Leah Bult Pulmonary Hypertension Research Fund.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (http://www.chestpubs.org/site/misc/reprints.xhtml).


© 2012 American College of Chest Physicians


Chest. 2012;141(4):944-952. doi:10.1378/chest.11-0205
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Background:  Myeloid-derived suppressor cells (MDSCs) are increased in inflammatory and autoimmune disorders and orchestrate immune cell responses therein. Pulmonary hypertension (PH) is associated with inflammation, autoimmunity, and lung vascular remodeling. Immature myeloid cells are found in the lungs of humans and animals with PH, and we hypothesized that they would be increased in the blood of patients with PH compared with control subjects.

Methods:  Twenty-six children with PH and 10 undergoing cardiac catheterization for arrhythmia ablation were studied. Five milliliters of fresh blood were analyzed using flow cytometry. Results were confirmed using magnetic bead sorting and immunofluorescence, while quantitative polymerase chain reaction and intracellular urea concentration assays were used as measures of MDSC arginase-1 activation.

Results:  Flow cytometry demonstrated enrichment of circulating MDSCs among patients with PH (n = 26; mean, 0.271 × 106 cells/mL ± 0.17; 1.86% of CD45+ population ± 1.51) compared with control subjects (n = 10; mean, 0.176 × 106 cells/mL ± 0.05; 0.57% of CD45+ population ± 0.29; P < .05). Higher numbers of circulating MDSCs correlated to increasing mean pulmonary artery pressure (r = 0.510, P < .05). Among patients with PH, female patients had a twofold increase in MDSCs compared with male patients. Immunofluorescence analysis confirmed the results of flow cytometry. Quantitative reverse transcription polymerase chain reaction assay results for arginase-1 and measurement of intracellular urea concentration revealed increased activity of MDSCs from patients with PH compared with control subjects.

Conclusions:  Circulating activated MDSCs are significantly increased in children with PH compared with control subjects. Further investigation of these cells is warranted, and we speculate that they might play significant immunomodulatory roles in the disease pathogenesis of PH.

Figures in this Article

Pulmonary hypertension (PH) is a progressive syndrome with a poor prognosis that results in right-sided heart failure.1 Although heterogeneous in its pathobiologic characteristics, it is distinguished in children and adults by cellular and structural changes in the full thickness of the walls of pulmonary arteries and by perivascular accumulation of inflammatory cells.2,3 Evidence for active, maladaptive inflammatory processes is manifest in both humans with PH and animal models of the disease.4 The scientific basis for inflammation in PH has recently been summarized5 and implicates innate and adaptive immune cells, mediators and effectors of inflammation, and potential triggering events. Of particular interest to us is the collection of data implicating T and B cells in the pathobiologic aspects of PH and their interactions with mononuclear phagocytic cells. It has been suggested that a subpopulation of monocyte-derived dendritic cells (DCs) is functionally impaired in idiopathic PH6 and that the presence of immature DCs may alter the T and B cell responses in the hypertensive lung.7

Myeloid-derived suppressor cells (MDSCs) compose a phenotypically diverse subpopulation of cells that includes mature (granulocytes, monocytes, macrophages, DCs) and immature (myelo-monocytic precursors) myeloid cells.8 MDSCs were described 20 years ago9 and regulate immune responses in both normal and disease settings.10 MDSCs participate in a multiplicity of processes, including innate and acquired immunity,10 autoimmunity,9 and nonimmunologic responses such as angiogenesis.11 Immunohistochemical analysis of lung sections from patients with PH indicates that immature DCs are present in and around areas of vascular remodeling.7 In the rat model of monocrotaline-induced PH, the DCs recruited to remodeled vessels display an immature myeloid phenotype.7 Additional studies reveal that monocyte-derived DCs from patients with PH are defective in their ability to stimulate T cells in an allostimulatory mixed-leukocyte reaction assay.6 Indeed, abnormalities of T lymphocyte subsets have been documented in patients with PH.12,13 Although a direct mechanistic role for MDSCs was not established in these studies, these functionally impaired immature myeloid cells or DCs appeared prior to the formation of, but remained constantly present within, areas of vascular remodeling.

We reasoned that MDSCs are participating in the immunomodulatory process apparently active in PH, and therefore might be in greater quantity in the peripheral blood of patients with PH. We found significant increases in MDSCs in these patients compared with control subjects and found correlation with increasing mean pulmonary artery pressure. We conclude that PH is associated with elevated levels of circulating active MDSCs. A more complete understanding of their putative roles in PH might lead to novel therapies.

Subjects, Clinical Data, and Blood Collection

The Colorado Multiple Institutional Review Board approved the study (10-0259), and consent (assent where appropriate) was obtained from all patients. Demographic data for all subjects are presented in Tables 1 and 2. Five milliliters of blood were drawn from the femoral vein during initial cardiac catheterization in all patients. The pulmonary arterial hypertension group consisted of children and young adults < 18 years of age at the time of diagnosis with PH, with World Health Organization classification group 1 defined as either idiopathic pulmonary arterial hypertension, heritable pulmonary arterial hypertension, or pulmonary arterial hypertension associated with congenital heart disease. The control group included children < 18 years of age with a structurally normal heart undergoing heart arrhythmia ablation, before the onset of the procedure. One control patient was included who was undergoing repair for a ventricular septal defect. All patients undergoing ablation were in normal sinus rhythm at the start of the catheterization when the blood was drawn and had a normal echocardiogram.

Table Graphic Jump Location
Table 1 —Data for Subjects With PH

Values are presented as mean ± SD. F = female; iPAH = idiopathic pulmonary arterial hypertension; M = male; MDSC = myeloid derived suppressor cell; mPAP = mean pulmonary artery pressure; n/a = not catheterized at the time; PH = pulmonary hypertension; + = medication taken; sPAH = secondary pulmonary arterial hypertension; WHO = World Health Organization.

Table Graphic Jump Location
Table 2 —Data for Control Subjects

Values are presented as mean ± SD. SVT = supra ventricular tachycardia; VSD = ventricular septal defect; WPW = Wolff-Parkinson-White syndrome. See Table 1 legend for expansion of other abbreviations.

Hemodynamics and echocardiographic data were obtained at the time of initial catheterization, then later at therapeutic assessment. Fresh, never-frozen blood (5 mL) was prepared without Ficoll gradient as described by Hong et al.14 Buffy coat cells were removed from blood centrifuged for 10 min at 1,200 rpm at room temperature. Following RBC lysis by the addition of ammonium chloride (0.15 M, pH 7.3), leukocytes were washed, verified for viability using trypan blue exclusion, and prepared to a concentration of 1 × 106 cells/mL for antibody labeling and flow cytometry.

Flow Cytometric Analysis

Cells were labeled using antibodies against CD45-Pacific Blue (PB986; BD Biosciences), CD33-FITC (340533; BD Biosciences), HLA-DR-PE (FAB4869P; R&D Systems), and CD11b-APC (340936; BD Biosciences), per manufacturer’s protocol. For flow cytometry, CXP software and an FC500 instrument (Beckman) were used. Negative thresholds for gating were set using isotype control-labeled cells from both patients with PH and control subjects, as described by Mathai et al.15 All samples were gated for the CD45+ region, then for expression of CD33, then analyzed for CD11b expression and absence of HLA-DR (MHCII). MDSC numbers (CD45+/CD33+/MHCII/CD11b+) were calculated and also expressed as a percentage of total leukocytes. More than 200,000 events were used to generate histograms.

Cytospin and Immunofluorescence

Flow-sorted cells from three patients with PH and three control subjects were centrifuged at 800 rpm and resuspended in 200 uL. Following cytospins, cells were fixed in cold acetone/methanol for 10 min. MDSCs were assessed as MHCII/CD33+/CD11b+. Antibodies used were pSTAT3 (Tyr705, Cell Signaling 9131), CD33 (Millipore WM53), CD11b (Abcam 2Q902), and MHCII (eBioscience 14-9956). Antibody isotype-negative controls were included with each sample group, as was preadsorption of antibody to its cognate antigen. Slides were mounted in Vectashield plus 4ʹ,6-diamidino-2-phenylindole medium (Vector), and images were acquired at room temperature using an Axiovert S100 (Zeiss) fitted with Zeiss 20 × 0.4 numerical aperture. and 10 × 0.3 numerical aperture objectives.

Immunomagnetic Bead Separation

Buffy coat cells were sorted per manufacturer’s instructions using the following separation sequences: CD33+ (130-045-501; Miltenyi), MHCII- (130-046-101; Miltenyi), and CD11b+ (130-049-601; Miltenyi). MDSCs were then immediately processed for either quantitative polymerase chain reaction or urea assay, as described in the “Quantitative Polymerase Chain Reaction” section.

Quantitative Polymerase Chain Reaction

Active MDSC expression increased transcripts for arginase-1 (Arg-1).16 Primer sequences used to assess Arg-1 gene transcripts were F: 5′-GGAACCCAGAGAGAGCATGA-3′; and R: 5′-TTTTTCCAGCAGACCAGCTT-3′. Bead-sorted MDSC total RNA isolation, RNA concentration, complementary DNA transcription, polymerase chain reaction cycling, and normalization were performed as previously described.17 Cells recovered from bead separations were pooled (n = 5 patients per group) prior to RNA analysis.

Urea Concentration Measurement

Stimulated MDSCs increase Arg-1 activity, as measured by urea production from the arginase metabolism of l-arginine to l-ornithine and urea.18 Pooled, bead-sorted MDSCs were counted, and an equivalent cell number (104) of lysates (in 10 mM Tris-HCl, pH 7.4 with 0.4% Triton X-100) from control subjects (n = 7) and from patients with PH (n = 21) were assayed colorimetrically in duplicate for urea, as previously described19 (Quantichrom Arginase Kit, DARG-200, Gentaur). Forty microliters of pooled sample were added to substrate buffer, and the difference in absorbance (520 nm) with and without pretreatment with urease (Sigma U1500) was compared with a urea standard curve.

Statistical Analysis

Values were expressed as mean ± SEM using Prism5 (GraphPad). Unpaired t tests were performed for two group comparisons, while Pearson correlation analysis was used to compare MDSC suppressor cell numbers with clinical and hemodynamic parameters. Significance was defined as P < .05.

MDSC Quantification

Circulating MDSCs expand in settings of autoimmunity and inflammation10 and following immunization with ovalbumin.20 PH is associated with inflammation21 and autoimmunity,22 and can be induced by antigenic challenge via airway ovalbumin.23 We hypothesized that individuals with PH would have increased circulating MDSCs. We found that they had increased MDSCs, expressed as cells per milliliter or as a percentage of CD45+ cells, compared with control subjects (n = 26; mean, 0.271 × 106 cells/mL ± 0.17; 1.86% ± 1.51%; vs n = 10; mean, 0.176 × 106 cells/mL ± 0.05; 0.57% ± 0.29%, respectively; P < .05). Analysis is shown in Figure 1, and quantification in Figures 2A, 2B. No differences were observed in MDSC numbers among those patients with idiopathic PH compared with those with secondary PH. Female patients with PH had higher MDSC percentages, but not cells per milliliter, compared with male subjects with PH (female subjects, n = 12; mean, 0.326 × 106 cells/mL ± 0.19; 2.68% ± 1.72%; vs male subjects, n = 14; mean, 0.226 × 106 cells/mL ± 0.13; 1.36% ± 0.97%; P < .01 for percentage data and P < .13 for cells per milliliter data) (Figs 2C, 2D).

Figure Jump LinkFigure 1. Scatter diagrams show flow cytometric analysis for myeloid-derived suppressor cells (MDSCs). A, Side scatter (SSC) vs forward scatter (FSC) analysis of ≥ 200,000 cells from representative control subject blood sample. B, SSC vs CD45 Pacific Blue (PB) isotype sample from control subject for gating. C, Isotype control used to set gating for the CD45+/CD33+ population from control subject blood sample. D, Isotype control used to set gating for the MHCII/CD11b+ population from control subject blood sample. E, This control subject had 0.5% of total CD45+ cells labeling as MHCII/CD11b+ (quadrant B4), corresponding to 0.17 × 106 cells/mL. F, Isotype control used to set gating for the MHCII/CD11b+ population from blood sample of patient with pulmonary hypertension (PH). G, This patient with PH had 2.1% of total CD45+ cells labeling as MHCII/CD11b+ (quadrant B4), corresponding to 0.24 × 106 cells/mL. Isotype controls were performed to set gates on each sample from control subjects and patients with PH, as described by Mathai et al,15 and all samples were run in triplicate. APC = allophycocyanin; FITC = fluorescein isothiocyanate; FL = channel; FS = forward scatter; INT LIN = integration linear; INT LOG = integration logarithmic; PE = phycoerythrin; SS = side scatter.Grahic Jump Location
Figure Jump LinkFigure 2. Scatter diagrams show that circulating MDSC counts are enriched in patients with PH compared with control subjects and correlate to mean pulmonary artery pressure. A and B, Mean MDSC counts in 26 patients with PH were mean, 0.271 × 106 cells/mL ± 0.17, 1.86% ± 1.51%; and counts in 10 control subjects were mean, 0.176 × 106 cells/mL ± 0.05; 0.57% ± 0.29%; P < .05 vs control subjects. C and D, Female subjects with PH (n = 12; mean, 0.32 × 106 cells/mL ± 0.19; 2.68% ± 1.72%) had higher MDSC counts than male subjects with PH (n = 14; mean, 0.23 × 106 cells/mL ± 0.13; 1.36% ± 0.97%); P < .05 vs control for percentage. E, MDSC numbers per milliliter in patients with PH positively correlate to mean PAP. (r = 0.510; P < .05 vs control subjects). PAP = pulmonary artery pressure. See Table 1 legend for expansion of other abbreviations.Grahic Jump Location
MDSC Correlation to Clinical and Hemodynamic Parameters

MDSCs are known to correlate to tumor burden, infection, inflammation, and autoimmunity.10 In our cohort of patients with PH, MDSC counts positively correlated to mean pulmonary artery pressure (r = 0.417 for percentage and 0.510 for cells per milliliter; P < .05) (Fig 2E), but not increasing age or duration of treatment.

Immunophenotyping and Activity Assessment of MDSC

We verified the sorted MDSC phenotype using immunofluorescence. MDSCs sorted from the blood of patients with PH and blood from control subjects strongly expressed CD33 and CD11b, but not MHCII, confirming the cytometry results (Figs 3A, 3B). Additionally, phospho-STAT3 was detected in MDSCs sorted from the blood of patients with PH but not blood from control subjects (Fig 3C). PhosphoSTAT3 is associated with the survival and proliferation of MDSCs.24 Arg-1 activity, along with that of inducible nitric oxide synthase, mediates the suppression of T-cell responses by MDSCs.18 To investigate differences in Arg-1 expression and activity, we performed quantitative polymerase chain reaction assays for Arg-1 transcripts and measured urea concentrations19 on pooled lysates from bead-sorted MDSCs. MDSCs from control subjects had minimal Arg-1 gene expression and activity, while those from patients with PH showed enhanced expression and activity of Arg-1 (Figs 3D-3E).

Figure Jump LinkFigure 3. Photomicrographs and charts display the expression and activity analysis of circulating MDSCs. A, Buffy coat cells were sorted using immunomagnetic beads conjugated to anti-CD33, CD11b, and MHCII antibodies. Cells were pooled (n = three per group) and probed using antibodies against CD33 or CD11b (red, Cy3) and either MHCII or pSTAT3 (green, Alexa-488). Neither CD33+ cells (B) nor CD11b+ cells (C) appreciably coexpress MHCII. D, Cells from patients with PH, but not cells from control subjects, contained detectable pSTAT3. E, Pooled (n = five per group) sorted CD33+/CD11b+/MHCII cells were analyzed using quantitative polymerase chain reaction assays for arginase-1 (Arg-1) mRNA. Patients with PH had increased Arg-1 levels compared with control subjects, normalized to the housekeeping gene HPRT. F, Arginase activity, as measured by intracellular urea concentration, was significantly enhanced in MDSCs from patients with PH compared with pooled control subjects. For F, the data represent the mean ± SD, n = 21 samples from patients with PH and n = seven samples from control subjects that were pooled following bead separation, from a representative group of three independently performed experiments. White scale bar = 10 μm. CTL = control sample; DAPI = 4ʹ,6-diamidino-2-phenylindole; n.d. = below level of detection. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location

We have previously shown that cells of mononuclear phagocytic origin accumulate in peribronchovascular regions in animal models of PH and contribute to vascular remodeling.4 Others have described immature myeloid cells in similar regions in humans and in animal models, often in proximity with T and B lymphocytes.7 Furthermore, observations of impaired DC function6 and increases in T regulatory (Treg) lymphocytes12,13 convinced us to test whether MDSC levels would be elevated in patients with PH compared with control subjects. We demonstrate an increase in circulating CD45+/CD33+/MHCII/CD11b+ MDSCs in patients with PH compared with control subjects. We found correlation between MDSC counts and mean pulmonary artery pressure. Our observations support the notion that the mobilization of MDSCs contributes to pathologic pulmonary vascular remodeling, since MDSCs are potent immunomodulatory cells functionally implicated in the pathobiologic aspects of chronic inflammatory disorders, cancer, and immunosuppression phenomena. This work extends (in humans) our previous findings of, in hypoxic models of PH, an abundance of circulating mononuclear cells that also appear in the pulmonary vasculature, are actively proliferating, and are profibrotic and proinflammatory. The current study identifies a unique subset of circulating mononuclear cells as myeloid suppressor cells with an activated phenotype.

MDSCs are a heterogeneous population of cells, and local contexts of inflammatory microenvironments greatly affect their tissue recruitment, retention, and immunomodulatory capabilities.25 There are significant differences between the pulmonary microvasculature and larger lung vessels in terms of cell constituency and specialization of cell function.26 Microenvironmental cues in these compartments may facilitate the recruitment and differentiation of unique MDSC subsets. Wang et al6 observed decreased levels of blood myeloid DCs in patients with PH compared with control subjects, which would appear to be in conflict with our data showing increased levels of MDSCs in patients with PH. However, the myeloid DCs in their study were HLA-DR+/CD11c+ cells, while we focused on MDSCs defined by several groups10 as HLA-DR/CD11b+. Furthermore, Perros et al7 determined that immature myeloid cells in lungs from the monocrotaline rat model of PH had an OX-62+/CD68/MHCII(HLA-DR)/CD11b+ phenotype characteristic of MDSC. Thus, unique circulating subsets of immature myeloid cells could vary in number and/or simultaneously differentiate and home in to distinct lung niches.

To effect immunosuppression, MDSCs act in proximity to T cells, perhaps requiring direct contact.27 Although we did not directly address MDSC-mediated immunosuppression, several lines of evidence offer clues as to how this might occur in the setting of PH. First, plasma IL-6 levels appear to be elevated in PH,28 and overexpression of IL-6 in mice induces PH.29 IL-6 potently activates pSTAT3 and augments survival in MDSCs,24 and we found increased pSTAT3 in MDSCs from patients with PH compared with control subjects. Second, expression of S100 proteins such as S100A4 contributes to cell motility and plexogenic arteriopathy in PH.30 Interestingly, the S100 family of inflammatory mediators, particularly S100A8/A9, participates in an autocrine feedback loop that sustains accumulation of MDSCs in mouse tumor models.31 Once retained in the lung, MDSCs may be able to induce FOXP3+ Treg lymphocytes, as they do in several models of inflammation and cancer.32 Two groups showed increased Treg cells in the peripheral blood of patients with idiopathic pulmonary arterial hypertension.12,13 Finally, while IL-4 upregulates immunosuppressive activity of MDSCs via Arg-1,33 it also drives Th2 immune responses in PH.23 Arg-1, in turn, is important for the development of secondary lymphoid organs such as bronchus-associated lymphoid tissues,34 which are critical sites for immune tolerance and self-antigen presentation potentially involving MDSCs. Intriguingly, bronchus-associated lymphoid tissues are more conspicuous in the lung tissues of humans with PH13 and in lungs from both the chronic hypoxia and monocrotaline rat models of PH (Michael E. Yeager, PhD, February 2011, unpublished data). Important next steps will be to determine whether the MDSCs in patients with PH are similar (or different) than the MDSCs in patients with cancers and autoimmune diseases, whether increases in blood MDSCs translate to increases in lung MDSCs, and what are the mechanisms of influx and retention.

In cancer, MDSCs inhibit innate and adaptive immunity and subvert the elimination of transformed cells.35 MDSCs suppress CD4+ and CD8+ T cells by mechanisms involving arginase, nitric oxide, and local depletion of cysteine.8 Further, MDSCs induce Treg cells, which then depress cell-mediated immunity,36 and they skew local immune responses from a T helper 1 to a T helper 2 program by IL-10 secretion.16 Last, MDSCs promote angiogenesis in models of cancer.37,38 In the context of PH associated with autoimmunity, MDSCs may be required to delimit the activation of autoreactive T and B cells. Some forms of PH have an autoimmune component, including production of autoantibodies.22,39 Our data showing increased levels of MDSCs in female patients with PH compared with male patients with PH may be related to the increased predilection for female patients to develop autoimmune disorders as well as PH.

Several potential limitations of our study should be pointed out. First, although our finding of increased activated MDSCs is novel, we lack lung-specific data. Therefore, the increase in MDSCs may be associative and not causal. We were unable to confirm the presence of MDSCs in the lungs of our patient cohort as a result of the scarcity of available tissue. Second, MDSCs are admittedly a heterogeneous population. Therefore, the marker set we chose may not encompass the chief subset (or subsets) of MDSCs influencing inflammation in the lungs of patients with PH. Third, we did not directly use assays to test for the immunosuppressive capability of the MDSCs because we had insufficient numbers to carry out such assays.

All-trans-retinoic acid has been used to attempt to mature MDSCs to attenuate their immunosuppressive function in patients with cancer.10 In the face of the extensive vascular remodeling present in patients with PH, it seems unlikely that therapeutically “braking” or modulating persistent inflammation would, by itself, lead to the restoration of normal lung vasculature. A better understanding of which subsets of mononuclear phagocytic cells (eg, fibrocytes, MDSCs, etc) are contributing to the pathologic characteristics of PH, and by what mechanisms, should greatly facilitate the future success of unique combinatorial therapies. Future studies will determine the extent of MDSC infiltration in the lungs of patients with PH, and using animal models, we will attempt to unravel their putative mechanistic contribution to inflammation-associated vascular remodeling, with particular emphasis on immune suppression phenomena.

Author contributions: Dr Yeager is the guarantor of the paper and takes responsibility for the integrity of the work as a whole, from inception to published article.

Dr Yeager: conceived of the study, conducted experiments, interpreted data, wrote and revised the manuscript, and approved the final article for publication.

Ms Nguyen: conducted experiments, helped draft and revise the manuscript, and approved the final article for publication.

Mr Belchenko: conducted experiments, helped draft and revise the manuscript, and approved the final article for publication.

Ms Colvin: conducted experiments, helped draft and revise the manuscript, and approved the final article for publication.

Dr Takatsuki: conducted experiments, helped draft and revise the manuscript, and approved the final article for publication.

Dr Ivy: provided patient access, interpreted data, provided funding, helped draft and revise the manuscript, and approved the final article for publication.

Dr Stenmark: interpreted data, provided funding, helped draft and revise the manuscript, and approved the final article for publication.

Financial/nonfinancial disclosures: The authors have reported to CHEST the following conflicts of interest: The University of Colorado, Denver has a contractual relationship with the pharmaceutical industry (Actelion, Gilead, Pfizer, United Therapeutics) for Dr Ivy to provide consultation. All monies are sent directly to the University of Colorado, Denver. Dr Stenmark has received pharmaceutical company grant monies from Pfizer. Drs Yeager and Takatsuki, Mss Nguyen and Colvin, and Mr Belchenko have reported that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Role of sponsors: The sponsor had no role in the design of the study, the collection and analysis of the data, or in the preparation of the manuscript.

Other contributions: We especially acknowledge the generosity and kindness of Robert Strieter, MD, in whose laboratory we learned the techniques used in this report. We also thank Marie Burdick, BS, from the Strieter Laboratory for teaching and graciously hosting us. We thank the University of Colorado Cancer Center Flow Cytometry Core for technical assistance.

Arg-1

arginase-1

DC

dendritic cell

MDSC

myeloid-derived suppressor cell

PH

pulmonary hypertension

Treg

T regulatory

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Rodrigues JC, Gonzalez GC, Zhang L, et al. Normal human monocytes exposed to glioma cells acquire myeloid-derived suppressor cell-like properties. Neuro Oncol. 2010;124:351-365 [CrossRef] [PubMed]
 
Humbert M, Monti G, Brenot F, et al. Increased interleukin-1 and interleukin-6 serum concentrations in severe primary pulmonary hypertension. Am J Respir Crit Care Med. 1995;1515:1628-1631 [PubMed]
 
Steiner MK, Syrkina OL, Kolliputi N, Mark EJ, Hales CA, Waxman AB. Interleukin-6 overexpression induces pulmonary hypertension. Circ Res. 2009;1042:236-244 [CrossRef] [PubMed]
 
Greenway S, van Suylen RJ, Du Marchie Sarvaas G, et al. S100A4/Mts1 produces murine pulmonary artery changes resembling plexogenic arteriopathy and is increased in human plexogenic arteriopathy. Am J Pathol. 2004;1641:253-262 [CrossRef] [PubMed]
 
Sinha P, Okoro C, Foell D, Freeze HH, Ostrand-Rosenberg S, Srikrishna G. Proinflammatory S100 proteins regulate the accumulation of myeloid-derived suppressor cells. J Immunol. 2008;1817:4666-4675 [PubMed]
 
Huang B, Pan PY, Li Q, et al. Gr-1+CD115+ immature myeloid suppressor cells mediate the development of tumor-induced T regulatory cells and T-cell anergy in tumor-bearing host. Cancer Res. 2006;662:1123-1131 [CrossRef] [PubMed]
 
Popovic PJ, Zeh HJ III, Ochoa JB. Arginine and immunity. J Nutr. 2007;1376 suppl 2:1681S-1686S [PubMed]
 
Enioutina EY, Bareyan D, Daynes RA. A role for immature myeloid cells in immune senescence. J Immunol. 2011;1862:697-707 [CrossRef] [PubMed]
 
Ostrand-Rosenberg S, Sinha P. Myeloid-derived suppressor cells: Linking inflammation and cancer. J Immunol. 2009;1828:4499-4506 [CrossRef] [PubMed]
 
Serafini P, Mgebroff S, Noonan K, Borrello I. Myeloid-derived suppressor cells promote cross-tolerance in B-cell lymphoma by expanding regulatory T cells. Cancer Res. 2008;6813:5439-5449 [CrossRef] [PubMed]
 
Gerber HP, Olazoglu E, Grewal IS. Targeting inflammatory cells to improve anti-VEGF therapies in oncology. Recent Results Cancer Res. 2010;180:185-200 [PubMed]
 
Bierie B, Moses HL. Transforming growth factor beta (TGF-beta) and inflammation in cancer. Cytokine Growth Factor Rev. 2010;211:49-59 [CrossRef] [PubMed]
 
Terrier B, Tamby MC, Camoin L, et al. Identification of target antigens of antifibroblast antibodies in pulmonary arterial hypertension. Am J Respir Crit Care Med. 2008;17710:1128-1134 [CrossRef] [PubMed]
 

Figures

Figure Jump LinkFigure 1. Scatter diagrams show flow cytometric analysis for myeloid-derived suppressor cells (MDSCs). A, Side scatter (SSC) vs forward scatter (FSC) analysis of ≥ 200,000 cells from representative control subject blood sample. B, SSC vs CD45 Pacific Blue (PB) isotype sample from control subject for gating. C, Isotype control used to set gating for the CD45+/CD33+ population from control subject blood sample. D, Isotype control used to set gating for the MHCII/CD11b+ population from control subject blood sample. E, This control subject had 0.5% of total CD45+ cells labeling as MHCII/CD11b+ (quadrant B4), corresponding to 0.17 × 106 cells/mL. F, Isotype control used to set gating for the MHCII/CD11b+ population from blood sample of patient with pulmonary hypertension (PH). G, This patient with PH had 2.1% of total CD45+ cells labeling as MHCII/CD11b+ (quadrant B4), corresponding to 0.24 × 106 cells/mL. Isotype controls were performed to set gates on each sample from control subjects and patients with PH, as described by Mathai et al,15 and all samples were run in triplicate. APC = allophycocyanin; FITC = fluorescein isothiocyanate; FL = channel; FS = forward scatter; INT LIN = integration linear; INT LOG = integration logarithmic; PE = phycoerythrin; SS = side scatter.Grahic Jump Location
Figure Jump LinkFigure 2. Scatter diagrams show that circulating MDSC counts are enriched in patients with PH compared with control subjects and correlate to mean pulmonary artery pressure. A and B, Mean MDSC counts in 26 patients with PH were mean, 0.271 × 106 cells/mL ± 0.17, 1.86% ± 1.51%; and counts in 10 control subjects were mean, 0.176 × 106 cells/mL ± 0.05; 0.57% ± 0.29%; P < .05 vs control subjects. C and D, Female subjects with PH (n = 12; mean, 0.32 × 106 cells/mL ± 0.19; 2.68% ± 1.72%) had higher MDSC counts than male subjects with PH (n = 14; mean, 0.23 × 106 cells/mL ± 0.13; 1.36% ± 0.97%); P < .05 vs control for percentage. E, MDSC numbers per milliliter in patients with PH positively correlate to mean PAP. (r = 0.510; P < .05 vs control subjects). PAP = pulmonary artery pressure. See Table 1 legend for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 3. Photomicrographs and charts display the expression and activity analysis of circulating MDSCs. A, Buffy coat cells were sorted using immunomagnetic beads conjugated to anti-CD33, CD11b, and MHCII antibodies. Cells were pooled (n = three per group) and probed using antibodies against CD33 or CD11b (red, Cy3) and either MHCII or pSTAT3 (green, Alexa-488). Neither CD33+ cells (B) nor CD11b+ cells (C) appreciably coexpress MHCII. D, Cells from patients with PH, but not cells from control subjects, contained detectable pSTAT3. E, Pooled (n = five per group) sorted CD33+/CD11b+/MHCII cells were analyzed using quantitative polymerase chain reaction assays for arginase-1 (Arg-1) mRNA. Patients with PH had increased Arg-1 levels compared with control subjects, normalized to the housekeeping gene HPRT. F, Arginase activity, as measured by intracellular urea concentration, was significantly enhanced in MDSCs from patients with PH compared with pooled control subjects. For F, the data represent the mean ± SD, n = 21 samples from patients with PH and n = seven samples from control subjects that were pooled following bead separation, from a representative group of three independently performed experiments. White scale bar = 10 μm. CTL = control sample; DAPI = 4ʹ,6-diamidino-2-phenylindole; n.d. = below level of detection. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location

Tables

Table Graphic Jump Location
Table 1 —Data for Subjects With PH

Values are presented as mean ± SD. F = female; iPAH = idiopathic pulmonary arterial hypertension; M = male; MDSC = myeloid derived suppressor cell; mPAP = mean pulmonary artery pressure; n/a = not catheterized at the time; PH = pulmonary hypertension; + = medication taken; sPAH = secondary pulmonary arterial hypertension; WHO = World Health Organization.

Table Graphic Jump Location
Table 2 —Data for Control Subjects

Values are presented as mean ± SD. SVT = supra ventricular tachycardia; VSD = ventricular septal defect; WPW = Wolff-Parkinson-White syndrome. See Table 1 legend for expansion of other abbreviations.

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Nefedova Y, Nagaraj S, Rosenbauer A, Muro-Cacho C, Sebti SM, Gabrilovich DI. Regulation of dendritic cell differentiation and antitumor immune response in cancer by pharmacologic-selective inhibition of the janus-activated kinase 2/signal transducers and activators of transcription 3 pathway. Cancer Res. 2005;6520:9525-9535 [CrossRef] [PubMed]
 
Dolcetti L, Peranzoni E, Ugel S, et al. Hierarchy of immunosuppressive strength among myeloid-derived suppressor cell subsets is determined by GM-CSF. Eur J Immunol. 2010;401:22-35 [CrossRef] [PubMed]
 
Cioffi DL, Lowe K, Alvarez DF, Barry C, Stevens T. TRPing on the lung endothelium: Calcium channels that regulate barrier function. Antioxid Redox Signal. 2009;114:765-776 [CrossRef] [PubMed]
 
Rodrigues JC, Gonzalez GC, Zhang L, et al. Normal human monocytes exposed to glioma cells acquire myeloid-derived suppressor cell-like properties. Neuro Oncol. 2010;124:351-365 [CrossRef] [PubMed]
 
Humbert M, Monti G, Brenot F, et al. Increased interleukin-1 and interleukin-6 serum concentrations in severe primary pulmonary hypertension. Am J Respir Crit Care Med. 1995;1515:1628-1631 [PubMed]
 
Steiner MK, Syrkina OL, Kolliputi N, Mark EJ, Hales CA, Waxman AB. Interleukin-6 overexpression induces pulmonary hypertension. Circ Res. 2009;1042:236-244 [CrossRef] [PubMed]
 
Greenway S, van Suylen RJ, Du Marchie Sarvaas G, et al. S100A4/Mts1 produces murine pulmonary artery changes resembling plexogenic arteriopathy and is increased in human plexogenic arteriopathy. Am J Pathol. 2004;1641:253-262 [CrossRef] [PubMed]
 
Sinha P, Okoro C, Foell D, Freeze HH, Ostrand-Rosenberg S, Srikrishna G. Proinflammatory S100 proteins regulate the accumulation of myeloid-derived suppressor cells. J Immunol. 2008;1817:4666-4675 [PubMed]
 
Huang B, Pan PY, Li Q, et al. Gr-1+CD115+ immature myeloid suppressor cells mediate the development of tumor-induced T regulatory cells and T-cell anergy in tumor-bearing host. Cancer Res. 2006;662:1123-1131 [CrossRef] [PubMed]
 
Popovic PJ, Zeh HJ III, Ochoa JB. Arginine and immunity. J Nutr. 2007;1376 suppl 2:1681S-1686S [PubMed]
 
Enioutina EY, Bareyan D, Daynes RA. A role for immature myeloid cells in immune senescence. J Immunol. 2011;1862:697-707 [CrossRef] [PubMed]
 
Ostrand-Rosenberg S, Sinha P. Myeloid-derived suppressor cells: Linking inflammation and cancer. J Immunol. 2009;1828:4499-4506 [CrossRef] [PubMed]
 
Serafini P, Mgebroff S, Noonan K, Borrello I. Myeloid-derived suppressor cells promote cross-tolerance in B-cell lymphoma by expanding regulatory T cells. Cancer Res. 2008;6813:5439-5449 [CrossRef] [PubMed]
 
Gerber HP, Olazoglu E, Grewal IS. Targeting inflammatory cells to improve anti-VEGF therapies in oncology. Recent Results Cancer Res. 2010;180:185-200 [PubMed]
 
Bierie B, Moses HL. Transforming growth factor beta (TGF-beta) and inflammation in cancer. Cytokine Growth Factor Rev. 2010;211:49-59 [CrossRef] [PubMed]
 
Terrier B, Tamby MC, Camoin L, et al. Identification of target antigens of antifibroblast antibodies in pulmonary arterial hypertension. Am J Respir Crit Care Med. 2008;17710:1128-1134 [CrossRef] [PubMed]
 
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