The role of cystatins in cells of the immune system
The cystatins constitute a large group of evolutionary related proteins with diverse biological activities. Initially, they were characterized as inhibitors of lysosomal cysteine proteases - cathepsins. Cathepsins are involved in processing and presentation of antigens, as well as several pathological conditions such as inflammation and cancer. Recently, alternative functions of cystatins have been proposed: they also induce tumour necrosis factor and interleukin 10 synthesis and stimulate nitric oxide production. The aim of the present review was the analysis of data on cystatins from NCBI GEO database and the literature, and obtained in microarray and serial analysis of gene expression (SAGE) experiments. The expression of cystatins A, B, C, and F in macrophages, dendritic cells and natural killer cells of the immune system, during differentiation and activation is discussed.
The role of cystatins in cells of the immune system
Department of Biochemistry and Molecular Biology, Joz
ef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia
Received 16 August 2006; revised 22 October 2006; accepted 24 October 2006
Available online 3 November 2006
Edited by Masayuki Miyasaka
Abstract The cystatins constitute a large group of evolutionary
related proteins with diverse biological activities. Initially, they
were characterized as inhibitors of lysosomal cysteine proteases
– cathepsins. Cathepsins are involved in processing and presenta-
tion of antigens, as well as several pathological conditions such
as inﬂammation and cancer. Recently, alternative functions of
cystatins have been proposed: they also induce tumour necrosis
factor and interleukin 10 synthesis and stimulate nitric oxide
production. The aim of the present review was the analysis of
data on cystatins from NCBI GEO database and the literature,
and obtained in microarray and serial analysis of gene expres-
sion (SAGE) experiments. The expression of cystatins A, B,
C, and F in macrophages, dendritic cells and natural killer cells
of the immune system, during diﬀerentiation and activation is
2006 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
Keywords: Cystatin; Cathepsin; Macrophages; Dendritic cells;
Natural killer cells
Cysteine cathepsins are long known to be responsible for
protein degradation in lysosomes. Recent studies show that
they are also involved in a number of other important cellular
processes such as antigen presentation , apoptosis, protein
processing , as well as several pathologies such as cancer
progression , inﬂammation  and neurodegeneration .
The role of lysosomal cathepsins in antigen presentation has
been reviewed recently . Proteinase activity in these pro-
cesses is highly regulated at the level of protease expression,
by regulation of zymogen activation and by expression of
endogenous inhibitors. Natural inhibitors that inhibit cysteine
cathepsins include cystatins, thyrophins and also some of the
The cystatins constitute a large group of evolutionary
related proteins acting as protease inhibitors of papain-like
cysteine proteases belonging to enzyme family C1 (see the
MEROPS database at http://merops.sanger.ac.uk), such as
cathepsins B, H, L, and S and legumain-related proteases of
the family C13 . Type 1 cystatins, steﬁns (A and B), are
polypeptides of 98 amino acid residues which possess neither
disulﬁde bonds nor carbohydrate side chains and are located
mainly intracellularly. Type 2 cystatins C, D, E/M, F, S, SN,
and SA are characterized by two conserved disulﬁde bridges,
a larger size (120 residues) and the presence of a signal
peptide for extracellular targeting . Type 3 cystatins, the
kininogens, are large (60–120 kDa) multifunctional plasma
proteins, containing three type 2 cystatin-like domains con-
taining a total of eight disulﬁde bridges. Although types 1
and 2 cystatins display considerable diﬀerences in amino acid
sequence, their tertiary structures are conserved and exhibit a
‘cystatin fold’ that is formed by ﬁve stranded anti-parallel b-
pleated sheet wrapped around a ﬁve-turn a-helix [12,13]. The
structure of human cystatin C in its dimeric form also shows
that each one of the two domains in the dimer adopt the typ-
ical monomeric ‘cystatin fold’ . Some type 2 cystatins (C,
E/M, and F) are also able to inhibit mammalian legumain,
an asparaginyl endopeptidase (AEP), using a binding site dis-
tinct from the family C1 interaction site . AEP has been
shown to be involved in class II major histocompatibility com-
plex (MHC) restricted antigen presentation  .
The present review focuses mostly on the expression of two
type 1 cystatins: steﬁns (cystatins) A and B and two type 2 cyst-
atins, cystatins C and F, in cells of the immune system upon
diﬀerentiation and activation. Two recently developed technol-
ogies, oligonucleotide or cDNA microarrays and serial analy-
sis of gene expression (SAGE), allow the determination of the
expression patterns of thousands of genes simultaneously
[17,18]. The gene expression omnibus (GEO) at the National
Center for Biotechnology Information (NCBI) is a large com-
pendium of gene expression data, addressing a wide range of
biological issues across many organisms  . The aim of the
present review is the identiﬁcation of some of the most interest-
ing questions regarding cystatins in cells of the immune system
on the basis of recent data collected in the NCBI GEO and the
2. Steﬁn A (cystatin A) in follicular dendritic cells (FDC)
Steﬁn A (cystatin A) has been isolated from epidermis, poly-
morphonuclear granulocytes, liver, and spleen [20–23]. SAGE
Abbreviations: BCR, B cell receptor; DC, dendritic cells; FDC, follic-
ular dendritic cells; GC, germinal centres; SAGE, serial analysis of
gene expression; MHC, major histocompatibility complex; PKC,
protein kinase C; TNF-a, tumour necrosis factor alpha; IL-4, inter-
leukin-4; IL-10, interleukin-10; IFN-c, interferon-gamma; NK, natural
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FEBS Letters 580 (2006) 6295–6301
studies showed that LPS stimulation decreased its synthesis in
monocytes . Steﬁn A was also found in the follicular den-
dritic cells (FDC) in germinal centres (GC) of human tonsils
. In contrast to antigen-presenting cells that present anti-
gens to T cells, FDC do not internalize, process and present
antigens in the context of major histocompatibility complex
class II (MHC II), but present intact antigen on their cell
surface [26,27]. GC form in lymphoid follicles of secondary
lymphoid organs and provide an essential microenvironment
for T cell-dependent humoral immune responses [28,29].
Within GC, antigen-speciﬁc B cells eﬃciently undergo clonal
expansion, isotype switching, somatic mutation, and aﬃnity
maturation leading to the generation of plasma and memory
cells [30–32]. Only B-cells with the highest aﬃnity B cell recep-
tor (BCR) bind to intact antigens on the surface of FDC and
receive survival signals from the FDC whereas low aﬃnity
BCR B cells and self-reactive B cell clones are eliminated by
apoptosis . Apoptosis in GC B-cells is mainly induced via
the death receptor pathway, by the rapid activation of cas-
pase-8 at the level of CD95 death-inducing signalling complex
(DISC) [33–35]. GC B cell apoptosis is dependent not only on
caspases but also endonucleases  and cathepsins . Re-
cently, van Nierop et al. showed that apoptosis in human
GC B-cells involved lysosomal destabilization, which was con-
trolled by caspase-8 activity. CD40 ligation provided resistance
to lysosomal destabilization, as well as binding of high-aﬃnity
B cells to FDC prevented lysosomal leakage and apoptosis in
GC B-cells . Van Nierop et al. speculated that besides cas-
pase-8 inhibition there was an additional mechanism to pre-
vent lysosomal instability in adhering GC B-lymphocytes:
steﬁn A, which is expressed at high levels in FDC may play
a role in the prevention of apoptosis, as previously proposed
by van Eijk et al. [37,39].
3. Cystatins B and C in dendritic cells and macrophages
Cystatin C is the most potent inhibitor of cysteine proteases
such as cathepsins B, H, L, and S, with apparent inhibition
constants even below the nanomolar range . Mature cysta-
tin C is synthesized as a preprotein with a 26 residue signal
peptide. Cystatin C is ubiquitously expressed in all tissues
and cell types, although mRNA levels vary several-fold be-
tween the tissues [41,42]. Oligomerization of cystatin C leads
to amyloid deposits in brain arteries at advanced age but this
pathological process is greatly accelerated in the mutant form
of cystatin C, responsible for hereditary cystatin C amyloid
angiopathy (HCCAA) . Extracellular monomeric cystatin
C was found to be internalized by Chinese hamster ovary
(CHO) cells and traﬃcked into lysosomes where it dimerized
. Cystatin C-deﬁcient mice have essentially a normal phe-
notype. Cystatin C-deﬁcient mice showed signiﬁcantly reduced
growth of melanoma lung metastases when compared to wild-
type mice . The reason for reduced growth of melanoma
lung metastases in cystatin C-deﬁcient mice is unknown, but
could be a consequence of an early proteolytic event by a cys-
teine proteinase during the ﬁrst hours after administration of
melanoma cells . Cystatin C-deﬁcient mice also showed
that cystatin C has a protective role in atherogenesis since cyst-
atin C-deﬁciency promotes atherosclerosis . It was shown
that a glycosylated form of cystatin C is a necessary cofactor
for ﬁbroblast growth factor 2 (FGF-2) induced mitogenic
activity on neural stem cells . Cystatin C N-glycosylation
was necessary to induce neural stem cell proliferation. The pro-
tease inhibitory domain of cystatin C was not directly involved
in the process . The unexpected consequences of cystatin
C deﬁciency on the spread of metastasis and atherosclerosis
could also be a consequence of alternative functions of cystatin
C, possibly as growth factor cofactor.
Dendritic cells (DC) are the professional antigen presenting
cells of the immune system. They are deﬁned functionally by
their ability to take up antigens such as microorganisms, pro-
cess them into short antigenic peptides, load the peptides onto
major histocompatibility complex (MHC) molecules and then
present the resulting complexes at the cell surface. Immature
DC are located in the periphery of the body and they take
up and process antigens. Activated DC lose their capacity to
capture and process antigens. Instead they migrate to the sec-
ondary lymphoid organs and present antigen to T cells
Self peptides derived from secretory membrane proteins that
are synthesized by the antigen-presenting cells themselves bind
to MHC class II molecules tightly, but normally do not acti-
vate T cells. Cystatin C peptide (amino acids 40–55) has been
found as one of such self peptides bound to MHC class II mol-
ecules, indicating that it is endocytosed and cleaved with the
antigenic material and then bind to MHC class II molecules
. Hashimoto et al.  observed that upon DC maturation
cystatin C transcripts were signiﬁcantly downregulated (http://
bloodsage.gi.k.u-tokyo.ac.jp/). The SAGE results of Hashim-
oto et al. were conﬁrmed at the protein level by Zavasnik-Ber-
gant et al. who observed a large increase in intracellular
cystatin C during the diﬀerentiation of monocytes to immature
DC , Upon DC maturation, intracellular cystatin C levels
decreased and following prolonged incubation of mature DC
in the presence of TNF-a, cystatin C was secreted from DC.
It has been proposed that cystatin C plays a pivotal role in
the control of cleavage and removal of the MHC class II
invariant chain (Ii) by regulating the activity of cathepsin S,
and hence in the formation of MHC class II-peptide complexes
. The work of El Sukkari et al. on DC isolated from cyst-
atin C-deﬁcient mice showed that cystatin C is neither neces-
sary nor suﬃcient to control MHC class II expression and
antigen presentation in DC and that its expression diﬀers be-
tween diﬀerent DC subsets . The absence of cystatin C
did not aﬀect the expression, subcellular distribution, or for-
mation of peptide-loaded MHC class II complexes in any of
the DC types, nor the eﬃciency of presentation of exogenous
antigens . Recent work by Kitamura et al. showed that
interleukin-6 (IL-6)-mediated signalling increased cathepsin S
activity, signiﬁcantly reduced cystatin C expression and re-
duced the H2-DM and MHC class II ab dimer levels in DC
. Overexpression of cystatin C in DC on the other hand sig-
niﬁcantly suppressed IL-6-mediated enhancement of cathepsin
S activity and reduction of MHC class II ab dimer, Ii, and H2-
DM levels in DC. The authors concluded that the IL-6-medi-
ated alteration of the balance between cystatin C and cathepsin
S levels is important for the status of MHC class II ab dimer,
Ii, and H2-DM levels in DC. At least in the system described,
cystatin C may regulate cathepsin S activity in immature and
mature DC . Murine spleen contains three major endo-
genous populations of DC. They are referred to as the CD8
, and CD8
subsets [55,56]. CD8
DC are distinct from CD8
DC on the basis of a number of
criteria and primarily direct a Th2 response by activating T
6296 N. Kopitar-Jerala / FEBS Letters 580 (2006) 6295–6301
cells to secrete cytokines such as interleukin-4 (IL-4) [57,58].
DC produce IL-12 upon stimulation and induce a Th1
response . Aﬀymetrix microarray gene analysis was used
to determine gene expression patterns among murine DC sub-
cells were analyzed directly
after sorting and after 2 h cultivation (NCBI GEO GDS352).
Cystatin C was upregulated in cultured CD8
GEO GMS4772, GMS4773), an observation that is in agree-
ment with the study of El-Sukkari et al. . DC also have
the capacity to take up, process and present exogenous anti-
gens in association with MHC class I molecules and this path-
way is termed cross-presentation [60–62]. CD8
DC have been
shown to be the principal DC subset involved in priming MHC
class I-restricted cytotoxic T cell immunity . Since cystatin
C is expressed predominately in CD8
DCs, it is possible that it
has a role in this process.
Steﬁn B (cystatin B) is a type 1 cystatin that is distributed
rather uniformly among diﬀerent tissues. In vitro steﬁn B binds
tightly to cathepsins H, L, and S, and less tightly to cathepsin
B . Mutations in the gene encoding steﬁn B are responsible
for the primary defect in Unverricht-Lundborg disease
(EPM1) [64–66]. Steﬁn B-deﬁcient mice display a phenotype
that is similar to the human disease with progressive ataxia
and myoclonic seizures . The mice exhibit apoptosis of cer-
ebellar granullar cells and show increased expression of apop-
tosis and glial activation genes . It was shown that removal
of cathepsin B from cystatin B-deﬁcient mice greatly reduced
neuronal apoptosis, but did not rescue animals from ataxia
and seizure . Thymocytes from steﬁn B-deﬁcient mice ex-
erted a markedly increased response when they were exposed
to staurosporin, a protein kinase C (PKC) inhibitor compared
to thymocytes from wild-type mice . We tested the possibil-
ity that steﬁn B interacts with the receptor for activated PKC
(RACK-1) in thymocytes and in this way interferes with PKC
signaling in the cells, but the interaction of RACK-1 with ste-
ﬁn B was not conﬁrmed. Preincubation of cells with E-64d did
not prevent apoptosis, indicating that staurosporin induces
apoptosis in a cathepsin-independent and caspase-dependent
manner. Brannvallet et al. reported that steﬁn B is localized
mainly in the nucleus of neural steam cells and in neurons,
while in glia cells it is also in the cytoplasm and in the lyso-
somes . Hashimoto et al. showed that gene transcripts
of steﬁn B were signiﬁcantly increased upon diﬀerentiation of
monocytes into macrophages . However, upregulation of
the expression of the inhibitor upon diﬀerentiation of macro-
phages does not result in co-localization or interaction with
cathepsins L, S or B (Kopitar-Jerala, unpublished observa-
tions). The SAGE studies showed that treatment with LPS
causes upregulation of steﬁn B expression in human mono-
cytes, whereas cystatin C is not aﬀected, indicating a possible
role of steﬁn B in innate immune response to bacterial infec-
Activated macrophages acquire antimicrobial activities
involving reactive oxygen species and reactive nitrogen metab-
olites. Chicken cystatin, cystatin C, and steﬁn B have been
implicated in nitric oxide (NO) production by interferon-c-
activated mouse peritoneal macrophages . Mouse perito-
neal macrophages activated with interferon-gamma (IFN-c)
and then stimulated with IFN-c plus chicken cystatin gener-
ated increased amounts of NO in comparison with macro-
phages only activated with IFN-c . The biological eﬀect
of cystatins as NO synergistic inducers is not related to inhibi-
tion of a cysteine proteinase activity since E-64 did not induce
any increase in NO. Increased NO was due to increased induc-
ible NO synthase protein synthesis. Further studies of Verdot
et al. suggested that chicken cystatin stimulated the release of
TNF-a and interleukin-10 (IL-10) by IFN- c-activated murine
peritoneal macrophages . This observation could be of bio-
logical importance as cystatin concentrations necessary to
upregulate TNF-a, IL-10 and NO synthesis are in the physio-
logical range, as found in human body ﬂuids. The cystatin C-
mediated release of TNF-a is probably responsible for the
increase in NO production by IFN-c-activated murine perito-
neal macrophages. The ﬁndings by Verdot et al.  point at
a new relationship between cystatins, cytokines, inﬂammation
and immune responses.
In vitro experiments in cell culture models described above
were conﬁrmed by experiments in Leishmania donovani in-
fected mice . L. donovani, the etiological agent for the
severe visceral form of leishmaniasis, multiplies in the phago-
lysosomes of macrophages of the infected host. Treatment of
L. donovani-infected murine peritoneal macrophages with a
combination of chicken cystatin and IFN-c induced increased
production of NO and did overcome the inhibition of NO
synthesis driven by L. donovani parasites. Mice treated with
chicken cystatin and IFN-c showed reduced splenomegaly, a
lowered parasite burden in the spleen and increased produc-
tion of NO . The infected mice treated with chicken cysta-
tin and IFN-c were cured by the induction of NO that killed
the parasite and the switched CD4
T cell-mediated immune
responses from disease-promoting Th2 cells to the protective
Th1 response shown by the increased production of IL-12
and decreased production of IL-4.
4. Cystatin F in NK cells
Cystatin F is expressed in a variety of tissues. Expression is
particularly high in the cells and tissues of the immune system:
thymus and spleen, monocytes, DC, T-cells and NK cells .
Mature cystatin F is composed of 126 amino acid residues. It is
synthesized as a preprotein with a 19 residue signal peptide and
possesses a unique extension of six amino acids at its N-termi-
nus. In addition to the two disulﬁde bridges common to all
type 2 cystatins, mature cystatin F has two cysteine residues
that, form an interinolecular disulﬁde bridge, as revealed in
the crystal structure of the cystatin F dimer . Cappello
et al. observred that in U937 cells cystatin F was secreted as
a disulﬁde bridge-linked dimer . Cystatin F dimer is inac-
tive as an inhibitor of papain like cathepsins and can be
activated by chemical reduction . As compared to other
cystatins, the protein exhibits a distinct inhibitory proﬁle. It
binds tightly to cathepsins F, K, L, and V, less tightly to
cathepsins S and H, and does not inhibit cathepsins B, C,
and X . Cystatin F can inhibit AEP, but with lower aﬃnity
as cystatin E/M and C . The recently elucidated crystal
structure of cystatin F revealed that two N-linked glycosyla-
tion sites of cystatin F may modulate its inhibitory properties,
in particular its reduced aﬃnity towards AEP as compared to
other cystatins .
Cystatin F has also been shown to be strongly upregulated
in LPS-stimulated monocyte-derived DC . In the U937
premyeloid cell line, both diﬀerentiation towards a granulo-
cytic pathway by all-trans-retinoic acid (ATRA) or towards
N. Kopitar-Jerala / FEBS Letters 580 (2006) 6295–6301 6297
a monocytic pathway by stimulation with phorbol ester (TPA)
resulted in marked downregulation of cystatin F expression
In U937 cells, cystatin F has been found to be secreted and
localized intracellularly in lysosome-like granules . Cap-
pello et al. found it in lysosomes in transfected HeLa cells,
but not in U937 cells. Sorting of cystatin F to lysosomes was
greatly enhanced when its C-terminal end was extended by
few amino acids . The authors concluded that under partic-
ular conditions, cystatin F can be sorted to the endocytic path-
way, but its unusual inhibitory function is mainly performed
extracellularly and probably controlled through dimerization
. Langerholc et al. showed that in U937 cells, cystatin F
was co-localized with lysosomal markers LAMP-2 and CD68
and when subcellular localization of cystatin F was compared
to that of cathepsins, cystatin F was found to be co-localized
with cathepsins X and H, but not with cathepsins L, B, C
. Further investigations on cystatin F localization, possibly
in other cells and in cystatin F-deﬁcient mice, will be necessary
to elucidate its biological function (see Fig. 1).
Gene expression analysis of human NK cells and CD8
lymphocytes revealed that transcripts of cystatin F were sign-
iﬁcantly upregulated in NK cells when compared to CD8
lymphocytes (http://bloodsage.gi.k.u-tokvo.ac.ip/) .
NK cells are innate immune lymphocytes that mediate two
major functions: recognition and lysis of cancer cells and
virus-infected cells and production of immunoregulatory cyto-
kines [83,84]. The activation of NK cells is controlled by com-
plex interactions between activating and inhibitory receptor
signals and can be modulated by cytokines . Human NK
cells comprise approximately 15% of peripheral blood lympho-
cytes and the majority of human NK cells are CD56
whereas a minority are CD56
function of CD56
NK cells is diﬀerent from that of
NK cells. CD56
NK cells can produce cytokines
more abundantly, consistent with their functional role as an in-
nate immunoregulator . In contrast, CD56
cells seem to be skewed toward homing to inﬂammation sites
and promoting immune responses, in addition to induction
of cytotoxicity .
Hanna and coworkers reported that cystatin F tran-
scripts were more abundant in CD56
NK cells and
in vitro activated CD56
NK cells than in
NK cells  (NCBI GEO GSM26200-5).
Although the microarray data do not give us any information
about inhibitory activity of cystatin F and have to be inter-
preted with caution, it is tempting to speculate that cystatin
F plays a speciﬁc role in the function of NK (CD56
Several structural and kinetic studies have given us insight
into interactions of cystatins and cysteine cathepsins in vitro
but in vivo very few interactions have been found. The present
review aims to identify the most interesting questions rather
than providing the deﬁnitive answers. The central question
remains what the targets of cystatins are that are diﬀerentially
regulated in cells of the immune system. Is it possible that cyst-
atins have diﬀerent roles in diﬀerent tissues like serpins? For
example, tPA is not just a ‘plasminogen activator’; it is now
Fig. 1. Steﬁn A (cystatin A) is expressed at high levels in FDC and may play a role in the prevention of apoptosis in GC B cells as proposed by van
Eijk et al. [37,39]. Several signalling molecules are involved in FDC-GC B cell contacts: intercellular adhesion molecule-1 (ICAM-1) and vascular cell
adhesion molecule-1 (VCAM-1) enhance cell-cell contact; B-cell-activating factor of the tumour necrosis factor family (BAFF/BLys) prevents
apoptosis of GC B cells and interleukin-15 (IL-15) stimulate GC B-cell proliferation .
6298 N. Kopitar-Jerala / FEBS Letters 580 (2006) 6295–6301
widely appreciated for its role in the central nervous system
[88,89]. Although it can act on its classical substrate, plasmin-
ogen, it also associates with other targets, and in some cases
can even act like a cytokine to activate microglial cells without
engaging its catalytic properties .
The real challenge that lies in front of us is to discover pro-
teinases (and possibly some other proteins) which interact with
cystatins that are diﬀerentially upregulated in cells of the im-
Acknowledgements: This work was supported by the Ministry of High
Education, Science and Technology of the Republic of Slovenia. Prof.
R.H. Pain is gratefully acknowledged for critically reading the manu-
script, giving useful comments and editing English. I also thank Prof.
B. Turk and Prof. V. Turk for reading the manuscript and giving use-
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