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IL-13 Acutely Augments HIV-Specific and Recall Responses from HIV-1-Infected Subjects In Vitro by Modulating Monocytes¹

Emmanouil Papasavvas^*, Junwei Sun^*, Qi Luo^*, Elizabeth C. Moore^*, Brian Thiel^*, Rob Roy MacGregor, Adrian Minty, Karam Mounzer, Jay R. Kostman^, and Luis J. Montaner^2,*

^* The Wistar Institute, Philadelphia, PA 19104; Infectious Diseases Division, University of Pennsylvania, Philadelphia, PA 19104; Molecular and Functional Genomics Department, Sanofi-Synthelabo Recherche, Labege, France; and Philadelphia Field Initiating Group for HIV-1 Trials, Philadelphia, PA 19107

	Abstract

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We show in this study that acute exposure of PBMCs derived from HIV-infected subjects to IL-13 results in increased recall T cell lymphoproliferative responses against HIV-1 p24 (n = 30, p < 0.0001) and other recall Ags (influenza, n = 43, p < 0.0001; purified protein derivative tuberculin, n = 6, p = 0.0299). This effect is due to a mechanism that acutely targets APC function in the adherent monocyte subset, as shown by the expansion of CD4⁺ T cell responses following coculture of IL-13-treated enriched CD14⁺ monocytes with donor-matched enriched CD4⁺ T cells and Ag. Exposure to IL-13 over 18–72 h resulted in a significant enhancement of monocyte endocytosis (n = 11, p = 0.0005), CD86 expression (n = 12, p = 0.001), and a significant decrease in spontaneous apoptosis (n = 8, p = 0.008). Moreover, IL-13 exposure induced a significant decrease of significantly elevated constitutive levels of PBMC-secreted TNF- (n = 14, p < 0.001) and IL-10 (n = 29, p < 0.001) within 18 h of exposure ex vivo, also reflected by decreased gene expression in the adherent cell population. Our data show that IL-13 is able to acutely enhance the function of the CD14⁺ cell subset toward supporting Ag-specific cell-mediated responses in chronic HIV-1 infection.

	Introduction

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Immune dysfunction in HIV-1 infection is associated with a loss of CD4⁺ T cell lymphoproliferative responses (1) in association with dysregulation of Ag presentation function to include impaired costimulatory molecule expression (CD80/CD86) (2) and cytokine production (3, 4, 5). Decreased levels of functional cell-mediated responses against HIV-1 and other recall Ags have been associated with delayed disease progression and remain a target of antiretroviral adjunct immunotherapy (6). However, direct modulation of Ag presentation with or without antiretroviral therapy has not been largely pursued by current immune-based therapy approaches, despite its central role during an immune response and its well-characterized impairment in HIV-1 infection (7).

IL-13 is secreted by activated T cells, basophils, mast cells, and NK cells (8). IL-13, unlike IL-4 or IL-10, has no direct regulation on T cell function (9) and modulates human monocytes/macrophages and B cells (10, 11). Regarding modulation of human monocytes/macrophages, exposure to IL-13 results in an increase of MHC class II expression (12), priming for increased IL-12 secretion upon stimulation (13), and direct inhibition of inflammatory cytokines such as TNF- and IL-1 (11, 14). In HIV-1 infection, secretion of IL-13 by CD4⁺ and CD8⁺ T cell subsets following anti-CD3/anti-CD28 stimulation ex vivo is decreased in parallel with IFN- secretion and the loss of CD4⁺ T cell count <500 (15). In vitro studies have also shown that IL-13 inhibits HIV-1 in monocyte-derived macrophages (16, 17) by blocking the completion of reverse transcription, decreasing virus production, and reducing the infectivity of progeny virus (17). Based on its anti-inflammatory activity and Ag presentation function modulation properties, we addressed the potential that a single short-term exposure of PBMC from HIV-infected persons to IL-13 would enhance the Ag presentation function of adherent monocytes and result in enhanced recall lymphoproliferative responses. We show that IL-13 significantly increases HIV-specific and recall CD4⁺ T cell-mediated responses in HIV-1 infection independently of IL-12 and in association with modulation of monocyte Ag presentation function, as reflected by effects on cell surface molecule expression, Ag uptake, apoptosis, and cytokine secretion.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Participants

A total number of 68 HIV^– and 175 HIV⁺ age-matched subjects participated in the study. As defined in Results, not all subjects were used among all assays performed. Regarding the HIV⁺ subjects in total, 73% were male and 19% were female; median age was 46 years (25th-75th interquartile (IQR):³ 42–53), while median year of HIV infection diagnosis was 1993 (25th-75th IQR: 1990–1996). Ethnic distribution for HIV⁺ subjects was 64.5% African American, 21% Caucasian, 6% Hispanic, and 0.5% Native American. Sixty-eight percent of patients were confirmed on antiretroviral therapy. Apart from six HIV⁺ subjects with confirmed tuberculosis, as described in Results, no additional information for opportunistic infection was available on subjects. Median CD4 count for the cohort was 392 cells/µl (25th-75th IQR: 232–604), while median plasma HIV-1 RNA was 1500 copies/ml (25th-75th IQR: 400–14,973). HIV-1-uninfected controls were derived from the Wistar Institute phlebotomy center that recruits/monitors HIV-1 and hepatitis C seronegative subjects without any known complicating health conditions. Demographic data for the HIV^– subjects used were as following: 54% were male, 69% African American, 23% Caucasian, 8% Hispanic, and 0% Native American.

Human experimentation guidelines of the US Department of Health and Human Services and the authors’ institutions were followed. All subjects provided signed informed consent. Venous peripheral blood of HIV^– and HIV⁺ subjects was collected in a cross-sectional pattern. Plasma HIV-1 RNA and CD4⁺ T cell count were determined by Quest Diagnostic at the time of blood collection.

Cell preparation

PBMC were isolated by standard Ficoll-Hypaque (Pharmacia), as previously described (18). Monocytes were prepared from PBMC by adherence to plastic and extensive washings to remove nonadherent cells, as previously described (19, 20). Unless indicated otherwise, PBL were removed by extensive washings following a 1 h culture of PBMC over tissue culture-treated plastic at 37°C. Adherent cells were subsequently harvested with cold 1xPBS and 45 mM EDTA and phenotyped for CD14 purity levels before each experiment. Routinely, we observed CD14 levels >87% in adherent fraction with <3% CD3⁺ T cells.

As indicated in Results, parallel enrichment for CD3⁺CD4⁺ T cells and CD14⁺ cells was performed for 11 separate donors by using the rosetteSep human CD4⁺ T cell and human monocyte enrichment mixtures (StemCell Technologies), according to the manufacturer’s instructions. The purity of the enriched populations was further confirmed by flow cytometry, as described below, showing preparations of CD3⁺CD4⁺ T cells enriched up to 92%, while same donor-matched CD14 enrichment was achieved to >82%.

Proliferation assays

Ag-specific proliferation was measured by lymphoproliferative assays (LPA) or by usage of CFSE.

Regarding LPA, PBMC (250,000 cells/well) were isolated and cultured on the day of collection in a six-replicate format, as previously described (18), in the presence or absence of IL-13 (20 ng/ml, as described by Montaner et al. (21)), added once and at the time of PBMC isolation along with no stimulation; positive control; Ag stimulation: 1) UV-inactivated influenza A virus PR8 H1/M1 (Flu) at 100 HAU/ml (Fisher UVXL-1000 UV Crosslinker, 1200 µW/cm², 15 min, 4°C), 2) purified protein derivative (PPD) tuberculin (5 µg/ml; Aventis Pasteur), 3) HIV-1 p24 (5 µg/ml; Protein Sciences) (18), 4) keyhole limpet hemocyanin (5 µg/ml; Sigma-Aldrich), or 5) PHA (5 µg/ml, positive control; Sigma-Aldrich). All Ags were added following 18 h PBMC isolation/IL-13 exposure. The number of Ags tested was decreased if PBMC yields were restricted, while PPD Ag was used only if a previous tuberculosis diagnosis was confirmed. Where indicated, neutralizing mouse anti-human IL-12 (10 µg/ml, in house production clone C8.6.2) was used in LPA. Analysis was done by stimulation index (SI) defined as: SI = Ag-stimulated mean cpm (with or without cytokine)/unstimulated mean cpm (with or without cytokine). An SI >3 was interpreted as positive.

For CD14⁺ and CD3⁺/CD4⁺ T cell enrichment experiments, LPA was performed in target cell populations pre-exposed or not to IL-13 (18 h, 20 ng/ml) and subsequently stimulated or not with Flu Ag, as indicated above. In addition, all IL-13-exposed cell populations were tested by either washout of cytokine at 18 h (time of addition of Ag) or its continuous presence. Briefly, the following conditions were tested: 1) CD3⁺/CD4⁺ T-enriched cells with or without IL-13 as negative controls, 2) CD14⁺-enriched cells with or without IL-13 as negative controls, 3) coculture of CD3⁺/CD4⁺ T-enriched cells without IL-13 and donor-matched CD14⁺-enriched cells without IL-13, and 4) coculture of CD3⁺/CD4⁺ T-enriched cells without IL-13 and donor-matched IL-13-exposed CD14⁺-enriched cells. All conditions were tested in the presence or absence of Flu (experimental conditions) using a similar method as that described for PBMC above. Cultures were initiated at a CD14⁺:CD3⁺/CD4⁺ T cell ratio as determined by CD14⁺ and CD3⁺/CD4⁺ cell subset ratio in donors’ PBMC at isolation. All conditions were tested in triplicate with a total cell content of 300,000 cells/well.

Regarding CFSE assays, measurement of proliferation was based on the Vybrant CFSE cell tracer kit (Molecular Probes). Briefly, cells were enriched for CD14⁺ and CD3⁺/CD4⁺ cell subsets; culture conditions were prepared, as described above; and cells were incubated with CFSE (1.5 µM) for 5 min at room temperature (RT) previous to Ag stimulation. At the end of the incubation with CFSE, 100 µl of FBS (Cansera) was added and cells were washed once with complete medium (RPMI 1640 (Mediatech) supplemented with 10% FBS, 100 U/ml penicillin/100 µg/ml streptomycin (Invitrogen Life Technologies), and 2 mM glutamine (Invitrogen Life Technologies)). Cells were resuspended at a final concentration of 10⁶ cells/ml and were either re-exposed or not to IL-13 (20 ng/ml), as described above, along with no stimulation or Ag stimulation (UV-inactivated Flu (100 HAU/ml)) for 4 days. Samples were analyzed by flow cytometry for cell-specific proliferative changes in the CD3⁺/CD4⁺ T cell subset via staining with Abs against CD14, CD4, and CD3, as described below. Live gates were set manually, and detection thresholds were set according to isotype-matched negative controls. Acquisition of data and analysis of CFSE dilution were performed on a DakoCytomation Cyan flow cytometer (DakoCytomation) using the FlowJo 4.5.9 (Tree Star) software package, as further described below.

Flow cytometry analysis

Flow cytometry was used for: 1) characterization of the cell subsets in proliferation assays described above, to assess CD3⁺/CD4⁺ T cell and CD14⁺ cell distribution in PBMC, as well as to assess purity of isolated adherent cells, PBL, CD3⁺/CD4⁺ T cell, and CD14⁺-enriched cell preparations; 2) measurement of cell surface expression of molecules associated with Ag presentation in adherent cell fractions derived from PBMC following culture for 18, 48, or 72 h with or without IL-13 (20 ng/ml added once and at the time of PBMC isolation); 3) cell-based quantification of endocytosis (further described below); and 4) cell-based measurements of apoptosis (further described below). Briefly, cells were washed with 1x PBS, blocked with blocking buffer (1x PBS, 0.1% gelatin, 0.1% NaN₃, 5% human AB serum, 5% mouse serum (Sigma-Aldrich)) for 15 min at RT, and then stained for 30 min at 4°C with the fluorochrome-conjugated mAbs (when needed, incubation with secondary Ab was done for an additional 30 min at RT and cells were washed three times in FACS washing buffer (1x PBS, 0.1% BSA, 0.02% NaN₃)). Following staining, cells were washed three times in FACS washing buffer, fixed in 1x PBS, 4% paraformaldeyde for 20 min at RT or in FACS Lyse (BD Biosciences) for 10 min at RT, washed once in FACS washing buffer, and resuspended in 100 µl of FACS washing buffer. Samples were analyzed as indicated on a BD Biosciences FACSCalibur flow cytometer using the CellQuest software package for acquisition and analysis, or on a DakoCytomation Cyan flow cytometer using the FlowJo 4.5.9 software package for acquisition and analysis. Either instrument was used consistently for data collection in respective applications of flow cytometry methods to avoid variability. Live cell gates were set manually, and detection thresholds were set according to isotype-matched negative controls. Results were expressed as mean fluorescent intensity (MFI) and percent positive.

The following anti-human mAbs from the following sources were used listed by source: 1) BD Biosciences: IgG1 CD8-allophycocyanin, IgG1 CD3-PE-Cy7, IgG1 CD4 allophycocyanin-Cy7, IgG2a CD14-PE, IgG2b HLA-DR-allophycocyanin, isotypes (mouse): IgG1-allophycocyanin, IgG1-PE-Cy7, IgG1-allophycocyanin-Cy7, and IgG2a-PE; 2) BD Pharmingen: IgG1 CD3-PE, IgG1 CD86-PE, IgG1 CD86-FITC, IgG1 CD40-TriColor, IgG2b HLA-DR-FITC, isotypes (mouse): IgG1-PE, IgG1-FITC, IgG2b-FITC, IgG1-allophycocyanin, and IgG1-TriColor; 3) Caltag Laboratories: IgG1 CD3-FITC, IgG1 CD14-FITC, IgG1 CD16-FITC, IgG1 CD19-FITC, IgG1-CD20-FITC, IgG1 HLA-DR-allophycocyanin, isotype (mouse) IgG1-FITC, and IgG2b-allophycocyanin; and 4) Beckman-Coulter: IgG1 HLA-DR-EnergyCoupledDye, IgG1 CD80, and isotype (mouse): IgG1-EnergyCoupledDye. Goat anti-mouse IgG-FITC (Sigma-Aldrich) was used as secondary Ab when needed.

Cytokine assays

Same day isolated PBMC (250,000 cells/well) were cultured with or without IL-13 (20 ng/ml, 18 h) before collection of cell-free supernatants. TNF- and IL-10 were measured by RIA in cell-free supernatants, as previously described, using the mAb pairs B154.9.2/B154.7.1 and 9D7/12G8, respectively (22, 23). Thresholds of TNF- and IL-10 detection were 1.69 and 0.8 pg/ml, respectively.

RNA isolation

PBMC from 11 HIV⁺ subjects under no antiretroviral therapy were isolated and PBL were removed following 1 h incubation at 37°C. For each of three experiments, adherent cells (with or without IL-13, 20 ng/ml, 5 h, 18 h) from three to four subjects were detached with cold 1x PBS and 45 mM EDTA and pooled together before isolation of nuclear RNA. Nuclear RNA was isolated using the TRI-REAGENT (Molecular Research Center), according to the manufacturer’s instructions.

RNase protection assay (RPA)

The Ambion’s RPA II kit (Ambion) with the hck2 and hck3 MultiProbe Template Set (BD Pharmingen) was used following the manufacturer’s instructions. Radioactivity was detected using the PhosphorImager 445 SI (Amersham Pharmacia Biotech). Data were analyzed using the IMAGEQUANT software version 5.0.

RT-PCR

cDNA was generated using Ambion’s RETROscript First-Strand Synthesis Kit for RT-PCR following the manufacturer’s instructions. Primers for human IL-10, the classic 18S primer set, and competitor primers were also obtained from Ambion. [³²P]dCTP was also included. Amplification was performed using a Peltier Model PTC 200 Thermal Cycler (MJ Research) and 0.2-ml thin-wall reaction tubes, as follows: 1) heat: 5 min, 95°C; 2) 23 cycles: 20 s, 94°C; 30 s, 56°C; and 40 s, 72°C; 3) hold: 5 min, 72°C. The amplified product was run on a 6% denaturing polyacrylamide gel, and radioactivity was detected using the PhosphorImager 445 SI. Data were analyzed using the IMAGEQUANT software version 5.0.

Cell population-based quantification of endocytosis

PBMC were isolated and PBL were removed following 1 h incubation at 37°C, as described. Adherent cells were cultured for 18, 48, or 72 h in the presence or absence of IL-13 (20 ng/ml, added once and at the initiation of the adherent cell culture). Endocytic uptake was measured with dextran Alexa 647 (Molecular Probes) or HRP (Sigma-Aldrich).

Measurement of cell-specific endocytosis by dextran Alexa 647 uptake was performed by incubating cells with dextran Alexa 647 (10,000 m.w., 500 µg/ml, 60 min) or dextran amino (Molecular Probes; 10,000 m.w., negative control); subsequently washed with 1x PBS and stained with surface Abs for CD3, CD4, CD14, CD19, and HLA-DR; and analyzed by flow cytometry, as described above. Monocytes were defined as CD3^–/CD4⁺/CD14⁺/HLA-DR⁺. Results were expressed as MFI and percent positive.

HRP endocytosis as a monocyte population-based assay was measured as follows: adherent cells were incubated with HRP (1 mg/ml, 60 min), washed six times with 1x PBS and 1% FBS, and lysed in 0.5% Triton X-100 (Boehringer Mannheim). Total protein in cell lysates was determined by Dc protein assay, according to the manufaturer’s instructions (Bio-Rad). The amount of HRP in the lysate was quantified by mixing each lysate with HRP substrate and analyzing in a kinetic absorbance reader at 460 nm (Rainbow Reader; STL), as previously described (21). HRP content (nanograms per milliliter) was determined using an HRP standard curve, correcting for any cell loss by expressing uptake values per microgram of adherent cells protein in lysates. Results were expressed as ng of HRP uptake/µg adherent cell protein in lysates.

Cell population-based measurement of spontaneous apoptosis

PBMC (10⁷ cells/well) were cultured for 48 h in complete medium with or without IL-13 (20 ng/ml, added once and at the time of PBMC isolation). PBL were subsequently removed, as described above, and adherent cells were detached with cold 1x PBS and 45 mM EDTA. PBL and donor matched-adherent cell apoptosis was qualified using the In Situ Cell Death Detection kit, Fluorescein (Boehringer Mannheim), according to the manufacturer’s instructions, and analyzed by flow cytometry. Fluorescein dUTP incorporated in the DNA strand breaks of the cells was measured on a BD Biosciences FACSCalibur flow cytometer using the CellQuest software package for acquisition and analysis. Results were expressed as MFI and percent positive.

Single cell imaging of endocytosis and apoptosis

Single cell imaging of endocytosis was performed by dextran uptake assays and confocal microscopy. Adherent cells in 5-cm² glass Gold Seal coverslips (BD Biosciences) were exposed to IL-13 (20 ng/ml, 48 h), pulsed with 70 K_d dextran-Texas Red (1 mg/ml, 60 min; Molecular Probes), washed six times with warm 1x PBS, and fixed with 2% paraformaldehyde (30 min, 4°C). Apoptosis was detected by in situ specific end labeling of the DNA fragments using the ApoptTag Fluorescein In Situ Apoptosis Detection kit (Serologicals), following the manufacturer’s instructions. Images were obtained at The Wistar Institute Microscopy Facility using rhodamine and FITC filters on a Leica Microsystems confocal microscope with Focus Imagecorder Plus software.

Statistical analysis

All descriptive analysis and statistical tests were performed using JMP 4.0 (SAS Institute), as previously described (24). All tests were two tailed, unless specified in text. All tests applied an of 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Single addition of IL-13 augments recall and HIV-1-specific lymphoproliferative responses

Exposure of PBMC from HIV^– subjects (n = 14) or HIV⁺ subjects (n = 43) to IL-13 and subsequent stimulation with Ag (Flu) resulted in significant increase in LPA response (p = 0.03 and p < 0.0001, respectively; Fig. 1A). In support of these findings, a significant increase of T cell LPA responses against HIV-1 p24 (n = 30, p < 0.0001) and PPD tuberculin (n = 6 HIV-1-infected patients with confirmed diagnosis of past tuberculosis, p = 0.0299) was also observed (Fig. 1B), indicating that IL-13 had a general effect in increasing LPA recall responses. IL-13-mediated increases in HIV-specific LPA were not directly associated with CD4⁺ T cell count or viral load (p > 0.05) at the time of analysis, even though a greater amount of LPA responses in the absence of IL-13 was observed at higher CD4⁺ T cell count, as previously noted by others (6) (data not shown). Although we did not find an association with viral load, we also addressed whether recall responses and the effects of IL-13 were different in subjects with or without antiretroviral therapy. The results of this analysis showed no difference in the ability of IL-13 to increase LPA responses (Flu: patients on therapy, n = 35, p < 0.0001, and patients without therapy, n = 6, p = 0.03; p24: patients on therapy, n = 19, p < 0.0001, and patients without therapy, n = 10, p = 0.01). Median CD4 count for patients without therapy was 510 cells/µl (25th-75th IQR: 379–709), while median plasma HIV-1 RNA was 1025 copies/ml (25th-75th IQR: 400-9519). The specificity of IL-13 in increasing recall and anti-HIV-1 responses was also supported by the lack of enhancement of non-Ag-specific PBMC responses to mitogen (PHA, n = 13, p = 0.094; Fig. 1B) or neoantigen stimulation (keyhole limpet hemocyanin, n = 8, p = 0.383; data not shown).

View larger version (15K): [in this window] [in a new window]   FIGURE 1. IL-13 significantly enhances recall CD4+ T cell responses against HIV-1 and recall Ags in HIV–- and HIV+-infected PBMCs. A, LPA responses (mean SI ± SE) against Flu ± IL-13 in HIV– (n = 14, left) and HIV+ (n = 43, right) PBMC are shown. B, LPA responses against HIV-1 p24 (n = 30), PPD (n = 6) or PHA (n = 13), ± IL-13 in HIV+ PBMC are shown. and in A and B show responses in the absence or presence of IL-13, respectively. Values of p are shown on top of each graph.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. IL-13 significantly enhances recall CD4+ T cell responses through modulation of CD14+ monocytes. Responses in A and B are shown following coculture of Flu Ag with isolated CD3+/CD4+ T cells (without IL-13) together with either: CD14+ monocytes without IL-13 (superscript a), IL-13-pre-exposed CD14+ monocytes in which IL-13 was washed out before subsequent coculture (superscript b), or IL-13 was maintained throughout coculture (superscript c). A, LPA responses of isolated cell subset cocultures (top) and CFSE-dye analysis of CD4+ T cells within cocultures (bottom) are shown for four representative HIV+ donors. B, LPA responses (top) and CFSE-dye analysis (bottom), as described above, are shown for representative HIV– donors. In bar graphs, and in A and B show responses (SI against Flu) in the absence or presence of IL-13, respectively. Due to cell number limitation, experimental conditions with the continuous presence of IL-13 upon coculture (conditions with superscript c) were not performed in all donors.

Because IL-12 has been reported to enhance cell-mediated Ag responses in HIV-1⁺ PBMC (25) and IL-13 can regulate for production of IL-12 following TLR stimulation (13), we tested whether the IL-13-mediated increase in lymphoproliferative responses was dependent on IL-12.

The role of IL-12 in the enhancement of cell-mediated Ag responses in HIV⁺ subjects was confirmed by our findings, because addition of the neutralizing anti-IL-12 Ab resulted in a significant decrease of recall responses compared with recall responses in the absence of IL-13 (n = 11, p = 0.01; Fig. 3A). However, a significant increase of recall response was still observed following exposure to IL-13 in presence of the neutralizing anti-IL-12 Ab when compared with PBMC in the presence of neutralizing anti-IL-12 Ab only (n = 11, p = 0.007; Fig. 3A). Importantly, no significant difference was observed between the level of IL-13-mediated induction in the presence or absence of neutralizing IL-12 Ab (n = 11, p = 0.2). Taken together, although our data do not exclude that IL-12 may play a role in the total induction of recall responses by IL-13, they suggest that IL-12 is not the predominant factor responsible for the IL-13-mediated effects on recall responses.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Effect of IL-13 on recall response is IL-12 independent, yet associated with an acute CD86 up-regulation. A, Flu SI ± IL-13 and neutralizing anti-IL-12. Gray area defines negative response (SI <3). Comparisons were performed by Wilcoxon/Kruskal-Wallis nonparametric test. Data shown as interquartile box plots (median and outliers) with significant p values on top (HIV+ subjects, n = 11, 18 h exposure). B, IL-13 modulation of CD86 in adherent cells isolated from HIV-1– (n = 10) and HIV-1+ (n = 12) PBMC is shown (left and right panels, respectively). MFI data (±IL-13, 18 h) shown as in A with p values (one-tail t test) shown on top of each graph.

TNF- and IL-10 secretion by HIV-1⁺ PBMC and corresponding monocyte gene expression was inhibited by IL-13

We investigated the effect of IL-13 on the constitutive PBMC secretion of TNF- and IL-10 over 18 h after isolation based on their well-documented up-regulation in HIV-1 infection (26, 27), their negative effects on APC function in association with membrane-bound TNF--induced apoptosis (4), and the inhibition of Ag presentation function by IL-10 (28, 29, 30). IL-13 decreased significantly the constitutive high secretion levels of TNF- (n = 14, p < 0.001) and IL-10 (n = 29, p < 0.001) by HIV-1⁺ PBMC over 18 h of culture (Fig. 4, right panel), while having no equally significant effect in the secretion of TNF- (n = 8, p < 0.461) and IL-10 (n = 11, p < 0.054) by HIV^– PBMC (Fig. 4, left panel). Although unpaired comparison between HIV^– and HIV⁺ for constitutive TNF- secretion levels showed a significantly higher TNF- secretion by PBMC from HIV⁺ as compared with HIV^– in the absence of IL-13 (p = 0.0002), no difference was observed between the two groups with regard to IL-10 secretion (p = 0.467). No significant correlation between TNF- or IL-10 production (prior or after exposure to IL-13) with each other, CD4⁺ T cell count, nor plasma HIV-1 RNA was observed (data not shown).

View larger version (14K): [in this window] [in a new window]   FIGURE 4. IL-13 decreases TNF- and IL-10 secretion in HIV+ PBMC within 18 h of exposure. Left panel, Shows constitutive PBMC secretion of TNF- (top, n = 8) and IL-10 (bottom, n = 11) ± IL-13 (18 h) in HIV– measured by in-house RIA. HIV+ subjects’ constitutive PBMC secretion of TNF- (top, n = 14) and IL-10 (bottom, n = 29) ± IL-13 (18 h) is shown on the right panel. Data shown as interquartile box plot (median and outliers), with significant p values on the top of each graph. Comparison between HIV– and HIV+ for TNF- and IL-10 secretion (±IL-13) revealed a significantly higher TNF- secretion in HIV+ when compared with HIV– in the absence of IL-13 (p = 0.0002).

View larger version (38K): [in this window] [in a new window]   FIGURE 5. IL-13 decreases TNF- and IL-10 gene expression in HIV+ adherent cells within 18 h of exposure. A, Representative RPAs showing the effect of IL-13 on TNF- (left) and IL-1r (right) mRNA induction at 5- and 18 h exposure, respectively. Image shows the hck-3 (left) and hck-2 (right) probe sets without treatment with RNases (lane 1, P), as well as the corresponding RNase-protected probes following hybridization with total RNA derived from HIV-1+ adherent cells nonexposed (lane 2) and exposed (lane 3) to IL-13. Note that each unprotected probe band (lane 1) migrates slower than its protected band (lanes 2 and 3) due to flanking sequences in the probes that are not protected by mRNA. B, RT-PCRs showing the effect of IL-13 in TNF- (top, 5 h exposure) and IL-10 (bottom, 18 h exposure) gene expression inclusive of control levels of 18S. Bar graphs at the right panel show pixel intensity of TNF- and IL-10 gene expression depicted at left panel, following normalization for 18S (, TNF- or IL-10 gene expression, respectively, in the absence of IL-13 exposure; , TNF- or IL-10 gene expression, respectively, following IL-13 exposure). Representative experiments of three independent experiments are shown.

Regarding Ag uptake function by adherent APCs, a single 72 h exposure of IL-13 significantly increased the pinocytic activity of HIV-1^– and HIV-1⁺ monocytes, as measured by soluble HRP (n = 4, p = 0.02 and n = 11, p = 0.0005, respectively; Fig. 6A). In general, uptake by HIV⁺ monocytes exposed to IL-13 rose to levels observed in IL-13-unexposed HIV^– monocytes as monocytes from HIV-1⁺ subjects had significantly lower endocytic activity at 72 h of culture as compared with HIV^– monocytes (HRP, p = 0.0001). Similar results were obtained by direct flow cytometry analysis of dextran uptake by IL-13-exposed vs non-IL-13-exposed adherent cells when analyzed for uptake levels within gated CD14⁺ cell subsets (HIV-1^–, n = 5; HIV-1⁺, n = 3; a representative donor from each group is shown in Fig. 6B). Enhanced endocytic uptake in conjunction with measurement of cell viability in the presence of IL-13 over 48 h was also documented via microscopy at a single cell level in cultured monocytes from HIV-1⁺ subjects coanalyzed with dextran-Texas Red uptake and ApoptTag Fluorescein staining (Fig. 6C).

View larger version (23K): [in this window] [in a new window]   FIGURE 6. IL-13 enhances endocytic uptake in HIV-1+ monocytes. A, Histograms show total HRP uptake (HRP nanogram per microgram of cell lysate) by HIV-1– (n = 4, left panel) and HIV-1+ (n = 11, right panel) adherent cell populations ± IL-13 (72 h). Significant p values are shown at the top of each graph, respectively. See Results for comparison of the endocytic uptake of HRP by adherent cells between HIV– and HIV+ monocytes. B, Histograms show IL-13-induced increase in endocytosis of dextran Alexa 647 within CD3–/CD4+/CD14+/HLA-DR+ cells from a representative HIV– (left panel, dextran Alexa 647 MFI: no IL-13 = 341, IL-13 = 579) and HIV+ (right panel, dextran Alexa 647 MFI: no IL-13 = 388, IL-13 = 1204) donor following 72 h cytokine exposure before endocytic assay. As a negative control for background fluorescence, each panel also includes the MFI data for endocytosis of dextran amino in the absence or presence of IL-13 (HIV– left panel, dextran amino MFI: no IL-13 = 1.92, IL-13 = 1.61; HIV+ right panel, dextran amino MFI: no IL-13 = 1.58, IL-13 = 1.79). C, Shows representative single cell fluorescence microscopy photomicrographs of HIV-1+ adherent cells (±IL-13, 48 h), followed by dextran-Texas Red uptake and ApopTag fluorescein staining. Top photomicrographs are at x40 (bar, 13 mm = 80 µm), while those on the bottom are at x100 (bar, 16 mm = 40 µm).

View larger version (15K): [in this window] [in a new window]   FIGURE 7. IL-13 decreases spontaneous apoptosis in HIV-1+ monocytes. Effect of IL-13 over 48 h of exposure in the apoptosis of adherent cells (top panel) and matching PBL (bottom panel) from HIV+ subjects (n = 8). Results measured by the TUNEL assay are expressed by MFI (left) and percentage of positive cells (right). Data shown as interquantile box plots (median and outliers), with significant p values on the top of each graph.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

A mechanism of action for IL-13 as primarily acting to modulate the CD14⁺ subset was directly shown following isolation and IL-13 exposure of this subset before washout and coculture with donor-matched CD3⁺/CD4⁺ T cells (Fig. 2). Results do not indicate IL-13 to have a single mechanism of action in augmenting recall responses, but rather a cumulative effect on multiple monocyte functions (CD86, apoptosis, endocytosis, cytokine regulation) that culminate in activation of CD4⁺ T cell recall responses. Our data document the normalization by IL-13 of potentially suppressive mechanisms by monocytes on CD3⁺/CD4⁺ T cell recall response activation such as an increased constitutive IL-10 and TNF- secretion, lower endocytic uptake, and decreased cell survival (Figs. 4–7). Constitutive TNF- and IL-10 PBMC secretion as well as monocyte viability have been associated with inhibitory mechanisms on the activation of CD4⁺ T cells in HIV infection due to TNF- receptor-mediated apoptosis (4, 34) and IL-10-mediated inhibition of Ag uptake and T cell activation (28, 29). However, our data do not exclude a role for IL-13 in independently augmenting positive mechanisms of Ag presentation function by monocytes, as shown by its significant increase of CD86 and Ag uptake in uninfected monocytes or its effect on activation of recall responses in uninfected PBMC. To our knowledge, this is the first report of an anti-inflammatory or Th2 cytokine acting to augment T cell-mediated responses by targeting the reversal of inhibitory mechanisms on Ag presentation in HIV-1 infection.

Cell-specific activity by IL-13 in increasing Ag presentation potential within HIV-infected PBMC has been shown with regard to its role in differentiating in vitro dendritic cell subsets when combined with GM-CSF after a prolonged culture with both cytokines over 7 days (35, 36). Our data now show that IL-13 can acutely and without GM-CSF, or a requirement for long-term culture conditions, act to quickly modulate monocytes from HIV-infected subjects, allowing for a greater potential to activate memory T cell responses. Consistent with the activity of IL-13 to modulate monocytes into dendritic-like cells after exposure, IL-13 has been shown to induce expression of dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin in human monocyte-derived macrophages (20, 37). Interestingly though, IL-13-mediated up-regulation of dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin was also observed to be an acute effect of exposure in the absence of GM-CSF and was not associated with an increased transmission of CXCR4 HIV-1 (20). In summary, in contrast to long-term differentiation of dendritic cell subsets in the presence of IL-13 and GM-CSF, our data show that short-term IL-13 effects can modulate monocyte function within 18 h of cytokine exposure and stress its association to activation of recall responses in HIV infection. Our results on IL-13 modulation of HIV-infected PBMC in vitro are also in accordance with in vivo immunoregulatory outcomes of decreased IL-10 expression in macaques infected with SIV for 22 mo and given daily IL-13 doses of 10 µg/kg/day, for 21 days before euthanasia (38). Interestingly, the previous descriptive in vivo study showed an increased recruitment of inflammatory cells and IFN- expression in the gut in association with an inflammation-induced shedding of villi observed in IL-13-treated SIV-infected, but not in IL-13-treated uninfected or untreated SIV-infected macaques (38). Based on this in vivo evidence for immune activation following IL-13 administration and the prominent role of gut-associated viral replication in SIV and HIV-1 pathogenesis (39, 40, 41, 42), further investigation would need to define the IL-13-dependent mechanisms in vivo that may have contributed to the activation of an otherwise down-regulated inflammatory response in the gastrointestinal track of SIV-infected animals.

Although we show that the effects of IL-13 were not associated with viral load or CD4 count in our cohort, it is important to highlight that the majority of our subjects were on therapy, and those that were not had a median CD4 count above 500 cells/µl to suggest that the level of immune function within the cohort tested may be at a higher level than otherwise observed if we had equal representation of subjects in end-stage disease. We interpret this to be the predominant reason that despite the presence of IL-13-mediated changes, no differences were noted at baseline values in the level of recall responses against Flu (Fig. 1), median IL-10 levels (Fig. 4), or apoptotic rates among lymphocytes between uninfected and infected groups despite significant differences in TNF- secretion (Fig. 4) levels and endocytic activity (Fig. 6). As our data reflect the patient cohort currently under medical care in center-city Philadelphia, where it is highly uncommon that subjects with a high viral load and a low CD4 count would be followed in a clinical setting in the absence of therapy, further analysis in subjects at end-stage disease would be needed to confirm the expectation that IL-13 would modulate monocytes at end-stage disease and augment recall responses in the presence of underlying Ag-specific CD4⁺ T cells.

In conclusion, the activity by IL-13 in increasing T cell LPA responses against HIV-1 and other recall Ags is shown to be associated with multiple changes in monocyte cell surface expression, function, and viability that are interpreted to collectively act to increase T cell-mediated responses. Based on IL-13’s lack of direct T cell modulation, further investigation will need to address whether IL-13 effects on monocytes could complement approaches targeted to expand T cells and cell-mediated responses in HIV infection (i.e., IL-2, IL-12, IL-15) (7, 25). Together with data already available on the antiviral activity of IL-13 (16), impaired secretion in HIV-1 infection (15), and its effect on augmenting Toll receptor-mediated IL-12 secretion by HIV-infected monocytes (13), our data further support IL-13 as a candidate antiviral cytokine of potential benefit to Ag presentation function and activation of cell-mediated T cell responses in HIV-1 infection.

	Acknowledgments

 
We thank each of the HIV^– and HIV⁺ participants; Matthew Farabaugh and Maxwell Pistilli for technical assistance; the Board and Staff of the Wistar Institute (D. D. Davis, J. S. Faust, and J. E. Hayden) and the Immunodeficiency Program Clinic at the Hospital of the University of Pennsylvania; and C. Gallo, J. Shull, and the Philadelphia Field Initiating Group for HIV-1 Trials.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
A. Minty is employed by Sanofi-Synthelabo Recherche, which holds claim rights to IL-13, studied in the present work.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by National Institutes of Health AI40379, the Philadelphia Foundation (Jacobs Fund), M. Stengel Miller’s support of the HIV-1 Partnership Program for Basic Research, and funds from the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health.

² Address correspondence and reprint requests to Dr. Luis J. Montaner, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address: montaner{at}wistar.org

³ Abbreviations used in this paper: IQR, interquantile; Flu, influenza A virus PR8 H1/M1; IL-1r, IL-1 receptor antagonist; LPA, lymphoproliferative assay; MFI, mean fluorescent intensity; PPD, purified protein derivative; RPA, RNase protection assay; RT, room temperature; SI, stimulation index.

Received for publication April 8, 2005. Accepted for publication August 2, 2005.

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