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  
J. Pathol. 189: 138–143 (1999)
THE DETECTION OF MAST CELL SUBPOPULATIONS
IN FORMALIN-FIXED HUMAN TISSUES USING A NEW
MONOCLONAL ANTIBODY SPECIFIC FOR CHYMASE
 . ,  .    . *
Immunopharmacology Group, University of Southampton, Southampton General Hospital, Southampton, U.K.
SUMMARY
Chymase is an important marker for human mast cells as well as a mediator of inflammation and matrix remodelling, but research
into chymase-containing mast cell subpopulations has been hampered by the lack of reagents suitable for use with formalin-fixed tissue.
A monoclonal antibody to chymase (designated CC1) was prepared by immunizing a mouse with chymase purified from human skin,
fusing the splenocytes with NS-1 myeloma cells, and screening the hybridoma supernatants by ELISA with recombinant human
prochymase isolated from a baculovirus expression system. This antibody bound to chymase in western blots and bound selectively to
cells with the morphology and distribution of mast cells in paraffin wax sections of skin, synovium, lung, and heart. In sequential sections
and with double-labelling experiments, chymase was localized to cells which contained mast cell tryptase; in contrast to previous reports,
no evidence was found for its presence in endothelial cells or any other cell type. The antibody permitted chymase-containing mast cells
to be detected in formalin-fixed tissues, and the numbers identified were similar to those in tissues fixed with Carnoy’s or ethanol
fixatives. Immunocytochemistry with antibody CC1 provides for the first time a sensitive and specific means for the detection of chymase
in routinely fixed tissues and should prove valuable in studying mast cell subsets in disease. Copyright 1999 John Wiley & Sons, Ltd.
KEY WORDS—chymase;
formalin; mast cell; monoclonal antibody; tryptase; immunocytochemistry
INTRODUCTION
Mast cells are present in almost all human tissues and
have been implicated in the control of blood flow,
angiogenesis, inflammation and fibrosis.1 Although frequently regarded as a single cell type, mast cells represent a highly heterogeneous population. Subpopulations
may differ in their responsiveness to various secretagogues,2,3 their susceptibility to pharmacological control
by anti-allergic drugs,2 and the extent to which they may
be stained using basic dyes following formalin fixation
of tissues.4,5 Identification of the major granule proteases, tryptase and chymase, has allowed biochemical
heterogeneity to be studied and using specific antibodies,
two distinct subpopulations have been distinguished.
Mast cells may contain both tryptase and chymase in
their secretory granules (MCTC cells) or tryptase but not
chymase (MCT cells).6
The MCTC population normally predominates at connective tissue sites and is most abundant in the tissues of
the skin,7 heart,8 gastrointestinal submucosa, and respiratory submucosa,7 while MCT cells are most numerous
in mucosal tissues.7,9 A selective deficiency in mast cells
of the MCT type has been noted in the gastrointestinal
mucosa of patients with AIDS,10 prompting suggestions
that the growth and survival of the MCT subset may
depend on intact T-lymphocyte function. Expansion of
*Correspondence to: Dr A. F. Walls, Immunopharmacology
Group, Level F, Mailpoint 837, Southampton General Hospital,
Tremona Road, Southampton SO16 6YD, U.K.
E-mail: [email protected]
Contract/grant sponsor: Action Research.
Contract/grant sponsor: Ministry of Agriculture, Fisheries and
Food.
Contract/grant sponsor: Arthritis Research Campaign.
CCC 0022–3417/99/100138–06$17.50
Copyright 1999 John Wiley & Sons, Ltd.
the MCT cell subpopulation has been associated with
inflammatory changes in seasonal allergic rhinitis,11
atopic dermatitis,12 vernal conjunctivitis,13 scleroderma,14 and rheumatoid arthritis,15 but has also
been noted in osteoarthritis,16,17 a condition in which
inflammation is not a prominent feature.
Chymase, one of the major secretory products of
MCTC cells,18 could alter cytokine bioavailability by
activating the interleukin-1â (IL-1â) precursor,19
degrading IL-4,20 and liberating membrane-bound stem
cell factor;21 could participate in matrix remodelling by
activating procollagenase;22 and could control blood
flow by generating angiotensin II.23 Furthermore, in
animal models, chymase increases microvascular
permeability24 and promotes the accumulation of
inflammatory cells.25
Despite these key mediator actions, the study of
chymase and its tissue distribution has been hindered by
a lack of suitable investigational tools. Antibodies
specific for chymase which are at present available
have proved unsuitable for use in western blotting or
in immunocytochemistry with routinely processed,
formalin-fixed tissues;6,7 and employing some reagents,
there has been uncertainty even as to the cellular sources
of chymase.26,27 We report here on the preparation and
characterization of a new monoclonal antibody which
overcomes these major disadvantages.
MATERIALS AND METHODS
Chymase and prochymase preparations
Chymase was purified from human skin extracts by
heparin agarose and gel filtration chromatography as
Received 27 July 1998
Revised 5 January 1999
Accepted 20 April 1999
139
A CHYMASE-SPECIFIC ANTIBODY FOR FORMALIN-FIXED TISSUES
described previously,28 except that an intermediate
step with p-aminobenzamidine agarose (Affinity
Chromatography, Ballasala, Isle of Man, U.K.) affinity
chromatography was incorporated to remove contaminating tryptase. Recombinant human prochymase (rhprochymase) was produced in a baculovirus expression
system and was purified as described previously.29
U.K.). Western blots were blocked with 3 per cent
gelatine, probed with test monoclonal antibody, or, for
comparison, rabbit antiserum specific for chymase,28
followed by treatment with the appropriate biotinylated
secondary antibody (Sigma) and Extravidin-alkaline
phosphatase conjugate (Sigma). Staining was developed
with 5-bromo-4-chloro-3-indolyl phosphate and nitro
blue tetrazolium (Sigma).
Preparation of monoclonal antibodies
Splenocytes from a BALB/c mouse immunized with
purified skin chymase were fused with myeloma cells of
the P3/NS1/1.Ag4.1 line (NS-1 cells) at a ratio of 2:1
using a standard somatic fusion protocol. Hybridoma
cells were screened by ELISA (see below) and a colony
secreting antibody reacting with rh-prochymase was
subjected to three rounds of subcloning. The antibody
isotype was determined using a commercially available
kit (Sigma, Poole, Dorset, U.K.). Immunoglobulins
were precipitated from cell supernatants with 45 per cent
saturated ammonium sulphate and purified on a
protein G-Sepharose column (Pharmacia, Uppsala,
Sweden). Antibody was eluted with 0·1  glycine (pH
2·7), neutralized with 2  Tris–HCl (pH 8·0), and
dialysed overnight against phosphate-buffered saline
(PBS) at 4C.
ELISA
Microtitre plates (Nunc Maxisorp, Life Technologies,
Paisley, U.K.) were coated with 20 ng/ml rh-prochymase
in 50 m sodium carbonate, pH 9·5, for 16 h at 4C and
blocked with 3 per cent BSA (Life Technologies). Hybridoma supernatant was applied for 100 min at 37C
and the plates were developed with biotinylated goat
antibodies to mouse immunoglobulins (Sigma),
Extravidin-horseradish peroxidase conjugate (Sigma),
and o-phenylene diamine (OPD). The reaction was
stopped with 3  H2SO4 and the plates were read at
490 nm. The potential for heparin to influence antibody
binding was examined by addition of porcine intestinal
heparin (molecular mass 13–15 kD; Calbiochem,
Nottingham, U.K.) to the coating rh-prochymase.
Possible cross-reactivity of the antibodies with tryptase
was investigated using plates coated with human lung
tryptase prepared as described previously.30
Assay of enzymatic activity
To determine the effect of antibody binding on catalytic activity, immunoglobulin was added to chymase in
ratios of 2:1 and 10:1 on a weight basis and enzymatic
activity was monitored spectrophotometrically at
410 nm using as substrate 0·7 m N-succinyl-Ala-AlaPro-Phe-p-nitroanilide (Sigma) in 1·5  NaCl, 0·79 per
cent dimethyl sulphoxide, and 0·3  Tris–HCl, pH 8·0 as
described previously.28
Electrophoresis and western blotting
Chymase was subjected to SDS-PAGE on 10 per cent
gels and silver-stained (Bio-Rad, Hemel Hempstead,
Copyright 1999 John Wiley & Sons, Ltd.
Immunocytochemistry
Specimens of foreskin (from boys at circumcision),
synovium [from the knees of patients with rheumatoid
arthritis (synovium 1), osteoarthritis (synovium 2 and 3)
or traumatic knee injury (synovium 4) undergoing synovectomy], macroscopically normal lung (removed at
bronchial resection from patients with carcinoma), and
the left ventricle of the heart (removed at autopsy,
approximately 1 day post-mortem) were fixed in neutral
buffered formalin, Carnoy’s fixative or 85 per cent
ethanol, dehydrated, and embedded in paraffin wax.
Sections (6 ìm) were dewaxed, rehydrated, and incubated for 10 min with 0·5 per cent H2O2 in methanol in
order to inhibit endogenous peroxidase activity. PBS
containing 1 per cent BSA was added for 1 h and the
same solution was employed as the diluent for the
antibodies added subsequently. Sequential sections of
skin, synovium or lung were incubated for 2 h with (1)
anti-tryptase monoclonal antibody AA1 (a 1/50 dilution
of culture supernatant), prepared as described previously;31 (2) the new monoclonal anti-chymase antibody (initially at a range of concentrations, before
selecting a standard concentration of 1 ìg/ml); or (3) an
IgG1 antibody to Aspergillus niger glucose oxidase
(clone X931; Dako, Glostrup, Denmark), which served
as a negative control.
Sections were washed between steps as described
previously.17 Biotinylated goat antibodies to mouse
immunoglobulins were applied for 1 h and Extravidinhorseradish peroxidase conjugate for 20 min. Staining
was developed over 4 min using 0·4 mg/ml 3-amino-9ethylcarbazole (Sigma) in 0·05  sodium acetate, pH 5·0,
containing 0·005 per cent H2O2. Sections were counterstained with Mayer’s haemalum. Double labelling with
antibodies to tryptase and chymase was performed as
described previously,17 but with the new antibody to
chymase. For each section, the number of AEC-stained
cells was counted in 20 (skin, synovium or lung) or 40
(heart) fields (area 0·25 mm2), using an eyepiece graticule. Sections were examined in the dermis of skin, along
the synovial lining layer of joint tissue or in random
fields of lung and heart parenchyma.
RESULTS
Characterization of antibody specificity
A hybridoma clone was selected for its ability to
produce antibody which reacted with rh-prochymase
in ELISA. The monoclonal antibody, which was
termed CC1, was of the IgG1 subclass. Heparin, at
J. Pathol. 189: 138–143 (1999)
140
M. G. BUCKLEY ET AL.
Fig. 1—Silver-stained SDS-PAGE gel of purified skin chymase (A)
and matching western blots probed with rabbit antiserum to chymase
(B) and monoclonal CC1 antibody (C). Arrow-heads indicate the
position and size of molecular mass marker proteins (kD)
concentrations of up to 640 ng/ml, had no effect on
the binding of CC1 antibody, suggesting that it does
not bind to the heparin-binding domain of chymase,
and CC1 antibody did not bind to plates coated with
800 ng/ml tryptase.
On western blots, antibody CC1 bound specifically to
chymase (Fig. 1). Incubation of CC1 with chymase
failed to alter the rate of cleavage of the chromogenic
substrate, suggesting that the epitope was distant from
the catalytic site.
Immunocytochemistry
Immunocytochemistry with CC1 antibody allowed
the detection of cells with the morphological appearance
and distribution of mast cells in sections of paraffinembedded tissues (Fig. 2). Staining was restricted to the
cytoplasmic granules and the identity of the cells as mast
cells was confirmed by staining adjacent sections with
antibody AA1 specific for mast cell tryptase (Figs 2A
and 2B) and, in certain experiments, by using a doublelabelling procedure. In no tissue was evidence found for
chymase in cells other than those which contained
tryptase.
In skin, chymase-containing mast cells were observed
to be distributed throughout the dermis, often in close
proximity to blood vessels (Fig. 2C), and the great
majority of mast cells were found to contain both
tryptase and chymase (Table I). In the synovial tissue
Copyright 1999 John Wiley & Sons, Ltd.
specimens, mast cells detected with CC1 were observed
in the superficial synovium, deeper synovium, and joint
capsule (Fig. 2D). Chymase-containing cells were most
numerous in areas of synovial membrane hyperplasia
(although not in the lining layer) and on the periphery of
lymphoid aggregates in inflamed synovium. Mast cells
staining with CC1 antibody were observed in the connective tissue of sections of lung parenchyma, as well as
in the walls of arterioles and sometimes, within or
beneath the bronchiolar epithelium (Fig. 2E), though
they were rare in the alveoli. Heart mast cells containing
chymase were distributed widely and were located
between myocytes and in connective tissue (Fig. 2F). In
the heart parenchyma, MCTC comprised between 17 and
55 per cent of mast cells, with the greatest proportion
being found in fibrotic tissue (Table I, heart 3). There
was no staining of endothelial cells or of any cell type
other than mast cells using CC1 antibody.
Of particular interest was the observation that CC1
antibody was able to detect mast cells in tissues which
had been fixed in neutral buffered formalin, as well as in
Carnoy’s fixative or 85 per cent ethanol. The overall
numbers of cells which stained in the formalin-fixed
tissues did not differ significantly from those in
Carnoy’s-fixed tissues (paired Mann–Whitney U-test).
The uneven distribution of mast cells within the tissues is
likely to account for much of the variability observed in
the numbers of cells detected in differently fixed tissues;
no consistent trends were apparent. Fixation-related
differences were not noted in the numbers of mast cells
stained with AA1 antibody, or in the proportions of
MCTC estimated.
DISCUSSION
This new monoclonal antibody prepared against
chymase should prove valuable in the study of this
important mediator and marker of human mast cells.
Antibody CC1 now opens the way for the reliable
detection of chymase in western blots, while in immunocytochemistry it offers the major advantage of allowing
chymase to be detected in formalin-fixed pathology
specimens. Immunocytochemistry with CC1 produced
intense staining of mast cells in each of the tissues
examined, regardless of their source or the fixative
employed.
Immunostaining was restricted to cells with the
morphology and distribution of mast cells and was
cytoplasmic. Moreover, in sequential sections and
double-labelling experiments it was found that the cells
with which CC1 reacted were bound by tryptase-specific
antibody. No evidence was found for chymase in cells
other than mast cells. It has been suggested previously in
studies employing chymase-specific rabbit antiserum
that chymase may be present in endothelial cells and in
various other cell types in addition to mast cells,26,27 but
no support was found for this in the present studies. The
mast cell would appear to be the principal cellular store
of chymase, but we cannot exclude the possibility that
other cells could synthesize and immediately secrete this
protease, or could ingest exocytosed mast cell granules.
J. Pathol. 189: 138–143 (1999)
141
A CHYMASE-SPECIFIC ANTIBODY FOR FORMALIN-FIXED TISSUES
Fig. 2—Staining of mast cells in formalin-fixed tissues with AA1 anti-tryptase antibody (A), or CC1 anti-chymase antibody (B–F). (A, B)
Sequential sections of synovial tissue were stained for tryptase (A) and chymase (B). Two cells staining for tryptase, but not chymase
(MCT cells) are marked with arrows. (C–F) Mast cells stained with CC1 antibody in the dermis of foreskin (C), in the subsynovium of
a patient with osteoarthritis (D), beneath the bronchial epithelium from uninvolved tissue of a patient who had undergone lung resection
for bronchial carcinoma (E), and in left ventricular heart tissue removed post-mortem (F). All sections were counterstained with Mayer’s
haemalum
Our failure, using the new antibody, to detect chymase
in cells other than those which contained tryptase conflicts with the observation by Weidner and Austen32 of
a tryptase-negative, chymase-positive mast cell subset
(MCC) in various human tissues when a tryptase-specific
Copyright 1999 John Wiley & Sons, Ltd.
monoclonal antibody was used in conjunction with
rabbit polyclonal antibody against chymase. However,
using different reagents, we have noted previously that
the appearance of the MCC phenotype may be an artefact associated with an inadequate staining procedure
J. Pathol. 189: 138–143 (1999)
142
M. G. BUCKLEY ET AL.
Table I—Number of cells staining for chymase in immunocytochemistry with antibody CC1, or tryptase
with antibody AA1, and the proportion of cells staining for both in formalin- and Carnoy’s-fixed tissue
Formalin
Tissue
Foreskin 1
Foreskin 2
Foreskin 3
Foreskin 4
Synovium 1
Synovium 2
Synovium 3
Synovium 4
Lung parenchyma
Lung parenchyma
Lung parenchyma
Lung parenchyma
Heart 1
Heart 2
Heart 3
CC1/mm
1
2
3
4
83
54
64
ND
0·2
20
5·6
34
1·0
0·2
4·8
15
3·1
0·6
4·8
2
AA1/mm
2
88
64
62
ND
130
33
110
100
110
72
47
53
12
3·5
8·8
Carnoy’s
% MCTC
95
85
100
ND
0·2
61
5·3
34
0·9
0·3
10
28
26
17
55
CC1/mm
16
ND
ND
46
0·4*
4·0*
5·0
9·2
1·2
9·6
2·8
4·6
ND
ND
ND
2
AA1/mm2
% MCTC
17
ND
ND
70
94*
61*
71
42
43
74
55
36
ND
ND
ND
92
ND
ND
66
0·4*
6·5*
7·0
22
2·8
13
5·1
13
ND
ND
ND
*Data from tissue fixed in 85 per cent ethanol rather than in Carnoy’s fixative.
ND=not done.
for tryptase.9 It also seems likely that the polyclonal
antibodies to chymase employed in certain previous
studies may have reacted with other antigens.
The finding that CC1 bound to mast cells in both
formalin and Carnoy’s-fixed tissues is of particular interest. There were some differences in the numbers of MCT
and MCTC cells between formalin- and Carnoy’s-fixed
specimens from the same patient, but these differences
were bidirectional for the group as a whole and are likely
simply to reflect variation within individual tissues. The
relative numbers of each mast cell subpopulation
detected in formalin-fixed tissues by immunocytochemistry using CC1 and AA1 antibodies were comparable to
those reported previously in Carnoy’s-fixed specimens
with other antibodies. In the present study, MCTC cells
accounted for a mean of 94 per cent of all mast cells in
foreskin. This compares with figures of 90 per cent
obtained using polyclonal rabbit antibody in a doublelabelling procedure,6 and 99 per cent in a subsequent
study by the same group with a monoclonal antibody.7
Overall, there was a mean of 25 per cent MCTC cells in
the synovial lining of patients with different forms of
arthritis, but considerable variation was noted between
tissues from different patients. Both a high degree of
variability and a very similar proportion of mast cells of
the MCTC phenotype (23 per cent) have been observed
previously using a different chymase-specific antibody in
immunocytochemistry with synovial tissues fixed with
ethanol.17 In lung parenchyma, we found that 10 per
cent of mast cells were of the MCTC subset, which
compares well with the 7 per cent of alveolar mast cells
reported by Irani et al.6 However, using our new monoclonal antibody CC1, only 17–55 per cent of cardiac
mast cells stained for chymase, which is much lower than
the figure of 90 per cent reported by Patella et al. using
polyclonal antisera in immunoelectron microscopy.8
Copyright 1999 John Wiley & Sons, Ltd.
The inability to detect chymase in tissues fixed with
the most widely employed fixatives has hindered understanding of the role of MCTC and MCT mast cell subsets
in disease and the lack of suitable reagents has led to
confusion over the extent to which chymase may be
present in cells other than mast cells. The application in
immunocytochemistry of the monoclonal antibody CC1
now provides a reliable means of detecting chymase
and allows mast cell heterogeneity to be studied in
tissues without the need to employ special conditions of
processing.
ACKNOWLEDGEMENTS
We are grateful to Dr Doreen Ashworth (Ferring
Research Institute, Southampton) for her help in the
preparation of the baculovirus containing the prochymase construct; Dr Trudy Roach, Department of
Orthopaedics, for the use of embedding and sectioning
equipment; and Drs Martin Glennie and Kevan Roberts
for helpful discussions on the production of monoclonal
antibodies. Financial support from Action Research;
the Ministry of Agriculture, Fisheries and Food; and
the Arthritis Research Campaign is gratefully
acknowledged.
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J. Pathol. 189: 138–143 (1999)
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