JP2001515716A - Viral infection of cells - Google Patents

Abstract

  (57) [Summary] The present invention comprises: a) collecting cells to be infected, b) treating cells collected with at least one cytokine, and c) infecting cells collected with a viral vector such as adenovirus. It relates to an in vitro method of infecting one or more cells with a viral vector capable of delivering exogenous or recombinant nucleic acid to the cells to be infected. Also disclosed are methods of infecting rheumatoid synovial cells and other cells that contain high levels of cell surface integrins.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to an improved in vitro method for infecting cells with a viral vector capable of delivering a recombinant nucleic acid to the cells. The present invention results in increased viral infection levels. The present invention also relates to cells infected by such a method.

[0002] For delivering exogenous or recombinant nucleic acids, eg, foreign genes, to cells,
The use of human adenoviruses or human retroviruses, eg, viruses such as human immunodeficiency virus (HIV) derivatives, as vectors is well known. Replication-deficient forms of human adenovirus have been used to deliver foreign genes to various cell types, including liver, muscle, nerve and airway epithelial cells. The success of adenoviruses as gene delivery vectors is based in part on their highly effective form of cell entry and their lack of need for host cell replication.

[0003] A major difficulty in using viral vectors to deliver recombinant nucleic acids to cells has been the difficulty in achieving high levels of infectivity in some cells. Due to the low levels of infection and therefore low gene transfer, any effects of the transgene on the cells can be difficult to observe as a result of the background of uninfected cells. High levels of gene transfer, for example, will allow investigation of intracellular mechanisms without having to remove uninfected populations of cells. This would allow, for example, the regulation of endogenous chromosomal genes to be investigated, and the elimination of the need to use ectopically expressed reporter gene constructs, which can be difficult to regulate. Alternatively, high levels of infection will avoid the need for cloning infected cells.

If an adenovirus is used to infect monocytes, for example, the maximum level of infection achieved is about 50%, which is up to 1000 plaque forming units /
The multiplicity of infection (MOI) as cells was required (Huang, 1995, J. Virol.
Commun., 69, 2257-63: Haddada, 1993, Biocem. Biophys. Res. Commun., 195, 1174-83). Typically, monocytes are obtained by isolating mononuclear cells from healthy adult donors, using a density gradient, washing the mononuclear cells by low speed centrifugation, and resuspending the mononuclear cells obtained from the growth medium. Prepared by turbidity. The cells are then allowed to adhere to the plastic tissue culture flask. The adherent cell population usually contains 90% monocytes. Then
Before infecting the obtained monocytes with the viral vector, granulocyte macrophage colony stimulating factor (GM-CSF) or macrophage colony stimulating factor (MC
SF). These cytokines increase the expression of integrins (αVβ3 and αVβ5) on the cell surface and are required for virus entry into cells.

[0005] The present inventors have demonstrated an improved method of infecting cells with viral vectors, which shows about 100% infection at very low viral vector concentrations. this is,
There is no need to remove uninfected cells, which means that less recombinant virus vector is needed. Thus, regulation of cellular pathways can be investigated.

[0006] We have found that inflammatory diseases, such as rheumatoid arthritis, respiratory diseases, and inflammatory diseases of the intestine, activate activated monocytes that are likely to express clearly high levels of integrins (αVβ3 and αVβ5). It was shown to be the result. Such cells have not previously been infected with a viral vector. We have shown that such cells can be successfully infected with adenovirus. This may identify biochemical mechanisms in the cells that are selectively investigated and new targets for clinical intervention to treat the disease.

[0007] This also means that such diseases can be treated with gene therapy. Adenoviruses are known to be used in gene therapy, the first case in which the use of adenoviruses to treat inflammatory diseases has been identified.

[0008] Accordingly, the present invention provides a) a step of collecting cells to be infected, b) a step of treating the collected cells with at least one cytokine, and c) a step of infecting the collected cells with a viral vector. An in vitro method for infecting one or more cells with a viral vector capable of delivering exogenous or recombinant nucleic acid to the infected cells, comprising:

Preferably, the cells are subjected to elutriation before harvesting. Preferably, centrifugal elutriation is used. This combines the steps of separation by washing, elutriation, and centrifugation of the sample to remove particles. Typically, the cell suspension is pushed into the funnel-shaped chamber at a preset flow rate. As liquid travels through the chamber, its velocity decreases as the chamber becomes wider, thus creating a velocity gradient from the narrow end of the chamber to its widest part. The cells move to the position of the velocity gradient where the effects of both the centrifugal field and the liquid velocity are balanced. Smaller cells are
An equilibrium is established at the centrifugal force field and at the slower elutriation boundary. Larger cells remain near the entrance to the centrifugal field and the faster chamber. By increasing the flow rate stepwise, cells can be washed out by size.

[0010] Cytokines are integrins on the cell surface, such as αVβ3 or αVβ5
Has been found to increase the concentration of We have shown that rheumatoid synovial cells have high levels of such integrins on their cell surface.
Thus, they were found to be able to infect rheumatoid meningeal cells at unexpectedly high concentrations of viral vectors. This alters the mode of action of such a pathway in the cell by inserting exogenous nucleic acid into the cell.

[0011] Accordingly, a further aspect of the present invention is an exogenous or recombinant form of the infected cells, comprising: a) recovering the rheumatoid synovial cells; and b) infecting the recovered cells with a viral vector. Provided is an in vitro method of infecting one or more rheumatoid synovial cells with a viral vector capable of transporting a recombinant nucleic acid.

For example, bronchial lavage from inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD), and intestinal biopsy for inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Other cells obtained from can also be infected by the methods of the present invention.

Preferably, the viral vector is an adenovirus, in particular a replication-defective adenovirus. M. O. I. Denotes plaque forming units / cell and may be 10-1000, preferably 35-100, especially 50-100.

[0014] Preferably, the cells to be infected are monocytes. Such cells can be isolated, for example, from a single donor platelet pheresis blood residue. Cells were partially purified by density gradient centrifugation prior to elutriation to obtain a cell suspension.

[0015] The isolated cells can be cultured in a suitable growth medium, but are preferably treated with the cytokine substantially directly after recovery from the elutriation machine with the cytokine.

[0016] Preferably, the cytokine is added to the medium in which the cells are cultured, and
The cells may be incubated for a period of time, typically 65-75 hours, preferably about 72 hours. The cytokine can be M-CSF. The cytokine concentration used is preferably between 100 ng and 1 μg / ml.

Preferably, the exogenous or recombinant nucleic acid in the viral vector is DNA. The recombinant nucleic acid preferably includes a sequence encoding the gene product of interest operably linked to appropriate regulatory sequences so that the gene can be expressed in infected cells.

[0018] Preferably, the foreign gene encodes a modulator of one or more biochemical pathways in the cell so that the cellular pathway can be investigated. An example of a modulator is IκB protein, an endogenous regulator, particularly IκBa, an inhibitor of NFκB. Alternatively, a mutant enzyme such as an enzyme with a disrupted catalytic activity (dominant negative configuration) can be used. Alternatively, a single-chain antibody or antibody fragment or antisense DNA can be encoded by a foreign gene. The invention also relates to cells infected by the virus in the method of the invention.

1. Infection of human monocyte-derived macrophages Materials and methods Isolation of peripheral blood monocytes. Single donor platelet pheresis blood debris
ndon Blood Transfusion Service (Colindale UK). Mononuclear cells were isolated by Ficoll-Hypaque centrifugation (specific gravity, 1.077 g / ml),
Monocytes were isolated on a Beckman JEL elutriator.

Typically, cells were harvested from the resuspended Ficoll gradient in Hanks and centrifuged at 2000 rpm for 10 minutes in a centrifuge. Falco pellets
Resuspended in an n tube at 50 ml and re-centrifuged at 1000 rpm for 15 minutes.

The resulting pellet was resuspended in RPMI supplemented with 10% FCS to 50 ml before injection into an elutriator. 1000x which is the maximum amount
10 ⁶ cells were added to the chamber. Typically, cells were elutriated according to the manufacturer's instructions.

The upper part of the elutriation machine was collected and 200 ml of 1% Etoxclean
And washed. This was followed by 1.5 L of sterile distilled water. Then, 300 ml of elutriation buffer (R / P with P / S and 1% low-concentration endotoxin FCS) was added.
PMI) was pumped. The sample containing the cells to be purified was then aspirated and centrifuged at 10 ° C., 2000 rpm. The fraction containing the cells of interest is
Collected in a con tube.

The degree of monocyte purification was directly conjugated to anti-CD45 and anti-CD14 antibodies (leukocytes, Becton D
ickinson, UK) and usually exceeded 90%. All media and monocyte media used in the separation were tested for endotoxin using the Limulus Modified Cell Lysate Test (Bio Whittaker Inc. Bethesda MD) and the endotoxin concentration exceeded 0.1 units / ml Avoided.

[0024] A recombinant adenovirus vector. E. FIG. A recombinant replication-defective adenovirus vector encoding E. coli β-galactosidase or lacking an insert (rAdΦ) has been described by A. et al. Courtesy of Dr. Byrnes (Oxford UK). E. FIG.
A second virus encoding the E. coli β-galactosidase gene (Ad / β-
gal) (Watanabe, 1996, Blood 87, 5032-9) from Canji, Inc. (San Diego CA)
And a vector encoding IκBα (rAD IκB) (Wrighton J. Exp. Med.
183, 1013-22) courtesy of Dr. de Martin (Sandoz, Vienna Austria). Virus was prepared, purified and titered as previously described (Watanabe, supra). 293 human embryonic kidney cell line
The virus was grown in an ell line) and purified by ultracentrifugation on a two-layer cesium chloride gradient. The virus strain titer was determined by limiting dilution plaque assay on 293 cells and was measured before dilution for use to be 2.9-5.8 × 10 ¹⁰ infectious units / ml. The virus was suspended in a buffer supplemented with 2% sucrose and 2 mmol / L MgCl ₂ and stored at -70 ° C.

[0025] Cytokine treatment of monocytes and adenovirus infection. Human monocyte or mouse R
AW 264.7 was RPMI 1640 supplemented with 5% (v / v) heat-inactivated fetal bovine serum containing 5 units / ml penicillin / streptomycin and 2 mM glutamine at 5 x 10 ⁵ / ml at 37 ° C. (Bio Whittaker inc.). Monocytes were treated with GM-CSF (5 units / ml) or M-CSF (1 μg / ml) for 72 hours. Integrin expression was determined by αVβ3 (100 μg / ml LM6
09, provided by IXSY (San Diego CA USA)) or αVβ5 (J. Gamble Smit
The undiluted active supernatant (kindly provided by Mr. h) was evaluated by indirect immunofluorescence and FACS analysis using a monoclonal antibody. Commercially available monoclonal antibody OX14 (100 μg / ml) was used as a negative control.

After cytokine treatment, monocytes were washed with RPM1640 and resuspended at 5 × 10 ⁵ cells / ml. Plaque forming units / cells indicating cells (multiplicity of infection-
m. o. i. ). Β-galactosidase expression in each cell was measured by FACS.

Preparation of cytosolic proteins. 4 × 10 ⁶ cells were plated on Petri dishes (Nunc) and cultured overnight. The cells were stimulated, washed with ice-cold PBS, detached by scraping from Petri dishes and lysed (20 mM Tris pH 8.0, 137 mM NaCl, 1 mM MgCl ₂ , 0.1% NP-40, 4 For 10 minutes). Lysates were centrifuged (1200 × g, 10 min, 4 ° C.), supernatants were retained and assayed for protein concentration by the commercial BCA method.

Preparation of nucleoprotein. Nuclear extracts were purified as previously described (Whiteside, 1992, Nuc.
Acids Res., 20, 1531-8). Briefly, cells were washed with ice-cold PBS, centrifuged (1200 × g, 30 seconds, 4 ° C.), and the aspirated pellet was washed with low salt lysis buffer (10 mM Hepes, 1.5 mM MgCl ₂ , 10 mM KCl, 0.5
mM DTT, 1 mM PMSF, 10 μg / ml aprotinin, 30 μg / ml
(Leupeptin) at 4 ° C. for 5 minutes, NP-40 is added to a final concentration of 0.125% and the cells are immediately vortexed. Centrifuge the sample (1200 × g, 10
Min, 4 ° C.), the cytosolic fraction is removed and the nuclear pellet is washed with nuclear extraction buffer (5 mM
Hepes, 25% w / v glycerol, 0.4 M NaCl, 1.5 ml MgCl ₂ , 0.2 mM EDTA, 1 mM DTT, 1 mM PMSF, 10 μg /
ml aprotinin, 10 μg / ml pepstatin, 30 μg / ml leupeptin) and left at 4 ° C. for 1 hour. Centrifuge the sample (1200 × g
, 10 minutes, 4 ° C), and kept the supernatant at -70 ° C.

[0029] Western blot of proteins. Mix the sample with the same volume of 2 × gel sample buffer (62.5 mM Tris pH 6.8, 2% SDS, 5% β-mercaptoethanol, 10% glycerol, 0.002% bromophenol blue) and 3 minutes Boiled. Equal amounts of protein were loaded into each lane and 10% w / v
Separated by SDS-PAGE using polyacrylamide gel and transferred to nitrocellulose (Boehringer Mannheim). The membrane was blocked with a 5% milk powder solution (20 mM Tris 150 mM NaCl pH 8.0, 0.1% Tween 20) and the protein was rabbit polyclonal antibody and horseradish peroxidase conjugated donkey anti-rabbit F (ab) ₂ fragment (Amersham). UK) and both were diluted in milk powder solution and visualized by enhanced chemiluminescence (Amersham UK).

Electrophoretic mobility shift assay. EMSA is described previously (Clarke, 1995
J. Immunol., 25, 2961-6). Data was analyzed on a Biorad GS-670 densitometer.

Measurement of TNFα production. RAW 264.7 cells were resuspended at 2 × 10 ⁵ cells / ml and plated at 500 ml / well in 24-well plates. LP cells
Various concentrations of cytokines were added and incubated for 16 hours in the presence or absence of S (10 ng / ml). The supernatant was WEHI for TNF levels.
164 (clone 13) bioassay (Espevik, 1986, J. Immunol. Methods,
95, 99-105).

Preparation of cytosolic mRNA. Plate 15 × 10 ⁶ cells, incubate overnight, stimulate with LPS and cytokines for 2 hours, wash with ice-cold PBS, 400 μl lysis buffer (10 mM Tris pH 7.9, 150 mM
NaCl, 1.5 mM MgCl ₂ , 0.65% NP-40, 10 minutes, 4 ° C.). The lysate was centrifuged (12000 × g, 5 minutes, room temperature). Add EDTA (1 μl, 0.25 M) and SDS (20 μl, 10% aqueous solution) to the required supernatant
Was added and the tube was mixed gently. The mRNA is purified by extraction with 1: 1 phenol-chloroform, precipitated with sodium acetate (pH 5.2) (−20 ° C., 30 minutes), brought to a final concentration of 0.3 M and separated into two columns with ethanol. Was. Spin down the precipitate (1200 × g, 15 minutes, 4 ° C.) and freeze 70%
Washed with ethanol and redissolved in water.

[0033] Northern blot. mRNA (10 μl, 1 μg / μl) was added to 2 μl H ₂ O,
5 μl 10 × Northern buffer (200 mM MOPS, 50 mM sodium acetate), 10 mM EDTA pH 7.0), and 25 μl deionized formamide were mixed and heated at 60 ° C. for 5 minutes. 10 μl of 6 × loading buffer (0
. 25% bromophenol blue, 0.25% xylenol cyanol 25%
Glycerol) and add the sample to an agarose gel (1% agarose, 20m
MMOPS, 5 mM sodium acetate, 1 mM EDTA, 6.5% formaldehyde pH 7.0, 100 mA, 4 hours). The mRNA was transferred to a Hybond-N membrane (Amersham UK) by capillary action and fixed by baking (80 ° C, 2 hours). Transfer the membrane to the hybridization solution (5
0% formamide, 5 × SSC, 0.05M sodium phosphate pH 6.6, 0
. 5 × Denhardt solution, 0.1 mg / ml salmon sperm DNA) at 42 ° C.
1-3x pre-incubated for 1 hour and dissolved in hybridization solution
Hybridization was carried out with a 10 ⁶ cpm / ml ³² P-labeled cDNA probe. The membrane was washed with 42 ° C., 0.2 × SSC, 0.1% SDS, and exposed to Hyperfilm MP at −70 ° C. for 1-3 days.

Results Effective gene transfer into primary macrophages after M-CSF treatment. E. FIG. Using an adenovirus vector encoding E. coli β-galactosidase, the effectiveness of this approach for gene transfer into primary human monocytes from peripheral blood was investigated. When using newly prepared monocytes, as others (Hddadd, supra, Huang, supra) have shown previously,
The present investigators m. o. i. Β-galactosidase expression could not be detected from these cells even after exposure to virus at> 1000 (results not shown). αV
It has long been shown that cofactors are necessary for infecting integrins with viruses (Huang, 1995, supra). Expression of αVβ3 and αVβ5 was found to be low in resting monocytes (FIG. 1), but when treated with GM-CSF or M-CSF for 48 hours, the expression of αVβ3 or αVβ5 respectively was reduced. Not adjusted. When treated with a combination of both cytokines, GM-CS
Integrin expression similar to F alone was shown (FIG. 1). For these observations, the infectivity with adenovirus was retested. With a 72 hour pretreatment with GM-CSF or M-CSF, which was found to be the optimal treatment, 100 m. o.
i. After the virus treatment, the expression of the β-galactosidase gene was observed in 52% or 90% of the cells, respectively (FIG. 2). For integrin expression, treatment with a combination of cytokines resulted similar to that of using GM-CSF alone (
52% of the cells were positive) (FIG. 2). Therefore, studies using only M-CSF were further performed. m. o. i. Was reduced to 50-100 with only a small effect on infection efficacy. Although 98% was 92% (FIG. 3), 25 m. o. i. Infectivity was significantly reduced (20%). Infection of monocyte / macrophage cell lines was also investigated. Human cell lines U937 and THP-1
Is completely resistant to infection by adenovirus even after treatment with GM-CSF or M-CSF, and in these cell lines, either cytokine
Vβ3 or αVβ5 expression could not be induced (results not shown). In contrast, the mouse macrophage cell line RAW 264.7 has a 100 m. o. i. Infected with adenovirus β-galactosidase at the same level as human monocytes (Fig. 4)
And m. o. i. Ten showed more than 60% infection (results not shown).
These cells do not need to be exposed prior to M-CSF (FIG. 4).

Adenovirus transduction of IκBα into primary monocytes. The present inventors have investigated the use of this system for the transfer of genes that could alter key intracellular signaling pathways with successful gene transfer to human monocytes. For this purpose,
The present inventors have determined that the CMV promoter rAD. Under the control of IκB (Wrighton, supra), an adenovirus expressing IκBα was used.

IκB is known to inactivate the NFκB transcription factor in cells. NF
Inhibition of κB may allow one to investigate factor-dependent suppression of protein expression. MC
Infection of both RAW 264.7 cells with SF-treated human primary macrophages resulted in significant overexpression of IκBα (FIG. 5). After infection with the control virus, IκBα
No change in expression was apparent. The anti-IκBα antibody used did not recognize mouse IκBα, thereby failing to detect endogenous IκBα in RAW 264.7 cells. As a control, p42MAP showing no change in expression after adenovirus infection
A Western blot for K was reported (FIG. 5). Since the IκBα construct also contains a nuclear localization sequence, the presence of nuclear IκBα can also be detected (FIG. 5C).

RAd. Infection with IκkB inhibits NFκB activity of human macrophages. Viral infection resulted in high levels of IκBα expression even after LPS treatment, unlike uninfected or control virus-infected cells in which IκBα was degraded (No. 5).
Figure). Investigations in nuclear extracts from the same cells showed that the NFκB p65 subunit, and to a lesser extent, the nuclear translocation of the p50 subunit, inhibited virus-infected cells. There is also some inhibitory effect on p50 nuclear translocation in control virus infected cells, but the reason for this is unknown. Iκ
Unlike Bα, IκBβ expression levels are not affected by viral infection. IκB
No LPS-induced degradation of β (FIG. 5b) was observed, and thus our findings are also observed for human monocytes (results not shown).

The effect of IκBα virus infection on nuclear NFκB DNA binding activity was also examined by EM
Investigation was conducted by SA (FIG. 6). By infecting monocytes with the virus, nuclear NFκ
All B DNA binding activities were dissociated. Such effects were observed in uninfected or control virus treated cells. AP-1 activity was also investigated. Unlike NFκB, AP-1 appeared essentially active in M-CSF treated cells and no LPS-inducing activity could be detected. However, compared to the control cell population, Iκ
It is noted that there was a somewhat lower level of decrease in nuclear AP-1 binding activity in Bα virus infected cells.

Infection of monocytes with IκBα adenovirus inhibits LPS-induced TNF expression. It was believed that activation of NFκB was essential for TNFα expression, however, this hypothesis had never been tested in primary human cells. Infection of monocytes with the IκBα virus resulted in inhibition of LPS-induced TNFα mRNA expression (FIG. 7a). No effect was observed at the GADPHα mRNA level. m. o. i. A similar effect was observed on TNFα protein expression, which was apparently closely dependent on (Fig. 7b). IκBα virus was also used on synovial cells obtained from rheumatoid joints. These cells essentially express TNF and other cytokines. Virus infection resulted in inhibition of TNF production by these rheumatoid synovial cells (FIG. 7c).

Depletion of Apoptosis Using IκB Since cell death has been reported to be a key anti-apoptotic mechanism of NFκB and that inhibition of NFκB induced by TNF results in cell death, cell depletion is associated with IκB virus. It can be described as an inhibitory effect. However, we have found that the m. o. i. No toxic effects of the virus could be observed, and further treatment of IκBα adenovirus infected macrophages did not undergo apoptosis after exogenous TNF treatment (results not shown).

Discussion It is shown that the method of the invention produces a high infectious effect on cells such as human primary macrophages by adenovirus.

We have a m.p. of 50-100. o. i. Have shown about 100% of infections. In a previous study (Haddada, 1993, supra, Huang, 1995, supra) a 1000 m.d. o. i. Only about 50% of the infections required were obtained. Our observation that high levels of gene transfer could be performed in the conditions of the present invention suggests that macrophages, such as eliminating any need to eliminate uninfected populations and avoid the need for cloning, Has important consequences for the investigation of the intracellular mechanism of E. coli. This allows for regulation of the endogenous chromosomal gene to be investigated and eliminates the need to use ectopic expression reporter constructs that can be differentially regulated.

High levels of adenovirus infection allow the insertion of a gene that expresses IκBα in cells to investigate its effect on NFκB on TNFα synthesis to be successfully investigated.

2. Demonstration of Selective Regulation of Cytokine Induction Materials and Methods Isolation of Peripheral Blood Monocytes Single Donor Platelet Pheresis by the North London Blood Transfusion Ser
vice (Colindale, UK). Mononuclear cells are transferred to Ficoll H
After isolation by centrifugation, monocytes were separated on a Beckman JEL elutriator. The purity of monocytes was assessed by flow cytometry and the purity was usually higher than 90%.

Adenovirus vector. E. FIG. The recombinant replication-defective adenovirus encoding E. coli β-galactosidase or lacking an insertion (Adv0) has been described by A. coli.
Dr. Byrnes and M.E. Provided by Dr. Wood (Oxford, UK). An adenovirus encoding porcine IκBα with the CMV promoter and nuclear localization sequence (AdvIκBα) is described in Courtesy of Dr. de Marin (Vienna, Austria). Virus was grown in a 293 human fetal kidney cell line and purified by ultracentrifugation using two cesium chloride gradients. Viral strain titers were determined by plaque assay on (24) 293 cells as described.

Infection techniques. Elutriated human monocytes were cultured in PRMI 1640 with 25 mM HEPES and 2 mM L-glutamine supplemented with 5% (v / v) heat-inactivated fetal bovine serum and 10 units / ml penicillin / streptomycin. Incubated at 2 × 10 ⁶ / ml. To optimize infection, purified human monocytes were pre-treated with M-CSF (100 ng / ml, Genetics Institute, Boston MA) for 48 hours to upregulate integrin αVβ5. It has previously been shown to be essential for adenovirus infection of monocytes (25). The cells were then replated in 100 mm Petri dishes and 10: 1 to 120: 1 in serum-free PRMI1640 (20: 1, 40: 1 or 80: 1 was used in most experiments). M. o. i. AdvIκBα or A
dv0 was infected for 2 hours. The cells were then incubated for 48 hours in PMRI 1640 supplemented above, resulting in significant overexpression of IκBα as assessed. Non-adherent cells were discarded during the replacement of the contained medium, resulting in further purification of monocyte-derived macrophages.

[0047] Cytokine analysis. For Northern analysis experiments, cells were replated in 5-10 × 10 ⁶ cells / 100 mm Petri dishes, LPS (10 ng / ml), PM
A (10 nM), zymosan (30 μg / ml), ionomycin (1 μM),
Or stimulated by UV irradiation. After 4 hours, the cells are collected, and mRNA is extracted to obtain Bu.
, et al. (Clin. Exp. Immunol., 73, 449 (1988)).

In the cytokine production assay, cells were replated at 5 × 10 ⁵ cells / well in 96 well dishes and stimulated for 4 or 16 hours as described above. The supernatant was analyzed by ELISA for TNFα (27), IL-1β, IL-6,
Analyzed for IL-8 (28), IL-10 (29, 30), IL-1ra and p55 and p75 soluble TNF receptor (31). Proteosome inhibitor Cbz-Ile-Glu (Ot-Bu) -Ala-leucine (P
SI) was obtained from Calbiochem (Nottingham, England). Statistical method. All statistical tests were performed using the paired comparison Student's t test.

Results Cytokine production upon virus stimulation. LPS was measured for all cytokines (TNFα, IL-1β, IL-6, IL-8) and inhibitors (IL-10, IL-10) evaluated in this study (Table 1, results are the average of 4-11 experiments). -1ra and soluble TNF receptor). Although the induction of TNFα mRNA by zymosan has previously been reported to be much weaker than LPS-induced TNFα mRNA in mouse peritoneal macrophages (32), the TNFα response to zymosan in M-CSF-treated human monocytes is equal to the TNFα response to LPS. (Table 1).

LPS was a much stronger inducer of IL-1β than any other stimulus used. Consistent with the observations in mouse macrophages of Bondeson J. and Sundler R. (1995, Biochem Pharmacol, 50, 1753), IL-6 was equally well induced by LPS and zymosan. IL-8 can be induced by the entire sequence of the stimulus. Zymosan and LPS are the only stimuli that induce IL-10 and p55 soluble TNF receptor (Table 1), and PMA can induce IL-1ra and p75 soluble TNF receptor.

Does IκBα Overexpression Inhibit LPS-Induced Proinflammatory Cytokines? We have previously observed that infection with a 20-80: 1 multiplicity of IκBα adenovirus results in high levels of IκBα expression. Thereby, NF
Blocking the κB function strongly inhibited LPS-driven TNFα induction in human macrophages (7). This was not due to cell loss due to apoptosis or other causes of cell death (7). The induction of IL-1β and IL-8 by LPS appears to be strongly NF-κB dependent, with already a strong inhibition of both these cytokines at 20: 1 AdvIκBα.

[0052]

[Table 1]

The results with supernatants collected after 4 hours of incubation were similar to those with 16 hours of incubation (not shown). In contrast to the other cytokines examined herein, LPS as well as other stimuli produced about 20-30% IL-6 production in Adv0 infected cells, despite no effect with adenovirus infection alone There was an enhancing effect (FIG. 8C). However,
IL-6 expression is strongly inhibited by infection with AdvIκBα in human macrophages (FIG. 8C).

[0054] TNFα is a cytokine of other types, such as IL-1β, IL-6, and IL-
Strong inhibition by AdvIκBα is due to the ability to induce the synthesis of
It can be a secondary inhibition of α. Culturing with exogenous TNFα partially eliminates the effect of IκBα overexpression on IL-1β production (62% inhibition without cytokine, but just 20% inhibition with 100 ng / ml TNFα) (9A (Figure), indicating that it is at least partially dependent on TNFα.
However, similar experiments have shown that TN in LPS-induced expression of IL-6 and IL-8
No major role of Fα was shown (results not shown).

To further investigate the relevant signaling mechanisms, we characterized whether TNFα-driven IL-1β would be less dependent on NF-κB than the IL-1β response induced by LPS. I investigated. One difficulty in accomplishing this is the fact that TNFα induces less IL-1β than LPS in our mechanism (Table 1). TNF in cells infected with AdvIκBα
As expected for restoration of IL-1β synthesis by α, TNFα-induced IL-1β I
κBα-induced inhibition was relatively less intense than the inhibition seen with TNFα-induced IL-6.

Does IκBα overexpression affect LPS-induced anti-inflammatory cytokines? The major anti-inflammatory cytokine produced by macrophages is IL-10
It is. In LPS-stimulated cells, inhibition gradually increased with increasing virus titer, but statistically significant inhibition (p <0.05) was due to 60: 1 or 80: 1 Adv.
Attention was paid only to IκBα (FIG. 10A). This inhibition is even more modest (up to 30%) and we have been shown to affect IL-10 expression because the human IL-10 promoter lacks a κB site. Whether the inhibitory effect of AdvIκBα infection, such as through the inhibitory effect of cytokines, was indirect was investigated. The combination of TNFα and LPS markedly enhanced the IL-10 response and partially eliminated inhibition by AdvIκBα (FIG. 10B). In contrast, IL-1 failed to have any significant effect (results not shown).

LPS-induced production of IL-1ra was slightly (は 3
0%) was inhibited (FIG. 11A). This response is only moderately affected by adding back IL-1 and TNFα (FIG. 11B). In human macrophages, LPS contains p75-soluble TNF receptor and lower
Induces 55 soluble TNF receptor production (Table 1). Infection with AdvIκBα observed inhibition of LPS-induced production of both of these lytic receptors (FIG. 12), and this response was not affected by adding IL-1 or TNFα back (results not shown). ).

Does IκBα Overexpression Inhibit Proinflammatory Cytokines Induced by PMA or UV Light? Induction of TNFα by PMA is very strongly inhibited by IκBα overexpression (〜90%), which is even more potent than LPS stimulated cells (〜80%) or UV light stimulated cells (〜80%) (Table 2). In addition, the results using the supernatant collected after 4 hours of incubation were similar to the results using the supernatant after 16 hours of incubation (results not shown). A series of Northern analysis experiments was performed using L
As in the experiment for PS-induced TNFα, T
NFα mRNA expression was shown to be eliminated by IκBα overexpression (first
3A-B).

As shown by TNFα, induction of IL-1β and IL-6 by PMA or UV light was NF-κB dependent (Table 2). In addition, AdvIκBα
Inhibition of IL-1β and IL-6 production by infection appears to be more potent in PMA-treated cells than in cells treated with LPS or UV irradiation.

When AdvIκBα infected cells were stimulated with PMA or UV light, IL-
8 Response inhibition (50-60%) is also present (Table 2). Ionomycin, a stimulator that does not induce large amounts of other cytokines of interest, induces a recognizable IL-8 response, which is also inhibited by IκBα overexpression (data not shown).

PMA does not induce detectable amounts of IL-10, but has detectable p75 soluble T
Induces the NF receptor response, which is inhibited by IκBα overexpression, similar to PMA-induced production of IL-1ra (40% inhibition with 40: 1 AdvIκBα). (75% at 40: 1 AdvIκBα).

[0062]

[Table 2]

Does IκBα Overexpression Affect Zymosan-Induced Cytokines? In contrast to previous stimulation, when macrophages were activated with zymosan, A
Infection with dvIκBα is 80: 1 m. o. i. Even had no effect on TNFα protein (Table 2) or mRNA expression (FIG. 13C). Induction of IL-1β and IL-8 by zymosan was also unaffected by IκBα overexpression (Table 2), but the inhibition of IL-6 was small (10-15% compared to uninfected cells). . Zymosan-induced IL-10, IL-1ra (Table 2) and soluble TNF receptor (not shown) were also resistant to inhibition by IκBα overexpression (Table 1).

The NFκB-independence of zymosan-induced TNF was further emphasized by investigations with the proteosome inhibitor PSI. Inhibition of proteosome function is
Inhibits κBα degradation and therefore NFκB nuclear translocation. PSI is LPS induction T
Very effective in blocking NF production but does not affect the response to zymosan (FIG. 14).

Discussion. The present inventors have shown that adenoviral gene transfer into macrophages provides a reliable, reproducible, and convenient method for investigating intracellular signaling pathways. The principle was established using NF-κB, a natural inhibitor of IκBα. Introduction of IκBα effectively inhibits NF-κB activity in human macrophages, mainly by overexpression of IκBα, which inhibits nuclear translocation of the p65 / p50 subunit of NF-κB. It blocked LPS-induced TNFα at both mRNA and protein levels. More interestingly, it was found that endogenous production of TNFα from rheumatoid synovial monocyte cell cultures was also inhibited.

The fact that LPS-induced TNFα, IL-1β and IL-6 were all NF-κB cytokines can be expected from much of the data from mouse cells and monocyte / macrophage cell lines. The results for LPS-induced cytokines are also
LPS-induced IL-1β in monocyte THP-1 cells stably modified by retroviral gene transfer of IκB Makar in IL-1β, IL-6 and IL-8
Very consistent with the results of ov et al. (Gene Therapy, 1997, 4, 46). Among the LPS-induced pro-inflammatory cytokines investigated here, IκBα overexpression
L-6 is most potently inhibited (> 85%), and AdvIκBα is highly potent (120
1) TNFα or IL-1β even in LPS-stimulated cells infected with
There is always some residual production of This is because a fixed amount of pre-prepared cytokine mR
Although it can be affected by NA, this cannot be shown in unstimulated cells (data not shown), and LSP, PMA or UV-induced (FIG. 13) TNFα mRNA
It was greatly down-regulated by Bα.

However, it cannot be ruled out that there is residual cytokine production arising from non-existent, non-infected cells, and studies are ongoing to elucidate this problem using intracytoplasmic staining of cytokines. Another undeniable hypothesis (discussed in more detail below) is that TNFα and other proinflammatory cytokines can be induced by both NF-κB-dependent and NF-κB-independent pathways.

Our finding that UV light induces a full spectrum of pro-inflammatory cytokines in an NF-κB dependent manner is novel. This is RAW 264. Early reports that UV-induced TNFα in 7 cells is not involved in NF-κB
(Bazzoni, J. Clin. Invest., (1994), 93, 56). Similarly, the finding that PMA induction of TNFα and other pro-inflammatory cytokines is greatly down-regulated by IκBα overexpression is consistent with several early studies in stably transfected human cell lines (Makarov, supra). do not do. On the part of the present inventors, this stimulus was the one most strongly dependent on NF-κB, as judged by% inhibition and reproduced in seven different tests. The discrepancy between the results obtained from human primary cells and the results obtained from various transfected cell lines is to study cytokine cell signaling that occurs in primary cells, at least in some cases, since the latter is the case in vivo. Is a suspicious model. In a way this is not surprising,
This is because there is an interaction between the enzymes and transcription machinery of the cell cycle machinery and the regulation of cytokine genes involved in cytokine activity, such as Rb regulation (Bassuk A. and Leiden JM, Adv. Immunol., (1997), 64, 65
).

With respect to macrophage signal transduction, one of the most noteworthy findings in this study is that zymosan is a very potent macrophage activator, but has been shown to induce inflammatory or anti-inflammatory cytokines in NF. It seems that -κB does not seem necessary. These findings would imply that in human macrophages, both the NF-κB-dependent and NF-κB-independent pathways are involved in TNFα and other pro-inflammatory cytokines. Iκ
The modest (15%) suspected significant inhibition of zymosan-induced IL-6 observed in Bα overexpressing cells was observed to be the strongest effect of this cytokine on IκBα overexpression and independent of stimulation (Table 2). See).

Another important finding is about human macrophages. IL-10 is present under complex regulation and in LPS-stimulated cells and appears to be driven at least in part through LPS-induced TNFα and IL-1. 40: 1 AdvIκB
It is interesting to note that even with α, IL-10 is not significantly inhibited when LPS-driven TNFα is eliminated by 60% or more (FIG. 10). At higher virus titers, even stronger inhibition of TNFα is observed, with some effects on IL-10, but even 80: 1 virus never exceeds 30%.
It is completely reversible by adding back TNFα, and LPS-induced IL-
10 implies that it is partially secondary driven by TNFα. This finding is in good agreement with previous reports, indicating that autocrine interactions can occur even in these short-term (16 hour) cultures.

Another interesting finding is that when TNFα is restored, IκBα-induced inhibition of LPS-induced production of IL-1β (FIG. 9A) (IL-6 or IL-8 or soluble TN
Some lack of F receptor production (data not shown) was also excluded, indicating that TNF-induced IL-1β is not largely dependent on NF-κB. This finding is also to a lesser extent than IL-10
May also be partially driven by LPS-induced TNFα. To a lesser extent, this result indicates that RA joint cells, in which TNFα block was found to inhibit IL-1 production and subsequently IL-6, IL-8, IL-10 and GM-CSF production. Similar to previous studies in culture, these have led to the concept of a TNFα-dependent “cytokine cascade” at sites of inflammation such as rheumatoid synovium.

Because of differences between cell lines (see above), detailed analysis of signaling pathways in normal primary cell lines is necessary, but using this adenovirus technique, This is possible within human macrophages from any of the specimens. In terms of cytokines, a human macrophage model infected with AdvIκBα yielded similar results to human synovial co-culture with the same virus (see below). The data show that while NF-κB is an important therapeutic target in chronic inflammatory diseases and does not directly affect the most important anti-inflammatory cytokines, IL-10 and IL-1ra, macrophage-produced pro-inflammatory cytokines It suggests that it can be greatly down-regulated.

[0073] 3. Demonstration of adenovirus infection of rheumatoid synovial cells Rheumatoid synovial cells produce high levels of cytokines and express high levels of integrins on their surface (Feldman M. et al., Ann. Rev. Immunol., (1996) , Volume 4). In vitro activated cells with spontaneous high levels of integrins and cytokines have been used to demonstrate that improved viral infection rates can be achieved. The use of such a system is also illustrated.

Methods Cells Synovium from rheumatoid arthritis patients who underwent joint surgery were separated by cutting into small pieces and digested with collagenase and DNAse. The whole cell mixture was cultured on RPMI 1640 containing 25 mM HEPES and 2 mM L-glutamine supplemented with 5% heat-inactivated fetal calf serum at 37 ° C on 24-well or 48-well plates.

Adenovirus vector A recombinant replication-defective adenovirus vector encoding E. coli β-galactosidase (Advβgal) or without insertion (Adv0). An adenovirus encoding porcine IκBα with the CMV promoter and nuclear translocation sequence (AdvIκBα) was provided above. The virus was grown in a 293 human fetal kidney derived cell line and purified by ultracentrifugation through a cesium chloride gradient of 2. Virus strain titers were determined by plaque assay on 293 cells. All viruses used were plaques purified from the master strain to prevent wild-type adenovirus contamination.

Infection Technique For experiments on cytokine or matrix metalloproteinase synthesis, freshly prepared rheumatoid synovial cells were placed in 12-well plates in 1 ml of serum-free RPMI 1640 at 2.5-4 × 10 ⁶ cells / ml. Resuspended. After 1 hour incubation, cells were uninfected or infected with AdvIκBα or Adv0 at a multiplicity of 40: 1. After 2 hours, the supernatant was removed and replated using 0.5 ml RPMI 1640 supplemented with 5% heat-inactivated fetal calf serum. Non-adherent cells were carefully centrifuged down and reintroduced into the rheumatoid cell co-culture in 0.5 ml R PMI 1640 supplemented with 5% heat-inactivated fetal bovine serum (therefore a total volume of 1 ml).

In some experiments, rheumatoid synovial cells were infected as described above,
The density was 0.4 × 10 ⁶ cells / well in a final volume of 0.2 ml on a 6-well plate. Cells of each category (uninfected, Adv0 infected and AdvIκBα infected) were plated in 4 wells. After 2 hours of incubation with adenovirus, one well of each category was untreated, one was 20 μl / ml of human recombinant I.
L-1 receptor antagonist (Cambridge Bioscience) treatment, one 20 μl
/ Ml of A2 anti-TNFα antibody (Centocor, Malvern, US) treatment, and one treatment with both of these cytokine inhibitors.

For infectivity testing, cells were resuspended at 1 × 10 ⁶ cells / well in 24-well plates in serum-free RPMI 1640. After a one hour incubation, cells were uninfected and infected with either Advβgal or Adv0 at a multiplicity of 40: 1. After 2 hours, the supernatant was removed and replated using 0.4 ml RPMI 1640 supplemented with 5% heat-inactivated fetal calf serum. Non-adherent cells are carefully centrifuged down and 0.2 ml RPMI 1 supplemented with 5% heat-inactivated fetal bovine serum.
640 (total volume 0.6 ml) was reintroduced into the rheumatoid cell co-culture.

Analysis of Infectivity After 48 hours of infection, the cells in the culture medium were scraped off the plate, centrifuged down and washed with FACS staining solution. Then uninfected, Adv0 infected or Adv
Each batch of βgal infected cells was resuspended in 25 μl of staining solution and 125 ng of anti-CD3 PerCP and 500 ng of anti-CD14PE (both Becton Dicki
nson, provided by San Jose CA) to make a total volume of 45 μl and incubated at 4 ° C. for 45 minutes. The cells were then incubated at 37 ° C. for 10 minutes, after which 45 μl of 2 mM fluorescein di- (β-galactopyranoside) (Sigma) was added and incubated for 1 minute. The reaction was stopped using an excess (10 ×) of ice-cold staining solution. Cell fluorescence was analyzed by FACS.

Cytokine and Metalloprotease Analysis In assays for cytokine production, after 48 hours of infection, supernatants were removed and non-adherent cells were removed. Cells were analyzed by ELISA for TNFα, IL-
1β, IL-6 and IL-8, IL-10 (), IL-1ra and p55
And analyzed for p75 soluble TNF receptor. Production of tissue inhibitors (TIMP-1) of MMP-1 (collagenase), MMP-3 (stromelysin), MMP-13 and metalloprotease was determined by Amersham (Little Chalfont, Buc).
ks., UK).

In some experiments, media was removed in 50 μl aliquots after 12, 24, 48, 72 and 110 hours. Remove non-adherent cells,
After reintroduction of the cells into the same volume of medium, the compartments were analyzed for cytokines and metalloproteases as described above.

Results Infectivity of Rheumatoid Synovial Cells with Adenovirus The infectivity of rheumatoid synovial cells was investigated using an adenovirus into which the β-galactosidase gene was introduced (AdvβGal). The 40: 1 AdvβGal is
We infected 95-100% of human macrophages and 30: 1 AdvβGal infected nearly 100% of human fibroblasts.
: 1 and 40: 1 AdvβGal were used. As demonstrated by FACS analysis, 40: 1 AdvβGal showed almost 10% of the total rheumatoid synovial cell population.
It was found that it infected 0% (FIG. 15A), but that it infected 60-70% at 10: 1 (not shown). Therefore, all subsequent experiments included a 40: 1 AdvβGa
1 and other adenoviruses.

It is interesting to distinguish between different cell types in rheumatoid synovial cell co-culture and study the infectivity of the major constructs (macrophages, T lymphocytes and synovial cells) separately. This is because the fluorescent antibody markers for CD3 and CD14 (CD3 + / C
D14- (T lymphocytes), CD14 + / CD3- (macrophages) and CD
3 // CD14- (can identify synovial fibroblasts)). By using human peripheral blood T cells and monocytes and human dermal fibroblasts (control), the frontal scatter / side scatter characteristics of these cell populations can be identified. Double gating was then used to confirm that the rheumatoid arthritis synovial cells selected to show T lymphocytes, macrophages and synovial cells had the correct size / particle and surface marker properties. As can be expected from the initial results, both macrophages (FIG. 15B) and fibroblasts (FIG. 15C) are easy to infect, and more surprisingly,
More than 90% of T lymphocytes were also infected with 40: 1 AdvβGal (first
5D).

IκBα Overexpression Inhibits Proinflammatory Cytokine Production We have previously shown that the spontaneous production of TNFα, assessed two days after infection, caused AdvIκBα as compared to mock- or Adv0-infected cells. 7 of cells infected with
0% was shown to be inhibited. IL-1β (FIG. 16A) and IL-8
(FIG. 16B) Both spontaneous productions were also inhibited by IκBα overexpression, but to a lesser extent (40% compared to uninfected and Adv0 infected cells).
). In contrast, IL-6 was very strongly inhibited (85%, FIG. 16C).
These results indicate that the most potent NF-κB-dependent proinflammatory cytokine
This is consistent with the above findings that pointed out L-6.

In a series of different experiments, equal sections of media were removed after 12, 24, 48, 72, and 110 hours for cytokine analysis. From day 1 to day 5, AdvIκBα-infected cells were found to remain almost quiescent, although TNFα and IL-6 production from uninfected or Adv0-infected cells increased gradually ( (FIG. 17), which indicates that once overexpression of IκBα has been achieved, sufficient to inhibit NF-κB activation, TNFα and IL-6
Is produced only slightly.

[0086] IκBα overexpression does not affect production of major anti-inflammatory cytokines. Previous results from human macrophages, which are major IL-10 producers in rheumatoid joints, indicate that large down-regulation of TNFα and IL-1β induced by NF-κB down-regulation is due to some secondary decreases. Nevertheless, both IL-10 and IL-1 receptor antagonists have been shown to be NF-κB independent in these cells. In rheumatoid synovial cells, spontaneous production of both IL-10 and IL-1 receptor antagonists is not affected by IκBα overexpression (FIGS. 18A-B). However, the production of p75 soluble TNF receptor is moderately inhibited (45%) (FIG. 18C).

IκBα inhibits metalloprotease production It was confirmed that IκBα strongly inhibited spontaneous production of both MMp-1 and MMP-3 in rheumatoid arthritis synovial cells (FIGS. 19A-B). On the other hand,
Spontaneous production of TIMP-1 was slightly enhanced after 2 days (FIG. 19C). In addition, while studying the time course of MMP production (FIGS. 20A-B), uninfected and A
Although the dv0-infected rheumatoid synovial cell co-cultures continue to produce these enzymes throughout the incubation, AdvIκBα-infected cells show significant Iκ
It appears that neither MMP-1 nor MMP-3 is produced after Ba overexpression. It is also in sharp contrast that there is a relatively strong TIMP-1 production after 3 or 5 days.

These results indicate that NF-κB is directly dependent on MMP-1 and MMP-3 production but not TIMP-1 production. Although both IL-1 and TNFα are known as inducers of MMP,
To rule out any effect on these two cytokine downregulations in MMP production, IL-1ra and / or TN immediately after infection of the cells.
A series of experiments in which F neutralizing antibody was added were performed. It was observed that the relative inhibition of MMPs by IκBα overexpression was largely unaffected by neutralization of either IL-1 or TNFα (FIG. 21). This suggests that MMP regulation tends to directly regulate NF-κB. Although MMPs partially drive TNFα, inhibition of TNFα production in our culture helps, as a major part of MMP reduction can be explained by the direct effects of their infection.

Discussion These results strongly indicate that NF-κB is a key therapeutic target in rheumatoid arthritis and possibly other inflammatory diseases. The unique spectrum of effects achieved by inhibition of this transcription factor includes strong inhibition of pro-inflammatory cytokines (especially TNFα and IL-6), but the key anti-inflammatory mediators IL-10 and IL-10 No effect on IL-1 receptor antagonist. This will help rebalance the disturbed balance between pro- and anti-inflammatory mediators in affected joint tissue. In addition, there is potent inhibition of the major disrupting enzymes MMP-1 and MMP-3, but instead enhances their effects on major inhibitors, which
-ΚB has also helped restore a disturbed balance between MMPs and their inhibitors.

[Brief description of the drawings]

FIG. 1 is a diagram of FIG. Differential induction of integrin αVβ5 in monocytes by GM-CSF and M-CSF. Untreated monocytes or GM-CSF (500 U / ml
) Or M-CSF (1 μg / ml) or both cytokines for 48 hours. Cells were then analyzed for aVβ3 (dashed line) or αVβ5 (dotted line) expression by FACS. Staining with control Ig (solid line) is also shown.

FIG. 2A. Effect of cytokine treatment on adenovirus infection of primary human monocytes. Monocytes were treated with GM-CSF (500 U / ml) or M-CSF (1 μg /
ml) for 72 hours, followed by 100 m. o. i. Including adenovirus (
(Dotted line) or β-galactosidase containing control virus (solid line).

FIG. 3B is a diagram showing FIG. After 72 hours (following the description of FIG. 2), various m. o. i.
Cells were analyzed by FACS for β-galactosidase expression against

FIG. 3; M-CSF treatment is not required for infection of RAW 264.7 cells. RAW 264.7 cells were incubated for 48 hours in the presence or absence of M-CSF (1 μg / ml), followed by control adenovirus or β-
It was infected with a vector containing galactosidase. After 4 days of culture, the cells were
Was assayed for β-galactosidase expression. Symbol: control virus, MC
No SF (thick line), control virus + M-CSF (fine line), β-galactosidase virus, no M-CSF (dashed line), β-galactosidase virus + M-CSF (dashed line).

5A to 4C. FIG. Expression of IκBα and IκBα adenovirus infecting monocytes and RAW 264.7 cells. RAW 267.4 cells (a) and M-CSF-treated monocytes (b) were isolated from a given m. o. i. , Or infected with the IκBα adenovirus vector. After 72 hours, cell lysates and the supernatant after the nucleus were analyzed for IκBα expression by immunoblotting. Equal amounts of protein were loaded on each track for a given cell type.
(C) Nuclear lysate was purified to the specified m. o. i. Prepared from M-CSF treated human monocytes infected with IκBα adenovirus or control virus for 72 hours. IκB
α expression was measured as described above and equal amounts of protein were loaded on each track.

FIG. 5; Expression of NFκB and IκB proteins in virus infected monocytes. Monocytes were treated with M-CSF for 48 hours. The cells were then used for control virus or 50 m. o. i. Of IκBα-containing adenovirus vector. Group 3 was untreated. After an additional 3 days treatment in M-CSF, cells were washed and half of each group was treated with 10 ng / ml LPS for 30 minutes. The cells were then lysed, cytosolic and nuclear extracts were prepared, and then analyzed by immuno Western blot for IκBα / β expression (cytosolic) or p65 / p50 expression (nuclear). 150 μg protein and 50 μg protein were analyzed as each cytosolic or nuclear sample.

FIG. 6; NFκB function inhibition in monocytes infected with IκBα virus. Monocytes were untreated or treated with IκBα or control adenovirus, followed by LPS (10 ng / ml) stimulation. Nuclear extracts were prepared and 20 μg of protein was analyzed for NFκB (upper panel) or AP-12 (lower panel) by EMSA.

FIG. 7A. IκBα virus infection inhibits LPS-induced TNF mRNA and protein. M-CSF treated cells were treated with untreated or various m. o
. i. With the IκBα virus or control virus. 3 days later,
Stimulated with LPS (10 ng / ml). Two hours later, cells were collected, mRNA was extracted, and analyzed for mRNA by Northern blotting using an induction probe.

FIG. 9B. After 24 hours (following the description of FIG. 8) the culture supernatant was removed and E
Assayed for TNFα by LISA. Cells were tested for toxic effects by MTT assay. Data shows% TNF production of uninfected control cells. The results show mixed data of cells from five different donors (= SD).
No significant toxicity was observed as determined by the MTT assay (not shown).

FIG. 7C is a diagram of FIG. Rheumatoid synovium-derived cells were cultured in 48-well plates at 1 × 10 cells (following the description of FIGS. 8 and 9), and cultured at 40-1 m. o. i
. Were infected with either AdvIκBα or Adv0. Remove the supernatant 4
ELISA assay performed for 8 hours; data collected from 5 patients are shown (± sd)
. The null hypothesis that there was no change in TNF production in whether cells were infected with Adv IκB or Adv0 can be ruled out using the Wilcoxon's signed rank test for combined differences compared to untreated cells in each patient. (P <0.05).

FIG. 8A to FIG. LPS-induced IL-1, I by human macrophages
AdvIκBα infection effect on L-6 and IL-8 expression. IL-1β (A)
AdvIκB in LPS-induced production of IL-8 (B) and IL-6 (C)
Infection effect of human monocyte-derived macrophages at various titers of α or Adv0. The percentage production of the cytokine of interest induced by LPS (10 ng / ml) in uninfected cells was shown. Error bars indicate the standard error of the mean (n = 7-10).

FIGS. 9a to 9c. Exogenous TN for IL-1β and IL-6 production
The effect of F. Pretreatment of TNFα at 20 ng / ml or 100 ng / ml eliminated the inhibitory effect of LPS-induced IL-1β production on Adv IκBα infection (A). TNFα-induced (20 ng / ml) IL-6 was strongly inhibited by Adv IκBα infection (B), whereas TNFα-induced IL-1 was only moderately inhibited (C). Error bars indicate the standard error of the mean (n = 6-8).

FIGS. 13a and 10b. Inhibition of NF-κB has only a low effect on IL-10 production. IL- induced by LPS (10 ng / ml)
The effect of infecting human monocyte-derived macrophages with different titers of AdvIκBα or Adv0 on the production of IL-10 by IL-10 by the same stimulation in uninfected cells
(A). In (B), pretreatment with 20 ng / ml TNFα eliminated the inhibitory effect of LPS-induced IL-10 production (pg / ml) on AdvIκBα infection. Error bars indicate standard error of the mean (n = 7 to 10).
.

FIG. 11A and FIG. 11B. Effect of IκBα infection on IL-1ra production. Different titers of AdvIκBα or A in LPS-induced IL-1ra production
The effect of infecting human monocyte-derived macrophages on dv0 was shown as a percentage of identical cytokine production in uninfected cells exposed to the same stimulus (A). IL-1
Alternatively, pretreatment with TNFα (20 ng / ml) alone had no effect, whereas treatment with both cytokines resulted in AdvIκB on LPS-induced production of IL-1ra.
The inhibitory effect of α infection is gently reversed (B). Error bars indicate standard error of the mean, n = 7-8.

FIG. 12A and FIG. 12B. AdvIκBα infection is LPS-mediated soluble T
Inhibits the NF receptor. The effect of infecting human monocyte-derived macrophages with different titers of Adv IκBα or Adv0 on LPS-induced production of p55 (A) and p75 (B) soluble TNF receptors in uninfected cells exposed to the same stimulus Identical cytokine production was expressed as a percentage. Error bars indicate standard error of the mean, n = 7-8.

FIGS. 13a to 13c. Inhibition of NF-κB selectively inhibits TNFα mRNA. Infection with 40: 1 AdvIκBα but not 40: 1 Adv0, as assessed by Northern analysis, resulted in PMA (A) or UV light (B
) Inhibits TNFα mRNA expression (upper panel). Zymosan-induced TNFα mRNA expression (C) is not affected by infection with 80: 1 AdvIκBα. The lower panel contains GADPH expression. This is three representative complete experiments.

FIG. 14 is a diagram. PS1 does not affect zymosan-induced TNFα LPS
Inhibits mediated TNFα. LPS induction (■) and zymosan induction (●) TNF
Effect of proteasome inhibitor PSI on α production. Error bars indicate standard error of the mean, n = 6-7.

FIG. 15A to FIG. More than 90% of rheumatoid synovial cells can be infected with adenovirus. Co-culture of whole rheumatoid synovial cells (A) or T lymphocytes (B), macrophages (C) or synovial cells in medium infected with 40: 1 Adv0 (solid line) or Advβgal (solid line) Β-galactosidase activity in the subpopulation with (D) was detected by double gating for size / particle and cell surface markers. Five representative experiments. In (E), the average of the number of β-galactosidase positive cells is plotted +/− the standard error of the mean, n = 5.

FIG. AdvIκBα infection inhibits spontaneous production of pro-inflammatory cytokines from rheumatoid synovial cells. 40: 1 Avd0 or AdvIκ for spontaneous production of IL-1 (A), IL-8 (B) and IL-6 (C)
Infection effect of rheumatoid synovial cells with any of Bα. Error bars indicate standard error of the mean, n = 6.

FIG. 17 is a diagram of FIG. IκBα overexpression permanently inhibits TNFα and IL-6 production. Infection effect of rheumatoid synovial cells with either 40: 1 Adv0 or AdvIκBα on spontaneous production of TNFα (A) or IL-6 (B) over time. Symbols are uninfected (), Adv0 infected (), or
Bα infected () cells are shown. Representative three independent experiments.

21a to 21c. Inhibition of NF-κB by AdvIκBα infection
Has little effect on spontaneous production of anti-inflammatory mediators. IL-10 (
A) Infection effect of rheumatoid synovial cells with either 40: 1 Adv0 or AdvIκBα on the spontaneous production of IL-1 receptor antagonist (B) and 75 soluble TNF receptor (C). Error bars indicate standard deviation of the mean, n = 6.

FIG. 19A to FIG. IκBα overexpression is associated with MMP-1 and MMP-
3 strongly inhibits production but slightly enhances TIMP-1 production. MMP-1
(A), 40 in spontaneous production of MMP-3 (B) and TIMP-1 (C).
1: Infectivity of rheumatoid synovial cells with either Adv0 or AdvIκBα. Error bars indicate standard deviation of the mean, n = 6.

FIGS. 20a to 20c; Effect of AdvIκBα infection by balance between MMP production versus TIMP-1 production over time. MMP-1 (A
), 40: 1 in the spontaneous production of MMP-3 (B) and TIMP-1 (C)
Effect of infecting rheumatoid synovial cells with either Adv0 or AdvIκBα.
The symbols indicate uninfected cells-, Adv0 infected cells {AdvIκBα cells}. Shown are three representative independent experiments.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] March 13, 2000 (2000.3.13)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 29/00 101 C12N 7/00 C12N 5/10 7/06 7/00 A61K 35/76 7/06 C12N 15/00 A // A61K 35/76 5/00 B (72) Inventor Foxwell, Brian, Maurice, John United Kingdom Middlesex Tidaburst 3 1 Luke Hounslow Rounds Down Road 23 (72) Inventor Brennan, Fionula , Mary United Kingdom Middlesex Tiedabourne 110 Arle Tedington Princess Road 41 (72) Inventor Bondeson, Jean United Kingdom London Esbourg 66 Tier Fulham Place Road 273

Claims (16)

[Claims] 1. A virus vector comprising the steps of: a) collecting cells to be infected, and b) infecting the collected cells with a viral vector, wherein the virus vector is capable of transporting an exogenous or recombinant nucleic acid to the cells to be infected. An in vitro method of infecting one or more cells with high integrin levels. 2. The method according to claim 1, wherein said cells have at least one
2. The method of claim 1, wherein the method is treated with a species cytokine. 3. The exogenous cytokine is GM-CSF and / or M
3. The method of claim 2, which is one or both of CSF. 4. The method according to claim 1, wherein the cells are suspended and subjected to elutriation prior to harvesting. 5. Sources of cells with high cytokine levels include rheumatoid synovial cells, bronchoalveolar lavage from inflammatory respiratory diseases such as asthma and chronic obstructive respiratory disease, and Crohn's disease and ulcerative colon 5. The method according to claim 1 or 4, wherein the method is selected from intestinal biopsy of inflammatory bowel disease such as inflammation. 6. The method of claim 1, wherein said cells are selected from one or more of macrophages, T lymphocytes, synovial cells, dendritic cells, chondrocytes, fibroblasts, epithelial cells, and monocytes. A method according to any one of the preceding claims. 7. The method according to claim 1, wherein the viral vector used is an adenovirus. 8. The method according to claim 7, wherein the viral vector used is a replication-defective adenovirus. 9. m. o. i. The method according to any one of the preceding claims, wherein is between 10 and 1000. 10. The method according to claim 1, wherein the exogenous or recombinant nucleic acid is DNA. 11. The exogenous or recombinant nucleic acid encodes a gene product of interest operably linked to one or more regulatory sequences so that the gene can be expressed in infected cells. The method according to any one of the preceding claims, comprising a nucleic acid sequence. 12. A cell infected with a viral vector capable of transporting exogenous or recombinant nucleic acid to an infected cell obtained by a method according to any one of the preceding claims. 13. Use of an in vitro method or cell according to any one of the preceding claims, wherein the effect of one or more exogenous compounds is examined on cells infected with a virus. 14. Use of a method according to any one of the preceding claims for gene therapy. 15. A method for infecting one or more cells having high integrin levels with a viral vector comprising an exogenous or recombinant nucleic acid operably linked to a promoter in the cells to be treated. Gene therapy methods. 16. An exogenous or combination in the manufacture of a medicament for treating inflammatory respiratory diseases such as rheumatoid arthritis, asthma and chronic obstructive pulmonary disease, and inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Use of a viral vector containing the replacement nucleic acid.