Neuroscience 155 (2008) 1227–1236 Reg-2 EXPRESSION IN DORSAL ROOT GANGLION NEURONS AFTER ADJUVANT-INDUCED MONOARTHRITIS S. AVERILL,a* J. J. INGLIS,b,f V. R. KING,a S. W. N. THOMPSON,c W. B. J. CAFFERTY,d P. J. SHORTLAND,a S. P. HUNT,e B. L. KIDDb AND J. V. PRIESTLEYa Small- and medium-sized dorsal root ganglion (DRG) cells comprise two neurochemically distinct subpopulations, defined according to their expression of neuropeptides and response to neurotrophic factors (Snider and McMahon, 1998; Priestley et al., 2002). Cells in the first population are commonly referred to as “peptidergic” because they constitutively express neuropeptides such as calcitonin generelated peptide (CGRP) and substance P. They also express the nerve growth factor (NGF) tyrosine kinase (trk) receptor trkA, and respond to NGF (Verge et al., 1995; Bennett et al., 1998). Cells in the second population mainly do not express neuropeptides but express receptors for the glial cell line– derived neurotrophic factor (GDNF) family, and respond to GDNF (Bennett et al., 1998; Wang et al., 2003; Averill et al., 2004). They are often referred to as “non-peptidergic” or as “IB4-positive” or simply “IB4 cells,” these latter terms referring to the fact that they bind the lectin Griffonia simplicifolia IB4. In both the peptidergic and IB4-positive populations the majority of cells are likely to be nociceptors (Michael and Priestley, 1999) and the role of the peptidergic population, especially in inflammatory pain, is reasonably well understood. Thus many peptidergic cells express the vanilloid receptor TRPV1 and respond to inflammatory factors such as NGF, bradykinin, eicosanoids and cytokines (Woolf and Costigan, 1999; Kidd and Urban, 2001; Hensellek et al., 2007). Inflammation leads to both sensitization and upregulation of TRPV1, together with upregulation of substance P and CGRP. These neuropeptides are released peripherally to cause vasodilation and extravasation, and are released centrally to contribute to sensitization in the spinal cord. In contrast to the peptidergic group, little is known about the function of the IB4-positive group. However some recent studies indicate that there may be circumstances in which these neurons are regulated by tissuederived factors. Inflammation causes a GDNF-dependent increase in TRPV1 expression in IB4 cells (Amaya et al., 2004; Breese et al., 2005) and GDNF sensitizes TRPV1 (Malin et al., 2006). In addition to GDNF receptors, IB4positive neurons express receptors for members of the interleukin-6 (IL-6) family of neuropoietic cytokines such as IL-6 and ciliary neurotrophic factor (CNTF) (Shuto et al., 2001), leukemia inhibitory factor (LIF) (Thompson et al., 1997; Gardiner et al., 2002) and oncostatin M (Tamura et al., 2003). The role of these cytokines in regulating DRG neurons is still poorly understood but there is good evidence, for example, that LIF contributes to the dramatic upregulation in galanin expression that occurs following axotomy (Sun and Zigmond, 1996; Corness et al., 1996; Thompson et al., 1998). In addition we have shown that a Neuroscience Centre, Institute of Cell and Molecular Science, Bart’s & The London School of Medicine & Dentistry, 4 Newark Street, Whitechapel, London E1 2AT, UK b Bone and Joint Unit, Bart’s & The London School of Medicine & Dentistry, Queen Mary University of London, London, EC1M 6BQ, UK c School of Biological Sciences, University of Plymouth, PL4 8AA, UK d Department of Neurology, Yale University School of Medicine, New Haven, CT 06520-8018, USA e Department of Anatomy and Developmental Biology, University College, London, WC1E 6BT, UK f Kennedy Institute of Rheumatology, Imperial College, London, W6 8LH, UK Abstract—Reg-2 is a secreted protein that is expressed de novo in motoneurons, sympathetic neurons, and dorsal root ganglion (DRG) neurons after nerve injury and which can act as a Schwann cell mitogen. We now show that Reg-2 is also upregulated by DRG neurons in inflammation with a very unusual expression pattern. In a rat model of monoarthritis, Reg-2 immunoreactivity was detected in DRG neurons at 1 day, peaked at 3 days (in 11.6% of DRG neurons), and was still present at 10 days (in 5%). Expression was almost exclusively in the population of DRG neurons that expresses the purinoceptor P2X3 and binding sites for the lectin Griffonia simplicifolia IB4, and which is known to respond to glial cell line– derived neurotrophic factor (GDNF). Immunoreactivity was present in DRG cell bodies and central terminals in the dorsal horn of the spinal cord. In contrast, very little expression was seen in the nerve growth factor (NGF) responsive and substance P expressing population. However intrathecal delivery of GDNF did not induce Reg-2 expression, but leukemia inhibitory factor (LIF) had a dramatic effect, inducing Reg-2 immunoreactivity in 39% of DRG neurons and 62% of P2X3 cells. Changes in inflammation have previously been observed predominantly in the neuropeptide expressing, NGF responsive, DRG neurons. Our results show that changes also take place in the IB4 population, possibly driven by members of the LIF family of neuropoietic cytokines. In addition, the presence of Reg-2 in central axon terminals implicates Reg-2 as a possible modulator of second order dorsal horn cells. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: rat, DRG, Reg-2, inflammation, LIF. *Corresponding author. Tel: ⫹44-020-7882-2284; fax: ⫹44-020-7882-2180. E-mail address: s.a.averill@qmul.ac.uk (S. Averill). Abbreviations: CFA, complete Freund’s adjuvant; CGRP, calcitonin gene-related peptide; CNTF, ciliary neurotrophic factor; DAPI, 4,6diamidino-2-phenylindole; DRG, dorsal root ganglion; GDNF, glial cell line– derived neurotrophic factor; IL-6, interleukin-6; LIF, leukemia inhibitory factor; NGF, nerve growth factor; TSA, tyramide signal amplification. 0306-4522/08 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.06.049 1227 1228 S. Averill et al. / Neuroscience 155 (2008) 1227–1236 Reg-2 is selectively upregulated in IB4 cells in the first few days after axotomy (Averill et al., 2002). Reg-2 (also known as PAPI, or peptide 23) is the rat homologue of human hepatocarcinoma–intestine–pancreas/pancreaticassociated protein (HIP/PAP) which has various functions (Lieu et al., 2006; Gironella et al., 2007) but which in neurons has been shown to be a Schwann cell mitogen and be regulated by members of the CNTF/LIF family (Livesey et al., 1997; Nishimune et al., 2000). Cytokines such as LIF increase markedly with inflammation (Zhu et al., 2001), and may therefore contribute to changes in DRG peptide expression. We have therefore used the complete Freund’s adjuvant (CFA) model of mono-arthritis to examine Reg-2 expression after inflammation. We show that Reg-2 is selectively upregulated in IB4 cells, and that Reg-2 expression in these cells can be induced by LIF treatment. EXPERIMENTAL PROCEDURES Animal treatments Thirty-two adult male Wistar rats (220 – 400 g body weight) were processed for immunocytochemistry. Sixteen of these were treated with an intraplantar injection of CFA into the right paw, 12 rats were injected with vehicle only (paraffin) and four rats were left untreated. CFA was prepared as a 10 mg/ml suspension of heat-attenuated Mycobacterium tuberculosis in paraffin oil. Inflammation was induced by a single intra-plantar injection (100 ␮l) of CFA into the right hind footpad of each animal. The rats survived for a further 1, 3, 7 or 10 days (three or four per group) before perfusion fixation. Footpad diameter, and mechanical (Von Frey) and thermal hyperalgesia (Hargreaves test), were assessed in the 10 day group prior to, and at 1– 4 days and 7 days post-CFA injection, using standard procedures (Kidd et al., 2003; Hargreaves et al., 1988). Statistical analysis of these parameters was performed by one-way ANOVA with post hoc Bonferroni multiple range testing and unpaired t-test or Mann-Whitney U test as appropriate. Rats were perfused under pentobarbitone anesthesia with a mixture of 2% paraformaldehyde, 1.36% L-lysine monohydrochloride and 0.213% sodium meta-periodate in 0.1 M phosphate buffer. An additional group of 13 rats was treated intrathecally with various growth factors. Under pentobarbitone anesthesia (40 mg/ kg, i.p., with sterile precautions), a small laminectomy was performed between L6 and S1 vertebrae and the dura was cut. A silastic tube of 0.6 mm outer diameter was introduced intrathecally so that its tip lay at the level of the lumbar enlargement of the spinal cord and the intrathecal tubing was attached to an Alzet mini-osmotic pump (Alzet, Alza corp., Palo Alto, CA, USA; type 2002) delivering at a rate of 0.5 ␮l/h. Animals received either a control infusion (n⫽4, saline with rat serum albumin, 1 mg/ml) or this vehicle plus recombinant human GDNF (n⫽4 at 12 ␮g/day), recombinant human NGF (n⫽3 at 12 ␮g/day), or recombinant human LIF (n⫽2 at 3.9 ␮g/day). These doses were chosen because they have been shown to be effective in previous studies (Averill et al., 2004; Bennett et al., 1998; Michael et al., 1997; Cafferty et al., 2001). Fourteen days later, animals were perfused with vascular rinse solution followed by 4% paraformaldehyde. All animal procedures were carried out according to Home Office regulations, conformed to international guidelines on the ethical use of animals and animal numbers and suffering were kept to a minimum. Immunocytochemistry Left and right L4 and L5 dorsal root ganglia and spinal cord were dissected out, postfixed for 1.5–2 h and frozen after cryoprotection. Cryostat sections (8 ␮m) were stained using single or dual color immunofluorescence or indirect tyramide signal amplification (TSA) fluorescence procedures. The Reg-2 polyclonal antibody was raised in rabbit against whole recombinant protein and used at 1:6000 for indirect immunofluorescence, and 1:200,000 with TSA amplification. For double labeling this antibody was combined with one of the following: guinea-pig anti P2X3 (Neuromics, USA, 1:250,000), anti substance P (Chemicon, CA, USA, 1:2000), anti trkA (Upstate, VA, USA, 1:8000), anti RET (1:500, for details see (Molliver et al., 1997) or Griffonia simplicifolia IB4 lectin (5 ␮g/ml biotinylated IB4, Sigma, UK). After incubation in FITC- or TRITC-labeled secondary reagents, sections were washed briefly in PBS and then mounted in PBS/glycerol (1:3) containing 2.5% (w/v) 1,4 diazobicyclo (2,2,2) octane (DABCO, antifading agent). In some cases sections were also counterstained with 100 ␮g/ml 4,6-diamidino-2-phenylindole (DAPI, Sigma) to reveal cell nuclei. Controls for double labeling included reversing the order of the primary antisera, as well as omitting the first or second primary antiserum. Photographs were taken using a Hamamatsu C4742-95 digital camera and plates assembled using Adobe Photoshop. Quantification Quantification of the proportion of Reg-2 expressing DRG neurons was determined by counting the number of immunoreactive and non-immunoreactive cells with visible nuclei. In doublelabeled sections the percentage of Reg-2 cells expressing a second marker was assessed by switching between DAPI, FITC and TRITC filter blocks. At least 250 labeled DRG neurons were examined for each marker and were counted on randomly chosen sections. Counts were compared using nonparametric statistical tests. Specifically, a Kruskal-Wallis analysis of variance was used to determine if there was a difference among groups for a measure of interest. Any significant difference found (P⬍0.05) was further analyzed by making pairwise comparisons using the Mann-Whitney U test. For cell size distributions and quantification, images were captured at 40⫻ objective magnification. Cells of interest were outlined manually using a computer mouse, and the size determined using Scion Image. RESULTS Immunoreactivity for Reg-2 was only present in single DRG neurons of occasional sections in normal lumbar ganglia (Fig. 1). By 24 h after CFA injection, mean Reg-2 expression had increased and was present in 1.6% of L4/L5 DRG neurons. Mean expression increased to 11.6% at 3 days, and then declined slightly at 7 and 10 days (Fig. 1, Table 1). At all time points studied, the Reg-2 cells were small to medium in size (Fig. 2). Within cell bodies, immunoreactivity appeared as prominent punctate structures (Fig. 1E). In addition, immunoreactivity was present in a few axons, where it was localized to small granules (Fig. 1E, F). In order to identify the types of small- and mediumsized cells that upregulate Reg-2, immunostaining at the 3, 7 and 10 day time points was combined with a range of markers for DRG subpopulations. Using IB4 and the purinoceptor P2X3 as markers for the IB4-positive population (Bradbury et al., 1998), a very high degree of coexistence with Reg-2 was observed (Fig. 3, Table 2). Thus at all time S. Averill et al. / Neuroscience 155 (2008) 1227–1236 1229 Fig. 1. Low (A–D) and high (E, F) magnification micrographs showing Reg-2 immunoreactivity in L4 DRG. At all time points after induction of inflammation, Reg-2 is expressed in small cells. Reg-2 immunoreactivity in ipsilateral (B–F) and naïve control (A) lumbar DRG at 3 days (B), 7 days (C, E, F) and 10 days (D) after CFA inflammation. At high magnification, Reg-2 immunoreactivity can be seen in isolated axons (arrowhead) and within punctuate structures (arrows) within cell bodies and axons. Scale bars⫽100 ␮m (A–D); 20 ␮m (E); 5 ␮m (F). points studied, greater than 93% of Reg-2 immunoreactive cells were labeled for IB4 or P2X3. A similar result was obtained when staining was carried out for Ret, the tyrosine kinase component of the GDNF receptor (Fig. 3, Table 2). In contrast to Reg-2, the number of cells labeled for IB4 and for P2X3 was either not changed (IB4, Ret) or only slightly changed (P2X3) by the CFA treatment (Table 2). To examine Reg-2 expression in the peptidergic population, staining was carried out for substance P and for trkA. Coexistence between Reg-2 and these markers was Table 1. Time course of Reg-2 expression after CFA inflammation % Naïve (n⫽3) 1 Day CFA (n⫽3) 3 Day CFA (n⫽3) 7 Day CFA (n⫽3) 10 Day CFA (n⫽3) % Reg-2 ⫹ve cells 0⫾0 1.6⫾0.2 11.6⫾0.7* 8.2⫾1.8* 5⫾0.82*† Reg-2 expression after CFA inflammation increases rapidly up to day 3 and then slowly declines. * Indicates significantly different from 1 day. † Indicates significantly different from 3 day (P⬍0.05). 1230 S. Averill et al. / Neuroscience 155 (2008) 1227–1236 DISCUSSION Fig. 2. Size distribution of Reg-2 immunoreactive sensory neurons in L4/5 DRG following CFA inflammation. DRG neurons can be subdivided into three size ranges (small, 0 – 600 ␮m2; medium, 600 – 1000 ␮m2; large, 1000 –3200 ␮m2) which approximate to the size ranges of cells with C, A␦, and A␣/␤ conduction velocities. Note that at all time points studied, Reg-2 expressing neurons are all predominantly small to medium sized. much less extensive (Fig. 4, Table 3). For example, at the various time points studied, less than 18% of Reg-2 cells expressed substance P. In addition to DRG cell bodies, CFA animals had Reg-2 immunoreactive terminals in the dorsal horn of the spinal cord (Fig. 5). Staining, which was concentrated in lamina II inner and confined to the ipsilateral termination zone of sciatic afferents, was present at all survival times studied but was most prominent in the 10 day CFA animals. Double labeling revealed that the Reg-2 immunoreactive terminals were not SP immunoreactive but were labeled for P2X3. As an aid to identifying a possible role for Reg-2, a range of functional endpoints was examined in the CFA treated animals. These included thermal and mechanical thresholds, and footpad diameter. For all parameters studied, and in agreement with previous reports (e.g. Inglis et al., 2005), changes were maximal by 1–3 days and were then maintained up to the 10 day time point (data not shown). The functional endpoints thus contrasted with the Reg-2 expression, which did not peak until 3 days and then declined steadily (Table 1). In order to identify growth factors that might be responsible for the changes in Reg-2 upregulation with inflammation, expression was examined after treatment with various growth factors. Animals treated with vehicle (Fig. 6A), GDNF (Fig. 6B) or NGF showed Reg-2 expression in only occasional cells and were similar to naïve controls. In contrast, animals treated with rhLIF showed strong Reg-2 immunoreactivity in many small- to medium-sized DRG neurons (Fig. 6C–H), in total comprising 39% of DRG neurons. In LIF-treated animals, a high degree of coexistence between Reg-2 and IB4 or P2X3 was observed (Fig. 6C–H, Table 4), although not quite as high as after CFA treatment (Table 2). For example, 71% of Reg-2 immunoreactive cells showed IB4 labeling in rhLIF-treated animals, compared with 98% in 3 day or 7 day CFA animals. RhLIF treatment caused a small increase in P2X3 expression but no change in IB4 labeling (Table 4). In this study we have shown that Reg-2 is upregulated in DRG neurons and their central terminals after inflammation, that this upregulation occurs selectively in the IB4/ P2X3 population of neurons, and that the neuropoietic cytokine LIF is a candidate regulator of this Reg-2 expression. Our results implicate for the first time Reg-2 and the IB4 population of cells in the response of DRG neurons to inflammation, and demonstrate some very novel aspects of DRG plasticity. Reg-2 is upregulated after nerve injury and axonally transported toward the injury site (Averill et al., 2002; Livesey et al., 1997). Our demonstration that IB4/P2X3 cells also upregulate Reg-2 after inflammation is unexpected because molecules that are upregulated by DRG neurons after inflammation are normally down-regulated after nerve injury. This is the case, for example, with substance P, CGRP and TRPV1 and is due to the fact that their expression is regulated by target-derived NGF (reviewed in Woolf, 1996; Priestley et al., 2002). NGF levels increase after inflammation (Donnerer et al., 1992) but fall after axotomy (Raivich et al., 1991) and expression of regulated molecules in DRG neurons changes in consequence. In contrast molecules which are upregulated after nerve injury, such as c-jun, ATF3, and neuropeptide Y, do not change following inflammation (Tsujino et al., 2000; Wakisaka et al., 1992). GAP-43 and galanin are slight exceptions in that they are upregulated after both axotomy and inflammation. The upregulation of galanin, however, is thought to be due to destruction of peripheral tissue, akin to an axotomy (Calza et al., 2000) while the GAP-43 upregulation is short-lived (peaks at 48 h, see Leslie et al., 1995). Reg-2 is therefore unique in being upregulated with a similar time course, and in the same population of cells, by both axotomy and inflammation. However there are some differences in the two responses. After axotomy, Reg-2 is seen initially (24 h) only in the IB4/P2X3 cells but at longer time points (5–7 days) appears in medium- and large-sized (IB4 negative) cells (Averill et al., 2002). In contrast, Reg-2 expression after inflammation was confined to IB4/P2X3 cells at all time points studied (3–10 days). In addition Reg-2 after axotomy is not transported centrally and does not appear in the dorsal horn (Averill et al., 2002), whereas Reg-2 immunoreactivity after inflammation does appear in the dorsal horn, presumably due to axonal transport into central terminals. It is therefore likely that Reg-2 in inflammation has a quite different role to that in nerve injury, where it is thought to promote regeneration (Livesey et al., 1997). For example, the axonal transport into central terminals raises the possibility that Reg-2 is released in the dorsal horn to modulate glial or neuronal function in the spinal cord. If this does occur, the lack of correlation that we observed between Reg-2 expression and either thermal or mechanical hyperalgesia would suggest that Reg-2 plays some other function than simply contributing to nociceptive transmission. Previous studies in mice in which the Reg2 gene has been knocked out have shown that pancreatic inflammation was more exten- S. Averill et al. / Neuroscience 155 (2008) 1227–1236 1231 Fig. 3. After CFA inflammation, Reg-2 is expressed selectively in the IB4-binding population of DRG neurons. (A, B) Low magnification micrograph showing Reg-2 and IB4 double labeling in an L5 DRG, 3 days after CFA treatment. The majority of Reg-2 immunoreactive cells are double-labeled (arrows). (C–H) High magnification micrographs showing Reg-2 and either IB4 (C, D), P2X3 (E, F) or Ret (G, H) double labeling at either 3 days (C, D) or 7 days (E–H) after CFA treatment. In each pair of micrographs, many Reg-2 positive cells are positive for the second marker (arrows). The arrowheads in C and E show rare Reg-2 immunoreactive cell that are not IB4 or P2X3 labeled. Scale bars⫽50 ␮m (A, B); 50 ␮m (C–H). sive and apoptosis more severe than in wild type mice (Gironella et al., 2007). Similarly in liver, Reg2 knockout mice have revealed that Reg2 is a critical mitogenic and anti-apoptotic factor (Lieu et al., 2006). If Reg2 had a similar role in the dorsal horn, it might protect neurons from excitotoxic damage and perhaps restrict activation of nonneuronal cells such as microglia which are thought to contribute to chronic pain states (Marchand et al., 2005). These possibilities deserve further investigation. Another unexpected aspect of our results is the identity of the cells that upregulate Reg-2 following inflammation. To date, most changes that take place in DRG neurons following inflammation have been reported to occur in the trkA/neuropeptide population of DRG neurons and are 1232 S. Averill et al. / Neuroscience 155 (2008) 1227–1236 Table 2. IB4-positive cells express Reg-2 after CFA inflammation % % % % % % % IB4 ⫹ve DRG neurons Reg-2 cells also IB4 ⫹ve P2X3 ⫹ve DRG neurons of Reg-2 cells also P2X3 ⫹ve RET ⫹ve DRG neurons Reg-2 cells also Ret ⫹ve Naïve (n⫽3) 3 Day CFA (n⫽3) 7 Day CFA (n⫽3) 10 Day CFA (n⫽3) 48.3⫾2.3 NA 32.7⫾0.8 NA 66.4⫾3.3 NA 49.5⫾1.9 98.2⫾0.2 38.0⫾0.9*† 96.5⫾1.8 73⫾2.7 100⫾0 49.9⫾3.5 98.3⫾1.7 44.6⫾1.0* 97.1⫾1.1 74.6⫾4.7 100⫾0 48.3⫾3.8 94.2⫾1.3 37.4⫾2.8† 93.2⫾1.4 72.4⫾2.7 100⫾0 Reg-2 expression after CFA inflammation occurs predominantly in the IB4-binding population of DRG neurons. Thus at all CFA time points studied, the vast majority of Reg-2 expressing cells show labeling for IB4, P2X3 or Ret. * Indicates significantly different from naive. † Indicates significantly different from 7 day (P⬍0.05). NA⫽not applicable. thought to be regulated by NGF. This is the case for peptides such as substance P and CGRP (Woolf et al., 1994), the neurotrophin brain-derived neurotrophic factor (Cho et al., 1997), receptors such as TRPV1 (Ji et al., 2002), the NaV1.8 Na channel (Kerr et al., 2001), the transcription factor oct-2 (Ensor et al., 1996), and GAP-43 (Leslie et al., 1995). Although cytokines are thought to play an important role in inflammation (Marchand et al., 2005), many of their effects on DRG neurons appear to be indirect and mediated by NGF (Woolf et al., 1997). It is clear that many IB4 neurons are nociceptors (Gerke and Plenderleith, 2001) but their exact role is unclear, and studies that have compared the responses properties of IB4-positive and IB4-negative neurons have identified only minor dif- Fig. 4. After CFA inflammation, Reg-2 is only expressed in a small proportion of the peptidergic population of DRG neurons. (A, B) Low magnification micrograph showing Reg-2 and substance P (SP) double labeling in an L5 DRG 3 days after CFA treatment. A few Reg-2 immunoreactive cells are SP immunoreactive (arrows) but the majority are not (arrowheads). (C–F) High magnification micrographs showing Reg-2 and SP (C, D) or trkA (E, F) double labeling 3 days after CFA treatment. In each pair of micrographs, some Reg-2 immunoreactive cells are positive for the second marker (arrows) but many are not (arrowheads). Scale bars⫽100 ␮m (A, B); 50 ␮m (C–F). S. Averill et al. / Neuroscience 155 (2008) 1227–1236 1233 Table 3. Neuropeptide cells show low Reg-2 expression after CFA inflammation % % % % % SP ⫹ve DRG neurons of Reg-2 cells also SP ⫹ve trkA ⫹ve DRG neurons of Reg-2 cells also trkA ⫹ve Naïve (n⫽3) 3 Day CFA (n⫽3) 7 Day CFA (n⫽3) 10 Day CFA (n⫽3) 21.3⫾0.8 NA 42.8⫾2.8 NA 18.3⫾1.0 9.5⫾0.8* 44.7⫾1.3 26.4⫾9.6 19.2⫾1.2 7.5⫾1.3* 44.3⫾3.0 22.3⫾2.4 19.4⫾1.5 17.6⫾2.3 42.2⫾2.8 16.1⫾3.7 Reg-2 expression after CFA inflammation shows low coexistence with substance P (SP) and trkA, markers for the peptidergic DRG subpopulation. Thus at all CFA time points studied, less than 26% of Reg-2 expressing cells show labeling for SP or trkA. NA, not applicable. * Indicates significantly different from 10 day (P⬍0.05). ferences (Stucky and Lewin, 1999; Petruska et al., 2000; Dirajlal et al., 2003; Liu et al., 2004; Breese et al., 2005). Recently, however, a role for IB4 cells in inflammation has been proposed based on data showing that TRPV1 expression increases in IB4-positive cells (Amaya et al., 2004; Breese et al., 2005) as well as in IB4-negative (trkA immunoreactive) cells (Amaya et al., 2004) during inflammation. Our study supports such a role, and in addition is the first to demonstrate a molecule (Reg-2) that is upregulated selectively in IB4 neurons in response to inflamma- tion. Our results also support the hypothesis that IB4 cells form a parallel pathway to the peptidergic/NGF-regulated one (Hunt et al., 1992) with distinctive properties and mode of regulation (see below). Although previous studies have shown that Reg-2 can be regulated by members of the CNTF/LIF family (Livesey et al., 1997; Nishimune et al., 2000), the very selective expression of Reg-2 in IB4/P2X3 cells implicates GDNF as a regulator. IB4/P2X3 cells show selective expression of GDNF receptors and many proteins expressed by IB4/ Fig. 5. Three days after CFA inflammation, Reg-2 immunoreactive terminals are visible in the ipsilateral dorsal horn of the spinal cord (B) but not on the contralateral side (A). Terminals are concentrated in the medial substantia gelatinosa (asterisk in B), corresponding to the sciatic innervation territory. Double labeling with P2X3 (C–E) reveals that the Reg-2 terminals are in lamina II inner, in register with the P2X3 terminals (7 day CFA). High magnification confocal analysis (F, G) reveals individual Reg-2/P2X3 double-labeled terminals (arrows). In the dorsal roots/tract of Lissauer (H) Reg-2 immunoreactivity is visible in individual axons (arrows, 10 day CFA). Scale bars⫽100 ␮m (A, B); 50 ␮m (C–E, H); 20 ␮m (F, G). 1234 S. Averill et al. / Neuroscience 155 (2008) 1227–1236 Fig. 6. After LIF treatment, Reg-2 is expressed in many DRG neurons and these are predominantly of the IB4-binding type. (A, B) Low magnification micrographs showing Reg-2 immunoreactivity after 2 weeks’ intrathecal vehicle (A) or GDNF (B) treatment. Only one or two Reg-2 immunoreactive cells are visible (arrows). (C–F) Low magnification micrographs showing Reg-2 and P2X3 (C, D) or IB4 (E, F) double labeling after 2 weeks’ intrathecal LIF treatment. Many double-labeled cells are visible (arrows). (G, H) High magnification micrographs showing Reg-2 and IB4 double labeling after LIF treatment. Many of the Reg-2 immunoreactive cells are also positive for IB4 (arrows), but some single-labeled Reg-2 cells are also present (arrowheads). Scale bars⫽100 ␮m (A–F); 50 ␮m (G, H). S. Averill et al. / Neuroscience 155 (2008) 1227–1236 Table 4. LIF treatment induces Reg-2 expression % % % % % % % % Reg-2 ⫹ve DRG neurons IB4⫹ DRG neurons P2X3 ⫹ve DRG neurons Reg-2 cells also IB4 ⫹ve of IB4 cells also Reg-2 ⫹ve of Reg-2 cells also P2X3 ⫹ve of P2X3 cells also Reg-2 ⫹ve Naïve (n⫽3) 2 Week rhLIF (n⫽2) 0⫾0 48.3⫾2.3 32.7⫾0.8 NA NA NA NA 38.5 47.5 40.7 71.4 50.2 58.1 62.3 Reg-2 expression after LIF treatment occurs predominantly in IB4and P2X3-labeled cells, markers for the IB4-binding population of DRG neurons. NA, not applicable. P2X3 cells are positively regulated by GDNF family members, including P2X3, TMP, somatostatin, TRPV1, and IB4 binding (Bennett et al., 1998; Bradbury et al., 1998; Amaya et al., 2004). In addition the injury-induced transcription factor, ATF3, is negatively regulated in IB4/P2X3 cells by GDNF (Averill et al., 2004). We did not determine whether GDNF can block the inflammation-induced upregulation of Reg-2, but we have shown that exogenous GDNF does not induce Reg-2 expression in naïve rats. In contrast, exogenous LIF upregulated Reg-2 in IB4⫹ cells. It was outside the scope of this study to establish whether endogenous cytokines of the CNTF/LIF family are responsible for the Reg-2 upregulation after inflammation, but our results suggest that this might be the case. LIF is upregulated in inflammatory states (Sugiura et al., 2000; Zhu et al., 2001) and interestingly appears to have both pro- and anti-inflammatory roles (Thompson et al., 1996; Banner et al., 1998; Sugiura et al., 2000; Zhu et al., 2001; Kerr and Patterson, 2004). However the pattern of Reg-2 expression in response to intrathecal LIF was not as selective for IB4⫹ cells as that seen with inflammation. Thus Reg-2 was upregulated in both IB4⫹ and IB4⫺ cells, consistent with the fact that some cells in both populations express LIF receptors (Thompson et al., 1997; Gardiner et al., 2002). 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