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,
*
Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, MA 02215; Departments of
Pediatrics and
Laboratory Medicine/Pathology, University of Minnesota, Minneapolis, MN 55455;
§
Laboratoire de Génétique Humaine des Maladies Infectieuses, Faculté de Médecine Necker, Paris, France; and
¶
Department of Adult Oncology, Dana-Farber Cancer Institute, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or proliferate in response to IL-12 despite
the expression of wild-type IL-12Rß1 and IL-12Rß2. The defect in
these functional responses to IL-12 was selective, as cytolytic
activity induced by IL-12 was intact, and lymphocytes were responsive
to stimulation by IL-2. An examination of cytokine signaling revealed
that STAT4 and extracellular regulated kinase 1 (ERK1) activation by
IL-12 was intact, whereas the activation of STAT1, -3, and -5 by IL-12
was lost. This impairment of STAT activation was specific for IL-12, as
STAT activation by IL-2, IL-15, and IFN- was unaffected. These
findings demonstrate that the activation of STAT4 alone is not
sufficient for IL-12-induced IFN- production and proliferation and
suggest that other STATs play a role in these responses to IL-12. While
the etiology of the impaired IL-12 signaling in this patient has not
yet been elucidated, the absence of mutations in IL-12Rß1 or
IL-12Rß2 and the preservation of STAT4 activation raise the
possibility that there may be a mutation in an as yet undiscovered
component of the IL-12 signaling complex that is normally required for
the recruitment and activation of STAT1, -3, and
-5. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-
production, T
and NK cell cytolytic activity, nitric oxide production, and
IFN--dependent antiangiogenesis (2).
The functional response of lymphocytes to IL-12 is dependent on the
expression of a high affinity IL-12 receptor that is comprised of two
low affinity subunits, IL-12Rß1 and IL-12Rß2 (3, 4).
While IL-12 has been shown to activate extracellular regulated kinase 1
(ERK1)3
(5), much of what is known about the intracellular
signaling that follows the binding of IL-12 to its receptor has been
attributed to activation of the Janus kinase (JAK)/STAT pathway
(6). Activation of the JAK/STAT pathway appears to be
central to the functional response of lymphocytes to IL-12 as well as
to a number of other cytokines that have both overlapping and unique
patterns of JAK/STAT utilization. For example, in T cells, IL-12 has
been shown to stimulate the tyrosine phosphorylation of JAK2, TYK2,
STAT1, STAT3, STAT4, and STAT5 (7, 8). IL-2, on the other
hand, activates JAK1, JAK3, STAT1, STAT3, and STAT5 (9),
whereas IFN- activates JAK1, JAK2, and STAT1 (10).
Exactly how these patterns of JAK/STAT activation control lymphocyte
functional responses to distinct cytokines is presently unknown. The
role of individual STATs in the lymphocyte response to select cytokines
has been elucidated using STAT knockout mice. Through this approach, it
has been shown that STAT1 is critical to IFN- responses
(11), while STAT5 plays a role in the proliferative
response of lymphocytes to IL-2 (12). The defective
response to IL-12 observed in lymphocytes from STAT4 knockout mice
(13) indicates that STAT4 is essential for lymphocyte
proliferation, IFN- production, and cytolytic activity mediated by
IL-12.
The severity of the phenotype of STAT4 knockout mice suggests that
STAT4 activation is the dominant mechanism controlling lymphocyte
responses to IL-12. This idea is supported by the observation that
cells reconstituted with the high affinity IL-12Rß1/IL-12Rß2 plus
various STATs are able to activate only STAT4 when stimulated with
IL-12 (14). However, there is now evidence to suggest that
STAT1 and STAT5 activation may also be important modulators of IL-12
responsiveness (8). When T cells activated into Th1 cells
with PHA and IL-12 were compared with T cells activated with PHA alone,
IL-12-induced IFN- production was greater in the Th1 cells. This
enhancement of IFN- production was associated with a large increase
in IL-12-induced STAT1 and STAT5 activation without any change in STAT4
activation. This finding suggested that STAT1 and -5 may be capable of
amplifying certain STAT4-dependent functional responses to IL-12 in T
cells.
Mutations in cytokine signaling pathways in humans have provided
important additional insights into the role that specific signaling
proteins play in the activation of lymphocytes. The majority of
mutations that have been reported involve cytokine receptor subunits,
leading either to deficiencies in receptor expression or abnormalities
in ligand binding (15, 16). For IL-12, mutations in the
IL-12Rß1 gene leading to loss of IL-12Rß1 expression have been
reported in children presenting with infections caused by atypical
mycobacteria and salmonella (17, 18). These patients
resembled those with IFN- receptor deficiencies, but their
infections resulted from diminished IFN- production rather than a
defect in IFN- signaling. Both T and NK cell responses to IL-12 in
these patients were greatly diminished, thus demonstrating the
importance of IL-12Rß1 in lymphocyte IL-12 responsiveness.
Here we report on a young patient with an atypical mycobacterial
infection, recurrent staphylococcal sinusitis, and multiple adverse
drug reactions whose T cells were unable to proliferate or produce
IFN- in response to IL-12 despite intact expression of wild-type
IL-12Rß1 and IL-12Rß2. While IL-12 was able to stimulate the
phosphorylation of STAT4 and ERK1 in her T cells, it was unable to
induce the activation of STAT1, -3, or -5, all of which are activated
by IL-12 in T cells from normal individuals. These findings provide new
insights into how STAT activation affects functional responses to IL-12
and suggests that the defect in this patient involves an as yet
undiscovered component of the IL-12 signaling complex distinct from
IL-12Rß1/IL-12Rß2 and necessary for STAT1, STAT3, and STAT5
activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The patient is a 3-year-old Caucasian girl of Northern European extraction with a history of multiple adverse reactions to various foods and antibiotics who presented at the age of 12 mo with fever and right cervical lymphadenitis. She responded partially to treatment with cephalexin, but 4 mo later had worsening cervical lymphadenitis with a strongly positive Mantoux test (30 x 30 mm erythema, 24 x 24 mm induration). One month later, she had a generalized tonic-clonic seizure, with a normal magnetic resonance imaging of the brain but an abnormal electroencephalogram, showing focal sharp waves in the left parieto-occipital region. She was started on phenytoin, but developed urticaria and a fine maculopapular rash and was changed to gabapentin. Because of her persistent cervical lymphadenitis and positive Mantoux test, she was begun empirically on rifampin and isoniazid, but these were discontinued soon afterward due to the development of a skin rash that was refractory to premedication and attempts at desensitization. She underwent surgical excision of multiple enlarged right cervical lymph nodes, which on histopathologic exam contained necrotizing granulomas. Cultures of the lymph nodes grew out Mycobacterium avium but were negative for Mycobacterium tuberculosis. A test for HIV was negative. The development of rashes to isoniazid, rifampin, and ciprofloxacin precluded the use of these antibiotics to treat the M. avium infection, and she was therefore started on an alternative regimen consisting of ampicillin plus amikacin.
An immune work-up revealed a normal complement of T, NK, and B cells
with normal surface immunophenotype, normal neutrophil chemotaxis and
nitro blue tetrazolium test, normal Ig levels (including IgM, IgA, IgE,
IgG, and IgG subtypes) and isohemagglutinin titers, and normal
proliferative response to mitogens such as PHA, Con A, and pokeweed
mitogen. IL-12 production by PBMC in response to mitogens such as Con A
and PHA was normal, but IFN- production in response to those same
mitogens was diminished compared with that in normal controls.
Her family history (Fig. 1
) was notable
for adverse reactions (fever, hives, Arthus-like local reactions) to
immunizations and antibiotics in all four of her siblings and death
from cardiomyopathy in one of her sisters. The deceased sister also had
a history of arthritis of unknown etiology and Staphylococcus
aureus sepsis. Her two surviving sisters had recurrent episodes of
S. aureus sinusitis. One of her surviving sisters also has
nonatopic asthma and had a history of septic arthritis caused by
Salmonella. Her mother had a history of breast cancer and
nonatopic asthma, while her father has allergic rhinitis. A maternal
aunt had a history of cervical lymphadenitis of unclear etiology (with
strongly positive Mantoux test), numerous adverse reactions to
medications, and recurrent sinusitis requiring multiple sinus
surgeries.
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production by PBMC in response to mitogens and
persistent swelling/drainage from the right cervical region, she was
begun on IFN- (1.5 x 106
U/m2 administered s.c. every other day). Over the
next few weeks her surgical wound stopped draining and healed, and her
lymphadenitis resolved. She has continued on chronic IFN- therapy,
and over the past year she has had no new sinus infections or
lymphadenitis. Both her growth and speech development have normalized,
and she no longer exhibits adverse reactions to various food Ags. In
addition, she has tolerated her childhood immunizations without adverse
reactions and has had normal Ab responses to diphtheria toxin, ProHib,
and Pneumovax. Abs and cytokines
The surface expression of IL-12Rß1 was detected using the
previously described 12Rß.3F12 Ab (19). A mAb
recognizing IL-12Rß2 (20) was provided by Dr. Jerome
Ritz. The Ab to the -chain (p55) of the IL-2R was purchased from
PharMingen (San Diego, CA). An anti-IL-12 Ab (C8.6.2) that is able
to bind IL-12 complexed to its receptor was provided by the
Bioanalytical Sciences Department of Genetics Institute (Cambridge,
MA). MOPC-21 (Sigma, St. Louis, MO) served as a mouse IgG1 negative
control Ab. Biotin-conjugated goat anti-mouse IgG1 and
streptavidin-PE were purchased from Southern Biotechnology
(Birmingham, AL).
The phospho-STAT1 Ab recognizes the tyrosine-phosphorylated forms of
STAT1 and STAT1ß and cross-reacts with the tyrosine-phosphorylated
form of STAT5 (8). The phospho-STAT5 and phospho-STAT3 Abs
specifically recognize the tyrosine-phosphorylated forms of STAT5 and
STAT3, respectively (8, 21). The Abs to STAT1, STAT3, and
STAT4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-phospho-ERK1/ERK2 and anti-ERK1/ERK2 were purchased from New
England Biolabs (Beverly, MA). The anti-phosphotyrosine Ab (4G10)
was a gift from Dr. Thomas Roberts (Dana-Farber Cancer Institute,
Boston, MA). HRP-conjugated rabbit anti-mouse and goat
anti-rabbit Abs were purchased from Calbiochem (San Diego,
CA).
Recombinant human IL-12 (sp. act., 1.7 x
107 U/mg) was provided by Genetics Institute, and
IL-2 (sp. act., 18 x 106 U/mg) was supplied
by Chiron (Emeryville, CA). IL-15 (sp. act., >2 x
106 U/mg) and IL-18 were purchased from Endogen
(Cambridge, MA), IFN- (sp. act., 1 x 107
U/mg) was obtained from R&D Systems (Minneapolis, MN), and IFN-2b
(sp. act., 2 x 108 IU/mg) was provided by
Schering-Plough (Madison, NJ).
Isolation and activation of PBMC
Heparinized blood samples were obtained from the patient and from healthy adult volunteers. The blood draws and immunologic studies involving cells obtained from the patient were performed as part of protocol 9603 M10871, approved by the institutional review board at the University of Minnesota. PBMC were isolated from blood samples through density gradient centrifugation using Histopaque-1077 (Sigma). For T cell activation studies, PBMC were cultured for 96 h in RPMI 1640 containing 15% FCS, 2% L-glutamine, 1% sodium pyruvate, 1% gentamicin, 1% penicillin-streptomycin, 5 µg/ml PHA (Sigma), and 100 U/ml IL-2. After 96 h of activation, cells were routinely >95% CD3 and CD56-.
Proliferation assays and measurement of IFN- production
Following activation with PHA plus IL-2 for 96 h, T cells
were incubated in 96-well U-bottom plates at 3 x
104 cells/well with medium alone or the indicated
concentration of cytokine at 37°C. Proliferation assays consisted of
a 96-h incubation, with 1 µCi of
[3H]thymidine (DuPont-NEN, Boston, MA) added
8 h before harvesting. For IFN- assays, supernatants were
harvested after a 72-h incubation, and the IFN- concentration was
assayed using an ELISA kit (Endogen, Cambridge, MA).
Cytotoxicity assays
A lactate dehydrogenase release assay (CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega, Madison WI) was used for measuring cytotoxicity. Whole PBMC were first incubated overnight in U-bottom 96-well plates with medium alone, 1 nM IL-12, 100 U/ml IL-2, or IL-12 plus IL-2 at 37°C. K562 cells were then added to the PBMC at a 10:1 E:T cell ratio. PBMC were incubated with the K562 targets for 4 h at 37°C, and the supernatants were harvested and assayed for lactate dehydrogenase release.
Immunoprecipitations and Western blots
T cells activated with PHA and IL-2 for 72 h were washed
and recultured for an additional 18 h in starvation medium
consisting of RPMI 1640 and 2.5% FCS before cytokine stimulation.
Activated T cells or unactivated PBMC were stimulated for 20 min at
37°C with medium alone, 1 nM rhIL-12, 100 U/ml IL-2, 10 ng/ml IL-15,
5 nM IL-18, 10 U/ml IFN-2b, or 100 U/ml IFN-. After stimulation,
cells were washed once with ice-cold PBS and then lysed on ice for 20
min in lysis buffer containing 1% Nonidet P-40, 50 mmol/L Tris (pH
8.0), 150 mmol/L NaCl, 2 mmol/L EDTA, 2 µg/ml aprotinin, 100 µg/ml
PMSF, 1 mmol/L sodium orthovanadate, and 1 mmol/L NaF. Aliquots of
whole cell lysates were mixed with reducing sample buffer and boiled,
and proteins were resolved on a polyacrylamide gel. For
immunoprecipitations, a STAT4 Ab was added to the lysates, which were
then incubated overnight at 4°C. Ab-protein complexes were
subsequently immunoprecipitated from the lysates by adding protein
A-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden) and incubating
with rotation at 4°C for 4 h. The beads were washed twice with
ice-cold PBS and then boiled in reducing sample buffer, and
precipitated proteins were resolved on a polyacrylamide gel.
For Western blots, fractionated proteins were transferred from polyacrylamide gels to nitrocellulose membranes by electroblotting, and membranes were then blocked for 30 min in TBS containing 0.1% Tween 20 (Bio-Rad, Hercules, CA) and either 5% BSA or 5% nonfat dried milk. Membranes were then incubated with dilutions of the indicated Abs for 1 h at room temperature, washed with TBST, incubated with either HRP-conjugated rabbit anti-mouse or goat anti-rabbit Abs for 1 h, washed again, and developed using enhanced chemiluminescence (Amersham, Aylesbury, U.K.). When reprobed, membranes were first stripped by incubating in a solution containing 2% SDS, 100 mmol/L 2-ME, and 62.5 mmol/L Tris-HCl, pH 6.7, for 30 min at 65°C.
Immunofluorescence analysis
Cytokine receptor staining was performed with a three-step method using a primary unconjugated Ab specific for the indicated receptor subunit, a biotin-conjugated anti-mouse IgG1 secondary Ab, and streptavidin-PE. An irrelevant unconjugated IgG1 Ab was used as the negative control in these experiments.
To assess IL-12 binding (22), T cells activated with PHA and IL-2 for 72 h were incubated on ice with either medium alone or 10 nM rhIL-12 for 1 h. Cells were then washed, incubated with an anti-IL-12 Ab, washed again, and incubated with a PE-conjugated goat anti-mouse IgG1 Ab. After staining, cells were fixed with 1% formaldehyde and analyzed by flow cytometry.
Sequencing of IL-12R subunits
IL-12Rß1. Extraction of total RNA from 20 x
106 T cells activated for 5 days with PHA and
IL-2, cDNA synthesis, and PCR were performed as previously described
(23). Primers for amplification of the IL-12Rß1 cDNA
coding region were 5'-TGAACCTCGCAGGTGGCAGA-3' (sense) and
5'-TCGGGCGAGTCACTCACCCT-3' (antisense). Sequencing was performed with
an ABI Prism dRhodamine Terminator kit and analyzed with an ABI Prism
377 DNA Sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA). A
series of nested primers was used for sequencing.
IL-12Rß2. RNA was isolated by the TRIzol
method from 20 x 106 T cells activated for
5 days with PHA and IL-2. The purified RNA was reverse transcribed
using oligo(dT)15 and mouse Moloney murine
leukemia virus reverse transcriptase. Ten percent of the RT mix was
used to PCR amplify the cytoplasmic region of IL-12Rß2 using a
reverse oligonucleotide (5'-ATTGGGGCAGGCTGTGCTGGTTGAG-3'; positions
3291–3267), which hybridized to the 3' untranslated region, and a
forward oligonucleotide (5'-GGTGGGCATTTTCTCAACGCATTAC-3';
positions 2551–2575), which hybridized to the transmembrane region.
The same RT mix was used to PCR amplify the extracellular region using
a reverse oligonucleotide (5'-AAGTAATGCGTTGAGAAAATGCCCACC-3';
positions 2578–2551), which hybridized to the 3' transmembrane region,
and a forward oligonucleotide (5'-CGTGGAAGAATACGGAGTTCTATACC-3';
positions 601–626), which hybridized to the 5' untranslated region.
The gel-purified products were sequenced (Tufts University Sequencing
Facility, Medford, MA) using the oligonucleotides above and the
additional sequential overlapping oligonucleotides to complete the
sequence shown in Table I.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production are impaired but
cytolytic activity is preserved in lymphocytes from the patient
To determine whether the diminished ability of PHA to stimulate
IFN- production from the patient’s PBMC was indicative of an
IL-12-based immunodeficiency, we examined the response of her
PHA-activated T cells to IL-12. As shown in Fig. 2 her T cells failed to proliferate or
produce IFN- in response to concentrations of IL-12 ranging from
10–1000 pM. To determine whether this represented a global abnormality
in functional responses to cytokines or was specific for IL-12, we
tested the response of her T cells to IL-2. While IL-2-induced
proliferation was intact (Fig. 2A), IFN- production in
response to IL-2 was greatly diminished (Fig. 2B). A similar
decrease in IL-2-induced IFN- production was observed when activated
T cells from a healthy control were stimulated with IL-2 in the
presence of a neutralizing IL-12 Ab (data not shown).
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production, we examined the
ability of IL-12 and IL-2 to stimulate cytotoxicity against the
NK-sensitive cell line K562. Although the baseline killing of K562 was
less with PBMC from the patient compared with control PBMC, the
augmentation of cytotoxicity by IL-12, IL-2, and IL-12 plus IL-2 was
similar for both patient and controls (Fig. 2
C). Diminished IL-12 responsiveness is not due to alterations in IL-12R subunit expression or primary structure
As the functional response of lymphocytes to IL-12 is dependent on
the expression of high affinity IL-12R, we examined whether the
selective loss of IL-12-induced proliferation and IFN- production in
our patient was due to a deficiency in the expression of the ß1
and/or ß2 subunits of the high affinity IL-12R. Cell surface staining
of PHA-activated T cells with Abs specific for IL-12Rß1 and
IL-12Rß2 revealed that T cells from the patient and control T cells
were comparable with respect to IL-12R subunit expression (Fig. 3A). A similar degree of IL-12
binding to activated T cells was also detected (Fig. 3B).
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IL-12 activates STAT4 in T cells from the patient but fails to activate STAT1, STAT3, or STAT5
As data from STAT4 knockout mice have shown that STAT4 is
necessary for lymphocyte IL-12 responsiveness, we examined whether the
inability of the patient’s activated T cells to proliferate or produce
IFN- in response to IL-12 was due to a loss of STAT4 activation by
IL-12. As shown in Fig. 4, IL-12 was able
to stimulate both the tyrosine and serine phosphorylation
(24) of STAT4 in activated T cells from the patient and
controls. However, although IL-12 could activate STAT1, STAT3, and
STAT5 in T cells from controls (Fig. 5A), it was unable to activate
STAT5 in the patient, and only weakly augmented the activation of STAT1
and STAT3 over the baseline levels (Fig. 5B). This
impairment of STAT1, STAT3, and STAT5 activation by IL-12 was not due
to diminished expression of these STATs (Fig. 5B).
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. As shown in Fig. 5B,
STAT activation by these cytokines was preserved, showing that the
JAK/STAT signaling pathways for IL-2, IL-15, and IFN- were intact in
the patient. In addition, STAT1 activation by IFN- was unaffected
(Fig. 5C). ERK activation by IL-12 is intact in T cells from the patient
While ERK1 activation by IL-12 has been observed in
mitogen-activated T cells, the role that ERK1 plays in mediating IL-12
functional responses remains undefined. In activated T cells from the
patient, ERK1 and ERK2 were expressed at levels comparable to those in
controls (Fig. 6). While the basal amount
of ERK1 phosphorylation was greater in activated T cells from controls
compared with the patient, the increases in ERK1 phosphorylation
stimulated by IL-2 and, to a lesser degree, IL-12 were similar in both
(Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As was observed with lymphocytes from patients with IL-12Rß1
mutations, the ability of IL-12 to stimulate lymphocyte IFN-
production and proliferation was completely abolished in our patient.
However, while IL-12Rß1 mutations also affected the ability of IL-12
to augment lymphocyte cytolytic activity (17), this IL-12
function was preserved in our patient with intact IL-12Rß1 and
IL-12Rß2 expression. This finding suggests that the production of
IFN- by T and NK cells and the resulting activation of macrophages
is more critical to the eradication of intracellular organisms such as
salmonella and mycobacteria than lymphocyte cytolytic activity. The
therapeutic effect of chronic IFN- administration in our patient
further underscores the role of IFN- in the control of mycobacterial
infections (26). In addition, the prevention of further
episodes of staphylococcal sinusitis by IFN- therapy suggests that
IFN- is an important component of immunity directed against
deep-seated infections by bacteria such as S. aureus.
IL-2-induced T cell proliferation and lymphocyte cytolytic activity
were normal in our patient. In contrast, IL-2-induced IFN-
production was impaired, suggesting a possible defect in the regulatory
sequences within the IFN- gene. However, her PBMC were able to
produce IFN- in response to PHA, albeit at a diminished level
compared with controls. Furthermore, a similar, selective defect in
IL-2-induced IFN- production was demonstrated for patients with
IL-12Rß1 mutations (18). This phenomenon probably
reflects the important role that IL-12 costimulation plays in IFN-
induction by IL-2 (27, 28) and suggests that endogenous
IL-12 contributes significantly to the ability of IL-2 to induce
IFN- production in vitro using activated PBMC. This is further
supported by our observation that a neutralizing IL-12 Ab impaired
IL-2-induced IFN- production in assays using activated PBMC from
controls. Therefore, it is not necessary to invoke a defect in the
IFN- gene per se to explain the failure of both IL-12 and IL-2 to
induce its expression. Rather, the decrease in IFN- production by
IL-2 is more likely a reflection of the defect in IL-12
responsiveness.
As STAT4 activation by IL-12 has been shown to be dependent on the
expression of both IL-12Rß1 and IL-12Rß2 (14), it was
not surprising to observe that IL-12 was able to stimulate both the
tyrosine and serine phosphorylation of STAT4 in activated T cells from
our patient. This finding provides further evidence that her
lymphocytes expressed functional high affinity IL-12R that could bind
IL-12. However, what was unexpected was that T cell IFN- production
and proliferation were not induced by IL-12, even though IL-12 was able
to activate STAT4 in T cells. It is unlikely that the function of STAT4
was impaired despite the ability of IL-12 to induce its
phosphorylation, for we also found that STAT4 DNA binding in response
to IL-12 was intact in the patient and have established that the STAT4
cDNA sequence was wild type (data not shown).
Importantly, the loss of T cell functional responses to IL-12 was
associated with the inability of IL-12 to activate STAT1, -3 and -5.
Therefore, while murine models have shown that STAT4 is necessary for
lymphocyte IL-12 responsiveness (13), our data indicate
that STAT4 activation alone may be sufficient for the induction of
cytolytic activity by IL-12 but may not be sufficient for the induction
of IFN- production or proliferation. Rather, the activation of
STAT1, STAT3, and/or STAT5 appears to be required in addition to STAT4
activation for IL-12 to elicit the full array of responses to IL-12. As
weak activation of ERK1 by IL-12 was observed in T cells from both the
patient and controls, it is doubtful that it plays a significant role
in IL-12-induced proliferation and IFN- production. It may, however,
play a role along with STAT4 in IL-12-induced cytolytic activity. Our
observation that the activation of STAT1, -3, and -5 and ERK1 by IL-2
was preserved in our patient is further evidence that a selective
defect in IL-12 signaling was responsible for the observed
immunodeficiency.
Although it appears likely that the loss of activation of STAT1, -3, and -5 by IL-12 was responsible for the impairment of lymphocyte IL-12 responsiveness, we have not yet elucidated the cause of this signaling defect. The history of cervical lymphadenitis and/or recurrent staphylococcal sinusitis in multiple family members raises the possibility that a heritable abnormality in the IL-12 signaling pathway may underlie the clinical phenotype of the patient. While these notable clinical features did not appear to exhibit a Mendelian mode of inheritance, an examination of T cell IL-12 responsiveness and IL-12-induced STAT activation in family members should further aid in determining whether a single genetic mutation may be operative in the susceptibility to infections.
As the cytoplasmic regions of IL-12Rß1 and IL-12Rß2 were intact in
the patient, the inability to activate select STATs is unlikely to have
been caused by an abnormality in STAT recruitment to those receptor
subunits. Furthermore, since the JAK/STAT pathways activated by IFN-
and IFN- were intact (29), both JAK2 and TYK2 must have
been present and functional. This is further supported by the ability
of IL-12 to activate STAT4. It is possible, therefore, that the
abnormality involves an as yet undiscovered component of the IL-12
signaling complex that is necessary for the recruitment and activation
of STAT1, -3, and -5 by IL-12. That such an entity exists is supported
by the finding that reconstitution of the IL-12R with IL-12Rß1 and
IL-12Rß2 renders cells capable of activating only STAT4 in response
to IL-12 (14). The future determination of the etiology of
the defect in IL-12 STAT signaling in this patient should therefore
yield new insights into both the structure of the IL-12R and the
genetic basis for her immunodeficiency.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jared A. Gollob, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, East Campus/Room KS-158, Boston, MA 02215.
3 Abbreviations used in this paper: ERK1, extracellular regulated kinase 1; JAK, Janus kinase.
Received for publication March 24, 2000. Accepted for publication July 13, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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: tyrosine phosphorylation of a 91 kD DNA binding protein. Science 258:1808. and interleukin-4 regulate T cell interleukin-12 responsiveness through the differential modulation of high affinity interleukin-12 receptor expression. Eur. J. Immunol. 27:647.[Medline]
receptor 1 deficiency in a child with tuberculoid bacillus Calmette-Guérin infection and a sibling with clinical tuberculosis. J. Clin. Invest. 100:2658.[Medline]
: evidence for the involvement of ligand-induced tyrosine and serine phosphorylation. J. Immunol. 157:4781.[Abstract]
: a preliminary report. N. Engl. J. Med. 330:1348. production by natural killer cell stimulatory factor: characterization of responder cells and synergy with other inducers. J. Exp. Med. 173:869. and interleukin-12 production by human peripheral blood mononuclear cells. J. Interferon Cytokine Res. 19:407.[Medline]
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T. Yoshimoto, M. Furuhata, S. Kamiya, M. Hisada, H. Miyaji, Y. Magami, K. Yamamoto, H. Fujiwara, and J. Mizuguchi Positive Modulation of IL-12 Signaling by Sphingosine Kinase 2 Associating with the IL-12 Receptor {beta}1 Cytoplasmic Region J. Immunol., August 1, 2003; 171(3): 1352 - 1359. [Abstract] [Full Text] [PDF]
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 H. Jyonouchi, S. Sun, A. Kelly, and F. L. Rimell Effects of Exogenous Interferon Gamma on Patients With Treatment-Resistant Chronic Rhinosinusitis and Dysregulated Interferon {gamma} Production: A Pilot Study Arch Otolaryngol Head Neck Surg, May 1, 2003; 129(5): 563 - 569. [Abstract] [Full Text] [PDF]
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 I. Sugawara, H. Yamada, and S. Mizuno Relative importance of STAT4 in murine tuberculosis J. Med. Microbiol., January 1, 2003; 52(1): 29 - 34. [Abstract] [Full Text] [PDF]
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 M. J. Scott, C. J. Godshall, and W. G. Cheadle Jaks, STATs, Cytokines, and Sepsis Clin. Vaccine Immunol., November 1, 2002; 9(6): 1153 - 1159. [Full Text] [PDF]
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H. Jyonouchi, S. Sun, H. Le, and F. L. Rimell Evidence of Dysregulated Cytokine Production by Sinus Lavage and Peripheral Blood Mononuclear Cells in Patients With Treatment-Resistant Chronic Rhinosinusitis Arch Otolaryngol Head Neck Surg, December 1, 2001; 127(12): 1488 - 1494. [Abstract] [Full Text] [PDF]
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C. J. Godshall, A. B. Lentsch, J. C. Peyton, M. J. Scott, and W. G. Cheadle STAT4 Is Required for Antibacterial Defense but Enhances Mortality during Polymicrobial Sepsis Clin. Vaccine Immunol., November 1, 2001; 8(6): 1044 - 1048. [Abstract] [Full Text] [PDF]
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R. Ortmann, R. Smeltz, G. Yap, A. Sher, and E. M. Shevach A Heritable Defect in IL-12 Signaling in B10.Q/J Mice. I. In Vitro Analysis J. Immunol., May 1, 2001; 166(9): 5712 - 5719. [Abstract] [Full Text] [PDF]
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