[international review of cytology] a survey of cell biology volume 252 || the biology of the gaucher...

The Biology of the Gaucher Cell:The Cradle of Human Chitinases

Anton P. Bussink,* Marco van Eijk,* G. Herma Renkema,{

Johannes M. Aerts,* and Rolf G. Boot**Department of Medical Biochemistry, Academic Medical Center,

University of Amsterdam, 1105 AZ Amsterdam, The Netherlands{Institute of Medical Technology, University of Tampere,

and Tampere University Hospital, FI‐33014 Tampere, Finland

Gaucher disease (GD) is the most common lysosomal storage disorder and is

caused by inherited deficiencies of glucocerebrosidase, the enzyme responsible

for the lysosomal breakdown of the lipid glucosylceramide. GD is characterized by

the accumulation of pathological, lipid laden macrophages, so‐called Gaucher

cells. Following the development of enzyme replacement therapy for GD, the

search for suitable surrogate disease markers resulted in the identification of a

thousand‐fold increased chitinase activity in plasma from symptomatic Gaucher

patients and that decreases upon successful therapeutic intervention. Biochemical

investigations identified a single enzyme, named chitotriosidase, to be responsible

for this activity. Chitotriosidase was found to be an excellent marker for lipid laden

macrophages in Gaucher patients and is now widely used to assist clinical

management of patients. In the wake of the identification of chitotriosidase, the

presence of other members of the chitinase family in mammals was discovered.

Amongst these is AMCase, an enzyme recently implicated in the pathogenesis of

asthma. Chitinases are omnipresent throughout nature and are also produced by

vertebrates in which they play important roles in defence against chitin‐containingpathogens and in food processing.

KEYWORDS: Lysosomal storage disorder, Gaucher disease, Gaucher cell,

alternatively activated macrophages, biomarker, chitinase, chitotriosidase,

AMCase, chi‐lectin, CCL18. � 2006 Elsevier Inc.

International Review of Cytology, Vol. 252 71 0074-7696/06 $35.00Copyright 2006, Elsevier Inc. All rights reserved. DOI: 10.1016/S0074-7696(06)52001-7

72 BUSSINK ET AL.

I. Introduction

In lysosomal storage diseases (LSD’s), accumulation within lysosymes takes

place of material that cannot be degraded in the organelle or of degradation

products that cannot be transported out of the organelle, giving rise to

numerous biochemical and physiological abnormalities. Investigations into

the molecular basis of these diseases have generally followed the same

approach. Firstly, the nature of the lysosomal storage material is identified.

Secondly, the defect in metabolism is determined, followed by the identifica-

tion of the deficient protein. Lastly, mutations in the genome causing the

deficiencies are determined, allowing for genetic screening of individuals

showing characteristics of one of the LSD’s. For most of these diseases, the

stages have all been accomplished, opening the possibility of therapeutic

intervention by replacement of the deficient protein.

For Gaucher disease (GD), the most common lysosomal storage dis-

order caused by deficiencies in glucosylcerebrosidase, enzyme replacement

therapy (ERT) has indeed proven to be successful and hence has served as

a model for other LSD’s. Gaucher disease (GD) is the most common lyso-

somal storage disorder and is caused by inherited deficiencies of glucocer-

ebrosidase, the enzyme responsible for the lysosomal breakdown of the lipid

glucosylceramide. The clinical features of the disease were first described in

detail by Philip E. Gaucher more than a century ago and the identification

of glucosylceramide (glucocerebroside) as the primary storage material in

Gaucher disease was accomplished early last century. GD is characterized by

the accumulation of pathological, lipid laden macrophages, so‐called Gauch-

er cells. Biochemical investigations into the biochemical abnormalities in GD

have led to valuable insights linking abnormal lipid metabolism to cellular

responses of all sorts, although the molecular mechanisms behind these

responses often remain to be determined. Among the findings was an enor-

mous increase in chitinase activity in plasma of patients suffering from GD,

providing an alternative biomarker which has proved to be of important

value in the clinical management of the disease. Biochemical investigations

identified a single enzyme, named chitotriosidase, to be responsible for this

activity and an excellent marker for lipid laden macrophages in Gaucher

patients. Chitinases are omnipresent throughout nature and are also produ-

ced by vertebrates in which they play important roles in defence against

chitin‐containing pathogens and in food processing. This review focuses on

the features and functions of chitinases with emphasis to the mammalian

enzymes. In addition, a recently discovered alternative biomarker of Gaucher

cells, the chemokine CCL18, is discussed.

BIOLOGY OF THE GAUCHER CELL 73

II. Lysosomal Storage Disorders and Gaucher Disease

A. Inherited Lysosomal Storage Disorders

The eukaryotic cell is characterized by the presence of numerous membrane‐enclosed compartments, of which a substantial part is dedicated to the

transport, secretion, and degradation of cellular macromolecules. Lysosomes

are generally considered to be end points of this so‐called vacuolar system, in

which the degradation of macromolecular compounds into their building

blocks takes place. Functional lysosomes are indispensable for normal turn-

over of basic cellular metabolites. The physiological importance of lysosomes

is illustrated by a group of inherited diseases in which deficiencies in one or

more catabolic pathways exist: the lysosomal storage disorders. Many of the

lysosomal storage disorders are caused by an inherited defect in a single

lysosomal hydrolase or cofactor. Lysosomal storage of natural compounds

may also occur because of a defect in a specific translocator in the lysosomal

membrane. For most lysosomal diseases the defective protein and the

corresponding gene have now been characterized and appropriate diagnostic

assays have been developed (Meikle et al., 2004; Vellodi, 2005).

B. Gaucher Disease

The most frequently encountered inherited lysosomal storage disorder in

humans is glucosylceramidosis, better known as Gaucher disease. The clini-

cal features of the disease were first described in detail by Philip E. Gaucher

more than a century ago (Gaucher, 1882). The identification of glucosylcer-

amide (glucocerebroside) as the primary storage material in Gaucher disease

was accomplished early last century (Aghion, 1934). Glucosylceramide is the

common intermediate in the degradation of gangliosides and globosides,

which takes place intralysosomally by the stepwise action of exoglycosidases.

In 1965 Patrick and Brady et al. showed independently that the primary

defect in Gaucher disease is a marked deficiency in activity of the lysosomal

enzyme glucocerebrosidase (Brady et al., 1965; Patrick, 1965). This hydrolase

(also known as acid b‐glucosidase or glucosylceramidase, EC 3.2.1.45)

catabolizes glucosylceramide to ceramide and glucose.

1. Pathology

The clinical presentation ofGaucher disease is quite heterogeneouswith respect

to age at onset and nature and progression of the symptoms, a phenomenon

that is also seen in some of the other lysosomal storage disorders. Historically,

a distinction between three variants is made on the basis of clinical features.

The main diVerence among the three types is the occurrence and progression

74 BUSSINK ET AL.

of neurological complications (Cox and Schofield, 1997; Sidransky, 2004).

The nonneuronopathic form of GD, referred to as type 1 GD, is by far the

most common. The incidence of this variant worldwide is about 1 in 50,000–

200,000 births (Gieselmann, 1995). A markedly increased incidence exists in

Ashkenazi Jewish populations (Beutler and Grabowski, 2001). The age at

onset and course of the disease vary enormously within this phenotype.

Clinical representation can occur at any age; the disease may manifest itself

within the first years, but hardly symptomatic individuals above the age of

70 have also been described. The major symptoms in type 1 GD result from

lipid‐laden macrophages in specific tissues, causing gross enlargement of

spleen and liver (hepatosplenomegaly), displacement of normal bone mar-

row cells, and damage to the bones (Beutler and Grabowski, 2001). The

acute neuronopathic manifestation of Gaucher disease is called type 2. This

variant of the disease is rare, and without ethnic predisposition. The average

age at onset of severe hepatosplenomegaly is about 3 months, which is

rapidly accompanied by progressive neurological complications, being

usually lethal within the first 2 years of life (Barranger and Ginns, 1989;

Beutler and Grabowski, 2001). Type 3, a subacute neuronopathic form of

GD, is also relatively rare and occurs panethnically. The neurological symp-

toms of this type are similar to those observed in type 2 GD, but with a later

onset and lesser severity. More recently it has become clear that a complete

lack of glucocerebrosidase activity results in the so‐called collodion baby

phenotype characterized by ichthyotic skin (Sidransky, 2004).

2. Genetics

In previous decades the precise nature of genetic lesions that underlie Gaucher

disease has been identified by analysis of the glucocerebrosidase gene (Beutler

andGrabowski, 2001). Numerous distinct mutations in the glucocerebrosidase

gene have been identified (Beutler and Gelbart, 1997; Horowitz and Zimran,

1994).Most of thesemutations are rare and someof themhave been found only

in one family. Not all the mutant glucocerebrosidases have been expressed and

subsequently characterized; therefore some of the claimed mutations may in

fact prove to be polymorphisms. Six mutant alleles account for more than 95%

of the defective glucocerebrosidase alleles in the Ashkenazi Jewish Gaucher

patient population and for about 70% of the mutant alleles in the various

non‐Jewish white Gaucher patient populations (Horowitz and Zimran, 1994).

Themost prevalentmutation in Jewish as well as non‐Jewishwhite populationsis the N370S mutation, the result of an adenine‐to‐guanine substitution at

cDNA position 1226 (Tsuji et al., 1988). This mutation leads to the synthesis

of normal amounts of enzyme that is routed normally to lysosomes (Ohashi

et al., 1991). However, the N370S enzyme is abnormal in catalytic features.

Under most conditions its specific activity is 10‐fold lower than normal.

However, at suYciently acidic pH and in the presence of activator protein the


N370S mutant glucocerebrosidase shows a considerable residual activity (van

Weely et al., 1993). Homozygosity or heterozygosity for this allele seems to

preclude the development of neurological symptoms, and is therefore always

associated with the type 1 form of the disease (Beutler and Grabowski, 2001;

Tsuji et al., 1988). It has been shown that a large part of the N370S homo-

zygotes have such a mild form of the disease that they do not seek medical

advice and remain therefore undiagnosed, the so‐called asymptomatic patients

(Aerts et al., 1993). The secondmost frequent mutation is the L444Pmutation,

often resulting in neurological symptoms in homozygotes. Unlike the N370S

protein, this mutation appears to result in impaired traYcking and priming for

degradation in the endoplasmic reticulum (ER) (Ohashi et al., 1991). Less is

known about the molecular consequences of other mutations in the glucocer-

ebrosidase protein and, in particular, their eVects on the phenotypic expression

of Gaucher disease. It is important to stress in this connection that although

some relation may exist between particular genotypes and phenotypes, clinical

manifestations can diVer markedly within the same genotype. Phenotypically

discordant identical twins with Gaucher disease have been documented (Cox

and Schofield, 1997; Lachmann et al., 2004). Such twin studies and the poor

predictive power of phenotype–genotype investigations in Gaucher disease

have clearly pointed out that epigenetic factors also play a key role in Gaucher

disease manifestation (Aerts et al., 1993).

3. Gaucher Cells

Although glucocerebrosidase activity is reduced in all cell types of Gaucher

patients, the lysosomal storage of glucosylceramide is restricted to cells of the

monocyte/macrophage lineage, at least in the type 1 variant. Although the

precise source of the lipid is not known, it has been speculated that a large

portion is derived from membranes of phagocytosed red and white blood

cells, explaining the predominant lipid accumulation in macrophages (Naito

et al., 1988; Parkin and Brunning, 1982). The glucosylceramide‐laden cells

show a characteristic morphology with an eccentric nucleus and a ‘‘wrinkled

tissue paper’’‐like appearance due to the massive presence of lipid in tubular

deposits. These storage cells are called Gaucher cells and are present in

various locations, predominantly the bone marrow, spleen, liver, and paren-

chyma of the lymph nodes. The massive accumulation of storage cells in

tissues causes abnormalities in architecture and function. In the bone mar-

row, displacement of the normal hematopoietic cells gradually occurs,

promoting pancytopenia. In other tissues, infiltration of Gaucher cells may

lead to fibrosis, infarction, necrosis, and scarring. The sheer presence of

storage cells probably does not fully explain the entire pathology of Gaucher

disease. Despite their abnormal large, swollen appearance, Gaucher cells are

not inert storage containers, but are metabolically active cells that are able to

produce and secrete proteins that drive pathophysiological processes.

76 BUSSINK ET AL.

Gaucher cells, although originating from common tissue macrophages,

have a specific phenotype. On the basis of their phenotype and immune

function two main types of macrophages are generally distinguished: classi-

cally and alternatively activated macrophages. Classically activated macro-

phages promote inflammatory responses, whereas alternatively activated

macrophages exert antiinflammatory responses (Goerdt and Orfanos, 1999;

Gordon, 2003; Mantovani et al., 2004). In situ analysis of Gaucher spleen

sections has revealed that the Gaucher cells resemble alternatively activated

macrophages. Gaucher cells express high levels of lysosomal acid phospha-

tase, HLA class II, CD68, the scavenger/lipid receptor CD36, and signal

regulatory protein (SIRP)‐a. Gaucher cells stain positive mainly for CD14,

but fail to show CD11b expression. Typical proinflammatory molecules such

as interleukin (IL)‐1b, IL‐1a, IL‐12 p40, tumor necrosis factor (TNF)‐a,interferon (IFN)‐g, and macrophage chemoattractant protein (MCP)‐1were not expressed. Gaucher cells strongly express interleukin‐1 receptor

antagonist and CCL18, which are typical markers of alternatively activated

macrophages (Boven et al., 2004). Histochemical analysis of Gaucher spleen

sections has revealed that storage lesions are characterized by a core of

mature, alternatively activated Gaucher cells surrounded by proinflamma-

tory macrophages that have apparently been recruited to the lesions.

4. Pathophysiology

It is generally believed that the complex mixture of factors, such as cytokines

and hydrolases, originating from storage cells themselves or from surround-

ing classically activated macrophages contributes to the characteristic

pathophysiology of Gaucher disease.

The activities of several common lysosomal hydrolases have been reported

to be elevated in Gaucher disease. As early as 1956 increased activity of

tartrate‐resistant acid phosphatase (TRAP) in plasma of type 1 Gaucher

patients was reported (Tuchmann et al., 1956). Further studies showed that

the increased activity was due to elevations in isoenzyme 5B (Lam and

Desnick, 1982). The finding of increased TRAP activity was followed by

reports on the presence of several lysosomal enzymes in plasma as well as

tissue samples (MoYtt et al., 1978; Ockerman and Kohlin, 1969). The

elevated acid phosphatase was used as a diagnostic marker before identifica-

tion of the genetic defect in Gaucher disease. Later it was suggested that the

levels of b‐hexosaminidase A and B could be used to screen for Gaucher

disease in Ashkenazi Jews who underwent testing for Tay‐Sachs disease car-riership, because Gaucher patients had clear elevations in b‐hexosaminidase

B, whereas b‐hexosaminidase A was relatively low (Nakagawa et al.,

1983). However, further studies highlighted the marked heterogeneity in

these enzyme activities, making this test unreliable for screening purposes

(Natowicz et al., 1991). The source of these elevated hydrolases is most likely


Gaucher cells or their macrophage precursors. Immunohistochemical studies

have indeed shown that TRAP can be localized to the storage cells and

surrounding activated macrophages in the spleen (Boven et al., 2004; Hibbs

et al., 1970). DiVerential gene expression techniques applied to Gaucher

spleen samples have identified increased levels of cathepsins S, C, and K

and again, by immunohistochemical staining, these hydrolases were found to

originate from the Gaucher cells (Moran et al., 2000). Two other commonly

elevated hydrolase activities in Gaucher patients deserve specific mention.

Serum angiotensin‐converting enzyme (ACE) levels are known to be often

increased, ranging from nearly normal to more than 10‐fold the control

median (Lieberman et al., 1976; Silverstein et al., 1980). Increased levels of

lysozyme activity have also been documented (Silverstein and Friedland,

1977). Although many attempts have been made to relate the elevated levels

of hydrolases to specific pathology in Gaucher disease or disease severity

in general, no direct relationship has been established. Of particular note,

cathepsin K, which is reported to have a pathogenetic role in osteolysis, and

TRAP, which is known to be secreted by osteoclasts, have been implicated

in the pathophysiology of Gaucher bone disease (Moran et al., 2000).

Various cytokines are produced by Gaucher cells and their surrounding

phenotypically diverse macrophages (Boven et al., 2004). Michelakakis and

co‐workers were the first to report on elevated levels of TNF‐a in the plasma

of type 2 and 3 Gaucher patients, and to a lesser extent in samples from type 1

Gaucher patients (Michelakakis et al., 1996). Allen and co‐workers could not

confirm the finding of elevated plasma TNF‐a in type 1 Gaucher disease, but

did observe increases in IL‐6 and IL‐10 (Allen et al., 1997). In another study,

Hollak and co‐workers did not detect consistent elevations of either TNF‐aor IL‐6 in the plasma of type 1 Gaucher patients (Hollak et al., 1997). The

same study, however, revealed that IL‐8 could be markedly increased in the

plasma of type 1 Gaucher patients. Analysis of plasma levels of TNF‐a and

genotyping for the –308 G!A polymorphism in the promoter of the TNF‐agene were performed in 17 patients with type 1 Gaucher disease. A significant

correlation was found between serum TNF‐a levels and TNF‐a genotypes

for homozygous versus heterozygous patients (p ¼ 0.02), with patients

homozygous for the polymorphism having the lower levels of serum

TNF‐a (Altarescu et al., 2005). Polymorphisms in promoter regions of the

TNF‐a (and IL‐6) genes may help to explain the heterogeneous findings on

plasma elevations of these proinflammatory cytokines in type 1 Gaucher

patients. A low‐grade inflammatory profile has been identified in Gaucher

disease. In comparison with a multivariable database from healthy controls,

patients with Gaucher disease were found to have significant elevations in

fibrinogen, an accelerated erythrocyte sedimentation rate, and elevated

C‐reactive protein (Rogowski et al., 2005). The authors pointed out that

these parameters were not influenced by enzyme therapy, which might imply

that these abnormalities are not directly related to the stored glycolipids but

78 BUSSINK ET AL.

could be the resul t of cytok ine or ch emokine relea se. Increas ed level s of

macrophag e colony ‐ stimula ting fact or (M ‐ CSF), 2‐ to 5‐ fold above normal ,

have been observed in the plasm a of most Gauch er patien ts ( Holl ak et al. ,

1997 ). In ad dition, the plasma of many Gaucher pa tients con tains up to 7 ‐foldincreased con centrations of the monocyt e/macrop hage activatio n marker ,

soluble CD 14 (sCD14 ). This finding supp orts the idea that activati on of

monocyt es/macroph ages oc curs in sympt omatic Gauch er pa tients. Anothe r

marker for macroph age activ ation is solubl e CD1 63 (sCD16 3). Increas ed

concentra tions have been measur ed in patients with infec tion an d myel oid

leukemias. The sCD163 plasma levels in type 1 Gaucher patients were found

to be far abo ve the level s in nor mal sub jects (Moller et al. , 2004).

5. Therapy

The concept of treating lysosomal storage disorders by supplementation with

the missing enzyme has already been proposed by De Duve in 1964, after his

discovery of lysosomes. Type 1 Gaucher disease is, in particular, an attractive

candidate for such a therapy, because the target cell is the macrophage and

there is no neurological involvement. Most cell types have specific receptors

by which they can internalize specific ligands by receptor‐mediated endo-

cytosis, followed by their eYcient delivery to lysosomes. One such receptor on

macrophages is the so‐called mannose receptor, which recognizes mannose,

fucose, and N‐acetylglucosamine terminal oligosaccharides on glycoproteins

(Lennartz et al., 1987). In the first attempts at enzyme supplementation

therapy for Gaucher patients, purified placental glucocerebrosidase was

administered intravenously. This enzyme preparation was found to result in

a reduction of glucosylceramide storage in the liver, without any major

clinical improvement (Brady et al., 1974; Furbish et al., 1978). It was found

that the placental enzyme, which contains variably sialylated, complex‐typeN‐linked glycans, was largely taken up by cells expressing the asialoglycopro-

tein receptor, such as hepatocytes. This led to the idea that macrophages

might be more selectively reached by modifying the oligosaccharide chains

in the placental enzyme, by enzymatically exposing the covered mannose

residues (Furbish et al., 1981). This concept resulted in the development of

alglucerase (Ceredase; Genzyme, Cambridge, MA). The eVect of intravenousadministration of the modified glucocerebrosidase was studied in a group of

type 1 Gaucher patients (Barton et al., 1990, 1991). In general, on enzyme

therapy a marked reduction of liver and spleen size occurs, accompanied by

improvement of hemoglobin levels and platelet counts. Skeletal improvement

is less prominent, and lags clearly behind the improvement in the other

symptoms. In the mid‐1990s, Ceredase was replaced by treatment with

recombinantly produced glucocerebrosidase from Chinese hamster ovary

(CHO) cells (Cerezyme; Genzyme), with similar therapeutic results

(Grabowski et al., 1995). Enzyme replacement therapy (ERT) has also been


tried for neuronopathicGaucher disease. In the case of type 2Gaucher patients

the results have not been positive. Visceral improvements occurred in these

patients, but there was no eVect on the lethal neuropathology (Erikson et al.,

1993). In contrast, positive results have been obtained for type 3 Gaucher

patients. Again, a marked improvement in visceral signs was noted as well as

arrest of neurological deterioration and even in some cases signs of neurological

improvements. It seems to be recommendable to initiate therapy in these

patients before the onset of neuropathology (Vellodi et al., 2001).

An alternative approach for therapeutic intervention of type 1 Gaucher

and other glycosphingolipidoses is substrate reduction therapy (SRT; also

termed substrate deprivation therapy). Radin first formulated the challeng-

ing concept (Radin, 1996). The approach aims to reduce the rate of glyco-

sphingolipid biosynthesis to levels that match the impaired catabolism. It is

conceivable that patients who have significant residual lysosomal enzyme

activity can gradually clear lysosomal storage material and therefore should

profit most from reduction of substrate biosynthesis.

Two main classes of inhibitors of glycosphingolipid biosynthesis have been

described, both of which inhibit the ceramide‐specific glucosyltransferase (alsotermed glucosylceramide synthase; GlcT‐1; UDP‐glucose:N‐acylsphingosineD‐glucosyltransferase, EC 2.4.1.80). The enzyme catalyzes the transfer of

glucose to ceramide, the first step of the biosynthesis of glycosphingolipids.

The first class of inhibitors is formed by analogs of ceramide. The prototype

inhibitor is D,L‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol(PDMP). More specific and potent analogs were subsequently developed

by substituting the morpholino group for a pyrrolidino function and by

substitutions at the phenyl group: 4‐hydroxy‐1‐phenyl‐2‐palmitoylamino‐3‐pyrrolidino‐1‐propanol (p‐OH‐P4) and ethylenedioxy‐1‐phenyl‐palmitoyla-

mino‐3‐pyrrolidino‐1‐propanol (EtDo‐P4) (Shayman et al., 2004). Studies in

a knockout mouse model of Fabry disease have shown that oral administra-

tion of the compounds can result in a marked reduction of the accumulating

glycosphingolipid globotriaosylceramide. The second class of inhibitors of

glucosylceramide synthase is formed by N‐alkylated iminosugars. Such

types of compounds were already in common use as inhibitors of N‐glycan‐processing enzymes and the potential application of N‐butyldeoxynojirimycin

(NB‐DNJ) as human immunodeficiency virus (HIV) inhibitor had been

studied in acquired immunodeficiency (AIDS) patients. Platt and co‐workersat the Oxford Glycobiology Institute (Oxford, UK) were the first to recognize

the ability ofN‐butyldeoxynojirimycin to inhibit glycosylceramide synthesis at

low micromolar concentrations (Platt et al., 1994). The same researchers

demonstrated in knockout mouse models of Tay‐Sachs disease significant

reductions in glycosphingolipid storage in the brain (Platt et al., 1997).

Preclinical studies in animals and the previous clinical trial in AIDS patients

have indicated (transient) adverse eVects in the gastrointestinal tract, probably

related to the ability of NB‐DNJ to inhibit disaccharidases on the intestinal

80 BUSSINK ET AL.

brush border. Overkleeft and co‐workers, in their search for inhibitors

of glucosidases, have serendipitously developed a more potent inhibitor

of glucosylceramide synthase. N‐(5‐Adamantane‐1‐yl‐methoxy‐pentyl)‐1‐deoxynojirimycin (AMP‐DNM) was found to inhibit glycosphingolipid bio-

synthesis at low nanomolar concentrations (Overkleeft et al., 1998) and able to

prevent globotriaosylceramide accumulation in a Fabry knockout mouse

model without overt side eVects (J. M. Aerts, unpublished observations).

The first clinical study of the use of NB‐DNJ to treat a glycosphingolipid

storage disorder has been reported (Cox et al., 2000). In anopen‐label phase I/IItrial 28 adult type 1 Gaucher patients received 100 mg of NB‐DNJ (OGT918;

OxfordGlycoSciences, Oxford,UK) three times daily. Improvements in viscer-

omegaly and hematological abnormalities as well as corrections of plasma

levels of glucosylceramide and biomarkers of Gaucher disease activity have

been described, although the extent of the response is less spectacular than

generally observed with high‐dose enzyme replacement therapy. As expected, a

dose–response relationship is demonstrable for NB‐DNJ in type 1 Gaucher

patients. It has been reported that administration of 50 mg of NB‐DNJ three

times daily is far less eVective (Heitner et al., 2002). NB‐DNJ (Zavesca; Acte-

lion, Allschwil, Switzerland) is now registered in Europe and the United States

for treatment of mild to moderately aVected type 1 Gaucher patients who are

unsuitable to receive enzyme replacement therapy (Cox et al., 2003).At present,

important insights have been gained regarding clinical eYcacy and safety. The

sustained eVects of prolonged substrate reduction therapy have been reported

(Elstein et al., 2004; Pastores et al., 2005). Provided that iminosugars or other

inhibitors of glucosylceramide synthase prove to be safe in the long term, they

should have an important role to play in the management of glycosphingolipid

storage disorders, including Gaucher disease.

6. Biomarkers

With the development of ERT and SRT, and because of the limited predictive

value of genotyping in Gaucher disease, an urgent need has developed for

surrogate markers of Gaucher cells. Such biomarkers would allow accurate

monitoring of the progress of the disease and eYcacy of therapy. The ideal

biomarker is detectable in plasma and directly reflects the presence of storage

cells. Although abnormalities in levels of tartrate‐resistant acid phosphatase

(TRAP), angiotensin‐converting enzymes, hexosaminidase, and lysozyme

have all been reported, none of these enzymes appears to meet this criterion

(Aerts and Hollak, 1997). Overlap between levels of these enzymes in patients

versus controls further restricts their use as biomarkers in Gaucher disease.

7. Discovery of Chitotriosidase

In an attempt to identify novel secondary biochemical abnormalities, a

thorough screening of plasma enzyme activities in the plasma of symptomatic


individuals versus a variety of substrates was conducted. This led to the

discovery of a 1000‐fold increased capacity of plasma samples from sympto-

matic Gaucher patients to hydrolyze the fluorogenic substrate 4‐methylumbel-

liferyl‐chitotrioside (Hollak et al., 1994). The responsible enzyme was named

chitotriosidase. Further studies revealed that plasma chitotriosidase originated

solely from the lipid‐laden macrophage. As a result of this, chitotriosidase

activity levels do not reflect one particular clinical symptom, but rather reflect

the total body burden ofGaucher cells. Although chitotriosidase activity can be

rapidly and sensitively measured using 4‐methylumbelliferyl‐chitotrioside as

substrate, the ability of the enzyme to transglycosylate complicates the enzyme

assay.As discussed inmore detail in Section IV.D, the use of a slightlymodified

substrate provides a much more convenient method for measuring activity of

chitotriosidase (Aerts et al., 2005; Aguilera et al., 2003).

As can be seen from Fig. 1, chitotriosidase activities are greatly increased

in glucocerebrosidase‐deficient individuals compared with controls, without

overlap of values between the cohorts. Strikingly, chitotriosidase values drop

sharply on ERT, coinciding with clinical improvements (Hollak et al., 1994).

To assess the utility of chitotriosidase activity measurements as a biomarker

for treatment eYcacy, the relationship between chitotriosidase activity and

clinical parameters has been studied (Hollak et al., 2001). On the basis of this

investigation, it has been proposed that in patients in whom initiation of

FIG. 1 Plasma chititriosidase levels in GD type 1 patients versus controls, showing the

relationship between chitotriosidase activity and genotype in patients and controls.

82 BUSSINK ET AL.

treatment is questionable, based solely on clinical parameters, a chitotriosi-

dase activity above 15,000 nmol/ml/h may serve as an indicator of a high

Gaucher cell burden and an indication for the initiation of treatment.

A reduction of less than 15% after 1 year of treatment should be a reason

to consider a dose increase. Furthermore, a sustained increase in chitotriosi-

dase at any point during treatment should alert the physician to the possibil-

ity of clinical deterioration and the need for dose adjustment, and hence

chitotriosidase activity has great potential in both diagnosis and monitoring

of the disease. The regular monitoring of plasma chitotriosidase levels in

Gaucher patients is presently used worldwide to assist in clinical manage-

ment of these patients (Cabrera‐Salazar et al., 2004; Deegan and Cox, 2005;

Deegan et al., 2005; Vellodi et al., 2005).

A pitfall regarding the use of chitotriosidase as Gaucher cell biomarker

results from the complete absence of the chitotriosidase activity in about 6%

of all individuals, including Gaucher patients. This results from homozygos-

ity for a null allele of the chitotriosidase gene (Boot et al., 1998). Plasma

chitotriosidase levels in heterozygotes for this mutation (about 35% of all

individuals) underestimate the actual presence of Gaucher cells in patients.

Determination of chitotriosidase genotype in Gaucher patients is therefore

recommended.

Although plasma chitotriosidase activity is now the most used biomarker

in GD, there is still a need for other biomarkers primarily because of the high

incidence of deficiency. A marked elevation of the chemokine CCL18 in GD

has been described (described in Section IV). Because both chitotriosidase

and CCL18 do not reflect a specific clinical symptom but rather total body

storage burden, the search for suitable symptom‐specific biomarkers remains

the subject of ongoing research.

The identification in Gaucher patients of chitotriosidase, the first chitinase

discovered in vertebrates, has stimulated clinical and fundamental research

interest in this endoglycosidase and in subsequently discovered related pro-

teins. In the next sections attention is focused on the features and functions of

the intriguing chitinase protein family.

III. Chitinases

A. Chitin and Chitinases

1. Occurrence and Synthesis of Chitin

Polysaccharides are present in most organisms for structural purposes. Chi-

tin, the linear polymer of b‐1,4‐linked b‐N‐acetylglucosamine (GlcNAc), is

the most abundant biopolymer in marine environments and, after cellulose,


the second most abundant in nature. Chitin is an insoluble polymer of

considerable mechanical and chemical strength, mostly because of hydrogen

bonding between the >NH group and the >C¼O group of the N‐acetylgroups of aligning chains (Fig. 2). A common modification of chitin, deace-

tylation to form chitosan, therefore considerably reduces its physicochemical

resilience. Hydrophobic stacking interactions between carbon‐bound hydro-

gen atoms (not shown in Fig. 2) further stabilizes the alignment of the chains.

Chitin exists in three crystalline forms: a, in which the chains align parallel;

b, in which the chain alignment is antiparallel; and g, a mixture of a and bforms. The properties of chitin are ideal for serving as a coating in a variety of

organisms, in which chitin is often covalently bound to other glycopolymers.

An example of this is the cell wall of many fungi, in which chitin is covalently

bound to a‐glucan (Debono and Gordee, 1994). Quantitatively, most chitin

is present in arthropods (a phylum that includes crabs, lobsters, the extinct

trilobite, spiders, and insects), which utilize chitin as the main constituent of

their exoskeleton, protecting them from environmental influences and

providing structural support. Furthermore, chitin is present in algae and

protists (Mulisch, 1993), in the septum between mother and daughter yeast

cells (Cid et al., 1995), and in the microfilarial sheaths of parasitic nematodes

(Araujo et al., 1993; Fuhrman and Piessens, 1985).

The sole substrate for chitin synthesis is UDP‐GlcNAc, which is the end

product of the hexosamine biosynthetic pathway to which part of the avail-

able glucose is diverted. Chitin synthases catalyze the stepwise addition of

GlcNAc to a growing chain of chitin. Interest in chitin synthases has been

considerable, partly because they are considered to be a potential target for

pest control and the development of antifungal agents (Cohen, 1990). Chitin

synthases have been identified and the corresponding genes have been cloned

from a variety of organisms, including Botrytis cinerea (Causier et al., 1994),

FIG. 2 The chemical structure of chitin. Note that the C–H bonds are not shown and the 1!4

glycosidic bonds are strictly in the b conformation. Because the far right sugar moiety

undergoes ring opening, this side of the chain is referred to as the reducing end; the left side is

referred to as the nonreducing end.

84 BUSSINK ET AL.

Aspergillus fumigatus (Mellado et al., 1995), Candida albicans (Mio et al.,

1996), and various other fungi (Bowen et al., 1992; Gow, 1994). One of the

best studied organisms in this respect is Saccharomyces cerevisiae, which

contains three distinct chitin synthase activities encoded by seven genes, all

fulfilling diVerent roles in yeast cell budding (Bulawa, 1993; Cid et al., 1995;

Roncero, 2002). The presence of several chitin synthase genes in a single

fungal species is not uncommon, underlining the complexity of chitin

synthesis in fungi.

Little information is available on the location of chitin synthesis, despite

numerous eVorts to unravel the exact mechanism. Investigations have fo-

cused on the existence of so‐called chitosomes, centers of chitin synthesis that

eventually release their cargo of small chitin molecules at the plasma mem-

brane and that can be visualized by electron microscopy (EM) (Bracker et al.,

1976; Leal‐Morales et al., 1988). However, chitin synthases are also

incorporated into the plasma membrane in insects (Locke and Huie, 1979;

Zimoch and Merzendorfer, 2002), at the interface between mother and

daughter cells in S. cerevisiae (Chuang and Schekman, 1996), and at the

plasma membrane in yeast (Duran et al., 1979) as has been determined by

both EM and immunostaining (Merzendorfer, 2006).

Chitin synthases belong to the large family of glycosyltransferases, a

ubiquitous group of enzymes catalyzing the transfer of sugar moieties to

specific acceptors. On the basis of sequence homology, chitin synthases have

been grouped into family 2 of the glycosyltransferases, a group with remark-

able homology within its active site sequence. In arthropods, the poly-

merization is believed to be initiated by a primer, covalently bound to the

enzyme. Although the identity of the primer is yet to be identified, experi-

mental evidence suggests that certain glycolipids, dolichol derivatives, or

chitooligomers can serve as acceptors for GlcNAc (Horst, 1983; Palli and

Retnakaran, 1999; Quesada Allue et al., 1975). As a consequence of a

covalently linked primer, release of the chitin chain must involve cleavage

of the bond linking the polymer and the enzyme.

Although the general consensus is that vertebrates are unable to synthesize

chitin, debate has focused on DG42, a protein from Xenopus laevis that

is diVerentially expressed at gastrulation and that shows sequence homology

with fungal chitin synthases. DG42, however, also shows sequence homology

with hyaluronan synthase, the enzyme responsible for synthesis of hyaluronic

acid, a common biopolymer in vertebrates that acts as a lubricating agent in

synovial fluid. Hyaluronic acid is synthesized at the plasma membrane in

contrast to most glycoaminoglycans, which are elongated in the Golgi com-

plex. It has been reported that mammalian cells, as well as S. cerevisiae,

transfected with DG42 cDNA are able to synthesize high molecular weight

hyaluronic acid (Meyer and Kreil, 1996). Surprisingly, it has also been shown

that DG42 in vitro can synthesize chitooligomers up to six GlcNAc residues


in size (Semino and Robbins, 1995). This was confirmed for the DG42

homologs of zebrafish and mouse, two proteins that are also diVerentiallyexpressed during embryogenesis (Semino et al., 1996). Because, to date, only

in vitro evidence is available regarding the synthesis of chitooligomers by

DG42, the possibility of an experimental artifact cannot be excluded. Alter-

natively, it has been proposed that DG42 is an enzyme that produces chit-

ooligomers that are subsequently used as primers for the synthesis of

hyaluronic acid (Varki, 1996). It might be considered that, even though

chitin synthase‐like proteins are present in vertebrates and plants allowing

for chitooligomers to be synthesized, cell physiology does not allow the

subsequent extension to longer strands.

2. Chitinolytic Enzymes

The degradation of chitin is mediated by chitinolytic hydrolases, among

which a number of distinct enzymes with diVerent specificities can be identi-

fied. First, most organisms contain b‐hexosaminidases that are able to

remove the terminal GlcNAc residue from the nonreducing end of the bio-

polymer (see Fig. 2 in Section III.A.1). Second, some eukaryotes contain a

chitobiase that can remove chitobiose from the reducing ends of intermedi-

ates in N‐linked glycan degradation and of chitin polymers (Charpentier and

Percheron, 1983; Gutowska et al., 2004). Finally, there are the chitinases that

are able to cleave within the chitin polymer. A distinction is made between

two types of chitinases: the more common endochitinases, and exochitinases.

Endochitinases are defined as enzymes splitting hydrolyzing glycosidic bonds

randomly within the chitin polymer, releasing mainly soluble, low molecular

weight chitooligomers (Sahai and Manocha, 1993), whereas exochitinases

catalyze the successive removal of chitobiose units from the nonreducing end

of chitin polymers (Robbins et al., 1988).

3. Functions in Nonvertebrates

Chitinases have been detected in chitin‐containing organisms and in species

that do not contain chitin, for example, in a variety of bacteria, plants,

vertebrates, and even viruses (Flach et al., 1992, Gooday, 1995; Sahai and

Manocha, 1993). Various biological functions have been attributed to the

chitinases in the various species.

a. Food Processing In marine environments alone, more than 1011 metric

tons of chitin is produced annually (Lutz et al., 1994), resulting in the

deposition of the biopolymer on the ocean floor, a phenomenon referred to

as ‘‘marine snow’’ in the early literature. Despite this continuous deposition,

only trace amounts of chitin are present in marine sediments, because of the

86 BUSSINK ET AL.

chitinolytic activity of bacteria that are able to utilize chitin as their only

source of carbon and nitrogen (Gooday, 1996).

Several chitinolytic bacterial species have been identified thus far, among

which are Pseudomonas, Bacillus, and Vibrio species. The degradation process

is complex, owing to the insolubility of chitin and the need therefore to secrete

chitinases into the extracellular environment. A model has been proposed for

chitin degradation by Vibrio species involving subsequent extracellular degra-

dation of chitin by secreted chitinases, nutrient sensing, chemotaxis, and

hydrolysis of chitooligomers by the combined action of several hydrolases,

after which the monomer GlcNAc can be converted into fructose 6‐phosphate,NH3, and acetate in the cytosol (Li and Roseman, 2004).

In the chitinolytic soil bacterium Serratia marcescens a chitin‐bindingprotein that lacks chitinase activity has been identified, which appears to be

essential for eYcient hydrolysis of natural chitin (Vaaje‐Kolstad et al., 2005).

Interestingly, homologs appear to exist in other chitin‐hydrolyzing organisms,

suggesting a ubiquitous mechanism adopted to increase chitin‐processingeYciency involving proteins other then chitinases. Several chitinolytic

fungi have also been described (Gooday, 1995) that have an important role

in recycling chitin in soil, whereas only one archaeon has been described

to date that utilizes chitin for nutritional purposes and has been named

Thermococcus chitinophagus (Andronopoulou and Vorgias, 2004; Huber

et al., 1995).

Insectivorous plants are known to employ chitinases for the digestion of

captured prey (Gooday, 1990). Some vertebrates that prey on insects have

chitinases in their guts, presumably as tools for food processing (Flach et al.,

1992; Gooday, 1995). In some cases, however, it is not clear whether these

chitinases are of endogenous origin, or alternatively are produced by the gut

microflora. It has been shown that fish without instruments such as teeth or

grinding gullets contain the highest chitinase activity in their intestinal tract

(Holm and Sander, 1994; Place, 1996), presumably for nutritional purposes,

as indicated by the ubiquitous presence of chitobiases, allowing for complete

degradation of chitin to absorbable nutritive monomers of GlcNAc

(Gutowska et al., 2004). However, for some species it has been demonstrated

that chitin is actually a poor nutrient (Lindsay et al., 1984). Seabirds, for

example, fed Antarctic krill, retain on average only half of the ingested chitin

(Place, 1996).

b. Host–Parasite Interaction Owing to the chemical and mechanical resil-

ience of chitin, several organisms have evolved to utilize chitin synthesis as a

means of internal and external compartmentalization. In response, however,

pathogens of chitinous organisms have adopted production of chitinases to

aid the penetration of the host. Various examples of bacteria and fungi that

penetrate through chitinous barriers of the insect gut, the exoskeleton of


insects and crustacea, employing chitinases, have been described (Flach et al.,

1992; Flyg and Boman, 1988; Gooday, 1990, 1995; Prehm, 1984; Sahai and

Manocha, 1993).

Blood‐sucking mosquitoes synthesize a chitinous peritrophic membrane

(PM) separating the bloodmeal from the midgut epithelium. Ookinetes of

Plasmodium, the malaria parasite, produce a chitinase in order to traverse

this chitinous barrier (Shahabuddin, 1995; Shahabuddin and Kaslow, 1993).

The finding that its inhibition by allosamidin blocks the sporogenic develop-

ment of Plasmodium falciparum and Plasmodium gallinaceum and conse-

quently parasite transmission has gained considerable attention because of

its implication for possible therapeutic intervention in malaria and has led to

the characterization of several Plasmodium chitinases (Shahabuddin and

Kaslow, 1994; Shen and Jacobs‐Lorena, 1997; Vinetz et al., 1999).Leishmania utilizes a similar mechanism (Shahabuddin and Kaslow, 1993);

however, experimental evidence suggests that the PM in early stages of

infection is in fact vital to survival of the parasite (Shahabuddin and Kaslow,

1994). Transmission of Leishmania to a vertebrate host is dependent on

disruption of the chitinous stomodeal valve (the main valve that maintains

the unidirectional blood flow during normal feeding), causing infected blood

to be regurgitated into the host (Schlein et al., 1992, 1991).

In Brugia malayi, a presumed function of its chitinase is the formation of

chitooligomers that act as decoy molecules for defense lectins present in the

gut (Fuhrman et al., 1992).

Interestingly, in tsetse flies susceptibility to Trypanosoma is thought to be

dependent on the load of a symbiotic rickettsia‐like organism (RLO) that

secretes chitinases producing the decoys (Shahabuddin and Kaslow, 1993;

Welburn et al., 1993).

For Entamoeba invadens, which infects reptiles and is used as a research

model for amoebic encystations, it has been shown that formation of chitin-

ous cysts is blocked by the chitinase inhibitor allosamidin, suggesting that its

chitinase is essential in the process of generating an infectious stage of these

protozoan parasites (Delavega et al., 1997).

Another spectacular example of the biological importance of a chitinase

is presented by Autographa californica nucleopolyhedrovirus (Hawtin et al.,

1995). The chitinase encoded by the viral genome, expressed in the late stages

of viral replication in insects, mediates not only the penetration of chitinous

barriers, but also results in liquefaction of cadavers, a pathogenic eVect thatenhances the dispersal of progeny virions. Deletion of the viral chitinase,

however, results in dry cadavers in which the viruses are largely contained

(Rao et al., 2004).

c. Defense Mechanisms Most plants are able to produce chitinases that are

implicated in the defense against chitin‐containing organisms, mainly fungi

88 BUSSINK ET AL.

and insects (Flach et al., 1992; Gooday, 1995; Lee and Hwang, 2005; Pirttila

et al., 2002; Sahai and Manocha, 1993). Plant chitinases are prominent

components of the plant pathogenesis‐related (PR) proteins (Kasprzewska,

2003), induced in the presence of pathogens (or extracts of pathogens) and

also after stress induction by wounding or exposure to certain chemicals

(Hamel and Bellemare, 1995; Taira et al., 2005). Another member of the

PR proteins is b‐1,3‐glucanase, which is generally coinduced with chitinase,

resulting in synergistic lysis of fungal cell walls that often contain both chitin

and b‐1,3‐glucan (Flach et al., 1992; Sahai and Manocha, 1993). The impor-

tance of chitinases in plants for their resistance against fungal infections has

been demonstrated by decreased susceptibility to fungi in plants overexpres-

sing a recombinant chitinase (Grison et al., 1996; Jach et al., 1995). Further-

more it has been reported that spraying of plants with a chitinase‐containingsubstance also results in decreased susceptibility to fungal infection (Hart

et al., 1993).

Chitinases may also mediate a defense response indirectly by releasing

chitooligomers that have been demonstrated to induce the expression of

several other proteins related to the defensive response, even in the absence

of pathogens (Felix et al., 1993; Hart et al., 1993; Kasprzewska, 2003).

d. Morphogenesis In chitin‐containing organisms chitinases fulfill obvious

roles in morphogenic processes involving their chitinous coatings. The vari-

ous types of arthropods utilize chitinolytic enzymes to digest chitin in their

exoskeleton during molting (Bassler et al., 1991; Gooday, 1990, 1995), and

nematodes during hatching from their chitin‐coated eggs (Arnold et al., 1993;

Schlein et al., 1991; Shahabuddin and Kaslow, 1993). A chitinase of

Hydractinia echinata, a member of the phylumCnidaria (encompassing sting-

ing jellyfish and corals), has been shown to be diVerentially expressed during

polyp formation (Mali et al., 2004).

A wealth of information is available concerning the involvement of chit-

inases in morphogenic processes in chitinous fungi, where they are required

for normal growth, hyphal formation, and spore germination (Barone et al.,

2003; Barrett‐Bee and Hamilton, 1984; Cabib et al., 1992; Flach et al., 1992;

Gooday, 1990, 1995; Sahai and Manocha, 1993; Zarain‐Herzberg and

Arroya‐Begovich, 1983). The morphogenesis is a result of ongoing chitin

synthesis and its degradation by chitinases, allowing for continuous remo-

deling of chitin microfibrils (Gooday, 1990; Sahai and Manocha, 1993).

Despite this delicate balance, chitin synthesis and chitinase activities in

Candida albicans and Saccharomyces cerevisiae appear to be independently

regulated (Selvaggini et al., 2004). Most chitinous fungi possess several

chitinases, up to 14 for the human pathogen Aspergillus fumigatus (Adams,

2004), adding to the complexity of the role of chitinases in fungi.


Interestingly, in plants that are thought to lack chitin a role for chitinases

in morphogenic processes has been postulated. The evidence for this is

largely based on the marked expression of chitinase genes during specific

morphogenic events such as flower formation (Kasprzewska, 2003; Sahai

and Manocha, 1993). Mutational studies in Arabidopsis thaliana have shown

that a lack of functional type II chitinase leads to severe phenotypic changes

(Zhong et al., 2002). Furthermore, in a temperature‐sensitive carrot mutant,

blocked in the embryonic globular stage, embryogenesis is rescued in the

presence of a chitinase (de Jong et al., 1992, 1993; Kragh et al., 1996).

Although the mechanism behind these observations remains to be deter-

mined, it has been proposed that the regulatory role of chitinases involves

degradation of oligosaccharides that can serve as messenger molecules

(Kasprzewska, 2003). Perhaps this occurs in a manner similar to the regula-

tion of the nodulation process in legume plants, in which chitinases have been

shown to hydrolyze lipo‐chitooligosaccharides, so‐called Nod factors (Day

et al., 2001; van der Holst et al., 2001). These factors are the major mediators

in the symbiotic relationship between leguminous plants and certain soil

bacteria (usually referred to as rhizobia) and are of major agricultural

importance because of their role in nitrogen cycling (D’Haeze and Holsters,

2002; Riely et al., 2004).

B. Biochemical Features of Chitinases

1. Detection and Measurement of Chitinase Activity

Analytical assessment of chitinase activity is hampered by the insolubility of

chitin, a problem that can be circumvented by using radiolabeled colloidal

chitin. However, even when using this substrate, precise quantification

is hampered by the fact that enzymatic activity is not only dependent

on the amount of radioactivity liberated but also on the exact size of the

fragments. As a convenient alternative to colloidal chitin, fluorogenic and

chromogenic substrates, such as 4‐methylumbelliferyl‐chitooligosaccharidesand p‐nitrophenyl‐chitooligosaccharides, are readily available and generally

employed to measure chitinase activity. Although they allow for extremely

sensitive detection, care must be taken to extrapolate results to natural

substrate kinetics, because enzymatic properties of chitinases toward these

artificial substrates may diVer markedly (Renkema, 1997). An additional

problem in measuring chitinase activity is caused by the ability of many

chitinases to transglycosylate (see Section III.C.4).

When using colloidal chitin or chitooligosaccharides as substrates for

chitinase activity analysis, measurement of both quantity and size of the

fragments is possible with a technique in which the fragments liberated are

90 BUSSINK ET AL.

chemically labeled by reductive amination with a fluorescent compound,

followed by analysis by high‐performance liquid chromatography (HPLC)

(Anumula and Dhume, 1998; Coles et al., 1985). This technique has been

used to study transglycosylation by chitinases of mammalian origin (A. P.

Bussink and K. Ghauharali‐van der Vlugt, unpublished observations).

Because chitinases are active after separation on native polyacrylamide

gels, enzymatic degradation of chitin can also be demonstrated with gels

containing glycol chitin, which appears in the gels as clearing zones that do

not stain with fluorescent brighteners such as Calcofluor White M2R (Escott

and Adams, 1995; Trudel and Asselin, 1989). Alternatively, overlay gels with

fluorescent substrates can be employed (Tronsmo and Harman, 1993).

Important tools for studying chitinase activity, both in vivo and in vitro, are

the specific inhibitors allosamidin and demethyl allosamidin (Nishihiro et al.,

1991). Allosamidin, originally isolated from a Streptomyces species (Sakuda

et al., 1987), is a structural analog of the transition state intermediate and has

proved to be particularly useful in elucidating the reaction mechanism of

chitinases.

2. Classification

Because of the enormous variety of reactions catalyzed by glycoside hydro-

lases, a preliminary classification system of the superfamily was proposed by

B. Henrissat, which was based on similarities in amino acid sequence rather

than on substrate specificity (Henrissat, 1991). Because of the direct relation-

ship between sequence and folding properties, this classification also

reflects structural similarities and, hence, reaction mechanism. After the

increase in available sequence and structural data on glycoside hydrolases,

the classification has been revised several times (URL: www.expasy.ch/cgibin/

lists?glycosid.txt), and has been shown to allow for fairly accurate predic-

tions (Henrissat et al., 1995), although not always reflecting evolutionary

relationships (Beintema, 1994). All chitinases with the exception of several

plant chitinases are grouped together in family 18 of the glycoside superfami-

ly, all of which contain the catalytic domain consensus sequence [LIVMFY]‐[DN]‐G‐[LIVMF]‐[DN]‐[LIVMF]‐[DN]‐X‐E (URL: www.expasy.ch/prosite/

PDOC00839), usually abbreviated to D‐X‐D‐X‐E. The same family includes

a number of eukaryotic chitobiases, involved in the degradation of N‐linkedglycans from proteins, and bacterial endoglucosaminidases capable of

releasing entire glycans from glycoproteins.

Family 18, the glycoside hydrolases, not only contains functional chiti-

nases of various species, it also encompasses a group of mammalian proteins

sharing high sequence homology with chitinases, especially chitotriosidase,

yet lacking chitinase activity. This can be explained from deviations from the

consensus sequence for active chitinases, D‐X‐D‐X‐E, because these proteins

http://www.expasy.ch/cgibin/lists?glycosid.txt

http://www.expasy.ch/cgibin/lists?glycosid.txt

http://www.expasy.ch/prosite/PDOC00839

http://www.expasy.ch/prosite/PDOC00839


lack the glutamate residue responsible for protonation of the glycosidic

bond. However, because the residues that allow for binding of the substrate

are conserved, these proteins are able to bind to chitin with considerable

aYnity and hence are named chitinoses or chi‐lectins. Because they are

diVerentially expressed on various environmental and chemical stimuli and

overexpressed in various pathologies, debate has arisen concerning their

function, as is discussed below in further detail below.

3. Reaction Mechanism

Although the reactions catalyzed by glycosyl hydrolases are diverse, their

reaction mechanisms are usually quite similar, following general acid–base

catalysis (Davies and Henrissat, 1995). A common feature of acid–base cataly-

sis is the presence of a nucleophilic residue, as is the case in lysozyme, for

example. Such amechanism requires a negatively charged residue in the vicinity

of the scissile bond stabilizing the carbonium reaction intermediate. However,

crystal structures of several family 18 chitinases did not reveal such a residue

(Hart et al., 1995; Rao et al., 1995; van Roey et al., 1994). This observation,

together with biochemical evidence that hydrolysis by family 18 chitinases

occurs with retention of stereochemistry (Armand et al., 1994; Davies and

Henrissat, 1995; Iseli et al., 1996), led to the proposition of a substrate‐assistedmechanism in which theN‐acetyl carbonyl group of the substrate stabilizes the

transition state mechanism (Fig. 3). Structural analysis of family 18 chitinases

complexed with allosamidin, an analog of the proposed reaction intermediate,

as well as theoretical and kinetic evidence, have well established the existence of

such a mechanism (Brameld et al., 1998; Honda et al., 2004; Rao et al., 2003;

Terwisscha van Scheltinga et al., 1995; van Aalten et al., 2001). An important

implication of this mechanism is that, although natural chitin generally also

contains glucosamine (deacetylated GlcNAc) units, family 18 chitinases can

hydrolyze fragments onlywhen anN‐acetyl group is present at the reducing endof the chitin strand (Sorbotten et al., 2005).

Most plant chitinases hydrolyze chitin with inversion of stereochemistry,

and do not share sequence homology with family 18 chitinases. Structural

analyses have shown they resemble lysozyme in common fold andmechanism

(Hart et al., 1995; Holm and Sander, 1994; Monzingo et al., 1996), although

the substrates that are hydrolyzed are the same as those of the family 18

members, suggesting these chitinases have evolved as a result of convergent

evolution. The lysozyme‐like chitinases are grouped together in family 19, the

glycoside hydrolases. Family 20 is composed of chitinolytic enzymes that can

cleave only small fragments of chitin (one or two units) by trapping the

substrate in a pocket instead of an extended groove via a mechanism thought

to be similar to that of family 18 chitinases. The classification of chitinolytic

enzymes is summarize d in Table I.

FIG. 3 Substrate‐assisted mechanism of family 18 chitinases. Protonation of the substrate by

the catalytic glutamate occurs, after which the partial positive charge on the anomeric carbon

atom is stabilized by the N‐acetyl oxygen atom of the substrate itself. One strand is released,

after which the oxazolinium intermediate is allowed to react with water, restoring the original

sugar conformation and reprotonating the glutamic acid. Note that the amino acid numbering

for the catalytic acid is that of the human chitotriosidase.

TABLE I

An Overview of the Classification of Chitinolytic Enzymes

Family 18 19 20

Members Chitinases

bacteria

fungi

protozoa

arthopods

virus

vertebrates

plant

Chitinases

plant

Chitobiases

bacteria

Hexosaminidases

eukaryotes

Chitobiases

eukaryotes

Endo-ß-N-acetyl-

glucosaminidases

Mammalian Chi-lectins

Several plant lectins

Catalytic mechanism Retention Inversion Retention

Catalytic domain structure Groove Groove Pocket

92 BUSSINK ET AL.


4. Chitin‐Binding Domain

Apart from the catalytic domain containing the D‐X‐D‐X‐E motif, many

family 18 chitinases have an additional chitin‐binding domain (CBD) located

at either terminus, separated from the catalytic region by a short linker region

(Blaiseau et al., 1992; Jekel et al., 1991; Yang et al., 1996). So far, four distinct

CBDs have been identified in chitinases and although the CBD is dispensable

for hydrolysis of soluble, artificial substrate, its presence alters kinetics toward

natural substrate. To compare activity toward insoluble chitin between the

full‐length and truncated enzymes (lacking only the CBD), Tjoelker and

co‐workers performed an agar diVusion assay, in which wells cut into

agarose‐containing crab shell chitin were loaded with equimolar concentra-

tions of either enzyme. Truncation of the protein was shown to result in a

major decrease in hydrolysis of colloidal chitin, as was measured by destaining

resulting from the disappearance of the white chitin particles, suggesting the

CBD to be essential for hydrolyzing insoluble chitin (Tjoelker et al., 2000).

Structural analysis of CBDs by means of both protein crystallography and

nuclear magnetic resonance (NMR) shows that only a few aromatic residues

are required for chitin binding (Ikegami et al., 2000; van Aalten et al., 2000).

In the case of Serratia marcescens chitinase B, the only two‐domain chitinase

for which the crystal structure of the full‐length protein is available, the

presenceof theCBDeVectively extends the chitin‐binding groove. Interestingly,in this chitinase, the linker does not appear to be very mobile, in contrast

to popular thought (van Aalten et al., 2000). The CBD C‐terminally bound

to human chitotriosidase contains six cysteine residues, all of which are

indispensable for chitin binding, suggesting the formation of three disulfide

bonds (Tjoelker et al., 2000).

Because of the strong CBD–chitin interaction, chitin binding can be

employed for research purposes. A cloning vector has become readily avail-

able that allows the expression of a target protein conjugated to a chitin‐binding domain from Bacillus circulans chitinase A1 (Ferrandon et al., 2003).

Furthermore, CBD conjugated to various fluorescent labels is also available,

allowing for sensitive detection of chitin.

C. Human Chitinases

1. Chitotriosidase: Molecular Features

Because chitin does not exist in mammals, it was initially assumed that the

presence of chitinases is also restricted to lower life forms. Even after chitinases

had been identified in bovine and goat sera (Lundblad et al., 1974, 1979),

similar activity in human serumwas still attributed to the activity of lysozymes,

which are also able to hydrolyze chitin albeit at slow rates. However, using

94 BUSSINK ET AL.

the artificial fluorescent substrate 4‐methylumbelliferyl (4MU)‐chitotetraosidefor lysozyme activity measurement, den Tandt, Overdijk, and co‐workersnoticed that human serum contains an enzyme that was able to hydrolyze

this substrate and was distinct from other hydrolases, including lysozyme

(den Tandt et al., 1993). This MU‐TACT hydrolase, named after the artificial

substrate it cleaved, was subsequently shown to have hydrolytic activity

toward chitin as well (Overdijk and van Steijn, 1994). Attempts to purify

the responsible enzyme from human plasma have been unsuccessful. A break-

through was the discovery by researchers at the Department of Biochemistry

of the University of Amsterdam (Amsterdam, The Netherlands) of markedly

increased chitotriosidase activity in Gaucher patients (Hollak et al., 1994).

This first allowed the purification and molecular characterization of a human

chitinase. Two major isoforms with molecular masses of 50 and 39 kDa have

been purified from the spleen of a Gaucher patient (Renkema et al., 1995).

Both purified isoforms were shown to be completely functional chitinases,

exhibiting activity toward colloidal chitin as well as artificial fluorogenic

substrates, activity that could be inhibited by allosamidin and demethyl

allosamidin, in a manner similar to bacterial chitinases (Renkema et al.,

1995). Using degenerate primers based on conserved regions in chitinases

from several species, the gene was cloned from a macrophage cDNA library

constructed from mRNA isolated from long‐term–cultured peripheral blood

monocytes that spontaneously diVerentiate into activated macrophages that

in turn produce large quantities of chitotriosidase (Boot et al., 1995). Sequence

alignments showed that chitotriosidase is remarkably homologous to

chitinases of various species, in particular the chitinases from nematode

B. malayi (54% similarity, 44% identity) and the insect Manduca sexta

(51% similarity, 41% identity). Furthermore, the catalytic region consensus

sequence (D‐X‐X‐D‐X‐D‐X‐E) is completely conserved.

Alignment of chitotriosidase with other chitinases also showed that the

enzyme consists of a 39‐kDa catalytic domain connected with a C‐terminal

chitin‐binding domain through a short linker region, again in a manner similar

to other chitinases (as discussed in Section III.B.4). When isoelectric focusing

was performed after neuraminidase treatment, a shift in isoelectric point was

observed. Because inhibitors of N‐glycosylation did not have an eVect, it wasdeduced that chitotriosidase contains O‐linked glycans, which were shown not

to aVect enzymatic activity (Renkema et al., 1997). The relationship between

both chitotriosidase isoforms has been elucidated by using cultured macro-

phages as amodel to study expression of the chitinase (Renkema et al., 1997). It

was found that the enzyme is synthesized as a 50‐kDa protein that is either

secreted into themediumor, alternatively, processed into the 39‐kDa enzyme in

the lysosome, where it accumulates. To a quantitativelyminor extent, a 39‐kDa

isoform containing only one extra C‐terminal residue can also be synthesized as

a result of alternative splicing (Fig. 4) (Boot et al., 1998; Renkema et al., 1997).

FIG. 4 The processing of chitotriosidase gene transcripts and protein. The majority of

transcription results in mRNA1 encoding the 50‐kDa isoform that is either secreted or

processed into the 39‐kDa protein. Alternative RNA splicing (mRNA2 and mRNA3) can also

result in the translation of isoforms similar to the proteolytically formed 39‐kDa protein,

diVering only at a few C‐terminal amino acids.


The locus of the chitotriosidase gene was assigned to 1q31‐32 by fluorescence

in situ hybridization, using the genomic clone as a probe (Boot et al., 1998).

The question remains whether the earlier described MU‐TACT hydrolase

activity is caused by chitotriosidase or yet another protein. Like chitotriosi-

dase, MU‐TACT hydrolase activity is dramatically increased in Gaucher

patients and it decreases on therapeutic intervention (Hollak et al., 1994).

Furthermore, in people deficient in chitotriosidase, MU‐TACT hydrolase is

absent. Nonetheless, the reported molecular mass of 17 kDa for partially

purified MU‐TACT hydrolase, as determined by gel filtration, clearly diVersfrom that of chitotriosidase (Overdijk et al., 1994).

After the discovery that chitotriosidase is highly expressed in Gaucher

disease, screening of patients for activity identified several individuals

completely deficient in enzymatic activity (Hollak et al., 1994). Subsequently,

broader studies revealed that a recessively inherited deficiency is commonly

encountered as a result of a 24‐base pair duplication resulting in aberrant

splicing. In fact, the observed carrier frequency of about 35% results in 6%

of individuals being absolutely deficient in chitotriosidase activity. It was

96 BUSSINK ET AL.

established that this duplication results in the formation of a 30 cryptic splice,generating an mRNA with an in‐frame deletion of 87 nucleotides.

2. Three‐Dimensional Structure

Crystal structures of the catalytic domains of several chitinases have been

solved, including that of chitotriosidase. All family 18 chitinases contain a so‐called TIM barrel, consisting of an 8‐fold repeat of a b/a unit, in which the

b sheets are surrounded by a helices, stabilized by hydrogen bonding. The

TIM barrel, first seen in triose‐phosphate isomerase (TIM), is one of the most

versatile folds known to date (Wierenga, 2001), encompassing proteins of

great variety in biological function throughout nature. Furthermore, it is the

single most represented fold in the structural database (Berman et al., 2000).

Besides the catalytic center consensus sequence, there are a number of highly

homologous stretches in the TIM barrel structure of family 18 chitinases.

Although their exact function is unknown, one of these homologous

sequence regions (K‐X‐X‐X‐S/A‐X‐G‐G) is postulated to be important for

stability and catalytic activity (Henrissat, 1990; Terwisscha van Scheltinga

et al., 1996). In family 18 chitinases, the TIM barrel serves as a scaVoldfor aromatic residues involved in substrate binding through stacking

interactions and the residues involved in catalysis.

The crystal structures of the native 39‐kDa human chitotriosidase and

complexes with a chitooligosaccharide and allosamidin have been studied in

detail (Fusetti et al., 2002; Rao et al., 2003). The structure consists of two

domains. The core domain has a (b/a)8 barrel as observed in the other family

18 chitinase structures for hevamine, chitinases A (ChiA) and B (ChiB) from

S. marcescens, and CTS1 from Coccidioides immitis, although helix a1 is

missing (Fusetti et al., 2002). An additional a/b domain, composed of six

antiparallel b strands and one a helix, is inserted in the loop between strand

b7 and helix a7, which gives the active site a groove character. Like all other

family 18 chitinases, the chitotriosidase has the D‐X‐D‐X‐Emotif at the end of

strand b4, with Glu‐140 being the catalytic acid. Two disulfide bridges were

observed between residues 26 and 51 and between residues 307 and 370.

The crystal structures reveal an elongated active site cleft, compatible with

the binding of long chitin polymers. Given the relatively open active site

architecture, chitotriosidase appears to function as an endochitinase rather

than an exochitinase. The complex with N,N0‐diacetylchitobiose (NAG2)

followed by modeling of a longer chitooligosaccharide revealed that the

active site would be able to accommodate longer chitin polymers, which

agrees with its ability to degrade various forms of polymeric chitin. The

crystal structures explain the inactivation of the enzyme through an inherited

genetic deficiency. The common mutation results in a completely inactive

enzyme with residues Val‐344–Gln‐372 missing. These residues correspond


to the C‐terminal half of helix a7, the entire strand b8, and almost the entire

b8–a8 loop. Deletion of these secondary structure elements could lead to

misfolded, and therefore inactive, protein. More importantly perhaps,

Trp‐358, which lies at the end of strand b8, is completely conserved in all

active family 18 chitinases. Inspection of chitinase structures in complex with

chitooligosaccharides shows that this tryptophan serves as an ‘‘anvil’’ onto

which the –1 sugar is pressed, whereas specific hydrogen bonds with other

residues may force the sugar into the boat conformation required for the

attack of the N‐acetyl group on the anomeric carbon. Thus, deletion of Trp‐358 could in itself lead to a completely inactive enzyme. Comparison with

YM1 and HC‐gp39 shows how the chitinase has evolved into these mamma-

lian lectins by the mutation of key residues in the active site, tuning the

substrate‐binding specificity (Renkema et al., 1998).

3. Design of Chitinase Inhibitors

Elucidation of the crystal structure of many chitinases of both human

pathogens, combined with the chitotriosidase structure, has prompted

attempts to design molecules that selectively inhibit chitinases. Although

allosamidin is a potent inhibitor of family 18 chitinases, its chemical synthesis

remains challenging because of the presence of the notorious b‐glycosidicbond, and poor bioavailability might pose a problem for therapeutic use. The

discovery of argifin, a cyclopentapeptide produced by a fungal species that

inhibits chitinases in the low micromolar range (Omura et al., 2000), has been

the basis of further structure‐based inhibitor design (Houston et al., 2002). It

was demonstrated that argifin and analogs inhibit chitotriosidase, as well as

family 18 chitinases from other organisms (Rao et al., 2005a). Methylxan-

thine derivatives, such as caVeine, also have been shown to inhibit chitinases

from various species (Rao et al., 2005b). To date, no chitinase inhibitors have

been tested in humans for antiparasitic eVect. The human enzyme appears to

have significant diVerences in the active site as compared with a chitinase

from the pathogenic fungus C. immitis, which may be exploitable in the

design of allosamidin derivatives or other inhibitors that show diVerentialspecificity toward human and pathogen chitinases.

4. Transglycosylation

Although chitotriosidase had been shown to exhibit high hydrolytic activity

toward 4MU‐chitooligosaccharide substrates, an inhibition of activity occurs

at high substrate concentrations. This remarkable phenomenon was finally

explained by the demonstration that chitotriosidase is not only capable of

catalyzing hydrolysis of the chitooligosaccharide substrate, but can also trans-

glycosylate the substrate. Although chitinases are not able to hydrolyze

4MU‐N‐acetylglucosaminide, it was demonstrated that fluorescent 4MU

98 BUSSINK ET AL.

was formed by recombinant chitotriosidase in the presence of p‐nitrophenyl(PNP)‐chitobioside or chitooligosaccharide, an observation that could be

explained only by the occurrence of transglycosylation (Aguilera et al., 2003).

This led to the design of a novel substrate that cannot be transglycosylated and

is equally well hydrolyzed: 4MU‐(4‐deoxy)‐chitobioside. With this novel sub-

strate, a convenient and more sensitive assay of enzymatic activity of chitinase

became feasible (Aguilera et al., 2003).

Despite the occurrence of transglycosylation, chitotriosidase can release

astonishing amounts of chitotriose from 4MU‐chitotriose. On the basis of the

specific activity toward this substrate (Renkema et al., 1995), each molecule

can catalyze hydrolysis more than 4000 times each second under standard

assay conditions. However, presumably because of a decrease in substrate

availability, activity toward chitin is thought to be considerably less.

Stereospecific transglycosylation as shown by chitotriosidase is a common

feature of glycoside hydrolases (Holtje, 1996). The fact that mammalian

chitinase also shows transglycosylation raises a question concerning whether

the phenomenon has any physiological importance or is a mere catalytic

imperfection. It has, in any case, opened the possibility of utilizing chitinases

for synthetic purposes, catalyzing the formation of the synthetically

challenging b‐glycosidic bond (Ochiai et al., 2004).

5. Acidic Mammalian Chitinase

The high incidence of the chitotriosidase deficiency in humans prompted

questions concerning the redundancy of chitotriosidase or, alternatively,

whether compensatory mechanisms exist. To investigate this matter, rat

and mouse tissue samples were screened for the existence of chitinolytic

activity other than that of chitotriosidase, using the artificial 4MU‐chitooli-gosaccharide substrates. Indeed, in extracts of intestines and stomach, high

levels of activity were measured and subsequent isoelectric focusing showed

chitotriosidase could not be responsible for this activity. Further purification

and protein sequencing of the N terminus allowed subsequent cloning of

both the mouse and human genes, encoding the second mammalian chiti-

nase, named acidic mammalian chitinase (AMCase) (Boot et al., 2001). Both

proteins were found to have chitinolytic activity toward chitin, releasing

mainly soluble chitobiose fragments, and were shown to be sensitive to

allosamidin. Like chitotriosidase, AMCase is synthesized as a 50‐kDa pro-

tein that contains a 39‐kDa catalytic domain, separated from a C‐terminal

chitin‐binding domain by a hinge region. Despite the high sequence similarity

between the human chitinases, AMCase exhibits a distinct pH activity

profile, being most active at acidic pH (Boot et al., 2001).

Although a crystal structure for AMCase is not yet available, several

insights into its acid activity can be obtained solely on the basis of its


sequence alignment with chitotriosidase. The presence of two additional

cysteine residues suggests formation of a third disulfide bond, which would

considerably inhibit unfolding under demanding conditions, such as ex-

tremely low pH. Indeed, when gel electrophoresed under nonreducing con-

ditions, AMCase moves slightly faster than chitotriosidase, providing

evidence of a diVerence in disulfide bonding (Boot et al., 2001). Furthermore,

the acid‐labile bond between aspartic acid and proline (Piszkiewicz et al.,

1970), present in chitotriosidase, is absent in both mouse and human

AMCase, indicating another mode of acid compatibility.

Rudimentary modeling studies of mouse AMCase, using the human

chitotriosidase structure as a template, suggest an acid adaptation by means

of substitutions of acid amino acids on or close to the surface of the molecule.

This may provide the molecule with a surface buVering capacity that, as

illustrated inFig. 5, results in a dramatically altered surface potential compared

with that of chitotriosidase. The catalytic cleft, however, appears unaltered.

D. Chitinases and the Immune System

In this section the contribution of the human chitinases chitotriosidase and

AMCase to immune responses is reviewed. Chitotriosidase has been linked to

innate immune responses against chitin‐containing fungi and AMCase has

been implicated in the pathogenesis of asthma. The homologous enzymati-

cally inactive proteins, the so‐called chitinase‐like lectins (chi‐lectins), such as

human chitinase‐3‐like protein‐1 (CHI3L1/HC‐gp39/YKL‐40; in mouse re-

ferred to as BRP39), human chitinase‐3‐like protein‐2 (CHI3L2/YKL‐39),oviductins, and mouse YM1 and YM2, are only briefly discussed. The chi‐lectins lack enzyme activity because of a mutation of the catalytic glutamate

in the active site of the enzyme. Some of the chi‐lectins such as CHI3L1 have

FIG. 5 Surface electrostatic potential of the 39‐kDa isoforms of human chitotriosidase (left)

and the model of murine AMCase (right). Positive and negative potentials are shown in blue

and red, respectively. Middle: The TIM barrel overall structure of human chitotriosidase is

depicted (the secondary structures are colored in succession), in which the acids involved in

catalysis (D‐X‐D‐X‐E) are shown in red.

100 BUSSINK ET AL.

been shown to bind strongly to chitin by virtue of their hypothetical catalytic

center (Houston et al., 2003; Renkema et al., 1998).

1. Chitinase and Chi‐Lectin Expression in the Immune System

Human chitotriosidase seems to be expressed exclusively by human phago-

cytes, such as macrophages and neutrophils (Escott andAdams, 1995; Hollak

et al., 1994). Monocytes do not express chitotriosidase, but in vitro diVeren-tiation of monocytes to macrophages results in the induction of mRNA and

protein after 4 to 10 days, depending on the donor used. Furthermore, lipid‐laden tissue macrophages express chitotriosidase as has, for instance, been

demonstrated in Gaucher disease and atherosclerotic plaques (Boot et al.,

1999; Boven et al., 2004; Renkema et al., 1995). Chitotriosidase is produced

by bone marrow–derived precursors of neutrophilic granulocytes and stored

in specific granules (van Eijk et al., 2005; and our unpublished observations).

In contrast to the situation in humans, mouse chitotriosidase seems to be

absent in phagocytes, but is expressed predominantly in the gastrointestinal

tract (stomach and tongue), brain, skin, bone marrow, testis, and kidney

(Boot et al., 2005; Zheng et al. 2005). However, diVerences seem to exist

between these diVerent studies with regard to the exact tissue distribution.

Human AMCase is expressed mainly in the stomach and to a lesser extent

in the lung, whereas mouse AMCase is expressed in tongue, stomach, and

alveolar macrophages (Boot et al., 2001, 2005; Suzuki et al., 2002). The fact

that in the mouse AMCase is found in alveolar macrophages whereas chit-

otriosidase is not could imply that the macrophage chitinase in mice is

AMCase instead of chitotriosidase.

The enzymatically inactive chitinase‐like proteins, the chi‐lectins, are also

cell‐type specifically expressed. In humans, HC‐gp39 is, among others,

expressed by articular chondrocytes and synovial cells, and as for human

chitotriosidase, HC‐gp39 expression is also observed in matured macrophages

and specific granules of human neutrophils (Hakala et al., 1993; Rehli et al.,

2003; Renkema et al., 1998; Volck et al., 1998). It has been suggested that it is

involved in tissue remodeling; it has been shown to dampen IL‐1b‐ and TNF‐a‐dependent production of matrix metalloproteinases (MMPs) and IL‐8 by

human skin fibroblasts and is a potent growth factor for these cells (Kirkpatrick

et al., 1997; Ling and Recklies, 2004; Recklies et al., 2002). Human chitinase‐3‐like protein‐2 (CHI3L2)/YKL‐39 expression is not observed in the immune

system, but in chondrocytes, followed by synoviocytes, lung, and heart

(Hu et al., 1996). However, like HC‐gp39 it has been found to serve as

autoantigen in arthritic disease (Sekine et al., 2001; Verheijden et al., 1997).

Oviductins, also known as oviduct‐specific glycoproteins, are a family of

high molecular weight glycoproteins that belong to the secretory class.

Oviductins are produced by nonciliated secretory cells of the mammalian


oviductal epithelium, glycosylated in the Golgi apparatus, and stored in the

secretory granules (Buhi, 2002). Oviductins consists of an N‐terminal domain

that is highly homologous to the members of the chitinase protein family and

a C‐terminal mucin‐like domain that is highly glycosylated (Malette et al.,

1995). Although oviductins are widely believed to be involved in the

process of mammalian fertilization, including spermatozoan function and

gamete interactions, based on experimental results obtained in vitro, their

physiological significance remains controversial. Oviductin gene null mice

have been described that showed no abnormalities in fertility, suggesting

that at least in mice this protein is not essential in the process of in vivo

fertilization (Araki et al., 2003).

Chi‐lectins of particular interest are YM1 and the highly homologous YM2

that to our knowledge have been identified only in mice and rats and seem to

be absent in humans. In mice the expression of the YM1 homolog is high in

lung and spleen and barely detectable in the stomach, whereas the expression

of the YM2 homolog is high in the stomach but barely detectable in lung and

spleen (Chang et al., 2001; Jin et al., 1998;Webb et al., 2001). YM1 expression

appears to be selectively produced and secreted by activated macrophages. It

has been purified after oral infection of mice with the parasite Trichinella

spiralis. Ascaris suum was equally potent in inducing YM1 accumulation in

the peritoneal cavity, whereas peritoneal activation of macrophages with

thioglycolate, Sephadex G‐50, and Cryptosporidium parvum resulted only in

mild induction. It has been suggested that YM1 is involved in controlling

inflammation in the peritoneal cavity as it is transiently induced on infection.

YM1 crystals have been detected in the lungs of immunodeficient mice, such

as the motheaten mouse; CD40 ligand‐deficient mice; p47phox‐deficient mice;

and a transgenic mouse with lung‐specific human tumor necrosis factor

receptor expression (Guo et al., 2000; Harbord et al., 2002), whereas crystals

of YM2 have been detected in the stomach (Ward et al., 2001).

Furthermore, it has been reported that YM1 is chemotactic for eosinophils

and is therefore also referred to as eosinophil chemotactic factor‐L (Falcone

et al., 2001; Owhashi et al., 2000). More detailed analysis of adult male ddY

mice showed that YM1 is present in immature neutrophils (Nio et al., 2004).

In conclusion, chitinases and chi‐lectins are broadly expressed in macro-

phages and neutrophils and seem to contribute to innate immunity and

allergic responses.

2. Induction of Chitotriosidase in Macrophages

In Fig. 6 an overview of the regulation of chitotriosidase expression in

macrophages is depicted. Lysosomal stress is an important inducer of chito-

triosidase in macrophages. By far the highest levels of chitotriosidase are

found in Gaucher disease, but other diseases characterized by lysosomal

FIG. 6 Regulation of chitotriosidase expression in human macrophages. Positive regulators of

the chitinase are depicted in green, and negative regulators in red. aam, alternatively activated

macrophages; cam, classically activated macrophages; iDC, immature dendritic cell; GSL,

glycosphingolipids.

102 BUSSINK ET AL.

accumulation of glycosphingolipids or other lipid species show increased

levels as well, albeit much lower. Examples of this are Niemann‐Pick A/B,

Niemann‐Pick C, Krabbe, GM1 gangliosidosis, cholesteryl ester storage

disease, Wolman disease, Morquio B syndrome, and Tangier disease (Aerts

et al., 2003; Boot et al., 2005; Guo et al., 1995). Elevated levels have also been

found in fucosidosis, galactosialidosis, glycogen storage disease IV, and

Alagille syndrome (Michelakakis et al., 2004). In atherosclerosis, chitotrio-

sidase and the chi‐lectin HC‐gp39 have been detected in lipid‐laden foam

cells (Boot et al., 1999). Moreover, also in b‐thalassemia, increased levels

of chitotriosidase have been found (Barone et al., 1999). Furthermore,

increased levels of chitotriosidase in cerebrospinal fluid are detected in mul-

tiple sclerosis, an autoimmune disease with accumulation of myelin in macro-

phages (Czartoryska et al., 2001). In the systemic granulomatous disorder

sarcoidosis, increased chitotriosidase activity in bronchoalveolar lavage fluid

and plasma has been reported as well (Grosso et al., 2004; Hollak et al. 1994).


Several cytokines and their eVect on chitotriosidase induction have been

studied. M‐CSF, a well‐studied macrophage maturation factor, does not

induce the enzyme when compared with maturation in medium alone. It

has been found that maturation of monocytes toward macrophages in the

presence of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)superinduces the chitinase, whereas both IFN‐g (classically activated) and

IL‐4 (alternatively activated) act as negative regulators. Monocyte‐derivedimmature dendritic cells, matured in the presence of GM‐CSF and IL‐4, failto induce the enzyme (van Eijk et al., 2005). Interestingly, no IL‐4 could be

detected in Gaucher spleen, which correlates well with the high chitotriosi-

dase levels observed in these individuals (Boven et al., 2004). AMCase is also

absent from human alternatively activated macrophages, whereas it is pres-

ent in murine alveolar macrophages, which can upregulate it after helper

T cell type 2 (Th2) stimulation (Boot et al., 2005; Raes et al., 2005; Zhu et al.,

2004). Restimulation of chitotriosidase‐expressing macrophages for a pro-

longed period of time (48 h) with IFN‐g and IL‐4 inhibits expression,

whereas an acute (2‐ to 4‐h) transient induction is observed after stimulation

with prolactin, IFN‐g, TNF‐a, but not with IL‐10 (Di Rosa et al., 2005;

Malaguarnera et al., 2004; van Eijk et al., 2005).

Mimicry of pathogen infection through triggering of Toll‐like receptors

(TLRs) on monocytes prevented induction of the enzyme. For TLR‐2 trigger-ing, peptidoglycan, lipoteichoic acid, zymosan, and the synthetic ligands

macrophage‐activating lipopeptide‐2 (MALP‐2) and Pam3CSK4 have been

used. For TLR‐3, poly(I:C) has been used. Stimulation of TLR‐4 and TLR‐9was accomplished with lipopolysaccharide (LPS) from Salmonella minnesota

andCpG, respectively (vanEijk et al., unpublishedobservations). In our hands,

restimulation of chitinase‐expressing macrophages for 5, 24, and 48 h did not

induce the enzyme, whereas acute induction has been previously found in a

studywithLPS (DiRosa et al., 2005; vanEijk et al., unpublished observations).

This discrepancymay be explained by diVerences in the experimental approach

or source of LPS. The results suggest that immediate activation of NF‐kB in

monocytes may serve as a negative regulator of chitotriosidase expression.

Interestingly, expression of high levels of chitotriosidase by Gaucher cells is

accompanied by a lack of TLR‐2 and ‐4 expression and as such NF‐kB activa-

tion is prevented. In addition, Gaucher cells express high levels of SIRP‐a,which on activation negatively regulates NF‐kB as well (Boven et al., 2004).

The precise signals that modulate chitotriosidase expression in macrophages

are being studied by detailed investigation of its promoter region.

3. Chitinases in Human Neutrophils

Polymorphonuclear neutrophils (PMNs) are important mediators of the first

line of defense against invading pathogens. Antimicrobial molecules are kept

104 BUSSINK ET AL.

inside granules until proper release triggers, such as cytokines or pathogen‐derived signals, are received by PMNs. Several types of granules can be

defined, namely, azurophilic, specific, gelatinase, and secretory granules

(Borregaard and Cowland, 1997). High chitinase activity was reported in

granulocytes shortly after the discovery of human chitotriosidase (Escott and

Adams, 1995). In a proteomic approach to released granule proteins it was

found that this chitinase activity indeed corresponds to chitotriosidase

(Boussac and Garin, 2000). More recently it has been described that chito-

triosidase is present in the specific granules of human neutrophils. In a

diVerential degranulation approach it has been shown that chitotriosidase

is released by a condition that triggers release of specific granules. Immuno-

gold electron microscopy experiments revealed that chitotriosidase and the

specific granule marker lactoferrin colocalize in these cells. This double

staining was not observed with the azurophilic granule marker myeloperox-

idase. In vitro stimulation of PMNs with GM‐CSF resulted in enzyme

release. In addition, it has been found that human subjects, injected with

GM‐CSF, show simultaneous release of chitotriosidase and lactoferrin (van

Eijk et al., 2005). In vivo administration of IL‐12 in chimpanzees resulted in

an increase in chitotriosidase after 24 and 48 h. As this was found in a

relatively short time period it could be caused by neutrophil‐derived activity;

however, the study could not elaborate on either production by macrophages

or release by neutrophils (Lauw et al., 1999). In Fig. 7 a schematic represen-

tation of induction of chitotriosidase release from PMNs is depicted. Patho-

gen infection has also been studied in vitro by stimulation of PMNs with

several TLR triggers. Of all stimuli tested, only peptidoglycan induced

release of the enzyme (up to 20% of total activity). However, it could not

be excluded that contamination of the preparation was responsible for this

release. Activation of several kinases was observed, including protein kinase

B (PKB), p38 mitogen–activated protein (p38MAP) kinase, and extracellular

signal–regulated kinase‐1/2 (ERK1/2). Release could by inhibited by 50% by

either phosphatidylinositol 3‐kinase (PI3 kinase) or p38MAP kinase inhibi-

tion, whereas simultaneous inhibition totally blocked release. ERK1/2 inhibi-

tion did not interfere with release of the chitinase. Mild induction of release,

less than 3% of total activity, was observed after stimulation of TLR‐2, ‐4,and ‐7/8 (van Eijk et al., unpublished observations). Thus, chitotriosidase

release from PMNs can occur as a result of mimicry of a pathogen infection.

4. Human Chitinase Activity in Innate Immunity and

Allergic Responses

a. Innate Immunity Because of the presence of chitin in coatings of several

pathogens, such as fungi and nematodes, it has been suggested that chito-

triosidase serves as a component of innate immune responses (Renkema

FIG. 7 Release of chitotriosidase from specific granules of human PMNs. On pathogen

mimicry or GM‐CSF stimulation chitotriosidase is secreted by human PMNs. Release among

others requires phosphorylation of phosphatidylinositol 3‐kinase (PI3 kinase), protein kinase B

(PKB)/Akt, and p38 mitogen‐activated protein kinase (p38MAPK).


et al., 1995). Clinical evidence pointing toward a role of this chitinase in

such responses can be summarized as follows. Chitotriosidase is raised in

plasma of children suVering from acute infection with Plasmodium falci-

parum malaria (Barone et al., 2003). Increases have also been found in the

sera of individuals suVering from visceral leishmaniasis (Hollak et al., 1994).

Susceptibility to Wuchereria bancrofti, which causes lymphatic filariasis, is

found to be associated with a genetic deficiency in chitotriosidase in southern

India (Choi et al., 2001). However, this correlation does not seem to occur

in Papua New Guinea (Hise et al., 2003). Virtually no heterozygotes and

homozygotes for the chitotriosidase gene defect seem to occur in parasite‐endemic areas such as the sub‐Sahara, suggesting its importance there

(Malaguarnera et al., 2003). Increases have also been reportedduring neonatal

herpesvirus infection (Michelakakis et al., 2004). Last, it has been found that

genetic variants in chitotriosidase are associated with gram‐negative bacter-emia in children undergoing therapy for acute myeloid leukemia (AML)

and that neonates with a bacterial infection show increases in chitotriosidase

106 BUSSINK ET AL.

activity ( Lehr nbecher et al. , 2005 ; Laba daridi s et al. , 2005). Thes e observed

associations suggest that chitotriosidase has more pleiotropic eVects in

innate immunity than previously appreciated. It can be envisioned that

chitotriosidase‐mediated chitin degradation, or degradation of a closely

related structure, results in the exposure of otherwise covered antigens,

which then become visible to the immune system and as such facilitate

recognition by antigen‐presenting cells.

The incidence of Candida sepsis has been reported not to be related to

deficiency in chitotriosidase, but as only survivors were included in the study

the evidence is not conclusive (Masoud et al., 2002). By contrast, strong

evidence in favor of an antifungal activity of chitotriosidase exists. First,

it is of interest to note that chitotriosidase activity has been found to be

elevated in the plasma of neonates with systemic candidiasis and aspergillosis

(Labadaridis et al., 1998, 2005). Second, chitotriosidase was found to inhibit

growth ofCryptococcus neoformans, to cause hyphal tip lysis inMucor rouxii,

and to prevent the occurrence of hyphal switch in C. albicans (van Eijk et al.,

2005). These data strengthen the earlier observed chitinolytic activity toward

cell wall chitin ofC. albicans (Boot et al., 2001). In addition, it has been found

that recombinant human chitotriosidase showed synergy with existing anti-

fungal drugs such as the polyene amphotericin B, the azoles itraconazole and

flucanozole, and cell wall synthesis inhibitors LY‐303366 and nikkomycin Z

(Stevens et al., 2000). Further proof of an important antifungal action has

been found in neutropenic mouse models of systemic candidiasis and sys-

temic aspergillosis, the main causes of mortality in immunocompromised

individuals. Recombinant human chitotriosidase clearly improved survival

in these mouse models (van Eijk et al., 2005). The observations made with

chitotriosidase are not entirely surprising given the well‐documented anti-

fungal role of chitinases in plants (Schlumbaum et al., 1986). It is possible

that recombinant chitotriosidase may be attractive from a clinical pers-

pective to treat life‐threatening fungal infections, especially because Gaucher

patients seem to tolerate 1000‐fold elevated serum levels. In addition,

AMCase also shows chitinolytic activity toward fungal cell wall chitin

(Boot et al., 2001). The discovery of this chitinase in humans has opened

the possibility that a deficiency in chitotriosidase might be partly compen-

sated for by the presence of the latter enzyme. Further studies are required to

elucidate on the potential contribution of AMCase to innate immune

responses.

b. Allergic Responses: Asthma It has been demonstrated that AMCase

is involved in the pathology of an aeroallergen‐induced asthma model,

with a crucial role of Th2 cytokines in the induction of this chitinase.

Interference with AMCase activity in this model, either by addition of

antiserum or addition of a chitinase‐specific inhibitor, alleviates pathology


(Zhu et al., 2004). Further analysis of bronchoalveolar lavage fluid from

these mice by proteomics revealed increased levels of lungkine, the chi‐lectinsYM1 and YM2, gob‐5, surfactant protein‐D, and AMCase (Zhao et al.,

2005). Furthermore, it has been found that increased AMCase mRNA

expression is present in human lung tissue of asthma patients (Zhu et al.,

2004). In addition, a study in human subjects points toward an association

between AMCase polymorphisms and asthma (Bierbaum et al., 2005).

The underlying mechanisms linking AMCase to asthma are still puzzling.

It is known that chitin induces a potent Th1 response and oral administration

of chitin to allergic mice results in a downregulation of serum IgE and lung

eosinophilia, thus complicating a straightforward Th2‐mediated induced

AMCase cause‐and‐eVect relationship (Shibata et al., 1997a,b, 2000, 2001).

One possibility might be that, because of improved hygiene and health care in

industrialized countries, fewer chitin particles are inhaled, resulting in a more

Th2‐prone microenvironment in the lung with increased AMCase and asth-

ma as a result (Holt, 2000). It has also been argued that pathogen infections

are associated with a switch toward a Th2 microenvironment because this

avoids continuation of an otherwise destructive Th1 response (Pearce and

MacDonald, 2002).

Although an appealing thought, inhibition of chitinase activity as a thera-

peutic approach to asthma must be dealt with carefully, as the risk exists that

important antipathogenic activities of chitinases are eliminated as a conse-

quence of total chitinase inhibition. Another complication might be that

chitinase expression diVers between mouse and human. In mice, chitotriosi-

dase is expressed mainly in the stratified squamous epithelium of the gastro-

intestinal tract and in Paneth cells in the crypts of Lieberkuhn. In contrast

to human chitotriosidase, a phagocyte‐specific expression of mouse chito-

triosidase was not detected (Boot et al., 2005). In humans AMCase does not

seem to be expressed by macrophages; the most likely source is epithelial cells

(Boot et al., 2005; Raes et al., 2005; Zhu et al., 2004).

In conclusion, the data on AMCase in asthma and the accumulating data

on defense functions of chitotriosidase toward bacteria, fungi, and nema-

todes, point toward an important role of the family of chitinase proteins in

the human immune system and as such are interesting targets or supplements

for therapeutic interventions.

IV. CCL18

A. CCL18 as Novel Biomarker in Gaucher Disease

In an attempt to identify novel factors that are secreted by Gaucher cells,

plasma samples of patients before and after several years of treatment were

108 BUSSINK ET AL.

analyzed by surface‐enhanced laser desorption/ionization (SELDI) time‐of‐flight (TOF) mass spectrometry (MS). In the plasma of symptomatic Gaucher

patients a peptide of 7856 Da was identified that is virtually absent in the

plasma of the same patients after several years of enzyme supplementation

therapy (Boot et al., 2004). The molecular mass and the isoelectric point of this

peptide were remarkably similar to those of pulmonary and activation‐regulated chemokine (PARC; systematic name, CCL18), of which the mRNA

was found to be upregulated in the spleen of a Gaucher patient (Moran et al.,

2000). Subsequent analyses revealed that this chemokine is produced by

Gaucher cells, that the plasma levels are on average 30‐fold elevated in sympto-

matic Gaucher patients, and that plasma concentrations decrease during

therapeutic intervention (Boot et al., 2004; Deegan et al., 2005). Moreover, it

was shown that plasmaCCL18 and chitotriosidase decreased proportionally in

treated patients, which suggests that both proteins are produced by the same

cells and that lowering the total body burden of Gaucher cells is reflected by

decreased plasma levels of these proteins (Boot et al., 2004;Deegan et al., 2005).

B. Chemokine CCL18

Chemokines are a family of low molecular mass and structurally related

proteins that are divided into C, CC, CXC, and CX3C subfamilies according

to their NH2‐terminal cysteine motifs, and are important players in directing

the migration and activation of leukocytes under normal physiological and

pathophysiological conditions (Rossi and Zlotnik, 2000; Zlotnik and Yoshie,

2000). CCL18 belongs to the subfamily of CC chemokines and was identified

independently by several diVerent groups and hence is known under a number

of diVerent names: pulmonary and activation‐regulated chemokine, PARC;

macrophage inflammatory protein‐4, MIP‐4; dendritic cell chemokine‐1,DC‐CK1; alternative macrophage activation‐associated CC chemokine‐1,AMAC‐1 (Adema et al., 1997; Hieshima et al., 1997; Kodelja et al., 1998;

Wells and Peitsch, 1997, respectively). The gene, which seems to be absent in

rodents, encodes a protein of 89 amino acids that after cleavage of its signal

peptide (20 amino acids) results in a 69‐amino acid mature protein that lacks

N‐linked glycans and has a calculated molecular mass of 7851.2 Da and an

isoelectric point of 9.2 (Adema et al., 1997; Guan et al., 1999; Hieshima et al.,

1997). The protein was shown to be expressed by several diVerent cell typessuch as monocytes/macrophages, alveolar macrophages, dendritic cells, eosin-

ophilic granulocytes, chondrocytes, fibroblasts, keratinocytes, and a number of

tumor cells either constitutively expressed or induced by diVerent cytokines(Schutyser et al., 2005). Therefore it belongs to the inflammatory/inducible

family of chemokines (Mantovani, 1999). However, because of its relatively

high concentration in the circulation of healthy individuals it is assumed that

CCL18 is also a constitutive/homeostatic chemokine (Mantovani, 1999).


C. Potential Role of CCL18 in GaucherDisease Pathophysiology

The potential role of CCL18 in the pathophysiology of Gaucher disease still

remains an enigma and detailed knowledge of the functions of this chemo-

kine may shed some light on this. The in vitro biological response of CCL18

seems to be restricted to a few diVerent cell types. For example, it has been

shown to trigger a response in certain T and B cells as well as dendritic cells,

fibroblasts, and haematopoietic progenitor cells (Schutyser et al., 2005, and

references therein). At physiological concentrations it has been shown to act

as a chemokine for naive resting (CD45RAþ) T lymphocytes, naive (CD38–)

B lymphocytes, helper (CD4þ) T lymphocytes, cytotoxic (CD8þ) T lympho-

cytes, germinal center (CD39–) B lymphocytes, and monocyte‐derived imma-

ture dendritic cells (Adema et al., 1997; Lindhout et al., 2001). As such it is

suggested to participate in the homing of these cells to the secondary lym-

phoid organs and, depending on the situation, could either assist in the

induction of tolerance (homeostatic conditions) or help in the induction of a

primary immune response in the case of inflammatory conditions (Schutyser

et al., 2005). Moreover, it has been suggested that CCL18 could also be

involved in B cell proliferation and plasma cell diVerentiation (Schutyser

et al., 2005). In this respect it is interesting to note the frequently encountered

abnormalities concerning serum immunoglobulins and other manifestations

of disturbed B cell and plasma cell function in Gaucher disease patients. An

increased incidence of B cell or plasma cell malignancies such as multiple

myeloma, leukemia, lymphoma, hypergammaglobulinemia, and systemic

amyloid light‐chain (AL) amyloidosis has been observed in Gaucher disease

patients (Airo et al., 1993; Bertram et al., 2003; de Fost et al., 2005; Fox et al.,

1984; Garfinkel et al., 1982; Kaloterakis et al., 1999; Marti et al., 1988; Pratt

et al., 1968). Although no correlation between CCL18 plasma levels and the

occurrence of a monoclonal gammopathy was observed, it could not be

excluded that the high plasma levels of this chemokine constitute a risk factor

for the development of disturbed B cell function with additional factors such

as time and/or other cytokines influencing the eventual outcome (Boot et al.,

2004). The finding that all symptomatic Dutch Gaucher patients have at least

10‐fold increased plasma levels of CCL18 and not all patients have disturbed

B cell function makes it diYcult to exclude a role for CCL18 in this process

(Boot et al., 2004). Research in this topic is even more hampered by the fact

that rodents seem to lack a CCL18 gene (Schutyser et al., 2005).

Although it appears that CCL18 signals via G protein–coupled receptors,

the identity of the agonistic receptor is currently unknown (Adema et al.,

1997; Lindhout et al., 2001). Interestingly, it has been reported that CCL18 at

physiological concentrations can act as a natural antagonist of the CCR3

110 BUSSINK ET AL.

chemokine receptor present on eosinophils, Th2 cell subsets, basophils, mast

cells, neural tissue, some epithelia, and CD34þ progenitor cells (Lamkhioued

et al., 2003; Nibbs et al., 2000; Olson and Ley, 2002; Wan et al., 2002).

Eosinophil chemotaxis induced by the most potent CCR3 agonists, such as

eotaxin and macrophage chemoattractant protein‐4 (MCP‐4), can be inhib-

ited by CCL18 at concentrations as low as 10 nM (Nibbs et al., 2000; Wan

et al., 2002). The CCL18 plasma levels in symptomatic Gaucher patients

exceed these inhibitory concentrations considerably and it seems likely that

tissues rich in Gaucher cells contain even higher CCL18 concentrations. At

this moment it is not clear whether Gaucher patients show abnormalities in

CCR3‐mediated chemotaxis of eosinophils or other cells. Moreover, it can-

not even be excluded that the high concentrations of CCL18 in plasma and

tissues also block other chemokine receptors, and hence might explain neu-

trophil chemotaxis abnormalities in Gaucher disease (Aker et al., 1993;

Zimran et al., 1993). Although it cannot be excluded that other factors are

involved, the decrease in CCL18 on therapeutic intervention is in agreement

with the observed correction of the chemotaxis defect in Gaucher patients

receiving enzyme replacement therapy (Boot et al., 2004; Zimran et al., 1993).

Several other human diseases have been reported to be accompanied by

elevated levels of CCL18, for example, atherosclerosis, sarcoidosis, active

hepatitis C infection, hypersensitive pneumonitis, allergic contact hypersen-

sitivity, septic as well as rheumatoid arthritis, ovarian carcinoma, gastric

carcinoma, and more recently Whipple disease, Niemann‐Pick type B

disease, and b‐thalassemia (Brinkman et al., 2005; Desnues et al., 2005;

Dimitriou et al., 2005; Goebeler et al., 2001; Kusano et al., 2000; Leung

et al., 2004; Mrazek et al., 2002; Pardo et al., 2001; Reape et al., 1999;

Schutyser et al., 2001, 2002). Various detection methods have been employed

in these previous studies; in some investigations CCL18 mRNA was detected

either by reverse transcription‐polymerase chain reaction (RT‐PCR) analy-

sis, microarrays, or in situ hybridization (Desnues et al., 2005; Goebeler et al.,

2001; Kusano et al., 2000; Mrazek et al., 2002; Pardo et al., 2001; Reape

et al., 1999), whereas in other studies enzyme‐linked immunosorbent assays

(ELISAs) were used to measure CCL18 protein in plasma or in synovial

or ascitic fluid (Boot et al., 2004; Dimitriou et al., 2005; Schutyser et al.,

2001, 2002).

In most cases macrophages seem to be the major source of CCL18 in the

various disorders described above. As in Gaucher disease, these macro-

phages show characteristics of alternatively activated macrophages that

exhibit antiinflammatory properties as opposed to classically activated

macrophages, which show more proinflammatory characteristics (Boven

et al., 2004). Alternatively activated macrophages express a molecular reper-

toire that leads to resolution of inflammation, scavenging of cellular debris,

promotion of angiogenesis, and tissue remodeling and repair. A common


theme in these situations is the removal of cellular debris, senescent cells, or

tumor cells by tissue macrophages that should lead to an increased flux

of particular lipids through these cells and hence to a transient or permanent

(depending on the underlying defect) accumulation of certain lipids in the

lysosomes of these cells. It is tempting to speculate that phagocytosis or the

accumulation of particular lipid species, for example, glycosphingolipids, in

the lysosomes of these cells leads to a form of alternative activation and

hence the expression of CCL18 and chitotriosidase. In this respect it is

interesting to note that in some of the diseases that are accompanied by

elevated expression of CCL18, increased expression of chitotriosidase is also

observed (Boot et al., 1999; Brinkman et al., 2005; Grosso et al., 2004; Hollak

et al., 1994). It is possible that, besides lipid accumulation, other signals such

as cytokines and cell–cell interactions also aid in producing the eventual

phenotypic characteristics of the macrophages in these diseases. It was

demonstrated that the phosphatidylinositol 3‐kinase (PI3 kinase) pathway

is important for the skewing of macrophages in the direction of alternative

activation. It was shown that elevated levels of phosphatidylinositol triphos-

phate (PIP3) predispose macrophage progenitors toward an alternatively

activated phenotype, and that Src homology 2‐containing inositol‐50‐phosphatase (SHIP) acts as a potent negative regulator of this skewing

(Rauh et al., 2005). It can be envisioned that lipid accumulation, either

transient or permanent, results somehow in increased cellular levels of PIP3

either directly by stimulation of PI3 kinase or by inhibition of SHIP and that

this will result in skewing of these cells in the direction of the alternatively

activated phenotype.

The potential role of CCL18 in the pathophysiology of Gaucher disease and

the eVect of lipid accumulation on the type of activation of macrophages

resulting in release of CCL18 and chitotriosidase warrant further investigation

of these matters and are the topic of current research.

V. Concluding Remarks

Gaucher disease has served as an inspiring and productive model for clinical

and fundamental research. It has acted as a testing ground for the develop-

ment of enzyme replacement and substrate reduction therapies, now being

copied for other inherited disorders. Furthermore, it has led to the discovery

of surrogate markers for pathological cells, resulting in laboratory tools that

assist in the clinical management of patients. Finally, it has led to the discov-

ery of mammalian chitinases. This versatile family of endoglycosidases and

related proteins is now intensively studied with emphasis on their potential

role in immune responses.

112 BUSSINK ET AL.

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