Leprosy Signal Transduction in Host Defense in a Role for IL-12

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

This information is current as Jenny Kim, Koichi Uyemura, Melissa K. Van Dyke, of September 25, 2021. Annaliza J. Legaspi, Thomas H. Rea, Ke Shuai and Robert L. Modlin J Immunol 2001; 167:779-786; ; doi: 10.4049/jimmunol.167.2.779

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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. Modlin2*†

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-12R␤1/IL-12R␤2 heterodimer, with the IL-12R␤2 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-12R␤2 was more highly expressed in tuberculoid lesions compared with lepromatous lesions. In contrast, IL-12R␤1 expression was similar in both tuberculoid and Downloaded from lepromatous lesions. The expression of IL-12R␤2 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-12R␤2 in lepromatous patients could be up-regulated by stimulation with M. tuberculosis. These data suggest that Th response to M. leprae determines IL-12R␤2 expression and function in host defense in leprosy. The Journal of Immunology, 2001, 167: 779–786. http://www.jimmunol.org/ he outcome of infection in murine models of infection and induced by IL-12 and IFN-␣ and inhibited by IL-4 (8, 9), suggest- in human infectious disease is regulated by the Th1 and ing that local cytokine milieu plays a role in determining IL-12 T Th2 T cell cytokine patterns. Th1 cells secrete IL-2 responsiveness of T cells during infection. and IFN-␥ and are generally associated with resistance to intra- Binding of IL-12 to the IL-12R results in activation of Janus cellular pathogens, whereas Th2 cells secrete IL-4 and IL-10 and kinases Tyk2 and Jak2, leading to tyrosine phosphorylation and are associated with progressive disease to these same pathogens. It DNA binding of Stat4 with subsequent IFN-␥ production by T has become increasingly evident that IL-12 is a pivotal regulator of cells (10, 11). Studies in both human and murine models have Th1 responses and is essential for promoting cell-mediated immu- shown that IL-12 induces tyrosine phosphorylation of Stat4 in Th1 nity (CMI)3 against intracellular microbial pathogens (1–5). cells but not in Th2 cells (8, 9, 12, 13), indicating a functional by guest on September 25, 2021 The ability of IL-12 to activate lymphocytes is mediated by the difference imparted by the expression levels of the IL-12R in these IL-12R, a heterodimer composed of IL-12R␤1 and IL-12R␤2 sub- T cell subsets. units (6, 7). The IL-12R␤1 is constitutively expressed on both Th1 To study the mechanism of responsiveness to IL-12 in human and Th2 cells. The IL-12R␤2, in contrast, is expressed more infectious disease, we chose leprosy as a model because of its strongly on Th1 cells as compared with Th2 cells, indicating a spectrum of clinical manifestations that correlate with the level of mechanism by which IL-12 differentially affects the growth of CMI to the pathogen Mycobacterium leprae. At one end of the these subsets. Studies involving the regulation of IL-12R have spectrum, patients with tuberculoid leprosy mount a strong CMI shown that Ag receptor triggering is sufficient to induce expression and are resistant to M. leprae. At the opposite end of the spectrum, of IL-12R␤1 and IL-12R␤2 (8). In humans, IL-12R␤2 chain is 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 *Division of Dermatology, and †Department of Microbiology and Immunology, Uni- IL-12, whereas T cells isolated from lepromatous patients do not versity of California School of Medicine, Los Angeles, CA 90095; ‡Section of Der- respond to IL-12 (14). Here, we present evidence that expression matology, University of Southern California School of Medicine, Los Angeles, CA and up-regulation of IL-12R␤2 correlates with CMI, being greater 90033; and §Division of Hematology-Oncology, University of California School of Medicine, Los Angeles, CA 90095 in tuberculoid than in lepromatous patients. IL-12 signaling in tu- Received for publication March 13, 2001. Accepted for publication May 14, 2001. berculoid leprosy patients correlates with the expression of the ␤ The costs of publication of this article were defrayed in part by the payment of page IL-12R 2 subunit. The lack of IL-12 responsiveness in leproma- charges. This article must therefore be hereby marked advertisement in accordance tous patients appears to be in part due to an Ag-specific unrespon- with 18 U.S.C. Section 1734 solely to indicate this fact. siveness to M. leprae and due to inappropriately differentiated Th 1 This work was supported by grants from the National Institutes of Health (AI22553, cells. Our data suggest that Th response to Ag determines IL- AR40312, and AI07118) and the United Nations Development Program/World Bank/ ␤ World Health Organization Special Program for Research and Training in Tropical 12R 2 expression and function in the generation of CMI to mi- Diseases (International Research Network on Immunology of Leprosy). J.K. is the crobial infection. recipient of a National Institutes of Health National Research Service Award fellowship. Materials and Methods 2 Address correspondence and reprint requests to Dr. Robert L. Modlin, University of Patients and clinical specimens California, Division of Dermatology, 52-121 Center for Health Sciences, 10833 Le Conte Avenue, Los Angeles, CA 90095. E-mail address: [email protected] Patients with leprosy were diagnosed at the Los Angeles County/Univer- 3 Abbreviations used in this paper: CMI, cell-mediated immunity; MFI, mean fluo- sity of Southern California Medical Center Hansen’s Disease Clinic (Los rescence intensity; hSIE, high affinity serum-inducible element. Angeles, CA) and classified according to the clinical and pathologic criteria

of Ridley and Jopling (15). After receiving informed consent, venous blood clone, D103.5 (19, 20), derived from a tuberculoid lesion and specific for was collected in heparinized tubes from tuberculoid and lepromatous lep- M. leprae 10-kDa protein, was used as a positive control. This T cell clone rosy patients. PBMC were isolated using Ficoll-Paque (Amersham Phar- produces IFN-␥ in response to IL-12 (data not shown). Cells were washed macia Biotech, Piscataway, NJ) differential centrifugation. with PBS and stimulated with and without IL-12 (20 ng/ml; R&D Systems, Skin biopsies from leprosy patients were also obtained, embedded in Minneapolis, MN) for 20 min before preparation of extracts, and EMSA OCT medium (Ames, Elkhart, IN), snap-frozen in liquid nitrogen, and was performed as previously described (21, 22). Briefly, whole cell extracts stored at Ϫ80°C until ready to use. 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 Antigens DTT, 0.1 mM sodium vanadate, 1 mM PMSF, 0.5 ␮g/ml leupeptin, and 3 ␮g of aprotinin per milliliter. Prepared extracts were incubated with a M. leprae and M. tuberculosis (strain H37Rv) were provided by P. Brennan 32P-end-labeled double-stranded oligodeoxynucleotide, high affinity se- (Colorado State University, Fort Collins, CO) and prepared by probe son- rum-inducible element (hSIE), 5Ј-GTCGACATTTCCCGTAAATCG ication (16). The level of endotoxin in M. leprae and M. tuberculosis was TCGA-3Ј (23) in binding buffer for 20 min at room temperature before measured quantitatively with a Limulus Amoebocyte Lysate assay (Bio- electrophoresis on 5% polyacrylamide gel. When used, 2 ␮l of anti-Stat4 Whittaker, Walkersville, MD) and found to be Ͻ0.1 ng/ml. and anti-Stat3 Abs (Santa Cruz Biotechnology, Santa Cruz, CA) were in- Polymerase chain reaction cubated with cell extracts for 30 min at room temperature before the addition of probe. Total RNA from skin biopsy specimens was isolated after lysing samples in guanidinium isothiocyanate buffer as previously described (17, 18), and Immunoprecipitation and SDS-PAGE cDNA was synthesized with reverse transcriptase (Life Technologies, 7 Gaithersburg, MD). Total cellular lysates of 2 ϫ 10 cells were immunoprecipitated with anti- cDNA samples were amplified in a DNA Thermocycler (Perkin-Elmer, Stat4 Ab as previously described (24) and were separated by electrophore-

San Diego, CA) for 40 cycles with each cycle consisting of denaturation at sis on 8% SDS-PAGE. After transfer to nitrocellulose, blots were probed Downloaded from 95°C for 20 s and annealing/extension at 65°C for 45 s. Each PCR mixture with anti-phosphotyrosine Ab, 4G10 (Upstate Biotechnology, Lake Placid, Ј Ј NY). Blots were then stripped and reprobed with anti-Stat4 Ab. 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, 5Ј and 3Ј, were as follows: IL-12R␤1, IFN-␥ production 5Ј-CTGTTTTCAGGACCCGCCATATCC-3Ј and 5Ј-AGAGTGTGACA Ninety-six-well ELISA plates (Corning Glass Works, Corning, NY) were GTGTACAGCACAG-3Ј; IL-12R␤2, 5Ј-GAGGGACTGGTACTGCTTA coated overnight at 4°C with 100 ␮l of mouse anti-human IFN-␥ (PharM- ATCGACTC-3Ј and 5Ј-CCTCACACAGGTTCATTATGTTAA-3Ј; and ingen) at 5 ␮g/ml per well. Plates were blocked with 200 ␮lof1%BSA http://www.jimmunol.org/ CD3␦,5Ј-CTGGACCTGGGAAAACGCATC-3Ј and 5Ј-GTACTGAG and 0.05% Tween 20 in PBS for2hatroom temperature. Aliquots (100 ␮l) CATCATCTCGATC-3Ј. cDNA concentrations were normalized to yield of each sample or of IFN-␥ standards (Endogen, Woburn, MA) were then equivalent CD3␦ PCR products. added to each well and incubated at room temperature for 2 h. Biotin- To verify IL-12R␤1 and IL-12R␤2 mRNA, PCR products were trans- conjugated mouse anti-human IFN-␥ mAb (PharMingen) was added to ferred to Hybond-N nylon membranes (Amersham Pharmacia Biotech) as each well and incubated for 1 h, followed by a 30-min incubation with previously described (17, 18) and probed with a labeled oligonucleotide streptavidin/peroxidase (Pierce, Rockford, IL). Peroxidase substrate solu- complementary to nucleotides within the sequences recognized by the PCR tion (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was used to amplification primers. Sequences of the oligonucleotide probes were as detect IFN-␥, and the plates were read at a wavelength of 405 nm in a 7520 follows: IL-12R␤1, 5Ј-GAGATGCTATCGGATATCCAGTGATCG-3Ј; microplate reader (Cambridge Technology, Cambridge, MA). All samples IL-12R␤2, 5Ј-TCTCCACTATCAGGTGACCTTGCA-3Ј; and CD3␦,5Ј- and standards were performed in duplicate. GCCGACACACAAGCTCTGTTGAGGA-3Ј. The relative intensity of by guest on September 25, 2021 PCR bands was assessed by densitometric analysis of the digitized image, performed on a Macintosh computer (Apple Computers, Cupertino, CA) Results using the public domain NIH Image program (developed at the U.S. Na- IL-12R expression in leprosy lesions correlates with CMI tional Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). To determine the relative expression of IL-12R subunits in leprosy patients, we performed RT-PCR on biopsy specimens from skin Flow cytometry lesions of patients with leprosy. RNA was extracted from biopsy PBMC (1 ϫ 106 cells/ml) from tuberculoid and lepromatous patients were specimens, then cDNA was synthesized and normalized to the cultured in complete medium (RPMI 1640, 0.1 mM sodium pyruvate, 2 amounts of CD3-␦ PCR products as a measure of total cellular ␮ mM penicillin, 50 g/ml streptomycin; Life Technologies, Grand Island, RNA in T cells. As shown in Fig. 1, the inducible IL-12R␤2 NY) supplemented with 10% human serum in a 24-well plate. Cells were stimulated with and without Ag for 5 days. Lymphocytes were recovered, mRNA was highly expressed in 10 of 10 tuberculoid lesions but washed twice in PBS, and resuspended in FACS buffer containing PBS only weakly expressed in 10 of 10 lepromatous lesions. In contrast, with 2% FBS and 0.1% sodium azide. Monoclonal Ab to IL-12R␤1 and the constitutively expressed IL-12R␤1 subunit was equally ex- ␤ IL-12R 2 (provided by D. H. Presky, Hoffmann-LaRoche, Nutley, NJ) or pressed in both tuberculoid and lepromatous lesions. These data isotype-matched control, IgG2a, (PharMingen, San Diego, CA) was added ␤ at a final concentration of 2 ␮g/ml to the cells and incubated at 4°C for 45 indicate that the expression of IL-12R 2 in leprosy correlates with min. The cells were washed twice with FACS buffer and incubated with host resistance to infection, greatest in the group of patients known biotin-conjugated anti-rat IgG (Jackson ImmunoResearch Laboratories, to manifest strong CMI to M. leprae. West Grove, PA) for 30 min, followed by final incubation with streptavidin-PE (Jackson ImmunoResearch Laboratories) for 30 min. After further IL-12R␤2 expression correlates with Ag responsiveness in washing, the cells were fixed in 2% paraformaldehyde, and flow cytometry analysis was performed using a FACScan (BD Biosciences, Mountain T cells View, CA). The expression of the IL-12R subunits was measured as flu- Because Ag stimulation has been shown to enhance the expression orescence intensity using WinMDI software (provided by J. Trotter, The of the IL-12R on T cells (7), we determined whether addition of M. Scripps Institute, La Jolla, CA). The up-regulation of the IL-12R␤1 and IL-12R␤2 expression by Ag stimulation was determined by calculating the leprae could modulate the expression of IL-12R subunits on T difference in the mean fluorescence intensity (⌬MFI) between Ag-stimu- cells from leprosy patients and explain the differential expression lated cells and unstimulated cells. ⌬MFI for IL-12R␤1 and IL-12R␤2 was in leprosy lesions. PBMC from tuberculoid and lepromatous pa- compared between tuberculoid and lepromatous patients using the Wil- tients were cultured with and without M. leprae for 5 days, and the coxon rank sum test for unpaired variables. Differences were considered ␤ ␤ statistically significant for p values Ͻ0.05. expression of IL-12R 1 and IL-12R 2 on the cell surface was measured by flow cytometry. When PBMC were cultured with EMSA medium alone, the expression of IL-12R␤1 and IL-12R␤2 was PBMC (5 ϫ 106) from tuberculoid and lepromatous patients were activated similar in both tuberculoid (n ϭ 9) and lepromatous (n ϭ 9) pa- with M. leprae (5 ␮g/ml) for 5 days in culture. A previously described Th1 tients, with IL-12R␤1 expression being slightly higher than the The Journal of Immunology 781 Downloaded from http://www.jimmunol.org/

FIGURE 1. IL-12 R␤1 and IL-12R␤2 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-12R␤1 and IL-12R␤2 mRNA. The PCR products were transferred to nylon membrane and probed with a radiolabeled internal oligonucleotide. The by guest on September 25, 2021 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.

expression of IL-12R␤2 in both patient groups. Representative ex- Although patients with lepromatous leprosy are unable to amples are shown in Fig. 2a. After stimulation with M. leprae,we mount the appropriate CMI response to M. leprae, this unre- found that in the tuberculoid patients both IL-12R␤1 and IL- sponsiveness is highly specific because these patients have been 12R␤2 were up-regulated. In contrast, in the lepromatous patient shown to have intact immune responses to Ags from a variety group the expression of IL-12R␤1 but not that of IL-12R␤2 could of other microbial agents, including M. tuberculosis (25–27). be up-regulated by M. leprae. Therefore, to determine whether the ability to up-regulate IL- In nine tuberculoid patients, Ag stimulation up-regulated the 12R␤2 was fundamentally altered in these patients, we asked ␤ ␤ ⌬ expression of both IL-12R 1 and IL-12R 2. The MFI between whether M. tuberculosis could up-regulate IL-12R␤2 expres- M. leprae-stimulated and unstimulated lymphocytes ranged be- sion in lepromatous patients. PBMC from tuberculoid and lep- ␤ ␤ tween 3 and 22 for IL-12R 1 (Fig. 2b). For IL-12R 2 expression, romatous patients were stimulated with either M. leprae or M. the ⌬MFI varied between 1.6 and 13.3. In lepromatous patients, tuberculosis for 5 days, and levels of IL-12R␤2 expression were the IL-12R␤1 expression was up-regulated by the addition of M. determined by FACS. Representative histograms from each pa- leprae in six of nine patients, and the ⌬MFI varied between 1.8 tient group are shown in Fig. 3. In tuberculoid patients (n ϭ 3), and 16.3. The up-regulation of IL-12R␤2 expression was even less both M. leprae and M. tuberculosis induced the expression of with only five of nine lepromatous patients showing a minimal ␤ change in the MFI (Ͻ1.1). The ⌬MFI for IL-12R␤1 expression in the IL-12R 2 when compared with medium alone. In leproma- ϭ tuberculoid patients (n ϭ 9) compared with lepromatous patients tous patients (n 3), M. leprae extract had no significant effect ␤ (n ϭ 9) was not significant ( p ϭ 0.07). In contrast, the ⌬MFI for on the expression of IL-12R 2, but the addition of M. tuber- IL-12R␤2 expression between the two groups of patients was sta- culosis extract to culture of PBMC increased the expression of tistically significant ( p ϭ 0.003). These results suggest that PBLs IL-12R␤2. In summary, although M. leprae could not induce in tuberculoid patients can respond to Ag stimulation and up-reg- IL-12R␤2 expression in PBMC from the lepromatous patients, ulate IL-12R␤1 and IL-12R␤2. In contrast, the response of lepro- activation by M. tuberculosis could lead to the up-regulation matous patients to M. leprae is limited to the up-regulation of IL-12R␤2. These data demonstrate that the inability of lepro- IL-12R␤1 expression, but not the up-regulation of IL-12R␤2 matous patients to up-regulate IL-12R␤2 correlates with their expression. ineffective Th response to M. leprae. 782 IL-12 RECEPTOR AND LEPROSY Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. Regulation of IL-12R␤1 and IL-12R␤2 expression by M. leprae. PBMC (1 ϫ 106) from tuberculoid and lepromatous patients were cultured in vitro in the absence of stimuli (medium) or with M. leprae (5 ␮g/ml) for 5 days. Cells were stained with IL-12R␤1, IL-12R␤2, or with an isotype control Ab, followed by PE-conjugated Ab. IL-12R␤1 and ␤2 expression was measured on gated lymphocytes. The M. leprae-activated T cells are mostly CD4ϩ T cells (25). a, Representative histograms showing the level of IL-12R␤1 and IL-12R␤2 expression on T cells from tuberculoid and lepromatous patients. Solid outline, staining with control Ab alone; bold outline, staining with IL-12R␤1 or IL-12R␤2 Ab. b, Summary bar graph demonstrating the modulation of MFI of lymphocytes stained with anti-IL-12R␤1 and anti-IL-12R␤2 Ab. Nine tuberculoid (BT1-BT9) and nine lepromatous (LL1-LL9) patients were tested. The value indicated is the difference in MFI (⌬MFI) in IL-12R␤1 and 12R␤2 expression between M. leprae-stimulated cells and unstimulated cells.

IL-12 induces Stat4-DNA binding in tuberculoid but not in (lanes 13–15). Arrow points to bands forming Stat4-containing lepromatous patients complexes. In contrast, PBMC from a lepromatous patient did not The IL-12 signaling pathway involves the activation of the Janus show any Stat4 binding activity (lanes 7–9) upon IL-12 stimula- kinase-STAT signal transduction pathway. Specifically, stimulation. Samples without IL-12 stimulation did not demonstrate any tion of the IL-12R by IL-12 leads to the phosphorylation and nu- binding activity in both tuberculoid (lanes 4–6) and lepromatous clear translocation of a transcription factor Stat4. Such activation (lanes 10–12) patients. Supershift experiments confirmed that of Stat4 has been shown to be essential for the IL-12-mediated these complexes contained Stat4 because Ab to Stat4 (lanes 3 and lymphocyte responses (12, 13), and this IL-12 responsiveness ap- 15), but not to Stat3 (lanes 2 and 14), interfered with this binding. ␤ pears to correlate well with the expression of IL-12R␤2 (8, 9). To These results suggest that low expression of IL-12R 2 in lepro- determine whether the low levels of IL-12R␤2 expression in lep- matous patients is not sufficient for IL-12 signaling. romatous patients is sufficient for IL-12 signaling, PBMC from tuberculoid and lepromatous patients were stimulated with M. lep- IL-12 induces Stat4 phosphorylation in tuberculoid but not in rae and activated with IL-12. Formation of Stat4-containing com- lepromatous patients plexes and binding to the oligonuclear probe hSIE, which contains Because the DNA-binding activity of STATs requires tyrosine a high affinity-binding site for several STAT proteins, were deter- phosphorylation (28), we next sought to determine whether IL-12 mined by gel shift analysis. As shown in Fig. 4, IL-12 induced could induce Stat4 phosphorylation in leprosy patients. PBMC binding activity to the hSIE probe in PBMC from a tuberculoid from four tuberculoid and four lepromatous patients were cultured patient (lanes 1–3) and in an IL-12-responsive Th1 clone, D103.5 with M. leprae for 5 days, then stimulated with IL-12. Total cell The Journal of Immunology 783 Downloaded from ␤ FIGURE 3. Modulation of IL-12R 2 expression by mycobacterial FIGURE 4. IL-12-induced patterns of hSIE binding complexes in M. ϫ 6 Ags. PBMC (1 10 ) from tuberculoid and lepromatous patients were leprae-stimulated T cells from leprosy patients. M. leprae-activated ␮ ␮ stimulated with M. leprae (5 g/ml) or with M. tuberculosis (10 g ml) PBMC (5 ϫ 106) from tuberculoid (lanes 1–6) and lepromatous (lanes for 5 days. Cells were harvested, and the cell surface expression of 7–12) patients were stimulated with IL-12 (20 ng/ml) for 20 min. Total ␤ IL-12R 2 was measured on gated lymphocytes by FACS. The results cell extracts were prepared and examined by EMSA using the hSIE shown are representative histograms demonstrating the level of IL- oligonucleotide probe. Arrow points to bands forming Stat4-containing

␤ http://www.jimmunol.org/ 12R 2 expression on T cells from three tuberculoid and three lepro- complexes. Anti-Stat4 Ab (lanes 3, 6, 9, 12, and 15) or anti-Stat3 Ab matous patients. Solid line, Staining with control Ab alone; bold line, (lanes 2, 5, 8, 11, and 14) was incubated with the cell extracts for 30 ␤ staining with IL-12R 2 Ab. 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 lysates were immunoprecipitated with Stat4 Ab, resolved by SDS- that gave similar results. 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 suggests that in lepromatous patients, the lack of IL-12 signaling is by guest on September 25, 2021 ␤ induce Stat4 phosphorylation in any of the four samples from the not likely due to a functional defect in the IL-12R 2. Instead, the lepromatous patients (Fig. 5a, lanes 5–8). Immunoblotting with lack of the IL-12 signaling in these patients may be due to the anti-Stat4 Ab demonstrated that the lack of phosphorylation in the inability of the lepromatous patients to mount an appropriate Th ␤ lepromatous samples was not due to the lack of Stat4 in these response to M. leprae, leading to insufficient level of the IL-12R 2 samples. expression. IL-12-induced IFN-␥ production correlates with Ag Activation of T cells by M. tuberculosis results in maintenance responsiveness in leprosy of functional IL-12 responsiveness in lepromatous patients Given that IL-12 is pivotal to the generation of a Th1 cytokine There are at least two possible explanations for the inability of response, and particularly IFN-␥ production, we studied whether IL-12 to activate Stat4 phosphorylation and DNA binding in lepromatous patients. Either the levels of IL-12R␤2 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-12R␤2, we sought to determine whether this up-regulation permitted IL-12 signaling. There- fore, 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 FIGURE 5. M. leprae and M. tuberculosis differentially regulate IL-12- anti-Stat4 Ab. induced Stat4 phosphorylation in leprosy patients. a, M. leprae-activated ϫ 7 In four of four tuberculoid patients, IL-12 induced Stat4 phos- PBMC (2 10 ) from four tuberculoid (1–4) and four lepromatous (5–8) patients were stimulated with IL-12 (20 ng/ml) for 20 min. Total cell ly- phorylation in both M. leprae- and M. tuberculosis-activated sam- sates were immunoprecipitated with Stat4 Ab, separated by SDS-PAGE ples (Fig. 5, a and b, lanes 1–4). In lepromatous patients, although (8%) gel, transferred to nitrocellulose, and probed with the anti- IL-12 could not induce Stat4 phosphorylation in four of four sam- phosphotyrosine (anti-PY) reagent RC20. After exposure, blots were ples activated by M. leprae (Fig. 5a, lanes 5–8), when cells were stripped and reprobed with anti-Stat4 Ab. b, Parallel experiment performed activated with M. tuberculosis IL-12 induced phosphorylation of with M. tuberculosis-activated PBMC from four tuberculoid (1–4) and Stat4 proteins in all four patients (Fig. 5b, lanes 5–8). Our finding four lepromatous (5–8) patients. 784 IL-12 RECEPTOR AND LEPROSY

12R␤2 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-12R␤2 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-12R␤2 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 Downloaded from IL-12R-deficient mice are immunodeficient, unable to produce FIGURE 6. IL-12 induction of IFN-␥ production in activated T cells cell-mediated immune responses to infection by sublethal doses of from leprosy patients. M. leprae- and M. tuberculosis-activated PBMC Listeria (29). Individuals with mutations in the IL-12R genes have (1 ϫ 106) from three tuberculoid (BT1–3) and three lepromatous patients increased susceptibility to infection by Mycobacteria and Salmo- (LL1–3) were stimulated with IL-12 (20 ng/ml). Culture supernatant fluids nella (30, 31). In studying patients with tuberculosis and sarcoid-

␥ http://www.jimmunol.org/ were collected after 24 h, and the level of IFN- was determined by osis, lung T cells were found to express high levels of IL-12R␤2 ELISA. IFN-␥ levels are expressed in nanograms per milliliter. f, M. in comparison to normal controlled subjects (32). Yet there is a leprae-activated cells; Ⅺ, M. tuberculosis-activated cells. recent report of an IL-12R␤1-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-12R␤2 expression, providing additional evidence for the IL-12 responsiveness correlated with the production of IFN-␥. importance of IL-12R in human infection. PBMC from tuberculoid and lepromatous leprosy patients were In this study, we did not investigate the differential regulation of activated with either M. leprae or M. tuberculosis for 5 days and ϩ ϩ stimulated with IL-12 for 24 h, and the level of IFN-␥ in cultured IL-12R on CD4 T cells in comparison to CD8 T cells, because by guest on September 25, 2021 previous studies have indicated that M. leprae-activated PBMC are supernatant fluids was determined. As shown in Fig. 6, PBMC ϩ ϩ ϭ mostly CD4 T cells (25). However, there is evidence that CD4 from tuberculoid patients (n 3) activated with M. leprae or M. ϩ tuberculosis produced significant levels of IFN-␥ upon IL-12 stim- and CD8 T cells regulate IL-12R expression by distinct mecha- ulation. In contrast, only M. tuberculosis- but not M. leprae-acti- nisms. A recent study by Elloso et al. (34) demonstrated that CD28 ␤ ϩ ϩ vated PBMC from lepromatous patients (n ϭ 3) produced signif- regulates IL-12R 1 expression in CD4 T cells but not in CD8 icant amounts of IFN-␥ upon IL-12 stimulation. Therefore, IL-12 T cells. Therefore, it would be important to study the regulation of signaling appears to regulate the production of IFN-␥ and corre- IL-12R expression and function in different T cell subsets. Addi- lates with IL-12R␤2 expression in leprosy patients. tional studies are needed to clarify the regulation of IL-12R in CD4ϩ and CD8ϩ T cells. Discussion Although the lepromatous patients lack the appropriate CMI re- In this study we investigated the expression and regulation of IL- sponse to M. leprae, most of these patients exhibit normal CMI to 12R to determine the mechanism of IL-12 responsiveness in pa- a variety of bacterial Ags, including M. tuberculosis (25–27). Our tients with leprosy. We found that the IL-12R␤2 subunit was results further support these previous findings because PBMC highly expressed in tuberculoid lesions but only weakly expressed from lepromatous patients exhibited intact IL-12 responsiveness in lepromatous lesions. In contrast, IL-12R␤1 expression was sim- when cells were initially stimulated with M. tuberculosis. M. tu- ilar in both tuberculoid and lepromatous lesions. We next deter- berculosis-activated lymphocytes derived from lepromatous pa- mined that in tuberculoid patients, but not in lepromatous patients, tients demonstrated increased IL-12R␤2 cell surface expression on the IL-12R␤2 could be up-regulated by stimulation of T cells with T cells. Furthermore, intact IL-12 signaling was demonstrated by M. leprae. Furthermore, IL-12 induced Stat4 phosphorylation and the phosphorylation of Stat4 in M. tuberculosis-activated cell ly- DNA binding in M. leprae-activated T cells from tuberculoid pa- sates. These data suggest that lepromatous patients do not have a tients but not from lepromatous patients. The ability of IL-12 to genetic defect in the structure or function of the IL-12R but rather induce signaling correlated with the production of IFN-␥ by T they are unable to respond to M. leprae and mount appropriate Th cells. These data suggest that IL-12R␤2 expression and function response to up-regulate the IL-12R expression. contribute to CMI and the presence of M. leprae-induced Th1 re- The mechanism behind the intact IL-12 signaling in T cells sponses in leprosy. from lepromatous patients stimulated with M. tuberculosis vs It has become increasingly evident that IL-12 is a key regulator M. leprae appears to be dependent on the T helper differentia- of Th1 cytokine responses. The ability of IL-12 to stimulate Th1 tion state. Human and animal studies have demonstrated that responses requires the expression of the IL-12R on T cells; how- Th1 cells up-regulate IL-12R␤2 whereas Th2 cells do not. In ever, the two receptor chains are differentially regulated. The IL- addition, intact IL-12 responsiveness to Ag occurs in Th1 cells 12R␤1 is constitutively expressed on T cells; in contrast, the IL- and not in Th2 cells (8, 9). Similarly, T cells from lepromatous The Journal of Immunology 785 patients produce IFN-␥ (35) when stimulated with M. tubercu- 7. Wu, C., R. R. Warrier, X. Wang, D. H. Presky, and M. K. Gately. 1997. Regu- ␤ losis Ags, and our data demonstrate that these Th1 cells have lation of interleukin-12 receptor 1 chain expression and interleukin-12 binding by human peripheral blood mononuclear cells. Eur. J. Immunol. 27:147. intact IL-12R function. In contrast, when T cells from lepro- 8. Rogge, L., L. Barberis-Maino, M. Biffi, N. Passini, D. H. Presky, U. Gubler, and matous patients are stimulated with M. leprae sonicate, there is F. Signaglia. 1997. Selective expression of an interleukin-12 receptor component little IFN-␥ production (14); we show here that these Th2 cells by human T helper 1 cells. J. Exp. Med. 185:825. 9. Szabo, S. J., A. S. Dighe, U. Gubler, and K. M. Murphy. 1997. Regulation of the do not respond to IL-12. Therefore, our findings support pre- interleukin (IL)-12R␤2 subunit expression in developing T helper 1 (Th1) and vious studies because Th1/Th2 differentiation appears to regu- Th2 cells. J. Exp. Med. 185:817. 10. Bacon, C. M., D. W. McVicar, J. R. Ortaldo, R. C. Rees, J. J. O’Shea, and late IL-12R expression and function in leprosy. J. A. Jonston. 1995. Interleukin 12 (IL-12) induces tyrosine phosphorylation of Exactly what factors influence Th1/Th2 differentiation remains JAK2 and TYK2: differential use of Janus family tyrosine kinases by IL-2 and unclear. The dose of Ag, route of Ag delivery, and genetic com- IL-12. J. Exp. Med. 181:399. 11. Zou, J., D. H. Presky, C. Y. Wu, and U. Gubler. 1997. Differential associations ponents including HLA haplotype influence Th1/Th2 develop- between the cytoplasmic regions of the interleukin-12 receptor subunits ␤1 and ment. One of the most clearly defined factors that influences Th1/ ␤2 and JAK kinases. J. Biol. Chem. 272:6073. Th2 pathway are cytokines present at the initiation of the immune 12. Jacobson, N. G., S. J. Szabo, R. M. Weber-Nordt, Z. Zhong, R. D. Schreiber, J. E. Darnell, and K. M. Murphy. 1995. Interleukin 12 signaling in T helper type response at the ligation of the TCR. 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator Two cytokines, IL-12 and IL-4, have been implicated as the of transcription (Stat)3 and Stat4. J. Exp. Med. 181:1755. critical inducer of the Th1 and Th2 pathway, respectively. IL-12 13. Bacon, C. M., E. F. Petricoin, J. R. 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