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Human synovial mast cells. I. Ultrastructural in situ and in vitro immunologic characterization

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Vol. 39, No. 7, July 1996. pp 1222-1233
0 1996, American College of Rheumatology
I. Ultrastructural In Situ and In Vitro Immunologic Characterization
Objective. To examine the ultrastructure of human synovial mast cells in situ, to identify immunologic
and nonimmunologic stimuli that activate these cells in
vitro, and to quantify a number of preformed and de
novo-synthesized mediators.
Methods. We conducted an ultrastructural study
of synovial mast cells in situ and performed immunoelectron microscopy localization of tryptase and chymase. Isolated synovial mast cells were analyzed biochemically, immunologically, and functionally in vitro and
compared with cells from human lung, heart, and skin.
Results. Ultrastructural study of synovial tissue
revealed mast cells with homogeneously dense, scrolled,
crystal, and mixed granules, and lipid bodies in the
cytoplasm. A small percentage of mast cells showed
evidence of degranulation. Immunoelectron microscopy
demonstrated the subcellular localization of tryptase
and chymase over granules of >90% of the mast cells,
which were of the MC,, subtype. Isolated synovial mast
cells released histamine in response to immunologic
(anti-IgE and anti-Fcs receptor I [anti-FcsRI]) and
nonimmunologic (substance P, recombinant human
stem cell factor, and 48/80) stimuli, but did not respond
to recombinant human C5a in vitro. Synovial mast cells
differed from those isolated from other human tissues,
in a variety of immunologic and biochemical features.
Supported by grants from CNR (Project FATMA, Prevention
and Control of Disease Factors, SP 2 no. 94.00607.PF.41 and no.
95.00856.PF.41) and the MURST (Rome. Italy) and by grant
030RFM87/1-IRCCS to Policlinico S. Matteo (Pavia, Italy) from the
Ministry of Health (Rome, Italy).
Amato de Paulis, MD, Isabella Marino, MD, Anna Ciccarelli,
MD, Gennaro de Crescenzo. MD, Gianni Marone, MD: University of
Naples Federico 11, Naples, Italy; Monica Concdrdi, PhAss, Laura
Verga. MD, Eloisa Arbustini, MD: University of Pavia, Pavia, Italy.
Address reprint requests to Gianni Marone, MD, Division of
Clinical Immunology and Allergy, University of Naples Federico 11.
Via S. Pansini 5, 80131 Napoli, Italy.
Submitted for publication November 28, 1995; accepted in
revised form February 20. 1996.
There was a linear correlation between the percentage of
histamine secretion and tryptase release (r = 0.79, P <
0.001) induced by cross-linking of FceRI. Cross-linking
of IgE with anti-IgE on synovial mast cells induced de
novo synthesis of prostaglandin D, (mean f SEM 87.5
f 4.9 ng/106 cells) and of leukotriene C, (57.6 & 17.8
ng/106 cells).
Conclusion. Mast cells ultrastructurally characterized in situ in synovial tissue were seen to differ from
mast cells previously isolated from other human tissues.
This raises the possibility that the local microenviroment influences their phenotype. Isolation of mast cells
from human synovia can be useful for studying their
role and their mediators in patients with arthritis.
It has been known for years that mast cells are
present in normal human synovium (1-3). The density of
synovial mast cells (SMC) is increased in juvenile rheumatoid arthritis (4), adult rheumatoid arthritis (RA) ( 5 ) ,
and osteoarthritis (OA) (6), and there is some evidence
of in situ partial degranulation, suggesting ongoing
mediator release (7,8). In addition, levels of mast cell
mediators, including histamine (9,10), heparin, chymase,
and tryptase (lO,ll), are increased in inflamed synovial
fluid. These findings suggest that mast cells and their
mediators may be involved in the onset and/or progression of inflammation of the human synovium.
Several investigators have sought to isolate, partially purify, and characterize human SMC (HuSMC) in
vitro (8). This work has provided a wealth of informative
data. It has been demonstrated, for instance, that isolated HuSMC can be activated in vitro to release
histamine in response to anti-IgE (7,12,13) and other
nonimmunologic stimuli (7,12-14). Bridges et a1 found
that IgE-mediated releasability of HuSMC was greater
in patients with RA than in those with OA (13).
Despite all these important findings, the immunologic and biochemical characterization of HuSMC is
still largely incomplete. In fact, most previous reports
have focused on the identification of only 1 chemical
mediator (histamine) and only 1 immunologic stimulus
(anti-IgE). It has yet t o b e established whether HuSMC
can be activated by such immunologic stimuli as anti-Fc,
o r C5a, o r nonimmunologic agents (substance P and
stem cell factor [SCF]) that are relevant to inflammatory
features of synovitis. T h e precise profile of chemical
mediators preformed (tryptase and chymase) o r d e novo
synthesized (leukotriene C, [LTC,] and prostaglandin
D, [PGD,]) by HuSMC has still not been fully characterized. Finally, 2 types of human mast cells have been
identified based o n their content of tryptase alone
(MC,.) or of tryptase together with chymase (MC,,)
(15), and it is not clear t o which type H u S M C belong.
The present study was designed t o further characterize
HuSMC ultrastructurally and by the immunogold technique in situ, a n d t o quantify a number of preformed and
d e novo-synthesized mediators released after their activation in vitro.
Reagents. C10, (60%) was purchased from Baker
Chemical (Deventer, The Netherlands). Bovine serum albumin (BSA), PIPES, hyaluronidase, chymopapain, elastase type
I, 48/80, recombinant human C5a, substance P, synthetic LTC,,
and PGD, were from Sigma (St. Louis, MO). Collagenase was
from Worthington Biochemical (Freehold, NJ). Hanks' balanced salt solution and fetal calf serum were from Gibco
(Grand Island, NY). DNase I and pronase were from Calbiochem (La Jolla, CA). RPMI 1640 with 25 mM HEPES buffer
and Eagle's minimum essential medium were from Flow
Laboratories (Irvine, Scotland). Percoll was from Pharmacia
(Uppsala, Sweden). 'H-LTC, (39.3 Ci/mmole) and 'H-PGD,
(210 Ci/mmole) were from New England Nuclear (Boston,
MA). Recombinant human c-kit receptor ligand (recombinant
human stem cell factor [rHuSCF]) was from Amgen (Thousand Oaks, CA). Protein A-gold complex was from Bio Clin
(Biochemical Services, Cardiff, Wales). Mouse anti-human
mast cell tryptase was from Chemicon International (Temecula, CA).
Rabbit anti-human FCEantibody was a generous gift
from Drs. Teruko and Kimishige Ishizaka (La Jolla Institute
for Allergy and Immunology, La Jolla, CA). A mouse monoclonal IgG anti-a chain of high-affinity receptor for IgE was
kindly donated by Dr. John Hakimi (Hoffman-La Roche,
Nutley, NJ). The rabbit polyclonal antichymase antiserum was
a generous gift from Dr. N. M. Schechter (University of
Pennsylvania, Philadelphia). The rabbit anti-sulfidopeptide
LTC, and rabbit anti-PGD, antisera were kindly donated by
Dr. L. M. Lichtenstein (The Johns Hopkins University, Baltimore, MD). The tryptase radioimmunoassay (RIA) kit
(Tryptase RIACT 50; Pharmacia) was donated by Kabi Pharmacia SpA (Milan, Italy).
Buffers. The PIPES buffer used in these experiments,
referred to as buffer P, was made up of 25 mM PIPES, pH 7.37,
110 mM NaC1, 5 mM KCI. P2CG contained, in addition to
buffer P, 2 mM CaCI, and 1 gm/liter dextrose (16); the pH was
titrated to 7.4 with sodium bicarbonate. PGMD (pH 7.37)
contained 0.25 gm/liter MgC1,. 6H,O, 10 mg/liter DNase, and
1 gm/liter gelatin in addition to buffer P.
Isolation and partial purification of HuSMC. The
synovial tissue used in this study was obtained from 72 patients
ages 27-66 years with OA (n = 58), RA (n = 7), or femur or
knee fracture (n = 7) undergoing synovectomy at the Division
of Orthopedic Surgery, University of Naples. Resected joint
tissue was immediately immersed in cold (4°C) buffer P and
was processed within 2 hours of removal, using a modification
of the technique of Kopicky-Burd et al (7). Two small samples
(-2 gm) representative of all tissue were obtained for microscopic examination of tissue mast cells and for measurement of
total histamine content. Fat, cartilage, and fibrous tissue were
The tissue was finely minced with scissors into 2-5-mm
fragments, suspended in buffer P (10 ml/gm of wet tissue), and
washed twice by centrifugation (150g for 8 minutes at 4°C then
22°C). The minced synovium was incubated for 45 minutes at
37°C in a shaking water bath with chymopapain (1 mg/ml) and
pronase (0.5 mg/ml) in 1 ml Tyrode's buffer/gm synovial tissue.
Fragments of remaining tissue were digested for another 45
minutes at 37°C with collagenase (1 mg/ml). The resulting cell
suspensions were pooled, filtered twice through 200ppore
Nytex cloth (Tetko, Elmsford, NY), centrifuged (20Og for 8
minutes at 22"C), and washed twice with buffer P. Cells were
cultured for up to 4 hours in RPMI 1640 containing 25 nM
HEPES, 2 mM L-glutamine, 1% gentamicin, and 10% fetal calf
At the end of the culture period, cells were harvested
(200g, 8 minutes, 22"C), washed 3 times in buffer P, and
resuspended in P2CG. Yields with this technique ranged from
0.1 X loh to 0.9 X 10" mast cells/gm of wet tissue, and purities
from 1% to 8%. Preparations of HuSMC were purified further
by countercurrent elutriation followed by discontinuous Percoll gradient. Isotonic Percoll was prepared by mixing 9 parts
Percoll and 1 part 1OX buffer P solution. This mixture was then
diluted with isotonic buffer P to give Percoll concentrations of
40%, 50%, 60%, 70%, 80%, and 90%. The cell suspension was
overlaid on the Percoll gradient in 50-ml polypropylene tubes,
and the mixture was centrifuged at 35% for 20 minutes at 22°C.
The cells found at the interface between 60-70% and 70-80%
fractions were removed, washed twice with buffer P, and
stained with Alcian blue to determine the mast cell purity. The
viability of mast cells was >95%. The purity of these populations ranged from 26% to 85%.
Isolation of human lung, heart, and skin mast cells.
Lung tissue from patients undergoing thoracotomy and lung
resection was dissected free of pleura, bronchi, and blood
vessels, minced into 5-10-mm fragments, and dispersed into
single cell suspension as previously described (17). Yields with
this technique ranged between 0.3 X lo6 and 1.2 x lo6 mast
cells/gm of wet tissue, and purities ranged between 1%and 8%.
Heart tissue from patients undergoing heart transplantation, mostly due to cardiomyopathy or ischemic heart disease, was dissected free of fat, large vessels, and pericardium,
minced into 2-5-mm fragments, and dispersed into single cell
suspension as previously described (18). Yields with this
technique ranged from 0.1 X lo6 to 0.6 X lo6 mast cells/gm of
wet tissue, and purities ranged from 0.1% to 8%.
Figure 1. Electron micrograph of human synovial mast cell in situ.
Note the close proximity of the mast cell to the capillary vessel wall.
The surface of the mast cell is adorned with numerous elongated
narrow surface folds. The cytoplasm contains numerous cytoplasmic
granules and a prominent lipid body (arrow). N = nucleus; L = lumen
(uranyl acetate and lead citrate stained; original magnification X
Skin obtained from patients undergoing either mastectomy for breast cancer or elective cosmetic surgery was separated from the subcutaneous fat by blunt dissection. The tissue
was cut into 1-2-mm fragments with scissors and the fragments
were dispersed into single cell suspension as previously described (19). Yields with this technique ranged between 0.1
and 0.9 X lo6 mast cells/gm of wet tissue, and purities were
between 1 % and 4%.
Determination of histamine content of human synovia,
lung parenchyma, heart, and skin. Samples of human synovium, heart, and skin were separated from fat by blunt
dissection. Macroscopically normal lung tissue was dissected
free of pleura, bronchi, and large vessels. Tissue samples were
weighed and boiled in 8% HCIO, for 30 minutes. The mixture
was filtered to remove particles, and supernatants were assayed
for histamine (19,20).
Histamine release assay. Cells (-3 X lo4 mast cells
per tube) were resuspended in P2CG, and 0.3 rnl of the cell
suspension was placed in 12 X 75-mm polyethylene tubes
(Sarstadt, Princeton, NJ) and warmed to 37°C; 0.2 ml of each
prewarmed releasing stimulus was added, and incubation was
continued at 37°C for 45 minutes (21). At the end of this step,
the reaction was stopped by centrifugation at 1,OOOg for 2
minutes at 2TC, and cell-free supernatants were stored at
-20°C for subsequent assay of histamine, tryptase, and LTC,
or PGD, content.
The cell-free supernatants were assayed for histamine
by an automated fluorometric technique (22). Total histamine
content was assessed by lysis induced by incubating cells with
2% HC10, before centrifugation. To calculate histamine release as a percentage of total cellular histamine, the "spontaneous" release of histamine from mast cells (2-14% of total
cellular histamine) was subtracted from both numerator and
denominator (23). All values are based on means of duplicate
o r triplicate determinations. Replicates differed in histamine
content by <lo%.
FUA of tqptase. Total tryptase content was assessed by
lysis induced by incubating cells with 100 p l of Triton X-100
(0.1%). Tryptase was analyzed by solid-phase RIA (Tryptase
RIACT 50). Duplicate samples o r tryptase standards were
added to plastic tubes coated with mouse monoclonal antitryptase antibodies. Then '251-radiolabeled antitryptase was
added to the tubes, which were incubated for 16-18 hours at
22°C (24). After washing the tubes 3 times with 2 ml of saline
at 22"C, bound radioactivity was counted, giving a linear
concentration-response curve within a range of 2-50 pdliter.
RIA of LTC, and PGD,. In some experiments, 200-p1
fractions were taken from the supernatant fluids for analysis of
PGD, and LTC,. The samples were stored at -70°C until
analyzed for PGD, and LTC, content. PGD, was assayed by a
previously described RIA (18) within 24 hours of the experiment, to minimize degradation of the compound (25). The
anti-PGD, antibody is highly selective, with <1% crossreactivity with other eicosanoids (26). The rabbit anti-LTC,
antiserum has been characterized and its cross-reactivity for
heterologous ligand described (27).
Ultrastructural study. Samples for ultrastructural
study were fixed in Karnovsky's solution at 4°C for 2 hours,
rinsed in sodium cacodylate buffer (0.2M, p H 7.3), postfixed
with 1 % osmium tetroxide in O.lM cacodylate buffer for 1 hour
at 4"C, dehydrated in ethanol and propylene oxide, and
embedded in Epon-Araldite. Ultrathin sections were stained
with uranyl acetate and Reynold's lead citrate. Stained sections
were examined under a Zeiss EM10 electron microscope (18).
Electron immunocytochemistry study. Ultrathin sections were deosmicated in aqueous saturated solution of 5%
sodium metaperiodate for 10 minutes, rinsed in 1% ovalbumin
in 0.01M Tris buffer-0.5M NaCI, p H 7.6 (TBS-Triton buffer)
and washed for 1 hour in TBS-Triton-lysine buffer, then
preincubated with 10% heat-inactivated normal goat serum
and subsequently incubated overnight with the antitryptase or
antichymase antiserum diluted 1:100 in TBS-1% BSA-0.5%
Na-azide buffer. The sections were then washed 3 times in
TBS-1% BSA-0.5% Na-azide buffer for 10 minutes each time,
and incubated for 1 hour with protein A-gold complex diluted
1:30 with TBS-1% BSA-0.5% Na-azide buffer (28). After a
2-hour wash in TBS-1% BSA-0.5% Na-azide buffer, the grids
were dried and stained for 15 minutes with aqueous uranyl
acetate ( 5 % ) and for 10 minutes with Reynold's lead citrate.
The stained sections were examined under a Zeiss EM10
electron microscope. In control tests, the first antibody layer
was omitted to eliminate immunoreactivity. As a negative
control, we used IgG from a nonimmunized rabbit instead of
rabbit polyclonal antichymase, and a murine myeloma directed
against an irrelevant antigen instead of monoclonal mouse
Figure 2. Ultrastructural features of human synovial mast cell secretory granules (A and B) and lipid bodies (C). A, Electron micrograph
of part of the nucleus (N)and cytoplasm of a synovial mast cell. Note
the abundance and heterogeneity of granules, some of which show
scroll-like structures (arrow). Pseudopodia are visible on the mast cell
surface. B, Ultrastructural features of secretory granules with a
lamellar pattern (arrowhead) or a swirl pattern (arrow). C, Two
cytoplasmic lipid bodies (arrows). Note the focal lucent area in 1 lipid
body (arrowhead). (Uranyl acetate and lead citrate stained; original
magnification X 30,000 in A, X 50,000 in B and C.)
Statistical analysis. Results are presented as the mean
SEM. The data subjected to linear regression were calculated by the least-squares method (y = a + bx) in which a was
the y-axis intercept and b the slope of the line. Correlations
were calculated by Spearman’s rank correlation (r,) (29).
Histamine content of human synovium, lung
parenchyma, heart, and skin. The histamine content in
12 preparations of human synovium (3.3 ? 1.1 pg/gm
wet tissue; n = 12) was lower than that in lung paren-
chyma (22.7 5 1.2 yg/gm wet tissue; n = 12) (P < O.OOl),
but did not differ significantly from that in heart (5.5 -+
0.3 mg/gm wet tissue; n = 25) or skin (5.8 f 0.6 pg/gm
wet tissue; n = 11).
In situ ultrastructural features of HuSMC. U1trastructural study of 8 synovia demonstrated mast cells
in close proximity to the capillary vessel wall, and fibrotic
interstitial areas (Figure 1). Mast cell nuclei were single
lobed; many had large nucleoli and partially condensed
chromatin. Cytoplasm contained numerous large,
membrane-bound granules and non-membrane-bound
lipid bodies. Cytoplasmic lipid bodies were usually adjacent to the nucleus and, on average, larger and less
numerous than the specific cytoplasmic granules. Additional ultrastructural features included cytoplasmic filaments, free ribosomes, and mitochondria. The surface of
synovial mast cells was occasionally adorned with numerous elongated narrow surface folds, as previously
reported in isolated HuSMC (7).
Cytoplasmic granules presented various ultrastructural patterns (Figure 2A): homogeneously dense
structure, scrolls, mixed granules, lamellar, and swirl
patterns (Figure 2B). Scrolled granules either had lucent
interiors or contained dense material, as previously
reported (30). Some granules had dense central nucleoids surrounded by a granule matrix, as in developing
mast cells. Non-membrane-bound lipid bodies were
present (Figure 2C), frequently with focal lucent areas;
small round structures with punctuated densities were
observed within them, as in gut, uterine, and heart mast
cells (18,30,31).
A small percentage (-5%) of HuSMC showed in
situ evidence of degranulation (Figure 3). HuSMC de-
Figure 3. Ultramicrograph of human synovial mast cell in situ, showing evidence of degranulation. Note the heterogeneity of the secretoxy
granules. Scroll granules are indicated by arrows. Large chambers (C)
contain granule matrix material (uranyl acetate and lead citrate
stained; original magnification X 50,000).
3x lo-’
Anti-lgE (pg/mll
Figure 4. Effect of increasing concentrations of anti-lgE on histamine
secretion from human synovial mast cells (HSyMC) from 9 donors.
Each symbol represents the results for a different donor; each point
indicates the mean of duplicate determinations.
granulation occurs through extrusion of altered,
membrane-free granules to the cell’s exterior, as do
intracytoplasmic granule solubilization, the presence of
large chambers containing granule matrix material, and
the opening of multiple intracytoplasmic degranulation
Histamine release from HuSMC induced by
cross-linking of IgE or of IgE receptors. Serum and
synovial fluid IgE levels tend to be high in patients with
RA (32), and antibodies to IgE and IgE rheumatoid
factors have been detected in serum and synovial fluid of
RA patients (10,33-36). Exposure of isolated HuSMC to
anti-IgE (0.1-3 pg/ml)-cross-linking IgE molecules resulted in mast cell activation. Figure 4 shows the results
of 9 experiments (of a total of 25 performed): release
ranged from 0% to 25%, with a maximum of 18.3 f
1.7% (n = 25), at a concentration of 3 mg/ml. A
monoclonal antibody against the a chain of the IgE
receptor (Fcs receptor I [FcsRI]) (18,37), at levels
between 0.1 pg/ml and 3 pg/ml, caused concentrationdependent histamine release from HuSMC. Histamine
release induced by anti-FcsRI reached a maximum of
18.6 -+ 1.8% (n = 22) at an anti-FcsRI concentration of
3 pg/ml.
Figure 5 illustrates the findings in 7 of 22 experiments with anti-FcsRI treatment: in 20% of the experiments, HuSMC did not release histamine after
anti-FcsRI challenge; in all other experiments, release
varied from 0% to 28%. The release of histamine
5 15
2 10
d o
3x 1 0-1
An ti-Fc,Rl
Figure 5. Effect of increasing concentrations of anti-Fc, receptor I
(anti-Fc,RI) on histamine secretion from human synovial mast cells
(HSyMC) from 7 donors. Each symbol represents the results for a
different donor; each point indicates the mean of duplicate determinations.
induced by anti-IgE and anti-FceRI from HuSMC was
highly dependent on extracellular C a + + and on an intact
glycolytic pathway. Optimal release occurred at 2 mM
extracellular Cat+, and when no extracellular Ca++ was
added, the responses to both anti-IgE and anti-FcsRI
were abolished (data not shown). The requirement for
glycolysis was evidenced by the >90% inhibition of
IgE-mediated histamine release after 15 minutes of cell
preincubation with 2-deoxy-~-glucose(10 mM) and antimycin A (1 pA4) (data not shown). All subsequent
experiments were performed with a physiologic concentration of extracellular Ca++ (2 mM).
Heterogeneous effects of C5a on human synovial,
heart, and skin mast cells. Complement activation and
anaphylatoxin formation (C3a and CSa) occur in the
synovial fluid of patients with various inflammatory
arthritides (38,39). In 12 experiments, recombinant human C5a (rHuCSa) (lo-’ to W 6 M )did not cause
histamine release from HuSMC (Figure 6). In contrast,
in 12 parallel experiments, rHuC5a (lop7 to 10-6A4)
caused a concentration-dependent release of histamine
from human skin mast cells and human heart mast cells.
Heterogeneous effects of substance P and 48/80
on HuSMC, human heart mast cells, human skin mast
cells, and human lung mast cells. The neuropeptide
substance P can be released into joint tissues from
primary sensory nerve fibers (40,41), and its levels are
increased in synovial fluids from patients with RA (42).
We therefore examined the effect of substance P on the
activation of HuSMC. Figure 7A shows the results of 11
parallel experiments in which HuSMC, human heart
mast cells, human skin mast cells, and human lung mast
cells were challenged with substance P (0.1-100 pA4). At
levels between 1 p M and 100 pM, substance P caused
concentration-dependent histamine release from
HuSMC. The maximum release ranged from 0 to 61%,
with an average of 33.3 2 8.6%. In these experiments,
substance P (1-10 pM) caused a concentrationdependent increase in histamine release from human
skin mast cells, but not from human heart mast cells or
human lung mast cells.
Compound 48/80, a classic activator of rodent
mast cells (43), induces histamine release from human
skin mast cells (25,44) and human heart mast cells ( 4 9 ,
but not from human lung mast cells (46). Its effect on the
activation of HuSMC is controversial (7,12-14). Figure
7B compares the effect of 48/80 (10-300 pg/ml) on
histamine release from the 4 cell types: it caused a
concentration-dependent increase in histamine release
from HuSMC, human heart mast cells, and human skin
mast cells, but not from human lung mast cells.
Histamine release induced by stem cell factor.
The c-kit ligand, also known as stem cell factor, is a
cytokine synthesized by fibroblasts and other cells (47).
SCF activates a membrane receptor present on human
skin, lung, and heart mast cells and induces histamine
release from these cells (18,48,49), but it has not been
rhC5a [pM)
Figure 6. Effect of increasing concentrations of recombinant human
CSa (rhC5a) on histamine secretion from human synovial mast cells
(HSyMC) (n = 12), human heart mast cells (HHMC) (n = 12), and
human skin mast cells (HSMC) (n = 12) from different donors. Values
are the mean and SEM (error bars are not shown when graphically too
small). = P < 0.01 compared with spontaneous release.
48/80 Ipg/ml)
Substance P (pM)
Figure 7. A, Effect of increasing concentrations of substance P on histamine secretion from human synovial mast cells (HSyMC) (n = l l ) , human
heart mast cells (HHMC) (n = Il), human skin mast cells (HSMC) (n = I l ) , and human lung mast cells (HLMC) (n = 11) from different donors.
B, Effect of increasing concentrations of compound 48/80 on histamine secretion from HSyMC (n = 16), HHMC (n = 14), HSMC (n = 16), and
HLMC (n = 13) from different donors. Each point represents the mean and SEM (error bars are not shown when graphically too small). = P <
0.01 compared with spontaneous release.
shown to affect HuSMC. In 8 experiments, low concentrations of rHuSCF (3 X lo-' ng/ml to 30 ng/ml)
induced the release of low but consistent percentages of
histamine from HuSMC as well as from human lung
mast cells, in a concentration-dependent manner (Table 1).
Mediators (PGD, and LTC,) synthesized de novo
from immunologically activated HuSMC. In 3 experiments, we measured the production of newly formed
lipid mediators (PGD, and LTC,) in response to
anti-IgE (0.1-3 pglml). The de novo synthesis of PGD,
was dose dependent at anti-IgE concentrations between
0.1 pg/ml and 3 pg/ml (Figure 8A). Maximum stimulation with anti-IgE led to the production of 79-96 ng of
PGD,/106 cells (mean t SEM 87.5 t 4.9). Figure 8B
illustrates 3 experiments in which anti-IgE induced
Table 1. Effects of increasing concentrations of recombinant human
stem cell factor (rHuSCF) on histamine release from human synovial
and lung mast cells*
% histamine release
3 x 10-1
mast cells
mast cells
1.5 ? 0.4
2.7 +- 0.7
6.2 +- 1.8
5.9 ? 1.6
4.5 +- 1.4
6.3 i- 2.0
8.0 i- 2.3
* Each result is the mean ? SEM of S experiments in which cells were
incubated with rHuSCF for 45 minutes at 37°C. ND = not done.
concentration-dependent de novo synthesis of LTC,
(0-91 ng/106 mast cells, with a mean -t SEM production
of 57.6 ? 17.8 ng/106 mast cells).
Tryptase content and subcellular localization of
tryptase and chymase in HuSMC. Either directly or
through the activation of proenzymes, mast cell proteases (tryptase, chymase, etc.), can cause significant
remodeling of extracellular matrix proteins (8,ll). Two
types of human mast cells, M C , and MC,,, have been
identified (15), and it is still not known to which subtype
HuSMC belong. A monoclonal antitryptase antiserum
was used to detect tryptase in HuSMC. After immunogold staining of human synovium, 10-nm gold particles
were present over all secretory granules of more than
95% of the HuSMC (Figure 9). Very few gold particles
were detected outside the cytoplasmic granules. Granules of HuSMC incubated with a murine myeloma
against an irrelevant antigen at concentrations similar
to those of the primary antibody lacked gold particles
(data not shown). The tryptase content in HuSMC
ranged between 1.1 and 9.3 pg/106 cells with a mean f
SEM of 5.0 t 0.85 pg/106 cells (n = 4), lower than that
in human skin mast cells (33.4 5 2.5 pg/106 cells; n = 8)
but similar to that in human lung mast cells (10.6 ? 1.9
p.8/106 cells; n = 7). Activation of HuSMC with anti-IgE
caused the release of tryptase in parallel to that of
histamine. There was a linear correlation between the
percentages of histamine secretion and tryptase release
observed when the first antibody was omitted (data not
80 -
In this study, we ultrastructurally characterized
HuSMC in situ. In addition, using mast cells enzymatically isolated from synovial tissue, we identified the
immunologic (anti-IgE and anti-FcsRI) and nonimmunologic (substance P, rHuSCF, and 48/80) stimuli that
induce the release of preformed (histamine and
tryptase) and newly generated (PGD2 and LTC,) mediators from HuSMC. Immunoelectron microscopy
60 -
80 -
gogs and the amount of mediators synthesized were
Exposure of HuSMC to anti-IgE or anti-FcsRI,
which cross-link either IgE molecules or the (Y chain of
FcsRI, resulted in mast cell activation. Interestingly,
histamine-releasing autoantibodies against FCE (antiIgE) and the a subunit of FcERI are found in the
circulation or synovial fluid of some patients with rheu-
Anti-lgE Ipg/mll
Figure 8. Effect of increasing concentrations of anti-IgE on the de
novo synthesis of prostaglandin D, (PGD,) (A) and leukotriene C,
(LTC,) (B) by human synovial mast cells (HSyMC). Values are the
mean t SEM of 3 experiments (error bars are not shown when
graphically too small).
induced by cross-linking of FcsRI (r = 0.79, P < 0.001)
(Table 2).
A previously described polyclonal antichymase
antiserum (50) was used to detect chymase in HuSMC.
After immunogold staining of human synovium, 20-nm
gold particles were present over all secretory granules in
the vast majority of HuSMC (>90%) (Figure lo). Very
were present Over nuclei, mitochonfew goid
surrounding the granules.
dria7 Or
Granules of HuSMC iIKlhated with
from nonimmunized rabbits at concentrations similar to those of the
Figure 9. Iminunogold staining of human synovial mast cell for
tryptase. Sections were stained for tryptase (mouse antitxyptase rnonoClonaI antibody; Protein A-10 nm gold complex, diluted 1:30) as
described in Materials and Methods. Gold particles that locate
tryptase are present over all secretory granules and not in the
perigranular cytoplasm. N = nucleus (uranyl acetate and lead citrate
stained; original magnification x SO,OOO).
Table 2. Effects of increasing concentrations of anti-lgE on tryptase and histamine release from human synovial mast cells*
Experiment 1
Experiment 2
% histamine
3 x 10-1
Experiment 3
% histamine
% histamine
* Each result is the mean of duplicate determinations in which cells were incubated with anti-IgE for 45 minutes at 37°C.
matic, inflammatory, or autoimmune disorders (35,51,
52). The wide variability in mediator release from synovial mast cells from different donors, prepared under
identical conditions and challenged with anti-IgE and
anti-FceRI, indicates that FceRI-mediated histamine
release may also depend on factor(s) that determine
mast cell releasability (53). In addition, the possibility
that the underlying disease of the cell donor (13) or the
drugs used before and during general anesthesia (26)
might have affected the in vitro response of synovial
mast cells cannot be excluded.
C5a, a clinically relevant stimulus (38,39), did not
cause histamine release from HuSMC. Thus, HuSMC
and human lung mast cells are the only human mast cells
identified so far that cannot be activated by anaphyla-
Figure 10. Immunogold staining of human synovial mast cell for
chymase. Sections were stained for chymase (rabbit antichymase
antibody; protein A-20 nm gold complex, diluted 1:30) as described in
Materials and Methods. Gold particles that locate chymase are present
over all secretory granules and not in the perigranular cytoplasm
(uranyl acetate and lead citrate stained; original magnification X
toxins (25). This negative finding is of some interest
because complement activation is a feature of RA (6),
and elevated levels of anaphylatoxins (C5a and C3a)
have been reported in synovial fluids from patients with
synovitis (38,39). Although we cannot rule out the
possibility of some specific damage to C5a receptors
caused by the enzymatic procedure used to isolate
HuSMC, the findings from our comparative studies with
mast cells isolated from other tissues tend to exclude this.
Exposure of HuSMC to the neuropeptide substance P resulted in activation. This might be clinically
important because several clinical and experimental
findings are consistent with nervous system involvement
in the pathogenesis of RA (40-42,54-56). Substance P
is one possible mediator, since it can be released into
joint tissues from primary sensory nerve fibers (40,41)
and increased levels have been detected in synovial
fluids of patients with RA (42). Thus, neurons and their
transmitters may affect synovial mast cells, raising the
possibility of reciprocal and/or feedback interactions
between the two cell types. This finding provides another
example of mast cell heterogeneity, because substance P
activates HuSMC and human skin mast cells, but not
human heart mast cells and human lung mast cells. Thus,
HuSMC and human skin mast cells are the only human
mast cells identified so far that can be activated by this
Another example of the unique biochemical
properties of HuSMC comes from studies of arachidonic
acid metabolism after immunologic stimulation. In response to anti-IgE, lung and gut mast cells synthesize
approximately the same amount of PGD, and LTC,
(24,57) (-60 ng/106 cells). Uterine mast cells synthesize
more PGD, (-90 ng/106 cells) than LTC, (-45 ng/106
cells) (31); skin mast cells also preferentially use the
cyclooxygenase pathway of arachidonic acid metabolism
(-40 ng PGD,/106 cells), with little metabolism through
the 5-lipoxygenase pathway (<5 ng LTC&06 cells)
(25,26). Immunologic activation of HuSMC leads to the
de novo synthesis of equal amounts of PGD, and LTC,
(-72.5 ng/106 cells). Thus, HuSMC might differ quantitatively or qualitatively from other types of human mast
cells with respect to arachidonic acid metabolism.
Our in situ ultrastructural study showed that
HuSMC are often found in perivascular locations, but
also are seen in loose connective tissue. As others have
observed with isolated HuSMC (7), some of these mast
cells showed in situ evidence of degranulation, which
raises the question of the mast cells’ pathophysiologic
role in human synovium. Our results suggest that
HuSMC have FcsRI and IgE bound to their surface.
Increased IgE levels (32) and IgE- and IgE rheumatoid
factor-containing immune complexes have been detected in the serum or synovial fluid of patients with RA
(33-36,58). Anti-IgE autoantibodies have been described in RA, systemic lupus erythematosus, and several other autoimmune diseases (10). Thus, specific
stimulation of HuSMC by the interaction of naturally
occurring autoantibodies against IgE might be involved
in the pathogenesis of arthritis in some patients.
The destruction of cartilage and other intraarticular tissue by proteolytic enzymes is a hallmark of
chronic synovitis. HuSMC are of the MC,, type, and
they synthesize and release neutral proteases such as
tryptase and chymase. Tryptase can activate rheumatoid
synovial collagenase (35) or up-regulate interleukin-8
(IL-8) (Holgate ST: personal communication) present in
inflamed synovial fluid. On the other hand, tryptase,
histamine, and peptide leukotrienes are mitogens for
fibroblasts, and they stimulate collagen accumulation
(59-61). Thus, tryptase and other mast cell mediators
could have a complex role in the metabolism of collagen
in rheumatoid synovium.
Our results demonstrate for the first time the
presence of chymase in the secretory granules of
HuSMC. Although the precise role of chymase in arthritis remains unclear, the chymase content of HuSMC
possibly influences synovial inflammation, given its
ability to convert inactive pro-IL-lp to the active form
(62). These findings suggest that the release of these
neutral proteases from perivascular and interstitial mast
cells might be relevant to the onset and progression of
the destructive inflammatory process involving human
Caution must be exercised in interpreting data
from synovia of patients undergoing surgery. Such donors are treated with multiple drugs, and some delay
before the tissue is processed is inevitable. Although
these are general concerns for all studies with mast cells
isolated from human tissues of patients undergoing
surgery, we cannot exclude the possibility that the pa-
tients’ clinical conditions and the drugs used before and
during anesthesia might have influenced some of our
results (13). This may explain why some HuSMC preparations were relatively nonresponsive to stimuli that, in
other preparations, induced mast cell mediator release.
In conclusion, synovial tissue contains mast cells
which are of the M G , type and differ from those
isolated from other human tissues in a variety of immunologic and biochemical features, raising the possibility
that the local microenvironment may influence their
phenotypic and biochemical characteristics. This experimental model could be useful for further defining the
role of synovial mast cells by identifying additional
mediators and cytokines synthesized and released, stimuli relevant to human pathophysiology, and pharmacologic agents that could selectively act on these cells.
We thank Prof. Domenico Marinb for his generous
cooperation in providing h u m an synovial specimens.
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