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A Role for IL-12 Receptor Expression and Signal Transduction in Host Defense in Leprosy¹

Jenny Kim^*,, Koichi Uyemura^*, Melissa K. Van Dyke, Annaliza J. Legaspi^*, Thomas H. Rea, Ke Shuai and Robert L. Modlin^2,*,

^* Division of Dermatology, and Department of Microbiology and Immunology, University of California School of Medicine, Los Angeles, CA 90095; Section of Dermatology, University of Southern California School of Medicine, Los Angeles, CA 90033; and Division of Hematology-Oncology, University of California School of Medicine, Los Angeles, CA 90095

	Abstract

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The generation of cell-mediated immunity against intracellular infection involves the production of IL-12, a critical cytokine required for the development of Th1 responses. The biologic activities of IL-12 are mediated through a specific, high affinity IL-12R composed of an IL-12R1/IL-12R2 heterodimer, with the IL-12R2 chain involved in signaling via Stat4. We investigated IL-12R expression and function in human infectious disease, using the clinical/immunologic spectrum of leprosy as a model. T cells from tuberculoid patients, the resistant form of leprosy, are responsive to IL-12; however, T cells from lepromatous patients, the susceptible form of leprosy, do not respond to IL-12. We found that the IL-12R2 was more highly expressed in tuberculoid lesions compared with lepromatous lesions. In contrast, IL-12R1 expression was similar in both tuberculoid and lepromatous lesions. The expression of IL-12R2 on T cells was up-regulated by Mycobacterium leprae in tuberculoid but not in lepromatous patients. Furthermore, IL-12 induced Stat4 phosphorylation and DNA binding in M. leprae-activated T cells from tuberculoid but not from lepromatous patients. Interestingly, IL-12R2 in lepromatous patients could be up-regulated by stimulation with M. tuberculosis. These data suggest that Th response to M. leprae determines IL-12R2 expression and function in host defense in leprosy.

	Introduction

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The outcome of infection in murine models of infection and in human infectious disease is regulated by the Th1 and Th2 T cell cytokine patterns. Th1 cells secrete IL-2 and IFN- and are generally associated with resistance to intracellular pathogens, whereas Th2 cells secrete IL-4 and IL-10 and are associated with progressive disease to these same pathogens. It has become increasingly evident that IL-12 is a pivotal regulator of Th1 responses and is essential for promoting cell-mediated immunity (CMI)³ against intracellular microbial pathogens (1, 2, 3, 4, 5).

The ability of IL-12 to activate lymphocytes is mediated by the IL-12R, a heterodimer composed of IL-12R1 and IL-12R2 subunits (6, 7). The IL-12R1 is constitutively expressed on both Th1 and Th2 cells. The IL-12R2, in contrast, is expressed more strongly on Th1 cells as compared with Th2 cells, indicating a mechanism by which IL-12 differentially affects the growth of these subsets. Studies involving the regulation of IL-12R have shown that Ag receptor triggering is sufficient to induce expression of IL-12R1 and IL-12R2 (8). In humans, IL-12R2 chain is induced by IL-12 and IFN- and inhibited by IL-4 (8, 9), suggesting that local cytokine milieu plays a role in determining IL-12 responsiveness of T cells during infection.

Binding of IL-12 to the IL-12R results in activation of Janus kinases Tyk2 and Jak2, leading to tyrosine phosphorylation and DNA binding of Stat4 with subsequent IFN- production by T cells (10, 11). Studies in both human and murine models have shown that IL-12 induces tyrosine phosphorylation of Stat4 in Th1 cells but not in Th2 cells (8, 9, 12, 13), indicating a functional difference imparted by the expression levels of the IL-12R in these T cell subsets.

To study the mechanism of responsiveness to IL-12 in human infectious disease, we chose leprosy as a model because of its spectrum of clinical manifestations that correlate with the level of CMI to the pathogen Mycobacterium leprae. At one end of the spectrum, patients with tuberculoid leprosy mount a strong CMI and are resistant to M. leprae. At the opposite end of the spectrum, patients with lepromatous leprosy have weak CMI and have a progressive form of the disease. Our earlier studies demonstrated that T cells from tuberculoid leprosy patients proliferate in response to IL-12, whereas T cells isolated from lepromatous patients do not respond to IL-12 (14). Here, we present evidence that expression and up-regulation of IL-12R2 correlates with CMI, being greater in tuberculoid than in lepromatous patients. IL-12 signaling in tuberculoid leprosy patients correlates with the expression of the IL-12R2 subunit. The lack of IL-12 responsiveness in lepromatous patients appears to be in part due to an Ag-specific unresponsiveness to M. leprae and due to inappropriately differentiated Th cells. Our data suggest that Th response to Ag determines IL-12R2 expression and function in the generation of CMI to microbial infection.

	Materials and Methods

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Patients and clinical specimens

Patients with leprosy were diagnosed at the Los Angeles County/University of Southern California Medical Center Hansen’s Disease Clinic (Los Angeles, CA) and classified according to the clinical and pathologic criteria of Ridley and Jopling (15). After receiving informed consent, venous blood was collected in heparinized tubes from tuberculoid and lepromatous leprosy patients. PBMC were isolated using Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ) differential centrifugation.

Skin biopsies from leprosy patients were also obtained, embedded in OCT medium (Ames, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at -80°C until ready to use.

Antigens

M. leprae and M. tuberculosis (strain H37Rv) were provided by P. Brennan (Colorado State University, Fort Collins, CO) and prepared by probe sonication (16). The level of endotoxin in M. leprae and M. tuberculosis was measured quantitatively with a Limulus Amoebocyte Lysate assay (BioWhittaker, Walkersville, MD) and found to be <0.1 ng/ml.

Polymerase chain reaction

Total RNA from skin biopsy specimens was isolated after lysing samples in guanidinium isothiocyanate buffer as previously described (17, 18), and cDNA was synthesized with reverse transcriptase (Life Technologies, Gaithersburg, MD).

cDNA samples were amplified in a DNA Thermocycler (Perkin-Elmer, San Diego, CA) for 40 cycles with each cycle consisting of denaturation at 95°C for 20 s and annealing/extension at 65°C for 45 s. Each PCR mixture contained 2.5 mM MgCl₂, 0.2 mM dNTP, 25 pM 5' and 3' oligonucleotide primers, and 2.5 U Taq polymerase (Life Technologies, Grand Island, NY). The sequences of the primer pairs, 5' and 3', were as follows: IL-12R1, 5'-CTGTTTTCAGGACCCGCCATATCC-3' and 5'-AGAGTGTGACAGTGTACAGCACAG-3'; IL-12R2, 5'-GAGGGACTGGTACTGCTTAATCGACTC-3' and 5'-CCTCACACAGGTTCATTATGTTAA-3'; and CD3, 5'-CTGGACCTGGGAAAACGCATC-3' and 5'-GTACTGAGCATCATCTCGATC-3'. cDNA concentrations were normalized to yield equivalent CD3 PCR products.

To verify IL-12R1 and IL-12R2 mRNA, PCR products were transferred to Hybond-N nylon membranes (Amersham Pharmacia Biotech) as previously described (17, 18) and probed with a labeled oligonucleotide complementary to nucleotides within the sequences recognized by the PCR amplification primers. Sequences of the oligonucleotide probes were as follows: IL-12R1, 5'-GAGATGCTATCGGATATCCAGTGATCG-3'; IL-12R2, 5'-TCTCCACTATCAGGTGACCTTGCA-3'; and CD3, 5'-GCCGACACACAAGCTCTGTTGAGGA-3'. The relative intensity of PCR bands was assessed by densitometric analysis of the digitized image, performed on a Macintosh computer (Apple Computers, Cupertino, CA) using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/).

Flow cytometry

PBMC (1 x 10⁶ cells/ml) from tuberculoid and lepromatous patients were cultured in complete medium (RPMI 1640, 0.1 mM sodium pyruvate, 2 mM penicillin, 50 µg/ml streptomycin; Life Technologies, Grand Island, NY) supplemented with 10% human serum in a 24-well plate. Cells were stimulated with and without Ag for 5 days. Lymphocytes were recovered, washed twice in PBS, and resuspended in FACS buffer containing PBS with 2% FBS and 0.1% sodium azide. Monoclonal Ab to IL-12R1 and IL-12R2 (provided by D. H. Presky, Hoffmann-LaRoche, Nutley, NJ) or isotype-matched control, IgG2a, (PharMingen, San Diego, CA) was added at a final concentration of 2 µg/ml to the cells and incubated at 4°C for 45 min. The cells were washed twice with FACS buffer and incubated with biotin-conjugated anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min, followed by final incubation with streptavidin-PE (Jackson ImmunoResearch Laboratories) for 30 min. After further washing, the cells were fixed in 2% paraformaldehyde, and flow cytometry analysis was performed using a FACScan (BD Biosciences, Mountain View, CA). The expression of the IL-12R subunits was measured as fluorescence intensity using WinMDI software (provided by J. Trotter, The Scripps Institute, La Jolla, CA). The up-regulation of the IL-12R1 and IL-12R2 expression by Ag stimulation was determined by calculating the difference in the mean fluorescence intensity (MFI) between Ag-stimulated cells and unstimulated cells. MFI for IL-12R1 and IL-12R2 was compared between tuberculoid and lepromatous patients using the Wilcoxon rank sum test for unpaired variables. Differences were considered statistically significant for p values <0.05.

EMSA

PBMC (5 x 10⁶) from tuberculoid and lepromatous patients were activated with M. leprae (5 µg/ml) for 5 days in culture. A previously described Th1 clone, D103.5 (19, 20), derived from a tuberculoid lesion and specific for M. leprae 10-kDa protein, was used as a positive control. This T cell clone produces IFN- in response to IL-12 (data not shown). Cells were washed with PBS and stimulated with and without IL-12 (20 ng/ml; R&D Systems, Minneapolis, MN) for 20 min before preparation of extracts, and EMSA was performed as previously described (21, 22). Briefly, whole cell extracts were prepared by lysing cells in a buffer containing 50 mM Tris (pH 8.0), 300 mM NaCl, 0.5% Nonidet P-40, 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.1 mM sodium vanadate, 1 mM PMSF, 0.5 µg/ml leupeptin, and 3 µg of aprotinin per milliliter. Prepared extracts were incubated with a ³²P-end-labeled double-stranded oligodeoxynucleotide, high affinity serum-inducible element (hSIE), 5'-GTCGACATTTCCCGTAAATCGTCGA-3' (23) in binding buffer for 20 min at room temperature before electrophoresis on 5% polyacrylamide gel. When used, 2 µl of anti-Stat4 and anti-Stat3 Abs (Santa Cruz Biotechnology, Santa Cruz, CA) were incubated with cell extracts for 30 min at room temperature before the addition of probe.

Immunoprecipitation and SDS-PAGE

Total cellular lysates of 2 x 10⁷ cells were immunoprecipitated with anti-Stat4 Ab as previously described (24) and were separated by electrophoresis on 8% SDS-PAGE. After transfer to nitrocellulose, blots were probed with anti-phosphotyrosine Ab, 4G10 (Upstate Biotechnology, Lake Placid, NY). Blots were then stripped and reprobed with anti-Stat4 Ab.

IFN- production

Ninety-six-well ELISA plates (Corning Glass Works, Corning, NY) were coated overnight at 4°C with 100 µl of mouse anti-human IFN- (PharMingen) at 5 µg/ml per well. Plates were blocked with 200 µl of 1% BSA and 0.05% Tween 20 in PBS for 2 h at room temperature. Aliquots (100 µl) of each sample or of IFN- standards (Endogen, Woburn, MA) were then added to each well and incubated at room temperature for 2 h. Biotin-conjugated mouse anti-human IFN- mAb (PharMingen) was added to each well and incubated for 1 h, followed by a 30-min incubation with streptavidin/peroxidase (Pierce, Rockford, IL). Peroxidase substrate solution (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was used to detect IFN-, and the plates were read at a wavelength of 405 nm in a 7520 microplate reader (Cambridge Technology, Cambridge, MA). All samples and standards were performed in duplicate.

	Results

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IL-12R expression in leprosy lesions correlates with CMI

To determine the relative expression of IL-12R subunits in leprosy patients, we performed RT-PCR on biopsy specimens from skin lesions of patients with leprosy. RNA was extracted from biopsy specimens, then cDNA was synthesized and normalized to the amounts of CD3- PCR products as a measure of total cellular RNA in T cells. As shown in Fig. 1, the inducible IL-12R2 mRNA was highly expressed in 10 of 10 tuberculoid lesions but only weakly expressed in 10 of 10 lepromatous lesions. In contrast, the constitutively expressed IL-12R1 subunit was equally expressed in both tuberculoid and lepromatous lesions. These data indicate that the expression of IL-12R2 in leprosy correlates with host resistance to infection, greatest in the group of patients known to manifest strong CMI to M. leprae.

View larger version (46K): [in this window] [in a new window]   FIGURE 1. IL-12 R1 and IL-12R2 mRNA expression in leprosy lesions. The cDNA derived from the skin lesions of 10 tuberculoid (1–10) and 10 lepromatous (11–20) patients were normalized to yield equivalent CD3- mRNA PCR products. PCR with specific primers was then used to detect IL-12R1 and IL-12R2 mRNA. The PCR products were transferred to nylon membrane and probed with a radiolabeled internal oligonucleotide. The intensity of PCR bands was assessed by densitometric analysis of the digitized image using the public domain NIH image program, and is expressed as PCR intensity.

IL-12R2 expression correlates with Ag responsiveness in T cells

Because Ag stimulation has been shown to enhance the expression of the IL-12R on T cells (7), we determined whether addition of M. leprae could modulate the expression of IL-12R subunits on T cells from leprosy patients and explain the differential expression in leprosy lesions. PBMC from tuberculoid and lepromatous patients were cultured with and without M. leprae for 5 days, and the expression of IL-12R1 and IL-12R2 on the cell surface was measured by flow cytometry. When PBMC were cultured with medium alone, the expression of IL-12R1 and IL-12R2 was similar in both tuberculoid (n = 9) and lepromatous (n = 9) patients, with IL-12R1 expression being slightly higher than the expression of IL-12R2 in both patient groups. Representative examples are shown in Fig. 2a. After stimulation with M. leprae, we found that in the tuberculoid patients both IL-12R1 and IL-12R2 were up-regulated. In contrast, in the lepromatous patient group the expression of IL-12R1 but not that of IL-12R2 could be up-regulated by M. leprae.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Regulation of IL-12R1 and IL-12R2 expression by M. leprae. PBMC (1 x 106) from tuberculoid and lepromatous patients were cultured in vitro in the absence of stimuli (medium) or with M. leprae (5 µg/ml) for 5 days. Cells were stained with IL-12R1, IL-12R2, or with an isotype control Ab, followed by PE-conjugated Ab. IL-12R1 and 2 expression was measured on gated lymphocytes. The M. leprae-activated T cells are mostly CD4+ T cells (25 ). a, Representative histograms showing the level of IL-12R1 and IL-12R2 expression on T cells from tuberculoid and lepromatous patients. Solid outline, staining with control Ab alone; bold outline, staining with IL-12R1 or IL-12R2 Ab. b, Summary bar graph demonstrating the modulation of MFI of lymphocytes stained with anti-IL-12R1 and anti-IL-12R2 Ab. Nine tuberculoid (BT1-BT9) and nine lepromatous (LL1-LL9) patients were tested. The value indicated is the difference in MFI (MFI) in IL-12R1 and 12R2 expression between M. leprae-stimulated cells and unstimulated cells.

Although patients with lepromatous leprosy are unable to mount the appropriate CMI response to M. leprae, this unresponsiveness is highly specific because these patients have been shown to have intact immune responses to Ags from a variety of other microbial agents, including M. tuberculosis (25, 26, 27). Therefore, to determine whether the ability to up-regulate IL-12R2 was fundamentally altered in these patients, we asked whether M. tuberculosis could up-regulate IL-12R2 expression in lepromatous patients. PBMC from tuberculoid and lepromatous patients were stimulated with either M. leprae or M. tuberculosis for 5 days, and levels of IL-12R2 expression were determined by FACS. Representative histograms from each patient group are shown in Fig. 3. In tuberculoid patients (n = 3), both M. leprae and M. tuberculosis induced the expression of the IL-12R2 when compared with medium alone. In lepromatous patients (n = 3), M. leprae extract had no significant effect on the expression of IL-12R2, but the addition of M. tuberculosis extract to culture of PBMC increased the expression of IL-12R2. In summary, although M. leprae could not induce IL-12R2 expression in PBMC from the lepromatous patients, activation by M. tuberculosis could lead to the up-regulation IL-12R2. These data demonstrate that the inability of lepromatous patients to up-regulate IL-12R2 correlates with their ineffective Th response to M. leprae.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Modulation of IL-12R2 expression by mycobacterial Ags. PBMC (1 x 106) from tuberculoid and lepromatous patients were stimulated with M. leprae (5 µg/ml) or with M. tuberculosis (10 µg ml) for 5 days. Cells were harvested, and the cell surface expression of IL-12R2 was measured on gated lymphocytes by FACS. The results shown are representative histograms demonstrating the level of IL-12R2 expression on T cells from three tuberculoid and three lepromatous patients. Solid line, Staining with control Ab alone; bold line, staining with IL-12R2 Ab.

The IL-12 signaling pathway involves the activation of the Janus kinase-STAT signal transduction pathway. Specifically, stimulation of the IL-12R by IL-12 leads to the phosphorylation and nuclear translocation of a transcription factor Stat4. Such activation of Stat4 has been shown to be essential for the IL-12-mediated lymphocyte responses (12, 13), and this IL-12 responsiveness appears to correlate well with the expression of IL-12R2 (8, 9). To determine whether the low levels of IL-12R2 expression in lepromatous patients is sufficient for IL-12 signaling, PBMC from tuberculoid and lepromatous patients were stimulated with M. leprae and activated with IL-12. Formation of Stat4-containing complexes and binding to the oligonuclear probe hSIE, which contains a high affinity-binding site for several STAT proteins, were determined by gel shift analysis. As shown in Fig. 4, IL-12 induced binding activity to the hSIE probe in PBMC from a tuberculoid patient (lanes 1–3) and in an IL-12-responsive Th1 clone, D103.5 (lanes 13–15). Arrow points to bands forming Stat4-containing complexes. In contrast, PBMC from a lepromatous patient did not show any Stat4 binding activity (lanes 7–9) upon IL-12 stimulation. Samples without IL-12 stimulation did not demonstrate any binding activity in both tuberculoid (lanes 4–6) and lepromatous (lanes 10–12) patients. Supershift experiments confirmed that these complexes contained Stat4 because Ab to Stat4 (lanes 3 and 15), but not to Stat3 (lanes 2 and 14), interfered with this binding. These results suggest that low expression of IL-12R2 in lepromatous patients is not sufficient for IL-12 signaling.

View larger version (73K): [in this window] [in a new window]   FIGURE 4. IL-12-induced patterns of hSIE binding complexes in M. leprae-stimulated T cells from leprosy patients. M. leprae-activated PBMC (5 x 106) from tuberculoid (lanes 1–6) and lepromatous (lanes 7–12) patients were stimulated with IL-12 (20 ng/ml) for 20 min. Total cell extracts were prepared and examined by EMSA using the hSIE oligonucleotide probe. Arrow points to bands forming Stat4-containing complexes. Anti-Stat4 Ab (lanes 3, 6, 9, 12, and 15) or anti-Stat3 Ab (lanes 2, 5, 8, 11, and 14) was incubated with the cell extracts for 30 min before addition of the probe. D103.5, an IL-12-responsive Th1 cell clone, was used as a positive control (lanes 13–15). The results shown were obtained from a single experiment with one tuberculoid and one lepromatous patient and are representative of two similar experiments that gave similar results.

Because the DNA-binding activity of STATs requires tyrosine phosphorylation (28), we next sought to determine whether IL-12 could induce Stat4 phosphorylation in leprosy patients. PBMC from four tuberculoid and four lepromatous patients were cultured with M. leprae for 5 days, then stimulated with IL-12. Total cell lysates were immunoprecipitated with Stat4 Ab, resolved by SDS-PAGE, and blotted with anti-phosphotyrosine and anti-Stat4 Abs. IL-12 induced tyrosine phosphorylation in samples from all four tuberculoid patients (Fig. 5a, lanes 1–4). In contrast, IL-12 did not induce Stat4 phosphorylation in any of the four samples from the lepromatous patients (Fig. 5a, lanes 5–8). Immunoblotting with anti-Stat4 Ab demonstrated that the lack of phosphorylation in the lepromatous samples was not due to the lack of Stat4 in these samples.

View larger version (39K): [in this window] [in a new window]   FIGURE 5. M. leprae and M. tuberculosis differentially regulate IL-12-induced Stat4 phosphorylation in leprosy patients. a, M. leprae-activated PBMC (2 x 107) from four tuberculoid (1–4) and four lepromatous (5–8) patients were stimulated with IL-12 (20 ng/ml) for 20 min. Total cell lysates were immunoprecipitated with Stat4 Ab, separated by SDS-PAGE (8%) gel, transferred to nitrocellulose, and probed with the anti-phosphotyrosine (anti-PY) reagent RC20. After exposure, blots were stripped and reprobed with anti-Stat4 Ab. b, Parallel experiment performed with M. tuberculosis-activated PBMC from four tuberculoid (1–4) and four lepromatous (5–8) patients.

There are at least two possible explanations for the inability of IL-12 to activate Stat4 phosphorylation and DNA binding in lepromatous patients. Either the levels of IL-12R2 are insufficient to permit IL-12 signaling or the lepromatous patients have some primary defect in their IL-12 signaling pathway. Because we observed that PBMC from lepromatous patients could respond to M. tuberculosis and up-regulate the IL-12R2, we sought to determine whether this up-regulation permitted IL-12 signaling. Therefore, we stimulated PBMC from tuberculoid and lepromatous patients with M. leprae or M. tuberculosis and cultured them for 5 days. Subsequently, the nonadherent cells were collected and stimulated with IL-12. Total cell lysates from these nonadherent cells were then used for immunoprecipitation with Stat4 Ab before analysis by immunoblotting with either anti-phosphotyrosine or anti-Stat4 Ab.

In four of four tuberculoid patients, IL-12 induced Stat4 phosphorylation in both M. leprae- and M. tuberculosis-activated samples (Fig. 5, a and b, lanes 1–4). In lepromatous patients, although IL-12 could not induce Stat4 phosphorylation in four of four samples activated by M. leprae (Fig. 5a, lanes 5–8), when cells were activated with M. tuberculosis IL-12 induced phosphorylation of Stat4 proteins in all four patients (Fig. 5b, lanes 5–8). Our finding suggests that in lepromatous patients, the lack of IL-12 signaling is not likely due to a functional defect in the IL-12R2. Instead, the lack of the IL-12 signaling in these patients may be due to the inability of the lepromatous patients to mount an appropriate Th response to M. leprae, leading to insufficient level of the IL-12R2 expression.

IL-12-induced IFN- production correlates with Ag responsiveness in leprosy

Given that IL-12 is pivotal to the generation of a Th1 cytokine response, and particularly IFN- production, we studied whether IL-12 responsiveness correlated with the production of IFN-. PBMC from tuberculoid and lepromatous leprosy patients were activated with either M. leprae or M. tuberculosis for 5 days and stimulated with IL-12 for 24 h, and the level of IFN- in cultured supernatant fluids was determined. As shown in Fig. 6, PBMC from tuberculoid patients (n = 3) activated with M. leprae or M. tuberculosis produced significant levels of IFN- upon IL-12 stimulation. In contrast, only M. tuberculosis- but not M. leprae-activated PBMC from lepromatous patients (n = 3) produced significant amounts of IFN- upon IL-12 stimulation. Therefore, IL-12 signaling appears to regulate the production of IFN- and correlates with IL-12R2 expression in leprosy patients.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. IL-12 induction of IFN- production in activated T cells from leprosy patients. M. leprae- and M. tuberculosis-activated PBMC (1 x 106) from three tuberculoid (BT1–3) and three lepromatous patients (LL1–3) were stimulated with IL-12 (20 ng/ml). Culture supernatant fluids were collected after 24 h, and the level of IFN- was determined by ELISA. IFN- levels are expressed in nanograms per milliliter. , M. leprae-activated cells; , M. tuberculosis-activated cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It has become increasingly evident that IL-12 is a key regulator of Th1 cytokine responses. The ability of IL-12 to stimulate Th1 responses requires the expression of the IL-12R on T cells; however, the two receptor chains are differentially regulated. The IL-12R1 is constitutively expressed on T cells; in contrast, the IL-12R2 is selectively expressed on Th1 but not Th2 cells. In our previous study, we demonstrated that M. leprae-specific T cells from tuberculoid leprosy lesions, which produce the Th1 cytokine pattern, proliferate in response to IL-12, whereas T cells from lepromatous leprosy lesions, which produce the Th2 cytokine pattern, do not (14). Furthermore, IL-12 could not reverse Ag unresponsiveness of PBMC from many lepromatous donors. In our present study, we also found that the loss of IL-12 responsiveness in lepromatous T cells correlated with the loss of IL-12 signaling, as measured by the lack of Stat4 phosphorylation and DNA binding activity. Thus, differential maintenance of IL-12R2 mRNA and cell surface protein expression correlates with the difference in IL-12 responsiveness observed between the two groups of patients. Together these data indicate that M. leprae-induced up-regulation of functional IL-12R2 occurs in tuberculoid but not in lepromatous patients and provide a mechanism for IL-12 responsiveness and unresponsiveness in leprosy.

Several recent studies have demonstrated the importance of IL-12R expression in mouse models and human diseases. IL-12- and IL-12R-deficient mice are immunodeficient, unable to produce cell-mediated immune responses to infection by sublethal doses of Listeria (29). Individuals with mutations in the IL-12R genes have increased susceptibility to infection by Mycobacteria and Salmonella (30, 31). In studying patients with tuberculosis and sarcoidosis, lung T cells were found to express high levels of IL-12R2 in comparison to normal controlled subjects (32). Yet there is a recent report of an IL-12R1-independent pathway of IL-12 responsiveness in human T cells (33). Here we demonstrate that unresponsiveness in lepromatous patients is in part due to insufficient IL-12R2 expression, providing additional evidence for the importance of IL-12R in human infection.

In this study, we did not investigate the differential regulation of IL-12R on CD4⁺ T cells in comparison to CD8⁺ T cells, because previous studies have indicated that M. leprae-activated PBMC are mostly CD4⁺ T cells (25). However, there is evidence that CD4⁺ and CD8⁺ T cells regulate IL-12R expression by distinct mechanisms. A recent study by Elloso et al. (34) demonstrated that CD28 regulates IL-12R1 expression in CD4⁺ T cells but not in CD8⁺ T cells. Therefore, it would be important to study the regulation of IL-12R expression and function in different T cell subsets. Additional studies are needed to clarify the regulation of IL-12R in CD4⁺ and CD8⁺ T cells.

Although the lepromatous patients lack the appropriate CMI response to M. leprae, most of these patients exhibit normal CMI to a variety of bacterial Ags, including M. tuberculosis (25, 26, 27). Our results further support these previous findings because PBMC from lepromatous patients exhibited intact IL-12 responsiveness when cells were initially stimulated with M. tuberculosis. M. tuberculosis-activated lymphocytes derived from lepromatous patients demonstrated increased IL-12R2 cell surface expression on T cells. Furthermore, intact IL-12 signaling was demonstrated by the phosphorylation of Stat4 in M. tuberculosis-activated cell lysates. These data suggest that lepromatous patients do not have a genetic defect in the structure or function of the IL-12R but rather they are unable to respond to M. leprae and mount appropriate Th response to up-regulate the IL-12R expression.

The mechanism behind the intact IL-12 signaling in T cells from lepromatous patients stimulated with M. tuberculosis vs M. leprae appears to be dependent on the T helper differentiation state. Human and animal studies have demonstrated that Th1 cells up-regulate IL-12R2 whereas Th2 cells do not. In addition, intact IL-12 responsiveness to Ag occurs in Th1 cells and not in Th2 cells (8, 9). Similarly, T cells from lepromatous patients produce IFN- (35) when stimulated with M. tuberculosis Ags, and our data demonstrate that these Th1 cells have intact IL-12R function. In contrast, when T cells from lepromatous patients are stimulated with M. leprae sonicate, there is little IFN- production (14); we show here that these Th2 cells do not respond to IL-12. Therefore, our findings support previous studies because Th1/Th2 differentiation appears to regulate IL-12R expression and function in leprosy.

Exactly what factors influence Th1/Th2 differentiation remains unclear. The dose of Ag, route of Ag delivery, and genetic components including HLA haplotype influence Th1/Th2 development. One of the most clearly defined factors that influences Th1/Th2 pathway are cytokines present at the initiation of the immune response at the ligation of the TCR.

Two cytokines, IL-12 and IL-4, have been implicated as the critical inducer of the Th1 and Th2 pathway, respectively. IL-12 responsiveness may depend on these local cytokines to modulate the expression of the IL-12R. During the Th2 development in BALB/c mice, early IL-4 production induced upon Leishmania major infection is thought to be responsible for the down-regulation of the IL-12R2 (36). In human leprosy infection, a similar mechanism may lead to the blocking of IL-12 signaling seen in lepromatous patients. However, the presence of IL-4 does not appear to be the primary mechanism behind the down-regulation of IL-12R2 expression, because a previous study from our laboratory found low levels of IL-4 when T cells from lepromatous patients were stimulated with M. leprae in vitro (14). Recently a number of Th1/Th2 specific transcription factors have been identified. GATA-3 is a Th2-specific transcription factor that appears to promote IL-5 and IL-13 production (37, 38). Furthermore, GATA-3 has been shown to inhibit IFN- production through down-regulation of IL-12R expression, thus leading T cells to IL-12 unresponsiveness (39). Whether GATA-3 is involved in down-regulation of IL-12R and inhibition of IL-12 signaling in lepromatous patients is unclear. In addition, the induction of T-bet, a Th1 specific transcription factor known to initiate Th1 immune response (40), may direct IL-12 responsiveness. Additional studies to elucidate factors that control the expression and function of IL-12R will be important in developing effective strategy in combating human infectious disease.

	Acknowledgments

 
We thank Dr. David H. Presky (Hoffmann-LaRoche) for providing the IL-12R1 and 2 Abs, Dr. Frederick Beddingfield for assistance with the statistical analysis, and Dr. Cheryl Hertz and Dr. Peter Sieling for helpful comments and suggestions.

	Footnotes

 
¹ This work was supported by grants from the National Institutes of Health (AI22553, AR40312, and AI07118) and the United Nations Development Program/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases (International Research Network on Immunology of Leprosy). J.K. is the recipient of a National Institutes of Health National Research Service Award fellowship.

² Address correspondence and reprint requests to Dr. Robert L. Modlin, University of California, Division of Dermatology, 52-121 Center for Health Sciences, 10833 Le Conte Avenue, Los Angeles, CA 90095. E-mail address: rmodlin{at}mednet.ucla.edu

³ Abbreviations used in this paper: CMI, cell-mediated immunity; MFI, mean fluorescence intensity; hSIE, high affinity serum-inducible element.

Received for publication March 13, 2001. Accepted for publication May 14, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Heinzel, F. P., D. S. Schoenhaut, R. M. Rerko, L. E. Rosser, M. K. Gately. 1993. Recombinant interleukin 12 cures mice infected with Leishmania major. J. Exp. Med. 177:1505.[Abstract/Free Full Text]
2. Sypek, J. P., C. L. Chung, S. H. E. Mayor, S. M. Subramanyam, S. J. Goldman, D. S. Sieburth, S. F. Wolf, R. G. Schaub. 1993. Resolution of cutaneous leishmaniasis: interleukin 12 initiates a protective T helper type 1 response. J. Exp. Med. 177:1797.[Abstract/Free Full Text]
3. Afonso, L. C., T. M. Scharton, L. Q. Vieira, M. Wysocka, G. Trinchieri, P. Scott. 1994. The adjuvant effect of interleukin-12 on a vaccine against Leishmania major. Science 263:235.[Abstract/Free Full Text]
4. Flynn, J. L., M. M. Goldstein, K. J. Triebold, J. Sypek, S. Wolf, B. R. Bloom. 1995. IL-12 increases resistance of BALB/c mice to Mycobacterium tuberculosis infection. J. Immunol. 155:2515.[Abstract]
5. Cooper, A. M., A. D. Roberts, E. R. Rhoades, J. E. Callahan, D. M. Getzy, I. M. Orme. 1995. The role of interleukin-12 in acquired immunity to Mycobacterium tuberculosis infection. Immunology 84:423.[Medline]
6. Presky, D. H., H. Yang, L. J. Minetti, A. O. Chua, N. Nabavi, C. Y. Wu, M. K. Gately, U. Gubler. 1996. A functional interleukin 12 receptor complex is composed of two -type cytokine receptor subunits. Proc. Natl. Acad. Sci. USA 93:14002.[Abstract/Free Full Text]
7. Wu, C., R. R. Warrier, X. Wang, D. H. Presky, M. K. Gately. 1997. Regulation of interleukin-12 receptor 1 chain expression and interleukin-12 binding by human peripheral blood mononuclear cells. Eur. J. Immunol. 27:147.[Medline]
8. Rogge, L., L. Barberis-Maino, M. Biffi, N. Passini, D. H. Presky, U. Gubler, F. Signaglia. 1997. Selective expression of an interleukin-12 receptor component by human T helper 1 cells. J. Exp. Med. 185:825.[Abstract/Free Full Text]
9. Szabo, S. J., A. S. Dighe, U. Gubler, K. M. Murphy. 1997. Regulation of the interleukin (IL)-12R2 subunit expression in developing T helper 1 (Th1) and Th2 cells. J. Exp. Med. 185:817.[Abstract/Free Full Text]
10. Bacon, C. M., D. W. McVicar, J. R. Ortaldo, R. C. Rees, J. J. O’Shea, J. A. Jonston. 1995. Interleukin 12 (IL-12) induces tyrosine phosphorylation of JAK2 and TYK2: differential use of Janus family tyrosine kinases by IL-2 and IL-12. J. Exp. Med. 181:399.[Abstract/Free Full Text]
11. Zou, J., D. H. Presky, C. Y. Wu, U. Gubler. 1997. Differential associations between the cytoplasmic regions of the interleukin-12 receptor subunits 1 and 2 and JAK kinases. J. Biol. Chem. 272:6073.[Abstract/Free Full Text]
12. Jacobson, N. G., S. J. Szabo, R. M. Weber-Nordt, Z. Zhong, R. D. Schreiber, J. E. Darnell, K. M. Murphy. 1995. Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4. J. Exp. Med. 181:1755.[Abstract/Free Full Text]
13. Bacon, C. M., E. F. Petricoin, J. R. Ortaldo, R. C. Rees, A. C. Larner, J. A. Jonston, J. J. O’Shea. 1995. Interleukin 12 induces tyrosine phosphorylation and activation of STAT4 in human lymphocytes. Proc. Natl. Acad. Sci. USA 92:7307.[Abstract/Free Full Text]
14. Sieling, P. A., X.-H. Wang, M. K. Gately, J. L. Oliveros, T. McHugh, P. F. Barnes, S. F. Wolf, L. Golkar, M. Yamamura, Y. Yogi, et al 1994. IL-12 regulates T helper type 1 cytokine responses in human infectious disease. J. Immunol. 153:3639.[Abstract]
15. Ridley, D. S., W. H. Jopling. 1966. Classification of leprosy according to immunity: a five-group system. Int. J. Lepr. 34:255.
16. Beckman, E. M., A. Melian, S. M. Beha, P. A. Sieling, D. Chatterjee, S. T. Furlong, R. Matsumoto, J. P. Sosat, R. L. Modlin, S. A. Porcelli. 1996. CD1c restricts responses of mycobacteria-specific T cells: evidence for antigen presentation by a second member of the human CD1 family. J. Immunol. 157:2795.[Abstract]
17. Yamamura, M., K. Uyemura, R. J. Deans, K. Weinberg, T. H. Rea, B. R. Bloom, R. L. Modlin. 1991. Defining protective responses to pathogens: cytokine profiles in leprosy lesions. Science 254:277.[Abstract/Free Full Text]
18. Yamamura, M., X.-H. Wang, J. D. Ohmen, K. Uyemura, T. H. Rea, B. R. Bloom, R. L. Modlin. 1992. Cytokine patterns of immunologically mediated tissue damage. J. Immunol. 149:1470.[Abstract]
19. Kim, J., A. Sette, S. Rodda, S. Southwood, P. A. Sieling, V. Mehra, J. D. Ohmen, J. Oliveros, E. Appella, Y. Higashimoto, et al 1997. Determinants of T cell reactivity to the Mycobacterium leprae GroES homologue. J. Immunol. 159:335.[Abstract]
20. Yamauchi, P. S., J. R. Bleharski, K. Uyemura, J. Kim, P. A. Sieling, A. Miller, H. Brightbill, K. Schilienger, T. H. Rea, R. L. Modlin. 2000. A role for CD40-CD40 ligand interactions in the generation of type 1 cytokine responses in human leprosy. J. Immunol. 165:1506.[Abstract/Free Full Text]
21. Khan, K. D., K. Shuai, G. Lindwall, S. E. Maher, Jr J. E. Darnell, A. L. Bothwell. 1993. Induction of the Ly-6A/E gene by interferon / and requires a DNA element to which a tyrosine-phosphorylated 91-kDa protein binds. Proc. Natl. Acad. Sci. USA 90:6806.[Abstract/Free Full Text]
22. Shuai, K., J. Liao, M. M. Song. 1996. Enhancement of antiproliferative activity of interferon by the specific inhibition of tyrosine dephosphorylation of Stat1. Mol. Cell. Biol. 16:4932.[Abstract]
23. Zhong, Z., Z. Wen, Jr J. E. Darnell. 1994. Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science 264:95.[Abstract/Free Full Text]
24. Greenlund, A. C., M. A. Farrar, B. L. Viviano, R. D. Schreiber. 1994. Ligand-induced IFN- receptor tyrosine phosphorylation couples the receptor to its signal transduction system (p91). EMBO J. 13:1591.[Medline]
25. Bach, M. A., D. Wallach, B. Flageul, F. Cottenot. 1983. In vitro proliferative response to M. leprae and PPD of isolated T cell subsets from leprosy patients. Clin. Exp. Immunol. 52:107.[Medline]
26. Sengupta, S. R., V. L. Yemul, T. N. Dhole. 1983. Lymphocyte transformation test in lepromatous patients and their healthy siblings. Lepr. India 55:261.[Medline]
27. Shankar, P., F. Agis, D. Wallach, B. Flageul, F. Cottenot, J. Augier. 1986. M. leprae and PPD-triggered T cell lines in tuberculoid and lepromatous leprosy. J. Immunol. 136:4255.[Abstract]
28. Shuai, K., C. M. Horvath, L. H. Huang, S. A. Qureshi, D. Cowburn, Jr J. E. Darnell. 1994. Interferon activation of the transcription factor Stat91 involves dimerization through SH2-phosphotyrosyl peptide interactions. Cell 76:821.[Medline]
29. Brombacher, F., A. Dorfmuller, J. Magram, W. J. Dai, G. Kohler, A. Wunderlin, K. Palmer-Lehmann, M. K. Gately, G. Alber. 1999. IL-12 is dispensable for innate and adaptive immunity against low doses of Listeria monocytogenes. Int. Immunol. 11:325.[Abstract/Free Full Text]
30. De Jong, R., F. Altare, I.-A. Haagen, D. G. Elferink, T. de Boer, P. J. C. van Breda Vriesman, P. J. Kabel, J. M. Draaisma, J. T. van Dissel, F. P. Kroon, et al 1998. Severe mycobacterial and Salmonella infections in interleukin-12 receptor deficient patients. Science 280:1435.[Abstract/Free Full Text]
31. Altare, F., A. Durandy, D. Lammas, J.-F. Emile, S. Lamhamedi, F. Le Deist, P. Drysdale, E. Jouanquy, R. Doffinger, F. Bernaudin, et al 1998. Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency. Science 280:1432.[Abstract/Free Full Text]
32. Taha, R. A., E. M. Minshall, R. Olivenstein, D. Ihaku, B. Wallaert, A. Tsicopoulos, A. B. Tonnel, R. Damia, D. Menzies, Q. A. Hamid. 1999. Increased expression of IL-12 receptor mRNA in active pulmonary tuberculosis and sarcoidosis. Am. J. Respir. Crit. Care. Med. 160:1119.[Abstract/Free Full Text]
33. Verhagen, C. E., T. de Boer, H. H. Smits, F. A. Verreck, E. A. Wierenga, M. Kurimoto, D. A. Lammas, D. S. Kumararatne, O. Sanal, F. P. Kroon, et al 2000. Residual type 1 immunity in patients genetically deficient for interleukin 12 receptor B1 (IL-12RB1): evidence for an IL-12RB1-independent pathway of IL-12 responsiveness in human T cells. J. Exp. Med. 192:517.[Abstract/Free Full Text]
34. Elloso, M. M., P. Scott. 2001. Differential requirement of CD28 for IL-12 receptor expression and function in CD4⁺ and CD8⁺ T cells. Eur. J. Immunol. 31:384.[Medline]
35. Shinde, S. R., S. V. Chiplunkar, R. Butlin, P. D. Samson, M. G. Deo, S. G. Gangal. 1993. Lymphocyte proliferation, IFN- production and limiting dilution analysis of T-cell responses to ICRC and Mycobacterium leprae antigens in leprosy patients. Int. J. Lepr. Other Mycobact. Dis. 61:51.[Medline]
36. Himmelrich, H., P. Launois, I. Maillard, T. Biedermann, F. Tacchini-Cottier, R. M. Locksley, M. Rocken, J. A. Louis. 2000. In BALB/c mice, IL-4 production during the initial phase of infection with Leishmania major is necessary and sufficient to instruct Th2 cell development resulting in progressive disease. J. Immunol. 164:4819.[Abstract/Free Full Text]
37. Zhang, D. H., L. Yang, A. Ray. 1998. Cutting edge: differential responsiveness of the IL-5 and IL-4 genes to transcription factor GATA-3. J. Immunol. 161:3817.[Abstract/Free Full Text]
38. Zhang, D. H., L. Yang, L. Cohn, L. Parkyn, R. Homer, P. Ray, A. Ray. 1999. Inhibition of allergic inflammation in a murine model of asthma by expression of a dominant-negative mutant of GATA-3. Immunity 11:473.[Medline]
39. Ouyang, W., S. H. Ranganath, K. Weindel, D. Bhattacharya, T. L. Murphy, W. C. Sha, K. M. Murphy. 1998. Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent mechanism. Immunity 9:745.[Medline]
40. Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman, L. H. Glimcher. 2000. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100:655.[Medline]

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