Involvement of Inflammatory Chemokines in Survival of Human Monocytes Fed with Malarial Pigment

Materials.Unless otherwise stated, reagents were obtained from Sigma-Aldrich, St. Louis, MO. Sterile plastics were from Costar, Cambridge, United Kingdom; Panserin 601 monocyte medium was from PAN Biotech, Aidenbach, Germany; percoll was from Pharmacia, Uppsala, Sweden; Dynabeads M-450 CD2 Pan T and M-450 CD19 Pan B were from Dynal, Oslo, Norway; Diff-Quik parasite stain was from Baxter Dade AG, Dudingen, Switzerland; 15-HETE was from Cayman, Ann Arbor, MI; the RNeasy Mini Kit was from Qiagen, Crawley, United Kingdom; the DNA-free kit was from Ambion, Austin, TX; the Panorama cDNA Labeling and Hybridization Kit and Panorama Human Cytokine Gene Arrays were from Sigma-Genosys, St. Louis, MO; the enhanced chemiluminescence (ECL) kit, [α-³³P]dCTP, and horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit secondary antibodies were from GE Healthcare, Milan, Italy; RNasin was from Promega, Milan, Italy; the PhosphorImager Screen, PhosphorImager Storm 860, and ImageQuant 5.0 software were from Molecular Dynamics, Sunnyvale, CA; avian myeloblastosis virus (AMV), Moloney murine leukemia virus (MMLV), oligo(dT), and primers for real-time reverse transcription (RT)-PCR were from Invitrogen Life Technologies, Carlsbad, CA; deoxynucleoside triphosphates (dNTPs) were from Applied Biosystems, Foster City, CA; IQ SYBR green Supermix, the iCycler instrument for real-time RT-PCR, the Geldoc computerized densitometer, and electrophoresis reagents were from Bio-Rad Laboratories, Hercules, CA; Beacon Designer 7.0 software was from Premier Biosoft International, Palo Alto, CA; the ApopTag in Situ Apoptosis Detection Kit was from Oncor, Eastleigh, United Kingdom; the TACS Annexin Kit for apoptosis detection by flow cytometry was from Trevigen, Gaithersburg, MD; the FACSCalibur cytometer and Cell Quest software were from BD Biosciences, San Jose, CA; the bicinchoninic acid protein assay was from Pierce, Rockford, IL; the anti-HSP27 polyclonal antibody (Ab) was from Stressgen, Ann Arbor, MI; and Mayer's hemallume was from Kaltek srl, Padova, Italy.

Culturing of P. falciparum and isolation of native HZ and dHZ. P. falciparum parasites (Palo Alto strain; mycoplasma free) were kept in culture as described previously (16). After centrifugation at 5,000 × g on a discontinuous Percoll-mannitol density gradient (16), HZ was collected from the 0-to-40% interphase, washed five times with 10 mM HEPES (pH 8.0) containing 10 mM mannitol at 4°C and once with phosphate-buffered saline (PBS), and stored at 20% (vol/vol) in PBS at −20°C or immediately used for opsonization and phagocytosis. For delipidized HZ (dHZ), lipid extraction was performed as previously reported (46).

Isolation of monocytes.Human monocytes were separated from freshly collected buffy coats (discarded from blood donations by healthy adult donors of both sexes) provided by the local blood bank (Associazione Volontari Italiani Sangue [AVIS], Turin, Italy) by Ficoll centrifugation and lymphocyte depletion with PanT/PanB-Dynal beads as described previously (49). Two milliliters of monocytes, resuspended at 1.25 × 10⁶ cells per milliliter of RPMI 1640 medium, was plated in 35-mm-diameter culture dishes. After a 1 h-incubation in a humidified CO₂/air incubator at 37°C, the dishes were washed three times with RPMI 1640 to remove nonadherent cells. Two milliliters of Panserin 601 medium was added to each dish, and the cells were cultured overnight. The Panserin medium was then removed, and 2 ml of RPMI 1640 was added to each dish before phagocytosis was started. In experiments of apoptosis detection by immunocytochemical staining, after isolation, monocytes were plated at 2 × 10⁶ to 4 × 10⁶ cells/plastic coverslip and treated as described above.

Phagocytosis of opsonized HZ, dHZ, and latex beads.HZ/dHZ, washed once and finely dispersed at 30% (vol/vol) in PBS, and latex beads (0.114-μm diameter) suspended at 5% (vol/vol) in RPMI 1640 were added to the same volume of fresh human AB serum (AVIS blood bank) and incubated for 30 min at 37°C as described previously (49). Phagocytosis was started by adding to the adherent monocytes opsonized HZ/dHZ (50 red blood cell [RBC] equivalents of heme content per monocyte) and latex beads (10 μl of a 100-fold dilution of the opsonized latex bead suspension per 10⁶ monocytes). After 2 h, phagocytosis was stopped by three washings with RPMI 1640. The amount of HZ phagocytosed by monocytes was quantified by luminescence as described previously (57, 58); on average, each monocyte ingested HZ equivalent to ∼8 to 10 trophozoites, in terms of ingested heme. The plates were then incubated in Panserin 601 medium in a humidified CO₂/air incubator at 37°C for the indicated times (0, 2, or 4 h for gene expression studies; 9 h for HSP protein expression analysis; 24 to 72 h for apoptosis detection). Under certain conditions for selected experiments, unfed monocytes were incubated as follows: with 1 μg/ml lipopolysaccharide (LPS) for 4 to 6 h (macroarray assay), with 10 μM 15-HETE for 6 h (real-time RT-PCR studies), and with 10 μM gliotoxin for 9 h (apoptosis detection and studies on HSP modulation).

Apoptosis detection by immunocytochemical staining and flow cytometry.After termination of 2 h of phagocytosis, the monocytes were further incubated with Panserin 601 monocyte medium in a humidified CO₂/air incubator at 37°C for 24 h (immunocytochemistry studies) or 72 h (flow cytometry studies). Alternatively, to obtain a positive control for apoptosis, monocytes were incubated with 10 μM gliotoxin for 6 h. Thereafter, monocytes adherent to coverslips were fixed in 4% (vol/vol) neutral buffered formalin for 10 min at room temperature. DNA fragmentation was determined using the ApopTag in Situ Apoptosis Detection Kit, according to the manufacturer's instructions. Briefly, the coverslips were equilibrated in equilibration buffer for 30 min and incubated with 54 μl of Working Strength TdT Enzyme (ApopTag kit) (for terminal deoxynucleotidyl transferase-mediated addition of digoxigenin-nucleotides to DNA). The reaction was blocked by transferring specimens to a Coplin jar containing Working Strength Stop/wash buffer (ApopTag kit). The incorporated nucleotides formed a random heteropolymer of digoxigenin-11-dUTP and dATP, detected with peroxidase-conjugated anti-digoxigenin antibody. The coverslips were then incubated in 0.05% (wt/vol) diaminobenzidine in PBS with 0.02% (vol/vol) hydrogen peroxide. Specimens were washed with water, counterstained with Mayer's hemallume for a few minutes, and then rinsed, dehydrated, and mounted for microscope analysis. Alternatively, monocyte apoptosis was evaluated by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide staining (TACS Annexin Kit), according to the manufacturer's instructions. Briefly, cells were washed with PBS with Ca²⁺ and then incubated for 15 min at 25°C with 0.025 μg FITC-conjugated annexin V and 0.5 μg propidium iodide before analysis with a FACSCalibur cytometer, using Cell Quest software. Live cells were distinguished from apoptotic or necrotic cells by appropriately gated light scatter characteristics. A total of 30,000 events were collected for each sample. Data analysis was performed with WinMDI software.

RNA isolation.After 2 h of phagocytosis, monocytes were incubated with Panserin 601 monocyte medium in a humidified CO₂/air incubator at 37°C for 0, 2, and 4 h. Total RNA was isolated from 15 × 10⁶ monocytes using an RNeasy Mini Kit in accordance with the manufacturer's instructions. A DNA digestion step with the DNA-free kit was included to avoid any genomic DNA contamination. In a typical experiment, 15 × 10⁶ monocytes yielded 4 to 8 μg of RNA.

Preparation of ³³P-radiolabeled probes.³³P-radiolabeled cDNA probes for array hybridization were prepared by reverse transcription. A Panorama cDNA Labeling and Hybridization Kit was used. To a solution of 2 μg of RNA in 7 μl of diethyl pyrocarbonate (DEPC)-treated water, 4 μl of human cytokine cDNA labeling primers was added. After incubation for 2 min at 75°C in a heating block, the solution was cooled to 42°C and then added to 6 μl of 5× reverse transcription buffer, 3 μl of dNTP mixture (10 mM [each] dATP, dGTP, and dTTP), 0.5 μl of dCTP (100 μM), 7 μl of 10 μCi/μl [α-³³P]dCTP (2,000 to 3,000 Ci/mmol), 0.5 μl of 40 U/μl RNasin (RNase inhibitor), and 2 μl of 25 U/μl AMV reverse transcriptase. Following 3 h of incubation at 42°C, the probes were purified by passage through Sephadex G-25 spin columns.

Human cDNA expression arrays.Panorama Human Cytokine Gene Arrays consisted of a matched set of charged nylon membranes containing PCR products spotted in duplicate. Each array contained 375 different human cytokine-related genes. Hybridization was carried out according to the manufacturer's instructions. Briefly, ³³P-radiolabeled first-strand cDNA probes were prepared as described above, and the arrays were prehybridized for 1 h at 65°C in hybridization solution (from the Panorama cDNA Labeling and Hybridization Kit). Filters were then incubated with the denatured, labeled cDNA for 15 h at 65°C in a hybridization oven. The filters were washed extensively under low- and high-stringency conditions in hybridization bottles and exposed to a phosphorimager screen for 24 h at 4°C, and the resulting hybridization signals were quantified using a PhosphorImager Storm 860 and ImageQuant 5.0 software. The intensity of each spot was corrected for background levels and normalized for differences in probe labeling using the average values for all genes. Genes showing a change of ≥1.5-fold in intensity were considered to be upregulated following phagocytosis.

Real-time RT-PCR studies.RNA (2 μg) was reverse transcribed into single-stranded cDNA using MMLV (200-U/μl final concentration) and oligo(dT) (25-μg/μl final concentration). The real-time RT-PCR experiments were performed on the iCycler instrument. The specific primers were designed with the Beacon Designer Software package (Table 1 shows the complete list). Oligonucleotide sequences were designed to be intron spanning, allowing differentiation between cDNA- and DNA-derived PCR products. Real-time RT-PCR assays were performed in a final volume of 25 μl containing 2 μl of cDNA diluted 1:5, primer pair concentrations as indicated in Table 1, and 12.5 μl of IQ SYBR green Supermix. DNA polymerase was preactivated for 2 min at 95°C, and amplification was performed with a 45-cycle PCR (94°C for 30 s, annealing temperature as indicated in Table 1 for 30 s, and 72°C for 30 s). The GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene was used as a housekeeping gene (primer sequences were from the Bio-Rad library). For quantification of the PCR results, expressed as fold variation over control (untreated cells), the efficiency-corrected quantification model was used (43). The C_T values were means of triplicate measurements. To validate the method, serial dilutions of cDNA from monocytes, stimulated for 6 h with LPS, were tested. The analyzed transcripts exhibited high-linearity amplification plots (r > 0.98) and similar PCR efficiencies (see Table 1), confirming that the expression levels of the genes could be directly compared to one another. The specificity of PCR was confirmed by melt curve analysis. The melting temperatures for each amplification product are expressed in Table 1.

Anti-HSP27 and anti-HSP70 Western blotting.After 2 h of phagocytosis, monocytes were further incubated with Panserin 601 monocyte medium in a humidified CO₂/air incubator at 37°C for 9 h. Subsequently, the cells were washed and lysed at 4°C in lysis buffer containing 300 mM NaCl, 50 mM Tris, 1% (vol/vol) Triton X-100, and protease and phosphatase inhibitors (50 ng/ml pepstatin, 50 ng/ml leupeptin, and 10 μg/ml aprotinin). The protein content of the lysate was measured by a bicinchoninic acid assay. Lysate samples (25 μg protein/lane) were separated by electrophoresis on 8% and 12% polyacrylamide gels under denaturing and reducing conditions, with addition of Laemmli buffer (100 mM Tris-HCl, pH 6.8, 2% [wt/vol] SDS, 20% [vol/vol] glycerol, 4% [vol/vol] β-mercaptoethanol) (28), blotted on a polyvinylidene difluoride membrane, and probed with 1:5,000 polyclonal rabbit anti-HSP27 and 1:2,000 monoclonal anti-HSP70 antibodies. After 5-min washes, the blot was incubated for 1 h with a 1:10,000 dilution of anti-rabbit or anti-mouse IgG horseradish peroxidase-labeled antibody, and immunoreactivity was detected with an ECL kit. Band densitometric analysis was performed using a Geldoc computerized densitometer.

Statistical analysis.For macroarray experiments, 2 array gene filters for each experimental condition were hybridized with ³³P-labeled cDNA synthesized from 2 different pools of RNA, each obtained from monocytes from 3 different donors (total number of donors, 6). All other data were obtained from three independent experiments with similar results. The results are shown as means plus standard errors of the mean (SEM) or as representative images. All data were analyzed by Student's t test, except those obtained from experiments with 15-HETE, which were analyzed by a one-way analysis of variance (ANOVA) and Tukey's test. A P value of <0.05 was considered significant.

Phagocytosis of HZ does not induce apoptosis in immunopurified human monocytes.Apoptosis was studied by immunocytochemical staining of digoxigenin-labeled genomic DNA in 24-h-incubated unstimulated (Fig. 1 A), 9-h gliotoxin-treated (Fig. 1B), and 24-h HZ-loaded (Fig. 1C) immunopurified monocytes. DNA fragmentation (brown cells) was rarely detected in unstimulated or HZ-fed monocytes (Fig. 1A and C), while it was clearly evident after 9 h in gliotoxin-treated monocytes (Fig. 1B). The absence of apoptosis after phagocytosis of HZ was confirmed up to 72 h after phagocytosis, as shown by viability studies performed through FACS analysis (Table 2). On average, apoptosis was detected in <4% of unfed and HZ-fed monocytes, necrosis was <6% under both conditions, and viable cells were ∼90% of total monocytes. Cells treated with gliotoxin showed 67.4% apoptosis, 11.7% necrosis, and 20.8% survival.

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FIG. 1.

Lack of short-term apoptosis in HZ-fed human monocytes. Immunopurified monocytes were incubated for 24 h following incubation with and without HZ and phagocytosis. Alternatively, cells were treated with 1 μM gliotoxin for 9 h. DNA fragmentation of immunopurified monocytes was detected by peroxidase immunocytochemical staining (brown). (A and C) Control (A) and HZ-fed (C) monocytes showed weak positivity with anti-digoxigenin antibody, indicating a predominantly poor DNA fragmentation. (B) Gliotoxin-treated monocytes were strongly positive with anti-digoxigenin antibody, indicating extensive DNA fragmentation. Representative images from three independent experiments are shown.

HZ modifies the expression of many genes in immunopurified human monocytes.Immunopurified monocytes were allowed to phagocytose HZ and latex particles (control meal) for 2 h and were further monitored at 0, 2, and 4 h after the end of the phagocytic period. As a positive control, we used cells treated with LPS for 2, 4, and 6 h. The cells were then exposed to macroarray analysis. Figure 2 shows two representative images of macroarrays obtained from unstimulated monocytes (Fig. 2A) and monocytes fed with HZ for 2 h and further incubated for 4 h (Fig. 2B). Fifteen genes were upregulated by HZ phagocytosis: 9 chemokines (interleukin 8 [IL-8], epithelial cell-derived neutrophil-activating peptide 78 [ENA-78], growth-regulated oncogene α [GROα], GRO-β, GRO-γ, monocyte chemoattractant protein 1 [MCP-1], macrophage inflammatory protein 1α [MIP-1α], MIP-1β, and myeloid progenitor inhibitory factor 1 [MPIF-1]), 4 cytokines (IL-1β, TNF-α, IL-1receptor antagonist [IL-1RA], and granulocyte-colony-stimulating factor [G-CSF]), 1 receptor (urokinase-type plasminogen activator receptor [uPAR]), and 1 matrix metalloproteinase (MMP-9). Analysis of normalized signal intensities of hybridized spots corresponding to induced genes is presented in Fig. 3: only genes showing at least a 1.5-fold increase were considered to be upregulated. Samples obtained after shorter incubation times following 2 h of HZ phagocytosis (0 and 2 h) did not show any modulation of gene expression (data not shown), except for IL-1β, which was time dependently upregulated from 0 h up to 4 h after phagocytosis of HZ, as shown in Fig. 3A; quantitation of genes upregulated 4 h after the end of phagocytosis of HZ (see above) is presented in Fig. 3B. Latex-fed monocytes, compared to unstimulated cells, displayed a moderate and transient upregulation of a restricted set of genes (ENA78, GROβ, IL-8, MCP-1, IL-1β, and IL-1RA) 2 h after phagocytosis, which totally disappeared 4 h after the end of phagocytosis; on the other hand, LPS (positive control) strongly induced a larger gene set than HZ at all times considered (data not shown). To validate the macroarray data, real-time RT-PCR for all 15 genes induced 4 h after phagocytosis of HZ was performed on RNA extracts. This approach confirmed that 12 genes were upregulated (Fig. 4) while 3 genes (MPIF-1, G-CSF, and uPAR) did not appear to be upregulated by HZ (data not shown).

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FIG. 2.

Differential gene expression of unfed and HZ-fed human monocytes as measured by cDNA macroarray. Unfed (A) and HZ-fed (B) immunopurified human monocytes were incubated for 4 h following 2 h of phagocytosis. After isolation, the pooled RNAs of monocytes obtained from 3 different healthy blood donors were converted into ³³P-labeled cDNA probes. The probes were hybridized to a Panorama Human Cytokine Gene Array filter containing 375 different human cytokine-related genes spotted in duplicate. Hybridization was detected using a phosphorimager. The most strongly upregulated genes are labeled. The data were obtained from one representative of two independent experiments.

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FIG. 3.

Quantitation of relative upregulated genes in HZ-fed versus unfed human monocytes as determined from the ³³P-labeled phosphorimages. Differential gene expression in HZ-fed/unfed immunopurified human monocytes incubated for 0, 2, and 4 h after phagocytosis is shown. Genes showing a change of 1.5-fold or more in intensity were considered to be upregulated. The IL-1β gene was the only gene upregulated at all three times of incubation after phagocytosis of HZ (A), while 15 genes showed 1.5-fold or greater induction at the longest time of incubation (4 h) after the end of phagocytosis of HZ (B). The results are expressed as means plus SEM of two independent experiments. The changes in expression of the upregulated genes shown were statistically significant (worst P value, <0.05) compared to the average values of changes in expression for all genes.

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FIG. 4.

Real-time RT-PCR validation of macroarray results. Unfed and HZ-fed immunopurified human monocytes were incubated for 4 h after phagocytosis. Threshold cycle values were normalized to GAPDH expression. The data are expressed as the ratio between relative gene expression levels of HZ-fed versus unfed monocytes and show only genes confirmed to be upregulated at least 1.5-fold. A representative experiment performed on the same pooled RNA utilized for the macroarray assay is shown.

15-HETE induces the transcription of chemokine genes in immunopurified human monocytes.To assess if 15-HETE could play a role in HZ-dependent upregulation of genes previously identified by macroarray analysis, immunopurified monocytes were treated with 1 μM and 10 μM 15-HETE for 6 h. Ten micromolar is the dose of 15-HETE that a monocyte might engulf with HZ, under the realistic assumption of 10 RBC equivalents per monocyte and a monocyte volume of 500 fl (55). Real-time RT-PCR analysis for chemokine (Fig. 5A) and IL-1RA (Fig. 5B) genes showed that 1 μM 15-HETE did not induce any significant increase of gene expression, while a significant 2- to 3-fold increase was obtained with higher doses of 15-HETE for ENA-78, GROα, GROβ, and IL-8 genes. All other genes were not significantly modulated, although some increase was observed occasionally in separate experiments. Additional experiments performed on monocytes treated with dHZ suggested a major role for the crystal moiety of HZ in the upregulation of MCP-1 and MIP-1α, while for GROα, cooperation between lipid and crystal moieties is likely (not shown).

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FIG. 5.

Real-time RT-PCR analysis of HZ-related chemokines and IL-1RA gene expression in 15-HETE-treated human monocytes. Immunopurified monocytes were treated for 6 h with different doses of 15-HETE (1 μM and 10 μM). The expression of ENA-78, GROα, GROβ, GROγ, IL-8, MCP-1, MIP-1α, and MIP-1β (A) and IL-1RA (B) genes was measured. Threshold cycle values were normalized to GAPDH, and the data are expressed as the ratio between relative gene expression levels of HETE-treated versus untreated monocytes. Measurements were done in triplicate, and the data are presented as means plus SEM. A one-way ANOVA and Tukey's test were used for statistical analysis: 1 μM 15-HETE-treated versus untreated cells, no significant increase (P > 0.05); 10 μM 15-HETE-treated versus untreated cells, significant increases for ENA-78 (P = 0.001), GROα (P = 0.012), GROβ (P = 0.040), and IL-8 (P = 0.001); all other genes were not significantly modulated (P > 0.05).

Phagocytosis of HZ enhances HSP27, but not HSP70, expression in immunopurified human monocytes.Expression of HSP27 and HSP70 proteins was studied by Western blotting and densitometric analysis in immunopurified, unstimulated, HZ-fed, and gliotoxin-treated monocytes after 9 h of incubation. As shown in Fig. 6 A, HSP27 protein expression increased by 50% in HZ-fed monocytes versus unstimulated monocytes, while it was almost totally degraded after gliotoxin treatment. HSP70 was not significantly modulated (Fig. 6B).

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FIG. 6.

Intermediate-term HSP27 and HSP70 protein expression in unfed and HZ-fed human immunopurified monocytes. Monocytes were incubated for 9 h following incubation with and without HZ and phagocytosis. Alternatively, cells were treated for 9 h with 1 μM gliotoxin. Thereafter, HSP27 (A) and HSP70 (B) proteins were detected in cell lysates by Western blotting (top; representative images) and analyzed by optical densitometry of 27- and 70-kDa bands (bottom; mean values plus SEM of arbitrary densitometric units from three independent experiments). The data were analyzed by Student's t test. (A) HZ-fed versus unfed cells, P < 0.05; gliotoxin-treated versus unfed cells, P < 0.005; HZ-fed versus gliotoxin-treated cells, P < 0.002. (B) No significant differences.