The following monoclonal Abs (mAbs) were purchased from eBioscience, Inc. (Santa Clara, CA): peridinin chlorophyll protein (PerCP)-conjugated rat anti-mouse F4/80 (BM8), r-phycoerythrin (PE)-conjugated rat anti-mouse CD11b (M1/70), biotin-conjugated rat anti-mouse CD86 (B7-2) (GL1), and fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD11c (N418). FITC-conjugated rat anti-mouse CD40 was purchased from BD Biosciences (San Diego, CA). Allophycocyanin (APC)-conjugated rat anti-mouse CD206 (MMR) and APC- or Brilliant Violet (BV) 421-conjugated rat anti-mouse CD163 were from BioLegend (San Diego, CA). Isotype control IgGs were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Ready-SET-Go cytokine ELISA sets for mouse IL-1β, TNF-α, and IL-6 were obtained from eBioscience, Inc. A polyclonal anti-mouse p190RhoGEF Ab (LF-r-gef) was described previously (5). A mouse mAbs for β-actin (C4), RhoA (67B9), glutathione S-transferase (GST, B-14) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse IgG (Bio-Rad, Hercules, CA) were used as secondary Abs for immunoblotting (IB). Enhanced chemiluminescence (ECL) reagents from Amersham Pharmacia Biotech Co. (Arlington Heights, IL) were used. TRIzol reagent was purchased from Life Technologies (Grand Island, NY), and Oligo(dT)_12-18 primer and Moloney murine leukemia virus reverse transcriptase were purchased from Promega (Madison, WI). DAPI (4′,6-diamidino-2-phenylindol dihydrochloride) was purchased from Roche Diagnostics GmbH (Mannheim, Germany). FITC- or rhodamine-phalloidin, LPS (Escherichia coli, serotype 055:B5), lucigenin, glutathione-agarose, and FITC-zymosan were purchased from Sigma-Aldrich Co. (St. Louis, MO). N-formylmethionine-leucyl-phenylalanine (fMLP) and rhosin from Calbiochem (La Jolla, CA) and 2’, 7’-dichloro-dihydrofluorescein diacetate (DCFDA) from Molecular Probes (Eugene, OR) were used. Recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF, 315-03) was obtained from PeproTech (Cranbury, NJ).

The generation of the p190RhoGEF TG mice was described previously (8). Mice were bred and maintained under specific pathogen–free conditions at the animal facility of the Ewha Laboratory Animal Genomic Center in accordance with institutional guidelines. Eight- to fourteen-week-old mice were used for all experiments. Mice were injected intraperitoneally with LPS (6 mg/kg) for 4 hours. The control animals were injected with the same volume of phosphate-buffered saline (PBS). All mouse protocols were approved by the Ewha Institutional Animal Care and Use Committee.

The peritoneal cavity was lavaged by injecting 1 ml of sterile PBS. Cells were harvested and resuspended in RPMI culture medium. Macrophages were allowed to adhere onto the culture dish overnight at 37°C in a 5% CO₂ incubator, and non-adherent cells were removed by washing twice with PBS. Macrophages collected using a scraper were washed twice with PBS before experiments.

Bone marrow cells were flushed from the femurs and tibiae of mice, as described previously (22). Those cells were cultured in RPMI 1640 medium containing 10% FBS and 10 ng/ml murine GM-CSF for 5–7 days. Cell purity was determined by FACS analysis for CD11b (> 96%).

Total RNA was extracted from purified macrophages using TRIzol reagent. Complementary DNA was prepared from an RNA template with oligo(dT) primers and was subjected to PCR. A fragment of either p190RhoGEF or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified, and the PCR products were separated on a 1.2% agarose gel. The primer sequences used were as follows. p190RhoGEF: 5’-GCCTCTAGATCTTCTCTGTGGATCGAC-3’, 5’-TCCAGCAGCCATCTAAGCAGG-3’; GAPDH: 5´-ATCACCATCTTCCAGGAGCGA-3´, 5´-ATGACCTTGCCCACAGCCTT-3´.

Quantitative real-time PCR (Q-PCR) was performed as described previously (6). An aliquot of cDNA template was amplified in a final volume of 10 μl of reaction mixture containing SYBR Green PCR Master Mix and each gene-specific primer using a StepOne Real-Time PCR System (Applied Biosystems, San Diego, CA). The fluorescence signals were quantified by the comparative cycle threshold method and normalized with GAPDH mRNA as an internal control gene. The primer sequences used were as follows. IL-6: 5′-GACTGATGCTGGTGACAACC-3′, 5′-AGACAGGTCTGTTGGGAGTG-3′; IL-1β: 5′-GACCTGTTTGAAGTTGACG-3′, 5′-GCGAGATTTGAAGCTGGATG-3′; TNF-α: 5′- GATGAGAAGTTCCCAAATGGC-3′, 5′-CAGGCTTGTCACTCGAATTTTG-3′; GAPDH: 5′-TCACGGCAAATTCAACGGCACA-3′, 5′-ATGGGCTTCCCGTTGATG ACAA-3′.

IB was performed as described previously (5). Whole cell lysates of BMDMs were prepared in 1% Nonidet P-40 lysis buffer containing protease and phosphatase inhibitors; 50 μg of the lysates were then subjected to 6% SDS-PAGE. IB analysis was performed with an anti-mouse p190RhoGEF polyclonal Ab (LF-r-gef) or an anti-mouse β-actin Ab, followed by an HRP-conjugated secondary Ab. Chemiluminescent detection was conducted with ECL reagents.

Macrophages (2 x 10⁵) were incubated in a petri dish overnight. The morphology of the macrophages attached to the dish was observed under a microscope with a 20X or 40X objective lens (Axiovert 200, Carl Zeiss, Inc.) at Ewha Fluorescence Core Imaging Center. Morphological differences were enumerated in 3 different fields in each sample, and the percentage of cells well spread (or the cellular area) was calculated. The area of each individual cell was determined using Image J analysis software. Macrophages (1 x 10⁵) attached on a cover glass were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature. After being blocked with 1% BSA for 1 hour, the samples were incubated with FITC- or rhodamine-conjugated phalloidin for 1 hour and then with DAPI for 5 minutes at room temperature. After being mounted, the samples were analyzed using a Zeiss Axiovert 200 inverted microscope that was equipped with an X40 Achroplan LD or X20 Plan-Neofluar objective, a Zeiss AxioCam HRc, and an Axio Vision Rel. 4.8 software (Carl Zeiss, Oberkochen, Germany).

Single-cell suspensions of isolated macrophages (2 x 10⁵/ml) were washed, blocked with the appropriate sera, and stained for specific cell surface markers as described previously (5). To stain actin, cells were fixed and permeabilized. F-actin was stained using FITC- or rhodamine-phalloidin. Three- or four-color flow cytometric analyses were then conducted for these cells. The data were acquired on a FACSCalibur or an LSR Fortessa cell analyser using CELLQuest software (BD Biosciences, San Jose, CA) at Ewha Fluorescence Core Imaging Center. Fluorescence signals were analyzed as dot plots of the fluorescence intensity. The percentage of the cell population in the gated area is represented graphically in the figures.

Intracellular ROS were measured using a fluorescent probe, DCFDA (23). Macrophages (2.5 x 10⁵) were incubated in serum-free medium for 1 hour at 37°C and then stimulated with LPS (100 ng/ml) for 30 minutes. DCFDA (5 μM) was added and incubated for 15 minutes before the fluorescent cells were read using a FACS. ROS production by macrophages (1 x10⁶) that followed LPS (1 μg/ml) treatment was also measured using a Nikon Eclipse Ts2R-FL inverted microscope with a 40X objective lens at Ewha Fluorescence Core Imaging Center. Fluorescence intensities were measured under excitation settings at 488 nm and emission at 515–540 nm. Fluorescence cell intensities that were collected with identical parameters, such as contrast and brightness, were compared between the control and LPS stimulation conditions. To measure ROS release in the media, macrophages were cultured in black 96-well plates (1.5 x10⁵/well) overnight and stimulated with either PBS or LPS (100 ng/ml). After adding lucigenin (200 μM), luminescence was measured for 60 minutes at 2 minute-intervals by a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA).

Macrophages (2.5 x 10⁵/ml) were starved in serum-free RPMI medium for 1 hour, and FITC-zymosan particles (20 μg/ml) were added. After being incubated at 37°C for 30 minutes, the cells were washed three times in PBS, and the FITC intake was analyzed using a FACS.

An in vitro chemotaxis assay was performed using Costar Transwell inserts (5 μm pores) in 24-well plates as previously described (8). Macrophages (2.5 x 10⁵) suspended in serum-free RPMI medium were put in the upper Transwell insert in a volume of 100 μl. The lower well contained 600 μl of serum-free RPMI medium containing a chemoattractant, fMLP (1 mM). The plates were incubated at 37°C for 2 hours, and the cells that migrated to the lower chamber were harvested and counted.

Ready-SET-Go cytokine ELISA sets were used according to the manufacturer’s instructions. Macrophages (2.5 x 10⁴) were stimulated with 200 μl of medium in the presence or absence of LPS (100 ng/ml or 1 μg/ml) in a 96-well U-bottomed culture plate for 24 hours or 1 hour. For IL-1β ELISA, cells were stimulated with ATP (2 mM) for 1 hour after 3-hour LPS (100 ng/ml) treatment. The culture supernatants collected were added to each well of 96-well flat-bottomed ELISA plates that were prepared for the assay. At the end of the assay, the plates were read at 450 nm using a microplate reader (Molecular Devices, Spectra MAX 190).

GST fusion protein containing the Rho-binding domain of Rhotekin (RBD-GST, the plasmid from Dr. G. Schmidt, Albert-Ludwigs-Universität Freiburg, Germany), which specifically recognizes the GTP-bound form of Rho proteins (24), was prepared as described elsewhere (25), and bound to glutathione-coated agarose beads. Lysates (400 μg) of PMs from LTM and TG mice were incubated for 1 hour at 4°C with 10 μg of RBD-GST fusion proteins immobilized on 10 μl of glutathione-coated beads. Proteins bound to the fusion proteins were separated by 8% SDS-PAGE and transferred to nitrocellulose membrane for IB with anti-RhoA, anti-GST, and anti-β-actin Abs.

Comparisons of multiple conditions were performed using one-way ANOVA with post hoc tests. Statistics were determined using Prism software (GraphPad Software, Inc., San Diego, CA). Values of p < 0.05 were considered significant. Asterisks indicate significant differences between different groups (*p < 0.05; **p < 0.01; ***p < 0.001).

Using p190RhoGEF TG mice that highly express the transgene in cells with class II MHC molecules, we previously reported that p190RhoGEF transgene is expressed in DCs that express class II MHC molecules (8). In this work, we tested whether p190RhoGEF transgene is also expressed in the macrophages of TG mice because macrophages also express class II MHC molecules. PMs were isolated from peritoneal cavity lavage of LTM control and p190RhoGEF over-expressing TG mice by removing non-adherent cells. BMDMs were also prepared from LTM control and p190RhoGEF TG mice. The transcriptional levels of p190RhoGEF were compared by RT-PCR (Figure 1A), and the protein expression was analyzed by Western blotting on macrophage cellular extracts using an anti-p190RhoGEF Ab (Figure 1B). As shown in the splenic DCs (8), endogenous expression of p190RhoGEF was also seen in macrophages isolated from the LTM control mice, and the over-expression of the p190RhoGEF transgene was confirmed in the macrophages isolated from the TG mice.

We examined the macrophage morphology and found that macrophages from the LTM and p190RhoGEF TG mice showed differences in shape. The LTM macrophages were spread well enough to show elongation, but the TG macrophages were roundish (Figures 2A, B). Polymerized actin was enriched at the edges of the attached TG macrophages (Figures 2C, D). These results could suggest that the p190RhoGEF over-expressed in TG macrophages activated changes in the RhoA-mediated actin cytoskeleton, which controls macrophage shape. We next examined whether these actin cytoskeletal changes also affected the macrophage subset population in the peritoneum of TG mice.

Flow cytometry analyses of PMs from LTM and p190RhoGEF TG mice showed that the total population of cells expressing F4/80 and CD11b (CD11b⁺F4/80⁺) was similar (Figure 3A), and subset populations (M1: CD11b⁺F4/80⁺CD11C⁺, M2: CD11b⁺F4/80⁺CD163⁺) were smaller in the PMs from TG mice, although the difference was not statistically significant due to individual variation among mice (Figure 3C). However, the ratio of subset populations of PMs was maintained similarly in LTM and TG mice (data not shown). To analyze the macrophage response during peritoneal inflammation elicited by LPS, changes in M1/M2 subset polarization were compared. Flow cytometry analyses showed that the M1 population (CD11b⁺F4/80⁺CD11C⁺) was significantly increased in the LPS-stimulated PMs of the LTM control mice, but not in those of the p190RhoGEF TG mice (Figures 3C). As a result, the LPS-stimulated PMs of the TG mice had a significantly smaller population of M1 subset PMs than the LTM control mice (Figures 3C). The M2 population (CD11b⁺F4/80⁺CD163⁺) was not significantly changed in the LPS-stimulated PMs of the LTM and the TG mice (Figure 3C). The expression of M1 markers was also significantly increased in the LPS-stimulated PMs of the control mice, but not in those of the TG mice (Figures 3A, B). Significant changes in the expression of M2 markers were not shown (Figures 3A, B). Those results suggest that over-expression of p190RhoGEF in macrophages might lead to a reduction in macrophage polarization toward the classical M1 subset during inflammation.

Previous studies indicated that M1 macrophages are roundish (12, 20, 21) and that macrophage shapes and functions depend on an actin cytoskeleton that is regulated by RhoGEF/RhoA activation pathways (18, 19). We therefore compared the characteristics of the M1 subset in macrophages from LTM and TG mice. Macrophages are polarized to the M1 phenotype in the presence of Th1 cytokines such as IFN-γ or bacterial products such as LPS (26, 27). M1 macrophages produce high levels of inflammatory cytokines such as TNF-α, IL-6, IL-1β, IL-12, and IL-23, as well as increased levels of ROS (10–13).

We first examined whether the expression of the p190RhoGEF transgene in macrophages affected their ability to produce ROS. To test that, PMs prepared from LTM control and p190RhoGEF TG mice were treated with LPS. Changes in the production of intracellular ROS were examined using DCF dye, which fluoresces in response to ROS. Representative fluorescence images of cells after a 30-minute stimulation are shown (Figure 4A); the fluorescence intensity is compared quantitatively (Figure 4B). The fluorescence in cells was also determined by flow cytometry (Figure 4C); the mean fluorescence intensity (MFI) is compared quantitatively (Figure 4D). These results demonstrate that ROS production was dramatically reduced in PMs from TG mice compared with that in cells from LTM control mice when cells were activated for 30 minutes with LPS. Similar results were also obtained by chemiluminescence detection using lucigenin when PMs from LTM control and p190RhoGEF TG mice were incubated in medium containing LPS. The luminescence caused by ROS release into the medium appeared 20 minutes after the stimulation, increased continuously for another 30 minutes, and then started to fade (Figure 4E). Less luminescence was observed from the PMs of the TG mice than in those from the LTM control mice (Figure 4E).

We next examined whether p190RhoGEF transgene expression altered the abilities of macrophages to engulf particles. PMs from the LTM control and p190RhoGEF TG mice were incubated with FITC-conjugated zymosan at 37°C for 30 minutes, and then the phagocytic capacity of the macrophages was compared. The macrophage population made fluorescent by zymosan internalization increased after LPS treatment, and that increase was significantly smaller in PMs from the p190RhoGEF TG mice than in the PMs from the LTM control mice (Figure 5A). To compare the chemotactic ability of PMs from LTM and TG mice, we analyzed the cells that migrated toward fMLP in the lower chamber of a Transwell plate. Macrophage migration increased significantly after LPS stimulation in the LTM macrophages, but not in the TG macrophages (Figure 5B).

We further studied the production of inflammatory cytokines, IL-6, IL-1β; and TNF-α; in PMs from LTM control and p190RhoGEF TG mice. The secretion of those pro-inflammatory cytokines increased significantly after stimulation with LPS (Figures 6A–C). However, the level of secretion in response to LPS was reduced significantly in the TG macrophages compared with the LTM control macrophages (Figures 6A–C). LPS-induced cytokine gene transcription was also reduced significantly in the PMs of TG mice (Figures 6D–F). These results indicate that the reduction of cytokine secretion in TG macrophages after LPS treatment was caused by a reduced level of cytokine gene transcription.

p190RhoGEF was identified as a specific GEF for RhoA activation, and RhoA is important in the regulation of the actin cytoskeleton (1, 14, 15, 28). We examined the level of RhoA activation in LTM and TG macrophages and found that endogenous level of RhoA activation in TG macrophages was similar to the RhoA activation stimulated by LPS in LTM macrophages (Figure 7A). Compared with LTM macrophages, TG macrophages showed little response to LPS (Figure 7A). To determine whether the characteristics of shape, subset polarization, and LPS response shown in the TG macrophages are caused by the over-expression of p190RhoGEF, we tested the effect of an inhibitor for RhoA, rhosin, which directly targets the Rho-GEF binding domain, preventing Rho from interacting with its GEFs (29, 30). After the rhosin treatment, the level of polymerized actin in the TG macrophages was reduced to that of the LTM macrophages (Figures 7B, C). Consequently, the TG macrophages were spread better, as in the LTM control macrophages, by the rhosin treatment (Figures 7D, E). Furthermore, LPS-induced ROS production and inflammatory cytokine gene transcription in the TG macrophages also recovered after rhosin treatment (Figures 8A–D). The effect of rhosin alone was similar to the control (data not shown). These results suggest that the over-expression of p190RhoGEF might regulate TG macrophages structurally and functionally.