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Blood, 1 May 2001, Vol. 97, No. 9, pp. 2688-2694
IMMUNOBIOLOGY
Missense mutation of the interleukin-12 receptor  1
chain-encoding gene is associated with impaired immunity against
Mycobacterium avium complex infection
Tatsunori Sakai,
Masao Matsuoka,
Manabu Aoki,
Kisato Nosaka, and
Hiroaki Mitsuya
From the Department of Immunopathophysiology and
Internal Medicine II, Kumamoto University School of Medicine; the
Laboratory of Virus Immunology, Research Center for AIDS, Institute for
Virus Research, Kyoto University, Japan; and the Experimental
Retrovirology Section, Medicine Branch, Division of Clinical Sciences,
National Cancer Institute, Bethesda, MD.
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Abstract |
Interleukin-12 (IL-12) plays an important role in the production of
interferon gamma (IFN- ) and is essential for protection against
intracellular pathogens such as Mycobacterium and
Salmonella. A 31-year-old man had disseminated
Mycobacterium avium complex (MAC) infection. The production
of IFN- by peripheral blood mononuclear cells stimulated with
phytohemagglutinin (PHA-PBMCs) was found severely impaired (40.7 pg/mL
compared with 833 ± 289 pg/mL for the patient's and healthy
subjects' (n = 3) PHA- PBMCs, respectively), and the
patient's PHA-PBMCs completely lacked surface IL-12 receptor  1
(IL-12R1) chain. The IL-12R1 gene transcript in his PHA-PBMCs had an R213W substitution in each allele. Family history showed that both parents were heterozygotes in the R213W substitution. Transfection of a human embryonal kidney cell line 293 (HEKC293) with
wild-type IL-12R1wt gene led to cell surface IL-12R1
expression; however, no expression was seen in HEKC293 transfected with
the mutated IL-12R1R213W gene. The
IL-12R1 gene transcript, but no IL-12R1 protein, was
detected in PHA-PBMCs and T cells, suggesting a post-translational
event(s), most likely a shortened turnover of the protein. The R213W
substitution was not detected in the cells of 32 healthy persons or of
25 patients with tuberculosis or MAC infection. Six amino acid
substitutions (Q214R, M365T, G378R, H438Y, A525T, and G594E) were
identified, but the incidences of such substitutions were not
significantly different between the groups. The Q214R substitution is
reportedly linked to IL-12R1 deficiency; however, the study showed
that 19 and 10 of 57 Japanese and 6 and 4 of 33 healthy white persons
were heterozygous and homozygous for Arg-214, respectively,
suggesting that the Q214R substitution represents a
polymorphism and is not related to IL-12R1 deficiency but that
the R213W substitution is responsible for IL-12R1 deficiency.
(Blood. 2001;97:2688-2694)
© 2001 by The American Society of Hematology.
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Introduction |
Interleukin-12 (IL-12) is a heterodimeric cytokine
composed of 2 disulfide-bound glycoprotein subunits, p35 and
p40.1 It is secreted from macrophages and dendritic cells
and exerts effects on T cells and natural killer cells,2
which, in response to IL-12, produce interferon gamma (IFN-) that
functions to activate macrophages. This cascade is critical to host
defense against intracellular pathogens such as Mycobacteria
and Salmonella. Indeed, an inherited deficiency of the
IFN-R gene presents susceptibility to even attenuated mycobacteria
such as bacille Calmette-Guérin and nontuberculous mycobacterial
infections.3,4
High-affinity receptors for IL-12 (IL-12R) are of heterodimer,
composed of 1 and 2 subunits. IL-12R1 chain is a type 1 transmembrane glycoprotein with a molecular size of approximately 100 kd and a cytoplasmic region shorter than that of IL-12R2 chain. Both
1 and 2 chains are essential to confer high affinity to IL-12 on
the receptor.2 The IL-12R2 chain has a long
intracytoplasmic region, which is necessary for IL-12 signaling through
the Jak/Stat pathway.5,6 Type 1 helper T-cell (Th1) clones
express mRNA for IL-12R2 chain, whereas Th2 clones do not, and the
IL-12R2 chain is expressed on human naive T cells during
differentiation to Th1 but not to Th2.7,8 Therefore, the
IL-12R2 chain is thought to be associated with Th1 development.
Inherited IL-12R1 deficiency has been identified recently in 7 patients with bacille Calmette-Guérin and nontuberculous mycobacterial infections.9,10 IL-12R1 chain deficiency
causes an illness characterized by a selective susceptibility to poorly pathogenic mycobacteria, resembling IFN- receptor 1 (IFN-R1) deficiency, attesting to the importance of IL-12R in the host defense
against intracellular pathogens. In patients with IL-12R1 chain
deficiency, which differs from complete IFN-R1 deficiency, the
formation of granulomatous lesion is observed, and its clinical course
is mild.
In this report, we describe a patient with IL-12R1 deficiency with
disseminated Mycobacterium avium complex (MAC) infection. A
missense mutation in the IL-12R1 chain was identified in this patient and apparently caused rapid proteolysis of IL-12R1,
resulting in null phenotype of the IL-12R1 chain. We believe that
this is the first report of a patient with IL-12R1 deficiency caused by a missense mutation.
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Patients, materials, and methods |
Case description
A 31-year-old man was referred to our hospital because of
persistent generalized lymphadenopathy. The patient was born of a
consanguineous marriage between cousins. On examination, enlarged elastic lymph nodes were palpated in the neck and the axillary and
inguinal regions. Computed abdominal tomography revealed swollen para-aortic lymph nodes as well. A lymph node biopsy from the neck
showed histiocytic granuloma. The gastric juice specimens (stained with
Ziehl-Neelsen basic fuchsin dyes) contained atypical Mycobacterium (Gaffky 5), and the same atypical
Mycobacterium was identified in biopsied neck lymph node
cells. The diagnosis was disseminated MAC infection. The patient was
seronegative for human immunodeficiency virus type 1 (HIV-1);
antibody-dependent cell cytotoxicity activity of his peripheral blood
mononuclear cells (PBMCs) scored 80% (normal range, 41%-72%), and
natural killer cell activity was 49% (18%-40%) assayed as previously
described.11,12 His serum immunoglobulin levels were
within normal ranges. Clarithromycin (400 mg/d), rifampicin (450 mg/d),
and ciprofloxacin hydrochloride (600 mg/d) were administered for 18 months when these drugs were discontinued because lymph node swelling
subsided. After 18 months, the lymph nodes in his neck enlarged again,
antibiotics were resumed, and subcutaneous injection of
5 × 105 U recombinant IFN- 3 times a week was
begun.13
Cells, cell lines, and method of transfection
Peripheral blood mononuclear cells from the patient and healthy
donors were isolated by density gradient centrifugation using Ficoll
Paque (Pharmacia Biotech, Uppsala, Sweden) and cultured with 5 µg/mL
phytohemagglutinin (PHA-P; Sigma, St Louis, MO) for 3 days.
PHA-activated lymphoblasts from the patient were co-cultured with
lethally irradiated (120 Gy) human T-cell leukemia virus type 1 (HTLV-1)-producing MT-2 cells and were propagated in the presence of
IL-2 as previously described.14 From this co-culture, TS-1HTLV-1, an HTLV-1-transformed T-cell line, was established. To
construct a vector carrying the IL-12R1-chain gene, wild-type and
mutated IL-12R1-chain genes were cloned into an expression vector,
pCEP4 (Invitrogen, Carlsbad, CA). A human embryonal kidney cell line,
HEKC293, was transfected with such expression vectors using Superfect
transfect reagent (Qiagen, Hilden, Germany) according to the
manufacturer's instructions. Stable transfectants were selected by
incubation in Dulbecco minimal essential medium-containing 0.5 mg/mL
hygromycin B (Sigma).
Flow cytometric analysis
Flow cytometric analysis was performed using an Epics XL-MCL
(Beckman Coulter, Fullerton, CA) as previously
described.15 In brief, cells (1 × 106) were
incubated with 1 µg anti-IL-12R1 monoclonal antibody (TOS mAb;
Pharmingen, San Diego, CA) or anti-IFN-R1 (Genzyme, Cambridge, MA)
or a control IgG-1 monoclonal antibody (0.516) followed by 50 µL of a 1:100 dilution of fluorescein isothiocyanate (FITC) goat-antimouse IgG (Cosmo Bio, Tokyo, Japan).
Reverse transcription coupled with polymerase chain
reaction
Total RNA was isolated using TRIzol reagent (Gibco BRL,
Gaithersburg, MD) according to the manufacturer's instructions and reverse-transcribed into cDNA using the Superscript II preamplification system (Gibco BRL) with oligo dT. cDNA of the human IL-12R1 (GenBank accession number U03187) and 2 (GenBank accession number U64198) chain genes were then amplified using primers as follows: IL-12R1, sense 5'-TGAACCTCGCAGGTGGCAGA-3' (nucleotides 7-26); IL-12R1 antisense, 5'-TCGGGCGAGTCACTCACCCT-3' (nucleotides 2070-2089); IL-12R2 sense, 5'-GGCGACACGTGGAAGAATAC-3' (nucleotides 594-613); and
IL-12R2 antisense, 5'-AGAGATGACAGCTGCTGGAG-3' (nucleotides 3303-3322). The polymerase chain reaction (PCR) reaction mixture contained 1.5 mM MgCl2, 0.2 µM each primer, 0.1 mM each
dNTP, 2 U LA Taq polymerase (Takara, Kyoto, Japan), and 3 µL reverse transcriptase (RT) reaction mixture. To increase the specificity of PCR
amplification, the hot-start method was performed with AmpliWax PCR Gem
50 (PerkinElmer, Norwalk, CT). Conditions of PCR amplification were as
follows: 95°C for 3 minutes, 35 cycles of 95°C for 60 seconds,
64°C for 60 seconds, 72°C for 120 seconds, and a final extension at
72°C for 4 minutes. PCR reaction was performed with a Robocycler
Gradient 40 (Stratagene, San Diego, CA).
Cloning of RT-PCR products and sequencing
PCR products were cloned into pGEM-T Easy vectors (Promega,
Madison, WI), and their sequences were determined using Big Dye Terminator (Applied Biosystems, Foster City, CA) with ABI 377 autosequencer (PerkinElmer Applied Biosystems). Sequencing primers used
were as follows: for IL-12R1, 5'-TGAACCTCGCAGGTGGCAGA-3' (sense,
nucleotides 7-26), 5'-TCGGGCGAGTCACTCACCCT-3' (antisense, nucleotides
2070-2089); 5'-GATAACCAGGTTGGTGCTGA-3' (sense, nucleotides 551-570),
5'-CGCAGTCGCCCAACTTCCAT-3' (antisense, nucleotides 604-623); 5'-GCCTACAACGTGGCTGTCAT-3' (sense, nucleotides 1001-1020),
5'-ATGCAATACGTCATGCTCTG-3' (antisense, nucleotides 1151-1170);
5'-CACCTGTCCCGGCGTCCTAA-3' (sense, nucleotides 1180-1499),
5'-CTGTTTGCTGTCTTCATCTC-3' (antisense, nucleotides 1521-1540); for
IL-12R2, 5'-GGCGACACGTGGAAGAATAC-3' (sense, nucleotides 594-613),
5'-AGAGATGACAGCTGCTGGAG-3' (antisense, nucleotides 3303-3322);
5'-GTCTGCAAACTGGCCTGTAT-3' (sense, nucleotides 938-957),
5'-TAAGTGGGTGTCTCGTCCTC-3' (antisense, nucleotides 1080-1099); 5'-GCAGGCTCTGGAATATGGTT-3' (sense, nucleotides 1431-1450),
5'-GTGCTCTCAATGATTCACTC-3' (antisense, nucleotides 1564-1583);
5'-GAGGGCATGGACAACATTCT-3' (sense, nucleotides 1934-1953),
5'-ATTGTAGGGTCGACTCCGTA-3' (antisense, nucleotides 2061-2080);
5'-CGAGTGACATATGTCCTGTG-3' (sense, nucleotides 2411-2430),
5'-GCTGGAAGTAATGCGTTGAG-3' (antisense, nucleotides 2563-2582); and
5'-AGCTGAGAGCAGACAACTGG-3' (sense, nucleotides 2911-2930).
Northern blot analysis
Expression of the IL-12R1 chain-encoding gene was examined
using Northern blot analysis as previously described.17 In
brief, total RNA (10 µg each) purified from various cell populations was subjected to 1% agarose-formaldehyde gel electrophoresis, blotted
onto a nylon transfer membrane (Hybond N+; Amersham Pharmacia Biotech,
Buckinghamshire, United Kingdom), and hybridized with a
[32P]dCTP-labeled whole IL-12R1 cDNA probe in a
hybridization solution (0.25 M Na2HPO4, pH 7.2, 7% sodium dodecyl sulfate [SDS]) at 65°C. After thorough washing,
hybridized RNA species were visualized by exposure to x-ray film
(Hyperfilm MP; Amersham Pharmacia Biotech) at  70°C.
Western blot analysis
Cells (107) were washed in phosphate-buffered saline
(PBS) and lysed in 200 µL RIPA buffer (1% NP40, 0.5% sodium
deoxycholate, 0.1% SDS in PBS). Cellular debris was removed by
centrifugation, and the cell extract was kept frozen at 80°C. Total
protein concentration was measured with the Bio-Rad (Hercules, CA)
protein assay. A loading solution (65 mM Tris, pH 6.8, 10% glycerol,
3.8% SDS, 5% 2-mercaptoethanol, 0.003% bromophenol blue) containing
proteins from TS-1HTLV-1, PHA-PBMC, and HEKC293 cells transfected with wild-type or mutated IL-12R1-chain gene was boiled for 5 minutes. Each sample (25 µL) was subjected to SDS-7.5% polyacrylamide gel electrophoresis (90 minutes), and proteins were transferred to the
polyvinylidene difluoride membrane (Millipore, Bedford, MA). The
membrane was blocked overnight in Tris-buffered saline containing 5%
skim milk (Wako, Osaka, Japan), then incubated at room temperature for
1 hour with a goat antibody (polyclonal) against the extracellular domain of human IL-12R1 protein (Genzyme/Techne, Cambridge, MA) or a
rabbit antibody (polyclonal) against the carboxy terminus of human
IL-12R1 protein (Santa Cruz Biotechnology, Santa Cruz, CA) (dilution
1:500). Then the membrane was washed with Tris-buffered saline
containing 0.05% Triton X-100 and incubated with the
peroxidase-conjugated antigoat or antirabbit immunoglobulin (dilution
1:1000; Santa Cruz Biotechnology) for 1 hour. Again the membrane was
washed, and the bands were detected using ECL-Plus (Amersham Pharmacia Biotech, Arlington Heights, IL) Western blotting detection reagents.
Statistical analysis
To examine whether the observed polymorphisms of IL-12R1 were
related to susceptibility to mycobacterial infection, the Fisher exact
probability test was performed. Statistical analysis was carried out
with Statview (Abacus Concepts, Berkeley, CA).
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Results |
Absence of IL-12R1 chains on PBMCs from the patient
Unusual disseminated MAC infection without HIV infection or any
apparent cause implied that a genetic alteration(s) was responsible for
the patient's immunodeficient state. Taking into consideration that
IL-12 and IFN- play major roles in the defense against mycobacterial infections, we first examined whether IL-12R1 chains were present on
PHA-PBMCs from the patient by using an IL-12R1-specific monoclonal antibody and flow cytometry. No IL-12R1 chains were seen on the surfaces of the PHA-PBMCs (Figure 1A),
though they were readily detected on those from healthy subjects
(Figure 1B). We immortalized the patient's T cells with HTLV-1 by
co-culturing with lethally irradiated HTLV-1-producing MT-2 cells in
the presence of IL-2 as previously described.14 The
resultant HTLV-1-transformed T cells (TS-1HTLV-1) were also found to
lack cell surface IL-12R1 chains (Figure 1C). Three unrelated T-cell
populations immortalized by HTLV-1 proved to fully express cell surface
IL-12R1 chains, verifying that immortalization by HTLV-1 per se does
not affect the expression of IL-12R1 chains (data not shown). We
next examined whether the patient's cells bore IFN-R1 on their
surfaces by using an IFN-R1-specific monoclonal antibody and flow
cytometry. As shown in Figure 1D-E, the patient's and a healthy
subject's PBMCs displayed comparable levels of IFN-R1.
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| Figure 1.
Expression of IL-12R1 chains and IFN- receptors in
various cells.
(A-C) PBMCs from the patient and a healthy subject were stimulated with
PHA, cultured for 3 days, washed, and incubated with a monoclonal
antibody against IL-12R1 chain (black) or a control IgG-1 monoclonal
antibody (0.5), stained with an FITC-labeled, goat-antimouse IgG,
and subjected to flow cytometry. The patient's HTLV-1-immortalized T
(TS-1HTLV-1) cells were treated and analyzed similarly. (D-E) PBMCs
from the patient and a healthy subject were stained with an
IFN-R1-specific monoclonal antibody and subjected to flow
cytometry.
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We also asked whether PBMCs from the patient were capable of producing
IFN- because they were stimulated with PHA in vitro. PHA-PBMCs from
all 3 healthy subjects produced substantial amounts of IFN- in vitro
(833 ± 289 pg/mL); however, those from the patient failed to produce
a significant level of IFN- (40.7 pg/mL). These data indicated that
the patient's PBMCs lacked the expression of IL-12R1 chains and had
an impaired ability to produce IFN-.
Genetic changes in the patient's IL-12R1
chain-encoding gene
We next asked whether the IL-12R1-encoding gene in cells from
the patient had a genetic change(s) and determined its entire nucleotide and amino acid sequences. As shown in Figure
2A, a missense mutation (C to T) at
nucleotide position 701, which results in the substitution of
arginine (CGG) with tryptophan (TGG) at amino acid position 213 (designated R213W), was identified. The human IL-12R consists of 2 distinct chains, 1 and 2, and forms high-affinity receptors to
IL-12. It was possible that the lack of cell surface expression of
IL-12R1 chains in cells from the patient was due to a mutation(s) in
the IL-12R2 chain-encoding gene. However, we found no substitutions
in the nucleotide or amino acid sequence of his IL-12R2
chain-encoding gene compared to a consensus sequence.6
Study of the family members of the index patient with respect to the
IL-12R1 chain-encoding gene revealed that both his parents were
heterozygous for R213W, whereas the expression level of IL-12R1
chain on their PHA-PBMCs was within a normal range (Figure 2B). The
patient's sister did not carry this mutation, and all family members
had no episodes of mycobacterial infections. It was possible that the
observed R213W substitution represented a polymorphism in the
IL-12R1 chain-encoding gene in the Japanese population; however,
the R213W substitution was not seen in cells from 32 healthy Japanese
subjects examined.
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| Figure 2.
Missense mutation in the
IL-12R1 chain-encoding gene identified in the patient and his
pedigree.
(A) A missense mutation identified in the patient: an amino acid
substitution from arginine (CGG) to tryptophan (TGG) at
position 213 (designated R213W). (B) Study of family members of the
patient. Family pedigree and electropherograms for nucleotide 701 position (amino acid 213 position) in the IL-12R1 chain-encoding
gene of the index patient, his parents, and sister are shown. Note that
the patient was homozygous for 701T (T/T), whereas both his parents
were heterozygous (C/T) and his sister did not carry R213W
(C/C).
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Altare et al9 have recently reported that certain patients
deficient in IL-12R1 chains and with impaired antimycobacterial immunity had a missense mutation at 214 (Q214R) that was associated with an impaired expression of IL-12R1 chains. However, we observed that, of 32 healthy Japanese subjects examined in this study, 6 and 13 were homozygous and heterozygous for the Q214R substitution, respectively (Table 1). Of 33 healthy
white subjects, 6 and 4 were heterozygous and homozygous for the Q214R
substitution, respectively. These data strongly suggest that the Q214R
substitution represents a polymorphism and is not directly linked to
the impaired cell surface expression of IL-12R1 chains.
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Table 1.
Nucleotide and amino acid substitutions identified in the
IL-12R1 chain-encoding gene in Japanese persons
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R213W substitution failed IL-12R1 chain expression
To determine whether the R213W substitution was directly
responsible for the absence of cell surface IL-12R1 chains, an
expression vector carrying a mutated IL-12R1 chain gene with the
R213W substitution (IL-12R1R213W) was transfected into a human
embryonal kidney cell line 293 (HEKC293) that completely lacked the
cell surface expression of IL-12R1 chains (Figure
3A, broken line). As shown in Figure 3A,
HEKC293 cells, as transfected with an expression vector carrying a
wild-type IL-12R1 chain-encoding gene, successfully expressed
IL-12R1 chains on their surfaces. However, when transfected with
IL-12R1R213W, no HEKC293 cells expressed IL-12R1 chains on their
surfaces (Figure 3B).
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| Figure 3.
Cell surface IL-12R1 chain expression in HEKC293
cells transfected with wild-type and mutated IL-12R1 genes.
An expression vector carrying the wild-type (A) or mutated (B)
IL-12R1 chain-encoding gene was transfected into HEKC293 cells.
Transfected HEKC293 cells were selected with hygromycin, stained with a
monoclonal antibody against IL-12R1 chain (black) or a control IgG-1
monoclonal antibody 0.5 (white), stained with an FITC-labeled,
goat-antimouse IgG, and subjected to flow cytometry.
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Northern and Western blot analyses of IL-12R1 chain
expression
Surface IL-12R1 chain-negative HEKC293 cells (broken line,
Figure 3A) lacked the expression of the IL-12R1 chain mRNA and proteins (Figure 4, lane 1; Figure
5A, lane 3). Unexpectedly, with Northern
blot analysis using a whole IL-12R1 cDNA probe, comparable levels of
transcripts of the IL-12R1 chain gene were detected in HEKC293 cells
transfected with either wild-type or mutated IL-12R1 chain gene
(Figure 4, lanes 2 and 3). In the patient's PHA-PBMCs, IL-12R1
transcripts were detected at distinct but lower levels than in those
from a healthy subject (Figure 4, lanes 4 and 5). However, in
TS-1HTLV-1 cells, which were completely negative for IL-12R1 chains
(Figure 1C), a substantial level of the transcripts was detected
(Figure 4, lane 6).
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| Figure 4.
Presence of transcripts of the IL-12R1 gene in the
patient's cells.
The presence of transcripts of the IL-12R1 chain gene was examined
with Northern blot analysis using a whole IL-12R1 cDNA probe. Lane
1, HEKC293 cells transfected with a vector only; lane 2, HEKC293 cells
transfected with the wild-type IL-12R1 chain gene; lane 3, HEKC293
cells transfected with the mutated IL-12R1 chain gene; lane 4, PHA-PBMCs from the patient; lane 5, PHA-PBMCs from a healthy subject;
lane 6, patient's HTLV-1-immortalized TS1HTLV-1 cells. Lower panel
shows 18S and 28S RNA species visualized by ethidium bromide staining,
confirming that an approximately equal amount of RNA was applied to
each lane. Note that the patient's PHA-PBMCs had IL-12R1
transcripts at a distinct but lower level, but TS-1HTLV-1 cells had a
substantial level of transcripts.
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| Figure 5.
Western blot analysis of the production of IL-12R1
chains.
(A) Cell lysates from various cells were subjected to Western blot
analysis using an antibody against the carboxy terminus of human
IL-12R1 protein (polyclonal), and the production of an IL-12R1
chain protein of approximately 100 kd was examined. Lane 1, HEKC293
transfected with wild-type IL-12R1wt gene; lane 2:
HEKC293 transfected with mutated IL-12R1R213W gene; lane
3, HEKC293 mock transfected with an expression vector only; lane 4, TS-1HTLV-1 cells; lane 5, cells from a healthy subject. The lower band
represents a nonspecific band. (B) Cell lysates were similarly treated,
but a different anti-IL-12R1 antibody (polyclonal) was used to
confirm the authenticity of the 100-kd band visualized. Lane 1, HEKC293
cells transfected with an expression vector carrying the wild-type
IL-12R1wt gene; lane 2, HEKC293 cells transfected with
expression vector carrying a mutated IL-12R1 gene; lane
3, HEKC293 cells transfected with expression vector only (mock); lane
4, TS-1HTLV-1 cells, an HTLV-I-immortalized T-cell line derived from
the index patient; lane 5, PHA-PBMCs from the patient; lane 6, PHA-PBMCs from a healthy subject. Note that the same 100-kd protein
profile was seen in panels A and B.
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We therefore examined whether IL-12R1 chain proteins were detected
in various cells with Western blot analysis. In the lysates of
PHA-PBMCs from a healthy subject and HEKC293 cells transfected with the
wild-type IL-12R1-chain gene, substantial levels of IL-12R1-chain proteins of approximately 100 kd were detected as
either of 2 different IL-12R1 chain-specific monoclonal antibodies was used (Figure 5A-B). However, no such proteins were detected in the
patient's PHA-PBMCs, TS 1HTLV-1 cells, or HEKC293 cells transfected
with IL-12R1R213W (Figure 5). These data strongly suggest that a
post-translational mechanism is at work for the null
IL-12R1-chain phenotype in the index patient.
Polymorphism of IL-12R1-chain gene
Polymorphism of the IL-4R subunit has been reported to be
linked to a gain-of-function mutation and to be associated with atopy.18 It is possible that certain polymorphisms, which
alter the function of IL-12R1 chain, may be associated with
increased susceptibility to mycobacterial infection. Although certain
genetic alterations linked to increased susceptibility to mycobacterial infection have been reported,19 it remains to be
determined whether a polymorphism within the IL-12R1 chain-encoding
gene affects sensitivity to mycobacterial infection. We therefore
determined the nucleotide sequence of the IL-12R1 chain encoding
gene in 32 healthy Japanese subjects, 19 Japanese patients with
pulmonary tuberculosis, and 6 Japanese patients with nontuberculous
mycobacterial infection. Six amino acid substitutions (Q214R, M365T,
G378R, H438Y, A525T, and G594E) were identified, as shown in
Table 1. There were no apparent accumulations in any of the observed
amino acid substitutions, and the incidences of the occurrence of such substitutions were not significantly different among the groups.
As described earlier, Q214R, which was thought to be a candidate
substitution responsible for the IL-12R1-chain
deficiency,9 was frequently found in persons examined in
this study. As shown in Table 1, 19 and 10 of 57 Japanese persons and 6 and 4 of 33 healthy white persons were heterozygous and homozygous for
the Q214R substitution, respectively. Moreover, PHA-PBMCs from 2 healthy subjects, who were homozygous for the Q214R substitution, fully expressed IL-12R1 chains on their surfaces (Figure
6), and those 2 PHA-PBMC preparations
produced IFN- (greater than 1000 and 3180 pg/mL) comparably, as did
PHA-PBMCs from those with wild-type IL-12R1 (833 ± 289 pg/mL;
n = 3). It should also be noted that the R213W substitution was not
found in any of the 90 healthy subjects we examined. Taken together,
the data presented in this study strongly suggest that the R213W
substitution is responsible for IL-12R1 deficiency in the present
patient, but the Q214R substitution represents a polymorphism and is
unlikely to be related to IL-12R1 deficiency.
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| Figure 6.
Expression of IL-12R1 chains in PHA-PBMC from 2 persons carrying the Q214R substitution.
PHA-PBMCs from 2 healthy Japanese persons who were homozygous for the
Q214R substitution were examined for the presence of cell surface
IL-12R1 chains, as described in the legend to Figure 1. Note that
both fully expressed IL-12R1 chains on their surfaces.
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Discussion |
Patients with IFN-R deficiency are highly susceptible to
mycobacterial infection at young ages and most often die
prematurely.3,4 It is noteworthy that the onset of
infection in the patient examined in this study did not occur until he
was 30 years of age, indicating that the production of IFN-
minimally required for protection against mycobacterial infection was
maintained until the age of 30 when mycobacterial infection became
frank. It is possible that an apparently normal level of natural killer
activity seen in this patient had been maintained by this minimally
retained level of IFN-. Indeed, Altare et al9 have also
described that patients with IL-12R1-chain deficiency had a mild
clinical course, as seen in our patient. Jouanguy et al20
recently reported that partial deficiency of IFN-R1, caused by a
missense mutation in its extracellular domain, presumably results in a
mild clinical course compared to that in patients with absolute
IFN-R1 deficiency. In fact, mycobacterial infection in patients with
such mild clinical courses can be well controlled by antibiotics
against such organisms, and IFN- supplementation often improves the
clinical outcome. In those with absolute IFN-R1 deficiency, however,
the formation of granulomatous lesions is absent in lymph nodes,
whereas granulomatous lesions are observed in patients with IL-12R1
chain deficiency, IL-12 deficiency,21 and partial
IFN-R1 deficiency.15 These findings suggest that the
production of even insignificant amounts of IFN- is sufficient for
granuloma formation and for preventing or delaying the contraction of
mycobacterial infection.
In the present patient, dinucleotide CG changed to TG, converting
arginine (CGG) to tryptophan (TGG). The CG dinucleotide is known as a
mutational hot spot that causes approximately one third of all
transition mutations.22 It is thought that cytidine of the
CG dinucleotide is often methylated, and the resultant 5-methylcytidine
is susceptible to spontaneous deamination, which yields thymidine.
Indeed, in a number of genes, including the p53 gene, the methylation
of CG sites is thought to be responsible for
mutations.23-25
The patient in this study did not show any disease susceptibility other
than MAC infection. Considering that patients with IFN-R1
deficiency, IL-12 deficiency, and IL-12R1-chain deficiency are
susceptible to Mycobacterium or Salmonella
infection though they do not contract viral, fungal, or bacterial
(except Salmonella) infections, it appears that IL-12 may
not be required for protection against the latter
pathogens.2,9,10,26,27 IL-12 is a pleiotropic cytokine
implicated in the development of type 1 helper T-cells (Th1), which are
apparently critical for developing and maintaining cell-mediated
immunity to certain pathogens and cancers. In fact, there are no
reports that patients with IL-12R1-chain deficiency experience
distinct viral or malignant diseases. One can pose at least 3 possibilities to explain this selective susceptibility to mycobacterial
infection. First, because the IL-12R2 chain is critical for the
development of Th1 cells, the expression of the IL-12R2 chain
without 1 chain is sufficient for the generation of Th1 cells. It is
argued, however, that the expression of IL-12R1 chain is essential
for the production of IFN- and that a reduction of IFN-
production results in an increased susceptibility to mycobacterial
infection. Second, the production of IFNs other than IFN- or a
minute amount of IFN- is sufficient for protecting a person against
pathogens other than intracellular microorganisms. Third, the
generation of Th1 cells sufficient for protecting a person against
infection from pathogens other than mycobacteria is possible without
IL-12 involvement.
Genetic polymorphism of cytokine receptors has been linked to certain
diseases. For example, polymorphism of the IL-4R subunit has been
shown to be associated with atopy.18 We hypothesized that
genetic predisposition to mycobacterial infection was possible and
examined the IL-12R1 chain-encoding gene in 32 healthy Japanese subjects, 19 Japanese patients with pulmonary tuberculosis, and 6 Japanese patients with nontuberculous mycobacterial infections. Six
amino acid substitutions (Q214R, M365T, G378R, H438Y, A525T, and G594E)
were identified, though the incidences of such substitutions were not
significantly different among the groups (Table 1). Of the 6 amino acid
substitutions, Altare et al9 have reported that the Q214R
substitution was singly responsible for IL-12R1 chain deficiency,
which was not in agreement with our present data. We found that 19 and
10 of 57 Japanese persons were heterozygous and homozygous for Arg-214,
respectively. Because this particular substitution is possibly a
polymorphism unique to the Japanese, we also examined the genotypic
profile in 33 healthy white persons. The results that 6 and 4 of the 33 white persons were similarly heterozygous and homozygous for Arg-214,
respectively, strongly suggested that the Q214R substitution represents
a polymorphism. It is possible that a substitution(s) responsible for
the illness other than Q214R was not identified in those patients
examined by Altare et al9 or that they had other
unidentified abnormality(ies) that caused IL-12R1 deficiency. It is
also possible that the abnormality caused by Q214R was compensated by
other concomitant amino acid substitution(s) in the IL-12R1 encoding
gene. However, among 32 healthy Japanese persons examined, one carried
the Q214R substitution (homozygous) with 2 substitutions (M365T and
G378R; both heterozygous), suggesting that the Q214R substitution is unlikely to be responsible for IL-12R1 deficiency.
By contrast, the R213W substitution identified in the index patient was
not seen in any of the 90 persons examined. Hypothesizing that the
R213W substitution observed in the patient was directly responsible for
the IL-12R1-chain deficiency, we transfected HEKC293 cells lacking
IL-12R1 mRNA or protein expression with either the wild-type or the
mutated IL-12R1 gene. The HEKC293 cells produced IL-12R1 protein
only when transfected with the wild-type gene but not with the mutated
gene transfected (Figure 3), strongly suggesting that the R213W
substitution was directly responsible for the IL-12R1 deficiency. It
was noted, however, that the patient's PHA-PBMC produced a limited but
distinct level of IL-12R1 mRNA and that the patient's T-cells
immortalized with HTLV-1 (TS-1HTLV-1), which do not bear surface
IL-12R1 chains, produced a significant amount of IL-12R1 mRNA.
This feature was corroborated by the observation that the HEKC293 cells
transfected with the mutated gene also produced IL-12R1 mRNA
comparable to that transfected with the wild-type gene (Figure 4, lanes
2 and 3).
These data suggest that the IL-12R1-chain deficiency seen in
the index patient was due mainly to a post-translational event(s), most
likely a shortened turnover of the protein, though an increased IL-12R1 mRNA turnover rate or a decreased IL-12R1 mRNA
translation rate may also be involved. Indeed, rapid intracellular
proteolysis has been implicated in several diseases causing
immunodeficiency, such as X-linked hyper IgM syndrome28
and chronic granulomatous disease.29 For example, in
patients with X-linked chronic granulomatous disease who are deficient
in the subunit of cytochrome b558, gp91phox amino acid
substitutions in the cybb gene have been shown not to affect
its mRNA expression but to cause rapid turnover of gp91phox proteins in
cells, resulting in a lack of NADPH-oxidase activity.29
Taken together, the observed R213W substitution is directly responsible
for the IL-12R1 deficiency seen in the index patient, and this
particular substitution most likely causes an intracellular rapid
protein turnover of the IL-12R1 chain in cells, though the exact
mechanism for such a proposed rapid proteolysis of mutant proteins
remains to be elucidated.
| |
Acknowledgments |
We thank Drs Hiroyuki Nunoi and Sadahiro Tamiya for helpful
discussions and comments.
| |
Footnotes |
Submitted August 7, 2000; accepted December 8, 2000.
Supported in part by a grant-in-aid for Scientific Research from the
Ministry of Education, Science, Sports and Culture of Japan.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Masao Matsuoka, Laboratory of Virus Immunology,
Research Center for AIDS, Institute for Virus Research, Kyoto
University, Kyoto Japan 606-8506, Japan; e-mail:
mmatsuok{at}virus1.virus.kyoto-u.ac.jp.
| |
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Abstract
Introduction