Interleukin-12 p40-homodimer production in sensory dorsal root ganglion neurons

Neuroscience 129 (2004) 75– 83

INTERLEUKIN-12 p40-HOMODIMER PRODUCTION IN SENSORY DORSAL ROOT GANGLION NEURONS N. HIKAWA,* Y. ISHIKAWA AND T. TAKENAKA

communications are commonly associated with release of humoral factors such as cytokines and hormones. These interactions between CNS and immune system are further augmented under pathological conditions such as autoimmune disease (ILucchinetti et al., 1996), traumatic CNS injury (Popovich et al., 1996), and CNS infections (Johnson et al., 1984). All of these disorders commonly show the recruitment of inflammatory immune cells to the pathologically changed tissue, and following local production of proinflammatory cytokines. In peripheral nervous system, the peripheral nerves are more closely related to immune systems in contrast to CNS. The afferent nerve terminals as well as efferent nerve terminals directly associate with immune cells by synapselike contacts or free nerve endings (Elfvin et al., 1992), which have receptors for cytokines or other molecules produced by immune cells in lymphoid organs. These functional innervations of lymphoid organs lead not only regulate local modulation of immune response by nerve terminals (Ruhl et al., 1994) but also regulation of nerve terminal function by local products of immune cells. The cutaneous sensory nerves also modulate contact dermatitis and other phenotypes of inflammation (Colpaert et al., 1983; Girolomoni and Tigelaar, 1990). These modulators of cell-mediated immune response produced by nerves are thought to be neurotransmitter or neuropeptides. The mechanism of this modulation, so-called neurogenic inflammation, is that chemical mediators such as prostaglandins, bradykinin, and tryptase induce the release of stored neurotransmitters or neuropeptides from nerve terminals (Maggi and Meli, 1988; Steinhoff et al., 2000). However, the effects of neuropeptides on immune cell functions are not so drastic compared with that of immune cytokines. Previous studies indicate that some types of peripheral neurons may produce cytokines (Freidin et al., 1992; Neumann et al., 1997), but the functions of cytokines in these neurons are not clear. Interleukin-12 (IL-12) is a heterodimeric cytokine consisting of a p35 and a p40 subunit that plays a crucial role in cell-mediated immunity (Trinchieri, 1998). A number of cell types such as monocytes and dendritic cells produce IL-12. The target cells for IL-12 include T cells and NK cells. Stimulation of naive T cells by IL-12 induces a differentiation to Th1-type cells. T cells and NK cells become activated and proliferate upon IL-12 stimulation and produce cytokines, in particular interferon-␥ (IFN-␥). With these effects IL-12 is known to play a central role in the immune response against infectious agents and tumor antigens. In contrast to these beneficial actions, IL-12 may have detrimental effects, particularly in autoimmune diseases (Leonard et al., 1996; McIntyre et al., 1996).

Department of Physiology, Yokohama City University, School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan

Abstract—Recently, the reports that sensory nerves contribute to induction and development of peripheral inflammation have been accumulating. Although neuropeptides have been thought to participate in modulation of inflammation, we supposed the involvement of cytokines. Interleukin-12 (IL-12) is a key regulator of cell-mediated immunity. IL-12 is heterodimer cytokine consisting of a p35 and a p40 subunit, but the results that some of immune cell types secrete p40homodimer have been reported. In this study, we investigated the expression and secretion of IL-12 in mouse sensory neurons in order to evaluate the involvement of sensory neurons in cell-mediated immunity. Expression of IL-12 p40 mRNA was detected and enhanced by interferon-␥ (IFN-␥), but another subunit of IL-12 p35 mRNA was not detected in sensory dorsal root ganglion (DRG) neurons in culture. IL-12 p40 molecule was detected in DRG neurons by immunocytochemistry. In addition, cultured DRG neurons secreted p40homodimer that inhibited IL-12-induced STAT4 phosphorylation in T cells. p40 mRNA expression was accumulated in DRG after administration of IFN-␥ into mouse footpad, and this enhancement was eliminated by a cut of sciatic nerve. These results suggest the possibility that p40-homodimer derived from sensory nerves suppresses the excessive peripheral inflammation. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: neural, neuroimmunology, cytokines, rodent, inflammation.

The immune system and the nervous system are both designed to interact with the body’s environment and to perceive and respond to physical or chemical stimuli generated within or outside of the organism. These complex responses require the well-tuned interaction between highly heterogeneous cell types that often exhibit remarkable plasticity. Although two systems have been traditionally thought to act separately, there is accumulating evidence of interactions between two systems. A large number of studies have been indicating that psychological and physical stress can compromise immunological factors (Aloe et al., 1994; Khansari et al., 1990). Sleep deprivation affects alterations of natural killer and cellular immune response (Irwin et al., 1996). These *Correspondence to: N. Hikawa, Laboratory of Physiology, Department of Food Science, Sagami Women’s University, 2-1-1 Bunkyo, Sagamihara, Kanagawa, Japan. Tel: ⫹81-42-742-1479; fax: ⫹81-42-7421479. E-mail address:[email protected] (N. Hikawa). Abbreviations: DIG, digoxigenin; DRG, dorsal root ganglion; IFN-␥, interferon-␥; IL-12, interleukin-12; ISH, in situ hybridization; SFM, serum-free medium.

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We proposed that cytokines in sensory neurons contribute to regulation of peripheral inflammation, and paid attention to IL-12, a key cytokine for cell-mediated immune response. In present study, we determined 1) IFN-␥induced expression of p40 but not p35 mRNA was detected in purified dorsal root ganglion (DRG) neurons in culture and in vivo; 2) responding to IFN-␥, DRG neurons secreted p40-homodimer that inhibited IL-12-induced phosphorylation of STAT4 in T cells.

EXPERIMENTAL PROCEDURES Animals We used 12- to 20-week-old C57BL/6 mice (male and female) obtained from SLC (Shizuoka, Japan). The housing and surgical procedures of mice were in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Yokohama City University School of Medicine. All efforts were made to minimize the number of animals used and their suffering.

Cultures General procedures. All cultures were performed at 37 °C in a humidified 5% CO2-95% air atmosphere. Ham’s F12 media were obtained from Invitrogen (CA, USA) and buffered with sodium bicarbonate. DRG neurons. DRG dissected from C57BL/6 mice were collected in F12 medium, transferred to centrifuge tubes and incubated in 0.25% collagenase (w/v) (Worthington Biochem., NJ, USA) in F12 at 37 °C for 90 min. The ganglia were washed twice with F12 and incubated in 0.2% trypsin (w/v) in F12 at 37 °C for 20 min. After incubation, soybean trypsin inhibitor (Sigma, MO, USA) was added to the solution to stop the reaction. The ganglia were dispersed to single cells by several passes through a Pasteur pipette. To remove myelin and non-neurons, dissociated cells were layered on 30% Percoll solution (Amersham Biosciences, NJ, USA) and centrifuged. The pelleted cells were washed twice with F12⫹10% heat-inactivated FCS and suspended in F12⫹10% FCS. Some non-neuronal cells remained in this suspension. The cells were seeded into 60-mm culture dishes at 2⫻104 cells per dish and cultured overnight. Non-adherent cells were collected the next day. Most non-adherent cells were neurons because nonneuronal cells were more adherent to the dishes than neurons. The cells were cultured in poly-L-lysine-coated dishes. Schwann cells. Schwann cells were prepared by the previous method with slight modifications (Zhang et al., 1995). Sciatic nerves were dissected from C57BL/6 mice and placed into F12 medium. After removal of the epineurium and connective tissue with forceps, the nerves were dissociated to single cells by incubation with 0.2% trypsin and 0.2% collagenase in F12 at 37 °C for 1 h and several passes with pipette. The cell suspension was put on 20% Percoll solution and centrifuged at 400 g for 10 min to remove myelin. The peletted cells were seeded onto poly-L-lysinecoated dish and cultured in F12⫹10% FCS for 1 day and cultured in serum-free medium (SFM) for the next 2 days. The loosely adherent cells on the dish were suspended by spraying F12 with a pipette and collected. They were dissociated in 0.02% trypsin solution and resuspended in F12⫹10% FCS. The suspension was incubated in culture dish for 60 min and then the non-adherent cells were replaced onto a new dish. After three replating procedures, the non-adherent cells were collected and cultured in polyL-lysine-coated dish in F12⫹10% FCS. At least 95% of cells in this culture reacted with antibodies against S-100 protein (data not shown).

Fibroblasts. Fibroblast cultures were prepared from sciatic nerves taken from C57BL/6 mice. The dissociated cells of sciatic nerves were obtained by the collagenase and trypsin incubation and Percoll centrifugation described in the Schwann cell preparation. The cells (fibroblasts and Schwann cells) were suspended in F12⫹10% FCS and incubated in culture dish for 60 min. Then the non-adherent and loosely adherent cells (most were Schwann cells) on the dish were removed by spraying F12. The cells on the dish were cultured in F12⫹10% for 1 day, detached using 0.1% trypsin and washed twice. To remove residual Schwann cells, the plating procedure described above was carried out more two times. The resulting cells were cultured in F12⫹10% FCS. The purity of the cultures was confirmed by phase-contrast microscopy and immunocytochemistry. Virtually, all cells had characteristic fibroblast morphology and reacted with anti-Thy-1.2 antibody. T cells. Splenic T cells were separated from spleen cells with the nylon wool columns. Spleens taken from C57BL/6 mice were placed in dishes containing F12, and cell suspensions were prepared by gently teasing the spleen with forceps. The suspensions were transferred to centrifuge tubes, allowed to stand for 5 min for large cell clumps to settle, and all but the settled cells were then transferred to clean centrifuge tubes. The cells were washed four times with F12 and resuspended in F12. The cell suspension was layered onto 63% Percoll solution in a centrifuge tube and centrifuged to remove red blood cells. After centrifugation, the cells of the white layer between F12 and Percoll solution were collected, washed twice with F12, and cultured (107 cells/ml) in F12⫹10% FCS. The columns were composed of 1 g of nylon wool in a 10-ml syringe equilibrated with F12⫹5% FCS at 37 °C. Spleen cells were applied to these columns and incubated for 60 min at 37 °C. The column-passed cells were eluted with 37 °C F12⫹5% FCS, applied to another column and incubated for 60 min at 37 °C. The eluted cells were washed with F12⫹10% FCS and used as splenic T cells. These T cells were stimulated by PHA described previously (Wu et al., 1997).

RNA preparation, cDNA synthesis and semi-quantitative RT-PCR Total RNA was extracted from cultured DRG neurons, Schwann cells, fibroblasts, and DRG with ISOGEN according to the manufacturer’s instructions (Nippon Gene, Tokyo, Japan). One microgram of total RNA or total amount of RNA isolated from one ganglion was reverse transcribed into cDNA in a 20 ␮l reaction using SuperScript II Reverse Transcriptase (Invitrogen) primed with oligo (dT) as recommended in the manufacturer’s protocol. To check that the same quantity of cDNA was used in the PCR, the relative amount of cDNA synthesized from each sample was assessed by PCR amplification using specific primers for ␤-actin or high-molecular neurofilament. One microliter of cDNA was amplified in 50 ␮l PCR reactions using a two-step amplification method. Reactions were cycled through one cycle at 95 °C for 15 min followed 33 (for cultured cells using p35 and p40 primers), 39 (for a DRG using p35 and p40 primers), 27 (for cultured cells using ␤-actin and neurofilament primers), or 30 cycles (for a DRG using neurofilament) at 94 °C for 20 s; 60 °C for 1 min and final extension at 72 °C for 5 min. To check that the reaction was within the exponential phase of amplification, one tenth of each reaction was analyzed on a 2% agarose gel electrophoresis at three cycle intervals. The sequences of primers for p40, p35, ␤-actin and neurofilament were described as follows: p40, 5=-CGTGCTCATGGCTGGTGCAAAG-3= (sense) and 5=-GATGAAGAAGCTGGTGCTG-3= (antisense); product: 269 bp; p35, 5=-GCAAGAGACACAGTCCTGGG-3= (sense) and 5=-TGCATCAGCTCATCGATGGC-3= (antisense); product: 618 bp; ␤-actin, 5=-CGTGGGCCGCCCTAGGCACCA-3= (sense) and 5=-TTGGCCTTAGGGTTCAGGGGGG-3= (antisense); product: 243 bp; neurofilament, 5=-GAGCAGGTGAAAAGTCCTGC-3= (sense) and 5=-GCCTTG-

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GTGTCTTCTGCTTC-3= (antisense); product: 428 bp. The reaction mixtures were applied to a 2% agarose gel, and the gel was stained with EtBr. Bands corresponding to PCR products were quantitated with the use of the FLA-3000 analysis system (Fuji Film Co., Tokyo, Japan).

were incubated for 3 days in the presence or absence of 100 U/ml IFN-␥ in SFM. The media were collected, and then concentrated to 50 times by ULTRAFREE-20 (Millipore, MA, USA). The concentrated media were used for p40 detection by immunoblot analysis and for inhibition of IL-12 activity.

In situ hybridization (ISH)

Inhibition of IL-12 activity by DRG neuronconditioned media

Single-stranded DNA probes for ISH were labeled with digoxigenin (DIG)-dUTP (Roche Diagnostics, Mannheim, Germany) in the asymmetric PCR described previously (Finckh et al., 1991). The PCR-amplified cDNA derived from LPS and DMSO-treated abdominal macrophages was used for the templates for the asymmetric PCR. RT-PCR products of p40 (M86771, position: 448 – 716) and p35 (M86672, position: 1– 618) from macrophages were separated by 2% agarose gel electrophoresis and extracted. Then, 100 ng of purified PCR products were brought to 25 ␮l in 1⫻ Expand High Fidelity buffer (Roche Diagnostics), 200 ␮M each of dATP, dCTP and dGTP, 130 ␮M dTTP, 70 ␮M DIG-11-dUTP, Expand High Fidelity polymerase (Roche Diagnostics) and 0.25 ␮M sense primer for the sense probe or 0.25 ␮M antisense primer for antisense probe. PCR cycles were run with 30 s at 94 °C, 30 s at 60 °C and 2 min at 72 °C for 30 cycles. The amount of product was quantitated using a UV spectrophotometer. Equal labeling efficiency of sense and antisense probes was checked by a dot blot procedure according to the instruction of Roche Diagnostics. For ISH, DRG neurons were cultured with 100 U/ml mouse recombinant IFN-␥ (Roche Diagnostics) in 3001 Falcon culture dishes for 2 days. The cells were fixed with 2% paraformaldehyde for 40 min at 4 °C, washed with PBS, treated with 0.5 ␮g/ml of proteinase K for 10 min at 37 °C, and then the reaction was stopped with 2mg/ml glycine in PBS. To inactivate endogenous peroxidase, the cells were treated with 2% H2O2 in methanol for 5 min. After washing three times in PBS and once in 2⫻ SSC for 5 min, the cells were incubated for 1 h at 37 °C in prehybridization buffer containing 50% formamide, 4⫻ SSC, 1⫻ Denhardt’s solution, 10% dextran sulfate, 500 ␮g/ml heat-denatured salmon sperm DNA and 250 ␮g/ml tRNA. Next, hybridization with DIGlabeled sense or antisense probe (100 ng/ml) was carried out in prehybridization solution at 40 °C overnight. Following hybridization, the cells were washed twice in 2⫻ SSC for 30 min, once in 0.5⫻ SSC for 60 min, once in PBS for 10 min, and then incubated with peroxidase-conjugated mouse anti-DIG antibody (Roche Diagnostics) for 60 min at room temperature. After washing, TSA system (NEN Life Sciences, MA, USA) was applied to the cells according to manufacturer’s protocol, and then incubated with Texas Red-conjugated avidin for 30 min at room temperature. The fluorescence was detected under fluorescent microscope.

Immunocytochemistry DRG neurons at 2 days in culture with 100 U/ml IFN-␥ were exposed to microwave for 15 s in 2% paraformaldehyde solution in a 500 W microwave oven and fixed for 40 min at room temperature. The cells were then washed with PBS three times and were incubated with 1 ␮g/ml anti-p40 mAb clone C17.8 (Genzyme, MA, USA) or anti-p35 mAb clone Red-T (Genzyme) overnight at 4 °C. After washing, cells were incubated with biotin-conjugated anti rat IgG antibody (Vector Laboratories, CA, USA) for 1 h at room temperature, and then incubated with Texas Red-conjugated avidin (Vector Laboratories) for 45 min at room temperature. The cells were observed under a fluorescent microscope.

To confirm the biological activity of p40-homodimer secreted from DRG neurons, we tested whether DRG neuron-conditioned media inhibited IL-12-induced STAT4 phosphorylation in PHA-stimulated T cells. T cells were preincubated with diluted ‘50⫻ concentrated media’ (final; ⫻16, ⫻4 and ⫻1) for 10 min and then added of IL-12 at the concentration of 10 ng/ml. After 20 min, the cells were collected and lysed in ice-cold lysis buffer (20 mM Tris–HCl at pH 7.5, 0.25 M sucrose, 2 mM EDTA, 0.5 mM EGTA, 50 mM 2mercaptoethanol, 1% NP40, 100 ␮g/ml leupeptin, 2 mM PMSF, 1 mM Na3VO4, 1 mM NaPPi, 20 mM NaF). The cell lysates were immunoprecipitated with rabbit anti-STAT4 antibody (Santa Cruz Biotech., CA, USA) at 4 °C overnight followed by incubation with protein G-agarose beads (Calbiochem, MA, USA) at 4 °C for 1 h. The beads were washed in PBS for three times and then boiled in SDS buffer (62.5 mM Tris–HCl at pH 6.8, 2% SDS, 5% glycerol, 0.02% BPB, 4% 2-mercaptoethanol) for 5 min. These samples were analyzed by immunoblotting.

Immunoblot analysis Immunoblot analysis was carried out according to standard procedure. Briefly, protein samples were run on a 12% SDSpolyacrylamide gel and blotted onto PVDF membranes (ATTO, Tokyo, Japan). The membranes were blocked in 2% BSA for 2 h at room temperature and incubated for 2 h at room temperature with a primary Ab against p40, p35, STAT4, phosphotyrosine PY20 (Transduc. Laboratory, KY, USA). Blots were incubated with horseradish peroxidase-conjugated secondary Ab (Amersham Biosciences) for 1 h at room temperature. Enhanced chemiluminescence (Pierce, Rockford, USA) on hyperfilm (Amersham Biosciences) was used to visualize labeled proteins. The images on the films were digitalized by image scanner and analyzed by Image Gauge software (Fuji Film Co.).

Injection of IFN-␥ and sciatic nerve transection The C57BL/6 mice were anesthetized with ether. Twenty microliters of IFN-␥ (5⫻104 U/ml) was injected into the right footpad and saline (20 ␮l) was injected into the left footpad. In some experiments, both right and left sciatic nerves were transected before injection. The sciatic nerve was exposed by making a small incision above the hip joint, cutting the fascia between the vastus lateralis and the biceps femoris muscles, and separating these muscles using blunt dissection. The nerve was transected proximal to its division into the tibial and common peroneal nerves. Control mice (0 h) were rapidly killed after injection by ether inhalation and L5 DRGs were removed. Other mice were allowed to survive for 24, 48, 72 and 96 h after injection. These mice were killed and L5 DRGs were removed. The change of p40 mRNA expression in the DRGs was analyzed by semi-quantitative RT-PCR.

RESULTS

Stimulation of DRG neurons by IFN-␥

Detection of p40 mRNA in DRG neurons

DRG neurons were incubated with 100 U/ml IFN-␥ for 8, 24, 48, and 72 h in SFM, at the end of which the cells were collected and used for RNA preparation. In other experiments, DRG neurons

Analysis of the expression of IL-12 p40 and p35 mRNAs in highly purified cultures of mouse DRG neurons, Schwann cells and fibroblast was investigated using RT-PCR (Fig.

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Fig. 1. Expression of mRNA for IL-12 p40, p35, and ␤-actin in cultured DRG neurons, Schwann cells, and fibroblasts. cDNA was synthesized from RNA extracted from highly purified these cells and analyzed by RT-PCR using primers for p40, p35, and ␤-actin.

1). IL-12 p40 mRNA was expressed in DRG neurons. In contrast, IL-12 p35 mRNA was not detectable in DRG neurons. In Schwann cell and fibroblast cultures, no mRNA for p40 or p35 was detected in this condition. Enhancement of mRNA expression for p40 in IFN-␥treated DRG neurons DRG neurons were stimulated with 100 U/ml IFN-␥ for 8, 24, 48, and 72 h, and were then examined for expression of IL-12 p40 mRNA using semi-quantitative RT-PCR (Fig. 2). IFN-␥ enhanced p40 mRNA expression in DRG neurons. The enhancement of p40 gene expression is evident 24 h after addition of IFN-␥, continues up to 48 h and then declines. The level of p40 mRNA in IFN-␥-stimulated DRG neurons was more than four-fold higher than the level of p40 mRNA in untreated DRG neurons. DRG neurons do

Fig. 3. IL-12 p40 and p35 mRNA expression in cultured DRG neurons. DRG neurons were cultured for 2 days with 100 U/ml IFN-␥, and then processed for ISH as described in Experimental Procedures. DRG neurons were hybridized with DIG-labeled antisense (a, b, e, f) or sense (c, d, g, h) probes to the p40 (a– d) or p35 (e– h) gene; a, c, e and g are phase-contrast micrographs, and b, d, f and h are fluorescent micrographs in the same field as a, c, e and g, respectively. Scale bar⫽50 ␮m.

Fig. 2. Semi-quantitative RT-PCR analysis of p40 mRNA expression in cultures of DRG neurons incubated with IFN-␥. RNA was extracted from DRG neurons that were treated with 100 U/ml IFN-␥ for 8, 24, 48, and 72 h, and were untreated as a control (0 h). RNA was reverse transcribed and amplified with primers for p40 and the housekeeping gene ␤-actin (a). mRNA expression for p40 was normalized to that for ␤-actin by densitometric analysis, and levels of p40 mRNA were determined as a ratio of density in IFN-␥-treated DRG neurons versus control DRG neurons (b). Data represents mean⫾S.D. For each time point, n⫽3 cultures.

not express IL-12 p35 mRNA both in basal culture conditions and after treatment with IFN-␥. In addition, neither p40 nor p35 mRNA was increased after stimulation by LPS (data not shown). ISH The culture of DRG neuron contains a small amount of other cell types. To confirm that p40 mRNA is derived from neurons, ISH was performed. Hybridization signal for the p40 mRNA was observed in cytoplasm of DRG neurons at 2 days in culture with 100 U/ml IFN-␥ (Fig. 3a, b). Seventy

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percent of DRG neurons were stained with antibody against p40. The signal was localized to the cell body, varicosities, and a part of neurites (Fig. 4a, b). In contrast, DRG neurons were not stained with anti-p35 antibody (Fig. 4c, d). Detection of p40-homodimer secretion by immunoblot analysis We next analyzed of p40 secretion by DRG neurons in culture. The neurons were treated for 3 days with 100 U/ml IFN-␥. The culture conditioning media were concentrated and analyzed by immunoblotting specific for p40 and p35. As shown in Fig. 5a, clear 10 5kD-band larger than IL-12-heterodimer and three faint bands around 40 kD were detected using anti-p40 Ab in the conditioning medium of IFN-␥-stimulated neurons. After treatment of conditioning medium with 2-ME, only bands around 40 kD were detected in the conditioning medium. These results strongly indicate that 105 kD-protein is p40-homodimer. No band was detected using anti-p40 Ab in the conditioning medium of unstimulated neurons. In contrast, p35 was not detected in culture conditioning media of DRG neurons (Fig. 5b). Biological activity of culture conditioning medium Fig. 4. Immunofluorescent distribution of p40 in DRG neurons cultured with IFN-␥. DRG neurons were cultured with 100 U/ml IFN-␥ for 2 days, and then stained with mAb against p40 (a and b) or p40 (c and d); a and c, phase-contrast micrograph; b and d, fluorescent micrograph. Scale bar⫽50 ␮m.

percent of DRG neurons exhibited the signal for the p40 mRNA. However, no signal was detected in neurons using sense probe for p40 gene (Fig. 3c, d). In addition, p35 mRNA was not detected (Fig. 3e– h). Immunocytochemistry To identify the p40 molecule and the localization of p40 in neurons, immunocytochemistry was carried out. DRG neurons were immunostained for anti-p40 Ab or anti-p35 Ab at 2 days in culture with 100 U/ml IFN-␥ (Fig. 4). Seventy

To examine the biological activity of culture conditioning medium of DRG neurons, we quantified the inhibiting activity for IL-12-inducing STAT4 phosphorylation in PHAstimulated T cells (Fig. 6). T cells were pretreated with conditioning media of IFN-␥-stimulated neurons at the indicated concentration in Fig. 6. After treatment, T cells were incubated with IL-12 and were then quantified of STAT4 phosphorylation by immunoblotting. The conditioning medium inhibited the phosphorylation of STAT4 with concentration-dependent manner. This effect is not derived from remained IFN-␥ in the conditioning media because IFN-␥ showed no effects on STAT4 phosphorylation by IL-12 (Fig. 6). This inhibitory effect on IL-12 activity is surely derived from p40-homodimer in the conditioning medium.

Fig. 5. Cultured DRG neurons treated with IFN-␥ secreted p40-homodimer. DRG neurons were cultured in the presence or absence of 100 U/ml IFN-␥ in serum-free medium. After 3 days in culture, conditioning medium was collected, concentrated to 50 times, and analyzed by immunoblotting using anti-p40 (a) or -p35 mAb (b). The conditioning medium and 20 ng of mouse rIL-12 (Genzyme) were reduced by 2-ME or unreduced before analysis.

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Fig. 6. Culture conditioning medium of DRG neurons inhibited IL-12-induced phosphorylation of STAT4 in PHA-activated T cells. PHA-activated T cells were incubated for 10 min in ⫻16, ⫻4, or ⫻1 conditioning medium of IFN-␥-treated DRG neurons prepared as described in Experimental Procedures, or in normal medium. T cells were then incubated with 10 ng/ml rIL-12, with 10 ng/ml rIL-12 and 100 U/ml IFN-␥, or with no cytokines (control). After 20 min, T cells were lysed and the lysates were immunoprecipitated with anti-STAT4 Ab. Three-fourths or one-fourth of samples were analyzed by immunoblotting with anti-phosphotyrosine Ab (PY20) or anti-STAT4 Ab, respectively (a). STAT4 phosphorylation was normalized to total STAT4 by densitometric analysis, and level of phosphorylation was determined as a ratio of density in IL-12-treated T cells versus control T cells (b). CM, conditioning medium; i.p., immunoprecipitation; IB, immunoblotting; P-STAT4, phosphoSTAT4; PY, phosphotyrosine. Data represent mean⫾S.D. (n⫽3 cultures).

Enhancement of p40 mRNA expression by IFN-␥ in DRG Since DRG contains some non-neurons, ␤-actin is not applicable as an internal standard of semi-quantitative RTPCR analysis in DRG. To determine whether neuronspecific high molecular neurofilament gene is applicable to this analysis, we amplified mRNA for neurofilament and ␤-actin in IFN-␥-treated DRG neurons using RT-PCR (Fig. 7). No change was found in neurofilament mRNA expression; thus, we used neurofilament instead of ␤-actin for RT-PCR in DRG. We examined IL-12 p40-inducing activity

of IFN-␥ in vivo. DRG (L5) were taken out at 24, 48, 72, and 96 h after injection of IFN-␥ into mouse footpad, and were then analyzed for p40 mRNA expression. The expression showed no changes after 24 h but significantly increased after 48 h to 96 h (Fig. 8a). To eliminate that injected IFN-␥ entered into the circulation and affected on DRG neurons, sciatic nerve was cut before injection. No changes were observed in this data. In addition, transection of sciatic nerve did not affect neurofilament mRNA expression (Fig. 8b).

DISCUSSION

Fig. 7. RT-PCR analysis of ␤-actin and neurofilament (NF) gene expression in cultured DRG neurons. DRG neurons were cultured with 100 U/ml IFN-␥ for 0, 8, 24, 48, or 72 h. After culture, cDNA was synthesized from RNA extracted from neurons and analyzed by RTPCR using primers for ␤-actin and neurofilament genes.

In this study, we have shown that IL-12 p40 but not p35 can be synthesized in DRG neurons. Moreover, following stimulation of IFN-␥, the production of p40 was enhanced and released as homodimer which exhibited IL-12antagonistic activity in vitro. This enhancement was also found in vivo. The expression patterns of p35 and p40 genes in DRG neurons were found to be different from other tissues. Recent reports indicated that expression of p35 gene is constitutive in a wide variety of cells and that of p40 gene is induced by IFN-␥ and/or LPS in immune cells (Trinchieri, 1998; Schoenhaut et al., 1992). In contrast, unstimulated DRG neurons expressed p40 mRNA and no p35 mRNA in

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Fig. 8. IFN-␥ enhanced mRNA expression for p40 in DRG. IFN-␥ was injected into the right footpad of C57BL/6 mouse, and saline was injected into the left footpad as a sham control (a). Sciatic nerve was cut before injection (b). L5-level DRG were removed at 0, 24, 48, 72, and 96 h after injection. RNA was extracted, and cDNA was synthesized. p40 mRNA expression was analyzed by semi-quantitative RT-PCR using internal standard neurofilament gene (NF). mRNA expression for p40 was normalized to that for NF by densitometric analysis, and level of expression was determined as a ratio of density in DRG at 24, 48, 72, or 96 h after injection versus DRG at 0 h after injection. Data represent mean⫾S.D. (n⫽3 mice).

our study. This expression pattern was reported in astrocyte (Aloisi et al., 1997). However, the accumulation of p40 mRNA was not observed after stimulation of IFN-␥ in astrocyte in contrast to our data. Additionally, p35 mRNA was not induced by IFN-␥ in DRG neurons. These findings in this study are identical to other studies that the expression of p35 and p40 gene is regulated in different mechanisms (Kato T. et al., 1996; Yamane et al., 1999). Although mRNA for p40 was detected by RT-PCR analysis, no p40 molecule was detected by immunoblotting in the conditioning medium of unstimulated DRG neurons. This result shows that unstimulated DRG neurons fail to produce the p40 molecules sufficient to be detectable, or fail to release them. Indeed, previous studies report that co-stimulation with IFN-␥ and LPS is necessary for microglial secretion of IL-12 (Aloisi et al., 1997). DRG neurons released p40homodimer by the stimulation of IFN-␥ alone in our study. These contradictory results may be explained by the possible involvement of autocrine intermediates in release of p40-homodimer after IFN-␥-stimulus. This possibility is supported by the observation that the enhancement of p40 mRNA expression by IFN-␥ was delayed in DRG neurons in vitro. The level of p40 mRNA in cultured DRG neurons increased after 8 h-exposure to IFN-␥ in our study. In other cell types, p40 mRNA was up-regulated 2 h after LPS or LPS/IFN-␥ treatment (Aloisi et al., 1997; Stalder et al., 1997). The delay of up-regulation suggests that IFN-␥ induces certain molecules responsible for up-regulation and release of p40 in DRG neurons in combination with IFN-␥. It is well known that both cell lines and normal monocytes can secrete p40-homodimer in addition to IL-12 heterodimer (D’Andrea et al., 1992; Podlaski et al., 1992). The production of p40 is five- to 500-fold higher than that of

p35 in these cells. The excess p40 results in the production of p40-homodimer which acts as an IL-12 antagonist that binds to the IL-12 receptor but does not mediate a biological response in vitro (Gillessen et al., 1995; Ling et al., 1995). Therefore, monocytes can simultaneously secrete physiological IL-12 antagonist as well as IL-12. Furthermore, recent papers show that the transplantation of p40hyper-expressing cells inhibited IFN-␥ production by IL-12 and delayed-type hypersensitivity in vivo (Kato K. et al., 1996), and that similar results was obtained in p40transgenic mice (Yoshimoto et al., 1998). In our study, IFN-␥-stimulated DRG neurons secreted only p40-homodimer which possessed IL-12-antagonistic activity. DRG neurons probably play a suppressor cell function for IL-12 functions in vivo. The enhancement of p40 mRNA in DRG after peripheral administration of IFN-␥ in mice was delayed compared with that in IFN-␥-stimulated DRG neurons in culture, and this enhancement was eliminated by cutting the nerve. The results show that the signal molecules derived from IFN-␥ injected into footpad were transported via axon to DRG distant from footpad and afterward, up-regulated gene expression of p40. A related result that neuropeptide Y expression was induced in DRG after peripheral inflammation was observed in another report (Ji et al., 1994). This evidence strongly suggests that a peripheral immune response can affect the production of biologically active molecules in neurons. Which cell types close to nerves can be the source of IFN-␥? It has been indicated that fetus rat DRG neurons may physiologically secrete IFN-␥ in auto/paracrine fashion (Neumann et al., 1997). Our data were obtained from adult mice. The convincing evidences of physiological IFN-␥ secretion in adult sensory nervous system have not

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been reported. In contrast, production of IFN-␥ correlates with Th1-type cells in pathological conditions. IFN-␥ and IL-12 are the key players in pathogenesis of atopic dermatitis (Grewe et al., 1998) and psoriasis (Yawalkar et al., 1998). In addition, free nerve endings in pathological skin lesions, especially in chronic lesions in which a Th1-type immune response is dominant, is increased in patients with these types of dermatitis (Naukkarinen et al., 1996; Sugiura et al., 1997; Urashima and Mihara, 1998). This evidence suggests that sensory nerves are involved in development and severity of this disease through secretion of IFN-␥-induced p40-homodimer antagonized to IL-12. Other reports demonstrate that peripheral nerve injury and demyelinating disease induced endoneurial expression of IFN-␥ (Hartung et al., 1992; Taskinen et al., 2000). In these situations, IFN-␥ is mainly produced and secreted by infiltrating immune cells. IFN-␥ can induce MHC class I molecules in adult DRG neurons in our previous study (Fujimaki et al., 1996). MHC class I-expressed nerves can be the targets for infiltrating immune cells in the local lesions of nerve injury and demyelination. However, autoimmune response against nerves is rare in these situations. The axons of injured or demyelinated nerves are bare because myelins surrounding axons are destroyed. Hence, these axons can bind to immune cell-derived cytokines, leading to release of active molecules. Although distribution of p40 molecules in DRG neurons in vivo is less clear, p40 molecule was found to be localized in neurites as well as cell bodies in our immunocytochemistry in vitro. When a local inflammation arises, the nerves may secrete p40homodimer responding to IFN-␥ in inflammatory lesions and may protect themselves from infiltrating cells by means of the inhibition of IL-12-activated cell-mediated immunity. The p40-homodimer can be released from any sites of neurons in inflammatory lesions such as neurites, nerve endings, and soma of neurons at both peripheral and spinal levels. Furthermore, it is known that these disorders tend to worsen with stress. The reports that stress leads to excessive release of neuropeptides from sensory neurons (Tsuchiya et al., 1996; Zhu et al., 1996) suggest that stress modified the function of sensory neurons. These evidences speculate that p40-homodimer derived from sensory nerves has an inhibitory role in deterioration of the disease. In summary, our present study evoked the hypothesis that sensory neurons control cell-mediated immune response by the secretion of IL-12 p40-homodimer. This is a neuro-immune interaction-mediated negative feedback regulation of inflammation, similar to the regulations of hormone level in serum, blood pressure, and body temperature; all of them act to maintain a homeostasis.

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(Accepted 27 July 2004) (Available online 23 September 2004)