Platelet-derived growth factor B (PDGF-B) plays an essential role in hepatic fibrosis. Inhibition of the PDGF-B signaling in chronically injured livers might represent a potential therapeutic measure for hepatic fibrosis. In this study, we assessed the effects of vaccination against PDGF-B on CCl4-induced liver fibrosis in BALB/c mice. The PDGF-B kinoid immunogens were prepared by cross-linking two PDGF-B-derived B-cell epitope peptides [PDGF-B16-(23–38) and PDGF-B16-(72–83)] to ovalbumin and keyhole limpet hemocyanin, respectively. Enzyme-linked immunosorbent assay, Western blotting, and NIH3T3 cell proliferation assay verified that immunization with the PDGF-B kinoids elicited the production of high levels of neutralizing anti-PDGF-B autoantibodies. The vaccination markedly alleviated CCl4-induced hepatic fibrosis, as indicated by the lessened morphological alternations and reduced hydroxyproline contents in the mouse livers. Moreover, immunohistochemical staining for proliferating cell nuclear antigen, α-smooth muscle actin, and desmin demonstrated that neutralization of PDGF-B inhibited both the proliferation and the activation of hepatic stellate cells in the fibrotic mouse livers. Taken together, this study demonstrated that vaccination with PDGF-B kinoids significantly suppressed CCl4-induced hepatic fibrosis in mice. Our results suggest that vaccination against PDGF-B might be developed into an effective, convenient, and safe therapeutic measure for the treatment of hepatic fibrosis.
Hepatic fibrosis, independent of the etiology, results mainly from the activation of hepatic stellate cells (HSCs). During the activation, HSCs undergo increased proliferation, altered cellular morphology to a more myofibroblast (MFB)-like cell type, and up-regulated extracellular matrix (ECM) expression. The activation of HSCs is induced by multiple profibrogenic factors in which transforming growth factor (TGF)-β1 and platelet-derived growth factor (PDGF) are the key stimuli for HSC activation (Pinzani, 2002; Friedman, 2008).
PDGF is a family of pleiotropic cytokines consisting of four polypeptide chains encoded by distinct genes. The PDGF isoforms are expressed as homo- and hetero-dimeric proteins, including PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD. The effects of the distinct cytokine dimers are triggered by binding to and dimerizing the cell membrane PDGF receptors (PDGFR) α and β, which have distinct expression patterns in tissues and various cell types. PDGF-BB triggers signaling by both PDGFRα and PDGFRβ, whereas PDGF-AA binds and dimerizes only PDGFRα. Activation of PDGFR results in phosphorylation of several tyrosine residues in the cytoplasmic domains of the receptors, which allows recruitment of Ras-extracellular signal-regulated kinase and phosphoinositol 3-kinase pathway signaling molecules to modulate the mitogenesis and chemotaxis (Pinzani, 2002; Bonner, 2004).
The relationship between PDGF signaling and hepatic fibrosis has been evidenced by a number of studies. Although all the four PDGF isoforms might be involved in hepatic fibrosis (Campbell et al., 2005; Czochra et al., 2006; Borkham-Kamphorst et al., 2007; Thieringer et al., 2008). PDGF-B signaling through PDGFRβ is considered to be most closely related to hepatic fibrosis. The expressions of PDGF-B and PDGFRβ are rapidly increased in both the experimental hepatic fibrosis in rats and human fibrotic liver (Pinzani et al., 1994, 1996; Wong et al., 1994; Borkham-Kamphorst et al., 2008) as well as in in vitro cultured HSCs (Pinzani et al., 1994, 1996; Borkham-Kamphorst et al., 2008). In vitro studies have demonstrated that PDGF-B is the most potent mitogenic factor for HSCs (Pinzani, 2002). Consequently, blockage of PDGF-B signaling inhibits experimental hepatic fibrosis (Borkham-Kamphorst et al., 2004a,b; Gonzalo et al., 2007; Chen et al., 2008).
Because of its pivotal roles in hepatic fibrosis, antagonizing the PDGF-B signaling in HSCs would offer an attractive strategy for the treatment of fibrotic liver diseases. Several approaches have been reported to block the PDGF-B signaling including reducing the synthesis of active PDGF-B (Borkham-Kamphorst et al., 2004b) or PDGFRβ (Chen et al., 2008) by gene silencing, neutralizing PDGF-B with specific antibodies (Abs) (Ogawa et al., 2010), decoying PDGF-B with soluble PDGF-B receptors (Borkham-Kamphorst et al., 2004a), and suppressing the postreceptor signal transduction pathways (Gonzalo et al., 2007). Although the efficacies of these measures have been validated in experimental hepatic fibrosis or in cultured HSCs, they are not seemingly possible to be employed in clinical practice. Some of the agents mentioned above have short half-lives that require repeated administration over a long time period to achieve therapeutic benefits. Measures involving genetic modification are associated with safety concerns. Furthermore, preparing these potential therapeutic agents is costly and arduous. Considering that clinical hepatic fibrosis is a persistent, chronic process, only a safe, effective, and convenient measure for the continuous elimination of PDGF-B is feasible for treating hepatic fibrosis. Here, we propose that vaccination against PDGF-B might provide a potentially feasible and effective measure for the prevention and retardation of hepatic fibrosis.
One of the advances in the therapeutic research field is the “anti-cytokine vaccines” (Zagury et al., 2001, 2003). By cross-linking or generating fusion proteins with carrier proteins, the normally nonimmunogenic cytokines or growth factors can be converted into immunogens to elicit the production of the specific autoantibodies (Dalum et al., 1999), which can further neutralize the abnormally overproduced cytokines or growth factors and thereby ablate their pathological effects. This notion has been validated in a number of disease models and clinical trials for some cytokine- or growth factor-related disorders (Dalum et al., 1999; Zagury et al., 1999; Holmgren et al., 2006; Le Buanec et al., 2006; González et al., 2007; Rad et al., 2007; Spohn et al., 2007, 2008; Delavallée et al., 2008; Neninger Vinageras et al., 2008; Tissot et al., 2008; Tohyama et al., 2008). In the present study, we prepared two PDGF-B-derived peptide-carrier protein heterocomplexes (kinoids) as PDGF-B vaccines, verified their antigenicity, and tested the suppressive effect of immunization with these two kinoids on CCl4-induced hepatic fibrosis in mice.
Materials and Methods
Preparation of PDGF-B Kinoids.
The two antigenic peptides (N-23VFEISRRLIDRTNANF38-C, PDGF-B-VF16; and N-72QVRKIEIVRKKPIFKK87-C, PDGF-B-QK16) were chosen from the human PDGF-B (CAA45383.1) according to the literatures in which these fragments were identified to be vital for PDGF-B binding to PDGFRβ (LaRochelle et al., 1989, 1992; Engström et al., 1992; Brennand et al., 1997; Patel et al., 1999). These two fragments are homologous to mouse PDGF-B and have no homology to other known PDGF isoforms. These polypeptides were synthesized on an automated peptide synthesizer (ABI433A; Applied Biosystems, Foster City, CA) and subsequently purified by high-performance liquid chromatography to reach a purity of 99%. The polypeptide-carrier heterocomplexes, QK16-keyhole limpet hemocyanin (KLH) and VF16-ovalbumin (OVA) were prepared with a 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) cross-linking kit (Imject Immunogen EDC Kit with KLH and OVA; Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. The conjugates were finally desalted by D-Salt dextran desalting columns (Thermo Fisher Scientific) and were stored at −20°C until use.
Animals and Experimental Protocol.
Specific pathogen-free, 6-week-old male BALB/c mice were provided by the Experimental Animal Center, School of Medicine, Xi'an Jiaotong University. All animals received humane care, and the experimental protocol was approved by the Committee of Laboratory Animal Research according to the institutional guidelines.
For evaluating the antigenicity of the kinoids, 24 mice were assigned to four equal groups: KLH, VF16-OVA, QK16-KLH, and phosphate-buffered saline (PBS). The mice in the two kinoid groups were given four intraperitoneally injections, two weeks apart, of the kinoids (50 μg in 0.2 ml). Complete Freund Adjuvant (Sigma-Aldrich, St. Louis, MO) was used for the first immunization, and Incomplete Freund Adjuvant (Sigma-Aldrich) was used for all the subsequent immunizations. The mice in the control groups were treated similarly, except that kinoid was replaced by KLH (50 μg in 0.2 ml) or PBS. Blood samples were collected from the tail vein for enzyme-linked immunosorbent assay (ELISA) immediately before each immunization. Two weeks after the fourth injection, three mice in each group were euthanized, and blood was collected to isolate the serum for Western blot and the neutralization assay. The remaining mice were maintained, and blood samples were taken every 2 weeks for ELISA detection of the Ab titer. Six months after the first immunization, the mice were euthanized. The liver, lungs, heart, and kidneys were harvested, fixed with 10% formalin, sectioned, and stained with hematoxylin and eosin to evaluate any adverse effects.
Forty BALB/c mice were subjected to the hepatic fibrosis experiment. The mice were assigned to five groups: VF16-OVA/CCl4 (n = 9), QK16-KLH/CCl4 (n = 9), KLH/CCl4 group (n = 8), CCl4 (n = 8), and normal control (NC) (n = 6). The mice were maintained and immunized as described in the above experiment except that the latter two groups were given an equal volume of PBS instead of immunogens. One week after the third immunization, the mice in the former four groups received intraperitoneal injections of CCl4 (1 ml/kg, dissolved in olive oil to reach a final concentration of 20%) twice a week for 6 weeks. The mice in the NC group were dosed with an equal volume of olive oil. After 6 weeks of CCl4 injection, the mice were euthanized. Blood samples were collected for ELISA detection of anti-PDGF-B Abs. The left lobe of the liver was fixed in 10% formalin for histological examination. Other liver tissue was snap-frozen in liquid nitrogen and stored at −70°C for hydroxyproline content determination.
ELISA Determination of Serum Anti-PDGF-B Abs.
Polystyrene microplates were coated with recombinant human PDGF-BB (20 ng/well; R&D Systems, Minneapolis, MN). Upon detection, the mouse serum was serially diluted (1:2, initiated at 1:200) with 1% bovine serum albumin/PBS/Tween 20 (0.02% Tween 20). Goat anti-mouse IgG (1:5000 diluted with PBS/Tween 20; Sigma-Aldrich) was used as the secondary Ab. A reaction was considered positive if the optical density is ≥2.1 times that of the negative control.
Five micrograms of prokaryotically expressed and purified recombinant human PDGF-B [expressed in Escherichia coli with pET-28 a(+) as the expression plasmid, generated by our group] was submitted to a 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto a nitrocellulose membrane. The membrane was incubated with the mouse serum that was diluted at 1:1000 at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG for 1 h. The bands were developed using an enhanced chemiluminescence reagent for 5 min.
For detection of the α-smooth muscle actin (SMA) expression in the liver tissues by Western blotting, the liver tissues were homogenized in radioimmunoprecipitation assay lysis buffer, and 100 μg of the total protein was applied to 12% SDS-PAGE. A mouse anti-α-SMA monoclonal Ab (mAb) (MS-113-P0; Lab Vision, Freemont, CA) was used as the primary Ab. The expression of β-actin was used as the internal control.
Growth assays with NIH3T3 cells were performed by measuring 5-bromo-2′-deoxyuridine (BrdU) incorporation (BrdU Cell Proliferation Assay kit; Calbiochem, San Diego, CA). NIH3T3 cells were plated on 96-well plates (3 × 103/well). The cells were starved in serum-free Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) for 48 h, and then the starvation medium was removed and replaced with fresh medium containing various dilutions of the antiserum. After a 30-min incubation period, recombinant human PDGF-BB (3 ng/ml) and BrdU were added to the medium. Forty-eight hours after treatment, the cells were washed with PBS, and the genomic DNA was fixed and denatured with the fixative/denaturing solution. BrdU incorporation in the cells was detected by an anti-BrdU Ab and quantified by ELISA according to the manufacturer's instructions.
Histology and Immunohistochemistry.
Five-micron-thick liver sections were processed by both hematoxylin and eosin staining and Masson's trichrome staining to assess the architectural alternations and hepatic collagen deposition (fibrosis). The degree of fibrosis was evaluated semiquantitatively by the Ishak system (Ishak et al., 1995).
Immunohistochemistry was performed using the Histostain-Plus SP kit (Invitrogen). After deparaffinization, rehydration, quenching endogenous peroxidase activity, and subsequent blockage with 10% (v/v) normal goat serum, the sections were processed by sequential reactions with the primary Abs [mouse anti-α-SMA mAb, 1:800 dilution; rabbit anti-desmin polyclonal Ab, 1:400 dilution; and mouse anti-proliferating cell nuclear antigen (PCNA) mAb, 1:400 dilution; Lab Vision], biotinized secondary Abs, and S-A/horseradish peroxidase. The negative controls were performed by replacing the primary Abs with preimmune mouse or rabbit serum.
Computer-assisted semiquantitative analysis was used to evaluate the areas of positive α-SMA and desmin staining. All images were quantified using Image-ProPlus version 4.5, a commercially available software package from Media Cybernetics, Inc. (Bethesda, MD). The imaging of the tissue sections was performed using an automated Image-Pro Plus macro that was calibrated for each microscope objective. The data for both α-SMA and desmin staining were expressed as the mean percentages of positively stained area over the total tissue section area. The parenchymal and mesenchymal cells were blindly counted (1000 cells analyzed in 10 randomly chosen fields centered on a centrilobular vein at 400× enlargement) for PCNA expression, which was represented as the PCNA labeling index (LI).
Hepatic Hydroxyproline Content.
The total hydroxyproline content in the liver was determined as described previously (Reddy and Enwemeka, 1996) and was expressed as μg/mg wet liver weight.
The quantitative data are expressed as the mean ± S.E.M.). To assess the statistical significance of intergroup differences in the quantitative data, Bonferroni's multiple comparison tests were performed after one-way analysis of variance, followed by Bartlett's test to determine the homology of variance. Nonparametric data were analyzed by the Mann-Whitney U test. p < 0.05 is considered statistically significant.
Immunization with the PDGF-B Kinoids Efficiently Elicits Specific Anti-PDGF-B-Neutralizing Abs in Mice.
The ELISA with recombinant human PDGF-BB-coated plates showed biweekly immunization with the PDGF-B kinoids resulted in the production of anti-PDGF-B Abs (Fig. 1A). The titers of anti-PDGF-B Abs reached 1:800 to 1600 after three immunizations and 1:6400 to 12,800 two weeks after the fourth boosting, respectively, in both QK16-KLH- and VF16-OVA-immunized mice.
To further confirm the specificity of the polyclonal Abs produced by immunization with PDGF-B kinoids, we performed Western blotting to test the reactivity of the antisera with the recombinant human PDGF-B. The result revealed that the antiserum from the mice immunized with both of the two PDGF-B kinoids reacted with membrane-bound PDGF-B (Fig. 1B). These results clearly indicated that the two PDGF-B kinoids efficiently and similarly elicited the production of anti-PDGF-B Abs.
NIH3T3 growth assay was performed to validate whether the Abs elicited by immunization with the PDGF-B kinoids could neutralize the bioactivity of mouse PDGF-BB. As shown in Fig. 1C, the antisera displayed dose-dependent inhibitory effects on the proliferation of NIH3T3 cells induced by 3 ng/ml of PDGF-BB, indicating that immunization with both QK16-KLH and VF16-OVA could produce neutralizing anti-PDGF-BB Abs in mice.
In addition, after immunization, the mice showed no behavioral abnormalities. When the immunized mice were euthanized 6 months after immunization, examination of the vital organs did not reveal any obvious adverse effects (data not shown).
Vaccination against PDGF-B Protects Mice from CCl4-Induced Hepatic Fibrosis.
Next, we examined the protective effect of immunization with the PDGF-B kinoids on CCl4-induced fibrosis in mice following the protocol illustrated in Fig. 2. The results of ELISA showed that the kinetic pattern of the production of anti-PDGF-B Abs in these mice was similar to that in the former experiment, even though the fourth immunization was performed 1 week after initiating CCl4 injection. The results suggest that CCl4-induced liver injury and fibrosis do not significantly influence the immune response to PDGF-B kinoid immunization in mice.
After 6 weeks of CCl4 injection, the mice were euthanized, and the livers were subjected to pathological examination. Masson's trichrome staining of the liver sections showed that 6 weeks of repeated injections of CCl4 induced obvious and uniform fibrosis in the livers of KLH/CCl4 and CCl4 mice. The severity of hepatic fibrosis in the two PDGF-B kinoid vaccination groups was obviously milder than that in the KLH/CCl4 and CCl4 groups (Fig. 3A). Semiquantitative evaluation by the Ishak system (Ishak et al. 1995) followed by statistical analysis indicated that the fibrosis scores of the two PDGF-B kinoid vaccination groups were significantly lower than that in either the KLH/CCl4 or the CCl4 group, although there were no notable differences between the former two groups or the latter two groups (Table 1).
The content of hydroxyproline, a specific indicator of fibrosis, was determined in the liver tissues. The results of the histological grading showed that the hepatic hydroxyproline contents of the two kinoid-immunization groups were similar and significantly lower than that of either the KLH/CCl4 or the CCl4 group, and those of the latter two groups were similar (Fig. 3B). These results collectively demonstrated that vaccination against PDGF-B inhibited CCl4-induced hepatic fibrosis in mice.
Vaccination against PDGF-B Inhibited the Proliferation of HSC/MFB in the Fibrotic Livers.
To evaluate the effects of vaccination with PDGF-B kinoids on the proliferation of mesenchymal cells and parenchymal cells, we performed immunohistochemistry to detect the expression of PCNA, which is an indicator of cell proliferation. The PCNA immunostaining and subsequent semiquantitative analysis showed that the mice in the CCl4 and KLH/CCl4 groups displayed markedly increased hepatic mesenchymal PCNA LIs compared with the mice in the NC group, whereas the hepatic mesenchymal PCNA LIs in the two PDGF-B kinoid immunization groups were significantly lower than that in either the CCl4 or the KLH/CCl4 group (Fig. 4, A and B). Because HSCs/MFBs are the main components of the mesenchyma in the fibrotic livers, this result essentially indicated that the proliferation of HSCs/MFBs was suppressed by the vaccination. However, although the PCNA LIs in the parenchyma (predominantly hepatocytes) were markedly increased by CCl4 injection, there was no significant difference among the four CCl4-injected groups (Fig. 4, A and C), indicating that the regeneration of the hepatocytes was not influenced by PDGF-B kinoid vaccination.
Vaccination with PDGF-B Kinoids Suppresses Activation of HSCs in Fibrotic Liver.
The expression of α-SMA, an indicator of activated HSCs, was assessed immunohistochemically in this study to evaluate the effect of vaccination against PDGF-B on HSC activation during hepatic fibrosis. In the NC mice, the expression of α-SMA was confined to the smooth muscle cells lining the portal and central veins and the large arteries within the liver. Six weeks of CCl4 injections led to a marked increase in the amount of α-SMA-positive cells distributing in clusters within the fibrous septa (Fig. 5A). The computer-assisted semiquantitative analysis revealed that the α-SMA-positive areas in the QK16-KLH/CCl4 and VF16-OVA/CCl4 groups were significantly decreased compared with that in either the CCl4 or the KLH/CCl4 group, although there was no significant difference either between the former two groups or between the latter two groups for the α-SMA-positive areas (Fig. 5B). This result was further confirmed by Western blotting detection of α-SMA in the liver tissues (Fig. 5C).
The expression of desmin, a marker for intermediately differentiated HSCs (Ballardini et al., 1988; Cassiman et al., 2002) was also detected in this study. The result showed that the changes in the expression of desmin were similar to those of α-SMA, except that the desmin-positive staining was observed at the rim of fibrous septa and in neighboring hepatocytes in the fibrotic livers (Fig. 6, A and B).
Vaccination against pathogenic cytokines and growth factors has been verified as a simple, safe, and efficient approach for the management of the relevant disorders. In this study, we demonstrated that the two PDGF-B kinoids prepared by cross-linking the PDGF-B-derived polypeptides to the carrier proteins elicited high levels of neutralizing anti-PDGF-B Abs and displayed marked antifibrosis effects on CCl4-induced hepatic fibrosis in mice. To our knowledge, this is the first study attempting to validate the suppressive effect of vaccination against a profibrogenic cytokine on hepatic fibrosis.
The Abs elicited by both preventive and therapeutic vaccines should have neutralizing abilities, although non-neutralizing Abs might also exert some inhibitory effects by facilitating the clearance of the antigens. In this study, immunization with the kinoids prepared with both PDGF-B-(23–38) (VF16) and PDGF-B-(72–87) (QK16) elicited high levels of neutralizing anti-PDGF-B Abs. Our results are in agreement with the previously reported results that these two fragments are critical for the binding of PDGF-B to PDGFRβ (LaRochelle et al., 1989, 1992; Engström et al., 1992; Brennand et al., 1997; Patel et al., 1999). Our subsequent results demonstrated that neutralizing PDGF-B by vaccination rendered marked attenuation of CCl4-induced hepatic fibrosis, which is in agreement with the previously reported results that inhibition of PDGF-B signaling suppressed hepatic fibrosis (Pinzani et al., 1994, 1996; Wong et al., 1994; Borkham-Kamphorst et al., 2008), and vaccination against pathogenic cytokines could efficiently alleviate the relevant disorders (Dalum et al., 1999; Zagury et al., 1999; Holmgren et al., 2006; Le Buanec et al., 2006; González et al., 2007; Rad et al., 2007; Spohn et al., 2007, 2008; Delavallée et al., 2008; Neninger Vinageras et al., 2008; Tissot et al., 2008; Tohyama et al., 2008).
The inhibitory effect of immunization against PDGF-B on hepatic fibrosis might be due to several mechanisms. First, neutralization of PDGF-B with anti-PDGF-B Abs is associated with decreased proliferation of HSC/MFB and, therefore, decreases the amount of ECM-producing cells. The second cause of this effect might be that the neutralization of PDGF-B weakens the profibrogenic activities of TGF-β1 and connective tissue growth factor because PDGF-B up-regulates the expression of TGF-β1 receptor I and II (Czuwara-Ladykowska et al., 2001) and connective tissue growth factor (Paradis et al., 2002). Third, it has been reported that PDGF-B has the ability to directly stimulate the activation of HSCs and the expression of ECM by HSCs/MFBs (Kinnman et al., 2003; Borkham-Kamphorst et al., 2004a, 2008; Czochra et al., 2006), so the neutralization of PDGF-B directly leads to the suppression of ECM expression. Consistent with this view, our results demonstrated that vaccination against PDGF-B not only decreased the PCNA LI in the hepatic mesenchyma but also reduced the expression of α-SMA and desmin, the markers for HSC activation, suggesting that PDGF-B possesses a capability of driving the production and deposition of ECM from HSCs/MFBs in addition to the direct mitogenic activity of HSCs/MFBs. The alleviation degree of CCl4-induced hepatic fibrosis resulting from vaccination against PDGF-B is not obviously lower than that resulting from vaccination against TGF-β1 (Z.-M. Hao, S. Li, X.-B. Fan, Y.-F. Lv, H.-Q. Su, H.-P.Jiang, and Q. N. Zhang, unpublished data), suggesting that the role of PDGF-B in hepatic fibrosis is as important as the role of TGF-β1.
One of the major concerns about the safety of vaccination against cytokines is that the neutralization of the cytokines by autoantibodies might impair nontargeted healthy tissues because cytokines are highly pleiotropic. This adverse effect was not observed in our current study or in investigations by others (Dalum et al., 1999; Zagury et al., 1999; Holmgren et al., 2006; Le Buanec et al., 2006; González et al., 2007; Rad et al., 2007; Spohn et al., 2007, 2008; Delavallée et al., 2008; Neninger Vinageras et al., 2008; Tissot et al., 2008; Tohyama et al., 2008). Possible explanations might include the following. 1) The affinities of the cytokines for their receptors are generally higher than for the autoantibodies raised by immunization. 2) The antigen-Ab reaction is a reversible reaction, and the neutralization cannot be so thorough that it completely eliminates the systemic cytokine. Because of the highly efficient biological activity of cytokines, their physiological functions are not likely to be impaired provided there is a residual level of cytokines. 3) The metabolism of a cytokine in the lesioned tissues is different from that in normal tissues. Cytokines exert biological functions principally in local tissues in an autocrine or paracrine manner. In normal tissues, there is an efficient feedback regulatory mechanism for the production of a cytokine. Removal of the cytokine will trigger the compensatory production of the cytokine, whereas in lesioned tissues, the production of a cytokine is dysregulated, and the cytokine is produced at a constant high rate. Neutralization of the excessively produced pathogenic cytokines does not lead to compensatory overproduction of the cytokine. 4) Pathologic tissues exhibit an abundant stromal lymph flow that facilitates the accumulation of local Abs, whereas normal tissues demonstrate a negligible lymph turnover flow where the poorly renewed Abs are unlikely to impair the short-distance and instant cytokine reaction occurring within the immunological synapse between tightly associated immune cells. Nevertheless, the safety of cytokine vaccines still needs to be investigated carefully in future studies.
In this preliminary study, we immunized the mice before the induction of hepatic fibrosis; thus, our results reflect the “preventive” rather than the “therapeutic” effect of vaccination against PDGF-B on experimental hepatic fibrosis. Because enhanced PDGF-B signaling not only promotes the initiation of hepatic fibrosis but also plays an important role in the persistence of hepatic fibrosis, and the humoral immune response both in animals and in patients with hepatic fibrosis is not seriously impaired (Cheong et al., 2006), we believe that vaccination against PDGF-B could produce a therapeutic effect on hepatic fibrosis/cirrhosis. Nevertheless, whether vaccination against PDGF-B could inhibit the advancement or further facilitate the reversal of established hepatic fibrosis/cirrhosis needs to be investigated in future studies.
In conclusion, our study verified that vaccination against PDGF-B with PDGF-B kinoids markedly inhibited CCl4-induced hepatic fibrosis, suggesting that this approach might be developed into an efficient, safe, simple, and convenient therapeutic strategy for managing chronic fibrotic liver diseases. Furthermore, it has been reported that PDGF-B signaling accelerates the carcinogenesis in the fibrotic livers (Maass et al., 2011). Therefore, vaccination against PDGF-B for chronic liver diseases might have dual benefits of both suppressing fibrosis and preventing carcinogenesis. In addition, because fibrosis of various organs shares a rather common underlying pathological mechanism in which multiple profibrogenic factors are involved, combined vaccination against more than one of these profibrotic cytokines should be considered for fibrosis of various organs and tissues.
Participated in research design: Hao and Fan.
Conducted experiments: Hao, Fan, S. Li, Su, Jiang, and H.-H. Li.
Contributed new reagents or analytic tools: Lv.
Performed data analysis: Hao and Fan.
Wrote or contributed to the writing of the manuscript: Hao and S. Li.
This study was supported by the National Foundation of Natural Sciences, China [Grants 30871144, 81070351].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- hepatic stellate cell
- extracellular matrix
- transforming growth factor
- platelet-derived growth factor
- platelet-derived growth factor receptor
- monoclonal antibody
- keyhole limpet hemocyanin
- phosphate-buffered saline
- enzyme-linked immunosorbent assay
- normal control
- polyacrylamide gel electrophoresis
- smooth muscle actin
- proliferating cell nuclear antigen
- labeling index
- 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride.
- Received March 17, 2012.
- Accepted June 15, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics