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in IFN-
-Induced Killing of Toxoplasma gondii and Salmonella typhimurium Contributes to Selective Susceptibility of Patients with Partial IFN- Receptor 1 Deficiency1
Departments of
* Infectious Diseases and
Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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- or IL-12-mediated
immunity are susceptible to infections with Salmonella
and non-tuberculous mycobacteria, but rarely suffer from infections
with other intracellular pathogens such as Toxoplasma
gondii. Here we describe macrophage and T cell function in
eight individuals with partial IFN- receptor 1 (IFN-R1)
deficiency due to a mutation that results in elevated cell surface
expression of a truncated IFN-R1 receptor that lacks the
intracellular domain. We show that various effector mechanisms
dependent on IFN-R signaling are affected to different extents.
Whereas TNF-
production was normally up-regulated in response to
IFN-, IL-12 production and CD64 up-regulation were strongly reduced,
and IFN--mediated killing of the intracellular pathogens
Salmonella typhimurium and T. gondii was
completely abrogated in patient’s macrophages. Since these patients
suffer selectively from infections with non-tuberculous mycobacteria
and Salmonella, but not T. gondii,
despite sero-immunity in six of eight patients, which indicates
previous contact with this pathogen, we next studied the role of
TNF- as a possible immune compensatory mechanism. IFN--induced
killing of T. gondii appeared to be partially mediated
by TNF-, and addition of TNF- could compensate for the abrogated
killing of T. gondii in the patient’s macrophages. In
contrast, IFN--mediated killing of S. typhimurium
appeared to be independent of TNF-. We propose that the divergent
role of TNF- in IFN--induced killing of T. gondii
and S. typhimurium may at least partially explain the
highly selective susceptibility of patients. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by NK cells and T cells. Binding of
IFN- to its receptors on macrophages activates these cells to
display enhanced microbicidal activity against intracellular pathogens.
Functionally active IFN- is a homodimer, which binds to two IFN-
receptor 1
(IFN-R1)3 subunits.
Upon cross-linking of these receptors binding sites for two IFN-R2
subunits are created. Upon formation of this complex Janus kinases 1
and 2 are recruited, resulting in phosphorylation of the IFN-R1
subunit and activation of Stat1. Phosphorylated Stat1 forms dimers that
translocate to the nucleus and induces the tran scription of genes involved in the activation of macrophage effector function (reviewed in Ref. 1).
Patients with defects in IFN-- or IL-12-mediated immunity are highly
susceptible to infections with non-tuberculous mycobacteria of low
virulence and Salmonella, and several mutations in genes
encoding receptors involved in type 1 responses have been described
(reviewed in Refs. 2 and 3). For instance,
patients with IL-12 (4) or IL-12R deficiency (5, 6) develop
disseminated infections with normally nonpathogenic
mycobacteria. A more severe immunodeficiency results from complete
IFN-R deficiency; children with complete absence of IFN-
responsiveness often succumb to mycobacterial or Salmonella
infection very early in life (7, 8, 9, 10). Patients with
partial IFN- deficiency are less severely affected (11, 12). The majority of these patients express an altered IFN-R1
chain on their cell surface (12). This dominant negative
mutation is the result of a frameshift mutation in the gene encoding
the IFN-R1 chain, which causes a premature stop codon and results in
the expression of a truncated receptor that lacks the intracellular
domain. The mutated receptor can bind IFN-, but a functionally
active signaling complex is not formed. Furthermore, the receptor lacks
the recycling domain and accumulates at the cell surface
(13). Mutations resulting in partial IFN-R1 deficiency
were described in 18 patients from 12 unrelated families. All affected
individuals were heterozygous for small deletions in an AT-rich region
of the IFN-R1 gene, which was then defined as a hot spot
for the occurrence of such mutations (12).
Although mycobacteria of low virulence are the most prevalent cause of
infection in patients with defects in type 1 immunity, 
30% of
patients described to date also suffered from Salmonella
infections (2). Infections with other intracellular
pathogens such as T. gondii, Legionella, and
Listeria are rare. Even though it is expected that a large
proportion of patients with defects in type 1 immunity have been
exposed to the intracellular pathogen T. gondii, active
T. gondii infection has never been observed in these
patients. This suggest that the dependence on type 1 immunity is more
stringent for mycobacteria and Salmonella than for T.
gondii, posing the intriguing question of why the infection
susceptibility of these patients is so selective.
We therefore studied macrophage function of patients from three
unrelated Dutch families who presented with disseminated mycobacterial
and Salmonella infections, and we identified the defect as
being partial IFN-R1 deficiency. We show that macrophages from these
patients indeed have a highly reduced capacity to kill the
intracellular pathogens Salmonella typhimurium and T.
gondii in response to IFN-. The role of TNF- as a
compensatory immune mechanism was investigated, and a divergent role
for TNF- was shown in the killing of these pathogens, which could
explain the highly selective susceptibility of patients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The three affected kindreds are displayed in Fig. 1
. Detailed case reports highlighting the
clinical aspects have been described previously (14), and
brief reports are presented below.
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Kindred B. The father in kindred B, born in 1949, developed disseminated bacille Calmette-Guérin (BCG) infection after vaccination in 1954. Since then, he did not suffer any infectious diseases. The daughter, born in 1985, presented in 1990 with abdominal pain, fever up to 39.7°C, and weight loss. Surgical biopsy of enlarged abdominal lymph nodes revealed acid fast rods and grew M. avium. The son, born in 1981, presented in 1998 with severe back pain and had a fever up to 39.3°C, night sweats, and 5-kg weight loss over 2 mo. A bone scan showed multiple hot spots in vertebrae, clavicula, femur, and ribs. A subsequent bone biopsy revealed acid fast rods and grew M. avium.
Kindred C. The mother in kindred C, born in 1940, developed facial skin lesions in 1968. A diagnosis of lupus vulgaris was made, and the patient received anti-tuberculous treatment. Since then she has had multiple recurrences of erythematous skin lesions in the face and a generalized lymphadenopathy, both due to M. asiaticum. The daughter, born in 1970, presented in 1999 with pain in the left hip joint and had a low grade fever. A bone scan showed multiple hot spots in vertebrae, left femur, and ribs. A biopsy of the inguinal lymph nodes revealed acid fast rods and grew M. avium.
Isolation and culture of human monocyte-derived macrophages
Human monocytes were isolated from heparinized blood using density centrifugation over a Ficoll-Hypaque gradient (Pharmacia Biotech, Uppsala, Sweden) and were cultured in DMEM (Life Technologies, Paisley, Scotland) supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (0.1 mg/ml), and 10% heat-inactivated pooled human serum. Cells were adhered onto plastic coverslips (Thermanox, Nunc, Naperville, IL) in 24-well tissue culture plates (Costar, Cambridge, MA) for 2 h. Nonadherent cells were removed by extensive washing, and cells were cultured for 6–10 days to obtain monocyte-derived macrophages. Any remaining nonadherent cells were washed away after this culture period, and relatively pure (>95%) monocyte-derived macrophage populations were obtained.
DNA manipulations
Genomic DNA and RNA was isolated from whole blood, and the
region of the IFN-R1 gene containing the hot spot for
mutation was amplified by PCR. PCR products were digested with
VspI and analyzed on a 12% acrylamide gel. Nucleotide
sequence analysis was performed using a genetic analyzer (Beckman,
Fullerton, CA).
Stimulation of whole blood
Blood collected in endotoxin-free tubes (Endotube ET,
Chromogenix, Molndal, Sweden) was diluted 1/10 and was stimulated
overnight with 100 ng/ml LPS in the presence or the absence of 500 IU
IFN-/ml. Supernatants were tested by ELISA for the presence of IL-12
p70 (R&D Systems, Minneapolis, MN) and TNF- (Central Laboratory of
The Netherlands Red Cross Blood Transfusion Service, Amsterdam, The
Netherlands).
T cell stimulation
PBMC were stimulated with anti-CD2 (5 µg/ml) and
anti-CD28 (5 µg/ml) (Central Laboratory of the Netherlands Red
Cross Blood Transfusion Service) for 3 days in IMDM (Life Technologies)
supplemented with 10% pooled, heat-inactivated normal human serum, 100
IU/ml penicillin, and 100 mg/ml streptomycin. The cultures were
incubated for an additional 16 h in the presence of
[3H]thymidine (0.5 µCi/well). Cells were
harvested, and incorporated radioactivity was measured by liquid
scintillation counting. Supernatants were collected from parallel
cultures and tested for the presence of IFN- by ELISA (UcyTech;
University of Utrecht, Utrecht, The Netherlands). Ag-specific T cell
proliferation and IFN- production in response to purified protein
derivative (PPD; 5 µg/ml) and M. avium sonicate (10
µg/ml) were measured after 6 days of culture.
FACS analysis
Fresh PBMC were washed with PBS containing 0.2% BSA and
incubated with mAbs directed to IFN-R1 (Genzyme, Cambridge, MA) or
CD64 in PBS/BSA. After washing, cells were incubated with
FITC-conjugated goat-anti-mouse F(ab)2. For each
sample 10,000 cells were analyzed using a FACScan (BD Biosciences,
Mountain View, CA). Surface expression of IFN-R1 was quantified
using the DAKO Quifikit (DAKO, Glostrup, Denmark) according to
the manufacterer’s instructions.
Stat1 binding assay
After stimulation of PMN with increasing amounts of IFN-
(0–100 U/ml) for 20 min, cellular extracts were prepared. Protein-DNA
complexes were detected by EMSA. Ten micrograms of extract was
incubated for 30 min at 4°C in a 10 mM HEPES buffer containing 60 mM
KCl, 1 mM EDTA, 1 mM DTT, 10 mM
Na3PO4, 10% glycerol, 1
µg poly(dI-dC), 0.5 µg sonicated herring sperm ssDNA, and 1 ng
32P-radiolabeled dsDNA probe corresponding to the
IFN- response region (5'-CATGTTATGCATATTCCTGTAAGT-3'; Santa Cruz
Biotechnology, Santa Cruz, CA). Supershift experiments were performed
by incubating formed complexes with Stat1-specific Ab E23 (Santa Cruz
Biotechnology) for 60 min on ice. Samples were separated by
electrophoresis on a 6% nondenaturing polyacrylamide gel. Gels were
fixed with 10% methanol and 10% acetic, dried onto Whatman 3M paper
(Clifton, NJ), and exposed to x-ray film.
Intracellular killing of S. enteritides serovar typhimurium (S. typhimurium) by monocyte-derived macrophages
S. typhimurium strain 14028S was cultured in
Luria-Bertoni medium at 37°C. Infection of macrophages was performed
as described by Verjans et al. (15) with some minor
adaptations. Briefly, 18 h before infection macrophages were put
onto DMEM supplemented with L-glutamine and 10%
FCS. IFN- (1–1000 U/ml) and anti-TNF- (5 µg/ml) were added
as indicated. Cells were infected at a multiplicity of infection of
10:1. After spinning the bacteria onto the cells for 10 min,
infection was continued for 30 min. After extensive washing,
extracellular bacteria were killed by incubation with 100 µg/ml
gentamicin, followed by three additional washes. Subsequently cells
were either lysed with H2O for determination of
CFU or maintained in medium supplemented with 10 µg/ml gentamicin in
the presence or the absence of IFN- and/or anti-TNF- and
lysed after 18 h. Serial dilutions were plated on Luria-Bertoni
plates to determine CFU.
Culture of T. gondii and intracellular killing of T. gondii by monocyte-derived macrophages
The virulent RH strain of T. gondii (16)
was grown by serial passage through CBA mice. Infections were performed
as described previously with minor modifications (17).
Briefly, 18 h after addition of IFN- (1–1000 IU/ml),
anti-TNF- (5 µg/ml), and TNF-, 5 x
105 T. gondii tachyzoites were added
to 1 x 105 adherent macrophages. After
incubation for 30 min at 37°C, extracellular tachyzoites were removed
by washing with prewarmed culture medium. Macrophages containing
ingested tachyzoites were incubated for another 20 h in the
presence or the absence of IFN-, anti-TNF-, and TNF-.
Immediately after infection (time zero) and 20 h after infection,
cells were fixed with methanol and stained with Giemsa, and the
percentage of positive cells, i.e., macrophages containing at least one
tachyzoite, and the number of tachyzoites per positive cell were
assessed by light microscopy. The intracellular replication of T.
gondii was expressed as the fold increase, which is the number of
tachyzoites per positive cell at 20 h divided by that at time
zero.
Patient material
PBMC were isolated from 40 ml heparinized patient blood. The
total number of monocytes present in the resulting PBMC suspension was
generally 0.6–1 x 107. For a standard CD64
up-regulation assay 2.5 x 106 monocytes
were used. For T. gondii and S. typhimurium
proliferation assays 2.5 x 106 and
5 x 106 monocyte-derived macrophages were
required, respectively. Stat1 activation assays were performed with
PMN, which also express the IFN- receptors at low levels, because
for each assay 4 x 107 cells were required,
and the use of monocytes was therefore not possible.
Statistics
Statistical analysis was performed using the Mann-Whitney test, and p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R1 internalization in patients’ monocytes
All patients displayed enhanced expression of the IFN-R1 (Fig. 2) as described previously for patients
with partial IFN-R1 deficiency (12). Sequence analysis
showed that eight patients from three unrelated families were all
heterozygous for the previously described 818del4 mutation (12, 14). Jouanguy et al. (12) postulated that
this mutation results in overexpression of the mutated receptor on the
cell surface due to the fact that it lacks the recycling domain.
Therefore, we measured receptor internalization after stimulation with
saturating amounts of IFN-. Incubation of control monocytes at
37°C with IFN- (500 U/ml) resulted in a 25–30% reduction of
IFN-R1 on the cell surface, as measured by staining with a
nonneutralizing Ab to IFN-R1. In patient monocytes only 3% of the
receptor was internalized (Fig. 2C). At 0°C no
significant amount of receptor was internalized in patients and
controls under these conditions. Together these data indicate that in
patients IFN- binding to the truncated receptor does not result in
receptor internalization, leading to a 5-fold greater surface
expression.
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responsiveness is reduced in patients’ monocytes
To investigate the IFN- responsiveness of patients, monocytes
were cultured in the presence of 1, 10, or 100 U/ml IFN-, and the
up-regulation of CD64 (FcR1) was measured by FACS analysis. The CD64
promoter contains a Stat1 binding site, and up-regulation of this cell
surface maker in response to IFN- stimulation is therefore a good
marker for the IFN- responsiveness of cells. Stimulation of control
monocytes with IFN- resulted in a dose-dependent increase in CD64
expression. Monocytes from patients showed a markedly decreased
responsiveness to IFN-. The concentration of IFN- required to
reach a similar level of CD64 up-regulation was 10-fold higher in
patients than in controls (Fig. 3A). For comparison, a patient
with complete IFN-R1 deficiency did not show any up-regulation of
CD64 in response to IFN- (data not shown). Reduced CD64
up-regulation was accompanied by reduced Stat1 activation. Nuclear
extracts of IFN--stimulated (1–100 U/ml) PMNs were added to a Stat1
probe representing part of the CD64 promoter. In controls, mobility
shifts were observed when as little as 1–10 U/ml IFN- was used to
stimulate PMNs (Fig. 3B). In patients, however, 10–100 U/ml IFN-
was required to observe Stat1 phosphorylation and dimerization (Fig. 3B). Total Stat1 levels, as determined by Western blot, were
similar in controls and patients (data not shown).
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responsiveness was confirmed when IL-12p40 and
IL-12p70 production of whole blood in response to LPS alone or LPS and
IFN- was measured. IFN--induced IL-12 production was 4-fold
decreased in patients compared with controls (Fig. 4). Remarkably, TNF- production was
not different in patients and controls. Results were similar when 10
U/ml IFN- was used for stimulation. Apparently, various IFN-
effector mechanisms have different thresholds for activation and are
therefore affected to different extents in patients.
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To assess whether T cell function was also affected in patients, T
cells were stimulated with a combination of anti-CD2 and
anti-CD28. Both proliferation and IFN- production were
comparable in patients and controls, although levels of IFN-
production were variable (Fig. 5).
Ag-specific T cell responses to PPD and M. avium sonicate
were measured for two patients (patients A and B) and two
BCG-vaccinated controls. Both patients and controls responded to the
two Ag preparations (Fig. 5, C and D), although
IFN- production in controls in response to PPD was higher than that
in patients. This is probably a reflection of the fact that both
controls were BCG vaccinated, whereas patients suffered from M.
avium infections, and the responses measured are only partly due
to the presence of cross-reactive Ags. In addition, patients showed
positive delayed-type hypersensitivity responses to PPD of M.
tuberculosis and other atypical mycobacterial extracts. For
instance, patient A had an induration of 40 mm to PPD, 58 mm to
M. avium, 12 mm to M. kansassii, and 28 mm to
M. scrofulacaeum. Taken together these data indicate that T
cell function in response to a polyclonal stimulus and to mycobacterial
Ags is normal in patients.
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-induced killing of S. typhimurium and
T. gondii
The experiments described above show that IFN- responsiveness
in patients is affected to a different extent depending on the effector
mechanism of IFN- measured. To study whether decreased IFN-
responsiveness also leads to differential defects in killing of
intracellular pathogens that might correlate with the clinical
syndromes in type 1 cytokine receptor deficiency, we chose to compare
IFN--induced killing of S. tyhimurium, a pathogen to
which patients with defects in type 1 immunity are susceptible
(12), and T. gondii, a pathogen to which they
are apparently not susceptible despite documented seroconversion
(14). Although the patients described here did not suffer
from severe Salmonella infection, Salmonella
infections have been described in patients with exactly the same
mutation (12). In addition, the mother of patient B
recalled a severe Salmonella infection in her son’s
childhood, but this could not be confirmed as clinical records from
this period were no longer available. In control macrophages S.
typhimurium or T. gondii multiplied intracellularly
(Fig. 6). Pretreatment of control
macrophages with IFN- inhibited T. gondii multiplication
and completely abrogated S. typhimurium multiplication. In
contrast, both pathogens readily multiplied in macrophages derived from
patients regardless of the presence of IFN- (Fig. 6). This shows
that IFN--mediated S. typhimurium killing and T.
gondii killing are not affected to different extents in patient’s
macrophages. The initial uptake of S. typhiurium and
T. gondii was not different for patients and controls, and
pretreatment of monocyte-derived macrophages with IFN- did not
affect initial uptake (data not shown). Initial uptake of S.
typhimurium, however, did differ up to 10-fold between, but not
within, experiments. Therefore, multiplication of S.
typhimurium is expressed as a percentage.
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-mediated T. gondii killing is partly
mediated by TNF-
The susceptibility of patients with defects in type 1 immunity to
infection with intracellular pathogens is highly selective. This is
also apparent in the eight patients described here. Six of them have
been exposed to T. gondii, as shown by the presence of
specific IgG1 Abs and the absence of IgM Abs, confirmed by a
Sabin-Feldman calibrated in-house immunoblotting assay (performed by L.
van Lieshout, Leiden University Medical Center), but did not suffer
from active infection. In our in vitro analysis no difference was
observed in the capacity of patients’ macrophages to eliminate
S. typhimurium or T. gondii. However, since
patients with defects in type 1 immunity do not develop T.
gondii infection, we were interested to determine whether TNF-
could compensate for the loss of IFN- responsiveness in patients.
First, T. gondii multiplication was studied in control cells
in the presence of both IFN- (100 U/ml) and anti-TNF-. Part
of the IFN- effect (46 ± 13%; n = 5) could be
reversed by the addition of anti-TNF-, indicating that
IFN--mediated T. gondii killing in normal controls is
partly mediated by TNF- (Fig. 7A).
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addition was studied in patients. Whereas
TNF- or IFN- alone did not have any effect on T.
gondii multiplication, addition of their combination resulted in
intermediate inhibition of T. gondii growth (Fig. 7
B). This indicates that when residual IFN-
responsiveness is present, TNF- synergizes to inhibit T.
gondii replication. To confirm this dependency the same experiment
was performed in a patient with complete IFN- unresponsiveness. In
this patient’s macrophages, the addition of IFN- and TNF- did
not have any effect on T. gondii multiplication (Fig. 7B). Thus, residual IFN- responsiveness in patients with
partial IFN-R1 deficiency is sufficient to inhibit T.
gondii multiplication in the presence of TNF-.
IFN--mediated S. typhimurium killing is not
mediated by TNF-
The role of TNF- was also studied in the killing of S.
typhimurium. Monocyte-derived macrophages from healthy controls
were preincubated with IFN- (1000 IU/ml) and/or anti-TNF- and
subsequently infected. IFN- blocked the persistence/replication of
S. typhimurium, but, in contrast to what we observed for
T. gondii replication, neutralization of TNF- had no
effect on the IFN--induced killing of S. typhimurium
(neutralization was 2 ± 2%; n = 4; Fig. 8A). The effectiveness of
anti-TNF- treatment was confirmed by assessing T.
gondii replication in parallel in two of four experiments. For
T. gondii, 30% of the IFN- effect induced by 1000 U/ml
could be neutralized with anti-TNF- (data not shown). This
indicates that there is a divergent role for TNF- in the killing of
T. gondii and S. typhimurium and suggests the
existence of a functionally different effector pathway for the killing
of these pathogens.
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was studied. Since it was difficult to
obtain sufficient monocyte-derived macrophages from patients (see
Materials and Methods), we studied the effect of TNF- in
control cells stimulated with suboptimal amounts of IFN-. When
control cells were stimulated with low amounts of IFN-, S.
typhimurium could still replicate, although replication was
somewhat inhibited. When TNF- was added to this suboptimal amount of
IFN-, no effect on replication was observed (Fig. 8B).
This indicates that in contrast to T. gondii killing,
IFN- induced S. typhimurium killing is not mediated by
TNF-a. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R1 gene. This results in overexpression of a dominant
negative form of the IFN-R1 that will bind IFN-, but lacks the
intracellular domain necessary for signaling. Since these families are
not related to each other or to any of the patients described
previously, our findings support the hypothesis that the region in the
IFN-R1 gene in which these mutations occur is a
mutational hot spot (12). In addition, there is aberrant
receptor internalization upon binding of IFN-, confirming the
explanation for the high receptor expression in these patients as
postulated by Jouanguy et al. (12).
We now show that various effector mechanisms of IFN- signaling are
affected to different extents in these patients. In patients’
monocytes stimulated with IFN-, the increase in TNF- production
was not affected, whereas IL-12 production and CD64-up-regulation were
reduced 4- and 10-fold, respectively. In contrast, the defect results
in complete abrogation of IFN--induced growth inhibition of the
intracellular pathogens S. typhimurium and T.
gondii, indicating that the defect in the patients is due to the
IFN- unresponsiveness of cells of the monocyte/macrophage lineage
and the subsequent inability to activate these cells. This is
consistent with findings in transgenic mice, where the dominant
negative form of the IFN-R1 was selectively expressed either on
cells of the macrophage lineage or on T cells (18). While
the former mouse strain was highly susceptible to T. gondii
and Listeria infection, the latter strain was unaffected.
Thus, in a murine model of partial IFN-R1 deficiency, increased
susceptibility to infections is only attributable to the lack of
macrophage responsiveness to IFN- (13, 18). T cell
function of patients was not affected in our studies. IFN-
production and proliferation in response to polyclonal or Ag-specific
stimulation appear normal, although our studies are not detailed enough
to conclude that the magnitude of the T cell response in patients and
controls is exactly the same. In addition, patients display normal
delayed-type hypersensitivity responses to mycobacterial Ags. These
findings together with the fact that patients with partial IFN-R1
deficiency do not have problems coping with other pathogens suggest
that T cell function is not grossly affected by the mutation and
strengthens the hypothesis that in these patients the functional defect
is restricted to the monocyte/macrophage lineage.
The fact that various IFN- effector mechanisms are affected to
different extents in patients indicates that each effector mechanism
has its own threshold for activation. We show that Stat1
phosphorylation in response to IFN- stimulation is highly reduced in
patients. Although the sensitivities of the various assays to detect
IFN- responsiveness are likely to be different, in whole blood
stimulation of TNF- production is normal in patients, whereas
IL12p40 production is highly reduced. An explanation for these findings
could be that the amount of phosphorylated Stat1 required to induce a
completely activated phenotype is different for each effector
mechanism. Alternatively, our findings in patients could be explained
by proposing that some IFN--activated pathways are induced in a
Stat1-independent manner and as a result have a different threshold for
activation. There is increasing evidence for such Stat1-independent
IFN- signaling pathways (reviewed in Ref. 19), and they
are known to be important in the defense against certain viruses
(20). However, these alternative signaling routes are
still dependent on the IFN-R, as they are not functional in
IFN-R-null macrophages (20). As a result they would
also be hampered in our patients’ macrophages. In addition, two
patients have been described with a dominant negative Stat1 mutation
(21). Both patients suffered from mycobacterial infection,
but not viral infections, which suggests that at least in mycobacterial
infection IFN--mediated Stat1-dependent pathways are crucial.
Patients with defects in type 1 immunity have a selective
susceptibility to infections with non-tuberculous mycobacteria and
Salmonella. The majority of patients described to date have
no problems coping with any other pathogens, although some severe
herpes virus infections have been described (22). The same
holds true for all but one patient with partial IFN-R1 deficiency
described to date (22). Given the mouse models, it is
intriguing that no problems with T. gondii infections have
been reported (23) in any patient with defects in type 1
immunity, even though IFN--induced T. gondii killing is
greatly reduced in our patients macrophages. Six of eight of our
patients have been exposed to T. gondii, as shown by the
presence of specific IgG1 Abs. We therefore reasoned that in T.
gondii infections, immune mechanisms other than IFN--induced
macrophage activation could be involved in eliminating the infection,
whereas infections with mycobacteria and Salmonella are
unique in that they rely so heavily on efficient IFN-
responsiveness. Our findings show that 50% of the IFN--induced
killing of T. gondii can be abrogated by neutralization of
TNF-. In patients with residual IFN- responsiveness a combination
of IFN- and TNF- can inhibit T. gondii proliferation,
and the growth inhibition reached by addition of TNF- and IFN- to
patient macrophages is between 40 and 50% of that in controls. This
shows that TNF- can partially compensate for the defect in IFN-
responsiveness in patients. In S. typhimurium infection, on
the other hand, IFN--induced killing is not mediated by TNF-.
Addition of TNF- to suboptimal amounts of IFN- also failed to
inhibit S. typhimurium survival/replication. Although
studies in murine models have shown that TNF- and TNF-R1 are
important in the host defense against S. typhimurium, it was
recently observed that in these models IFN--induced killing of
S. typhimurium is not affected by neutralization of
exogenous TNF- (24). Mastroeni et al. (25)
also showed that neutralization of TNF- early in an S.
typhimurium infection model had no effect on the microbicidal
activity of the macrophage, although late effects on granuloma
formation were observed. As in our human macrophage system, there is
apparently no synergy between exogenously added IFN- and TNF-.
While IFN- is essential for killing of S. typhimurium,
the IFN- requirement in T. gondii killing appears to be
less stringent, because suboptimal IFN- responsiveness can still
result in growth inhibition when TNF- is present. Interestingly, we
also show that TNF- production in response to LPS and IFN- is
hardly affected in these patients. It is important to note, however,
that only part of the effect of IFN- on T. gondii
replication is mediated by TNF- and that IFN- responsiveness
itself is also essential. When IFN- responses are suboptimal in our
in vitro system, an extra source of TNF- is required to inhibit
T. gondii replication. In vivo, however, other cell types
could provide this extra amount of TNF-. Our data are also
consistent with observations in murine models where killing of T.
gondii is highly dependent on TNF- (17, 26) and
TNF- receptors (27). Taken together, our data indicate
that the different roles for TNF- in IFN--mediated killing of
T. gondii and S. typhimurium might explain the
highly selective susceptibility of patients to certain pathogens.
Even though all patients with partial IFN-R1 deficiency described to
date are more susceptible to non-tuberculous mycobacteria and
Salmonella infections the severity of disease is highly
variable between individual patients. Some of the patients described
previously have even suffered fatal infections (12). Other
patients have suffered one severe infection and remained disease free
for years afterward. For instance, one of our patients suffered severe
BCG infection as a child, but is now in his early fifties and has been
free of serious infections ever since. These differences in clinical
manifestation of the defect could, of course, be attributable to
differences in exposure to mycobacteria and Salmonella among
individual patients, although exposure to these pathogens is expected
to be similar in the same family. Alternatively, these differences may
suggest that there are compensation mechanisms, which in some patients
could partially overcome the defect. We have previously described
residual IL-12 responsiveness in patients with IL-12R
1 deficiency,
which could act as a partial compensation mechanism to produce low
levels of IFN- (28). Better insights into such
compensation mechanisms and their roles in patients with distinct
clinical presentation may provide new clues for treatments of such
patients. The intriguing question of whether immune compensation
mechanisms can be so efficient that some people with defects in type 1
immunity remain disease free for their entire lives remains to be
answered.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jaap T. van Dissel, Department of Infectious Diseases C5-P, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail: j.t.van_dissel{at}lumc.nl
3 Abbreviations used in this paper: IFN-R1, IFN- receptor 1; BCG, bacille Calmette-Guérin; PPD, purified protein derivative.
Received for publication May 7, 2002. Accepted for publication July 29, 2002.
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,
Affected family members. Individuals that were not available for
genetic analysis are indicated with a question mark. Individuals not
available for analysis are indicated with a slash.
) and conjugate
controls (
) were stimulated with
anti-CD2 and anti-CD28, and proliferation (A)
and IFN-
