CCR-2 neutralization augments murine fresh BMC activation by Staphylococcus aureus via two distinct mechanisms: at the level of ROS production and cytokine response

Maintenance of experimental animals

All experiments involving Swiss albino mice were conducted according to the protocols that had been approved by the Institutional Animal Ethics Committee (IAEC), Department of Physiology, University of Calcutta, under the guidance of CPCSEA (Approval Number: IAEC/IV/Proposal/BB-1/2014 dated 26.08.2014), Ministry of Environment and Forest, Government of India. Wild type male Swiss albino mice were used throughout the study.

Preparation of bacteria

The S. aureus strain AG-789 was obtained from Apollo Gleneagles Hospital, Kolkata, West Bengal, India, and was maintained in our laboratory. This bacterial strain is of catalase and coagulase-positive. S. aureus (AG-789) grown overnight in Luria Bertani broth, and was diluted with fresh broth and cultured until mid-logarithmic phase of growth. Bacteria were adjusted to the desired inoculum spectrophotometrically before infection (OD₆₂₀ = 0.2 for 5.0 × 10⁷cells/ml for S. aureus) and the colony-forming unit (CFU) count of the desired inoculum was confirmed by serial dilution and culture on blood agar. ⁴⁸ Among the three clinical isolates (AG-886, AG-591 and AG-789) used in earlier studies from our laboratory, it was observed that all of the three strains of S. aureus (AG-886, AG-591 and AG-789) showed the expression of both the TSST-1 and coagulase genes in all phases of their growth, and induction was maximum during the log and stationary phases of their growth. Furthermore, from the biochemical analysis and biotyping for characterization of the S. aureus (AG-789) isolate, it was found that S. aureus (AG-789) was catalase-positive, coagulase-positive, could coagulate bovine plasma and was resistant to methicillin. This MRSA was found to be sensitive to ciprofloxacin, chloramphenicol and azithromycin. The results of the checker board assay showed no synergistic interaction between the antibiotics tested. Antagonism was observed with methicillin interacting with ciprofloxacin or azithromycin. In our laboratory, several other clinical isolates of S. aureus also have been extensively studied in the mouse model of arthritis with short term but non-lethal infection.

Isolation and stimulation of murine FBMCs

The Swiss albino mice were used at 6–8 wk of age and fed standard laboratory chow and water. Murine resident FBMCs were isolated from femurs of Swiss albino mice according to the Marim et al. ²³ The femurs from Swiss albino mice were dissected using scissors, cutting through the tibia below the knee joints, as well as through the pelvic bone close to the hip joint. Muscles connected to the bone were removed using clean gauze, and the femurs were placed into a polypropylene tube containing sterile PBS on ice. In a tissue culture hood, the bones were placed in 70% ethanol for 1 min, washed in sterile RPMI 1640 and then both epiphyses were removed using sterile scissors and forceps. Briefly, bone marrow was flushed into a 50-ml conical tube with RPMI 1640, 100 IU/ml penicillin with 100 µg/ml streptomycin, 20% (v/v) FBS a using a 10-ml syringe and a 26-G needle. Cells were then passed through a 23-G needle and cultured in 20 ml RPMI in 95-mm diameter Petri dishes at 37℃ in humidified 5% CO₂ in air. ⁴⁹ FBMCs were cultured 200 µl of culture medium (RPMI-FBS) in a well and incubated under different conditions (with S. aureus, inhibitors, Abs, etc.).

Cell viability assay

Cell viability was determined by the MTT assay. FBMCs were cultured 200 µl culture medium in a well and incubated under different conditions (with S. aureus, inhibitors, Abs, etc.). Ten µl MTT (10 mg/ml) were added to each well, and the mixture was incubated for 4 h at 37℃. SDS was then added to the mixture at 10% and incubated for 3 h. Culture medium was then replaced with DMSO to dissolve formazan crystals. After shaking at room temperature (23–25℃) for 10 min, absorbance of each well was determined at 570 nm using a microplate reader (Bio-Rad, Richmond, CA, USA). Samples were measured in three replicates and each experiment was repeated at least twice. ⁵⁰

Immunophenotyping of BMCs by FACS analysis

The cell suspension generated thereafter was considered as FBMC. CD11b, known as the integrin alpha M chain, is implicated in different adhesive interactions of monocytes, macrophages and granulocytes. ⁵¹ It has been shown that the spleen and peripheral blood monocyte progenitors share phenotypic markers with bone marrow progenitors, but differ in their expression of CD11b, which was low in bone marrow but high in the periphery. It was also reported that in the bone marrow populations, these peripheral populations contain common progenitors for osteoclast, macrophages and dendritic cells. These progenitors share phenotypic markers with the bone marrow progenitor, but differ in their expression of CD11b. ⁵² Therefore, characterization of FBMC by CD11b staining seems valid. Further phenotypic studies based the expression of F4/80 and CD11b could suggest that they might present a monocyte/macrophage phenotype; however, we did not do it in this case. Therefore, these FBMCs were definitely monocyte/macrophages and devoid of neutrophils. FBMCs were used, when > 90% of cells were positive for CD11b, a monocyte/macrophage marker.

FBMCs, pooled from six mice were used for immunolabelling. Trypsinized cells, washed with PBS, were incubated for 30 min with FITC-conjugated IgG2b rat anti-mouse CD11b (1 µg/ml, FITC-conjugated; BD Bioscience, Oxford, UK) on ice. After washing twice with PBS containing 1% FCS, cells were filtered through a 70-µm cell strainer (BD Biosciences) and were analyzed on a fluorescence-activated cell sorter (FACS Aria^III; BD Biosciences) for measurement of cell size (forward scatter) and complexity or granularity (side scatter). Results were considered positive if 20% or more of the cells expressed a particular Ag. ¹⁹ Importantly, it has been shown earlier that bone marrow-derived macrophages obtained from fresh or frozen BMCs did not differ in expression of surface molecules, such as CD80 and CD86, either in response to LPS or live intracellular bacteria Legionella pneumophila; therefore, description of CD80/CD86 surface molecules after S. aureus infection is not relevant. Samples of the cell cultures were tested for viability by MTT assay and were >93 % viable.

Blocking Ab reagents and culture conditions

For CCR-2/CCL-2R blocking assays, rabbit polyclonal Abs against CCR-2 (Cat. No: orb 10487; Biorbyt, Cambridge, UK) or control IgG isotype polyclonal Ab (Cat. No: ab 37355; Abcam, Cambridge, UK) were added at 5 µg/ml to isolated BMCs and were incubated for 1 h at 37℃ in a 5% CO₂ incubator.^53,54 This anti-CCR-2 polyclonal Ab is reactive against human (Swissprot: P25024), mouse, rat, cow, dog and rabbit, and was recommended as quite homologous to murine CCR-2. Then, both anti-CCR-2 Ab-treated or untreated murine fresh BMCs (5 × 10⁶ cells/ml) were infected with S. aureus (5 × 10⁶ CFU/ml) for 30 min, 60 min and 90 min at 37℃ in 5% CO₂ incubator. The same rabbit polyclonal Abs against CCR-2 (Cat. No: orb 10487; Biorbyt) were also used in a separate in vivo study for neutralization of endogenous CCR-2 in Swiss albino mice.

Assays for colony-forming ability of engulfed bacteria and determination of survival index

For the in vitro study, cells were combined from a minimum of six mice to obtain the requisite amount of individual cells (5 × 10⁶/ml), counted using a hemocytometer and were used for each experiment. ⁵⁵

Murine FBMCs (5 × 10⁶ cells/ml), were mixed with S. aureus (5 × 10⁶ CFU/ml) in a 1:1 cell:bacterium ratio and incubated for 30 min, 60 min and 90 min at 37℃ in a cell culture incubator in the presence or absence of anti-CCR-2 Ab, as well IgG isotype control Ab. Extracellular S. aureus were removed by washing the suspension four times in RPMI by centrifugation at 250 g at 4℃ for 5 min. After centrifugation, cell culture supernatants were collected and stored at –80℃ for further assay. Then, the pellets were disrupted in sterile water containing 0.01% BSA by vigorously vortexing to release intracellular bacteria in the lysate. The lysate–containing bacteria were plated at serial dilutions on mannitol agar plates. The plates were incubated at 37℃ for 1–2 d and the number of colonies was determined. ⁴⁹

In addition to the percentage of viable intracellular bacteria at time t was calculated according to the formula: Survival index = (CFU/ml)_t/(CFU/ml)₀ × 100. ⁵⁶ It has been previously demonstrated that extracellular bacteria are sufficiently removed by washing and that at least 98% of the remaining bacteria are intracellularly located.

Labeling of S. aureus with FITC

Briefly, bacteria were grown overnight (16–18 h) at 37℃ in nutrient broth, then diluted 1:100 in fresh nutrient broth and grown at 37℃ with shaking for 3 h. Bacteria were then collected by centrifugation and washed twice in PBS and then bacterial density was adjusted spectrophotometrically. Bacteria were then labeled by incubation with 0.1% FITC (Cat. No: ab 145325; Abcam) in 50 mM sodium carbonate buffer, pH 9.0, for 1 h at 37℃, protected from light. ²⁴

Immunofluorescence study for engulfment of bacteria by resident FBMCs

FBMCs were suspended in RPMI 1640 medium supplemented with 10% FBS, 100 IU/ml penicillin and 100 µg/ml streptomycin A at prior to infection and 5 × 10⁵ cells were seeded in six-well plates (total volume: 3 ml) (Invitrogen, Carlsbad, CA, USA). Two hundred µl FITC-labeled S. aureus was mixed with equal volumes of murine FBMCs and incubated for 90 min at 37℃. However, extracellular S. aureus were removed by washing the suspension in RPMI (note that we were unable to use antibiotics to kill bacteria present outside of macrophages because engulfed bacteria died quickly during the period necessary for the action of antibiotics). After the invasion procedure, 2–4% paraformaldehyde fixative solution was added and incubated for 20 min at room temperature. Then, the coverslips containing fixed cells were washed twice with PBS. After rinsing, FBMC cell nuclei were stained using Nuclear yellow (Hoechst S769121; ab13903) at 250 µg/ml dilution for 2 h. Finally, specimens were mounted on slides in 10% glycerol and visualized under a 60 × oil immersion lens. The excitation and emission filters for FITC were 495/519 nm and for Hoechst were 355/495 nm, respectively. Images were acquired by an Olympus Laser Confocal Scanning Microscope (Spectral type inverted Microscope IX81) and then analyzed with Olympus FV1000 viewer software. Co-localization was accessed by Pearson’s correlation coefficient. Higher co-localization indicates more S. aureus engulfed within the BMCs. ²⁴

Assay of hydrogen peroxide production

Hydrogen peroxide (H₂O₂) released in the supernatant and lysate at 30, 60 and 90 min of phagocytosis by BMCs infected with S. aureus pre-treated with or without exogenous catalase (catalogue no SC-50508) (166.7 µg/ml) was measured according to the method as described earlier, with slight modification.^48,57 Briefly 70 µl lysate of different groups of BMCs, 20 µl HRP (500 µg/ml) and 70 µl of phenol red (500 µg/ml) were added in each of the microtiter plates and was allowed to react at 37℃. The reaction was stopped by adding 25 µl 2 N NaOH and the absorbance was read at 620 nm. A standard H₂O₂ curve was plotted and the amount of H₂O₂ released in lysate was evaluated and expressed in µM/10⁶ cells.

Assay of superoxide anion production

Superoxide anion (O₂^–) release assay measures the change in color of cytochrome C (cytC), when reduced by O₂^– released from the stimulated BMCs infected with S. aureus pre-treated with or without exogenous SOD (catalogue no SC-11407) (2.78 µg/ml). The difference between the amounts of cyt C reduced in the presence and absence of SOD represents the amount of O₂^– generated during the incubation. The instrument was blanked on the samples containing cytC plus SOD and instructed sequentially to read the absorbance of all the samples at 550 nm in reference to the blank. The absorbances read in this manner are closest to the true SOD inhibitable cytC reduction. Cell supernatant and lysate obtained after the time-dependent phagocytosis by BMCs, infected with S. aureus, were incubated in the presence of cytC (100 µl at 2 mg/ml). The production of O₂^– was monitored spectrophotometrically at 550 nm with reference to the blank.^49,57 The amount of O₂^– production was calculated by the following formula: micromoles of superoxide anion = (mean absorbance at 550 nm × 15.87).

Assay of NO production

The amount of NO released in the supernatant and lysate was determined by the Griess reagent assay. Supernatant and lysate (50 µl), obtained after dependent phagocytosis by BMCs infected with S. aureus, were incubated with 40 µM Tris (pH 7.9) containing 40 µM of the reduced form of NADP, 40 µM flavin adenine dinucleotide and 0.05 U/ml nitrate reductase at 37℃ for 15 min. Then, reduced samples were incubated with an equal volume of Griess reagent consisting of sulfanilamide [0.25% (w/v)] and N-1-naphthylethylenediamine [0.025% (w/v)] for 10 min and the absorbance at 550 nm was measured. The total nitrate/nitrite concentration was determined by comparison with a reduced NaNO₃ standard curve. ⁵³

Intracellular ROS production by flow cytometry

Flowcytometric data acquisition and analysis were performed on a BD FACS Verse. The flow cytometer was equipped with an iron laser with excitation at 488 nm and 15-mW output power. A single cell suspension was prepared with a density of 10⁵–10⁶ cells/ml and a minimum of 10,000 cells were measured. Non-fluorescent 2’,7’-dichlorofluorescin diacetate (DCFH-DA) that upon esterase activity within the cells become DCFH, which, after oxidation by ROS, converted to fluorescent product (DCF). The amount of DCF was proportional to the amount of ROS present inside the cell.

Briefly, 20 mM DCFH-DA stock solution prepared in DMSO was diluted in the cultured medium from different groups of BMCs, to yield a 20-µM working solution. Then the cells were incubated with for 30 min, in the dark, at 37℃. Finally, cells were suspended in PBS and ROS generation was measured by determining the fluorescence intensity at 530 nm. ⁵³

TNF-α, IFN-γ, IL-6, IL-10, IL-1β and CCL-2 ELISA assays

Sandwich ELISA was used to determine cytokine concentrations from cell culture supernatant. Supernatants from different groups were normalized to the protein content by the method of Lowry et al. ⁵⁵ before the assay, and the levels of major pro-inflammatory cytokines—TNF-α, IFN-γ, IL-6, IL-1β and CCL-2 along with an anti-inflammatory cytokine IL-10—were determined as per the manufacturer’s guidelines, in a BioRad ELISA Reader at 450 nm (Raybiotech, Norcross, GA, USA). The reproducibility of cytokine kits are intra-assay coefficient of variation (CV) < 10% and interassay CV < 12%.

Western blot analysis for CCR-2 expression

Western blot analysis for CCR-2 expression was performed by standard methods. In brief, the cell lysate from different groups of BMCs, were lysed with RIPA-NP40 and normalized to the protein content following the method of Lowry et al. ⁵⁵ Sixty µg of the lysate was separated on a 10% SDS-PAGE gel and blotted onto nitrocellulose membrane. The membranes were blocked with 5% BSA in TBST for 3 h at room temperature, washed and incubated with primary anti-mice CCR-2 Abs in 1/500 dilution overnight at 4℃. The membranes were washed with TBST and incubated with the appropriate HRP-conjugated secondary Ab in 1/5000 dilution for 1 h at room temperature. Detection of antigen was performed using the enhanced chemiluminescent detection method (Thermo Scientific, Waltham, MA, USA). We used β-tubulin as loading control for Western blot to ensure equal loading throughout the gel as it is a housekeeping gene that exhibits high level, constitutive expression in the sample. It also has a different molecular mass than our protein of interest, i.e. CCR-2, to help distinguish between both bands. ²⁴

Preparation of whole staphylococcal cell lysate for demonstration of staphylococcal catalase and SOD enzyme activity

The bacteria that survived after 30, 60 and 90 min of phagocytosis by FBMCs, as obtained from the plates of respective incubation times, were washed twice in PBS, vortexed and finally diluted to an OD at 600 nm of 0.6 and re-suspended in lysis buffer. ⁵⁸ One hundred µl of this crude bacterial lysate was used to estimate the whole staphylococcal cell catalase and SOD enzyme activity. Catalase activity was determined spectrophotometrically by measuring the decrease in H₂O₂ concentration at 240 nm. Catalase activity was expressed in terms of mmol/min/mg protein.

For, SOD enzyme activity assay, 100 µl of the bacterial crude lysate was mixed separately with 1.5 ml of a Tris-EDTA-HCl buffer (pH 8.5), then 100 µl of 7.2 mmol/l pyrogallol was added and the reaction mixture was incubated at 25℃ for 10 min. The reaction was terminated by the addition of 50 µl of 1 M HCl and measured at 420 nm. The activity was expressed as unit/mg protein. ⁵⁹

Assay of catalase activity of 3 amino-1,2,4-triazole pre-treated or non-treated FBMC infected with S. aureus with or without anti-CCR-2 Ab

One mM 3 amino-1,2,4-triazole (ATZ) solution, an intracellular BMCs catalase inhibitor, was added to the FBMCs, incubated at 37℃ for 30 min and washed thoroughly with PBS to remove excess ATZ from the medium. Then, catalase enzyme activity in supernatant or in lysate of ATZ pre-treated or non-treated FBMCs after infection with the recovered S. aureus from different groups was determined spectrophotometrically by measuring the decrease in H₂O₂ concentration at 240 nm, as described in an earlier section. Catalase activity was expressed in terms of mmol/min/mg protein. ⁶⁰

Assay of SOD activity of diethyl-dithiocarbamic acid pre-treated or non-treated FBMCs infected with S. aureus with or without anti-CCR-2 Ab

One mM diethyl-dithiocarbamic acid (DDC) solution, an intracellular SOD inhibitor of BMCs was added to the fresh BMCs, incubated at 37℃ for 30 min and washed thoroughly with PBS to remove excess DDC from the medium. Then, SOD activity in supernatant, as well as in the lysate of DDC pre-treated or non-treated FBMCs after infection with S. aureus, was determined as mentioned earlier. ⁵⁹ The activity was expressed as unit/mg protein.

Immunoblot analysis for SOD

Bacterial suspensions were lysed with 1 mM disodium EDTA, pH 7.2, 0.5 mM PMSF and sonicated on ice at 8 µm for 90 s (six 15-s bursts with 15-s cooling periods) and then centrifuged at 16,000 g for 45 min at 4℃. One hundred µl of this crude bacterial lysate was used to estimate the whole staphylococcal cell SOD expression. Bacterial cell lysate samples were re-suspended in SDS-PAGE sample loading buffer, subjected to SDS-PAGE and transferred onto BioTrace PVDF membrane, as described elsewhere. ⁶¹ Membranes were probed at room temperature with primary Abs against catalase (Cat. No. SC50508; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and SOD (Cat. No. SC11407; Santa Cruz Biotechnology) at a 1:3000 dilution, and immunoreactivity was detected with a goat anti-rabbit IgG HRP-conjugated Ab (Biorbyt). Blots were developed with a chemiluminescent substrate (Thermo Scientific) according to the manufacturer’s instructions. We used β-tubulin as a loading control to ensure equal loading throughout the gel as it is a housekeeping gene that exhibits high-level, constitutive expression in the sample.

RNA extraction and PCR

S. aureus strains were extracted by the cetyltrimethylammonium bromide method after pre-treatment of bacteria with lysostaphin (1 mg/ml) for 1 h at 37℃ in Tris/EDTA/sucrose. ⁶² Total RNA was prepared by the method of Kornblum et al. ⁶³ Finally, RNA samples were dissolved in 30 µl RNase-free water. RNA integrity was tested with electrophoresis on 1% agarose gel. RNA quantification was performed measuring the absorbance at 260 nm. Nucleic acid purity was assessed measuring A260/A280 ratio (acceptable ratio was between 1.8 and 2.0).

cDNA synthesis using RT-PCR

A sufficient quantity of RNA thus obtained was used to synthesize first-strand cDNA by using the RevertAid H Minus First Strand cDNA synthesis kit (Thermo Scientific) (lot no. 00110899, Cat. No. K1632). The 20 µl reaction mixture, according to the manufacturer’s instructions, contained 11 µl RNA templates, 1 µl primers (equivalent to 15–20 pmol), 4 µl 5 × reaction buffer, 1 µl RiboLock RNase inhibitor (20 U/µl), 2 µl 10 mM deoxynucleoside triphosphate mixture and 1 µl reverse transcriptase enzyme (200 U/µl). The nucleotide sequence of the forward primer for the SODM (sodM) was CCG GAA GCG ATG AGG ATG TCA GTC, and the sequence for the reverse primer was TGC CCC ACT GCG CTT TGA TGT C. ⁶⁴ The nucleotide sequence of the forward primer for the SODA (sodA) was TGC ACG CTT TGG TTC AGG TTG GG, and the sequence for the reverse primer was GCG CCA ATG TAG TCA GGG CGT TTG. ⁶⁴ For the catalase gene (katA) a primer set comprising the nucleotide sequence of the forward primer for the (katA) 5′^-ATGTCACAACATGATAAAAA-3′ and the sequence for the reverse primer 5′-TTATTTTTTAAAGTTTTCGTA-3′ were used. ⁶⁵ The primers were designed using S. aureus MSHR1132 (GenBank accession number FR821777.2) catalase as a reference.

PCR was carried out in a 50 µl volume containing 50 ng cDNA with reagents and protocols supplied by the manufacturer. Thermocycler reaction conditions were 1 min at 94℃, 1 min at 52℃, and 1 or 1.5 min at 72℃ for 30 cycles. All PCR amplifications included preliminary denaturation at 94℃ for 10 min and a final incubation at 72℃ for 10 min. ⁶⁶ The PCR products were run on 2% agarose gels containing 0.01% ethidium bromide and viewed in a Bio-Rad Gel Doc Imager. The images were analyzed using the Image Lab software from Bio-Rad Laboratories.

Immunoblot analysis of CCR-2 expression, NF-κB expression and CCL-2 chemokine production after blocking of intracellular NF-κB and p38 MAPK

To elucidate the roles of NF-κB and p38 MAPK on CCR-2 expression, as well as CCL-2 release, pyrrolidine dithiocarbamate [PDTC; Cat No. SC-203224 (Santa Cruz Biotechnology)] an inhibitor of NF-κB activation, and pyridinyl imidazole (Cat. No. SB203580; Santa Cruz Biotechnology) an inhibitor of p38 MAPK activation, were separately added to the cultured FBMCs 30 min prior to S. aureus infection in the presence of anti-CCR-2 Ab.^67,68 After 30 min, 60 min and 90 min of phagocytosis, followed by centrifugation, the cell supernatants were used for CCL-2 ELISA assaym ⁶⁹ and cell lysates were prepared for CCR-2 and NF-κB immunoblot analysis using primary Abs using anti-CCR-2 and anti-NF-κB (p65) (Abcam) as mentioned previously. ⁷⁰

Transwell migration assay

This assay was performed in 6.5-mm Transwell plates with 8-µm pore inserts. FBMCs (1 × 10⁵ cells) were re-suspended in 100 µl chemotaxis buffer (RMPI 1640 plus 0.02% BSA). In this experiment, cells were first pre-treated with IgG isotype or anti-CCR-2 Ab for 30 min at 37℃ prior to S. aureus infection. Cells were added to the upper chamber, and 600 µl migration medium, with or without CCL-2 as chemotactic factors (10 ng/ml), ⁷¹ was added to the lower chamber. Cells were allowed to migrate through the insert membrane for 3 h at 37℃ under a 5% CO₂ atmosphere. The inserts were then washed with PBS, and non-migrating cells remaining on the upper surface of the insert were removed with a cotton swab. The migrated cells on the insert were fixed, stained with Giemsa, and mounted on glass slides. Migration was measured visually by counting of cells using a light microscope at 40 × magnification. The mean number of cells in 10 randomly chosen microscopic fields was calculated for each treatment. Results were expressed as the mean number of migrated cells/10 microscopic fields at 40 × magnification. ⁷²

In vivo S. aureus infection of either anti-CCR-2 Ab-treated or IgG isotype Ab pre-treated mice and clearance of bacterial load from blood, spleen and synovial tissue

Most of the earlier studies used MC21 as the neutralizing Ab to CCR-2 in vivo,^73,74 but we used the same rabbit polyclonal Ab against CCR-2 (Cat. No. orb 10487; Biorbyt) to neutralized CCR-2 in vivo, as well as in vitro study to demonstrate whether this Ab could also reduce inflammation due to S. aureus infection by determination of bacterial burden in blood, spleen and synovial tissue. Swiss albino mice were pre-treated with either anti-CCR-2 Ab or control IgG isotype (500 µg per mouse) in PBS the day before S. aureus infection. ⁷³

We did not find any reference in favor of the dose of the antiCCR-2 Ab purchased from Biorbyt (Cat. No. orb 10487), and we used 500 µg per mouse of this Ab as opposed to the in vivo dosing of the control IgG isotype. This approach was based on the earlier in vivo study of Vergunst et al., ⁷⁵ where treatment with CCR2-blocking Ab reduced the levels of free CCR2 on CD14⁺ monocytes by at least 57% and up to 94%, demonstrating the biologic activity of the compound in patients with active rheumatoid arthritis. ⁷⁵ A separate dose-dependent study using this antiCCR-2 Ab purchased from Biorbyt (Cat. No. orb 10487) will give a better indication for endogenous neutralization of CCR-2 after determining the CCL-2 level.

Next, live S. aureus (10⁶ CFU/mouse of average body mass of 20 g) were administered in 0.1 ml saline via the tail vein. The mice were monitored daily. Blood was obtained on d 3, 9 and 15 after infection by retro-orbital sinus bleeding before sacrifice. The blood from each infected mouse was plated on mannitol salt agar selective media. Then, the mice were sacrificed, and spleen tissues were aseptically removed and homogenized with 3 ml sterile RPMI-1640. Synovial tissues from joints of each mouse were removed, weighed and homogenized in RPMI-1640 medium (1 ml per 100 mg joint mass). After homogenization, all tissue samples were diluted and plated in triplicate on mannitol agar, and the results were expressed as the number of CFU per ml of blood and per whole organ. To avoid false-positive results due to contamination, an isolate was considered positive when 15 or more S. aureus colonies were present. ⁵⁹

Sample preparation for estimation of serum CCL-2

Blood was obtained on d 3, 9 and 15 after infection by retro-orbital sinus bleeding before sacrifice. The blood (0.2 ml) was transferred to micro-centrifuge tubes and allowed to clot at 4℃. Blood samples were then centrifuged at 300 g for 5 min at 4℃. The supernatant, pale yellow-colored serum, was pipetted out carefully with the help of micropipettes into fresh micro-centrifuge tubes, labeled and stored at –80℃ for CCL-2 analysis. In each experiment, the mice were coded to ensure that the observer was blinded. Serum from different groups were normalized to the protein content by the Bradford method before the assay and levels of CCL-2 were determined by Sandwich ELISA, according to the manufacturer’s instructions, in a BioRad ELISA Reader. ⁷⁶

Statistical analysis

Isolated FBMCs from six mice were combined to obtain the requisite number of macrophages (5 × 10⁶ cells/ml) and the different parameters were measured. This was repeated three times for each parameter and then the mean values of these triplicate experiments were taken for calculation. Data are expressed as mean ± SD. One-way model 1 ANOVA was performed between the groups. In ANOVA, observed variance is partitioned into components owing to different explanatory variables. A level of P < 0.05 was considered significant. Significant differences of the means between the groups were performed by one-way ANOVA. Scheffe’s F-test was done as a post-hoc test for multiple comparisons of means of different groups when a significant F value was found. A Scheffe’s F-, post-hoc test for multiple comparisons of the different groups was done when significant P-values were obtained. A P-value < 0.01 was considered significant. All analyses were done using OriginPro 8 software (Origin Lab, Northampton, MA, USA)

Immunolabeling of BMCs by FACS analysis

BMCs were studied for surface antigen expression using monoclonal Abs directly conjugated with fluorochrome. CD11b-positive immune phenotype was observed in > 80% of the gated area (Figure 1). FBMCs were tested for viability by MTT assay and > 93% were found to be viable. It is reported that the spleen and peripheral blood monocyte progenitors share phenotypic markers with bone marrow progenitors, but differ in their expression of CD11b, which is low in bone marrow but high in the periphery. It is also reported that in the bone marrow populations, these peripheral populations contain common progenitors for osteoclasts, macrophages and dendritic cells. As these progenitors share phenotypic markers with the bone marrow progenitor, but differ in their expression of CD11b, CD11b staining of FBMCs definitely indicates monocyte/macrophages lineages and no neutrophils. OPEN IN VIEWER

Phagocytic activity of murine FBMCs after neutralization of CCR-2 during acute S. aureus infection

We used live S. aureus to investigate whether CCR-2 receptor blocking alters the phagocytic activity of murine FBMCs. As shown in Table 1, a significant difference (P < 0.05) was found in the degree of phagocytic engulfment of S. aureus between the BMCs of Ab-untreated and CCR-2-blocked BMCs at 60 and 90 min post S. aureus infection.

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Table 1.

Phagocytic activity of murine FBMCs with or without neutralization of cell surface CCR-2 during acute S. aureus infection by plate count method.

Groups	Fresh bone marrow cells (CFU/ ml of lysate)
	30 min	60 min	90 min
S. aureus-infected FBMCs	208 ± 12.62	272 ± 18.59	360 ± 26.04
IgG isotype Ab-treated FBMCs + S. aureus infection	210 ± 10.19	269 ± 14.88	359 ± 19.11
Anti CCR-2 Ab-treated FBMCs + S. aureus infection	284 ± 21.45a,b	316 ± 23.05a,b	420 ± 28.08a,b

Determination of intracellular killing of S. aureus

We sought to establish whether there is any alteration in the intracellular killing of S. aureus inside murine FBMCs after in vitro exposure to S. aureus (AG-789) in presence or absence of anti-CCR-2 Ab. The mean survival index (%) (Table 2) of S. aureus was found to be higher in CCR-2-neutralized FBMCs infected with S. aureus after 30, 60 and 90 min of re-incubation time than in S. aureus-infected FBMCs pre-treated with or without control IgG isotype Ab.

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Table 2.

Intracellular killing of S. aureus within murine FBMCs with or without neutralization of cell surface CCR-2 during acute S. aureus infection.

Groups	Survival index (%)
	30 min	60 min	90 min
S. aureus-infected FBMCs	72 ± 2.12	58 ± 2.79	37 ± 1.94
IgG isotype Ab-treated FBMCs + S. aureus infection	70 ± 3.40	62 ± 1.31	39 ± 1.95
Anti-CCR-2 Ab-treated FBMCs + S. aureus infection	80 ± 3.12a,b	68 ± 2.65a,b	48 ± 2.88a,b

Immunofluorescence study

In order to investigate the internalization of S. aureus into murine FBMCs pre-treated with or without anti-CCR-2 Ab, we performed an immunofluorescence study. S. aureus cells were found to be efficiently engulfed by CCR-2-intact BMCs at all time points, as indicated by a time-dependent increase in co-localization signals in this group. However, low co-localized signals in CCR-2-blocked FBMCs after time-dependent phagocytosis of S. aureus indicate less engulfment in this group (Figure 2). Also, co-localized signals in CCR-2 FBMCs containing engulfed S. aureus were found to be significantly higher at 30, 60 and 90 min of phagocytosis as compared with CCR-2-blocked FBMC. OPEN IN VIEWER

Alteration in H₂O₂ release by murine FBMCs infection with S. aureus due to CCR-2 blocking

We tested whether H₂O₂ produced by FBMCs in supernatant or in cell lysate was CCR-2-dependent during short-term S. aureus infection. Both anti-CCR-2 Ab-untreated and anti-CCR-2 Ab-treated BMCs were exposed to S. aureus and incubated for 30, 60 and 90 min at 37℃. Anti-CCR-2 Ab treatment significantly (P < 0.05) inhibited H₂O₂ production, both in supernatant and lysate, by S. aureus-infected BMCs at all three time points after infection compared with the Ab-untreated and infected FBMCs (Figure 3a, b). However, no such differences in H₂O₂ production were observed in S. aureus-infected BMCs with or without IgG isotype control Ab treatment. However, S. aureus-infected BMCs, pre-treated with anti-CCR-2 Ab, showed a significant decrease (P < 0.05) in H₂O₂ production at all three time points, with respect to IgG isotype-treated BMCs during S. aureus infection. OPEN IN VIEWER

Alteration in O₂^– free radical release by murine FBMC infection with S. aureus due to CCR-2 blocking

Murine FBMCs were infected in vitro with S. aureus, as described earlier, or left uninfected. At 30, 60 or 90 min after infection, the amount of O₂^– produced was estimated. Both in culture supernatant (Figure 3c) and in lysate (Figure 3d), S. aureus-infected BMCs produced significantly (P < 0.05) higher amounts of O₂^– than uninfected BMCs after 30, 60 and 90 min infection, whereas infection of CCR-2-neutralized BMCs after S. aureus infection produced lower levels of O₂^– than BMCs infected with S. aureus alone at also 30, 60 and 90 min in culture medium, as well as in lysate. However, no such differences in O₂^– production were observed in S. aureus-infected BMCs with or without IgG isotype control Ab treatment. However, S. aureus-infected BMCs, pre-treated with anti-CCR-2 Ab showed significant decrease (P < 0.05) in O₂^– production at all three time points, with respect to IgG isotype-treated BMCs during S. aureus infection.

Alteration in NO production by murine FBMCs infection with S. aureus due to CCR-2 blocking

To investigate the crucial role of CCR-2 in acute in vitro infection of FBMCs with S. aureus for 30, 60 and 90 min, we determined the NO production in the supernatant and lysate (Figure 3e, 3f) at 30, 60 and 90 min in the presence or absence of anti-CCR-2 Ab. A significant decrease in NO production in the lysate and supernatant (P < 0.05) was found, owing to CCR-2 blocking in S. aureus-infected FBMCs after 30, 60 and 90 min compared with only S. aureus-infected BMCs. However, no such differences in NO production were observed in S. aureus-infected BMCs with or without IgG isotype control Ab treatment. However, S. aureus-infected BMCs that had been pre-treated with anti-CCR-2 Ab showed a significant decrease (P < 0.05) in NO production at all three time points, with respect to IgG isotype-treated BMCs during S. aureus infection.

Alteration in H₂O₂ and O₂^– free radical release by adding exogenous pure SOD and catalase in the medium

To find out the specificity of reaction by H₂O₂ and O₂^–, we investigated H₂O₂ and O₂^– production during acute staphylococcal infection in the presence of exogenous catalase and SOD enzyme, respectively. A significant (P < 0.05) decrease in absorbance values was observed for H₂O₂ production in CCR-2-neutralized S. aureus-infected FBMCs pre-treated with exogenous catalase compared with both S. aureus-infected FBMCs and IgG isotype pre-treated S. aureus-infected FBMCs along with catalase treatment suggested the specificity of the reaction by H₂O₂ only. A significant (P < 0.05) decrease in absorbance values observed for O₂^– production in CCR-2 neutralized S. aureus-infected FBMCs pre-treated with exogenous SOD compared with both S. aureus-infected FBMCs and IgG isotype pre-treated S. aureus-infected FBMCs along with SOD treatment suggested the specificity of the reaction by O₂^– only (Figure 4a, b). OPEN IN VIEWER

Alteration in cellular ROS production by murine FBMC infection with S. aureus due to CCR-2 blocking

As it has been found that infection of BMCs with S. aureus from anti CCR-2-treated BMCs group led to a significant decrease (P < 0.05) compared with anti-CCR-2-untreated BMCs in H₂O₂, O₂^– and NO production, we further determined the intracellular ROS production by flow cytometry at 30, 60 and 90 min post-infection. Following incubation with DCF-DA, it has been found that the macrophages infected with S. aureus from CCR-2 neutralized groups showed a significant decrease (P < 0.05) in ROS production after only 60 and 90 min post-infection than that of their respective CCR-2 non-neutralized groups (Figure 5a, b). OPEN IN VIEWER

Alteration in cytokine (TNF-α, IFN-γ, IL-6, IL-1β, CCL-2 and IL-10) production by murine FBMCs after infection with S. aureus due to CCR-2 blocking

Acute infection of BMCs for 30, 60 and 90 min with S. aureus in presence of anti-CCR-2 Ab resulted in a significant decrease (P < 0.05) in IFN-γ and IL-6 release (Figure 6b, c) compared with that amount secreted by the CCR-2-untreated BMCs groups. However, S. aureus-infected BMCs, pre-treated with anti-CCR-2 Ab, showed a marked increase (P < 0.05) in CCL-2 and IL-10 production at 30, 60 and 90 min compared with the Ab-untreated but S. aureus-infected group (Figure 6e, f). A similar scenario was also observed in case of TNF-α and IL-1β (Figure 6a, d) where anti-CCR-2 Ab pre-treated BMCs infected by S. aureus had insignificantly decreased TNF-α and IL-1β production with respect to Ab-untreated but S. aureus-infected BMCs. However, no such differences in cytokine or chemokine production were observed in S. aureus-infected BMCs with or without IgG isotype control Ab treatment. S. aureus-infected BMCs, pre-treated with anti-CCR-2 Ab, showed a significant decrease (P < 0.05) in the release of TNF-α and IFN-γ but increase the release of CCL-2 and IL-10 at all three time points, with respect to IgG isotype-treated BMCs infected with S. aureus. OPEN IN VIEWER

Western blot analysis for CCR-2 expression of FBMCs during acute S. aureus infection

Receptor expression was studied in murine FBMCs infected with S. aureus for 30, 60 and 90 min in the presence or absence of anti-CCR-2 Ab, to get a wider overview of the end product. The data presented in Figure 7 show that stimulation of anti-CCR-2 Ab-treated BMCs infected by S. significantly reduced expression of CCR-2 (P < 0.05), particularly after 60 and 90 min post-infection compared with CCR-2 non-neutralized BMCs. Additionally, no such reduction in CCR-2 expression was observed in IgG isotype Ab-treated, S. aureus-infected BMCs. However, there was a significant decrease (P < 0.05) in CCR-2 expression in anti-CCR-2 Ab pre-treated BMCs prior to S. aureus infection compared with IgG isotype pre-treated BMCs prior to S. aureus infection. OPEN IN VIEWER

Alteration in the bacterial catalase activity from the recovered S. aureus after time-dependent phagocytosis

Whether any changes occur in bacterial antioxidant enzyme activity after CCR-2 blocking followed by S. aureus infection, bacterial catalase activity was measured. A significant increase (P < 0.05) in the activity of catalase was observed in S. aureus recovered from CCR-2-unblocked BMCs compared with their mother strain (AG-789) at all time points, whereas a significant increase (P < 0.05) in the activity of catalase was observed in S. aureus recovered from CCR-2-blocked BMCs after 60 and 90 min of phagocytosis compared with S. aureus recovered from CCR-2 unblocked BMCs (Table 3).

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Table 3.

Bacterial catalase activity from the recovered S. aureus after time-dependent phagocytosis.

Groups	Recovered bacteria after phagocytosis (time)	Bacterial catalase activity (mmol/min/mg bacterial protein)
S. aureus (AG-789)	30 min	10.21 ± 0.87
S. aureus-infected FBMCs		19.42 ± 0.92a
S. aureus-infected FBMCs	60 min	26.80 ± 2.04a
S. aureus-infected FBMCs	90 min	34.17 ± 1.91a
Anti-CCR-2 pre-treated FBMCs + S. aureus infection	30 min	17.05 ± 1.98
Anti-CCR-2 pre-treated FBMCs + S. aureus infection	60 min	37.88 ± 2.44b
Anti-CCR-2 pre-treated FBMCs + S. aureus infection	90 min	42.89 ± 3.52b

Alteration in the bacterial SOD activity from the recovered S. aureus after time-dependent phagocytosis

A similar scenario has also been found in the case of bacterial SOD activity. A significant increase (P < 0.05) in the activity of SOD was observed in S. aureus recovered from CCR-2-unblocked BMCs compared with their mother strain (AG-789) at all time points, whereas a significant increase (P < 0.05) in the activity of SOD was observed in S. aureus recovered from CCR-2-blocked BMCs after 60 and 90 min of phagocytosis compared with S. aureus recovered from CCR-2-unblocked ones (Table 4).

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Table 4.

Bacterial SOD activity from the recovered S. aureus after time-dependent phagocytosis.

Groups	Recovered bacteria after phagocytosis (time)	Bacterial SOD activity (unit/mg bacterial protein)
S. aureus (AG-789)	30 min	7.37 ± 0.62
S. aureus-infected FBMCs		11.68 ± 1.03a
S. aureus-infected FBMCs	60 min	17.17 ± 1.97a
S. aureus-infected FBMCs	90 min	22.33 ± 1.72a
Anti-CCR-2 pre-treated FBMCs + S. aureus infection	30 min	20.01 ± 2.94
Anti-CCR-2 pre-treated FBMCs + S. aureus infection	60 min	36.36 ± 2.46b
Anti-CCR-2 pre-treated FBMCs + S. aureus infection	90 min	41.18 ± 3.42b

Alteration in catalase activity of ATZ-pretreated and -non-treated BMCs infected with S. aureus in the presence or absence of anti-CCR-2 Ab

On the basis of the above findings, activity of bacterial catalase in the supernatant and lysate of ATZ-pretreated and -non-treated BMCs was further assessed to investigate the role of catalase in the intracellular survival of S. aureus during CCR-2 blocking. A significant increase (P < 0.05) in the activity of catalase was observed both in the supernatant and lysate of murine BMCs (Table 5) when anti-CCR-2 Ab-treated BMCs were infected with S. aureus compared with BMCs infected with S. aureus alone without any ATZ pre-treatment at 60 and 90 min, and with ATZ pre-treatment at 30, 60 and 90 min of phagocytosis.

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Table 5.

Catalase activity of the ATZ-pre-treated or -untreated FBMCs infected with S. aureus in the presence or absence of anti-CCR-2 Ab.

Groups	Catalase activity from culture supernatant (mmol/min/mg protein)	Catalase activity from cell lysate (mmol/min/mg protein)
Without ATZ pre-treatment
Control FBMCs	9.14 ± 0.73	10.18 ± 0.55
Anti-CCR-2-pre-treated FBMCs	8.73 ± 0.61	8.63 ± 0.57
FBMCs + S. aureus infection (30 min)	10.05 ± 0.86	11.38 ± 0.93
FBMCs+S. aureus infection (60 min)	15.11 ± 0.90a	17.16 ± 1.53a
FBMCs + S. aureus infection (90 min)	21.18 ± 1.92a	24.26 ± 2.71a
Anti-CCR-2 pre-treated FBMCs + S. aureus infection (30 min)	12.76 ± 1.28	13.95 ± 1.96
Anti-CCR-2 pre-treated FBMCs + S. aureus infection (60 min)	27.21 ± 1.72b	32.50 ± 2.40b
Anti-CCR-2 pre-treated FBMCs + S. aureus infection (90 min)	36.02 ± 3.68b	40.14 ± 3.60b
With ATZ treatment
Control FBMCs	4.09 ± 0.55	4.41 ± 0.57
Anti-CCR-2-pre-treated FBMCs	3.93 ± 0.58	4.11 ± 0.62
FBMCs + S. aureus infection (30 min)	7.41 ± 0.76a	8.04 ± 0.9a
FBMCs + S. aureus infection (60 min)	14. 02 ± 1.30a	20.04 ± 2.11a
FBMCs + S. aureus infection (90 min)	17.28 ± 2.31a	23.03 ± 2.33a
Anti-CCR-2-pre-treated FBMCs + S. aureus infection (30 min)	10.92 ± 1.01b	15.25 ± 1.75b
Anti-CCR-2-pre-treated FBMCs + S. aureus infection (60 min)	23.87 ± 1.83b	28.98 ± 2.01b
Anti-CCR-2-pre-treated FBMCs + S. aureus infection (90 min)	31.27 ± 2.33b	39.07 ± 2.66b

Alteration in SOD activity of DDC-pretreated and -non-treated murine BMCs infected with S. aureus in the presence or absence of anti-CCR-2 Ab

As SOD played an important role in the CCR-2 receptor-mediated intracellular survival of S. aureus within the FBMCs, we investigated whether there any alterations occur in antioxidant enzyme activity when the anti-CCR-2 Ab-treated BMCs were infected with S. aureus, either in the presence or absence of DDC and SOD. A marked increase (P < 0.05) in the activity of SOD was observed both in the supernatant and lysate of murine BMCs (Table 6) when anti-CCR-2 Ab-treated BMCs were infected with S. aureus compared with cells infected with S. aureus alone without any DDC pre-treatment at 60 and 90 min, or with DDC pre-treatment at 30, 60 and 90 min of phagocytosis.

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Table 6.

SOD activity of the DDC-pretreated or -untreated FBMCs infected with S. aureus in the presence or absence of anti-CCR-2 Ab.

Groups	SOD activity from culture supernatant (unit/mg protein)	SOD activity from cell lysate (unit/mg protein)
Without DDC pre-treatment
Control FBMCs	8.54 ± 1.03	9.18 ± 1.08
Anti-CCR-2 pre-treated FBMCs	7.31 ± 0.71	8.55 ± 0.59
FBMCs + S. aureus infection (30 min)	10.97 ± 0.94	11.85 ± 1.71
FBMCs + S. aureus infection (60 min)	17.14 ± 2.05a	20.11 ± 2.31a
FBMCs + S. aureus infection (90 min)	28.25 ± 2.42a	28.98 ± 2.80a
Anti-CCR-2-pre-treated FBMCs + S. aureus infection (30 min)	13.34 ± 1.98	14.01 ± 1.93
Anti-CCR-2-pre-treated FBMCs + S. aureus infection (60 min)	25.75 ± 1.86b	26.07 ± 2.92b
Anti-CCR-2-pre-treated FBMCs + S. aureus infection (90 min)	35.92 ± 2.83b	38.17 ± 2.90b
With DDC treatment
Control FBMCs	4.09 ± 0.29	4.12 ± 0.24
Anti-CCR-2-pre-treated FBMCs	3.89 ± 0.45	3.89 ± 0.82
FBMCs + S. aureus infection (30 min)	9.94 ± 0.66a	10.08 ± 0.37a
FBMCs + S. aureus infection (60 min)	15.17 ± 1.02a	17.07 ± 1.16a
FBMCs + S. aureus infection (90 min)	22.84 ± 2.01a	24.37 ± 1.93a
Anti-CCR-2-pre-treated FBMCs + S. aureus infection (30 min)	14.02 ± 1.06b	16.96 ± 1.15b
Anti-CCR-2-pre-treated FBMCs + S. aureus infection (60 min)	21.84 ± 2.03b	27.27 ± 2.61b
Anti-CCR-2-pre-treated FBMCs + S. aureus infection (90 min)	29.61 ± 2.13b	29.87 ± 2.01b

Alteration in the expression of SOD from recovered S. aureus after time-dependent phagocytosis

Crude bacterial lysate from the recovered bacteria after time-dependent phagocytosis in the presence or absence of anti-CCR-2 Ab-mediated receptor blocking was prepared as described earlier. A marked increase (P < 0.05) in the expression of bacterial SOD protein was observed in the cell-free lysate of bacteria recovered from anti-CCR-2-pre-treated BMCs at 30, 60 and 90 min of phagocytosis, compared with that of Ab-untreated BMCs (Figure 8a, b). OPEN IN VIEWER

Alteration in S. aureus-specific gene expression (mRNA) of sodA, sodM and katA from the recovered bacteria after time-dependent phagocytosis in presence or absence of anti-CCR-2 Ab

In order to assess if the changes in SOD activity take place at the transcription level, we performed estimation of sodA and sodM gene expression levels in S. aureus recovered after time-dependent phagocytosis. Both sodA and sodM gene expression levels were found to be significantly higher in S. aureus recovered from CCR-2-blocked BMCs after 60 and 90 min of phagocytosis compared with S. aureus recovered from CCR-2-unblocked BMCs (Figure 9a). Similar patterns of results were also observed when we measured katA expression in S. aureus recovered from FBMCs in the presence or absence of anti-CCR-2 Ab after time-dependent phagocytosis (Figure 9b). OPEN IN VIEWER

Alteration in CCR-2 expression, NF-κB expression and CCL-2 chemokine production in BMCs due to intracellular NF-κB and p38 MAPK blocking during acute S. aureus infection in the presence or absence of anti-CCR-2 Ab

CCR-2 receptor expression (Figure 10a, b), NF-κB expression (Figure 10c) and CCL-2 chemokine production (Table 7) were studied in the lysate and supernatant of anti-CCR-2 Ab-pre-treated or untreated murine BMCs infected with S. aureus for 30, 60 and 90 min during NF-κB and p38 MAPK blocking. Figure 10 shows that NF-κB, as well as p38 MAPK blocker-treated BMCs with S. aureus infection in the presence of anti-CCR-2 Ab, significantly reduced (P < 0.05) the expression of CCR-2 and NF-κB, particularly 60 and 90 min post-infection compared with the CCR-2-non neutralized condition. However, Table 7 shows that the production of CCL-2 was markedly increased (P < 0.05) after 60 and 90 min of phagocytosis in the case of anti-CCR-2 Ab-treated BMCs in presence of both NF-κB and p38 MAPK blocker than that of their only anti-CCR-2 Ab-treated one during acute S. aureus infection. OPEN IN VIEWER

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Table 7.

CCL-2 chemokine production in FBMCs due to intracellular NF-κB and p38 MAPK blocking during acute S. aureus infection in presence of anti-CCR-2 Ab.

Groups	CCL-2 (pg/ml of supernatant) at 30 min post-S. aureus infection	CCL-2 (pg/ml of supernatant) at 60 min post-S. aureus infection	CCL-2 (pg/ml of supernatant) at 90 min post-S. aureus infection
Control FBMCs	264 ± 10.31	284 ± 15.14	307 ± 19.24
FBMCs + NF-κB blocker+S. aureus infection	323 ± 23.12	347 ± 25.96	401 ± 28.72
FBMCs + anti-CCR-2 Ab + NF-κB blocker + S. aureus infection	396 ± 28.26	570 ± 32.11a	610 ± 40.03a
FBMCs + p38 MAPK blocker + S. aureus infection	428 ± 29.06	495 ± 35.35	501 ± 39.19
FBMCs + anti-CCR-2 Ab + p38 MAPK blocker + S. aureus infection	493 ± 44.69	796 ± 53.22b	809 ± 63.63b

CCL-2 release was measured in the supernatant by the FBMCs, which were pre-treated with either NF-κB or p38 MAPK blocker during S. aureus infection in the presence or absence of anti-CCR-2 Ab by utilizing ELISA according to the manufacturer’s recommendations and expressed as pg/ml of supernatant. Values are expressed as mean ± SD of three independent experiments. ^aP < 0.05 compared with NF-κB-blocker-treated FBMCs after S. aureus infection; ^bP < 0.05 compared with p38 MAPK blocker-treated FBMCs after S. aureus infection.

Anti-CCR-2 Ab suppresses BMC migration in response to S. aureus infection

To investigate the contribution of CCR-2 to S. aureus-infected BMC chemotaxis to CCL-2, in vitro transwell migration assays were performed following pre-incubation with neutralizing anti-CCR-2 Ab and control isotype Ab. Cell migration assays with CCL-2 and control serum-free medium were performed and demonstrated that CCL-2 has a pronounced recruitment effect on the migration of FBMCs. A significant increase (P < 0.05) in migration was observed in S. aureus-infected BMCs compared with uninfected BMCs. However, S. aureus-infected BMCs pre-treated with anti-CCR-2 Ab showed a significant decrease (P < 0.05) in migration when compared with only S. aureus-infected BMCs. Additionally, we observed no significant increase in the migration of BMCs with or without S. aureus infection but pre-treated with control IgG isotype Ab as compared with control group or S. aureus-infected BMCs, respectively. However, S. aureus-infected BMCs pre-treated with anti-CCR-2 Ab showed a significant decrease (P < 0.05) in migration when compared with only S. aureus-infected BMCs pre-treated with IgG isotype control (Figure 11). OPEN IN VIEWER

Recovery of bacteria from the blood, spleen and synovial tissue of mice after intravenous injection of S. aureus

Mice were infected (in vivo) with S. aureus either prior to in vivo CCR-2 neutralization or control IgG isotype treatment. As shown in Table 8, bacterial burden in blood, spleen and synovial tissue of mice infected with S. aureus were found to be significantly lower (P < 0.05) even after 9 and 15 d of infection as compared with CCR-2-neutralized S. aureus-infected mice, whereas no such changes were observed in S. aureus-infected mice pre-treated with control IgG isotype.

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Table 8.

Bacterial density in blood, spleen and synovial tissue of mice infected with S. aureus (AG-789) either prior to CCR-2 neutralization or treatment with control IgG isotype.

	Blood (CFU/ml)			Spleen (CFU/g tissue)			Synovial tissue (CFU/g tissue)
Groups	3 d	9 d	15 d	3 d	9 d	15 d	3 d	9 d	15 d
Control mice	0	0	0	0	0	0	0	0	0
S. aureus-infected mice	680 ± 20.12	190 ± 11.19	82 ± 6.13	3270 ± 82.16	974 ± 43.15	533 ± 30.33	3020 ± 66.19	1160 ± 48.32	483 ± 42.11
Control IgG isotype Ab Ab-S. aureus-infected mice	692 ± 27.93	179 ± 10.14	93 ± 7.14	3176 ± 57.12	1011 ± 54.12	620 ± 22.11	2963 ± 61.66	1124 ± 62.18	517 ± 36.23
Anti-CCR2 Ab-S. aureus-infected mice	711 ± 24.73	474 ± 23.27a,b	286 ± 17.16a,b	3466 ± 71.56	1709 ± 49.02a,b	993 ± 59.12a,b	3210 ± 74.22	1720 ± 53.81a,b	1070 ± 49.12a,b

Alteration in serum CCL-2 production in mice after intravenous injection of S. aureus

Mice were infected (in vivo) with S. aureus either prior to in vivo CCR-2 neutralization or control IgG isotype treatment. Serum CCL-2 production (Table 9) in mice treated with anti-CCR-2 Ab was found to be significantly higher after 9 and 15 d of intravenous S. aureus infection as compared with S. aureus-infected mice pre-treated with or without control IgG isotype Ab. Additionally, serum CCL-2 production in only S. aureus-infected mice did not alter significantly, owing to pre-treatment of control IgG isotype.

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Table 9.

Serum CCL-2 chemokine production in mice infected with S. aureus (AG-789) either prior to CCR-2 neutralization or treatment of control IgG isotype.

	CCL-2 (pg ml-1) (mean ± SD)
Groups	3 d	9 d	15 d
Control mice	32 ± 2.21	36 ± 2.38	34 ± 1.92
S. aureus-infected mice	60 ± 4.81a	56 ± 3.19a	52 ± 4.13a
Control IgG isotype Ab-S. aureus-infected mice	58 ± 3.02	55 ± 3.13	50 ± 2.14
Anti-CCR2 Ab-S. aureus-infected mice	76 ± 4.86a,b	70 ± 4.05a,b	66 ± 3.16a,b