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and Results in Cell Apoptosis Through TNF-1
* Graduate Institute of Biochemistry, Medical College, National Cheng Kung University, Tainan, Taiwan; and
Chi-Mei Medical Center, Tainan, Taiwan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. It also induced mouse monocyte apoptosis and the
production of reactive oxygen species. Taken together, our results
indicate that mouse IL-19 may play some important roles in inflammatory
responses because it up-regulates IL-6 and TNF- and induces
apoptosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-6, it is a normal endogenous feedback factor for
the control of immune responses and inflammation (2, 3).
Autoimmune models of rheumatoid arthritis, thyroiditis, and
collagen-induced arthritis and a model of herpetic stromal keratitis
all suggest negative regulatory roles of IL-10 in limiting
inflammation and immunopathology (3). However, IL-10 is
also a stimulatory factor for mast cells, B cells, and thymocytes
(4, 5, 6), and has been shown to be pleiotropic and to act on
many other cell types, including monocytes/macrophages, T cells, NK
cells, neutrophils, endothelial cells, and PBMC (7, 8).
Several new members of the IL-10 family, including IL-19, IL-20, IL-22,
melanoma differentiation-associated gene (MDA)3-7 (IL-24),
and AK155 (IL-26), have only recently been discovered. The
IL-19, IL-20, and MDA-7 (IL-24) genes have been mapped on
chromosome 1q31–32, a region where IL-10 is located. The two other
IL-10-related cytokines, AK155 (IL-26) and IL-22, are on chromosome
12q15 (9). Overexpression of IL-20 in transgenic mice
causes neonatal death as well as skin abnormalities, including aberrant
epidermal differentiation (10). IL-22 was originally
identified as a gene induced by IL-9 in murine T lymphocytes.
Stimulation of HepG2 human hepatoma cells with IL-22 up-regulated the
production of acute phase reactants like serum amyloid A,
1-antichymotrypsin, and haptoglobin (11, 12). Expression of MDA-7 was up-regulated in wound healing and
during the in vitro differentiation of a melanoma cell line (13, 14). AK155 is induced by transformation of T lymphocytes with
Herpesvirus saimiri, but its biologic activities and
receptor remain poorly understood (9, 15). Little is known
about the biologic function of IL-19, except that it is expressed by
LPS- or GM-CSF-activated monocytes (16). Our aims,
therefore, were to understand the gene regulation of human IL-19 and
the biologic function of IL-19. For these aims, we isolated the human
genomic clone and determined its promoter sequence. We also isolated
the mouse IL-19 cDNA and identified the biologic functions of the IL-19
recombinant protein.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A homology screening of the National Center for Biotechnology Information human high throughput genome database (http://www.ncbi.nlm.nih.gov) using the human IL-19 cDNA sequences as a query was conducted using a basic Blast search. The human genomic clone (clone identification, RP11-237C22) was identified (accession number AF276915) and purchased from Research Genetics (Huntsville, AL). The genomic DNA was isolated from the BAC clone and used in the PCR amplification of the promoter fragments.
Isolation of full-length human cDNA clone
The full-length human cDNA clone4 was obtained by repetitive 5' RACE using anchor primers and the gene-specific antisense primers: 5'-gatatagctgattaatca-3' (reverse transcription primer); 5'-taaactccccatctccatgcaa-3' (first PCR); 5'-caattctatgtccatgcagaaaaat-3' (second PCR). After three rounds of 5' RACE, the 5' end of exon 1 were determined.
Construction of promoter-luciferase fusion gene
Five different regions upstream of exon 1 of the human IL-19 gene were amplified by PCR from the DNA of the BAC clone RP11-237C22. Five fragments (pA, pB, pC, pD, pE) containing different lengths of sequences upstream of exon 1 and 247 bp (1 to 247) of exon 1 were ligated into the vector of the promoterless luciferase gene (pGL3 enhancer). pA contains 2105 bp (from -1858 to 247). pB contains 1365 bp (from -1118 to 247). pC contains 1085 bp (from -838 to 247). pD contains 713 bp (from -466 to 247). pE contains 394 bp (from -147 to 247). We generated five fusion genes by cloning these fragments into the SacI-XhoI site of the pGL3 enhancer plasmid vector containing the entire coding sequences of firefly luciferase and SV40 enhancer (Promega, Madison, WI).
Transfection and luciferase activity assay
The fusion gene was used along with the promoterless pGL3
enhancer plasmid DNA in the transfection of the canine kidney
epithelial-like Madin-Darby canine kidney (MDCK)4
cells. Cells at a density of 3 x 105/well
in a six-well plate were transfected with 1 µg of plasmid DNA from
the fusion gene and 0.4 µg of the 
-galactosidase (-gal) gene
which was used as an internal transfection efficiency control by using
1 µl of LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA).
Twenty-four hours after transfection, the medium was replaced with
fresh medium. Forty-eight hours after transfection, the cells were
collected, and the luciferase activity was analyzed according to the
protocol of the luciferase assay system (Promega). To obtain internal
control of -gal gene transfection, the cell lysate was also used for
-gal activity analysis. The luciferase activity from each
promoter-fusion gene was divided by -gal activity to obtain the true
representation of luciferase activity from each promoter-luciferase
fusion gene.
Isolation of mouse IL-19 cDNA
A partial murine cDNA clone was isolated by PCR amplification from mouse embryo cDNA (Clontech, Palo Alto, CA). A pair of primers (sense primer, 5'-agagccatccaagctaaggacacctt-3'; and antisense primer, 5'-gcattggtggcttcctgcctgcagt-3') designed from human cDNA sequences was used in PCR amplication. The full-length cDNA clone was further obtained by 5' and 3' RACE.
Protein expression and purification
A cDNA clone coding for the human and mouse IL-19 sequences from leucine to histidine (aa 25–176) was inserted into pET32 EK/LIC (Novagen, Madison, WI). The protein was found mostly in the inclusion bodies and was purified to >95% by a series of affinity chromatography and refolding. Before in vitro use, all preparations of IL-19 recombinant protein were found to contain <2 ng/ml LPS endotoxin by the detection methods of Limulus amebocyte lysate. The human IL-19 was also expressed in the yeast vector of Pichia pastoris, and the protein was purified by a series of affinity chromatography.
Isolation of mouse monocytes
Mouse monocytes were prepared from the spleen of 8- to 10-wk-old male mice. Spleen cells were depleted of erythrocytes. Monocytes were allowed to adhere for 30 min at 37°C and 5% CO2. The nonadherent cells were then removed by three washes with warm medium. The monocytes were >95% pure, as determined by Liu’s staining and contained >98% viable cells.
In vitro biological function analysis
The monocytes were cultured at a concentration of 5 x
106 cells/ml in a six-well plate and then treated
with different concentrations of mouse IL-19 protein for 8 h.
After incubation, the monocyte supernatants were collected, and the
production of IL-6 and TNF- was measured by ELISA kits (R&D,
Minneapolis, MN). To analyze induction of IL-6 and TNF- transcript
by IL-19, the monocytes were treated with mouse IL-19 (100 ng/ml) or
LPS (50 ng/ml) for 4 h. Total RNA was isolated from the monocytes.
RT-PCR was performed with IL-6- or TNF--specific primers using total
RNA as templates. Amplified PCR fragments were run on gel
electrophoresis. Specific primers for -actin were also used to
amplify a PCR fragment and run on gel as an internal control.
IL-6-specific primers used are 5'- tgt gca atg gca att ctg at-3'
(sense) and 5'-gga aat tgg ggt agg aag ga-3' (antisense).
TNF--specific primers used are 5'-ccc caa agg gat gag aag tt-3'
(sense) and 5'-gtg ggt gag gag cac gta gt-3' (antisense).
Mouse -actin-specific primers used are 5'-ggg aat ggg tca gaa gga
ct-3'(sense) and 5'-ttt gat gtc acg cac gat tt-3'(antisense).
To analyze interaction of IL-19 and IL-10, monocytes were pretreated
with IL-10 (50 ng/ml) or IL-19 (50 ng/ml) for 2 h, and then the
other cytokine, either IL-19 or IL-10, was added to the culture. Six
hours after coincubation with both cytokines, monocyte supernatants
were collected together with the controls (PBS or single cytokine
treatment). Production of IL-6 and TNF- from these monocytes was
analyzed.
In the experiment to test the effect of cycloheximide, 1 h after IL-19 (100 ng/ml) had been added to the culture, cycloheximide was added at a concentration of 0.3 mM and incubated with cells for another 7 h.
Cell viability analysis
Cell viability was analyzed by propidium iodide (PI) staining
and annexin V assay (Clontech). Mouse monocytes were treated with PBS
or mouse IL-19 (100 ng/ml) alone or combined with TNF- Ab for
12 h. After treatment, cells were stained with PI and then
analyzed by flow cytometry (FACScan; BD Biosciences, San Juan, CA). For
the annexin V assay, cells were treated as above, harvested, and then
resuspended in 1x binding buffer at a concentration of 1 x
106 cells/ml. Annexin V-FITC (5 µl) was added
to 100 µl of the cell solution. The cells were gently vortexed,
incubated in the dark for 15 min at room temperature, and then analyzed
by flow cytometry (FACScan).
DNA fragmentation by gel electrophoresis
Using the method described by Oren and Prives (17), mouse lymphocytes (5 x 106 cells/well) were treated with mouse IL-19 for 12 h. After treatment, the culture medium was removed, and the cells were washed twice with PBS and harvested. Then the cells were fixed with 1 ml 70% ethanol. After storage overnight at 4°C, the ethanol was removed, and the cells were resuspended in 1 ml phosphate-citric acid buffer with 0.2 M Na2HPO4 and 0.1 M citric acid (pH 7.8) and maintained in this solution at room temperature for 60 min with occasional shaking. This treatment extracts low molecular mass DNA from apoptotic cells but has no effect on the DNA content of nonapoptotic cells (18). The cell suspension was centrifuged at 2000 rpm for 5 min. The supernatant containing low molecular mass DNA was collected for analysis of internucleosomal DNA degradation by agarose gel electrophoresis.
Measurement of reactive oxygen species (ROS) produced from monocytes
To evaluate the effect of mouse IL-19 on ROS production by monocytes in vitro, mouse monocytes (1 x 106 cells) were incubated at 37°C with different concentrations of mouse IL-19 (0–50 ng/ml) for various times. The ROS activities were determined at the end of the incubation.
Monocytes were collected and resuspended in 0.2 ml PBS. The
chemiluminescence (CL) count was measured in a completely dark chamber
of the Chemiluminescence Analyzing System. After a 100-s background
level determination, 0.5 ml of 25 mM luminol in PBS (pH 7.4) was
injected into the sample. The CL was monitored continuously for an
additional 600 s. To analyze the effect of TNF- Ab on ROS
production, the Ab was added to monocytes 30 min before or after the
addition of IL-19 (100 ng/ml), or else both reagents were added at the
same time. After incubation with both reagents for another 30 min, ROS
production from monocytes was measured by CL count.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human IL-19 genomic clone (clone identification, RP11-237C22) was
identified by homology search against a human high throughput genome
database. The 5' end of untranslated sequences of the human cDNA was
obtained by a series of repeated 5' RACE. After obtaining the
full-length cDNA clone, the cDNA sequence was compared with the human
genomic sequences to locate the exon/intron boundaries. The 5'
untranslated sequence and the potential promoter sequence is shown in
Fig. 1
. The locations of the introns in
this region are indicated by the two arrows at nt 250 and nt 937
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). Therefore, human IL-19
gene contains seven exons and six introns. As Fig. 2 shows, the
exon/intron junctions conform to the GT/AT rule. The human IL-19
protein is encoded by exon 3 to exon 7.
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Promoter activity of human IL-19
To characterize the DNA sequences involved in the human IL-19 gene
expression, we used PCR to amplify five potential promoter fragments
(A, B, C, D, and E) by using a human genomic clone as a template. The
sizes of the PCR fragments ranged from 2.1 kb to 400 bp upstream of
exon 1 (Figs. 1
and 3A). These
potential promoter regions were subcloned into the pGL3 enhancer vector
containing the luciferase gene along with the SV40 enhancer to generate
five fusion genes: pA, pB, pC, pD, and pE.
During our isolation of the full-length cDNA clone, we isolated partial
cDNA sequences from human kidney RNA. We also performed Northern blots
to analyze tissue distribution of IL-19 and found that the 1.35-kb
transcript of IL-19 was expressed in heart, brain, liver, and kidney.
In these four tissues, kidney and heart expressed higher amount than
brain and liver. Therefore, we used the MDCK cells and human embryonic
kidney 293 cells for the analysis of promoter activity. After
transfection of the MDCK cells with the fusion gene, the luciferase
activities were analyzed. All five promoter fragments contained at
least one or several TATA boxes. All showed some promoter activity,
with the pE fusion gene the highest, 7- to 8-fold higher
than the negative control of the promoterless pGL3 enhancer vector
(Fig. 3B). This experiment was repeated five times with
similar results. The luciferase activity in 293 cells was similar to
that of MDCK cells. The promoter region4 2.1 kb contained
several transcription factor-binding sites: several copies of
keratinocyte-enhancer; TATA box; NF-
B; AP-1; AP-2; E1A-CS; GATA-1;
SP-1; P53; and C/EBP (Fig. 1). Previous study has shown that IL-19 is
inducible by LPS (16). We added LPS into the transfectants
and found that luciferase activity was not inducible by LPS. This could
be due to the constitutive expression of IL-19 in kidney cells.
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The full-length mouse cDNA clone4 (
1 kb long) was
isolated by 5' and 3' RACE. The 3'-untranslated region contained only
one ATTTA mRNA-destabilizing segment. Hydropathic analysis predicts a
hydrophobic signal peptide of 24 aa. Beginning with leucine (residue
25), the mature protein, which contains 152 aa, has a predicted
molecular mass of 17 kDa. Three potential N-linked
glycosylation sites were detected in the amino acid sequences, only two
of which, NVT and NAT, are identical with those in human IL-19. The
third, NCS, is not present in human IL-19. The protein contains six
cysteines the positions of which are identical with those in human
IL-19 (Fig. 4). The amino acid sequences
of mouse IL-19 showed 75% similarity and 71% identity with those in
human IL-19, and the genomic structure of mouse IL-19 is similar to
that of human IL-19. Locations of exon/intron boundaries in the mouse
gene are also indicated in Fig. 4.
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To express the recombinant IL-19 in Escherichia coli,
we constructed an expression vector that contained a coding region from
leucine (residue 25) to histidine (residue 170) downstream of the
fusion protein sequence (thioredoxin). The predicted molecular mass of
mouse IL-19 containing fusion protein (thioredoxin) is 35 kDa.
Treatment of mouse IL-19 fusion protein with enterokinase to cleave off
thioredoxin resulted in the disappearance of the 35-kDa band and the
formation of a single 17-kDa band on the SDS-PAGE after protein was
purified and reduced with 2-ME (data not shown). Recombinant human
IL-19 was similarly expressed and showed the same purification pattern
as mouse IL-19. Both mouse and human recombinant protein after a series
of chromatography was found to contain <2 ng/ml LPS endotoxin. Human
IL-19 was also expressed in the yeast P. pastoris. The
recombinant protein produced from P. pastoris showed three
bands on SDS-PAGE after affinity chromatography purification. Amino
acid determination of the three bands by mass spectrophotometry showed
that all three proteins were human IL-19 (data not shown).
Mouse IL-19 stimulated monocytes to produce IL-6 and TNF-
To determine the effects of mouse IL-19 on the production of
cytokines by monocytes, the monocytes were incubated with various
concentrations of mouse IL-19 for 8 h, after which the level of
cytokine production in the supernatant of monocytes was determined by
ELISA. As shown in Fig. 5, monocytes
incubated in PBS alone at 37°C did not produce IL-6 and TNF-.
However, the amount of IL-6 (Fig. 5A) and TNF- (Fig. 5B) produced by monocytes increased with the addition of
mouse IL-19. The increase of these two cytokines was dosage dependent
on IL-19.
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production. To prove that the production of IL-6 and TNF- from IL-19
treatment was not due to the contamination of LPS endotoxin in the
recombinant protein, the protein was heat-denatured at 100°C for 10
min, a condition under which LPS endotoxin cannot be denatured. The
heat-denatured protein was added to monocytes to test its biologic
activity. The result showed that the heat-denatured protein had lost
its activity (Fig. 5). Therefore, the activities we observed were not
due to contamination of the LPS endotoxin. Human IL-19 was shown to
have the same activities on human monocytes as mouse IL-19 has on mouse
monocytes (data not shown). Mouse IL-10 has been shown to be inactive on human monocytes. In contrast, we found that mouse IL-19 protein is active on human monocytes but that human IL-19 is inactive on mouse monocytes (data not shown). This result also provided the evidence that the activity observed from IL-19 treatment is not due to LPS endotoxin.
To investigate whether induction of IL-6 and TNF- was regulated at
the transcriptional level, total RNA was isolated from IL-19- or
LPS-treated monocytes. The levels of IL-6 and TNF- transcripts were
analyzed by RT-PCR. As shown in Fig. 6, both IL-6 and TNF- transcripts were induced in monocytes stimulated
with IL-19. Induction of IL-6 and TNF- transcripts may not require
de novo protein synthesis because the addition of cycloheximide did not
inhibit the induction (data not shown).
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production in monocytes. Therefore, interaction between IL-10 and IL-19
was analyzed by treating monocytes with both cytokines at different
times. As shown in Fig. 7, pretreatment
of monocytes with IL-10 for 2 h followed by IL-19 abolished both
IL-6 and TNF- production by IL-19 (Fig. 7, A and
B, column C). However, treatment of monocytes with IL-19
followed by IL-10 blocked only part of IL-19 activity on IL-6
induction, whereas a majority of TNF- production was inhibited (Fig. 7, A and B, Column D). This result demonstrated
that interactions between IL-19 and IL-10 in the regulation of IL-6 and
TNF- may be different.
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. Therefore, we also added
LPS and IL-19 together to the monocytes and analyze whether both have
any synergistic effect. The result demonstrated there was no
synergistic effect (data not shown). IL-19-induced monocyte apoptosis
During the incubation of monocytes with IL-19, trypan blue
staining showed a decrease in cell viability. We therefore further
analyzed cell apoptosis using three methods. Mouse monocytes were
treated with IL-19 for 12 h, and then cell viability was measured
by PI staining, annexin V staining, and DNA fragmentation. As shown in
Fig. 8A, monocytes treated
with 100 ng/ml IL-19 resulted in 33% cell death (Fig. 8Ac),
whereas the control showed only 13–16% cell death (Fig. 8A,
a and b). LPS endotoxin produced 23% cell death (Fig. 8Ad). In contrast, heat-denatured IL-19 also showed only
17% cell death (Fig. 8Ae). Early in apoptosis, the
phosphatidylserine in the inner membrane translocates to the outer
surface of the plasma membrane and has a high affinity for annexin V,
which makes annexin V staining an alternative method to demonstrate
cell apoptosis. Fig. 8B shows that treatment of monocytes
with mouse IL-19 increased the population of annexin V-stained dead
cells (Fig. 8Bb). LPS endotoxin also induced cell death
(Fig. 8Bc). This apoptotic effect of IL-19 may be due to the
production of TNF-, because addition of both IL-19 and TNF-Ab
abolished the apoptotic effect of IL-19. To further verify the
apoptotic effect of IL-19, after IL-19 treatment, DNA fragmentation
analysis was performed. The results showed that mouse IL-19 induced DNA
fragmentation of monocytes and that the extent of DNA fragmentation was
dosage dependent on IL-19 (lanes 3-7) in
Fig. 8C.
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Exposure to certain cytokines induces marked transient increases
in the intracellular level of ROS. For example, exposure to TNF- or
IL-1 increases intracellular levels of ROS in NIH3T3 fibroblasts,
which suggests that ROS may act as signaling intermediates for TNF-
and IL-1. These highly reactive ROS molecules regulate many
important cellular events in response to TNF-, including
transcription factor activation (NF-B), cellular proliferation, and
apoptosis. Mouse IL-19 induced TNF- production and resulted in cell
apoptosis. To test whether this effect was mediated through the
production of ROS, we treated mouse monocytes with various
concentrations of IL-19 for various lengths of time. Monocytes treated
with IL-19 for 6 h showed an increase in ROS formation in a
dose-dependent manner (Fig. 9A). When monocytes were
treated with IL-19 at the concentration of 25 ng/ml, ROS production
increased with time (Fig. 9B). However, production of ROS
decreased rapidly after 12 h incubation (data not shown). To
analyze whether production of ROS depends on TNF-, monocytes were
treated with both TNF- Ab and IL-19, and ROS production was
monitored. As shown in Fig. 9C, when monocytes were treated
with TNF- for 30 min followed by IL-19 stimulation for another 30
min (Fig. 9C, column C and curve C), ROS production was
partially inhibited. However, if monocytes were treated with IL-19 for
30 min followed by incubation with TNF- Ab, ROS production was not
inhibited (Fig. 9C, column D and curve D). If both IL-19 and
TNF- were added at the same time (Fig. 9C, column E and
curve E), the extent of inhibition on ROS production was not as great
as when TNF- Ab was added first. These results indicate that ROS
production may not be completely dependent on TNF- production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The analysis of the human IL-19 promoter region showed a fragment of 394 bp (pA), including 247 bp of exon 1 that supports luciferase activity at a level 7- to 8-fold greater than that of the negative control. The fluctuation of promoter activity in pA, pB, pC, pD, and pE may be due to some repressor or enhancer sequences located in these regions, such as the sequences between -1858 and -147. We will perform an EMSA to determine the positive or negative sequence on the promoter region and potential transcription factors in the cell lysate involved in the regulation.
The promoter region of IL-10 has been shown to be involved in many mechanisms of IL-10 activities. For example, AP-1 and SP-1 were shown to play an important role in regulating IL-10 function (19). Several diseases have been shown to be associated with polymorphism of the IL-10 promoter region. It was shown that high IL-10 production associated with autoimmune disease, including rheumatoid arthritis and systemic lupus erythematosus, may be a genetic risk factor for disease susceptibility (20). The basis for a heritable difference in IL-10 production is not well known. The IL-10 gene promoter is polymorphic, and promoter-reporter studies have identified several positive and negative regulatory sequences within the 1.3-kb region upstream of the transcription start site. Several single-nucleotide polymorphisms (SNP) have also been identified in this region. All these results demonstrate that IL-10 promoter sequences may be a central mediator of its function. Searching the human genome SNP database, we found four potential SNPs on the 2105 bp of the pA fragment in the human IL-19 promoter region. Whether these four potential SNPs of human IL-19 are also associated with any inflammatory diseases awaits further study.
Analysis of the MDA-7 (IL-24) gene, another member of the IL-10 family, has demonstrated that AP-1 and C/EBP transcription factor contributed to its promoter activity during human melanoma differentiation (21). This finding provided insights into the regulation mechanism of the MDA-7 gene during induction of terminal cell differentiation in human melanoma cells. The IL-19 promoter region also contains AP-1- and C/EBP-binding sites. Whether or not IL-19 and MDA-7 share some similar mechanism in gene regulation by the regulatory element of AP-1 and C/EBP awaits detailed study of these sequences.
In the promoter region of IL-19, there are several copies of keratinocytes enhancer element (AARCAAA). Another member of IL-10 family, IL-20, has been shown to be involved in the proliferation of keratinocytes (10). It will be interesting to study whether IL-19 also has any association with keratinocytes function by the keratinocyte enhancer element in its promoter region.
In addition to expression of IL-19 in E. coli, we expressed IL-19 in the yeast P. pastoris system. Protein purified from the culture medium of the yeast showed three different sizes of the molecule with identical amino acids analyzed by mass spectrophotometry. The heterogeneity of the protein may be due to different levels of glycosylation, which is similar to what was observed in the mammalian expression system (16).
We also observed dimerization of recombinant IL-19 protein when the pure protein was run on a nonreduced gel. This indicated that, as with IL-10, a homodimer of IL-19 may be the functional unit.
IL-10 suppresses the release and function of a number of
proinflammatory cytokines, such as IL-1, IL-6, and TNF- (2, 3). We found, however, that both human and mouse IL-19, a
homologue of IL-10, stimulated monocytes to produce IL-6 and TNF-.
Therefore, IL-19 may play an immune response role different from that
of IL-10. The data shown in Fig. 7 also demonstrated that IL-10
completely inhibited the induction of IL-6 and TNF- by IL-19 if
monocytes were pretreated with IL-10 followed by IL-19. However, if
IL-10 was added to monocytes 2 h after IL-19, it only partially
inhibited IL-6 production (12% activity). In contrast, TNF-
induction can be completely inhibited because level of TNF- is same
as that of IL-10 treated alone. These results indicated that induction
of IL-6 and TNF- may occur at different stages of incubation with
IL-19. Alternatively, regulation of IL-6 production by IL-19 and IL-10
may take different mechanisms in signal transduction.
Moreover, IL-6 attenuates the synthesis of the proinflammatory
cytokines while having little effect on the synthesis of
anti-inflammatory cytokines, such as IL-10 and TGF-. IL-6
promotes the synthesis of IL-1 receptor antagonist and soluble TNF-R
release in humans (22). We may then speculate that IL-19
plays an important role in inflammation.
The result illustrated by Fig. 9C demonstrated that IL-19
induced ROS production which could be only partially inhibited by
TNF- Ab. It indicated that ROS production induced by IL-19 may not
completely depend on TNF-. Moreover, when monocytes were
preincubated with IL-19 for 30 min followed by the addition of TNF-
Ab, production of ROS was not only not inhibited but also enhanced. The
reason for this was not clear. We also found that a high level of ROS
production could be induced rapidly during the first 30 min when a
higher concentration of IL-19 (200 ng/ml) was used to stimulate
monocytes (data not shown). Therefore, we may speculate that ROS
production induced by high concentration of IL-19 is not mediated
through TNF-, because 30 min was not long enough for TNF- protein
to be synthesized.
IL-10 has been shown to inhibit generation of ROS in macrophages
(23). Treatment of macrophages with LPS caused activation
of NF-B and rapid proteolysis of IB- degradation in the cells.
IL-10 pretreatment inhibited both NF-B activation and IB-
degradation. Both processes were also inhibited by ROS scavengers,
demonstrating that inhibition of IL-10 on NF-B activation and
IB- degradation was mediated through ROS production. When
IL-10-deficient mice were used to test whether IL-10, through ROS,
protects endothelial cells from inflammation, it was shown that IL-10
protects endothelial function after an acute inflammatory stimulus by
limiting local increases in superoxide (24). Our results
demonstrated that IL-19 induced production of ROS in monocytes,
contrary to what was observed with IL-10. Taken together, IL-19,
sharing some homology with IL-10, demonstrated biologic functions quite
distinct from those of IL-10.
In summary, we have isolated the full-length human IL-19 cDNA and identified its genomic clone. The human IL-19 gene consists of seven exons and six introns and is encoded by exons 3–7. Several different transcripts were generated by alternative splicing at their 5'-untranslated region. These transcript variants seemed to encode the same amino acid sequences as the mature protein (16). A fragment containing 394 bp upstream of exon 1 showed promoter activity 7- to 8-fold greater than that of the negative control. A fragment (from -1858 bp to -147 bp) further upstream of 1712 bp may contain repressor or enhancer sequences, because the presence of these sequences affected the promoter activity of 394 bp.
IL-19 can induce IL-6 and TNF- production in monocytes and result in
cell apoptosis. Because our assay system was an in vitro analysis, cell
apoptosis was observed in a pure population of monocytes (95% pure).
The observation of apoptosis on the monocytes themselves may not
reflect the true activity in vivo. It is possible that monocytes may
use the production of TNF- to cause apoptosis in other cells as a
defense mechanism against infection or in tumor cell eradication after
receiving a signal from IL-19. In other words, it may be a paracrine
and not an autocrine effect. Our results also demonstrated that IL-19
induced monocytes to produce ROS. Whether or not ROS production plays
an important role as a mediator of the functions of IL-19 awaits
further comprehensive study. In contrast to IL-19, IL-10 inhibits
production of TNF-, IL-6, cell apoptosis, and ROS production. These
results demonstrate that the activities and functions of IL-19 and
IL-10 are different. IL-10 was used as an anti-inflammatory drug in
clinical trials. We therefore speculate that the antagonist of IL-19
may be the molecule to be applied in clinics, if IL-19 can be
demonstrated to be associated with any immune disorder diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ming-Shi Chang, Graduate Institute of Biochemistry, National Cheng Kung University, College of Medicine, Tainan, Taiwan 70. E-mail address: mschang{at}mail.ncku.edu.tw
3 Abbreviations used in this paper: MDA, melanoma differentiation-associated gene; MDCK, Madin-Darby canine kidney; ROS, reactive oxygen species; SNP, single-nucleotide polymorphism; -gal, -galactosidase; CL, chemiluminescence; PI, propidium iodide.
4 The nucleotide sequences reported in this paper have been submitted to GenBank database. Human IL-19 promoter sequence, AF454433; human IL-19 cDNA, AF453946; mouse IL-19 cDNA, AF453945.
Received for publication March 14, 2002. Accepted for publication August 20, 2002.
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References |
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. 
, Location of
mouse IL-19 introns.
