The authors have declared that no competing interests exist.
Conceived and designed the experiments: TC IM PK. Performed the experiments: TC. Analyzed the data: TC MM GT IM PK. Wrote the paper: TC IM PK.
The mechanisms resulting in progressive immune dysfunction during the chronic phase of HIV infection are not fully understood. We have previously shown that arginase, an enzyme with potent immunosuppressive properties, is increased in HIV seropositive (HIV+) patients with low CD4+ T cell counts. Here we show that the cells expressing arginase in peripheral blood mononuclear cells of HIV+ patients are low-density granulocytes (LDGs) and that whereas these cells have a similar morphology to normal-density granulocyte, they are phenotypically different. Importantly, our results reveal that increased frequencies of LDGs correlate with disease severity in HIV+ patients.
Since the initial report of AIDS in 1981, approximately 60 million people have become infected with HIV of which more than 30 million have died. Although depletion of CD4+ T cells explains much of the immune suppression in HIV infected individuals, the precise reasons for the onset of immunopathology during HIV infection are not yet fully understood
Arginase, an enzyme of the urea cycle, can also be expressed in cells of the immune system and has been shown to exert potent immunoregulatory functions: a reduction in the bioavailability of L-arginine by arginase results in impaired T cell responses, characterized by down-regulation of T cell proliferation, reduced expression of CD3ζ and cytokine production
Transport of extracellular L-arginine into the cells by cationic amino acid transporter (CAT)2B to make it accessible for catabolization by arginase results in depletion of L-arginine in the microenvironment
Arginase can be released by neutrophils into the extracellular milieu, where it binds L-arginine and thereby reduces the level of free L-arginine available to T cells
We have previously shown that arginase activity is higher in peripheral blood mononuclear cells (PBMCs) isolated from HIV-1 infected individuals with low CD4+ T cell counts and that this coincided with lower levels of L-arginine
Thirtytwo HIV seropositive (HIV+) treatment-naïve individuals (mean age 42.1±12.2 years) were recruited from St Mary’s Hospital and 11 healthy volunteers (mean age 35±4.7 years) were recruited as control subjects. The study was approved by the National Research Ethics Service (05/Q0410/93) and all individuals gave written, informed consent before participation.
Twenty ml of peripheral blood was collected in EDTA tubes and PBMCs were isolated by density gradient centrifugation on Histopaque®-1077 (Sigma). Neutrophils were isolated from the erythrocyte fraction by dextran sulphate sedimentation
The following antibodies were used: CD14FITC, CD15PE (BD Pharmingen), Arginase1Alexa Fluor® 647 (Hycult Biotechnology), CD11bPerCP-eFluro710, CD16eFluro450, CD33PE-Cy7 (eBioscience), CD13APC-Cy7 (Biolegend), CD66bFITC and CD63FITC (Beckman Coulter) (
Antigen | Fluorophore | Clone |
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CD15 | PE | H198 | mouse IgM | BD Pharmingen | 555402 | 20 | NP |
CD11b | PerCP-eFluor710 | ICRF44 | mouse IgG1 | eBioscience | 46–0118 | 3 | 0.075 |
CD13 | APC-Cy7 | WM15 | mouse IgG1 | Biolegend | 301710 | 7 | NP |
CD16 | eFluor 450 | eBioCB16 | mouse IgG1 | eBioscience | 48–0168 | 3 | 0.15 |
CD33 | PE-Cy7 | WM-53 | mouse IgG1 | eBioscience | 25–0338 | 3 | 0.15 |
CD66b | FITC | 80H3 | mouse IgG1 | Beckman Coulter | IM05310 | 3 | NP |
LDGs and NDGs were isolated as described in materials and methods and the expression levels of phenotypic markers were determined by flow cytometry.
NP = Not provided by manufacturer.
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CD15 | PE | H198 | mouse IgM | BD Pharmingen | 555402 | 20 | NP |
CD13 | APC-Cy7 | WM15 | mouse IgG1 | Biolegend | 301710 | 7 | NP |
CD16 | eFluor 450 | eBioCB16 | mouse IgG1 | eBioscience | 48–0168 | 3 | 0.15 |
CD63 | FITC | CLBGran/12 | mouse IgG1 | Beckman Coulter | IM1165U | 14 | NP |
Arginase 1 | Alexa Fluor® 647 | 6G3 | mouse IgG1 | Hycult biotech. | HM2162 | 2.75 | 0.275 |
LDGs and NDGs were isolated as described in materials and methods and the expression levels of phenotypic markers were determined by flow cytometry.
NP = Not provided by manufacturer.
CD3+ and CD14+ cells were removed by positive selection, CD15+ cells were incubated with anti-CD15PE and selected using EasySep PE Positive Selection Kit (EasySep, Stem Cell Technologies, France). Purity of CD15+ cells was checked by flow cytometry (>95%). The remaining cells were transferred onto a microscope slide (Thermo Scientific, United Kingdom) using a cytospin centrifuge and stained with hematoxylin and eosin (H&E) (Reagena, Gamidor, United Kingdom).
LDGs and NDGs were isolated as described in materials and methods and their morphology was compared after H&E staining. Data show the results of one representative experiment out of five independent experiments.
LDGs and NDGs were isolated as described in materials and methods (n = 22) and the expression levels of CD11b (A), CD15 (B), CD33 (C), CD66b (D), CD16 (E), CD13 (F), CD63 (G) and arginase 1 (H) were determined by flow cytometry. Isotype controls: <1%. Statistical significance was determined by a two-tailed Mann-Whitney test. Box = interquartile range and median; whiskers = range.
Data were evaluated for statistical differences using a Wilcoxon signed rank test, a two-tailed Mann-Whitney test and a Spearman’s rank test when appropriate (GraphPad Prism 5); differences were considered statistically significant at
Controls | Controls | HIV+ patients | ||
Phenotypic markers | LDGs (MFI) | NDGs (MFI) | % change | % change |
CD11b | 3267±587 | 4334±183 | −6.9±10.4 | 29.0±9.2% |
CD13 | 305±200 | 2196±167 | −65.4±11.0 | −7.5±10.0% |
CD15 | 5313±270 | 2267±201 | 106.1±12.2 | 239.4±46.6% |
CD16 | 384±81 | 15678±1197 | −97.2±0.4 | −46.4±8.4% |
CD33 | 1385±327 | 506±95 | 176.4±19.1 | 82.8±12.1% |
CD66b | 11954±929 | 8393±326 | 58.0±9.7 | 61.2±17.3 |
CD63 | 2077±129 | 987±68 | 111.8±18.7 | 84.7±19.3 |
Arginase 1 | 11899±592 | 16463±884 | −29.5±5.1 | −22.8±4.5 |
LDGs and NDGs were isolated as described in materials and methods and the expression levels of phenotypic markers were determined by flow cytometry (median±SEM). The percentage increase or decrease in MFI was calculated for controls (n = 11) and HIV+ patients (
We have previously shown that arginase activity is significantly higher in PBMCs of HIV+ individuals with low CD4+ T cell counts. Furthermore, we showed that the cells expressing arginase in the PBMC fraction are granulocytes
To establish whether this phenotype differs from that of healthy individuals, we measured the expression levels of the same panel of cell surface markers on LDGs and NDGs from healthy individuals. As shown in
The blood from the controls and HIV+ patient with high or low CD4+ T cell counts was processed immediately and in the exact same way, therefore excluding that any differences observed were due to the handling procedure.
These results show that NDGs and LDGs are morphologically mature neutrophils, but that they differ in the expression level of markers of polymorphonuclear cells.
In neutrophils, arginase is localized in azurophilic granules
Markers of disease severity in HIV-1 infection include CD4+ T cell count and viral load. Consequently we assessed whether the frequency of LDGs in healthy controls and HIV patients correlated with markers of disease severity. First, we compared the frequency of LDGs in healthy controls and HIV patients. The frequency of LDG was significantly lower in healthy controls as compared to HIV+ patients with CD4+ T cell counts >350 cells/µl (0.24±0.3 vs 0.60±0.52,
PBMCs from HIV+ patients with CD4+ T cell counts >350 (n = 14) or <350 cells/µL (n = 18) were isolated by Ficoll gradient and the frequency of CD15+ arginase+ cells was determined by flow cytometry, statistical significance was determined by a two-tailed Mann-Whitney test (A). Correlation between %LDGs and CD4+ T cell counts (B) or viral load (C), statistical significance was determined by a Spearman’s rank test. Isotype controls: <1%.
Arginase-induced L-arginine catabolism is a well-established mechanism of immune suppression
Following density gradient centrifugation, LDGs co-purify in the PBMC fraction, whereas NDGs sediment with erythrocytes. This difference could result from i) different degrees of maturation and ii) different activation and degranulation states:
During neutrophil maturation there is a change in expression of surface antigens
The degree of activation of neutrophils and degranulation depends on the strength of the activating signal: the order of granule release follows a strict hierarchy requiring increasing activation: 1) secretory granules; 2) gelatinous (tertiary) granules; 3) specific (secondary) granules and 4) azurophilic (primary) granules. From the panel of markers used in the present study, the following markers were significantly increased on LDGs: CD11b, present in the membrane of secretory vesicles, gelatinase granules and specific granules; CD63, found in the membrane of azurophilic granules; and CD66b, detected in the membrane of specific granules. Further, the intensity of arginase, which is present in azurophilic granules
The presence of CD15+ granulocytes in the PBMC fraction has been demonstrated in a number of different conditions including cancer, pregnancy, trauma and SLE, each of which is frequently accompanied by a degree of immune suppression. We have shown that arginase-induced L-arginine depletion and the subsequent T cell inhibition is a mechanism of immune suppression that is restricted to the site of pathology
The impact of arginase-induced L-arginine depletion on T cell effector functions has been well established
More work is needed to understand the causes of the observed neutrophil activation and the subsequent release of arginase, as it is not yet not possible to answer whether it is the virus or the immune system or a combination of both that accounts for the increased frequency of activated low-density granulocytes in HIV+ patients. A better understanding of the complex mechanisms leading to progressively impaired immune functions might prove helpful in improving the existing treatment not only for HIV+ individuals, but also for other chronic infectious diseases such as tuberculosis and leishmaniasis, in which a degree of immune suppression may be caused by abnormal arginase activity.
We thank Drs E. Riley, M. Simon, C. Bangham and S. Herath for helpful discussions and critical reading of the manuscript.