(Received for publication, September 16, 1996, and in revised form, November 15, 1996)
From the Schools of Pharmacy and ¶ Medicine,
West Virginia University, Health Sciences Center, Morgantown, West
Virginia 26506 and the
National Institute of Occupational Safety
and Health, Morgantown, West Virginia 26505
Tumor necrosis factor- (TNF
) has been shown
to play an important role in the pathogenesis of silicotic fibrosis. In
this study, antisense oligonucleotides targeted to TNF
mRNA were
used to inhibit silica-induced TNF
gene expression in alveolar
macrophages. To achieve macrophage-specific oligonucleotide delivery, a
molecular conjugate consisting of mannosylated polylysine that exploits endocytosis via the macrophage mannose receptor was used. Complexes were formed between the mannosylated polylysine and oligonucleotides and added to the cells in the presence of silica. Enzyme-linked immunoadsorbent assay showed that the complex consisting of the conjugate and antisense oligomer effectively inhibited TNF
production, whereas the oligomer alone had much less effect. Reverse
transcriptase-polymerase chain reaction analysis revealed that the
reduction in TNF
secretion was associated with specific ablation of
targeted TNF
mRNA. The conjugate alone or conjugate complexed
with inverted or sense sequence oligonucleotide had no effect. The
promoting effect of the conjugate on antisense activity was shown to be
due to enhanced cellular uptake of the oligomer via mannose
receptor-mediated endocytosis. Cells lacking mannose receptors showed
no susceptibility to the conjugate treatment. These results indicate
that effective and selective inhibition of macrophage TNF
expression
can be achieved using the antisense mannosylated polylysine system.
Tumor necrosis factor- (TNF
)1 is
a macrophage-derived peptide that has been shown to play an important
role in the pathogenesis of pulmonary fibrosis (1-3). Several
fibrogenic agents such as crystalline silica and asbestos stimulate
TNF
mRNA expression and protein synthesis in macrophages (2, 3).
Elevated TNF
levels stimulate fibroblast proliferation and
production of collagen matrix, leading to fibrosis (2-4). Although the
role of TNF
in the pathogenesis of pulmonary fibrosis has been well
studied, relatively little is known about its molecular regulation
during the fibrotic process. This information is important not only for the understanding of the molecular mechanism of disease pathogenesis but also for the development of potential therapeutic
interventions.
The ability to regulate expression of an individual gene by the use of an antisense oligonucleotide (ON) complementary to a specific sequence of mRNA provides a powerful tool for elucidating the role of a particular gene and may allow therapeutic intervention when that gene is overexpressed (5). The strong binding affinity of ONs to their mRNA targets makes these compounds potent and specific inhibitors of gene expression (6). To take advantage of this specificity, however, the ONs must also be delivered selectively to the intended target cells, where they can find and bind to their target mRNA sequences. The lung represents a major target for antisense therapy for diseases such as cancer and fibrosis. However, the complex anatomy of the lung as well as the presence of numerous lung cell types make ON targeting in this organ difficult. Furthermore, ONs are structurally bulky and highly charged, so their cellular uptake is generally low (7) and must be enhanced.
Receptor-mediated endocytosis offers the potential to target a selected
cell population and enhance cellular uptake of ONs. This method has
been used successfully to aid ON delivery in selected cell types
(8-11). For example, asialoorosomucoid-polylysine conjugates have been
used to target ONs to HepG2 and chloramphenicol acetyltransferase cells
(8, 9), and transferrin and folic acid conjugates have been employed to
aid ON delivery to rapidly growing cells such as HL-60 cells (10, 11).
In the lung, alveolar macrophages (AMs) represent a major target for
dust-induced pulmonary fibrosis. These cells are also major sources of
TNF production in the lung. The AMs possess mannose-specific
membrane receptors that can recognize and internalize glycoproteins
bearing mannose residues (12, 13). On this basis, mannosylated
glycoproteins could potentially be used to target and enhance ON uptake
by the AMs. Targeted delivery of several cytotoxic drugs and antiviral
agents by glycoproteins specific to macrophages has been demonstrated
previously (14, 15). Moreover, 6-phosphomannosylated glycoproteins have
also been used to target antisense ONs to peritoneal macrophages (16). In this study, we utilized mannosylated polylysine (MPL) as a drug-targeting vector to aid the cellular delivery of ONs to the AMs.
The effectiveness of this system in promoting antisense activity and
its mechanism of enhancement were evaluated.
Nuclease-resistant
phosphorothioate ON with a sequence complementary to the initiation
codon of TNF mRNA (17) (5
-TGTGCTCATGGTGTCTTT-3
, AS-ON), its
inverted sequence (5
-TTTCTGTGGTACTCGTGT-3
, INV-ON), and sense
sequence (5
-AAAGACACCATGAGCACA-3
, S-ON) were synthesized on an
automated solid phase synthesizer using standard phosphoramidite chemistry (OligoTherapeutics Inc., Wilsonville, OR). A fluorescent label was sometimes attached to the terminal 5
linkage group by the
use of 5
-carboxyfluorescein phosphoramidite. The ONs were purified by
high performance liquid chromatography and were >98% pure.
The MPL was
synthesized according to the method previously described (18). Briefly,
poly-L-lysine (Sigma, 0.25 µmol,
Mr 10,000) was dissolved in 0.15 M NaCl solution. The pH of the solution was adjusted to 9.0 using 0.1 N NaOH.
4-Isothiocyanatophenyl-
-D-mannoside (Sigma, 12.5 µmol) was added in small portions to
the magnetically stirred protein solution. After a 6-h reaction, the
resulting solution was refrigerated overnight, and on the following day the pH was adjusted to 7.0. Unreacted mannoside and polylysine were
removed by centrifugal filtration through a dialysis membrane filter
(DuraporeTM CL3K, Millipore Corp.) at 5000 × g for 30 min. The degree of sugar substitution was
determined by the resorcinol sulfuric assay (19). The percentage of
sugar in MPL was determined using the equation X% = n × Mo/(Mp + n × M), where n is the number of
mannose residues per polylysine molecule and Mo, Mp, and M are the molecular weights of
mannose, polylysine, and isothiocyanatophenylmannoside,
respectively. The MPL prepared under this condition was found to
contain
25% sugar.
In a typical experiment, the complex was prepared by adding 25 µl of 50 µg/ml MPL to 25 µl of 50 µg/ml ON in culture medium for 1 h prior to use. In studies designed to evaluate the dose effect of ON, various concentrations of ON (25-100 µg/ml) were used. Complex formation was verified by gel electrophoresis using a 6% polyacrylamide, 7 M urea gel. Using a dual fluorescence labeling technique, we previously found that both the ON and MPL, when prepared as a complex under this condition, were co-internalized by the AMs (20). In a separate study, the stability of ON and ON·MPL complex was studied in culture medium using gel electrophoresis. After a 7-h incubation at 37 °C, no detectable degradative products of ONs were observed.
Cell PreparationsThe AMs were harvested from male Sprague-Dawley rats (200-250 g) by bronchoalveolar lavage. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (0.2 g/kg of body weight). The trachea was cannulated and the lungs lavaged 10 times with 8-ml aliquots of Ca2+- and Mg2+-free Hanks' balanced salt solution (145 mM NaCl, 5 mM KCl, 1.9 mM NaH2PO4, 5.5 mM glucose, pH 7.4). Lavaged cell suspensions were centrifuged at 500 × g for 10 min at 4 °C. The cell pellets were washed twice by alternate resuspension and centrifugation in macrophage-serum-free medium, pH 7.4 (Life Technologies, Inc.). Cell number and purity of the macrophage preparations were determined using a Coulter electronic cell counter with a cell-sizing attachment (Coulter Instrument, Hialeah, FL). The average values for yield and purity were 6.2 ± 0.3 × 106 cells/rat and 92.5 ± 0.4%, respectively. Cell viability, measured via trypan blue exclusion, was >95%. Aliquots of 0.1 ml containing 105 cells were added onto a 96-well plate (Costar, Cambridge, MA) and incubated at 37 °C in a humidified atmosphere at 5% CO2.
In studies involving the use of alveolar epithelial cells, the lungs were lavaged as described above. They were then excised and incubated for 20 min at 37 °C with phosphate-buffered saline containing elastase (40 units/ml, Type I, U. S. Biochemical Corp.) and DNase (0.006%, Sigma). After enzymatic digestion, the lungs were finely minced and the digestion was arrested by incubation for 5 min in phosphate-buffered saline containing 25% fetal bovine serum and 0.006% DNase (to help prevent cell clumping). The crude extract was sequentially filtered through 160- and 45-µm screens and centrifuged, and the resulting cell pellet was spun on a sterile Percoll density gradient. The second cell band from the surface was collected, washed twice, and resuspended in Dulbecco's modified Eagle's medium. The cell suspension yielded 15-20 × 106 cells/rat with viability greater than 95% as determined by the Coulter counter and trypan blue exclusion.
Cell CultureJ774.1 cells, a macrophage cell line deficient in mannose receptors (21), were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 0.1 µg/ml streptomycin. They were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Prior to use, the cells were washed and resuspended in macrophage-serum-free medium culture medium.
Silica Exposure and Antisense Inhibition StudiesCells were
plated on a 96-well plate at the density of 105 cells/well.
They were treated with heat-sterilized silica (Mil-U-Sil, endotoxin-free, Pennsylvania Glass and Sand Corp.) or titanium dioxide
(a nonfibrogenic dust control) (Fisher), both at final concentrations
of 10-100 µg/ml. To test the effect of ONs on TNF expression, the
cells were simultaneously treated with free oligomers (AS-ON, INV-ON,
or S-ON) (25 µg/ml) or oligomers complexed with MPL (ON·MPL, 25:25
µg/ml). After a 7-h incubation at 37 °C, the culture medium was
collected and analyzed for TNF
using a Genzyme TNF
enzyme-linked
immunoadsorbent assay kit (Genzyme Corp., Cambridge, MA) according to
the manufacturer's instructions. This assay is specific for TNF
and
does not cross-react with other cytokines, as stated by the
manufacturer.
Analysis of TNF mRNA was also performed using RT-PCR assay.
Total RNA was extracted from cells following treatments. Lysis buffer
(500 µl) containing 0.15 M NaCl, 10 mM Tris,
pH 7.5, 1.5 mM MgCl2, 0.65% Nonidet P-40 was
added to a cell pellet containing approximately 106 cells.
The mixture was incubated for 5 min at 4 °C. After centrifugation, the supernatant was mixed with an equal volume of urea-SDS buffer (7 M urea, 10 mM Tris, pH 7.5, 10 mM
EDTA, 1% SDS). Then protein precipitation was carried out in the
presence of 2 M NaCl. The tubes were centrifuged and RNA
was precipitated from the supernatant with 2 volumes of ethanol.
Reverse transcription and PCR (2 µg of RNA) were performed using a
Clontech RT-PCR kit according to the manufacturer's instructions
(Clontech Laboratories). Reactions were performed on a Perkin-Elmer
thermal cycler (model 480). As an internal standard,
-actin cDNA
was also amplified. Samples were denatured for 2 min at 94 °C and
then amplified for 25 cycles, with each cycle consisting of three
heated segments: 94 °C for 30 s, 55 °C for 30 s, and
74 °C for 90 s. Samples were held for a further 10 min at
74 °C on the final cycle. The primers used were based on those
previously reported (22). Reaction products were precipitated and
separated on a 7% polyacrylamide gel and visualized by ethidium
bromide staining.
Cellular uptake studies were conducted using fluorescently labeled AS-ON. The cells (105/well) were plated on a 96-well plate and incubated for 4 h at 37 °C in culture medium containing AS-ON (25 µg/ml) or AS-ON·MPL complex (25:25 µg/ml). The cells were washed with cold medium containing excess unlabeled ON and/or MPL to remove surface-bound labeled ON. The cells were then measured for their fluorescence intensity using a fluorescence microplate reader at the excitation and emission wavelengths of 490 and 520 nm, respectively. For competitive inhibition studies, cells were treated with the AS-ON·MPL complex in the presence of 1 mg/ml mannosylated BSA (a specific competitor for mannose receptors) (Sigma) or BSA (a nonspecific competitor).
Cell ViabilityCell viability was determined using
fluorescence propidium iodide assay. After specific treatments the
cells were incubated with 1 µg/ml propidium iodide in culture medium
for 10 min at 37 °C. Cellular fluorescence intensity was then
measured at the excitation and emission wavelengths of 490 and 600 nm,
respectively. Cell survival was calculated as percentage cell
survival = 100 percentage cell death.
![]() |
The maximum signal is the fluorescence signal obtained in the presence of Triton X-100 (0.1%), which was used to permeabilize the cells. The minimum signal is the background autofluorescence signal.
Statistical AnalysisData were analyzed by analysis of variance with the use of the Newman-Keuls test for multiple comparisons. Statistical significance was considered at p < 0.05.
Exposure of the AMs to crystalline silica
(10-100 µg/ml) resulted in a dose-dependent increase in
TNF secretion over nontreated controls (Fig. 1).
Treatment of the cells with nonfibrogenic dust titanium dioxide at
similar concentrations had no significant effect on TNF
secretion
(Fig. 1). In order to assess the potential inhibitory effect of
antisense ONs on silica-stimulated TNF
release, AS-ON (25 µg/ml)
or AS-ON·MPL complex (25:25 µg/ml) was added to the cells
simultaneously with silica (100 µg/ml). As controls, noncomplementary
ON sequences (INV-ON and S-ON) and their respective MPL complexes
(INV-ON·MPL and S-ON·MPL) at the same concentrations were also
used. Our results, shown in Fig. 2, indicated that
treatment of the cells with free AS-ON had no significant effect on
TNF
level (p < 0.05). However, when the AS-ON was
given as a complex (AS-ON·MPL), >90% of the TNF
secretion was
inhibited. Non-antisense ONs, given either alone or in the form of
complexes, had no effect on TNF
level (p < 0.05).
The latter results suggested that the inhibitory effect of AS-ON·MPL
complex was not due to MPL and that the presence of AS-ON was required
for the anti-TNF
effect. Studies using MPL alone (see below) further
confirmed this point.
The ability of AS-ON·MPL complex to suppress TNF production at the
cellular level leads to the question of its molecular mechanism of
TNF
inhibition. To address this query, AS-ON·MPL complex (25:25
µg/ml) was added to the AMs in the presence of silica (100 µg/ml),
and 4 h later mRNA for TNF
was recovered and assessed by
RT-PCR analysis. As controls, AS-ON (25 µg/ml), INV-ON (25 µg/ml),
or MPL (25 µg/ml) alone were also tested (Fig. 3). The
addition of AS-ON·MPL complex reduced the expression of silica-induced TNF
mRNA, whereas all other control treatments had no effect. These results are consistent with the earlier
enzyme-linked immunoadsorbent assay studies and suggest that the
mechanism of TNF
inhibition by the AS-ON complex is due to
translational inhibition of TNF
mRNA.
The lack of the inhibitory effect of free AS-ON is somewhat surprising
but may be attributed to its poor cellular uptake and the inability to
reach its intracellular target. Because previous studies have
established that cellular uptake of ONs is dose-dependent (6), we therefore investigated the effect of ON concentration (25-100
µg/ml) on silica-induced TNF production (Fig. 4).
Our result showed that an increase in ON concentration (AS-ON), as expected, resulted in a corresponding increase in TNF
inhibition. A
significant inhibitory effect (
35%) was observed at the AS-ON concentration of 75 µg/ml (p < 0.05). At the highest
concentration tested (100 µg/ml),
50% inhibition was observed.
The control INV-ON had no significant effect on TNF
production at
all concentrations tested except at 100 µg/ml, at which concentration
a minimal but significant effect was observed (
15%)
(p < 0.05). This latter result suggested the possible
nonsequence-specific effect of ON, which has been reported in other
systems when high concentrations of ONs are used (23, 24). The
potential cytotoxic effect of ONs at high concentrations was also
examined using fluorescence propidium iodide assay. The percentage of
cells viable after treatment with 100 µg/ml AS-ON was 94 ± 5 and 92 ± 4% for INV-ON. These values were not significantly
different from that of the nontreated control (95 ± 4%)
(p < 0.05, n = 4).
Antisense Oligonucleotide Cellular Uptake Mediated by Mannosylated Polylysine
To further examine the promoting effect of MPL on
antisense activity of AS-ON, cellular uptake studies were conducted. In these studies, 5 fluorescently labeled AS-ON was used to allow direct
detection of cellular uptake. AS-ON (25 µg/ml) or AS-ON·MPL complex
(25:25 µg/ml) was added to the AMs, and after indicated time
intervals the cells were washed and analyzed for their fluorescence intensity. As shown in Fig. 5, the MPL greatly promoted
the cellular uptake of AS-ON. At 4 h of incubation, the uptake of
the complex was found to be
17-fold greater than that of the free
AS-ON. This result indicated that the MPL carrier system was highly
effective at promoting the cellular uptake of AS-ON and that the AS-ON
alone was poorly taken up by the AMs. The saturable nature of the
complex uptake is indicative of receptor-mediated endocytosis.
To test whether the cellular uptake of the MPL complex occurred via
mannose receptor-mediated endocytosis, cellular uptake of the
AS-ON·MPL complex in the presence of competition for mannose receptors was carried out. In these experiments, the AS-ON·MPL complex was incubated with the AMs in the presence of an excess amount
of a specific mannose receptor competitor, mannosylated BSA, or a
nonspecific competitor, BSA (Fig. 6). These results demonstrated that the cellular uptake of the AS-ON·MPL complex was
inhibited by mannosylated BSA but not by BSA, thus indicating that
the ON uptake mediated by MPL occurred via the mannose receptor pathway.
To further confirm the mechanism of AS-ON complex uptake and to test the potential targeting ability of the MPL system, uptake studies were repeated in the J774.1 macrophage cell line, which is known to express a low level of mannose receptors (21), and in alveolar epithelial cells, which lack mannose receptors (Fig. 6). Both cell types exhibited a much lower complex uptake compared with the AMs (p < 0.05), thus confirming the mannose receptor-mediated complex uptake and the targeting ability of the MPL carrier system.
Pulmonary macrophages are known to be sources of inflammatory
mediators and fibrogenic factors and are one of the first cells to
respond to inhaled particles in the lung. Several studies have shown
that TNF plays an important role in the pathogenesis of silicotic
fibrosis. Driscoll et al. (1) demonstrated an increase in
TNF
production by the AMs in rats treated with silica. Piguet et al. (2) later showed that this increase in TNF
production was associated with increased expression of TNF
mRNA
in the lung, although the cellular origin of TNF
was not identified
in this study. The work by Piguet et al. (2) also
demonstrated that pretreatment of the animals with anti-TNF
IgG or
exogenous recombinant TNF
reduced and augmented, respectively,
silica-induced fibrosis. Thus, these studies strongly indicate the role
of TNF
in silicotic fibrosis.
There are, however, a number of studies that have reported
contradictory results. Mohr et al. (25) and Lemaire (26)
showed that silica treatment in rats had no effect on TNF production by the AMs. Bissonnette and Rola-Pleszczynski (27) similarly reported
the lack of silica effect in mice. The discrepancies between test
results by different groups may be attributed to differences in
experimental conditions, i.e. the dose and type of silica
used and the mode of silica instillation. With regard to the dose,
Driscoll et al. (1) showed that silica stimulation of TNF
release occurred only at doses greater than 50 mg/kg. Lemaire (26) also
reported the lack of silica effect on TNF
release at a low silica
dose of 15 mg/kg. In agreement with these in vivo data, our
in vitro results indicated a dose-dependent effect of silica on TNF
production by the AMs. Silica directly stimulated TNF
secretion and mRNA expression, effects that were not observed with the nonfibrogenic dust titanium dioxide. These results confirm and extend those of Gosset et al. (28) and
Driscoll et al. (29), who reported a direct effect of silica
on TNF
production in isolated human and rat AMs, and those of Savici et al. (30), who reported an elevated TNF
gene expression
in THP-1 myelomonocytic cells following silica treatment.
The regulatory role of TNF in fibrogenesis and its potential
therapeutic intervention can be studied at the molecular level using
antisense ONs. These compounds have the potential to interfere selectively with cellular protein synthesis by sequence-specific hybridization to DNA or RNA molecules. Since the gene sequence encoding
TNF
had been previously identified (17), we predicted that specific
antisense sequences could be synthesized and used to inhibit TNF
expression and therefore fibrosis. To investigate this possibility, we
utilized an antisense ON specific to the initiation codon of the TNF
mRNA (AS-ON) and tested its effect on the macrophage cytokine gene
expression. Our results indicate that AS-ON can be used to inhibit
TNF
expression. However, when used alone this compound is relatively
ineffective, and high concentrations are required to elicit the effect.
At a high concentration of 100 µg/ml, AS-ON causes
50% inhibition
of TNF
production. Because high concentrations of ONs are commonly
associated with nonsequence-specific effects (23, 24), innovative
approaches to improving potency of ONs are desirable.
It has been reported that ONs, due to their polyanionic nature, poorly
permeate cell membranes to reach their intracellular targets (31, 32).
Several research groups (23, 33, 34) also observed that in the absence
of appropriate delivery systems ONs exhibited no antisense activity,
whereas in the presence of these delivery systems, i.e.
liposomes, ONs showed strong activity. These observations suggest that
ONs do not normally enter the cells to a significant extent, and
therefore approaches to improve their cellular uptake are important for
their effective use. In agreement with previous findings, our results
indicated that the cellular uptake of free ON by the AMs is very low,
and this may account for the observed low activity of the AS-ON. This
conclusion is supported by a dramatic and parallel increase in cellular
uptake and anti-TNF activity of the AS-ON when given as a complex
with the MPL. At a low concentration of AS-ON (25 µg/ml), the MPL (25 µg/ml) promoted a 17-fold increase in cellular uptake of the ON and
caused
90% TNF
inhibition. At this concentration level, the AS-ON
alone had no significant effect on TNF
. The effect of AS-ON·MPL
complex on TNF
activity was associated with specific ablation of
targeted mRNA as shown by RT-PCR. Control studies also showed that
the promoting effect of MPL was not associated with loss of cell
viability as determined by fluorescence propidium iodide assay.
Furthermore, MPL alone or complexed with non-antisense ONs (INV-ON and
S-ON) had no effect on TNF
activity. Thus, these results suggested
that the promoting effect of MPL was most likely due to enhanced ON
uptake.
To further confirm these observations and to test the mechanism of ON uptake mediated by the MPL, a series of cellular uptake experiments was conducted. Our results indicated that MPL promoted the cellular uptake of AS-ON via mannose receptor-mediated pathway since competition for mannose receptors by mannosylated BSA inhibited the uptake of the AS-ON·MPL complex and the noncompetitor BSA had no effect. Furthermore, cells lacking mannose receptors (epithelial cells) or bearing few receptors (J774.1 cells) did not appreciably take up the AS-ON·MPL complex. The latter results also indicated the potential targeting ability of the MPL carrier system for the AMs.
In conclusion, we have demonstrated that antisense ON targeted to the
TNF mRNA can be used to inhibit TNF
expression in AMs. The
findings in this study have a direct implication on the therapeutic
utilization of ONs in pulmonary fibrosis. A similar strategy may be
employed to aid the study of specific cytokine functions and their role
in disease pathogenesis. Effective utilization of antisense ONs
requires development of appropriate delivery systems capable of
effective and selective transfer of ONs to the target cells. With
regard to the AMs, we have shown that the MPL delivery system is
effective and selective in promoting the cellular delivery and
antisense activity of ON. The system exploits the efficient
receptor-mediated endocytosis to achieve cell-specific antisense
delivery. Because the system utilizes a naturally occurring cellular
process, it is potentially nontoxic. Although our in vitro
studies indicate the relative safety of this system, further in
vivo studies are needed. Demonstration of its safety and efficacy will allow further development of this system for therapeutic purposes.