Normal expression of IFN-gammaR in four patients with uncommon mycobacterial infection phenotypes

Abstract

Several primary immunodeficiency diseases affecting the interleukin 12/interferon gamma (IFN-gamma) pathway have been identified, most of them characterized by recurrent and protracted infections produced by intracellular microorganisms, particularly by several species of mycobacteria. In the present study we analyzed the expression of IFN-gamma receptor (IFN-gammaR) and signal transducer and activator of transcription 1 (STAT-1) in 4 children with Mycobacterium tuberculosis infection of uncommon clinical presentation. These molecules were evaluated by flow cytometry and Western blotting in B cells transformed with Epstein-Barr virus and mutations were scanned by single-strand conformational polymorphisms and DNA sequencing. The expression of IFN-gammaR1 was normal in all 4 patients. The genetic analysis of IFN-gammaR1 and IFN-gammaR2 coding sequences did not reveal any mutation. The expression of the STAT-1 molecule was similar in patients and healthy controls; however, when the phosphorylation of this transcription factor in response to IFN-gamma activation was evaluated by Western blot, a significant lower signal was evident in one patient. These data indicate that there are no alterations in the expression or function of the IFN-gammaR chains in these patients. However, the low level of STAT-1 phosphorylation found in one of these patients might be explained by a defect in one of the molecules involved in the signal transduction pathway after IFN-gamma interacts with its receptor. In the other three patients the inability to eliminate the mycobacteria may be due to a defect in another effector mechanism of the mononuclear phagocytes.

Mycobacterial disease; IFN-gamma; IFN-gamma receptor; STAT-1; SSCP-PCR

Braz J Med Biol Res, September 2004, Volume 37(9) 1353-1363

Normal expression of IFN- g R in four patients with uncommon mycobacterial infection phenotypes

M.T. Rugeles^1,2, B. Rincón¹, C. Rugeles¹, C.J. Montoya¹, M. Hernández¹, C. Estrada¹, M.M. Olivares¹ and P.J. Patiño¹

¹Grupos de Inmunodeficiencias Primarias and ²Inmunovirologia, Facultad de Medicina, Corporación Biogénesis, Universidad de Antioquia, Medellín, Colombia

^{Correspondence and Footnotes}

Abstract

Several primary immunodeficiency diseases affecting the interleukin 12/interferon gamma (IFN-g) pathway have been identified, most of them characterized by recurrent and protracted infections produced by intracellular microorganisms, particularly by several species of mycobacteria. In the present study we analyzed the expression of IFN-g receptor (IFN-gR) and signal transducer and activator of transcription 1 (STAT-1) in 4 children with Mycobacterium tuberculosis infection of uncommon clinical presentation. These molecules were evaluated by flow cytometry and Western blotting in B cells transformed with Epstein-Barr virus and mutations were scanned by single-strand conformational polymorphisms and DNA sequencing. The expression of IFN-gR1 was normal in all 4 patients. The genetic analysis of IFN-gR1 and IFN-gR2 coding sequences did not reveal any mutation. The expression of the STAT-1 molecule was similar in patients and healthy controls; however, when the phosphorylation of this transcription factor in response to IFN-g activation was evaluated by Western blot, a significant lower signal was evident in one patient. These data indicate that there are no alterations in the expression or function of the IFN-gR chains in these patients. However, the low level of STAT-1 phosphorylation found in one of these patients might be explained by a defect in one of the molecules involved in the signal transduction pathway after IFN-g interacts with its receptor. In the other three patients the inability to eliminate the mycobacteria may be due to a defect in another effector mechanism of the mononuclear phagocytes.

Key words: Mycobacterial disease, IFN-g, IFN-g receptor, STAT-1, SSCP-PCR

Introduction

The protective immune response against intracellular microorganisms such as mycobacteria, salmonella and leishmania depends on immune mechanisms mediated by different cells. Perhaps the most relevant mechanism is the activation of mononuclear phagocytes by cytokines, particularly by interferon gamma (IFN-g). This cytokine is produced as a consequence of antigenic activation by natural killer (NK) and T cells, especially TH1, and its production is increased by antigen-presenting cell-derived cytokines such as interleukin 12 (IL-12) and 18 (IL-18). IFN-g has regulatory effects on the innate and adaptive immune response, which include activation of mononuclear phagocytes and NK cells, increased expression of major histocompatibility complex I and II, inhibition of TH2 cell differentiation, induction of CD8+ lymphocyte maturation into cytotoxic cells, and promotion of the immunoglobulin isotype switch in B cells towards immunoglobulin G1 (IgG1) and IgG3 (1-5).

The IFN-g receptor (IFN-gR) is present in almost every human cell and is composed by two different integral membrane proteins: IFN-gR1 (a chain or CD119w), a 90-kDa protein that binds IFN-g with high affinity, and IFN-gR2 (ß chain or accessory factor 1), a 62-kDa molecule that has a less active role in binding IFN-g but that is essential for the signal transduction triggered by IFN-g. These proteins are encoded by genes located in chromosome 6 and chromosome 21, respectively. Before activation, IFN-gR1 and IFN-gR2 are separated in the plasma membrane; however, when the IFN-g homodimer binds two IFN-gR1 molecules, two IFN-gR2 molecules interact with the IFN-g/IFN-gR1 complex (1,5-9).

Two tyrosine kinases called Janus kinase 1 and 2 (JAK1 and JAK2) are bound to resting IFN-gR. JAK1 is bound to a four-amino acid motif (₂₆₆LPKS₂₆₉) located in the intracellular domain of IFN-gR1, whereas JAK2 is bound to a proline-rich sequence (₂₆₃PPSIPLQIEEYL₂₇₄) also in the intracellular region of IFN-gR2. The formation of the IFN-g/IFN-gR1-IFN-gR2 complex allows the interaction between JAK1 and JAK2 and their reciprocal transphosphorylation, which induces the phosphorylation of the IFN-gR1 440 tyrosine. This phosphorylated domain (₄₄₀YDKPH₄₄₅) is a dock site for the SH2 domain of the signal transducer and activator of transcription 1 (STAT-1) molecule; the binding of STAT 1 to this site promotes its own phosphorylation, homodimerization and later translocation to the nucleus. The interaction of activated STAT 1 with different DNA consensus sequences induces the transcription of a first set of IFN-g-regulated genes including the IFN-g-regulating factor 1. In turn IFN-g-regulating factor 1 activates the transcription of a second set of genes induced by IFN-g, which play an important role in antiviral activity, apoptosis, antigen processing, expression of major histocompatibility complex, and development of TH1 cells. Furthermore, in the murine model there is clear evidence that the inducible nitric oxide synthase gene is up-regulated by IFN-g. This enzyme is essential for the microbicidal activity of macrophages; however, in humans the data are still controversial (1,5,7,10-19).

Recently, the first cases of IFN-gR deficiency have been described in humans, caused by mutations in the IFN-gR1 or IFN-gR2 gene (Online Mendelian Inheritance in Man, 209950). The phenotype of these genetic defects has a main characteristic, i.e., an abnormal susceptibility to infections caused by non-pathogenic mycobacteria such as bacillus Calmette Guerin (BCG) and non-tuberculosis mycobacteria. These patients exhibit systemic infections and inability to develop mature granulomas in response to mycobacteria and frequently die as a consequence of this infection. Microorganisms other than mycobacteria such as Salmonella and some viruses also produce severe infections (20-26).

In the present study we report the functional and molecular characterization of IFN-gR in 4 patients followed at the Primary Immunodeficiency Diseases Clinic at the School of Medicine of the University of Antioquia, who presented uncommon manifestations of mycobacterial infections suggesting a defect in the immune response against this type of intracellular microorganism.

Material and Methods

Patients

A deficiency in the IL-12/IFN-g/IFN-gR/STAT-1 pathway was considered in these patients because their phenotypes agreed with the criteria recommended by the Primary Immunodeficiency Committee of the World Health Organization, which indicate that this diagnosis must be considered in patients with severe infection by an intracellular microorganism who present normal numbers of T, B and NK cells (Table 1) and normal levels of serum immunoglobulins. The study was approved by the Ethics Committee of the Research Medical Center of the University of Antioquia and patients were submitted to clinical evaluation and medical examination after giving written informed consent.

Table 1.
Lymphocyte populations in peripheral blood of patients quantified by flow cytometry.

Case reports

Patient LM, a female, at 4 months of age presented severe abdominal symptoms that required surgical treatment. She also developed respiratory manifestations and Mycobacterium tuberculosis was identified in tracheal secretion. Diagnosis of miliary tuberculosis was established and treatment was started. When she was two years old, tuberculosis reactivation was confirmed by an intestinal biopsy positive for M. tuberculosis. The patient was treated by surgical removal of the affected region and by administration of ethambutol, ethionamide and isoniazid, with an appropriate response. Currently the patient is in good condition with prophylactic treatment with trimethoprim-sulfamethoxazole (TMP-SMX). She had been vaccinated with BCG during the first week after birth.

Patient BA, a boy, at 2 months of age presented an abscess in the left thigh after application of anti-hepatitis B vaccine. In the following three years, four surgical procedures were performed to remove the abscess. The biopsy demonstrated multiple granulomas, some of the solid and others of caseous consistency, and the Ziehl Nielsen stain was positive for mycobacteria. M. tuberculosis was identified by culture. This patient was under anti-tuberculosis treatment and is currently in prophylaxis with TPM-SMX. He received the BCG vaccine after birth.

Patient AR, a 10-year-old boy, presented respiratory distress due to a granulomatous lesion in the oropharynx. The biopsy revealed a lesion characterized by reactive acanthosis, a necrotic center and a plasmocytoid infiltrate. One of the serial Koch's bacillus analyses from this lesion was positive; however, the mycobacterium species could not be identified. Despite the institution of anti-tuberculosis treatment the patient developed a severe deterioration of his general condition and died. This patient had not received the BCG vaccine.

Patient JF, a boy who presented jaundice, choluria and acholia at 2 months of age. Liver gammagraphy indicated bile tree atresia and a liver biopsy revealed a soft granuloma. A chest X-ray showed enlarged lymph nodes and infiltration of the hilum. The tuberculin test was positive (11 mm) and the biopsy of a cervical lymph node revealed chronic granulomatous inflammation compatible with tuberculosis. When he was 5 months old he presented purulent secretion from axillary and supraclavicular lymphadenitis, and M. tuberculosis was identified by culture of this secretion. Functional tests of phagocytic cells were normal. The patient received anti-tuberculosis treatment followed by improvement and currently is in good condition. The patient was vaccinated with BCG.

Analysis of IFN- g R expression

The expression of IFN-gR1 was determined in Epstein-Barr virus transformed B (EBV-B) cells from patients and normal subjects. EBV-B cells were obtained after incubation of 10⁷ blood mononuclear cells with B95-8 cell line supernatant (ATCC #CRL 1612) for 3 to 4 weeks. IFN-gR1 was analyzed in 5 x 10⁵ EBV-B cells by incubation for 20 min with 1 µg monoclonal antibody against IFN-gR1 labeled with rhodamine (a gift of Dr. Steve Holland, NIAID, NIH). Cells were washed with cytometry buffer (PBS, 1% fetal calf serum (Gibco, Rockville, MD, USA) and 0.1% sodium azide) at 300 g for 7 min and fixed with fixation buffer (PBS, 1% paraformaldehyde and 1% sodium azide) and median fluorescence intensity was measured in an Epics XL flow cytometer (Coulter, Miami, FL, USA).

Analysis of STAT-1 expression by flow cytometry

The intracellular expression of STAT-1 was determined by flow cytometry in EBV-B cell lines obtained from our patients; 5 x 10⁵ EBV-B cells were permeabilized with CITO Fix/PERM (Becton-Dickinson, San Jose, CA, USA) and incubated for 30 min at 4ºC in the dark with 1 µg anti-STAT-1 monoclonal antibody (New England Biolabs, Beverly, MA, USA). Cells were washed with cytometry buffer (PBS, 1% fetal calf serum (Gibco) and 0.1% sodium azide) at 600 g for 7 min and 1 µg FITC-labeled anti-rabbit IgG (Santa Cruz Company, Santa Cruz, CA, USA) was added; then cells were fixed with fixation buffer (PBS and 1% paraformaldehyde) and median fluorescence intensity was measured with an Epics XL flow cytometer (Coulter).

Detection of STAT-1 and phosphorylated STAT-1 by Western blotting

To assess the function of the IFN-g/IFN-gR activation pathway the phosphorylation of STAT-1 was analyzed in EBV-B cells from patients. Briefly, 10⁷ cells were incubated with 100 ng/ml recombinant human IFN-g (rhIFN-g) for 15 min at 37ºC, the cell phosphorylation was stopped by adding a 4ºC solution containing 2 mM EDTA and 400 mM sodium orthovanadate (O-NaVO4; Sigma, Saint Louis, MO, USA), and cells not incubated with rhIFN-g were used as negative control. Cells were centrifuged at 600 g for 10 min at room temperature and the cell pellets were lysed with 50 µl lysis buffer containing 0.5% Triton X-100 (Sigma), 50 mM HEPES, pH 7.2 (BioWhittaker, Walkersville, MD, USA), 150 mM NaCl, 5 mM EDTA (Sigma), 1 mM O-NaVO4, 10 mM NPGB (Sigma), 100 µg/ml aprotinin (Sigma), leupeptin (Sigma), 100 µg/ml PMSF (Sigma) and 100 µg/ml chymostatin (Boehringer Mannheim, Indianapolis, IN, USA).

The cell lysis product was centrifuged at 122 g for 15 min at 4ºC and proteins in the supernatant were quantified by the BCA method (Pierce, Rockford, IL, USA). Then, 20 µg protein was diluted in loading buffer containing 50 mM Tris, pH 7.2 (Bio-Rad, Hercules, CA, USA), 10% glycerol (Sigma), 2 mercaptoethanol (Sigma), bromophenol blue (Sigma), and 5% SDS (Sigma), and finally boiled for 5 min. Protein samples were submitted to 8% SDS-PAGE and later transferred to PVDF membranes (Bio-Rad). For immunodetection, membranes were blocked with 5% BSA for 60 min, washed three times with PBS/Tween 20, 1 min each wash, incubated with 1 µg antibody to phosphorylated STAT-1 (New England Biolabs) for 1 h, and washed again as before and 1 µg anti-rabbit IgG peroxidase-labeled antibody (Santa Cruz Company) was added. The reaction was developed by chemiluminescence using the ECL kit (Amersham Life Science, Buckinghamshire, UK) and exposed to X-Omat RX film (Eastman Kodak Co., Rochester, NY, USA). Non-phosphorylated STAT-1 was detected in non-stimulated cells using an antibody against non-phosphorylated STAT-1 (New England Biolabs).

PCR-SSCP for IFN- g R genes

To find possible mutations in the IFN-gR1 and R2 genes, PCR fragments were analyzed by single-strand conformational polymorphisms (SSCP). Each exon of these two genes was amplified with the oligonucleotides listed in Table 2. The amplification was carried out with 100 ng genomic DNA, 0.5 µM of each primer, 200 µM dNTPs, 1.25 mM Mg²⁺, and 2U Taq DNA polymerase (Promega, Madison, WI, USA). The reaction was carried out for 25 cycles at 94ºC/30 s, 58ºC/45 s and 72ºC/40 s and a final incubation at 72ºC/5 min was performed using a 9600 PE thermocycler (Perkin Elmer, Foster City, CA, USA). To check for the proper amplification, PCR products were run on agarose gel and visualized by UV light after ethidium bromide staining. Next, 5 µl of each product was mixed with 1 µl of 6X loading buffer (formamide, xylene cyanol, and bromophenol blue), denatured at 80ºC/2 min and placed on ice. These fragments were then electrophoresed on 6% polyacrylamide gel under non-denaturing conditions at 35 W/4 h at 25ºC. To identify DNA bands, the gel was stained with silver (Bio-Rad).

Table 2.
Synthetic oligonucleotides used for amplification of IFN-gR1 and IFN-gR2 genes from genomic DNA.

DNA sequencing

All PCR products exhibiting an altered electrophoretic pattern in the PCR-SSCP for IFN-gR genes were sequenced. Direct sequencing of purified PCR products was performed using the ABI Big Dye Terminator Cycle Sequencing Ready Reaction DNA kit (Perkin Elmer) and the sequenced products were resolved on an ABI 310 automated sequencer (Perkin Elmer).

In patient AR the STAT1 cDNA was analyzed for a mutation in the region encoding the tail segment of the protein. Sense (5'-TCGACAGTCTTGGCACCTAACGTGC-3') and antisense (5'-TGCTATCAACAGGT TGCAGCG-3') primers were used to amplify the complete cDNA. The STAT1 region encoding L706 was amplified with sense (5'-TCGGTTGATGGAAAGCGTA-3') and antisense (5'-CTCTTCTGTGTTCACTTAC-3') primers. Genomic DNA from EBV-B cell lines was also amplified. The products were sequenced as previously described.

Results

Expression of IFN- g R1 in EBV-B cell lines from patients

The expression of IFN-gR1 in these patients was evaluated by flow cytometry in peripheral blood B cells transformed with EBV. This assay was repeated twice in each patient with cells transformed from two different blood samples. As shown in Figure 1, the percentage of EBV-B cells expressing IFN-gR1 was similar for patients and control subjects: 65.8 ± 5.2 for controls, 60.2 ± 8.1 for AR, 72 ± 11 for JF, 78.3 ± 16.1 for BA, and 62.8 ± 10.9 for LM.

Figure 1.
Analysis of interferon-gamma receptor 1 (IFN-gR1) expression by flow cytometry in EBV-B cells using a rhodamine-labeled monoclonal antibody against IFN-gR1. The control (C) value for the percentage of cells expressing the receptor was obtained from cell lines from five different healthy subjects; the patients were analyzed twice and their mean values are presented. No statistically significant differences were observed (unpaired t-test).

Evaluation of the expression of STAT-1 and STAT-1 phosphorylation in EBV-B cell lines from patients

When the level of expression of STAT-1 was determined by flow cytometry using intracellular staining, no differences in the percentage of positive cells for this molecule were detected between EBV-B cells from patients and control subjects: 71.8 ± 18.8 for controls, 85.6 ± 13.5 for AR, 65.5 ± 10.7 for JF, 76.3 ± 9.6 for BA, and 61.4 ± 11.4 for LM (Figure 2); as for IFN-gR1 analysis, this assay was repeated twice in each patient with cells transformed from two different blood samples. Similarly, when the expression of STAT-1 protein was analyzed by Western blotting no differences between cells from patients and healthy controls were demonstrated (Figure 3).

To evaluate the integrity of the IFN-gR intracellular signaling pathway in the patients of this study, the phosphorylation of STAT-1 protein in Tyr701 was determined after EBV-B cell stimulation with recombinant human IFN-g. The assay revealed that one of the patients (AR) had an evident reduction in the phosphorylation of STAT-1 when compared with other patients or with a healthy control after cell activation with rhIFN-g (Figure 4). An even loading of protein extract indicated that there was less phosphorylation of the STAT-1 tyrosine residue in cells of the patient AR.

Figure 2.
Analysis of intracellular signal transducer and activator of transcription 1 (STAT-1) expression determined flow cytometry in EBV-B cell lines obtained from our patients. The control (C) value of percentage of cells expressing intracellular STAT-1 was obtained from cell lines from five different healthy subjects while the patients were analyzed twice. The mean values are presented. No statistically significant differences were observed (unpaired t-test).

Figure 3.
Detection of non-phosphorylated signal transducer and activator of transcription 1 (STAT-1) by Western blotting in resting EBV-B cell lines from patients and two healthy controls (C) using an antibody against the non-phosphorylated form of STAT-1. This assay was repeated at least once for each patient.

Figure 4.
Evaluation of signal transducer and activator of transcription 1 (STAT-1) phosphorylation in response to interferon-gamma (IFN-g) in EBV-B cells from two patients and one healthy control (C). Cells were incubated with 100 ng/ml rhIFN-g for 15 min/37ºC, then an antibody to P-Tyr701 was used to identify phosphorylated STAT-1 in the lysis extract after electrophoresis and blotting onto a PVDF membrane. In order to confirm an equal load of protein extract, the membrane was removed and incubated with the antibody against the non-phosphorylated form of STAT-1. This assay was repeated twice to confirm the result.

SSCP analysis and DNA sequencing of genomic DNA corresponding to the IFN- g R1 and IFN- g R2 genes

SSCP analysis of genomic DNA obtained from the present patients revealed an abnormal electrophoretic pattern in some of the PCR fragments spanning the coding region of the IFN-gR1 gene. One patient (AR) exhibited an abnormal shift in the fragment corresponding to exon 2. When PCR fragments amplified from exon 7 were analyzed, patients AR and LM showed aberrant electrophoretic patterns (Figure 5). However, when these three PCR fragments were sequenced in order to identify a possible mutation explaining the SSCP data, no sequence alterations with respect to the known sequence of the IFN-gR1 gene were demonstrable (data not shown). Furthermore, in PCR fragments amplified from genomic DNA corresponding to the coding region of the IFN-gR2 gene, SSCP analysis did not demonstrate any abnormal pattern in PAGE carried out under non-denaturing conditions (data not shown).

As described above, patient AR exhibited a reduction in the phosphorylation of STAT-1 at Tyr701; therefore, in order to determine whether this patient had a mutation in the codon encoding this amino acid or in other near residues critical for STAT-1 function, as recently described (27), the DNA segment encoding the tail segment of STAT-1 protein was sequenced in this patient. After sequencing both strands of cDNA and genomic DNA no mutations were detected (data not shown).

Figure 5.
Representative pictures of SSCP analyses of the PCR fragments corresponding to exons two and seven of the IFN-gR1 gene in different patients. The arrows indicate the PCR products that presented an abnormal migration pattern after 6% native electrophoresis. These analyses were performed on two different occasions in order to confirm the findings. C = control.

Discussion

In this study we report the immunologic and molecular characterization of the IFN-gR/STAT-1 activation pathway in 4 patients suffering uncommon infections produced by mycobacteria. Our hypothesis was that these patients might present a genetic defect in one of the molecules involved in the IL-12/IFN-g pathway based on their age and clinical presentation of these defects; furthermore, no other primary or acquired defects of the immune system such as severe combined immunodeficiency or HIV infection were detected. To determine the cell surface expression of IFN-gR1, the presence of this protein in EBV-B cells was assessed by flow cytometry. The expression of IFN-gR1 observed in all patients was similar to that found in healthy controls. Since this result did not rule out the presence of a defect that could affect the function of IFN-gR1 but not its structure, the IFN-GR1 was characterized at the genomic level. The SSCP analysis of coding regions for this gene revealed some abnormal migration profiles in exons 2 and 7 in 2 patients; however, the DNA sequencing of these fragments did not show any nucleotide change.

Different possibilities might explain the inconsistency of these results. During PCR prior to SSCP analysis some DNA fragments exhibiting a different conformation during electrophoresis might have been generated. The altered electrophoretic mobility could also be a consequence of the thermal and physical conditions of electrophoresis (28). In other cases, the changes in DNA responsible for the altered mobility in the SSCP might be located in the sequence complementary to the primers used during PCR, in regions very difficult to define by the sequencing reaction used.

Based on these results, the next step was the molecular analysis of the IFN-GR2 gene; in this case the SSCP did not indicate any possible sequence alteration of the PCR fragments spanning the coding region of this gene.

Since alterations were not found in the IFN-GR1 or IFN-GR2 genes, we decided to evaluate the expression and phosphorylation of STAT-1, a molecule essential for the signal transduction pathway in response to IFN-g. In the 4 patients the intracellular levels of STAT-1, assessed by flow cytometry and Western blotting, were similar to those found in healthy controls; however, the phosphorylation of cellular STAT-1 in response to human IFN-g was found to be substantially reduced in one patient (AR). A recent report described two unrelated kindred affected by a heterozygous dominant STAT-1 mutation with a leucine substitution for a serine at amino acid position 706 (L706S), which is a loss-of-function mutation that severely impairs the phosphorylation of tyrosine 701 (27). In order to determine whether the alteration observed in our patient could be explained by a mutation in one of the nucleotides encoding these critical tyrosines, the DNA segment encoding the tail segment of the STAT-1 protein was sequenced. After sequencing the DNA region spanning these critical residues, no mutations were detected.

The protective immune response to intracellular pathogens such as mycobacteria essentially depends on adequate activation and function of mononuclear phagocytic cells. In order to achieve a full effector response, these cells need soluble signals from the microenvironment; of particular importance is the production of IFN-g by NK and T helper cells. IFN-g secretion is induced by IL-12, a cytokine produced by macrophages and dendritic cells. IFN-g activates different effector mechanisms of phagocytic cells, which ensure appropriate control and elimination of phagocytized microorganisms. The immune response against intracellular microorganisms also involves lysis of infected cells by cytotoxic T and NK cells (2,5,6,20).

The importance of the immune effector mechanisms related to IL-12 and IFN-g has been clearly demonstated by a growing group of patients with Mendelian susceptibility to mycobacterial infections, a primary immunodeficiency disease caused by genetic defects in the IL-12/IFN-g activation pathway. Mutations in five different genes produce this disorder: IFN-GR1, IFN-GR2, STAT1, IL12B, and IL12RB1. The main clinical characteristic of these patients is their higher susceptibility to the development of disseminated mycobacterial disease or local recurrent infections by non-tuberculosis mycobacteria and, in some cases, chronic infections by other agents such as Salmonella species and certain viruses (20,22-27,29-36). Many of these characteristics are similar to the clinical features observed in our patients (Table 3); therefore, a defect in one of the molecules involved in the IL-12/IFN-g/IFN-gR/STAT-1 activation pathway is a possible explanation for their phenotype.

Patients with defects in the IL-12/IFN-g pathway exhibit differences in susceptibility to mycobacterial disease. Complete abrogations of IFN-gR1 or IFN-gR2 are strongly associated with an early onset of severe and often fatal infection with low virulence mycobacterial species, such as non-tuberculosis mycobacteria or BCG. In these patients the infection continues despite the instauration of an appropriate anti-tuberculosis treatment; furthermore, the lesions reveal poor granuloma formation and are rich in mycobacteria. In contrast, partial deficiency of IFN-gR1 or IFN-gR2 is associated with a milder clinical presentation and an impaired but not abolished response to IFN-g. Most of the time these patients develop infections by more aggressive mycobacteria such as M. avium or M. tuberculosis; the granulomas found in the lesions of these patients are mature, well formed and with few mycobacteria; moreover, they respond very well to anti-tuberculosis treatment (2).

Patients who present mutations affecting the expression or function of IL-12p40 or IL-12Rß1 molecules generally develop disseminated but curable mycobacterial infections with non-tuberculosis mycobacteria or BCG, a similar phenotype to that observed in patients with partial deficiency of IFN-gR1 or IFN-gR2. This less severe clinical picture seems to be explained by a residual IL-12-independent IFN-g secretion (25,32,37-39). Several patients with mutations in STAT-1 gene have been recently described; however, they show differences in their clinical presentation. As mentioned before, two kindred with the same heterozygous mutation affecting the dimerization of STAT-1 to form gamma-activated factor were susceptible to mycobacteria but resistant to viruses, with a phenotype similar to that of patients with partial IFN-gR (28). A more recent report described two other children who developed disseminated BCG after vaccination, with remission occurring with antibiotic treatment. However, both patients died from disseminated viral disease, produced in one of them by herpes simplex virus 1, while no virus was identified in the other (40). In these patients two different homozygous mutations, which produced a complete deficiency of STAT-1, were found, affecting both the formation of gamma-activated factor and the formation of the STAT-1/STAT-2/p48 trimer (transcription factor IFN-stimulated gene factor 3) that is essential for the cellular response to IFN-a/ß.

The present results indicate that molecules that constitute the IFN-gR/STAT-1 pathway were intact and functioning in at least three of the 4 patients studied: normal surface expression of IFN-gR1, no mutations found in the IFN-GR1 or IFN-GR2 genes, and normal expression and phosphorylation of STAT-1 in 3 patients (Table 3). The phenotype of these 3 patients might be explained by a defect in IL-12/IL-12R that precludes an adequate activation of mononuclear phagocytes or by an alteration in a step downstream from STAT-1 activation; furthermore, an IFN-g-independent anti-mycobacterial mechanism could be affected in any of these patients. In order to identify the genetic defect associated with the phenotype of these 3 patients it is necessary to continue studying the molecules involved in the activation of mononuclear phagocytes, particularly IL-12/IL-12R.

On the other hand, in patient AR the reduced phosphorylation of STAT-1 might have led to a defective activation of effector mechanisms required for a full activation of mononuclear phagocytes in response to mycobacterial infection and therefore may have been the reason for the fatal course of the infection in this patient. The molecular mechanism responsible for this defect remains to be elucidated; however, different alternatives can be proposed: a mutation in the IFN-GR1 or IFN-GR2 genes not detected by our SSCP method, an intrinsic alteration of STAT-1 that affects its phosphorylation in a region of the protein different from the one analyzed in this patient or a defect in any other molecule involved in the response to IFN-g but upstream from STAT-1 activation, for instance JAK1 or JAK2. Therefore, we consider important to continue the analysis of these different possibilities in the EBV-B cells from this patient.

Table 3.
Summary of the characteristics, clinical outcome and experimental findings for four patients with uncommon presentation of mycobacterial infections.

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Address for correspondence: P.J. Patiño, Laboratorio de Inmunología, Facultad de Medicina, Universidad de Antioquia, A.A. 1226, Medellín, Colombia (S.A.). Fax: [phone]. E-mail: [e-mail]

Research supported by Fundación para la Promoción de la Investigación y la Tecnología del Banco de la República, by Instituto Colombiano para el Desarrollo de la Ciencia y la Tecnología, and by Comité para Investigación de la Universidad de Antioquia. Received August 27, 2003. Accepted May 3, 2004.

Correspondence and Footnotes

Publication Dates

• Publication in this collection
  16 Sept 2004
• Date of issue
  Sept 2004

History

• Accepted
  03 May 2004
• Received
  27 Aug 2003