Cationic liposomes enhanced the rate of transduction of target cells with retroviral vectors. The greatest effect was seen with the formulation DC-Chol/DOPE, which gave a 20-fold increase in initial transduction rate. This allowed an efficiency of transduction after brief exposure of target cells to virus plus liposome that could be achieved only after extensive exposure to virus alone. Enhancement with DC-Chol/DOPE was optimal when stable virion-liposome complexes were preformed. The transduction rate for complexed virus, as for virus used alone or with the polycation Polybrene, showed first-order dependence on virus concentration. Cationic liposomes, but not Polybrene, were able to mediate envelope-independent transduction, but optimal efficiency required envelope-receptor interaction. When virus complexed with DC-Chol/DOPE was used to transduce human mesothelioma xenografts, transduction was enhanced four- to fivefold compared to that for virus alone. Since the efficacy of gene therapy is dependent on the number of cells modified, which is in turn dependent upon the balance between transduction and biological clearance of the vector, the ability of cationic liposomes to form stable complexes with retroviral vectors and enhance their rate of infection is likely to be important for in vivo application.
We are developing retroviral-mediated gene transfer to human fibroblast-like synovial cells (FLS) as one approach to characterizing genetic pathways involved in synoviocyte pathophysiology. Prior work has suggested that FLS are relatively refractory to infection by Moloney murine leukemia virus based vectors. To determine if viral titer influenced the transduction efficiency of FLS, we optimized a rapid, efficient, and inexpensive centrifugation method to concentrate recombinant retroviral supernatant. The technique was evaluated by measurement of the expression of a viral enhanced green fluorescent protein transgene in transduced cells, and by analysis of viral RNA in retroviral supernatant. Concentration (100-fold) was achieved by centrifugation of viral supernatant for four hours, with 100% recovery of viral particles. The transduction of FLS increased from approximately 15% with unconcentrated supernatant, to nearly 50% using concentrated supernatant. This protocol will be useful for investigators with applications that require efficient, stable, high level transgene expression in primary FLS.
enhanced green fluorescent protein; fibroblast-like synovial cell; gene therapy; retrovirus; titer
Retrovirus-mediated gene transfer offers the potential for stable long-term expression of transduced genes in host cells subsequent to integration of vector DNA into the host genome. Using a murine amphotropic retrovirus vector containing an interleukin-2 receptor (IL-2R) gene as a reporter and a neomycin phosphotransferase (neor) gene as a dominant selectable marker, we measured the efficiency of retrovirus-mediated gene transfer and the stability of transduced gene expression in a cystic fibrosis tracheal epithelial cell line (CFT1). The use of the IL-2R cell surface marker as a reporter of infection permitted both quantitation of vector gene expression and flow cytometric sorting of cells transduced with the vector. In initial studies, the optimal conditions for retrovirus-mediated gene transfer were determined. The presence of a polycation was required for optimal transduction efficiency. The efficiency of infection of CFT1 cells was increased by repetitive exposure to virus such that it was possible to transduce approximately 80% of the cells following three successive daily exposures. The long-term stability of expression of the non-selected IL-2R gene was also evaluated. A slow decline in the percentage of cells expressing IL-2R was seen with cells that were maintained under constant selection pressure for expression of the neor gene, which was expressed from an internal promoter. Similar results were obtained when cultures were selected initially for neor gene expression and maintained without selection thereafter. In contrast, stable expression was observed in CFT1 cells for at least one year following multiple infections in the absence of G418 selection. In conclusion, (i) transduction of foreign genes into human airway epithelial cells using an amphotropic retrovirus vector can be highly efficient in the presence of appropriate polycations and multiple exposures; and (ii) stable expression of a non-selected gene in these epithelial cells is better maintained without selection.
The colony formation assay is the most commonly used titration method for defining the concentration of replication-incompetent murine leukemia virus-derived retroviral vectors. However, titer varies with target cell type and number, transduction time, and concentration of polycation (e.g., Polybrene). Moreover, because most of the viruses cannot encounter target cells due to Brownian motion, their short half-lives, and the requirement for target cell division for activity, the actual infectious retrovirus concentration in the collected supernatant is higher than the viral titer. Here we correlate the physical viral particle concentration with the infectious virus concentration and colony formation titer with the help of a mathematical model. Ecotropic murine leukemia retrovirus supernatant, collected from the GP+E86/LNCX retroviral vector producer cell line, was concentrated by centrifugation and further purified by a sucrose density gradient. The physical concentration of purified viral vectors was determined by direct particle counting with an electron microscope. The concentrations of fresh and concentrated supernatant were determined by a quantitative reverse transcriptase activity assay. Titration of all supernatants by neomycin-resistant colony formation assay was also performed. There were 767 ± 517 physical viral particles per infectious CFU in the crude viral supernatant. However, the infectious viral concentration determined by mathematical simulation was 143 viral particles per infectious unit, which is more consistent with the concentration determined by particle counting in purified viral solution. Our results suggest that the mathematical model can be used to extract a more accurate and reliable concentration of infectious retrovirus.
We have used cationic liposomes (Lipofectin) to facilitate retrovirus infection of cells lacking the homologous viral receptor. Ecotropic murine leukemia virus and packaged retroviral vectors were shown to infect mink cells, and amphotropic packaged retroviral vectors were shown to infect hamster cells in the presence of Lipofectin but not in the presence of Polybrene. Lipofectin-mediated infection of cells lacking the homologous receptor results in a titer approximately 0.1% of the titer in cells with the homologous receptor, using the standard Polybrene protocol. The use of Lipofectin may provide a simple means to experimentally infect a wide variety of cells with viruses not normally infectious for the species, tissue, or cell type of interest.
This paper provides the first evidence of a clinical response to gene therapy in human arthritis. Two subjects with rheumatoid arthritis received ex vivo, intraarticular delivery of human interleukin-1 receptor antagonist (IL-1Ra) cDNA. To achieve this, autologous synovial fibroblasts were transduced with a retrovirus, MFG-IRAP, carrying IL-1Ra as the transgene, or remained as untransduced controls. Symptomatic metacarpophalangeal (MCP) joints were injected with control or transduced cells. Joints were clinically evaluated on the basis of pain; the circumference of MCP joint 1 was also measured. After 4 weeks, joints underwent surgical synovectomy. There were no adverse events in either subject. The first subject responded dramatically to gene transfer, with a marked and rapid reduction in pain and swelling that lasted for the entire 4 weeks of the study. Remarkably, joints receiving IL-1Ra cDNA were protected from flares that occurred during the study period. Analysis of RNA recovered after synovectomy revealed enhanced expression of IL-1Ra and reduced expression of matrix metalloproteinase-3 and IL-1β. The second subject also responded with reduced pain and swelling. Thus, gene transfer to human, rheumatoid joints can be accomplished safely to produce clinical benefit, at least in the short term. Using this ex vivo procedure, the transgene persisted within the joint for at least 1 month. Further clinical studies are warranted.
Several factors are thought to limit the efficiency of retroviral transduction in clinical gene therapy protocols that target hematopoietic stem cells. For example, the level of expression of the amphotropic receptor Pit-2, a phosphate symporter, appears to be low in human and murine hematopoietic stem cells. We have previously demonstrated that transduction of hematopoietic cells in the presence of the fibronectin (FN) fragment CH-296 is extremely efficient (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876–882, 1996). To examine functionally whether the retrovirus receptor is a limiting factor in transduction of hematopoietic cells, we performed competition experiments in the presence of FN CH-296 with retrovirus vectors pseudotyped with the same or a different envelope protein. We demonstrate in both human erythroleukemia (HEL) cells and primary human CD34+ hematopoietic cells inhibition of efficient infection due to receptor interference when two vectors targeting the amphotropic receptor are used simultaneously. Receptor interference lasted up to 24 h. No interference was demonstrated when vectors targeting the amphotropic receptor and the gibbon ape leukemia virus (GALV) receptor Pit-1 were used concurrently. In contrast, simultaneous infection with vectors targeting both Pit-1 and Pit-2 yielded transduction efficiencies consistently higher than with either vector alone in both HEL cells and human CD34+ hematopoietic cells. These data demonstrate that the use of FN CH-296 leads to amphotropic receptor saturation in these cells. Simultaneous infection with vectors targeting both amphotropic and GALV receptors may prove to be of additional benefit in the design of gene therapy protocols.
Using a model recombinant retrovirus encoding the Escherichia coli lacZ gene, we have found that medium conditioned with NIH 3T3 cells and packaging cell lines derived from NIH 3T3 cells inhibits infection. Most of the inhibitory activity was greater than 100 kDa and was sensitive to chondroitinase ABC digestion, which is consistent with the inhibitor being a chondroitin sulfate proteoglycan. Proteoglycans secreted by NIH 3T3 cells and purified by anion-exchange chromatography inhibited amphotropic retrovirus infection. Pretreatment of amphotropic retrovirus stocks with chondroitinase ABC boosted the level of transduction efficiency by more than twofold. The implications of these findings with respect to retrovirus-cell interactions and the production of high-titer retroviral stocks are discussed.
Transduction by murine leukemia virus-based retrovirus vectors is limited in certain cell types, particularly in nondividing cells. But transduction can be inefficient even in cells that divide rapidly. For example, exposure of 208F rat embryo fibroblasts to an excess of an amphotropic retrovirus vector encoding alkaline phosphatase results in a transduction efficiency of only about 10%, even though these cells divide rapidly. Here we show that transduction of 208F cells is limited by cell surface retrovirus receptor levels; overexpression of the amphotropic retrovirus receptor Pit2 markedly improved the transduction efficiency to 50%. To characterize receptor levels and binding affinity, we synthesized a fusion protein that joins the amino terminus of the amphotropic envelope protein to the Fc region of a human immunoglobulin G1 molecule for use in binding assays. In comparison to the parental cell line, the modified cell line showed an order of magnitude increase in binding sites of from 18,000 to 150,000 per cell. Thus, efficient transduction by an amphotropic retrovirus vector requires high-level expression of the retrovirus receptor Pit2. These results provide the rationale for further examination of the role of receptor levels in inefficient transduction, especially with regard to target cells for gene therapy, where a high transduction rate is often crucial.
We constructed several retroviruses which transduced a mutant dihydrofolate reductase gene that was resistant to methotrexate inhibition and functioned as a dominant selectable marker. The titer of dihydrofolate reductase-transducing virus produced by virus-producing cells could be increased to very high levels by selection of the cells in increasing concentrations of methotrexate. Helper virus-free dihydrofolate reductase-transducing virus was also generated by using a broad-host-range amphotropic retroviral packaging system. Cell lines producing helper-free dihydrofolate reductase-transducing virus with a titer of 4 X 10(6) per ml were generated. These retroviral vectors should have general utility for high-efficiency transduction of genes in cultured cells and in animals.
Background: Gene therapy of the joint has great potential as a new therapeutic approach for the treatment of rheumatoid arthritis (RA). The vector chosen is of crucial importance for clinical success.
Objective: To investigate the tropism and transduction efficiency in arthritic joints in vivo, and in synovial cells in vitro, using five different serotypes of recombinant adeno-associated virus (rAAV) encoding ß-galactosidase or green fluorescent protein genes.
Methods: rAAV was injected into the ankle joints of rats with adjuvant arthritis after the onset of disease. Synovial tissue was examined at different time points for ß-galactosidase protein and gene expression by in situ staining and polymerase chain reaction (PCR) analysis, respectively. In addition, the ability of rAAV to transduce primary human fibroblast-like synoviocytes from patients with RA was investigated in vitro.
Results: Intra-articular injection of the rAAV5 serotype resulted in the highest synovial transduction, followed by much lower expression using rAAV2. Expression of the transgene was already detectable 7 days after injection and lasted for at least 4 weeks. Only background staining was seen for serotypes 1, 3, and 4. Importantly, there was a minimal humoral immune response to rAAV5 compared with rAAV2. Additionally, it was found that both rAAV2 and rAAV5 can efficiently transduce human fibroblast-like synoviocytes obtained from patients with RA.
Conclusion: Intra-articular rAAV mediated gene therapy in RA might be improved by using rAAV5 rather than other serotypes.
Human mesenchymal stem cells (hMSCs) can be engineered to express specific genes, either for their use in cell-based therapies or to track them in vivo over long periods of time. To obtain long-term expression of these genes, a lentivirus- or retrovirus-mediated cell transduction is often used. However, given that the efficiency with these viruses is typically low in primary cells, additives such as polybrene are always used for efficient viral transduction. Unfortunately, as presented here, exposure to polybrene alone at commonly used concentratons (1–8 µg/mL) negatively impacts hMSC proliferation in a dose-dependent manner as measured by CyQUANT, EdU incorporation, and cell cycle analysis. This inhibition of proliferation was observable in culture even 3 weeks after exposure. Culturing the cells in the presence of FGF-2, a potent mitogen, did not abrogate this negative effect of polybrene. In fact, the normally sharp increase in hMSC proliferation that occurs during the first days of exposure to FGF-2 was absent at 4 µg/mL or higher concentrations of polybrene. Similarly, the effect of stimulating cell proliferation under simulated hypoxic conditions was also decreased when cells were exposed to polybrene, though overall proliferation rates were higher. The negative influence of polybrene was, however, reduced when the cells were exposed to polybrene for a shorter period of time (6 hr vs 24 hr). Thus, careful evaluation should be done when using polybrene to aid in lentiviral transduction of human MSCs or other primary cells, especially when cell number is critical.
Pathogenicity of the encephalomyocarditis (EMC) virus for adult mice was increased when polycations of diverse type were mixed with virus and inoculated by the subcutaneous or intraperitoneal routes. Diethylaminoethyl (DEAE) dextran, hexadimethrine (polybrene), polymyxin B, polylysine, and calf thymus histone in various concentrations stimulated multiplication of virus in tissues at the injection site and enhanced entry of virus into the blood. The nonpathogenic r+ variant of EMC which grows locally in tissues but fails to disseminate after subcutaneous and intraperitoneal inoculation was used in most experiments. This virus caused viremia and fatal central nervous system disease only when the polycations were included in the inoculum. DEAE dextran and polybrene stimulated the release of interferon in infected tissues but had no effect in the absence of virus multiplication. Histological studies of tissues from the injection site showed that polycations provoke a mononuclear cell reaction and alter the integrity of connective tissue. However, the mechanism by which the substances enhance virus growth and dissemination was not defined.
We report here on the development and characterization of a novel herpes simplex virus type 1 (HSV-1) amplicon-based vector system which takes advantage of the host range and retention properties of HSV–Epstein-Barr virus (EBV) hybrid amplicons to efficiently convert cells to retrovirus vector producer cells after single-step transduction. The retrovirus genes gag-pol and env (GPE) and retroviral vector sequences were modified to minimize sequence overlap and cloned into an HSV-EBV hybrid amplicon. Retrovirus expression cassettes were used to generate the HSV-EBV-retrovirus hybrid vectors, HERE and HERA, which code for the ecotropic and the amphotropic envelopes, respectively. Retrovirus vector sequences encoding lacZ were cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retrovirus titers between 106 and 107 transducing units/ml, while infection of the same cells with amplicon vectors generated maximum titers 1 order of magnitude lower. Retrovirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retrovirus vectors even at the same transduction efficiencies. Infection of human and dog primary gliomas with this system resulted in the production of retrovirus vectors for more than 1 week and the long-term retention and increase in transgene activity over time in these cell populations. Although the efficiency of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery.
Currently, treatment for rheumatoid arthritis and other inflammatory arthropathies is often ineffective in ameliorating the progression of the disease, particularly the invasive destruction of cartilage and bone by rheumatoid synovium. Multiple aspects of this inflammatory process are mediated by the synovial lining cells (synoviocytes). Genetic modification of these cells in vivo represents a potential method for the treatment of these conditions. In this report, we describe a novel technique for the genetic transduction of synovial lining cells in vivo using recombinant adenoviral vectors and intraarticular injection techniques. Purified high titer suspensions of a recombinant adenoviral vector containing the gene for Escherichia coli beta-galactosidase (AdCMVlacZ) were directly injected into the hind knees of New Zealand white rabbits. Synovial tissues were then examined for transgenic lacZ expression using a combination of in situ staining for beta-galactosidase activity, immunohistochemical staining, and transmission electron microscopy. High efficiency gene transfer and lacZ expression was observed in both type A and type B synoviocytes throughout the articular and periarticular synovium of the rabbit knee, with continued expression of transgenic lacZ detected for > or = 8 wk after infection.
Currently, amphotropic retroviral vectors are widely used for gene transfer into CD34+ hematopoietic progenitor cells. The relatively low levels of transduction efficiency associated with these vectors in human cells is due to low viral titers and limitations in concentrating the virus because of the inherent fragility of retroviral envelopes. Here we show that a human immunodeficiency virus type 1 (HIV-1)-based retroviral vector containing the firefly luciferase reporter gene can be pseudotyped with a broad-host-range vesicular stomatitis virus envelope glycoprotein G (VSV-G). Higher-efficiency gene transfer into CD34+ cells was achieved with a VSV-G-pseudotyped HIV-1 vector than with a vector packaged in an amphotropic envelope. Concentration of virus without loss of viral infectivity permitted a higher multiplicity of infection, with a consequent higher efficiency of gene transfer, reaching 2.8 copies per cell. These vectors also showed remarkable stability during storage at 4 degrees C for a week. In addition, there was no significant loss of titer after freezing and thawing of the stock virus. The ability of VSV-G-pseudotyped retroviral vectors to achieve a severalfold increase in levels of transduction into CD34+ cells will allow high-efficiency gene transfer into hematopoietic progenitor cells for gene therapy purposes. Furthermore, since it has now become possible to infect CD34+ cells with pseudotyped HIV-1 with a high level of efficiency in vitro, many important questions regarding the effect of HIV-1 on lineage-specific differentiation of hematopoietic progenitors can now be addressed.
Certain glycosaminoglycans (GAGs), including heparin, inhibit infection by murine leukemia virus (MLV). We now show that this is due to inhibition of virus attachment independent of the interaction between viral envelope proteins (Env) and their cellular receptors. Heparin blocked the binding of both Env-deficient and amphotropic MLV (MLV-A) particles to NIH 3T3 fibroblasts, CHO cells which lack the amphotropic retroviral receptor Pit-2, and CHO cells transfected with Pit-2 (CHO-Pit-2). Heparin also inhibited the transduction of NIH 3T3 cells by MLV-A over a similar concentration range. This effect was observed within 15 min of exposure to retrovirus. Preloading target cells with heparin had no effect on transduction and both MLV-A and Env-deficient retrovirus bound efficiently to heparin-coated agarose beads, suggesting that heparin interacts with the virus rather than the target cell. This requires both a strong negative charge and a specific structure since GAGs with different charge and carbohydrate composition inhibited virus infection variably. The specificity of GAG-virus interaction also depends on the producer cells, since virus packaged by murine GP+EnvAM12 cells was 1,000-fold more sensitive to inhibition by chondroitin sulfate A than was virus packaged by human FLYA13 packaging cells. No evidence for an interaction between MLV and cell surface proteoglycans was found, however, since the attachment of MLV-A and envelope-defective virus to proteoglycan-deficient CHOpgsA-745 cells was similar to that seen with both wild-type and CHO-Pit-2 cells. Although the molecular mechanism is unclear, this study presents evidence that Env receptor-independent attachment is an important step in MLV infection.
Human NK-like cell lines are difficult to transfect using standard mammalian expression vectors and conventional transfection protocols, but they are susceptible to retroviral transduction as a means to introduce cDNAs. Our lab has exploited this technique to study a number of receptors in human NK cell lines. The method utilizes a bicistronic retroviral vector that co-expresses either drug resistance or enhanced green fluorescent protein (EGFP) in parallel with the gene of interest. After a single infection with recombinant retrovirus, transduced NK cells can be sorted for expression of EGFP or the transduced cell surface marker. Alternatively, cells expressing the transduced cDNAs can be selected for by treatment with neomycin, puromycin or hygromycin. Using this method, the sorted/selected cells uniformly express the gene of interest and the expression is stable for many weeks of culture.
retroviral transduction; NK cell lines; EGFP
Nonviral producer cell proteins incorporated into retroviral vector surfaces profoundly influence infectivity and in vivo half-life. We report the purification and concentration of lentiviral vectors using these surface proteins as an efficient gene transduction strategy. Biotinylation of these proteins and streptavidin paramagnetic particle concentration enhances titer 400- to 2,500-fold (to 109 CFU/ml for vesicular stomatitis virus G protein and 5 × 108 for amphotropic murine leukemia virus envelope). This method also uses newly introduced membrane proteins (B7.1 and ΔLNGFR) directed to lentiviral surfaces, allowing up to 17,000-fold concentrations. Particle conjugation of lentivirus allows facile manipulation in vitro, resulting in the transduction of 48 to 94% of human acute myeloid leukemia blasts.
Primary human T lymphocytes are powerful targets for genetic modification, although the use of these targets in human gene therapy protocols has been hampered by low levels of transduction. We have shown previously that significant increases in the transduction of hematopoietic stem and progenitor cells with retroviral vectors can be obtained by the colocalization of the retrovirus and target cells on specific fibronectin (FN) adhesion domains (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876–882, 1996). We studied the transfer of genes into primary T lymphocytes by using FN-assisted retroviral gene transfer. Activated T lymphocytes were infected for three consecutive days on the recombinant FN fragment CH-296 with a retroviral vector encoding the murine B7-1 protein. Transduced lymphocytes were analyzed for murine B7-1 expression, and it was found that under optimal conditions, 80 to 89% of the CD3+ lymphocytes were transduced. Gene transfer was predominantly augmented by the interaction between VLA-4 on the T lymphocytes and the FN adhesion site CS-1. Adenosine deaminase (ADA)-deficient primary T lymphocytes transduced on CH-296 with a retrovirus encoding murine ADA (mADA) exhibited levels of mADA activity severalfold higher than the levels of the endogenous human ADA protein observed in normal human T lymphocytes. Strikingly, the long-term expression of the transgene was dependent on the activation status of the lymphocytes. This approach will have important applications in human gene therapy protocols targeting primary T lymphocytes.
The purpose of this study was to determine the capacity of cells transduced with human β-defensins (HBDs) to express antimicrobial peptides, since sufficient expression level is required for effective antimicrobial activity. Retroviral vector pBabeNeo and lentiviral vector SIN18cPPTRhMLV (SIN18) carrying HBDs were utilized to transduce non-HBD-expressing cells such as fibroblasts or HBD-producing oral epithelial cells. We found that HBD-3 gene transfer to fibroblasts was possible not via retrovirus but by direct vector transfection. SIN18 had high transduction efficiencies (80.9–99.9%) and transduced cells expressed higher amounts of HBD-2 than those by pBabe-Neo. Primary human gingival epithelial cells (HGECs) expressed greater amounts of HBD-2 than primary fibroblasts after lentiviral transduction. Additionally, HBD-2 secretion from transduced HGECs cells was further increased when stimulated with IL-1 or TNFα. Our data indicate that while HBD-2 expression is limited in primary fibroblasts, its expression in HGECs may be maximized by gene transduction plus cytokine induction.
HBD-2; HBD-3; Lentiviral vectors; Retroviral vectors; IL-1; TNFα; Gingival epithelial cells
The ER chaperone GRP78/BiP is crucial for the development of rheumatoid arthritis.
An accumulation of misfolded proteins can trigger a cellular survival response in the endoplasmic reticulum (ER). In this study, we found that ER stress–associated gene signatures were highly expressed in rheumatoid arthritis (RA) synoviums and synovial cells. Proinflammatory cytokines, such as TNF and IL-1β, increased the expression of GRP78/BiP, a representative ER chaperone, in RA synoviocytes. RA synoviocytes expressed higher levels of GRP78 than osteoarthritis (OA) synoviocytes when stimulated by thapsigargin or proinflammatory cytokines. Down-regulation of Grp78 transcripts increased the apoptosis of RA synoviocytes while abolishing TNF- or TGF-β–induced synoviocyte proliferation and cyclin D1 up-regulation. Conversely, overexpression of the Grp78 gene prevented synoviocyte apoptosis. Moreover, Grp78 small interfering RNA inhibited VEGF165-induced angiogenesis in vitro and also significantly impeded synoviocyte proliferation and angiogenesis in Matrigel implants engrafted into immunodeficient mice. Additionally, repeated intraarticular injections of BiP-inducible factor X, a selective GRP78 inducer, increased synoviocyte proliferation and angiogenesis in the joints of mice with experimental OA. In contrast, mice with Grp78 haploinsufficiency exhibited the suppression of experimentally induced arthritis and developed a limited degree of synovial proliferation and angiogenesis. In summary, this study shows that the ER chaperone GRP78 is crucial for synoviocyte proliferation and angiogenesis, the pathological hallmark of RA.
Mast cells have been implicated in the pathogenesis of the matrix degradation observed in the cartilaginous and osseous structures of the rheumatoid joint. We previously reported that human mast cell tryptase, a 134-kD granule-associated neutral protease, is present in rheumatoid synovium and can activate collagenase in crude culture medium in vitro. the present study attempts to depict the precise mechanism of this activation. To express full activation of latent collagenase, matrix metalloproteinase 3 (MMP-3) or stromelysin, can be activated by tryptase in a time and dose-dependent manner. Tryptase was not capable of generating active collagenase in the crude media from cultured rheumatoid synoviocytes depleted of proMMP-3 by immunoadsorption. In addition, the function of the tissue inhibitor of metalloproteinases (TIMP) was not altered by tryptase, and SDS-PAGE analysis revealed no degradation of TIMP by tryptase. The tryptase dependent activation of synoviocyte procollagenase thereby appears to be entirely dependent upon its ability to activate proMMP-3.
To understand the accumulation of plasma cells within RA synovium, the ability of rheumatoid synoviocytes to support the differentiation of B cells into plasma cells was explored. Tonsillar B lymphocytes cultured over confluent monolayers of synoviocytes, secreted threefold more Igs (mainly IgM) than B cells cultured directly on plastic well. More importantly, synoviocytes enhanced by 14-fold the production of Igs (mainly IgG) by B cells costimulated with Staphylococcus aureus Cowan (SAC) particles. IL-10 and, in a lower extent, IL-2 increased Ig secretion in cocultures, and their combination was synergistic. In the presence of SAC, IL-2, and IL-10, synoviocytes increased by 13-884-fold the production of IgG, which reached 0.19 ng/cell per day. RA as well as normal synoviocytes were more potent than other adherent cell lines to support terminal B cell differentiation. Synoviocyte activity involved both a support of B cell survival, and an induction of the terminal differentiation of B cells into mature plasma cells with typical morphology, high levels of intracytoplasmic Igs, and CD20- CD38high surface expression. The present observation should permit the identification of molecules involved in the maturation of B cells into plasma cells, and in their accumulation in rheumatoid synovium.
Retroviral vectors were engineered to express either sense (MoTiN-TRPsie+) or sense and antisense (MoTN-TRPsie+/-) RNAs containing the human immunodeficiency virus type-1 (HIV-1) trans -activation response (TAR) element and the extended packaging (Psie) signal. The Psie signal includes the dimer linkage structure (DLS) and the Rev response element (RRE). Amphotropic vector particles were used to transduce a human CD4+ T-lymphoid (MT4) cell line. Stable transductants were then tested for sense and antisense RNA production and susceptibility to HIV-1 infection. HIV-1 production was significantly decreased in cells transduced with MoTiN-TRPsie+ and MoTN-TRPsie+/-vectors. Efficient packaging of sense and most remarkably of antisense RNA was observed within the virus progeny. Infectivity of this virus was significantly decreased in both cases, suggesting that the interfering RNAs were co-packaged with HIV-1 RNA. Vector transduction was not expected to occur and was not observed. Inhibition of HIV-1 replication was also demonstrated in human peripheral blood lymphocytes transduced with retroviral vectors expressing antisense RNA. These results suggest that (i) both sense and antisense RNAs were co-packaged with HIV-1 RNA, (ii) the co-packaged sense and antisense RNAs inhibited virus infectivity and (iii) the co-packaged sense and antisense RNAs were not transduced. Sense and antisense RNA-based strategies may also be used to co-package other interfering RNAs (e.g. ribozymes) to cleave HIV-1 virion RNA.