Lysosomes andthe connective tissue diseases · Lysosomes andthe connective tissue diseases LUCILLE BITENSKY Fromthe Cellular Biology Division, KennedyInstitute ofRheumatology, Bute

J. clin. Path., 31, Suppl. (Roy. Coll. Path.), 12, 105-116

Lysosomes and the connective tissue diseasesLUCILLE BITENSKY

From the Cellular Biology Division, Kennedy Institute of Rheumatology, Bute Gardens, London

Lysosomes are small intracellular organelles presentin most or all cells of animals of widely differentevolutionary development. In general their diametermay vary from 0(2 to 0(5 ,tm so that they overlap thedimensions of mitochondria. In the original differen-tial centrifugation studies they were isolated as the'light mitochondrial' fraction. It is difficult to givean exact size to these organelles because the term'Iysosome' covers a wide range of structures from thesmall primary lysosomes budded off from the Golgiapparatus, to secondary lysosomes caused by thefusion of primary lysosomes with endocytoticvacuoles, to complex fusion-structures such as het-erolysosomes and autophagic vacuoles. Indeed,de Duve (1969) suggested that lysosomes are only apart of the intracellular vacuolar system. Con-sequently the term 'Iysosome' includes granules ofvarious sizes, specific densities, and enzymaticcontent.

Possibly these structures have different comple-ments of enzymes in the various tissues of the sameanimal. The lysosomes of polymorphonuclear cells,the azurophil, and possibly the specific granules(Goldstein, 1974) are peculiar in both their relativelygreat size and enzymic complement. Seemingly,however, the main cellular content of hydrolyticenzymes, particularly those acting optimally at anacidic pH, is within the lysosomes and is one of thecharacterising features of these organelles. They maybe defined as small intracellular organelles that con-tain acid hydrolases bounded by a semi-permeablemembrane which is said to control the latency of theintralysosomal enzymes and to allow the organellesto behave as osmometers (de Duve, 1959). Byelectron microscopy they are seen to be bounded by asingle membrane (Daems et al., 1969; Schellenset al., 1977).Lysosomes can be seen when living cells are

examined by phase-contrast microscopy, whentheir greater refractility distinguishes them from thesmaller mitochondria or chondriosomes. Indeed, itis now apparent that the lipochondria of the oldermicroscopists probably correspond to lysosomes(Lane, 1968; Munro et al., 1964). Such bodies wereseen to congregate around the food vacuole inamoebae shortly after the uptake of food. Cor-

respondingly, in phagocytic cells primary lysosomesbecome attached to endocytic vacuoles and releasetheir hydrolytic enzymes into these vacuoles, digest-ing the endocytosed matter (Cohn and Fedorko,1969).

Methods for studying lysosomes

BIOCHEMICALThe term 'Iysosome' was coined by de Duve (deDuve et al., 1955; de Duve, 1969) for particlesisolated by homogenisation and differential centri-fugation that contained acid hydrolases in a latentform. The latency of the enzymic activities was themost surprising feature of this organelle. Now thesestructures are usually separated from mitochondriaby recentrifuging the crude 'mitochondrial' pellet toequilibrium in a medium that has a gradient ofdifferent specific gravities. The lysosomes equilibrateat a specific gravity of about 1-22 whereas the mito-chondria accumulate at a specific gravity of about1 19 (Beaufay, 1969). Occasionally the specificgravity of the lysosomes is deliberately altered byusing their ability to concentrate material of lowdensity such as Triton WR-1339 or material of highdensity such as Dextran 500, iron, or gold (Dean andBarrett, 1976; Dean, 1977b).

If lysosomes were completely stable possibly theactivities of none of their characteristic enzymescould be demonstrated biochemically because allwould be sequestered behind the semi-permeablemembrane of the organelle. Treatment with acetateat pH 5 0 at 37°C, or more crudely with a surfactantsuch as Triton X-100, modifies or otherwise disruptsthe lysosomal membrane and releases the enzymes,which then express their full biochemical activity.When there is no 'free' activity, so that the activityof the intralysosomal enzymes is totally latent, themembrane is considered fully stable. This type offinding led to the idea that these organelles were'suicide particles' by analogy with the suicidepastilles that could safely be held in the mouth untilthe casing was broken. However, it was then foundthat in different physiological and pathologicalconditions more or less of the enzymic activitieswere present in the fluid in which the purified lyso-

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somal fraction was isolated. This gave rise to manystudies (for example, de Duve, 1959; Peters et al.,1975) in which the 'free' activity was measuredseparately from that activity which still remainedlatent, or bound, within the organelles and whichcould be measured only after the particles had beenphysically or chemically disrupted. This 'free-to-bound' ratio seemed to relate to physiological orpathological activity in the cells.

ELECTRON MICROSCOPYLysosomes and related structures are recognised bythe characteristic single outer membrane bounding astructure that may be electron-dense and containsacid hydrolase activity. The application of electronmicroscopy to the study of lysosomes has been fullyreviewed by Daems (Daems et al., 1969; Schellenset al., 1977)

CYTOCHEMISTRYLysosomes can be recognised by their concentrationof acridine orange so that they appear as vivid redgranules when viewed by fluorescence microscopy(Robbins and Marcus, 1963; Allison and Young,1969). They can usually be stained histochemically bya suitable method for acid phosphatase activity(Holt, 1959; Bitensky, 1963a, b) because the lyso-somes of most but not of all cells contain active acidphosphatases. The presence of lysosomal enzymes,in characteristic particles, can also be visualised byimmunohistochemical methods (Weston and Poole,1973).

Quantitative cytochemical methods applicable tothe activity of many characteristically lysosomalenzymes are now well established (Bitensky andChayen, 1977). Two of these techniques allow theassessment of 'freely manifest' and of 'bound', orlatent, activity, approximating to the free-to-boundratios used by biochemists. These cytochemicaltechniques are considerably more sensitive than thebiochemical tests for assessing the function of thelysosomal membranes. For example, they haveshown that thyrotrophin, acting on thyroid folliclecells, increases the instability of the lysosomal mem-branes as a prelude to lysosomal involvementin the production of thyroid hormones.The cytochemical bioassay of thyrotrophin

(Bitensky et al., 1974a), 10 000 times more sensitivethan the radioimmunoassay, is now the micro-bioassay recommended by the World HealthOrganization. The cytochemical ratio of freelyavailable to total activity is altered in many patho-logical states and during physiological conditionswhich apparently depend on lysosomal intervention(Bitensky, 1963b; Bitensky et al., 1973). One of theadvantages of cytochemical investigations is that the

lysosomal activity in histologically identified cellscan be measured in individual cells by micro-densitometry.

Contents of lysosomes and role in infmmation

Lysosomes in different cell-types contain differentenzymes and bioactive molecules, and possibly thelysosomes of any one cell type are not homogeneous.Nevertheless, the lysosomes of most if not all cellscontain an array of hydrolytic enzymes includingthose that will hydrolyse proteins, nucleic acids,polysaccharides, and phospholipids. The pH opti-ma of all these enzymes, with a few exceptions, arein the acidic range. Barrett and Dean (1976; Barrettand Heath, 1977) have listed all the lysosomalenzymes that have been encountered. The possiblerole of the proteinases in the degradation of theprotein matrix of cartilage in rheumatoid arthritishas been much studied (Dingle, 1962; Weissmann,1966). The phospholipases are thought to have apotential function in the liberation of unsaturatedfatty acids that initiates the formation of prosta-glandins (Anderson et al., 1971; Zurier, 1974).Prostaglandins alone can cause resorption of bone(Robinson et al., 1975) and their production can beblocked at a very early stage by glucocorticoids(Kantrowitz et al., 1975) which influence lysosomalfunction (see later).Lysosomes of many types of cell also contain

amino-acid arylamidases capable of generatingbradykinin or other inflammatory amines from theirinactive precursors (Hopsu-Havu et al., 1966). Apartfrom such enzymes lysosomes of polymorphonuclearleucocytes contain a number of inflammatory media-tors (Goldstein, 1974) including cationic proteins(Janoff and Zweifach, 1964), an activator of plas-minogen (Lack and Ali, 1964), a protease that causeschanges in the permeability of capillaries (Movatet al., 1964; Uriuhara et al., 1965), as well as haemo-lysins (Desai and Tappel, 1965). Thus, not sur-prisingly, when the contents of lysosomes isolatedbiochemically were injected under the skin or intojoints (Weissmann et al., 1969) they proved highlyinflammatory. Repeated intra-articular injection oflysates of lysosomes isolated from rabbit leucocytesproduced hypertrophy and hyperplasia of the cellslining the synovium, round cell infiltration of thesynovium close to blood vessels, the formation of apannus, and erosion of cartilage (Weissmann et al.,1969; Page-Thomas, 1969). Moreover, the fact thatmany anti-inflammatory agents appreciably stabiliselysosomal membranes (Weissmann, 1968, 1969;Chayen et al., 1972; Bitensky et al., 1974b) alsoindicates a strong lysosomal component in inflam-mation.

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Lysosomes and the connective-tissue diseases

Intracellular digestion

There is now a wealth of information about theintracellular digestion, mediated by lysosomes, ofmaterial endocytosed by cells (Dean and Barrett,1976). There is also clear evidence from electronmicroscopy that lysosomes participate in the intra-cellular digestion of parts of the cell such as mito-chondria. This process, known as autophagy, isrelated to the digestion of secretory granules(crinophagy)-seen, for example, in the parathyroidand pituitary glands-which apparently regulates theamount of hormone available for secretion. Crino-phagy can be induced in the appropriate cells in theanterior lobe of the pituitary gland if secretion of thetrophic hormone is blocked (Farquhar, 1969).However, autophagy and possibly crinophagy may

represent pathological roles of the intracellularlysosomal digestive system.Over and above these morphologically identifiable

processes, lysosomes in normal cells are probablyconcerned with the regular degradation of cellularcomponents and the turnover of cellular proteins.Thus the normal rate of degradation of intracellularproteins in the liver is retarded by causing a specificinhibitor of carboxyproteinases, supplied in lipo-somes, to be incorporated into liver lysosomes (Dean,1975). Further evidence of lysosomal participationin the normal turnover of cellular proteins is givenby Dean (1977a; La Badie et al., 1976). The indirectevidence that lysosomes are concerned in the turn-over of other chemical components of cells comes

from the study of inborn errors of metabolism (seelater; also M. F. Dean (Dean, 1978) at page 120).

Extraceliular digestion

The extracellular release of lysosomal enzymes intothe lacunar space has been implicated in the de-struction of bone by osteoclasts (Vaes, 1969). There isevidence that lysosomal cathepsin D released fromcells participates in the early stages of the breakdownof connective tissues (Dingle, 1971). Dingle (1968)suggested that the release of lysosomal enzymes fromcells could be as packages released by exocytosis.The earlier work of Fell and coworkers (Fell and

Thomas, 1960) is still the clearest evidence that lyso-

somal enzymes released from living cells can degradecartilage and other connective tissue. In particular,studies on cells in tissue or organ culture have shownthat addition of vitamin A (which labilises lysosomes)or of non-digestible material (which accumulateswithin lysosomes) results in the release into theculture medium of lysosomal enzymes and in theloss of metachromasy in the cartilage (Fell andDingle, 1963; Dingle et al., 1969; Dingle, 1969).

Other, more circumstantial, evidence will be dis-cussed later. Nevertheless, these studies indicatedthat degradation of the matrix of connective tissuecould result from the extracellular release of lyso-somal enzymes from still-viable cells.

Nature of lysosomal enzymes

INTRALYSOSOMAL pHThere has been some concern over the fact that mostof the enzymes located within lysosomes act op-timally at pH values around pH 5 or even lower, andthat some are totally inactive at what is considered tobe the 'physiological pH'-namely, about pH 7.

Several attempts have been made to explain howthe pH within these organelles can be so differentfrom that of their environment. Some have adducedevidence for an active proton pump (de Duve et al.,1974; Mego, 1973) to explain why the measurableintralysosomal pH is appreciably lower than thepH of the surrounding medium. Goldman (1976)found it to be 1-6 units of pH lower; Reijngoud andTager (1973) found it to increase from pH 4 9 to 6-9as the pH of the external medium was raised from5 0 to 8-5. Moreover, Koenig (1974) showed thatlipoproteins make up about half of the total proteincomplement of lysosomes isolated from rat liver andkidney.The influence of these acidic, ionisable phosphate

moieties could play some part in maintaining arelatively acidic hydrogen-ion activity (see below) atthe active sites of the intralysosomal enzymes. Deanand Barrett (1976) considered that such fixed anionsin the interior of the organelles could be sufficient toaccount for the maintenance of the acidic intra-organelle pH that has been recorded (Goldman,1976) without recourse to active proton pumps.Among the fixed anions they listed were sialic acidon the inside of the membrane, acidic lipoproteinsand phospholipids, and an excess of amino-acidsproduced by hydrolysis of peptide bonds.

pH OF EXTRACELLULAR DIGESTIONMany workers are disquieted by the fact that lyso-somal enzymes act optimally at acidic pH valueswhile the digestion of extracellular matrix occurs,ostensibly, at physiological pH values-namely,around pH 7TO. Because purified cathepsin D hadno detectable action on a protein-polysaccharidecomplex or proteoglycan subunit at physiologicalpH, Woessner (1973) concluded that it and, presum-ably, other cathepsins with acidic optimal activitieswere unlikely to have any significant effect on thematrix of cartilage at pH values greater than pH 6-0.Any degradation of cartilage must therefore be dueto the influence of neutral proteases. Such proteases

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have been found in the granules of polymorpho-nuclear leucocytes (Goldstein, 1974) and in othercells. Lysosomal carboxypeptidase B and cathepsinC have been shown to have pH optima of 6'2 and5-6 respectively (Barrett and Heath, 1977).Some lysosomal endopeptidases, such as acrosin

(pH optimum: 8) and cathepsin G (pH optimum:7'5), act optimally at around neutral pH, as do someother enzymes such as aspartyl glucosyl aminase(E.C. 3.5.1.26) which hydrolyses the amide link inaspartamido-(N-acetyl-) glucosamine, and whichmay be relevant to the breakdown of cartilage-matrix (Barrett and Heath, 1977). Furthermore,even such endopeptidases as cathepsin B, whichacts optimally at pH values of between 3'5-6'0, maystill have significant activity at higher pH values,even at pH 7-0.The lowest mean pH of sterile inflammatory

exudates has been measured as 7'049 (Edlow andShelden, 1971), leading Goldstein (1974) to concludethat the pH was far above the range at which acidcathepsins could be active. Similarly the pH offluid from rheumatoid joints was 7'1-7'28 (Barnettetal., 1961; Cummings and Nordby, 1966). However,these arguments are somewhat academic. There issufficient neutral lysosomal protease activity toaccount for degradation of tissue matrix at neutralpH, and polymorphonuclear leucocytes, when in-volved, contain an array of neutral hydrolyticlysosomal enzymes (Barrett and Heath, 1977).Moreover, the whole argument is based on the pH-namely the hydrogen-ion concentration in solution-which operates when solubilised enzymes act ontheir substrate in solution.

Small (1954) emphasised that at surfaces we areconcerned not with pH but with hydrogen-ionactivity. McLaren and Packer (1970) reviewed thewhole question of how pH optima can be modifiedwhen the enzyme becomes bound to a solid matrixor when a soluble enzyme acts to digest a solidmatrix, as it must when digesting extracellularconnective tissue components. Several examplesare now known of enzymes, such as chymotrypsin ortrypsin, whose pH optimum may be changed asmuch as 2 pH units depending on whether they actin solution or attached to a solid matrix (McLarenand Packer, 1970).Dean and Barrett (1976) reviewed work on

enzymes linked to insoluble model membranes inwhich extensive changes in the pH optima and evenenzymatic characteristics have been recorded. Theygave as one example a lysosomal proteolytic activitythat acts optimally at pH 6'5 when bound to a mem-brane but at pH 4-5 when solubilised. It is by nomeans inconceivable that isolated and purifiedlysosomal enzymes which act optimally at an acidic

pH of, for example, 4'5 may in fact act optimallyclose to neutrality when adsorbed on to extracellularconnective-tissue components or even when presentwithin the matrix of the lysosome. A typical exampleof the former is given by Quarles and Dawson(1969). They studied the action of isolated phos-pholipase D acting on micelles of phospholipids andshowed that the pH optimum could be shifted 1'5 pHunits towards neutrality, depending on the nature ofthe micelles. They pointed out that the pH in thebulk phase (pHB) was related to the pH in the surfacephase (pHs) adjacent to a negatively charged inter-face by an equation derived from the Boltzmannequation:

pHs = pHB + (eb/2'3KT)where K is the Boltzmann constant, T is the absolutetemperature, E is the electronic charge, and l is thepotential of the surface phase (Davies and Rideal,1961). Although it is difficult to calculate l thereis every reason to expect a strong shift towardsneutrality.

Nature of lysosomal membranes

Electron microscopy shows that lysosomes arebounded by a single membrane, and biochemicalinvestigations on the osmotic properties of lysosomes(Dean and Barrett, 1976) added weight to the viewthat these organelles are encased in a semi-permeablemembrane. Some biochemical studies indicated thatsulphydryl groups in the membrane are importantin the labilisation of these membranes by thiols andcertain metals (Lucy, 1969) and by vitamin A (Lucyand Lichti, 1969). Lysosomal membranes can belabilised by some steroids and by retinol andstabilised by other steroids(Dean and Barrett, 1976).Dingle and Barrett (1968) isolated a peculiar phos-pholipid component of lysosomal membranes thatmay play a significant role in some of the cyto-chemical tests made on these organelles (Bitenskyand Chayen, 1977).Proof that changes in the permeability of lyso-

somal membranes, which are sometimes reversible,may play a significant part in cellular physiology andpathology depended on the adequate measurementoflysosomal membrane function. These changes wereshown first by cytochemistry (Bitensky, 1963b;Allison and Mallucci, 1964, 1965; Bitensky andCohen, 1965; Bitensky et al., 1963) but havenow beenshown biochemically by Peters and Seymour (1976)and by Burton and Lloyd (1976). Both cytochemi-cally and biochemically, the effects measured areestimates of the permeability or stability of thelysosomal membranes. Although there are dissentingvoices (Baccino and Zuretti, 1975; Koenig, 1969),

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the weight of evidence favours the concept thatreversible changes do occur in the permeability oflysosomal membranes under different physiologicaland pathological conditions and when tissues aresubjected to drugs such as steroids (Weissmann,1969; Allison, 1968a; Bitensky et al., 1974b) ordimethyl sulphoxide (Misch and Misch, 1975).Pathogenetic role of lysosomesLysosomes were first thought of as 'suicide capsules'.When they burst they would release their cytolyticenzymes into the cytosol and digest the cell in whichthey were present. Although there was some evidencethat this might occur in morphogenesis (Weber,1969), it is now clear that this is remarkably rare andoccurs only in a few particular situations. One suchsituation was shown to be the uptake of toxicparticles by macrophages (Allison et al., 1966;Allison, 1971). Silica was taken up by these cells,lysosomes fused with the phagosome and releasedlysosomal enzymes into the so-formed secondarylysosomes. The toxic particles, such as those ofsilica, reacted with the membranes of the lysosomes(Allison, 1968a, b), allowing the lysosomal enzymesto escape into the cytoplasm. Macrophages that hadingested such particles were killed within 24 hourswhereas those that ingested non-toxic particles (suchas diamond dust or silica coated with either alu-minium or polyvinyl-pyridine-N-oxide), incapableof disrupting the lysosomal membranes, werenot killed.

It now seems likely that the common effect oflysosomes is to release their enzymes into theexternal milieu of the cell. Dingle (1968) suggestedthat perturbation of the lysosomal membranesallowed the lysosomes to fuse with similarly per-turbed regions of the plasma membrane and so toextrude packages of lysosomal enzymes by a formof exocytosis. Lysolecithin, produced locally, couldmediate this phenomenon (Lucy, 1970). An alterna-tive mechanism, which might be called 'regurgita-tion during feeding', has been postulated byWeissmann et al. (1971a, b). This suggests that thephagocytic vacuole in the leucocyte may remain openat the external surface of the cell and that the lyso-somes fuse with the inner boundary of such vacuolesand release their enzymes into these vacuoles, which,being still open to the outside medium, allow theloss of lysosomal enzymes into the environment ofthe cell.

Evidence from electron micrographs is consistentwith such a mechansim (Zucker-Franklin andHirsch, 1964; Henson, 1971). Indeed, there isconsiderable biochemical evidence for the selectiverelease of lysosomal enzymes from leucocytes andfrom macrophages (Cardella et al., 1974; Schorlem-

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mer et al., 1977) when these cells are exposed to avariety of stimuli, including immune complexes.This can occur even in the absence of any signs ofloss of viability of the cells, monitored by the releaseof cytosolic enzymes such as lactate dehydrogenase.A particular example of the release of nine lysosomalenzymes from cells which remain viable is the studyby Hosli and Vogt (1977) on fibroblasts grown frompatients with cystic fibrosis.A system of particular interest in inflammation

is the activation of complement by lysosomes.Various workers (Goldstein and Weissmann, 1974;Schorlemmer and Allison, 1976) have shown thatwhen mononuclear leucocytes are stimulated byagents that induce chronic inflammation they secretehydrolytic enzymes. These enzymes degrade con-nective tissue and interact with the complementsystem to generate mediators of inflammation. Thecleavage products of complement can then furtherstimulate macrophages (Schorlemmer et al., 1976) toperpetuate the inflammatory response. Manyworkers have shown that this activation of com-plement is by the alternative pathway resulting in thecleavage of C3 and the generation of C3b, whichcauses the secretion of hydrolases from these cells.This work, and the concepts arising out of it, havebeen reviewed by Schorlemmer et al. (1977).

Given that lysosomal enzymes can be exteriorisedfrom the cell, conceivably they could have thefollowing influences.

Firstly, body fluids contain many hydrolytic anddegradative enzymes which occur in a latent form,often as a proenzyme or in combination withinhibitors. For example, there is much work onprocollagenases and proelastases (Vaes, 1972;Woessner, 1977; Sellers et al., 1977). These can beactivated by proteolytic enzymes (Birkedal-Hansenet al., 1976; Dayer, 1976; Eeckhout and Vaes, 1977).Glynn (1977) and Reynolds et al. (1977) suggestedthat such activation of collagenase, possibly bylysosomal enzymes extruded from still viable cells,could participate in the degradation of cartilage inosteoarthrosis and rheumatoid arthritis. Similarly,cathepsin B can inactivate the circulating xl-pro-teinase inhibitor, allowing proteases, previouslylatent because of the presence of this inhibitor, to befree to act on connective tissue. (For the possiblerelationship between this circulating protease in-hibitor and emphysema see Galdston et al., 1973;Goldstein, 1974.) Equally lysosomal enzymes canact on kallidin-10 to form bradykinin (Hopsu-Havuet al., 1966; Goldstein, 1974).

Secondly, if the release of lysosomal enzymeswere to occur in the synovium or in the joint theycould attack the connective tissue matrix, providedthat the pH or hydrogen-ion activity was suitable.


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It could cause a cascade phenomenon of inflamma-tion by the alternative pathway of complementactivation; it could influence the permeability of thecapillaries; and, by the release of chemotacticsubstances known to be present in lysosomes(Goldstein, 1974), it could exacerbate the cellularinfiltration in the synovium or joint. We have seenthat the injection into joints of lysates of lysosomesapparently has all these effects. It is also known thatwhen cells ingest certain substances they releaselysosomal enzymes (Dingle et al., 1969). Page-Thomas (1969) showed that the injection into a jointof disazo dyes or polysaccharides produced anarthritis, apparently due to excessive synthesis oflysosomes and lysosomal enzymes. The ingestionof immune complexes has a similar effect in thesecretion of lysosomal enzymes from leucocytes andmacrophages (Weissmann et al., 1971b; Cardellaet al., 1974).A different aspect of the extrusion of matter from

lysosomes concerns the handling of immune com-plexes by these organelles. Antigens appear to betaken up into lysosomes by endocytosis and to be'processed' by them (Weissmann, 1964; Allison,1968c). The fate of these proteins seems to bedetermined by the state of the lysosomal membranes.As reviewed by Chayen and Bitensky (1971), themore stable lysosomes (as may occur in poly-morphonuclear leucocytes) are said to be capable ofmore complete digestion of these antigens than areless stable lysosomes, as found in macrophages(Allison, 1968c). Treatment which labilised lyso-somal membranes prolonged the 19S antibodyresponse (Weissmann, 1967), implying that theantigen had not been degraded under these con-ditions (Uhr, 1964). Weissmann (1964; 1966) arguedthat this type ofphenomenon could play some part inautoimmunity. The hypothesis requires cells thatare undergoing autophagy and endocytosis. If thelysosomal membranes are labile there will be in-complete degradation with retention of antigenicity.The selective release of these incompletely degradedantigens, whether by 'regurgitation,' exocytosis, orany other mechanism, will result in an immuneresponse either to the original cell or to the exo-genous immune complex. Similar hypotheses havebeen propounded by Page-Thomas (1969) and byHollander (Hollander et al., 1965; Rawson et al.,1965; Restifo et al., 1965). Such hypotheses wouldaccount for the chronic nature of rheumatoid arthri-tis, as discussed by Chayen and Bitensky (1971).Possible role of lysosomes in certain diseases ofconnective tissues

STORAGE DISEASESMany diseases caused by inborn errors of meta-

Lucille Bitensky

bolism are now known. They have been reviewedfully by Hers and van Hoof (1973). In many asingle specific lysosomal enzyme is absent. The factthat undigested matter accumulates in giant, swollenlysosomes, from which phenomenon the name'storage diseases' is derived, indicates that lysosomesmay well be involved in the normal turnover of thesesubstances. In Tay-Sachs disease there is a deficiencyof lysosomal hexosaminidase A which wouldnormally cleave the terminal N-acetyl-,B-D-galacto-samine from ganglioside GM2. This results in hugelysosomes filled with cytoplasmic membranousbodies presumably of ganglioside material (O'Brien,1973).Type II glycogenosis, also called type II glycogen-

storage disease or Pompe's disease, is ascribed to acomplete defect of x-glucosidase; this results in theintralysosomal accumulation of intact glycogenwhich causes the lysosomes to swell to up to 8,um-diameter (Hers and de Barsy, 1973). Cardiacand skeletal muscle hypertrophy follow. In three ofthe mucopolysaccharidoses (Hurler's, Hunter's, andSanfilippo's disease) there may be a complete defectof one of the degradative enzymes-for example,iduronidase (which liberates sulphate ions fromdermatan sulphate) in Hurler's disease, sulphatase,or fl-glucuronidase-or partial defects of a- orfl-galactosidases (Hers, 1973). Gaucher's disease,with its hepatosplenomegaly and bone erosion, isassociated with a nearly complete defect of gluco-cerebrosidase (f-glucosidase). Gaucher cells arecharacterised by lysosomes filled with membrane-bound inclusions (Brady and King, 1973).

These and other lysosomal defects discussed byHers and van Hoof (1973) indicate how importantlysosomes may be in the normal turnover ofmaterialsin life, so that defects in their metabolic activitybecome apparent in these diseases. Surprisingly nogenetic disease due to deficiencies of lysosomalproteolytic activity seem to have been recorded.Whether this is because such defects are lethal orbecause the wide array of proteolytic potency oflysosomes can cover-up the effect of a single deletionis not determined.

GOUT AND CRYSTAL SYNOVITISLong, needle-shaped crystals of monosodium uratemonohydrate are found in the fluid from jointsduring acute attacks of gout (see J. T. Scott (Scott,1978) at p. 205, and P. A. Dieppe (Dieppe, 1978)at p. 214). It was known in the last century thatcrystals occurred at the sites of gouty necrosis andthat the subcutaneous injection into animals ofmicrocrystals of various urates, xanthine, hypox-anthine, and creatinine-and even of calciumcarbonate-provoked acute inflammatory responses


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and the phagocytosis of the crystals by mononuclearand polymorphonuclear leucocytes (Chayen andBitensky, 1971). These effects were rediscoveredby Seegmiller and Howell (1962) and by McCarty(1968). Seegmiller and Howell (1962) suggested thatthe urate crystallises in tissue or synovial fluid andthat the crystals are phagocytosed by leucocytes.They assumed that in this process lactic acid wasproduced locally. This would lower the pH and so

cause further precipitation of urate from the exu-date, which would be in equilibrium with thehyperuricaemic blood. The last step is probably anover-simplification because Watts et al. (1971) foundurate crystals in tissues of patients in whom theblood levels of urates were well below saturation.McCarty (1968) emphasised that inflammation

induced by crystals either of urates, as in gout, or ofpyrophosphate, as in pseudogout or chondrocal-cinosis, depended on the presence of polymorpho-nuclear leucocytes. Phelps and McCarty (1966) andChayen and Bitensky (1971), however, reportedplenty of urate crystals in some synovial fluids inwhich very few of these cells could be found. For allthat, the general view is that the crystals are phago-cytosed by polymorphonuclear leucocytes into anendocytic vacuole. The cells degranulate, injectingtheir lysosomal enzymes into this vacuole. Theseenzymes then leak from the cells either by the re-

gurgitation mechanism discussed previously or bysome other selective release, very much as when suchcells ingest particles of silica (as discussed above).Indeed, there is probably no call to invoke selectiverelease of lysosomal contents from viable cells in thisinstance and possibly the fluids that containedcrystals but very few polymorphonuclear leucocytescould have been sampled at a time when these cellshad already disintegrated, before the inflow of freshcells.

Probably, therefore, the inflammation associatedwith gout, chondrocalcinosis, and possibly the othercalcium phosphate synovitides (Dieppe et al., 1976)is brought about by the lysosomal contents ofleucocytes that have ingested these particles. Whensuch crystals are deposited in muscle they do notproduce an inflammatory response but may beassociated with muscle pain, as in the cases ofxanthinuria reported by Chalmers et al. (1969) andpossibly when urates of various forms crystallise inthe muscle of patients with gout (Watts et al., 1971).

RHEUMATOID ARTHRITISRheumatoid arthritis is a systemic disease ofunknown origin. The earliest joint changes are in thesynovium, where the established lesion is character-ised by villous enlargement, hyperplasia of thelining cells, infiltration by inflammatorycells, deposi-

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tion of fibrin interstitially, and the formation ofpannus and erosion of cartilage of the joint surface(Lack, 1969). The chronic inflammation and erosionof cartilage point to lysosomal participation. Thisview is strengthened by the fact that an experimentalarthritis can be induced by injecting lysates of lyso-somes into the joint (Weissmann et al., 1969) andthat it can be producedexperimentally(Page-Thomas,1969) by injecting various substances that interferewith lysosomes, such as those which rupture lyso-somes, or disazo dyes which, by inhibiting some lyso-somal enzymes, may cause excessive production ofother enzymes, as has been found in many of thestorage diseases.

If lysosomes participate in the pathogenesis ofrheumatoid arthritis it is necessary to identifywhich cells are involved. In gout there seems littledoubt that the lysosomal dysfunction is in thepolymorphonuclear leucocytes. These are unlikelyto be the source of lysosomal enzymes and inflam-matory substances in the rheumatoid joint because,were they the source, the erosive influences would beliberated into the synovial fluid and the erosion of thecartilage would begin uniformly over the wholesurface of the cartilage. Ball (1968) has shown thaterosion begins at the articular cartilage junction.This location implicates the synovium itself, whichoriginates at this site-at which it also shows itsmost florid appearance in completely normal joints.Muirden (1972) showed that the severity of jointerosion did not correlate with the degree of in-flammatory cell infiltrate in the synovium but withthe amount of hyperplasia of the synovial liningcells. Ghadially and Roy (1967) found a strikingincrease in the number of lysosomes and autophagicvesicles, particularly in Type A synoviocytes, inrheumatoid synovial tissue (Lack, 1969).From this evidence it is possible that the lysosomes

of rheumatoid synoviocytes are abnormal andcapable of extruding their contents on to thecartilage, to which these cells are already in closeapposition. But, as Page-Thomas (1969) hasremarked: 'The fibrous nature of synovial membraneand the cellular heterogeneity of the diseased tissuehave deterred workers from serious biochemicalstudies of subcellular fractions'. Thus this was anideal system for analysis by the techniques ofquantitative cytochemistry (Bitensky et al., 1973;Bitensky and Chayen, 1977).

Cytochemical analysis of the functional state ofthe lysosomal membranes, based on a comparison ofthe free and latent activities of two intralysosomalenzymes, showed that the lysosomes of non-rheumatoid synoviocytes contained between 28%and 60% latent activity, the lower values beingfound in recently traitmatised joints. The total


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activity found in rheumatoid synoviocytes was atleast as great, or greater, than that found in non-rheumatoid synoviocytes. But the striking findingwas that not more than 5% of this activity was latent,showing that the lysosomal membranes in therheumatoid cells were relatively functionless, atleast with regard to these substrates (Chayen et al.,1971). Glucocorticoids administered to synovialtissue maintained in vitro improved the amount oflatent activity up to 21 %. A similar improvement inlatent activity was found in eight out of 13 patientswho had received at least 5 mg of prednisolonedaily. It was not found in patients receiving lessprednisolone (Bitensky et al., 1974b). These levelsof prednisolone correlated well with the findingsof Chamberlain and Keenan (1976). In a double-blind trial patients receiving prednisolone 3 mg/daygained little clinical benefit whereas some clinicalimprovement was apparent in those treated with 5mg/day.

Conclusion

Since lysosomes were first described as a biochemicalentity in 1955 (de Duve et al., 1955) they have beena favoured candidate for a pathogenic role inconnective tissue diseases. Today it is clear that theyhave important functions in these diseases Theirimportance in intracellular metabolic processes ishighlighted by the damage caused by the deletion ofa single intralysosomal enzyme, as in the storagediseases. Their abundance in polymorphonuclearleucocytes and macrophages and their behaviour inresponse to inflammatory stimuli implicate lysosomalenzymes in acute and chronic inflammation. Such afunction seems clear in gout and probably in othercrystal synovitides.

There is strong evidence that extracellular lyso-somal enzymes are concerned in the normal turnoverof bone (Vaes, 1969) and of the extracellular com-ponents of connective tissue (Reynolds, 1969;Dingle, 1969). How much they participate in thepathogenesis of rheumatoid arthritis and osteo-arthrosis still awaits full clarification. But there isstrong circumstantial evidence that their part is amajor if not decisive one.

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Lysosomes andthe connective tissue diseases · Lysosomes andthe connective tissue diseases LUCILLE BITENSKY Fromthe Cellular Biology Division, KennedyInstitute ofRheumatology, Bute