Neuropeptide Y- and vasoactive intestinal polypeptide-like immunoreactivity in adjuvant arthritis: Effects of capsaicin treatment

Neuropeptides (1995) 29, 33-43 © Pearson Professional Ltd 1995

Neuropeptide Y- and Vasoactive Intestinal Polypeptide-like Immunoreactivity in Adjuvant Arthritis: Effects of Capsaicin Treatment M. AHMED*, A. BJURHOLM*, E. THEODORSSON$, M. SCHULTZBERGI and A. KREICBERGS*

*Department of Orthopaedics, Karolinska Hospital, S-171 76 Stockholm, Sweden, fDepartment of Clinical Neuroscience and Family Medicine, Geriatric Medicine, Novum, KFC, S- 141 86 Huddinge, Sweden and ¢Department of Clinical Chemistry, Karofinska Hospital, S-171 76 Stockholm, Sweden (Reprint requests to MA)

Abstract--The occurrence of the neuropeptides vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) in ankle joints and dorsal root ganglia (L2-L6) was analyzed in normal and arthritic Lewis rats. In addition the effect of capsaicin pretreatment was investigated. The study included 92 rats consisting of 4 groups, 23 rats in each; normal rats, normal rats given capsaicin, arthritic rats and arthritic rats pretreated with capsaicin. The localization of the neuropeptides was assessed by immunohistochemistry and the tissue concentrations were determined by radioimmunoassay (RIA). In the arthritic rats, there was a slight increase in NPY immunoreactive nerve fibres in the ankle joint synovium and bone marrow, as compared to normal rats. Notably, there was an intense fluorescence and significant increase (p <0.01, 41%) in the number of NPY-positive megakaryocytes in the tibial bone marrow of arthritic rats. RIA showed that the concentration of NPY-like immunoreactivity (LI) was increased by 50% in the ankle joint. Pretreatment with capsaicin did not affect the increased level of NPY-LI in the ankle joint of arthritic rats. The concentration of NPY-LI in the dorsal root ganglia was not altered in arthritic rats, nor was it affected by the capsaicin treatment. No NPY immunoreactive cells could be detected in the dorsal root ganglia. The number of VIP immunoreactive nerve fibres observed in ankle joints of arthritic and normal rats did not differ. However, RIA measurements showed an 11% increase in the VIP concentration in arthritic rats, which was unaffected by capsaicin treatment. In dorsal root ganglia, RIA disclosed a 21% increase in VIP-LI, although no VIP-positive cells could be detected. Capsaicin treatment did not affect the increased concentration of VIP-LI in the dorsal root ganglia.

Date received 17 November 1994 Date accepted 2 M a r c h 1995

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NEUROPEPTIDES

The present study showed an up-regulation of VIP-LI in ankle joints and dorsal root ganglia of arthritic rats, whereas NPY-LI increased in ankle joints and bone marrrow. Pretreatment with capsaicin did not affect the levels of these neuropeptides in ankle joints or dorsal root ganglia. The main observation that there is an increase in VIP- and NPY-LI in adjuvant arthritis, which is unaffected by capsaicin, indicates that the autonomic nervous system participates in the pathophysiology of the disease, to some extent independently of the sensory nervous system.

Introduction

A number of experimental and clinical studies indicate that the nervous system is implicated in the pathophysiology of arthritis.l-14 On the basis of substantial evidence, the main interest has so far focused on the sensory nervous system? 9However, also the autonomic nervous system has been suggested to participate in the development of arthritis. 9-~vThus, spontaneously hypertensive rats with increased sympathetic activity have been shown to develop more severe adjuvant arthritis than normotensive rats. 9 In rat adjuvant arthritis, sympathectomy by guanethidine or reserpine has been found to attenuate the signs of inflammation.9'1° In patients with rheumatoid arthritis (RA), regional sympathectomy by surgery ll or by intravenous infusion of guanethidine and reserpine was reported to be of therapeutic value. ~2'13 Moreover, propranolol, a /~-adrenergic receptor antagonist, decreased the severity of joint inflammation both in patients with RA 14and in rats with adjuvant arthritis, l° Altogether, these findings suggest an involvement of the autonomic nervous system in arthritis. We recently observed an up-regulation of sensory neuropeptides, notably, substance P (SP) and calcitonin gene-related peptide (CGRP), in ankle joints and the corresponding dorsal root ganglia in adjuvant arthritis. 18 Pretreatment with the neurotoxin capsaicin was found to mitigate this upregulation of SP and CGRP as well as the signs of inflammation.~9 However, it has yet to be shown whether a total inhibition of the sensory nervous system would prevent entirely the development of arthritis. Thus, it remains unknown to what extent the sensory nervous system contributes to the development of arthritis. Conceivably, also the autonomic nervous system takes part in the pathophysiology of arthritis, either independently or in interaction with the sensory system.

In the present study the occurrence of neuropeptide Y (NPY-) and vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) was analyzed in ankle joints and dorsal root ganglia of arthritic rats by immunohistochemistry and radioimmunoassay (RIA). In an attempt to isolate the autonomic neuropeptide response from the influence of the sensory, the occurrence of NPYand VIP-LI was also studied after capsaicin treatment.

Materials and methods

The study included 92 female Lewis rats (bw 230250g). The rats were divided into 4 experimental groups: 1) normal rats, 2) normal rats treated with capsaicin, 3) arthritic rats and 4) arthritic rats pretreated with capsaicin. The animals were housed 5/cage at 21°C in a 12 h light/dark cycle with water and pellets ad lib according to the Karolinska Institute protocol.

Experimental animals Forty-six rats, deeply anaesthetized with ether, were injected subcutaneously (s.c.) with capsaicin (50 mg/kg bw), dissolved in 10% ethanol and 10% Tween-80 in isotonic saline, for 4 consecutive days (total dose 200 mg/kg). To reduce the respiratory symptoms, the animals received theophylline (5 mg/kg), intraperitoneally (i.p.) prior to each capsaicin injection. The remaining 46 rats received s.c. injections of vehicle (isotonic saline). 23 of the capsaicin injected rats and 23 of the rats receiving vehicle were inoculated with heat-killed mycobacteria (Mycobacterium butyricum) in paraffin oil (10 mg/ml) to induce arthritis, z° The mycobacterial suspension (0.05 ml) was injected intradermally into the base of the tail 1 week after the last capsaicin injection, i.e. on day 11. The remaining 46 rats (23

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A U T O N O M I C NEUROPEPTIDES A N D A R T H R I T I S

capsaicin injected + 23 vehicle injected) received an intradermal injection of paraffin oil. The end-point of the experiment was set at day 29, given that the day of mycobacteria inoculation was defined as day 0; control rats were killed on day 29 after inoculation with paraffin oil and arthritic rats on day 29 after inoculation with mycobacteria. Capsaicin treatment for 4 days and a free interval of 7 days preceded the inoculation with mycobacteria or paraffin oil; altogether the experiment covered a period of 40 days (4 + 7 + 29 days).

and interassay coefficients of variation were 7 and 12%, respectively. VIP-LI was analyzed using antiserum VIP2 raised against conjugated natural porcine VIP. The antiserum does not cross-react with gastrin, pancreatic polypeptide, glucagon, NPY or neurotensin. HPLC purified 125I-porcine VIP was used as radioligand and porcine VIP was used as standard. The detection limit of the assay was 3 pmol/1. Intra- and interassay coefficients of variation were 9 and 13%, respectively.

Radioimmunoassay

Immunohistochemistry

18 rats from each experimental group were killed 5 rats of each experimental group were anaeby decapitation during ether anaesthesia. In each sthetized by i.p injection of sodium pentobarbitone rat the ankle joint including the tarsal joints was (60 mg/kg bw). Intra-arterial perfusion with phosdissected as one specimen bilaterally. Thus, the phate buffered saline (PBS) preceded perfusion with specimen consisted of capsular/synovial tissue as fixative, consisting of 4% paraformaldehyde in 0.2 well as the distal tibial epiphysis, the whole talus tool/1 S6rensen phosphate buffer, pH 7.3, conand calcaneus, and the tarsal bones. Each joint taining 0.2% picric acid. specimen was prepared and analyzed separately. In each rat the ankle joints and dorsal root ganThe dorsal root ganglia (L2-L6) were also dissected glia were excised and analyzed separately. Each separately. However, for analysis they were pooled joint specimen included the same tissue components from each rat as left and right specimens, i.e. 5 as described before (see RIA). All samples were ganglia in each pooled sample, yielding a total of immersed in the fixative for 2 days at 4°C. The joint 18 right and 18 left samples from each experimental specimens containing bone were subjected group. This permitted correlative studies of the to demineralization in a 4% EDTA solution at neuropeptide concentrations in a given ankle joint pH 7.3 for approximately 3 weeks as described preand the ipsilateral dorsal root ganglia. All dissected viously.23 tissues were immediately frozen on dry ice and kept All specimens were rinsed for at least 2 days in at - 7 0 ° C until neuropeptide extraction. 20% sucrose in 0.1 tool/1 S6rensen phosphate Each frozen sample was weighed before extrac- buffer, pH 7.2, containing sodium azide and baction. The neuropeptides were extracted and quant- itracin (Sigma Chemicals, St Louis). The tissues ified as described recently, zl Briefly, the samples were cut on a Leitz cryostat to a section thickness were cut into small pieces, boiled for 10 min in of 15 #m. The sections were mounted on chrom10 volumes of 2 mol/1 acetic acid in 4% ethylene alum-gelatin coated slides and processed for diamino-tetraacetic acid (EDTA), homogenized in indirect immunofluorescence histochemistry as a Polytron (15 s), sonicated (30 s) and centrifuged described by C o o n s . 24 Briefly, the sections were 3000 x g for 15 rain. The supernatants were freeze rinsed for 15 rain in 0.01 tool/1 PBS, pH 7.3, and dried and diluted in 10 ml RIA buffer and kept at then incubated overnight in a humid atmosphere at - 2 0 ° C until analysis. The analysis was performed +4°C with antiserum to NPY (1:200) and VIP using the following antisera: (1 : 200) NPY-LI was analyzed using antiserum N12~ The NPY and VIP antisera were raised in rabbits which cross-reacts 0.1% with avian pancreatic poly- against the synthetic porcine NPY and VIP, respecpeptide, but not with other peptides. HPLC purified tively (Peninsula Laboratories Europe Ltd, St 125I-porcine NPY was used as radioligand and syn- Helens, UK). After incubation with the primary thetic porcine NPY was used as standard. The antisera, the sections were rinsed in PBS for 2 x 10 detection limit of the assay was 11 pmol/1. Intra- rain, and then incubated with fluorescein iso-

36 thiocyanate (FITC)-conjugated goat anti-rabbit antibodies (diluted 1:10, Amersham Sweden AB, Solna) for 30 min at 37°C. A final rinse in PBS for 15 rain preceded mounting in a mixture of glycerol and p-phenylenediamine}5 Controls were performed by omitting the primary antisera, or addition of 50/~g/ml peptide to the corresponding antiserum prior to application on tissue sections. A Nikon epifluorescence microscope was used to analyze the sections, and T-Max black and white film (Kodak, Rochester) was used for photography. The identity of NPY immunoreactive cells in the bone marrow was determined by staining with cresyl violet after photography. The number of NPY immunoreactive cells per cm2 was determined in 3 sections (counting cells in 4 fields in each section) of the distal tibia of each of 4 control rats and 4 arthritic rats. Statistics The mean and one standard deviation were used as measures of central tendency and variation. Data were tested for normality using the Anderson-Darling test. 26 Mann-Whitney non-parametrical analysis of variance was used to test for differences between 2 groups. The differences in the number of NPY immunoreactive cells in normal and arthritic bone marrow was calculated by using MGLM module of SYSTAT for windows version 5.02 systat INC. Evansaton, IL. Spearman's rank coefficient was used to analyze the correlation between groups. P values < 0.05 were considered significant.

Results

In rats inoculated with mycobacteria, signs of inflammation appeared from day 9 to 13 in the ankle joints. There was bilateral hind paw swelling, warmth and redness, which persisted until the end of the experiment. Rats pretreated with capsaicin before inoculation with mycobacteria, similarly showed signs of inflammation, although less pronounced compared to the arthritic rats not pretreated with capsaicin. Histological analysis confirmed these observations. Thus, there was synovial hypertrophy, oedema and round cell infiltration in both experimental groups given mycobacteria. Comparison of the ankle joint

NEUROPEPTIDES

Table 1. Comparison of ankle joint weights Control rats

Arthritic rats

Capsaicin + arthritic rats

Ankle-R 211.94___18.24 438.33_+66.80*** 316.11_+75.24"** Ankle-L 204.44_+ 18.86 433.89_+56.37*** 302.78_+72.58*** Comparisons of ankle joint weights were made between the control, arthritic and capsaicin pretreated arthritic rats. Each value (mg) corresponds to the m e a n + s t a n d a r d deviation, n = 18. Differences are expressed by asterisks according to the level of significance: ***p < 0.001, R = right, L = left.

weights (left and right sides combined) showed a significant increase (110%) in arthritic rats compared to control rats (Table 1). In arthritic rats pretreated with capsaicin, the increase in weight was 44% compared to control rats, reflecting a significant reduction (40%, p < 0.001) in ankle joint weights of arthritic rats pretreated with capsaicin compared to untreated arthritic rats (Table 1). Radioimmunoassay NPY-LI consistently occurred at higher (about 10 fold) concentrations than VIP-LI in the different tissues analyzed. In both normal and arthritic rats capsaicin treatment failed to affect the concentration of NPY and VIP in ankle joints and dorsal root ganglia. NPY. In arthritic rats, NPY-LI was significantly increased in the ankle joints compared to control rats. The increase as assessed by comparing the means of left and right ankle joints, respectively, was 50% (Table 2). No increase in NPY-LI was noted in the dorsal root ganglia of arthritic rats. Capsaicin treatment did not affect the concentration of NPY in arthritic rats, nor in control rats. Thus, NPY-LI remained increased (40%) in ankle joints of arthritic rats despite pretreatment with capsaicin. Comparative analysis of the NPY concentrations in ipsilateral ankle joints and dorsal root ganglia showed no correlation. VIP. In arthritic rats, there was a slight increase in VIP-LI in the ankle joints compared to control rats. The increase as assessed by comparing the means of left and right ankle joints, respectively, was 11% (Table 3). In the dorsal root ganglia of arthritic rats, there was a 21% mean increase in VIP-LI. Capsaicin treatment did not affect the con-

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AUTONOMIC NEUROPEPTIDES AND ARTHRITIS Table 2. Tissue concentrations of NPY-LI in ankle joints and dorsal root ganglia Control

Ankle-R Ankle-L DRG-R DRG-L

1.76_+0.43 1.77_+0.31 13.68_+2.73 13.41+ 1.63

Capsaicin

Arthritic

1.81 _+0.25NS 1.80_+0.39NS 12.97 +_3.09NS 13.24_+2.63NS

Capsaicin ÷ arthritic

2.64_+0.60*** 2.67__+0.84*** 13.56_+1.96NS 13.45_+1.75NS

2.54_+0.71"** 2.50_+0.67*** 13.67_+ 1.86NS 13.72_+ 1.85NS

Comparisons were made between the control and capsaicin treated rats, between control and arthritic rats, between control and capsaicin pretreated arthritic rats and between capsaicin and capsaicin pretreated arthritic rats. The values were obtained from RIA measurements in tissue extracts from individual animals as described in Materials and methods, and each value (pmol/g) corresponds to the mean _+standard deviation, n = 18. Differences are expressed by asterisks according to the level of significance: ***p < 0.001, NS = nonsignificant, R = right, L = left, DRG = dorsal root ganglia.

c e n t r a t i o n o f V I P - L I in a n k l e j o i n t s a n d d o r s a l r o o t g a n g l i a o f a r t h r i t i c rats, n o r in c o n t r o l rats. C o m p a r a t i v e analysis o f the V I P c o n c e n t r a t i o n s in ipsilateral a n k l e j o i n t s a n d d o r s a l r o o t g a n g l i a s h o w e d no c o r r e l a t i o n . Immunohistochemistry

I n the a n k l e j o i n t s , N P Y i m m u n o r e a c t i v e nerve fibres were identified in the s y n o v i u m , p e r i o s t e u m , b o n e a n d b o n e m a r r o w in b o t h c o n t r o l a n d arthritic rats. V I P - p o s i t i v e fibres were f o u n d only in the s y n o v i u m a n d p e r i o s t e u m in c o n t r o l rats, whereas in arthritic rats some fibres o c c u r r e d also in bone. In general, N P Y - p o s i t i v e fibres were m o r e a b u n d a n t t h a n V I P - p o s i t i v e fibres in the tissues analyzed. Abundant NPY i m m u n o r e a c t i v e cells were o b s e r v e d in the tibial b o n e m a r r o w o f c o n t r o l a n d a r t h r i t i c rats. A f t e r cresyl violet staining, the N P Y i m m u n o r e a c t i v e cells were f o u n d to exhibit c h a r a c teristic m o r p h o l o g i c a l features o f m e g a k a r y o c y t e s , such as m u l t i p l e nuclei. N o N P Y - o r V I P - p o s i t i v e cells were d e t e c t e d in the d o r s a l r o o t g a n g l i a o f

either c o n t r o l o r arthritic rats. A d m i n i s t r a t i o n o f c a p s a i c i n did n o t affect the density o f nerve fibres i m m u n o r e a c t i v e to N P Y a n d V I P in the a n k l e joints, neither in arthritic n o r in c o n t r o l rats. VIP. I n c o n t r o l rats, o c c a s i o n a l V I P i m m u n o r e a c t i v e nerve fibres were f o u n d in the ankle j o i n t s y n o v i u m a n d p e r i o s t e u m (Fig, 1A). The fibres were predominantly non-vascular, No VIP-positive fibres c o u l d be detected in b o n e or b o n e m a r r o w . I n the d o r s a l r o o t ganglia, neither cells n o r fibres with V I P - L I were found. There was no effect o f c a p s a i c i n t r e a t m e n t on VIP-L1 in the ankle joints. The d i s t r i b u t i o n a n d the n u m b e r o f V I P i m m u n o r e a c t i v e nerve fibres in the s y n o v i u m (cf Fig. 1A & B) a n d p e r i o s t e u m o f the ankle j o i n t s was essentially the same in arthritic rats as in c o n t r o l rats. In a d d i t i o n there were a few fibres in bone. N e i t h e r cells n o r nerve fibres with V I P - L I c o u l d be seen in the d o r s a l r o o t ganglia. T h e c a p s a i c i n t r e a t m e n t h a d n o visible effect o n V I P i m m u noreactive structures in the a n k l e joints. N P Y . In c o n t r o l rats, N P Y i m m u n o r e a c t i v e

Table 3. Tissue concentrations of VIP-LI in ankle joints and dorsal root ganglia

Ankle-R Ankle-L DRG-R DRG-L

Control

Capsaicin

Arthritic

0.18__+0.02 0.18_+0.02 1.05+0.19 1.09_+0.31

0.19_+ 0.02NS 0.19_+ 0.02NS 0.99_+ 0.23NS 1.01 -+0.26NS

0.20_+0.06* 0.20_+0.05* 1.32_+0.37" 1.28-+0.33"

Capsaicin+ arthritic

0.19± 0.03NS 0.20_+ 0.04NS 1.22_+0.23" 1.19-+0.18"

Comparisons between the groups were made as in Table 2. The values were obtained from RIA measurements in tissue extracts from individual animals as described in Materials and methods, and each value (pmol/g) corresponds to the mean+standard deviation, n = 18. Differences are expressed by asterisks according to the level of significance: *p <0.05, NS = non-significant, R = right, L = left, DRG = dorsal root ganglia.

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Fig. IA-D Immunofluorescence micrographs of sections of rat ankle joints after incubation with antisera to VIP (A, B) and NPY (C, D). No obvious difference was observed in the number of nerve fibres (arrows) immunoreactive to VIP in the synovial membrane of control (A) and arthritic (B) rats. NPY immunoreactive nerve fibres are observed (arrows) in the synovial membrane of control (C) and arthritic (D) rats. Note the slightly increased number of NPY-positive fibres in the arthritic synovium (D). The magnification is 250 x in all micrographs, s = synovial membrane, b = bone.

nerve fibres were easily identified in the s y n o v i u m a n d b o n e m a r r o w o f a n k l e j o i n t s (Fig. 1C) T h e fibres were exclusively vascular; f o r m i n g a n e t w o r k in the b l o o d vessel walls. I n the b o n e m a r r o w , m a n y cells o f m e g a k a r i o c y t i c a p p e a r a n c e also e x h i b i t e d N P Y - p o s i t i v e staining (Fig. 2A). V a s c u l a r N P Y i m m u n o r e a c t i v e fibres were also f o u n d in the perio s t e u m a n d bone. I n the d o r s a l r o o t ganglia, there were no d e t e c t a b l e N P Y - p o s i t i v e cells. H o w e v e r , a few N P Y - p o s i t i v e fibres were f o u n d in r e l a t i o n to b l o o d vessels. C a p s a i c i n t r e a t m e n t was n o t f o u n d to alter the o c c u r r e n c e o f N P Y - L I in either ankle j o i n t s o r d o r s a l r o o t ganglia. In a n k l e j o i n t s o f arthritic rats, the n u m b e r o f N P Y i m m u n o r e a c t i v e nerve fibres was slightly increased in the s y n o v i u m (cf Fig. I C & D ) a n d

Fig. 2A & B Immunofluorescence micrographs of sections of rat tibial bone marrow after incubation with antiserum to NPY. NPY immunoreactive perivascular nerve fibres (arrows) in the distal tibial bone marrow of control (A) and arthritic (B) rats. Slightly increased NPY-positive fibres are observed in arthritic bone marrow (D). Increased numbers of NPY immunoreactive megakaryocytes are observed in the tibial bone marrow of arthritic (B) rats as compared to control (A) rats (arrow heads). The magnification is 125 x in A and B. m = bone marrow.

b o n e m a r r o w (cf Fig. 2 A & B) o f the ankle j o i n t s c o m p a r e d to c o n t r o l rats. T h e fibres were exclusively vascular. N o increase in N P Y - p o s i t i v e fibres was n o t e d in p e r i o s t e u m a n d bone. T h e r e was a 4 1 % increase (p < 0 . 0 1 ) in the n u m b e r o f m e g a k a r y o c y t e s , stained with N P Y , in the tibial b o n e m a r r o w o f arthritic rats (1.68 _+ 0.69/cm 2) c o m p a r e d to c o n t r o l rats (1.19 _+ 0.28/cm2). N o N P Y - p o s i t i v e cells were identified in the d o r s a l r o o t ganglia, a n d there was no d e t e c t a b l e increase in v a s c u l a r N P Y positive fibres in the d o r s a l r o o t ganglia. C a p s a i c i n t r e a t m e n t h a d no effect o n the extent o f N P Y i m m u n o r e a c t i v e fibres in either a n k l e j o i n t s o r d o r s a l r o o t ganglia.

A U T O N O M I C NEUROPEPTIDES AND A R T H R I T I S

Discussion

The present study shows that there is an increase in NPY- and VIP-LI in ankle joints of arthritic rats. There is also a slight increase in VIP-LI in the corresponding dorsal root ganglia, but no increase in NPY-LI. Capsaicin treatment does not affect the occurrence of these neuropeptides in ankle joints or dorsal root ganglia. An involvement of the autonomic nervous system in inflammatory disorders of the locomotor system has been suggested in a number of reports. 9 17 Surgical and chemical sympathectomy has been shown to reduce the severity of pain and inflammation in patients with RA and reflex sympathetic dystrophy. 11 13,16,17In experimental arthritis, chemical sympathectomy by reserpine, guanethidine or 6-hydroxydopamine (6-OHDA) have been shown to reduce the inflammatory signs as assessed by radiography and histology. 9,~°,27 However, the underlying mechanisms behind the therapeutical effect of sympathectomy remain unclear. Data on autonomic messengers presumed to be involved in inflammatory disorders is still very limited. In arthritis, only a few studies have focused on the role of autonomic neuropeptides. In a recent immunohistochemical study of joint tissues from RA patients by M a p p e t al, 28 a decrease in NPY-positive fibres was noted in the synovial membrane. Seemingly contradictory findings were reported in a RIA study by Larsson et al, 29 who found a significant increase in NPY-LI in synovial fluid from patients with RA. Hence, it remains unclear whether there is an up- or down-regulation of autonomic neuropeptides in arthritis. Moreover, it is unclear whether the change in expression of autonomic neuropeptides in arthritis occurs independently or in interaction with the sensory nervous system suggested to be involved in the pathophysiology of arthritis. In the present study we found an increase in NPY-LI by 50% and in VIP-LI by 11% in the ankle joints of arthritic rats according to RIA. This would seem to reflect that autonomic neuropeptides are up-regulated in arthritis. However, this up-regulation was only partly supported by our immunohistochemical observations. Thus, there was a slight increase in NPY-positive fibres in the bone marrow and synovial membrane of arthritic rats,

39 whereas the distribution of VIP-fibres appeared the same in arthritic and normal rats. The most conspicuous immunohistochemical finding was the increase in non-neuronal NPY. Thus, in the bone marrow of arthritic rats, the immunofluorescence of the NPY-positive megakaryocytes appeared much more intense than in the control rats and there was a 41% increase in the number of NPY immunoreactive megakaryocytes. The increased concentration of NPY-LI in the ankle joints of arthritic rats may prove to predominantly originate in bone marrow cells rather than neuronal structures. Further studies are necessary to evaluate the importance of an increased production of this autonomic neuropeptide in arthritis, and whether it acts as a neuronal or immunogenic messenger. An interaction between the immune system and the autonomic nervous system has been suggested in a number of studies. Thus, noradrenergic (NA) nerve fibres were shown to innervate both primary (bone marrow and thymus) and secondary (spleen and lymph nodes) lymphoid organs. 3°-32 The NA nerve terminals end in close apposition to T-lymphocytes, 33 and NA receptors have been identified on lymphocytes, monocytes and macrophages. 3° Sympathetic denervation of lymphoid organs in rodents has been shown to abrogate primary immune responses. 32,34 VIP receptor binding has been detected on T-lymphocytes and macrophages, 35 and VIP was shown to suppress lymphocyte proliferation and killer cell activity. 36 High levels of NPY were demonstrated in megakaryocytes and platelets, 37 and notably, in the spleen of autoimmune mouse strains? 7 In the present study, the increase (RIA) in autonomic neuropeptides in the arthritic ankle joints seemed to have occurred predominantly in the bone marrow (immun ohistochemistry). Presumably, the increased NPY-LI observed in the megakaryocytes reflects enhanced NPY-synthesis. Whether this enhancement occurs in response to or independently of a neuronal stimulus remains to be clarified. In addition to the increase in NPY- and VIP-LI in the ankle joints of arthritic rats, there was a 21% increase in VIP-LI in the dorsal root ganglia. An earlier study on m R N A for VIP in arthritis 3a did not reveal any changes in transcription in the dorsal root ganglia. In agreement with previous studies, 39

40 there was no change in NPY-LI in the dorsal root ganglia. Recently, an increased expression of NPY m R N A was observed in dorsal horn neurons of the spinal cord during inflammation. 4° In a previous study on SP and C G R P we observed a parallelity between the concentration changes in the dorsal root ganglia and the ipsilateral ankle joints in arthritic rats. 18 Such paratlelity could not be seen for VIP. Immunohistochemical analysis of the dorsal root ganglia failed to substantiate the increase in VIP-LI. In fact, neither VIP nor NPY-LI could be detected in the ganglion cells of normal or arthritic rats. The reason for the failure of demonstrating immunohistochemically VIP-LI in the dorsal root ganglia, despite the observed increase in concentration (RIA) in arthritis, should be attributed to the design of the study. Thus, previous studies showing the presence of VIP- and also NPYpositive ganglion cells are based on manipulation by either colchicine treatment 41 or peripheral nerve section. 42'43 Presumably, the 20% increase in VIPLI observed in the dorsal root ganglia in arthritis is too low to be detected by immunohistochemistry. The physiological significance of the observed increase in VIP-LI in the dorsal root ganglia is obscure. It has been speculated that the conditional occurrence of VIP-LI in the dorsal ganglia reflects a facultative function elicited when the availability of sensory neuropeptides is inadequate i.e., VIP takes over the role of tachykinins after axotomy. 44 Although research on the pathophysiological mechanisms leading to arthritis mainly has focused on the immune system, there are a number of recent reports suggesting an important role of the sensory nervous system. 3-9 An altered expression of the sensory neuropeptides SP and C G R P claimed to be involved in nociception and inflammation has been demonstrated in several studies on arthritis .6 8,18,29,45 The therapeutic effect of sympathectomy in arthritis may reflect that there is an interaction between the sensory and autonomic nervous systems. Thus, the autonomic nervous system has been suggested to potentiate the nociceptive effect of the sensory nervous system. 4~48 From these studies, it may be assumed that the expression of sensory neuropeptides is influenced by autonomic messengers and vice versa. In the present study, we found an upregulation of NPY- and VIP-LI in ankle joints of

NEUROPEPTIDES arthritic rats. In order to investigate whether or not this upregulation is a secondary phenomenon to the altered expression of sensory neuropeptides in arthritis the expression of autonomic neuropeptides was analyzed also after capsaicin treatment. This was expected to isolate the autonomic system from the sensory or at least reduce the effect of the latter. Notably, capsaicin had no effect on the concentration of NPY and VIP in the ankle joints and dorsal root ganglia of arthritic rats. Although this is the first study on autonomic neuropeptides in ankle joints of arthritic rats given capsaicin, the results are in close agreement with those reported from similar studies of other tissues such as gastrointestinal tract, lung, spleen and lymph nodes. 49-53 Our observations indicate that the increased expression of autonomic neuropeptides in arthritis is not closely linked to that of sensory neuropeptides. This in turn implies that neuronal therapy in arthritis should be targeted against both the sensory and autonomic systems. Assuming that the simultaneous up-regulation of sensory and autonomic neuropeptides in arthritis occurs independently, it seems likely that the stimulation of both systems is related to the same factor, possibly nerve growth factor (NGF). N G F has been suggested as an essential factor for the survival and function of both sensory and sympathetic neurons. 53'54In adult rats, N G F treatment resulted in increased synthesis of SP and C G R P in dorsal root ganglia. 55'56In addition, N G F deprivation was shown to cause a down-regulation of sensory neuropeptides. 57,58 Similarly, the concentration of tyrosine hydroxylase, the rate limiting enzyme in the synthesis of NA, was elevated in response to N G F administration and markedly decreased after N G F deprivation. 59 Furthermore, there was an up-regulation of VIP in neonatal rats by N G F . 6° In addition to its general role in the control of cell number in sympathetic ganglia, N G F is suggested to regulate the degree of axonal branching. The administration of N G F in vivo induces profuse axonal branching and dendritic arborization. 61 Notably, the levels of N G F are increased in the synovial fluid and membrane of arthritic rats. 27'62'63 Recently, a simultaneous upregulation of N G F and sensory neuropeptides was found in the sciatic nerve in rats with paw inflammation. 64 Therefore, it might be speculated that the increase in autonomic neuro-

41

AUTONOMIC NEUROPEPTIDES AND ARTHRITIS peptides seen in the present study of adjuvant a r t h r i t i s is i n d u c e d b y N G F , m o s t l i k e l y i n d e pendently of the sensory nervous system.

14. 15.

Acknowledgements This study was supported by grants from the Royal 80 Year Funds of King Gustaf V, Uggla's Foundation, Ake Wibergs Foundation, the Karolinska Institute Research Funds, Lundbergs Foundation, the Swedish Association Against Rheumatic Disease, the Swedish Medical Research Council (12X-08652-01, 12X-07464) and the Swedish Society for Medical Research.

16.

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