Annals of the Rheumatic Diseases 1995; 54: 398-403

Innervation of the synovium

P I Mapp

This review will describe the normalinnervation of the joints, in particular thesynovium, and then show how this changes inthe rheumatoid arthritis (RA) joint in thehuman. Substance P will be described in detail,as an example of a particular neuropeptide.The concepts surrounding neuropeptide releaseand the role of neuropeptides in inflammatoryjoint disease (neurogenic inflammation) willthen be explored. The in vitro and in vivoeffects of substance P will be described. Thereceptors for substance P and the effects ofantagonists, peptide and synthetic, for thereceptor on inflammatory models will also bediscussed. The synovial distribution of theenzyme systems that break down neuro-peptides and how this may lead to functionalcompartmentalisation will be described. Finally,some mechanisms by which pain may arise willbe presented.

Normal innervation ofthe jointJoints are supplied by both primary andaccessory nerves. Primary nerves are branchesof peripheral nerves passing near to the joint,whilst accessory nerves are branches of intra-muscular nerves crossing the joint capsule.Some joints such as the knee and ankle alsoreceive a nerve supply from cutaneous nervesin the overlying skin. The nerves to any oneparticular joint always arise from more thanone level in the spinal cord. About 50% of theaxons that comprise articular nerves are lessthan 5 Vim in diameter; those that are less than2 im in diameter are unmyelinated. Thesefibres carry nociceptive information with aslow conduction velocity. Until relativelyrecently, the nerve supply to the synovium wasthought to be sparse. However, it is now knownthat the synovium contains a good supply ofthinly myelinated or unmyelinated nerve fibres(table). These are of two types: postganglionicsympathetic adrenergic fibres located aroundthe larger blood vessels are responsible for thecontrol of articular blood flow; unmyelinated Cfibres are responsible for pain transmission.This latter group of fibres are not normallyactive and are believed to fire only during

Diameters of nerve fibres, their classification and location

Afferentfibre diameter Conduction velocity Class Location FunctionOWS)

Myelinated10-18 ,um 60-100 I (Aac) Ligaments Proprioceptive5-12 p.m 20-70 II (A,B) Capsule Proprioceptive1-5 p.m 2-5-20 III (Ab) Capsule Nociceptive

SynoviumUnmyelinated

<1 .Lm <1 IV (C) Capsule NociceptiveSynovium

tissue damage, either mechanical or chemical.They are therefore sometimes referred to asnociceptive fibres. They are not responsive tonormal ranges of movement. However, duringan inflammatory response, these fibres may besensitised by mediators such as prostaglandinE2, whereupon even movement in the normalrange can cause them to discharge, signallingpain.

INNERVATION OF RHEUMATOID SYNOVIUM

The rheumatoid synovium is a chronicallyinflamed tissue showing villous hypertrophy ofthe synovial layer, with an underlying infiltrateof inflammatory cells. Perivascular cuffs oflymphocytes are seen and the tissue mayorganise itself to resemble a lymphoid follicle.The innervation of the synovium in rheum-

atoid arthritis is markedly different from thatof the normal synovium. The initial observationsin the human synovium, outlined below, weremade by Gronblad et al, subsequently extendedMapp et al,2 and confirmed independently byPereria da Silva and Carmo-Fonseca.3

Whilst the deeper vessels in the synoviumhave a nerve population surrounding them, thesuperficial vessels are apparently not innervated.Also, the number of free fibres is greatlyreduced compared with normal synovium. Noimmunoreactive fibres are seen in intenselyinflamed and 'lymphoid' areas of the synovium.Free sensory fibres are not seen in the intimallayer, or in the tissue immediately underlyingthe intima. Staining for specific peptides showsthat the free sensory fibres present in theintimal layer of normal synovium are absentin rheumatoid specimens. The perivascularinnervation of the deeper blood vessels is pre-dominantly sympathetic efferent fibres. Similarchanges in the innervation of the synoviumhave been reported in rat models of arthritis.4

In summary, it would appear that in rheu-matoid synovium sensory and sympathetic nervefibres are absent from the superficial synoviumand areas of intense inflammation, whilst thedeeper synovium retains its innervation.Staining for specific neuropeptides in the deepsynovium is weaker and shows a more varicosedistribution than in normal controls, and it ispossible that this reflects an increased releaseof these peptides. The absence of nerve fibresin the superficial synovium may reflect damageto the peripheral terminals of nerve fibrescaused by mediators released during theinflammatory response. However, it is possiblethat the proliferating synovium outstrips thecapacity of the nerve supply to innervate it.How these changes in the nerve supply affectthe ability of the nervous system to maintainjoint homeostasis is a key question, and is the

Bone and JointResearch Unit,London HospitalMedical College,Whitechapel,London El 2AD,United KingdomP I Mapp

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subject of much debate and research. Thesensory and sympathetic fibres exert some oftheir influences by the release of neuropeptidesand this concept is described below.

SENSORY FIBRES CONTAIN NEUROPEPTIDES

Unmyelinated C fibres arise from cell bodieswhich are located in the dorsal root ganglion,close to the spinal cord. In addition to itsperipheral projection, the cell body also has acentral projection to the spinal cord. The finediameter nerve fibres terminate in the super-ficial layers of the spinal cord, laminae I andII of the dorsal horn, from where they synapsewith ascending fibres in the spinal cord. Thecell bodies are the site of manufacture ofneuropeptides which are transported bothcentrally and peripherally along the nerveaxons. The functions of these peptides are notclearly understood in the central nervoussystem but some, for example substance P, arethought to be involved in the modulation ofpain transmission. Peripherally, where theeffects of these peptides are much easier toinvestigate, they are clearly involved in theinflammatory process-both its induction andits modulation. They have the capacityindividually or in synergism with other neuro-peptides or mediators to modulate, mediateor prime for an inflammatory response. Inaddition, they have an effect on vascular tone.

SENSORY AFFERENT FIBRES CAN HAVE AN

EFFERENT FUNCTION

In addition to their sensory afferent function,the small unmyelinated fibres present in thesynovium, and throughout the body, are ableto release the neuropeptides they contain whenchallenged by a nociceptive stimulus. Theproduction of a nociceptive stimulus within asensory afferent fibre results in an orthodromicelectrical signal (in the direction of the spinalcord) and results in a sensation of a dullpain. However, within the extensive peripheralbranches of that same fibre, an antidromicelectrical signal (away from the spinal cord) ispropagated and this leads to the peripheralrelease of neuropeptides. This effect is knownas the axon reflex. The peripheral release ofneuropeptides stimulates an acute reaction-neurogenic inflammation.6 The principalpeptides known to be involved in the inductionof neurogenic inflammation are substance Pand calcitonin gene related peptide (CGRP).These peptides have a direct effect on thevasculature, causing vasodilatation and oedema,the classical weal and flare reaction. Inaddition, they may generate indirect effectsmediated by stimulation of effector cells,particularly mast cells, with the release ofhistamine and other inflammatory mediators.

Substance PIn 1931, von Euler and Gaddum extractedfrom equine brain and intestine a materialwhich was hypotensive and smooth musclestimulating.7 They designated this activity

preparation P (for powderable) and in laterpublications referred to it as substance P. It wasnot until 1971 that the peptide was purified,8sequenced and later synthesised.9

Substance P is a small polypeptide of 11amino acids and is one of a family of peptides,the tachykinins, which share four commonamino acids at the carboxyl terminal. Thereappear to be two preprotachykinin (PPT)genes, the SP/NKA PPT I gene and the NKBor PPT II gene. Substance P is encoded bymRNAs from the PPT I gene, alternative RNAsplicing resulting in the production of three(at, P and y) mRNAs, all ofwhich may producesubstance P.'0 Substance P is distributed widelythroughout the central and peripheral nervoussystem. In the central nervous system, it isbelieved to play a role in pain transmission,whilst in the periphery, vascular and numerousother effects have been reported.

IN VITRO INVESTIGATIONS

In vitro, the effects of substance P on a widevariety of cell types have been investigated.These data show release of inflammatorymediators from various cell types, includingthe degranulation of mast cells," the simu-lation of prostaglandin E2 and collagenase fromsynoviocytes, 12 (Lotz et al 1987), secretionof prostaglandins'3 and thromboxane'4 frommacrophages, and the modulation of immuno-globulin production from lymphoid tissue.'5Substance P is also reported to induce therelease of interleukin-1 from cultured mousemacrophages.'6 Thus tackykinins may contri-bute to the maintenance of chronic arthritis.These activities of substance P required dosesof the peptide in the nanomolar range, whichis consistent with published figures for thedissociation constant of substance P with itsreceptor (0-5-2-0 nmolI1). Other cells, such asneutrophils, appear to be activated directlyonly by much larger concentrations of thepeptide (>10 limoIlA), suggesting a lack ofphysiological relevance. However, smaller con-centrations of substance P, in the nanomolarrange, appear to be able to prime neutrophilsto respond to other mediators. In particular,neutrophils have been shown to react,synergistically with substance P, to otherchemotactic peptides such as complement-derived C5a or bacterial N-formyl-methionyl-leucyl-phenylalanine.17 Collectively these resultssuggest that, in addition to a direct effect ofsubstance P on several cell types, a secondmodulatory mechanism is also operative. Thenervous system may therefore be capable ofpriming cells to respond to smaller doses ofcertain agents than might otherwise be the case,leading to an apparent hypersensitivity. Thismechanism may be particularly important inallergic responses, such as that seen in asthma.

IN VIVO INVESTIGATIONS

As neuropeptides are synthesised in the dorsalroot ganglion cell bodies, an alternativeapproach to determine the involvement ofneuropeptides in arthritis would be to induce

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an inflammatory response in a single joint andthen monitor changes in neuropeptide levels inthe dorsal root ganglia that supply that joint.In the case of the knee joint, nerve tracingstudies indicated that L4 to L6 are theappropriate ganglia to examine. Using thismethod, Smith et al'8 found a 70% increase inimmunoreactive substance P in the ipsilateraldorsal root ganglia. In a Freunds adjuvantmodel of hind paw inflammation, substance Pconcentration was found to be increased by30 40% in the L4 to L6 dorsal root ganglia,five days after the induction of the inflam-mation.'9 In the same study the substance Pcontent of the sciatic nerve supplying theinflamed paw was increased by 60-75%. Theincreased biosynthesis of substance P inthe dorsal root ganglia of the monoarthritic ratis reflected in the finding of increases in themRNA levels of preprotachykinin A, theprecursor of substance P.20 These data suggestthat, in the early stages of the inflammatoryresponse, the amounts of substance P bothsynthesised and transported to the inflam-matory site are increased.The involvement of substance P in arthritis

has also been studied directly in animalmodels. Levine et al2' found that infusion ofsubstance P into rat knees increased theseverity of adjuvant induced arthritis. Thiswork was extended to show that the substanceP receptor was directly involved in this effect.Further experiments will be described in thesection on receptors.

EVIDENCE FOR RELEASE OF SUBSTANCE P IN

HUMAN JOINT INFLAMMATIONOne of the most obvious ways to implicatesubstance P, or any other neuropeptide, inarthritis is to demonstrate that the peptide ispresent in the inflammatory joint fluid. Manygroups have reported the presence of substanceP in synovial fluid, but agreement between thevalues obtained is poor. Of concern is thereliance on antisera prepared from rabbitplasma for the radioimmunoassay; becausedifferent studies have used a different anti-serum, the results are not strictly comparable.Of even greater concern must be the handlingof the specimens after withdrawal from thejoint, as there are numerous enzymes withinthe joint which are capable of the degradationof substance P (see later). These enzymes areoften unaffected by broad spectrum enzymeinhibitors, such as aprotinin, and may thereforecontinue to be active ex vivo. A furthercontribution to the confusion surroundingsuch measurements may be that the substanceP is bound to serum albumin22 in the jointfluid, and therefore the nature of the extractionprocedure for the peptide is critical to the finalamount observed. These reservations aresubstantiated by data demonstrating thatconcentrations of substance P in joint fluids areless than 4-7 pg/ml, which was the detectionlimit of the assay.23 The study showed that theinhibition of degrading enzymes and correctextraction procedures were all-important inobtaining reliable results.

RECEPTORS FOR SUBSTANCE P

There are currently three pharmacologicallydistinct receptors for tachykinins, termedNK1, NK2, and NK3. They are usuallydefined by the rank order of potency oftachykinins in binding studies. Substance P hasthe greatest affinity for the NK1 receptor,24although it may interact with NK2 and NK3receptors at higher concentrations. SubstanceP has effects on many different cell types, fromwhich it may be concluded that each cell typepossess a receptor, either constitutive orinducible.

Peptide receptors can be localised and theirkinetics studied by quantitative in vitroreceptor autoradiography with computerisedimage analysis.25 Specific high affinity, lowcapacity binding sites are present on theendothelial cells of human synovial bloodvessel. The ratio of the IC50 values for thesesites for substance P, neurokinin A, and neuro-kinin B is 1-25:175:>1000, respectively, and ischaracteristic of the NK1 class of tachykininreceptor.26 The interaction of substance P withNK1 receptors on endothelial cells may beimportant in the regulation of vascular tone.Vasodilatation induced by substance P hasbeen shown to be endothelium dependent andis inhibited by N-monomethyl-L-arginine,indicating a probable mediation by nitric oxide,the endothelium derived relaxing factor.27The distinct localisation of substance P

receptors on endothelial cells in humansynovium suggests that proinflammatory actionsof substance P may be predominantly via directvascular action.

PEPITIDE ANTAGONISTSThe involvement of substance P and itsreceptors in inflammation and arthritis hasbeen studied using several animal models.Levine et a12' found that infusion of substanceP into rat knees increased the severity ofadjuvant induced arthritis, but administrationof the substance P analogue (D-Pro2, D-Trp7'9)-substance P, a putative substance P receptorantagonist,28 only produced moderate softtissue swelling and osteoporosis. Lam andFerrell29 showed that the carrageenan model ofacute joint inflammation could be virtuallyabolished by prior administration of thesubstance P antagonist D-Pro4, D-Trp7'9",0-substance P (4-1 1). It has also been shown thatinjection of capsaicin, the hot component ofthe chilli pepper, into the synovial cavity of therat knee inhibited the inflammation seen afterinjection of substance P. Capsaicin causesthe depolarisation of afferent C fibres, leadingto the release of substance P and thedegranulation of mast cells, but in this casemay have been acting by causing a depletionof substance P receptors in the target tissue.30

NON-PEPTIDE ANTAGONISTS

Until recently, antagonists of the NK1 receptorwere limited to peptide analogues, such asthose described in the previous section. How-ever, the commercial pressure to develop non-

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metabolisable synthetic antagonists has led tothe development of a number of compounds.The first to be described was CP-96,345(from the Pfizer Company), arising from a highthroughput chemical file screening strategyusing a 3H-substance P binding assay as theprimary screen.3' This compound is a potent,reversible and competitive antagonist of theNK1 receptor. In vivo studies show that thecompound lacks agonist activity and is highlyselective. Investigations using CP-96,345 haveshown that NK1 receptors display hetero-geneity: CP-96,345 has a greater affinity forhuman and guinea pig NK1 receptors thanfor those in the rat.32 33 FK888 (FujisawaPharmaceuticals, Osaka, Japan) has similarlybeen found to have a greater affinity for guineapig receptors than for rat receptors.34 There isalso evidence for differences in NK1 receptorspecificities between different tissues of asingle species,35 and suggestions that receptorexpression or affinity may be influenced byinflammation.36 Results from studies ondifferent tissues in different species or indifferent pathological states should thereforebe interpreted with caution.

Degradation ofpeptidesThe local activities of regulatory peptidesdepend on a combination of release andclearance from the vicinity of their receptors.Many peptides have a short half lives, rapidclearance being attributable to membranebound peptidases. These enzymes havecharacteristic regional distributions, and theiractivities may vary during inflammation.Understanding the local topography of mem-brane bound peptidases and their relation tothe sites of release and action of those peptidethat may act as substrates is therefore essentialto understanding peptidergic regulatorysystems in the normal and diseased synovium.

Several peptide degrading enzymes havebeen localised to the synovium, includingneutral endopeptidase (NEP), angiotensin con-verting enzyme (ACE), dipeptidyl peptidase IV(DPPIV), and aminopeptidase M (APM).

NEUTRAL ENDOPEPTIDASE

NEP (EC 3.4.24.1 1) is capable of thehydrolysis of many peptides, including sub-stance P37 and CGRP.38 It is responsible for themajority of the degradation of substance P inthe human synovium. This enzyme has beenshown to be identical with the common acutelymphoblastic leukaemia antigen (CALLA)39and is the CD 10 determinant.NEP is an integral membrane metallo-

proteinase with a characteristic zinc bindingmotif in the extracellular carboxyterminaldomain.40 The enzyme hydrolyses peptidebonds at the amino side of hydrophobic aminoacids,4' and is believed to inactivate regulatorypeptides at the cell surface. NEP has beenhypothesised to modulate neurogenic inflam-mation, for example in the lung.42 Its activityhas been demonstrated in the synovium ofpatients with chronic arthritis,43 and was found

to be greater in all patients with rheumatoidarthritis and some patients with degenerativejoint disease, compared with traumatic arthritiscontrols. Since mature B and T lymphocytesand macrophages, which constitute the majorinflammatory cell populations in rheumatoidarthritis, contain no detectable NEP activity,44it has been speculated that synovial fibroblastsmay be the source of this enzyme. Our owndata show the localisation of this enzyme inhuman synovium, as determined by immuno-cytochemistry, and support the hypothesisthat fibroblasts are its source, a restrictedpopulation of cells surrounding blood vesselsbeing responsible for the majority of theactivity.45 These may represent a specialistsubpopulation of fibroblasts.As the function of NEP is probably the

degradation of locally released regulatorypeptides, its presence around the blood vesselsmakes it ideally located to inactivate vasoactivepeptides, such as substance P, which arereleased from perivascular nerve fibres. Thereis good evidence from in vivo investigationsto support this hypothesis. Substance P ininduced oedema in guinea pig skin ispotentiated by the NEP inhibitors phos-phoramidon and thiorphan,46 and attenuatedby coadministration of NEP. Furthermore,NEP may also limit the activity of endogen-ously released regulatory peptides. The bron-chial contraction induced by capsaicin, whichacts by releasing neuropeptides, includingsubstance P, from peripheral nerve endings, isalso enhanced by phosphoramidon.47 48

ANGIOTENSIN CONVERTING ENZYME

ACE acts as a peptidyl peptidase, inactivatingbradykinin and activating angiotensin I byremoval of C-terminal dipeptides. In addition,ACE can act as an endopeptidase, cleavingsubstance P to liberate the C-terminal di- ortripeptide, and subsequently as a peptidylpeptidase, cleaving successive dipeptides fromthe unprotected C-terminus.49 The localisationof ACE to the endothelia of all vessels, withthe most intense staining on capillaries andarterioles, is similar to that seen in othertissues.

DIPEPTIDYL PEPTIDASE IV

The seine protease DPPIV is identical tothe leucocyte antigen CD265° and cleavesunprotected N-terminal dipeptides where thepenultimate residue is proline. In the case ofsubstance P, this may not abolish biologicalactivity, which resides at its C-terminus, butrenders it susceptible to further inactivation bysuccessive cleavage of further N-terminalpeptides by APM.

AMINOPEPTIDASE MAPM has been identified as the leucocyteantigen CD 13 and also participates in theinactivation of enkephalins.5' In the inflamedsynovium, APM is found in a distributionsimilar to that of NEP-that is, in spindle

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shaped fibroblast like cells surrounding theblood vessels, and also in similarly shaped cellsin a layer underlying the hypertrophic intimalcell layer.

FUNCTIONAL COMPARTMENTALISATION

The localisation of membrane peptidases in thehuman synovium suggests a role, not only inlimiting the duration of action of regulatorypeptides, but also in localising their activities tothe vicinity of their release. This functionalcompartmentalisation5" of vascular and stromalregions in synovium is essential to the localregulatory function of peptides and is likely toinfluence responses to exogenous peptideadministered into non-physiological com-partments in experimental investigations.Depletion of neuropeptide immunoreactivenerves in the chronically inflamed synoviumimplies a loss of normal neurovascularregulation, and the abundance of membranepeptidases in inflamed synovial tissue would beexpected to exacerbate this situation further.Inhibition of specific peptidases may have atherapeutic role in restoring the protectiveeffects of vasoactive regulatory peptides.Inhibitors of aminopeptidases have anti-inflammatory activity in vivo and the specificACE inhibitor, captopril, may have slow anti-rheumatic activity.53

PainInflammatory mediators may directly stimulateor sensitise the normally silent nociceptivefibres to perceive and react to pain. Suchmediators include bradykinin, 5-hydroxy-tryptamine (serotonin), histamine, and protons(reviewed recently54). Undoubtedly, pain inarthritis may arise by such a mechanism. How-ever, if peripheral nerve terminals are damagedby the inflammatory response, other mech-anisms may be operative. One such possibilityis described below.As previously mentioned, the nociceptive C

fibres have their input into laminae I and II ofthe dorsal horn of the spinal cord, giving riseto the notion that this anatomical area of thespinal cord is responsible for pain sensation. Inrheumatoid arthritis the peripheral terminals ofnociceptive fibres are damaged. The centralterminals may also be affected; in animalmodels of axotomy, peripheral nerve damageleads to the withdrawal of central terminals ofnociceptive fibres.55 Furthermore, the laminaeI and II become occupied by nerve fibres whichsprout in from the adjacent laminae III and IV;such nerve fibres are AP fibres and, in the joint,are responsible for proprioception. Thus thepossibility arises that proprioceptive nervefibres have an input into a nociceptive area ofthe spinal cord. The result of this may be painon normal range of movement.

ConclusionThis review has considered selected aspects ofneuropeptides as they relate to inflammationwithin the joint. Arguments persist as to the

relevance of acute effects of neuropeptides, asopposed to their contribution to chronicdisease such as rheumatoid arthritis. Further-more, it is not clear if some aspects of theiraction are protective or deleterious. Forinstance, there is evidence to suggest thatoedema formation in adjuvant arthritis ofthe rat is protective, as increasing oedemacorrelates with a decrease in severity of jointdisease, as judged by radiographic scores. Theoverall view is that the acute effects of neuro-peptide release are protective. The chronicaspects of the involvement of the nervoussystem in joint disease remain poorly under-stood, but may well be characterised bythe loss of the protective features of acuteinflammation.

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2 Mapp P I, Kidd B L, Gibson S J, et al. Substance P-,calcitonin gene-related peptide- and C flanking peptideof neuropeptide Y-immunoreactive fibres are presentin normal synovium but depleted in patients withrheumatoid arthritis. Neuroscience 1990; 37: 143-53.

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