The first family (kindred A) with XR-MSMD has been described elsewhere (1
). None of the family members were vaccinated with BCG. Severe M. avium
infection was documented in four maternally related male family members in two successive generations (; patients II.1, II.4, III.7, and III.8). The clinical features of patient 1 (P1, II.4), the index case, have been described elsewhere (1
). In brief, at the age of 13 yr, P1 presented granulomatous cutaneous lesions, thought to be sarcoid. Extensive erosive lesions on the face and arm were subsequently found to be the result of M. avium
complex infection. Treatment with rifampicin, ethambutol, clofazimine, isoniazid and streptomycin was initiated. After 2.5 yr of therapy, smears and cultures of skin from the patient's face and arm remained positive for M. avium
complex X cluster. Intensive antibiotic and IFN-γ therapy has led to periods in which the skin has healed and cultures have tested negative, but this patient has frequently displayed intermittent mycobacteremia over the last 10 yr. Three other family members also developed disseminated M. avium
complex infection. Patient II.1 was cured of miliary tuberculosis at the age of 6 yr. Disseminated M. avium
infection occurred in this patient at the age of 40 yr, and was never completely eradicated. The patient died of Enterobacter
bacteremia complicating parenteral nutrition at the age of 48 yr. Patient III.7 was successfully treated for disseminated M. avium
infection at the age of 5 yr, but died in an automobile accident at the age of 10. His brother, patient III.8, had recurrent Haemophilus influenzae
bacteremia at the age of 6 yr. At the age of 14 yr, he presented disseminated M. avium
complex infection involving abdominal lymph nodes and blood. Overt signs of ectodermal dysplasia (conical teeth, hypodontia, hypotrichosis, abnormal hair whorl) were evaluated. This patient has remained mycobacteremic despite treatment with multiple antibiotics and IFN-γ. Sparse teeth were the only developmental abnormality on physical examination, and long went unrecognized in patient 1 (, left). Cells of the various blood lineages and lymphoid subsets (CD3, CD4, CD8, CD20, CD16, CD56) were present in normal numbers. T cells proliferated normally in vitro in response to mitogenic (PHA) and antigenic (recall antigens) stimulation. In P1, serum levels of Ig isotypes (IgG, IgM, IgA, IgE, IgD) were normal for age, and there was a normal Ab response to protein and polysaccharide antigens.
The second family (kindred B) included a single patient (P2, II.1), an 8-yr-old boy, born to unrelated parents of Italian and Serbian descent living in France (). At the age of 2 mo, this child was vaccinated with BCG. At the age of 2 yr, he was hospitalized for persistent low-grade fever (38°C) with night sweats, cough, and cervical and inguinal lymphadenopathy. Serum C-reactive protein (CRP) levels (31 mg/l) and erythrocyte sedimentation rate (ESR) were high (32 mm/h), as was leukocyte count, at 15,200 leucocytes/mm3. Chest X-ray and pulmonary function tests were normal. Urine, gastric, and blood cultures for mycobacteria were negative, but the patient had a strongly positive tuberculin skin test (TST) (24-mm induration). The patient was treated with isoniazid and rifampicin for 6 mo, and his condition improved. Approximately 1 yr after the end of treatment, he was hospitalized for cervical lymphadenitis and prolonged fever. The TST was again positive (10 mm). Four lymph nodes were excised and a biopsy revealed granulomas with no visible acid-fast bacilli. Cultures were negative. The patient also had diarrhea, with Salmonella enteritidis identified in the stools, from which he recovered spontaneously. He is now 8 yr old and clinically well with no treatment. Conical decidual incisors were the only developmental abnormality on physical examination, and this abnormality was long unrecognized (, middle). Permanent teeth were normal in shape and number, as shown by clinical examination and mandibular X ray (unpublished data). Cells of the various blood lineages and lymphoid subsets (CD3, CD4, CD8, CD19, CD16, CD56) were present in normal numbers. T cells proliferated normally in vitro in response to mitogenic (PHA) and antigenic (recall antigens) stimulation. The NBT assay and chemioluminescence of PMNs were normal. Serum levels of Ig isotypes (IgG, IgM, IgA, IgE, IgD) were normal for age, and there was a normal Ab response to protein antigens. Serum titers of allo-hemagglutinins were normal (group B). The patient had no detectable antibodies against polysaccharide antigens at the age of 6 yr, when he was immunized with 23-valent nonconjugated pneumococcal vaccine, but mounted a normal response at the age of 7 yr, after two injections of nonconjugated pneumococcal vaccines.
The third family (kindred C) included a single patient (P3, II.1), an 11-yr-old boy, born to unrelated German parents living in Germany (). He was not vaccinated with BCG. At the age of 1 yr, he was hospitalized for a cervical abscess caused by Haemophilus influenzae b, which responded well to surgery and antibiotic treatment. At 9 yr of age, he was hospitalized for persistent low-grade fever (38°C) of unknown origin. He presented splenomegaly, marked hypergammaglobulinemia, and granulocytopenia. After his discharge from the hospital, he suffered recurrent infections, including bronchitis and pneumonitis. At the age of 10 yr and 4 mo, he had a strongly positive TST (20-mm induration), whereas negative results had been obtained for this test on three previous occasions, at the ages of 7, 8, and 9 yr. At the same time, an ELISPOT assay for IFN-γ after ESAT-6 stimulation was negative. Chest X-ray showed no mediastinal lymph node enlargment, but did show infiltration of the lower parts of both lungs. A tentative diagnosis of mycobacterial disease was made, and the patient was treated with isoniazid for 3 mo. This treatment was stopped as it seemed to cause headaches, but the patient was subsequently treated with cefpodoxime prophylaxis. No developmental abnormalities, not even conical teeth, were observed on physical examination (, right). Mandibular X-ray indicated an absence of hypodontia. Cells of the various blood lineages and lymphoid subsets (CD3, CD4, CD8, CD19, CD16, CD56) were present in normal numbers. T cells proliferated normally in vitro in response to mitogenic (PHA) and antigenic (recall antigens) stimulation. The results of NBT and dihydrorhodamine tests were normal. Serum levels of the immunoglobulin IgG, IgM, and IgD isotypes were high (maximum titers: IgG 70.85 g/liter, IgM 1.54 g/liter, and IgD 304 IU/ml). Serum IgA and IgE levels were normal. There was a normal Ab response to protein antigens. There were no allo-hemagglutinins (group A). The patient had detectable, but very low titers of antibodies against polysaccharide antigens at the age of 11 yr, before vaccination. After vaccination with 23-valent nonconjugated vaccine at the age of 11 yr, titers rose and reached normal levels.
Mutations in the five known MSMD-causing autosomal genes (IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1) were excluded in the three kindreds by means of genetic and immunological assays (unpublished data). Other patients with well-defined genetic defects were enrolled in our study, including patients with IL-12p40 deficiency, IL-12Rβ1 deficiency, IFN-γR1 deficiency, CD40 deficiency, and CD40L deficiency. Their genetic lesions are available upon request. Our study was conducted according to the principles expressed in the Helsinki Declaration and was approved by our Institution Review Boards. An informed consent was provided by all patients studied, or by their parents, in the case of children.
Blood cell culture and stimulation.
Whole blood samples were diluted 1/2 in RPMI 1640 (GIBCO BRL) and infected by incubation with live M. bovis BCG (Pasteur substrain), at a multiplicity of infection of 20:1, alone or with recombinant IFN-γ (5,000 IU/ml; Imukin, Boehringer Ingelheim) or recombinant IL-12p70 (20 ng/ml; R&D Systems), LPS (from Salmonella minnesota, 1 μg/ml; Sigma-Aldrich), TNF-α (20 ng/ml; R&D Systems), IL-1β (10 ng/ml; R&D Systems), phorbol 12-myristate 13-acetate (PMA) (10−7 M; Sigma-Aldrich) with ionomycin (10−5 M; Sigma-Aldrich) and supernatants were recovered after 14 and 48 h. Cytokine production was normalized according to the number of PBMCs in the individual tested. PBMCs were obtained from patients and healthy donors by centrifuging heparin-treated blood diluted 1/2 in RPMI 1640 on Ficoll-Paque Plus (GE Healthcare), followed by isolation in mononuclear cell medium. PBMCs were cultured in RPMI 1640 medium (GIBCO BRL) supplemented with 10% heat-inactivated pooled FBS (GIBCO BRL), referred to as complete RPMI 1640. Cells (106 PBMCs/ml/well) were activated by incubation with phytohemagglutinin-P 1/700 (PHA, Bacto PHA-P; Becton Dickinson), alone or in combination with recombinant IFN-γ (5,000 IU/ml; Imukin, Boehringer Ingelheim) or recombinant IL-12p70 (20 ng/ml; R&D System) or IL-23 (20 ng/ml; R&D Systems), anti-CD3 antibody (10 ng/ml, OKT3, OrthoPharmaceutical) and LPS (from Salmonella minnesota, 1 μg/ml; Sigma-Aldrich) for 14 and 48 h.
Culture and stimulation of cell lines.
Fibroblast cell lines were derived from dermal biopsy samples from patients and healthy donors and immortalized by transformation with a plasmid-containing SV40 large T antigen. We added 105 fibroblasts to DMEM (GIBCO BRL) supplemented with 10% (vol/vol) heat-inactivated FBS (GIBCO BRL) in each well of a 24-well plate. Fibroblasts were stimulated with TNF-α (20 ng/ml; R&D Systems), IL-1β (10 ng/ml; R&D Systems), or PMA (10−7 M; Sigma-Aldrich) plus ionomycin (10−5 M; Sigma-Aldrich) and supernatants were recovered after 24 h of activation. B lymphocytes were immortalized with Epstein-Barr virus (EBV-transformed B cells) and cultured in complete RPMI 1640. EBV-transformed B cells were stimulated with imidazoquinolone R-848, an agonist of TLR-7 and TLR-8 (provided by R. Miller, 3M Pharmaceuticals and 3M Innovative Properties Company, St. Paul, MN) for 24 h.
Genomic DNA was isolated by phenol/chloroform extraction from blood cells, which were washed in TE 10:1 buffer (10 mM Tris, 1 mM EDTA, pH 7.6) and lysed in extraction buffer (10 mM Tris, 0.1 M EDTA, 0.5% SDS, and 10 mg/ml proteinase K) overnight at 37°C. RNA was extracted from EBV-transformed B cell lines or SV40-transformed fibroblasts with TRIzol (Invitrogen), according to the kit manufacturer's instructions. We used 1–5 μg total RNA for direct reverse transcription with Oligo-dT (Invitrogen). PCR was performed using Taq polymerase (Invitrogen), and the GeneAmp PCR System 9700 (Applied Biosystems). Primer sequences for the amplification of NEMO exons and cDNA are available upon request. PCR products were purified by centrifugation through Sephadex G-50 Superfine resin (GE Healthcare) and sequenced with the BigDye Terminator Cycle sequencing kit (Applied Biosystems). Sequencing products were purified on Sephadex G-50 Superfine resin and sequences were analyzed on a 3100 ABI Prism Genetic Analizer (Applied Biosystems).
Western blot and EMSA.
For EMSAs, nuclear extracts were prepared from SV40-transformed fibroblasts, after incubation with or without IL-1β (10 ng/ml) and TNF-α (20 ng/ml) for 20, 40, and 60 min. We used the Bio-Rad Laboratories protein assay to adjust protein concentrations such that all samples contained similar amounts of protein. EMSA was performed with a [32P]-labeled NF-κB–specific DNA probe (5′-GATCATGGGGAATCCCCA-3′, 5′-GATCTGGGGAATTCCCCCAT-3′). For Western blotting, total protein was extracted from SV40-transformed fibroblasts and EBV-transformed B cells. Cytosolic extracts were prepared from SV-40–transformed fibroblasts with or without stimulation with IL-1β (10 ng/ml) and TNF-α (20 ng/ml). Protein fractions were separated by reducing SDS-PAGE and electrotransfered onto nitrocellulose membranes (Schleicher & Schuell). Membranes were blocked by incubation in 5% nonfat milk powder (Nestle USA) in 0.05% Tween-20 in PBS for 60 min at room temperature. NEMO was detected using rabbit polyclonal anti-NEMO antibodies (3328 and p18, gifts from G. Courtois, INSERM, Saint Louis Hospital, Paris, France), rabbit antibodies against GAPDH (SC-25778, Santa Cruz Biotechnology, Inc.), rabbit anti-IκBα (C-21, sc-37, Santa Cruz Biotechnology, Inc.), and mouse antibodies against Stat-3 (Santa Cruz Biotechnology, Inc.). Membranes were washed several times in 0.05% Tween-20 in PBS, and antibody binding was detected by incubation with horseradish peroxidase–conjugated anti–rabbit secondary antibodies (GE Healthcare), using the ECL system (GE Healthcare).
Purification of peripheral T cells and monocytes.
For T cell isolation, we depleted peripheral PBMCs of adherent cells and performed negative immunomagnetic depletion using a cocktail of antibodies against CD14, CD16, CD19, CD36, CD56, CD123, and glycophorin A (Macs; Miltenyi Biotec), according to the kit manufacturer's instructions. The preparation was >95% pure, as assessed by flow cytometry with FITC-CD2 and PE-CD3 staining (BD Biosciences). Monocytes were isolated from peripheral PBMCs by negative immunomagnetic depletion, using a cocktail of antibodies against CD3, CD7, CD16, CD19, CD56, CD123, and glycophorin A (Macs; Miltenyi Biotec), according to the kit manufacturer's instructions. Monocytes were >80% pure, as assessed by flow cytometry with PE-CD14 (BD Biosciences) staining.
Coculture of monocytes and T cells.
We plated 105 monocytes per well in complete RPMI 1640 medium in 24-well plates (Nunc) and incubated them for 3 h at 37°C in an atmosphere containing 5% CO2. The nonadherent cells were washed off and the adherent cells were incubated overnight with 400 μl of complete RPMI 1640. The following day, T cells were purified as described in a previous paragraph and 5 × 105 T cells were added to the monocytes. Selected wells were stimulated with a 1/700 dilution of PHA (Bacto PHA-P; Becton Dickinson), alone or in combination with recombinant IFN-γ (5,000 IU/ml; Imukin, Boehringer Ingelheim, France), recombinant IL-12p70 (20 ng/ml; R&D Systems), and recombinant IL-23 (20 ng/ml; R&D Systems). Supernatants were collected 14 and 48 h after activation.
Generation of dendritic cells from purified monocytes.
MDDCs were obtained from purified monocytes as described in a previous paragraph. They were incubated for 2 h in complete RPMI 1640 medium at 37°C under an atmosphere containing 5% CO2 to remove all nonadherent cells, thereby increasing purity. The adherent cells were cultured in complete RPMI 1640 in the presence of GM-CSF (50 ng/ml; R&D Systems) and IL-4 (10 ng/ml; R&D Systems). Optimal conditions were maintained by splitting these cultures every 2 d. Cells were collected on day 7, when the cultures contained >95% immature CD1a+ (BD Biosciences) dendritic cells.
Mouse fibroblastic L-cells transfected with the human ligand of CD40 (L-cell-hCD40L) were used to induce CD40 activation on MDDCs. Nontransfected L-cells were used for control cultures. L-cells were first treated with 10 μg/ml mitomycin (Sigma-Aldrich) in sterile 1 x PBS for 2 h at 37°C in 5% CO2
. L-cells were washed five to seven times in 1 x PBS to remove all trace of mitomycin. We then plated 104
L-cells per well in 24-well plates and incubated them for 2 d in complete RPMI 1640 at 37°C in an atmosphere containing 5% CO2
. We incubated 2 × 105
MDDCs in complete RPMI 1640 with mitomycin-treated L-cells and recovered cell-free supernatants for cytokine analysis and MDDCs for cell surface phenotyping of CD40, CD80, and CD86 (BD Biosciences) by flow cytometry 24 h later. CD40 was activated on B cells with a mixture of soluble recombinant CD40L (500 ng/ml; Amgen) and IL-4 (100 IU/ml; R&D Systems). In brief, PBMCs were cultured for 5 d for proliferation (assessed by [3
H]thymidine incorporation) assays and 12 d for the measurement of IgE production (assessed by ELISA) as previously described (43
For the staining of NEMO in EBV-transformed B cells, fibroblastic cell lines, and highly purified monocytes and T cells isolated from PBMCs, we fixed 2 × 105 cells with 4% paraformaldehyde (PFA, Fluka) in 1 x PBS and permeabilized the cells by incubation in 0.1% saponin (Sigma-Aldrich) in PBS. The cells were incubated with anti-NEMO (611306; BD Biosciences) antibody and the corresponding mouse IgG1 isotype (BD Biosciences), and primary antibody binding was detected by incubation with Alexa Fluor 488–labeled anti–mouse antibodies (Invitrogen). Cell surface phenotyping was performed by flow cytometry, in MDDCs incubated with L-cell or L-cell-hCD40L, using directly labeled antibodies. Dual-color fluorescence assays were performed with PE-CD1a (BD Biosciences) in combination with FITC-CD40, FITC-CD80, and FITC-CD86 (all from BD Biosciences). The respective isotype controls were performed with mouse PE-IgG2k (BD Biosciences) and mouse FITC-IgG1k (BD Biosciences). Fluorescence was analyzed with a FACScan apparatus (Becton Dickinson), using CELLQuest software.
Cytokine concentrations in supernatants were measured by two-sided sandwich ELISA, according to the kit manufacturer's instructions: IL-12p40, IL-12p70, G-CSF (R&D Systems), and TNF-α, IFN-γ, IL-10, IL-6, IL-8 (Sanquin). Multiplex cytokine assays were also performed on culture supernatants, using a cocktail of 17 cytokines: IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17, TNF-α, IFN-γ, GM-CSF, G-CSF, MCP-1, and MIP-1β (Bio-Plex Suspension Array System).
Structural modeling of the NEMO oligomerization domain.
The NEMO sequence encompassing the CC2-LZ region is not >30% of identity to any reference protein in the Protein Data Bank. We therefore used experimental data to search for compatible structure references. The NEMO oligomerization domain forms a globular trimer by equilibrium sedimentation with a hydrodynamic radius of 26.1 Å, as shown by gel filtration (30
) and dynamic light scattering (unpublished data), and the CC2 and LZ coiled-coil subdomains interact by fluorescence polarization (30
). Limited proteolysis in combination with mass spectrometry identified a trypsin-sensitive site at residue Lys302, coinciding with the loop connecting the CC2 and LZ coiled-coils (unpublished data). Finally, the CD spectrum of the trimer showed it to contain 92% α-helix at 277 K (unpublished data). Based on these data, we used the HIV-1 gp41 ectodomain (PDB no. 1F23; reference 31
) as a structure reference to provide atomic coordinates. The trimeric CC2-LZ domain of NEMO was modeled using the Insight II program (Accelrys, Inc.). The model was constructed by manually docking the Cα backbone of the CC2 coiled-coil (residues 260–292) with that of the LZ coiled-coil (residues 306–344) in an antiparallel manner. During docking, we looked for unfavorable and favorable contacts between the CC2 and LZ atoms, by calculating electrostatic and van der Waals interaction energies, using the Docking module of Insight II. The resulting energy-minimized model is shown in .
MDDCs were allowed to differentiate and were activated by CD40L-expressing fibroblasts, as previously described. MDDCs were immediately fixed upon activation, by incubation in 4% formaldehyde/PBS at 4°C for 20 min, followed by surface staining with mouse anti–huamn CD1a (BD Biosciences) at 4°C for 15 min. Cells were washed in PBS and incubated with goat Cy3-conjugated anti–mouse IgG antibody (Zymed Laboratories). For intracellular staining, MDDCs were permeabilized by incubation in 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 15 min. Cells were washed twice in PBS, blocked by incubation with FcR-blocking reagent (Macs; Miltenyi Biotec) for 20 min and washed once with PBS. MDDCs were incubated with 1 μg primary antibody/PBS, rabbit anti-p65 (C20, Santa Cruz Biotechnology, Inc.) and rabbit anti–c-Rel (C; Santa Cruz Biotechnology, Inc.) at 4°C for 30 min and were washed with PBS. Cells were incubated with Alexa 488–conjugated goat anti–rabbit IgG antibody (1:300 dilution) (Invitrogen) and with 4,6-diamidino-2-phenylindole (DAPI) (1/10,000 dilution; Invitrogen) for 15 min. They were washed at least three times with PBS. MDDCs were resuspended in 100 μl of PBS and plated on lysine-coated slides (VWR), using the Cytospin system. The coverslips were mounted on glass slides with Mowiol. Slides were viewed under an LSM 510 confocal microscope.