Search tips
Search criteria 


Logo of jbbJournal's HomeManuscript SubmissionAims and ScopeAuthor GuidelinesEditorial BoardHome
J Biomed Biotechnol. 2006; 2006: 28945.
Published online 2006. doi:  10.1155/JBB/2006/28945
PMCID: PMC1510939

Adenovirus-Mediated In Vivo Silencing of Anaphylatoxin Receptor C5aR


C5a, one of the most potent inflammatory peptides, induces its inflammatory functions by interacting with C5a receptor (C5aR) that belongs to the rhodopsin family of seven-transmembrane G protein-coupled receptors. C5a/C5aR signaling has been implicated in the pathogenesis of many inflammatory and immunological diseases such as sepsis and acute lung injury. Widespread upregulation of C5aR has been seen at both the protein level and transcriptional level under pathological conditions. Here, we show that C5aR gene expression can be specifically suppressed by siRNA, both in vitro and in vivo. A panel of chemically siRNA oligonucleotides was first synthesized to identify the functional siRNA sequences. The short hairpin RNAs (shRNAs) were also designed, cloned, and tested for the silencing effects in C5aR transfected cells. The effective shRNA expression cassettes were then transferred to an adenovirus DNA vector. ShRNA-expressing adenoviruses were intratracheally administered into mouse lung, and a significant in vivo silencing of C5aR was obtained four days after administration. Thus, C5aR shRNA-expressing adenoviruses appear to be an alternative strategy for the treatment of complement-induced disorders.


The complement system was initially identified as an important innate immune mechanism of host defense to eradicate microbial pathogens. Recently, complement activation has been implicated in the pathogenesis of many inflammatory and immunological diseases, including sepsis [1], acute respiratory distress syndrome [2], rheumatoid arthritis [3], glomerulonephritis [4], multiple sclerosis [5], ischemia-reperfusion injury [6], and asthma [7]. Complement activation exerts its harmful roles through the generation of complement protein split products, especially C3a and C5a (also known as anaphylatoxins). C5a induces its inflammatory functions by interacting with C5aR that belongs to the rhodopsin family of seven-transmembrane G protein-coupled receptors [810]. Traditionally, C5aR expression was thought to be present only on hemopoietic cells, bone marrow cells [11], neutrophils [12], monocytes [13], and eosinophils [14]. However, recent studies have demonstrated the presence of C5aR on nonmyeloid cells, including cells in human lung and liver [1517], rodent type II alveolar epithelial cells [18], astrocytes [19], kidney tubular epithelial cells [20], mesangial cells [21], and hepatocyte-derived cell lines [22, 23]. Widespread upregulation of C5aR has been seen in organs (heart, liver, lungs, kidneys) from septic animals [24].

Due to the detrimental effects of complement activation under pathologic conditions, interventions aimed at blocking C5a/C5aR signaling represent promising targets for therapeutic treatment in the inflammatory disorders. Peptide antagonist (C5aRa) to the C5aR markedly reduced the lung permeability index (extravascular leakage of albumin) in mice after intrapulmonary deposition of IgG immune complexes [25]. C5aRa treatment substantially reduced I/R-induced pathological markers [26, 27]. In addition, mice injected at the start of CLP with a blocking antibody to C5aR showed dramatically improved survival [24].

RNA interference (RNAi) is an emerging technology that specifically inhibits target gene expression in vitro and in vivo. Tuschl and colleagues demonstrated that exogenously introduced short (19–23 nt) synthetic RNA oligonucleotides can silence genes in somatic cells without activating nonspecific suppression by dsRNA-dependent protein kinases [28]. Successful gene silencing has been achieved in vivo by intravenous injection of siRNA oligos in a large volume of saline solution [2931] or by injecting smaller volumes of siRNAs that are packaged in cationic liposomes [32]. However, these strategies are limited by the in vivo stability of siRNA molecules and the efficiency with which they are taken up by target cells and tissues. DNA vector-based siRNA expression system would facilitate transfection experiments in cell cultures, and allow the use of transgenic or viral delivery systems [3336]. Several viral vectors have been used to induce RNAi silencing in cultured cells and in experimental animals, including lentivirus [37, 38], retrovirus [33], adenovirus [39, 40] and adeovirus-associated viruses (AAV) [41, 42]. Adenoviruses can infect a wide range of cells and have been shown to silence gene expression in vivo [39, 43, 44]. In this study, we demonstrated that systemic application of an adenovirus expressed siRNA can specifically inhibit C5aR gene expression in vivo.


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 (MDPIDNSSFEINYDHYGTMDPNIPADGIHLPKRQPGDC) [45]. 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 EcoR I and Bgl II and then cloned into pCMV- HA, a mammalian expression vector that contains the hemagglutinin epitope (PYDVPDYA).

siRNA oligos

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 Table 1 (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 from Qiagen.

Table 1
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 (Amersham Pharmacia).

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

  1. forward primer: 5′-GTTGCAGCCCTTATCATCTAC-3′,
  2. 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

  1. forward primer: 5′-ACCACCATGGAGAAGGCTGC-3′,
  2. 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 amplifications.

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 lens.

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 Table 2 (only the top strands are shown).

Table 2
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.


siRNA duplexes efficiently inhibited endogenous C5aR in MHS cells

The 21- to 23-nucleotide siRNAs were generated by ribonuclease III through cleavage of longer dsRNAs. They have been shown to act as the mediators of post-transcriptional gene silencing in cells and animals [49, 50]. For the initial screening of the functional siRNA sequences of mouse C5aR, we used synthesized 21-nucleotide siRNA duplexes with 3′-(dTT) overhangs (Table 1) to transiently transfect MHS cell, a cell line that expresses C5aR mRNA endogenously.

Similar to other macrophages, none of the standard tranfection methods (e.g., calcium phosphate, lipid, or electroporation) can efficiently transfer DNA plasmids into MHS cells. As a minimum, seventy percentage transfection efficiency is required to study the silencing effects. We used rhodamine-labeled control siRNA to determine the transfection efficiency. Different from larger DNA plasmids, short siRNA oligos could be efficiently transferred into MHS cells by a lipid-mediated method (TransIT-TKO). All of the cells showed red fluorescence eight hours after transfection (Figure 1(a)). No fluorescence was observed for the control cells without TransIT-TKO reagent (data not shown). To confirm that these oligos actually entered the cells, a Z-stack protocol of confocal microscopy was performed and the scanning results confirmed the cytosolic localization of the fluorescence-labeled siRNA.

Figure 1Figure 1
Screening of functional mouse C5aR siRNA oligos in MHS cells. MHS cells in 6-well plates were transfected with 30 pmol of control siRNA and four C5aR siRNA oligos using Mirus TransIT-TKO transfection reagent. (a) Eight hours after transfection, cells ...

All four synthesized siRNA duplexes showed silencing effects on the endogenous C5aR expression as examined by semiquantitative RT-PCR (Figure 1(b)). SiC5aR-210 and siC5aR-297 had moderate inhibition effects, whereas siC5aR-656 and siC5aR-888 suppressed 90 percentage of the endogenous gene. No silencing effects were observed for the control siRNA (scrambled sequences). The specificity of these siRNA oligos was verified by BLAST search against the gene bank.

Cotransfection of siRNA duplexes inhibits C5aR protein expression in HEK293 cells

C5aR is a member of the seven transmembrane receptor superfamily and is ubiquitously expressed on neutrophils, macrophages, thymocytes, epithelial, and endothelial cells. However, in vitro cultured cell lines have very low or nondetectable expression of the receptor. To determine if these siRNA duplexes could also suppress C5aR protein expression, full-length mouse C5aR cDNA was cloned into a HA-tagged mammalian expression vector and transfected into HEK293 cells. Immunocytochemistry analysis showed that this C5aR construct showed a cortical pattern of expression on the membranes of HEK293 cells (Figure 2(a)). Western-blot analysis using anti-mouse C5aR antibody revealed a ~ 45 kDa band, which is consistent to the size of the receptor expressed in tissues and primary cells (Figure 2(b)) [51].

Figure 2Figure 2
Oligo siRNA inhibition of C5aR protein expression in HEK293 cells. HEK293 cells were cotransfected with HA-tagged C5aR plasmid, control siRNA, or C5aR siRNA oligos. (a) Forty-eight hours after transfection, transfected cells were fixed in paraformaldehyde ...

In the cotransfection experiment, 0.8 μg C5aR plasmid was transfected with 40 pmol of control siRNA or the C5aR-siRNAs. Two days after transfection, these cell lysates were analyzed by Western blot. Compared to control group, both siRNAs (siC5aR-656 and siC5aR-888) could significantly inhibit the protein expression of C5aR in HEK293 cells (Figure 2(b)).

Hairpin RNA constructs inhibit C5aR expression

Based on the identified C5aR siRNA oligonucleotide sequences, we designed short hairpin RNAs (shC5aR-656 and shC5aR-888) according to the design rules suggested by the manufacturer (BD PharMingen) and inserted them into a U6 promoter-driven shRNA expression donor vector, pSiren-DNR-dsRed. For the most efficient transcription initiation of RNA polymerase III, an extra “G” was added at the 5′ end of the sense sequence (Table 2). Sense- and antisense nucleotides were separated by a 9 nt spacer and five consecutive Ts were added at the 3′ end for the termination of short RNA transcripts.

To evaluate the silencing effects of these short hairpins, a 1:10 (HA-C5aR to hairpin RNA) ratio of plasmids was used for the cotransfection experiment in HEK293 cells. A luciferase short hairpin construct pSiren-shLuc served as negative control. Unexpectedly, neither one of the C5aR hairpin constructs (pSiren-shC5aR-888 and pSiren-shC5aR-656) efficiently inhibited C5aR expression (Figure 3).

Figure 3
Silencing effects of plasmid-derived short hairpin RNAs. HEK293 cells in 6-well plates were transfected with 4 μg of a 10:1 mix of pSIREN-DNR-DsRed-shRNA to HA-tagged C5aR together with 0.3 μg gfp plasmid. Forty-eight hours after transfection, ...

Different from synthesized siRNA oligos, the effects of DNA vector-based hairpin RNAs are regulated by multiple components. Target sequence selection is an important component, while other factors such as the transcription efficiency, the cleavage efficiency of hairpin RNA into siRNA by Dicer [33], and the subcellular localization of the short transcript [52], can also affect the efficacy of a hairpin RNA. To select an effective hairpin RNA structure that could be used for our in vivo adenoviral delivery, four additional plasmids, pSiren-shC5aR-300, pSiren-shC5aR-420, pSiren-shC- 5aR-517 and pSiren-shC5aR-831 were constructed. pSiren-shC5aR-831 (third bar) and pSiren-shC5aR-517 (fifth bar) strongly inhibited C5aR expression in HEK293 cells, while pSiren-shC5aR-420 (sixth bar) and pSiren-shC5aR-300 (seventh bar) had little inhibitory effects (Figure 3). No extra “G” was added to pSiren-shC5aR-420, -517 and -831 as the target sequence itself start with a “G”. An extra “G” was added to pSiren-shC5aR-300 (Table 2).

In vitro and in vivo silencing effects of adenovirus-expressed siRNA

pSiren-DNR-DsRed is an intermediate vector of adenoviral DNA. After we identified two functional shRNAs (C5aR-517 and C5aR-831), the U6 promoter and the hairpin cassette in the donor vector were transferred to a promoterless adenoviral acceptor vector by cre-loxp mediated recombination. The adenoviral DNAs were then transfected into HEK293 cells to produce infectious viruses. Two adenoviruses (adeno-shC5aR-517 and adeno-shC5aR-831) and one control virus (adeno-shLuc) were generated for in vivo gene silencing.

Macrophages that express C5aR endogenously were used to test the silencing effects of these viruses. However, these cells do not express coxsackie receptor [53, 54] and they internalize adenovirus about 100- to 1000-fold less than receptor-expressing cells, such as epithelial cells [55]. To identify an optimal infection condition, we tested a range of infectious units (100, 500, 1000, and 2000 MOI) and found that ~ 80% of the cells could be infected (as shown by the DsRed marker in the adenoviral DNA) at 2000 MOI (Figure 4), whereas less than half the cells were infected at 1000 MOI (data not shown). In addition to the high infectious units, we also used a low volume of medium during the incubation to enhance other virion uptake pathways (endocytosis or phagocytosis). C5aR mRNA expression in infected MHS cells were examined by semiquantitative RT-PCR. Both adeno-shC5aR-517 and adeno-shC5aR-831 effectively inhibited endogenously expressed C5aR and the inhibition effect of adeno-shC5aR-831 appeared to be stronger (Figure 4(b)).

Figure 4Figure 4
In vitro silencing by siRNA-expressing adenovirus. Both MHS cell and peritoneal macrophages were infected with 2000 MOI of adenovirus. (a) Sixty hours after infection, expression of dsRed protein in the cells was visualized by fluorescence microscope. ...

To evaluate the ability of virally expressed siRNAs to diminish target gene in vivo, mice were injected intratracheally with 1 × 109 plaque-forming unit (pfu) infectious units of recombinant adenovirus expressing shC5aR-831 or the control virus adeno-shLuc. Four days after infection, RNAs were isolated from mouse lungs and subjected to RT-PCR analysis. As shown in Figure 5(b), the luciferase control virus infection did not change C5aR expression in the lung tissue. However, adeno-shC5aR-831effectively inhibited C5aR expression in the lung tissue. To test the effect of adeno-shC5aR-831 in disease condition, sepsis was induced by CLP in mice that had received adenovirus for four days. Twenty four hours after CLP, RNAs from lungs were analyzed for C5aR and GAPDH expression. As shown in Figure 5(c), the inhibitory effect of adeno-shC5aR-831 is still effective under sepsis condition. These data indicate that complement receptor C5aR could be suppressed in vivo by an adenovirus-mediated siRNA knock-down strategy under both normal and disease conditions.

Figure 5Figure 5Figure 5
In vivo silencing by adenovirus delivey of siRNA. (a) The predicted shRNA transcript from the adenovirus vector and the expected Dicer processing products in vivo. Semiquantitative RT-PCR analysis with whole lung RNAs from control (b) and septic mice ...

RNA interference is a powerful tool to silence gene expression post-transcriptionally. Different from gene knock out, the inhibition efficiency of siRNAs could vary dramatically by employing a different delivery method and sequence design strategy of siRNA oligonucleotides or short hairpins. In this study, it is noted that the vector-based siRNA sequences could not be simply derived from chemically synthesized oligo sequences. It may be due to the fact that the functionality of shRNAs depends on more complicated intracellular mechanisms. In fact, none of the current design rules guarantee an effective siRNA and a functional siRNA can only be identified experimentally. Another important factor that affects the application of siRNA is the efficiency and the effectiveness of delivery routes. Here we demonstrated the intratracheal administration of siRNA-expressing adenovirus that could efficiently knock down C5aR expression. Thus, C5aR siRNA-expressing adenovirus shall not only serve as a useful tool for studying the mechanisms of complement activation in inflammation, but may also have important therapeutic applications.


This work is supported by the National Institutes of Health (Grants GM-61656 and HL-31963).


1. Ward PA. The dark side of C5a in sepsis. Nature Reviews Immunology. 2004;4(2):133–142.
2. Robbins RA, Russ WD, Rasmussen JK, Clayton MM. Activation of the complement system in the adult respiratory distress syndrome. American Review of Respiratory Disease. 1987;135(3):651–658. [PubMed]
3. Linton SM, Morgan BP. Complement activation and inhibition in experimental models of arthritis. Molecular Immunology. 1999;36(13-14):905–914. [PubMed]
4. Welch TR. Complement in glomerulonephritis. Nature Genetics. 2002;31(4):333–334. [PubMed]
5. Ffrench-Constant C. Pathogenesis of multiple sclerosis. Lancet. 1994;343(8892):271–275. [PubMed]
6. Arumugam TV, Shiels IA, Woodruff TM, Granger DN, Taylor SM. The role of the complement system in ischemia-reperfusion injury. Shock. 2004;21(5):401–409. [PubMed]
7. Hawlisch H, Wills-Karp M, Karp CL, Kohl J. The anaphylatoxins bridge innate and adaptive immune responses in allergic asthma. Molecular Immunology. 2004;41(2-3):123–131. [PubMed]
8. Gerard NP, Gerard C. The chemotactic receptor for human C5a anaphylatoxin. Nature. 1991;349(6310):614–617. [PubMed]
9. Amatruda TT, III, Gerard NP, Gerard C, Simon MI. Specific interactions of chemoattractant factor receptors with G-proteins. Journal of Biological Chemistry. 1993;268(14):10139–10144. [PubMed]
10. Siciliano SJ, Rollins TE, Springer MS. Interaction between the C5a receptor and Gi in both the membrane-bound and detergent-solubilized states. Journal of Biological Chemistry. 1990;265(32):19568–19574. [PubMed]
11. Chenoweth DE, Goodman MG, Weigle WO. Demonstration of a specific receptor for human C5a anaphylatoxin on murine macrophages. Journal of Experimental Medicine. 1982;156(1):68–78. [PMC free article] [PubMed]
12. Chenoweth DE, Hugli TE. Demonstration of specific C5a receptor on intact human polymorphonuclear leukocytes. Proceedings of the National Academy of Sciences of the United States of America. 1978;75(8):3943–3947. [PubMed]
13. Werfel T, Oppermann M, Schulze M, Krieger G, Weber M, Gotze O. Binding of fluorescein-labeled anaphylatoxin C5a to human peripheral blood, spleen, and bone marrow leukocytes. Blood. 1992;79(1):152–160. [PubMed]
14. Gerard NP, Hodges MK, Drazen JM, Weller PF, Gerard C. Characterization of a receptor for C5a anaphylatoxin on human eosinophils. Journal of Biological Chemistry. 1989;264(3):1760–1766. [PubMed]
15. Haviland DL, McCoy RL, Whitehead WT, et al. Cellular expression of the C5a anaphylatoxin receptor (C5aR): demonstration of C5aR on nonmyeloid cells of the liver and lung. Journal of Immunology. 1995;154(4):1861–1869.
16. Schieferdecker HL, Rothermel E, Timmermann A, Gotze O, Jungermann K. Anaphylatoxin C5a receptor mRNA is strongly expressed in Kupffer and stellate cells and weakly in sinusoidal endothelial cells but not in hepatocytes of normal rat liver. FEBS Letters. 1997;406(3):305–309. [PubMed]
17. Schlaf G, Schieferdecker HL, Rothermel E, Jungermann K, Gotze O. Differential expression of the C5a receptor on the main cell types of rat liver as demonstrated with a novel monoclonal antibody and by C5a anaphylatoxin-induced Ca2+ release. Laboratory Investigation. 1999;79(10):1287–1297. [PubMed]
18. Riedemann NC, Guo R-F, Sarma VJ, et al. Expression and function of the C5a receptor in rat alveolar epithelial cells. Journal of Immunology. 2002;168(4):1919–1925.
19. Gasque P, Chan P, Fontaine M, et al. Identification and characterization of the complement C5a anaphylatoxin receptor on human astrocytes. Journal of Immunology. 1995;155(10):4882–4889.
20. Fayyazi A, Scheel O, Werfel T, et al. The C5a receptor is expressed in normal renal proximal tubular but not in normal pulmonary or hepatic epithelial cells. Immunology. 2000;99(1):38–45. [PubMed]
21. Braun M, Davis AE., III Cultured human glomerular mesangial cells express the C5a receptor. Kidney International. 1998;54(5):1542–1549. [PubMed]
22. Buchner RR, Hugli TE, Ember JA, Morgan EL. Expression of functional receptors for human C5a anaphylatoxin (CD88) on the human hepatocellular carcinoma cell line HepG2: stimulation of acute-phase protein-specific mRNA and protein synthesis by human C5a anaphylatoxin. Journal of Immunology. 1995;155(1):308–315.
23. McCoy R, Haviland DL, Molmenti EP, Ziambaras T, Wetsel RA, Perlmutter DH. N-formylpeptide and complement C5a receptors are expressed in liver cells and mediate hepatic acute phase gene regulation. Journal of Experimental Medicine. 1995;182(1):207–217. [PMC free article] [PubMed]
24. Riedemann NC, Guo R-F, Neff TA, et al. Increased C5a receptor expression in sepsis. Journal of Clinical Investigation. 2002;110(1):101–108. [PMC free article] [PubMed]
25. Huber-Lang MS, Riedeman NC, Sarma VJ, et al. Protection of innate immunity by C5aR antagonist in septic mice. FASEB Journal. 2002;16(12):1567–1574. [PubMed]
26. Arumugam TV, Shiels IA, Strachan AJ, Abbenante G, Fairlie DP, Taylor SM. A small molecule C5a receptor antagonist protects kidneys from ischemia/reperfusion injury in rats. Kidney International. 2003;63(1):134–142. [PubMed]
27. Arumugam TV, Shiels IA, Woodruff TM, Reid RC, Fairlie DP, Taylor SM. Protective effect of a new C5a receptor antagonist against ischemia-reperfusion injury in the rat small intestine. Journal of Surgical Research. 2002;103(2):260–267. [PubMed]
28. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001;411(6836):494–498. [PubMed]
29. McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA. RNA interference in adult mice. Nature. 2002;418(6893):38–39. [PubMed]
30. Lewis DL, Hagstrom JE, Loomis AG, Wolff JA, Herweijer H. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nature Genetics. 2002;32(1):107–108. [PubMed]
31. Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nature Medicine. 2003;9(3):347–351.
32. Ge Q, Filip L, Bai A, Nguyen T, Eisen HN, Chen J. Inhibition of influenza virus production in virus-infected mice by RNA interference. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(23):8676–8681. [PubMed]
33. Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell. 2002;2(3):243–247. [PubMed]
34. Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & Development. 2002;16(8):948–958. [PubMed]
35. Sui G, Soohoo C, Affar el B, Gay F, Shi Y, Forrester WC. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proceedings of the National Academy of Sciences of the United States of America. 2002;99(8):5515–5520. [PubMed]
36. Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proceedings of the National Academy of Sciences of the United States of America. 2002;99(9):6047–6052. [PubMed]
37. Hwang JI, Fraser ID, Choi S, Qin XF, Simon MI. Analysis of C5a-mediated chemotaxis by lentiviral delivery of small interfering RNA. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(2):488–493. [PubMed]
38. Schomber T, Kalberer CP, Wodnar-Filipowicz A, Skoda RC. Gene silencing by lentivirus-mediated delivery of siRNA in human CD34+ cells. Blood. 2004;103(12):4511–4513. [PubMed]
39. Xia H, Mao Q, Paulson HL, Davidson BL. siRNA-mediated gene silencing in vitro and in vivo. Nature Biotechnology. 2002;20(10):1006–1010.
40. Boden D, Pusch O, Lee F, Tucker L, Ramratnam B. Efficient gene transfer of HIV-1-specific short hairpin RNA into human lymphocytic cells using recombinant adeno-associated virus vectors. Molecular Therapy. 2004;9(3):396–402. [PubMed]
41. Kay MA, Nakai H. Looking into the safety of AAV vectors. Nature. 2003;424(6946):251. [PubMed]
42. Thomas CE, Ehrhardt A, Kay MA. Progress and problems with the use of viral vectors for gene therapy. Nature Reviews Genetics. 2003;4(5):346–358.
43. Arts GJ, Langemeijer E, Tissingh R, et al. Adenoviral vectors expressing siRNAs for discovery and validation of gene function. Genome Research. 2003;13(10):2325–2332. [PubMed]
44. Zhao LJ, Jian H, Zhu H. Specific gene inhibition by adenovi-rus-mediated expression of small interfering RNA. Gene. 2003;316(1-2):137–141. [PubMed]
45. Riedemann NC, Neff TA, Guo R-F, et al. Protective effects of IL-6 blockade in sepsis are linked to reduced C5a receptor expression. Journal of Immunology. 2003;170(1):503–507.
46. Gerard C, Bao L, Orozco O, Pearson M, Kunz D, Gerard NP. Structural diversity in the extracellular faces of peptidergic G-protein-coupled receptors: molecular cloning of the mouse C5a anaphylatoxin receptor. Journal of Immunology. 1992;149(8):2600–2606.
47. Sun L, Bittner MA, Holz RW. Rab3a binding and secretion-enhancing domains in Rim1 are separate and unique. Studies in adrenal chromaffin cells. Journal of Biological Chemistry. 2001;276(16):12911–12917. [PubMed]
48. Speyer CL, Neff TA, Warner RL, et al. Regulatory effects of iNOS on acute lung inflammatory responses in mice. American Journal of Pathology. 2003;163(6):2319–2328. [PubMed]
49. Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell. 2000;101(1):25–33. [PubMed]
50. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes & Development. 2001;15(2):188–200. [PubMed]
51. Guo R-F, Ward PA. Role of C5a in inflammatory responses. Annual Review of Immunology. 2005;23:821–852.
52. Paul CP, Good PD, Li SX, Kleihauer A, Rossi JJ, Engelke DR. Localized expression of small RNA inhibitors in human cells. Molecular Therapy. 2003;7(2):237–247. [PubMed]
53. Huang S, Endo RI, Nemerow GR. Upregulation of integrins alpha v beta 3 and alpha v beta 5 on human monocytes and T lymphocytes facilitates adenovirus-mediated gene delivery. Journal of Virology. 1995;69(4):2257–2263. [PMC free article] [PubMed]
54. Kaner RJ, Worgall S, Leopold PL, et al. Modification of the genetic program of human alveolar macrophages by adenovirus vectors in vitro is feasible but inefficient, limited in part by the low level of expression of the coxsackie/adenovirus receptor. American Journal of Respiratory Cell and Molecular Biology. 1999;20(3):361–370. [PubMed]
55. Greber UF, Willetts M, Webster P, Helenius A. Stepwise dismantling of adenovirus 2 during entry into cells. Cell. 1993;75(3):477–486. [PubMed]

Articles from Journal of Biomedicine and Biotechnology are provided here courtesy of Hindawi