Cells and antibodies
Mouse alveolar macrophages (MHS cell line) were purchased from
ATCC and was cultured in RPMI1640 medium (Life Technologies)
supplemented with 10% fetal calf serum as well as 2 mM
L-glutamine, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM
sodium pyruvate, penicillin (100 U/ml) and streptomycin
(100 μg/ml), and 0.05 mM 2-mercaptoethanol. HEK293
cell was cultured in DMEM medium (Life Technologies) supplemented
with 10% fetal calf serum.
Anti-mouse C5aR polyclonal antibody was generated against a 37 aa
peptide spanning the N terminus of the mouse C5aR
]. The antipeptide specific Ab was
purified by affinity chromatography using the synthetic peptide coupled to cyanogen
bromide-activated Sepharose 4B (Amersham Pharmacia Biotech, Piscataway, NJ). HA
antibody (12CA5) was obtained from BABCO (Berkeley Antibody Company).
Cecal ligation puncture-induced sepsis
C57BL/6 male mice (6 to 8 wk of age weighing
25–30 g; Jackson Laboratories, Bar Harbor, ME) were used in all
experiments. Mice were anesthetized with ketamine. A 1 cm long
midline incision was made to expose the cecum and adjoin
the intestine. With a 4–0 silk suture, the cecum was tightly
ligated below the ileocecal valve without causing bowel
obstruction. The cecum was punctured through with a 21
gauge needle and gently squeezed to extrude luminal
contents, ensuring patency of the two puncture holes. The
abdominal incision was then closed with a 4–0 nylon suture and
skin metallic clips (Ethicon, Somerville, NY). Sham-operated
animals underwent the same procedure except for ligation and
puncture of the cecum.
Cloning of mouse C5aR
According to the mouse C5aR sequence [46
two primers (forward primer:
5′-CGG AAT TCC
GAT GGA CCC CAT AGA TAA CAG C-3′; reverse primer:
5′-GAA GAT CTT
CTA CAC CGC CTG ACT CTT CCG-3′) were designed to amplify mouse C5aR
from mouse liver RNA using reverse transcription-polymerase chain
reaction. PCR products were digested with Eco
R I and Bgl
II and then cloned into pCMV- HA, a mammalian expression vector
that contains the hemagglutinin epitope (PYDVPDYA).
The 21 nt sense and antisense siRNA oligomers targeting
against mouse C5aR mRNA were designed and synthesized by Qiagen.
Their locations and sequences are shown in
(only the sense sequences are shown). The oligos were numbered
based on the nucleotide position within the coding region of mouse
C5aR sequence. Sense and antisense oligos were annealed in HEPES
buffer (100 mM potassium acetate, 30 mM HEPES-KOH,
2 mM magnesium acetate, pH 7.4) to obtain siRNA duplexes.
Rhodamine labeled control (nonsilencing) siRNA was also purchased
Sequences and locations of siRNA oligos.
Cell transfection and western blot
For MHS cell transfection, cells were plated in 6-well plates
(8 ×105/well) and transfected with 6 μ l of TransIT-TKO (Mirus) and 30 pmol of siRNA
duplexes. Silencing effects were detected by semiquantitative RT-PCR two days
after transfection. For HEK293 cell transfection, cells plated in 35 mm
dishes (5 × 105 cells/dish) were transfected with HA-tagged C5aR
using Lipofectamine 2000(Invitrogen). Two days after transfection,
cells were placed in lysis buffer containing 50 mM HEPES, pH
7.4, 1% Triton X-100, 2 mM MgCl2, 150 mM
NaCl, 1 mM dithiothreitol,
and 1 mM PMSF. Thirty microliters of the whole cell lysates were
electrophoresed in 10% SDS-PAGE and then transferred to a nitrocellulose
membrane. Nonspecific binding sites were blocked with TBST (40 mM
Tris-HCl, pH 7.4, 300 mM NaCl,
0.1% Tween 20), containing 5% nonfat dry milk for 1 hour at
room temperature. The membrane was then incubated with anti-mouse C5aR serum (1:500
dilution) overnight at 4°C. After three washes in TBST,
the membrane was then incubated in a 1:10 000 dilution of
horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham
Pharmacia). The membrane was developed by enhance
chemiluminescence according to the protocol of the manufacturer
Detection of C5aR mRNA by semiquantitative RT-PCR
Total RNA was isolated from cells or lung tissue with the Trizol
reagent according to the manufacturer's instructions (Invitrogen).
Digestion of any contaminating DNA was achieved by treatment of
samples with RQI RNase-free DNase (Promega). RT-PCR was performed
with 1 μg of total RNA using the one-step RT-PCR system
(Invitrogen) according to the protocol of the manufacturer.
Primers for C5aR were
- forward primer: 5′-GTTGCAGCCCTTATCATCTAC-3′,
- reverse primer: 5′-TTCCGGGTTGAGGTGTCGTCTG-3′.
The primers were designed for a 908 bp DNA fragment
amplification (nucleotides 112-1019). The primers for the
“housekeeping” gene GAPDH were
- forward primer: 5′-ACCACCATGGAGAAGGCTGC-3′,
- reverse primer: 5′-CTCAGTGTAGCCCAGGATGC-3′.
After a reverse transcription step for 30 min at
50°C, 25–35 cycles were used for amplification with a
melting temperature of 94°C, an annealing temperature of
60°C, and an extending temperature of 72°C,
each for 30 seconds, followed by a final extension at 72°C for
7 min. RT-PCR products were confirmed by electrophoresis of
samples in 1% agarose gel. To ensure that DNA was detected at
the linear part of the amplification curves, PCR was performed
with different cycle numbers for C5aR and GAPDH primers. Thirty
cycles were used for C5aR amplification in CLP mice, and
thirty-two cycles were used in control mice. Twenty five cycles
for GAPDH were found to be in the linear range of PCR
Immunocytochemistry and confocal microscopy
HEK293 cells were plated on glass bottom 6-well plates (no. 1
thickness coverslips). Two days after transfection, cells were
fixed in paraformaldehyde. Fluorescence microscopy was performed
as previously described [47
HA-tagged C5aR was visualized with the affinity purified anti-mouse C5aR antibody (1:500
dilutions) and goat anti-rabbit Alexa 568 (Molecular Probe)
secondary antibody (1:1000 dilutions) in the lissamine-rhodamine
channel. Cells were imaged on a LSM 510 laser scanning confocal
microscope (Zeiss, Oberkochen, Germany) with a 63 × water
Plasmids expressing short hairpin RNAs
Vectors that express C5aR short hairpin RNAs (shRNAs) under the
control of U6 promoter were constructed by inserting pairs of
annealed DNA oligonucleotides into the linearized RNAi-Ready
pSIREN-DNR-DsRed-Express Vector (BD knockout adenoviral system 2)
between the BamH I and EcoR I sites. Sequences and locations of
shRNAs are shown in (only the top strands are shown).
Sequences and locations of short hairpin RNAs (note: “G” indicates
an extra nucleotide added to the target sequence).
Generation of siRNA-expressing adenoviruses
U6-driven shRNA cassettes and the CMV-driven DsRed expression
cassette in pSIREN-DNR-DsRed donor vector ware transferred to the
adenoviral acceptor vector pLP-Adeno-X-PRLS by cre-loxP mediated
recombination according to the protocol of the manufacturer.
HEK293 cells were transfected with Pac I-digested
adenoviral DNA using lipofectamine 2000. One week after
transfection, cytopathic effect (CPE) was detected and cells were
spun down and lysed in 500 μl PBS with three consecutive freeze-thaw cycles. Supernatants
containing infectious adenoviruses were amplified twice by infecting larger scale of
HEK293 cells. Viruses were purified by column (Puresyn, Inc) and
concentrated by YM-50 centricon (Millipore). Titers of the viruses
were determined by Adeno-X rapid titer kit (BD clontech).
Isolation of peritoneal macrophages and adenovirus infection
Macrophages were isolated from the peritoneal cavities of 4- to
6-week-old C57BL/6 mice 4 days after intraperitoneal injection of
0.5 ml 3% thioglycollate, yielding ≥ 95%
macrophages as demonstrated by cytospin and differential stain
analysis. The cells were seeded at a density of 2 × 106
cells/ml and plated into 6-well plates at
2 ml/well [48
] in the same culture
medium as MHS cells.
MHS cells and peritoneal macrophages plated in 6-well plates were
infected with 100- to 2000-MOI of adenoviruses in a volume of
150 μl of culture medium for one-hour. During the one
hour incubation, plates were shaked occasionally at a 15 min
interval. Cells were changed to 2 ml fresh medium after the
incubation and cultured for another two days for the examination
of silencing effects.
Adenovirus-mediated siRNA delivery in animals
Eight- to 10-week-old C57BL/6 mice (weighing 25–30 g) were
used in this study. The 50 μl viral suspensions with a dosage of
1 × 109 plaque-forming units (pfu) were injected intracheally into mouse lungs.
Four days after the injection, mouse lung were extensively flushed with DPBS, and
frozen in liquid nitrogen. The 2 ml Trizol reagent was added
into one lung for RNA isolation procedure.