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Human synovial mast cells. II. Heterogeneity of the pharmacologic effects of antiinflammatory and immunosuppressive drugs

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ARTHRITIS & RHEUMATISM
Vol. 40, No. 3, March 1997, pp 469-478
0 1997, American College of Rheumatology
469
HUMAN SYNOVIAL MAST CELLS
11. Heterogeneity of the Pharmacologic Effects of Antiinflammatory and
Immunosuppressive Drugs
AMATO DE PAULIS, ANNA CICCARELLI, ISABELLA MARINO, GENNARO DE CRESCENZO,
DOMENICO MARINO, and GIANNI MARONE
Objective. To evaluate the in vitro effects of 4
antiinflammatory and 5 immunosuppressive agents on
the release of preformed and de novo-synthesized mediators from human synovial mast cells (HSyMC) activated by immunologic and nonimmunologic stimuli.
Methods. The effects of antiinflammatory and
immunosuppressive agents were evaluated on the in
vitro release of histamine and tryptase and the de novo
synthesis of prostaglandin D, (PGD,) and leukotriene
C, (LTC,) by HSyMC challenged with anti-IgE and
substance P.
Results. Nimesulide, a sulfonanilide nonsteroidal
antiinflammatory drug (NSAID) chemically unrelated
to other acidic NSAIDs (such as acetylsalicylic acid
[ASA], diclofenac, and piroxicam) inhibited in a
concentration-dependent manner the release of preformed (histamine and tryptase) mediators from
HSyMC challenged with anti-IgE. In contrast, diclofenac and piroxicam had little or no effect on HSyMC
activated by anti-IgE. ASA, diclofenac, piroxicam, and
nimesulide caused a concentration-dependent inhibition of IgE-mediated PGD, release from HSyMC. Nimesulide, but not diclofenac or piroxicam, also inhibited
the de novo synthesis of LTC, by HSyMC challenged
with anti-IgE. Nimesulide, diclofenac, and piroxicam
had no effect on HSyMC activated by substance P.
Supported by grants from CNR (Project FATMA, Prevention
and Control of Disease Factors, SP 2 nos. 94.00607.PF.41 and
95.00856.PF.41) and the MURST, Rome, Italy.
Amato de Paulis, MD, Anna Ciccarelli, MD, Isabella Marino,
MD, Gennaro de Crescenzo, MD, Domenico Marino, MD, Gianni
Marone, MD: School of Medicine, University of Naples Federico 11,
Naples, Italy.
Address reprint requests to Gianni Marone, MD, Division of
Clinical Immunology and Allergy, University of Naples Federico 11,
Via S. Pansini 5, 80131 Naples, Italy.
Submitted for publication August 1, 1995; accepted in revised
form October 14, 1996.
Cyclosporin A (CSA) inhibited histamine release from
HSyMC challenged with anti-IgE, whereas cyclosporin
H (CSH) had no effect. FK-506 also inhibited histamine
release from HSyMC activated by anti-IgE, whereas
rapamycin had no effect. Neither CSA, CSH, FK-506,
nor rapamycin inhibited the release of histamine from
HSyMC induced by substance P. Methotrexate had no
effect on the release of mediators from these cells,
whereas adenosine (R-phenylisopropyl adenosine and
5'-N-ethylcarboxamide adenosine) enhanced histamine
release from immunologically activated HSyMC in a
concentration-dependent manner.
Conclusion. Mast cells isolated from human synovia display 4 levels of pharmacologic heterogeneity with
regard to 1) the inhibitory effects of 4 antiinflammatory
drugs; 2) the capacity of different immunosuppressive
drugs to exert antiinflammatory activity; 3) the inhibition of the release of different mediators; and 4) the
capacity of antiinflammatory and immunosuppressive
drugs to modulate HSyMC activated by different stimuli. This complexity of pharmacologic modulation of
HSyMC in vitro might help explain the different activity
of the compounds used to treat various pathophysiologic
aspects of the inflammatory arthritides.
There is compelling evidence that human synovial mast cells (HSyMC) and their mediators may be
involved in the onset and progression of inflammation of
the human synovium and, consequently, of diverse arthritides (1-4). It has been demonstrated, for instance,
that isolated HSyMC can be activated in vitro to release
histamine and other proinflammatory mediators in response to anti-IgE (5-7) and other nonimmunologic
stimuli (5-8). However, HSyMC differ from mast cells
isolated from other human tissues in a variety of immunologic and biochemical ways (4,9). This implies that the
DE PAULIS ET AL
470
local microenvironment influences their phenotypic and
biochemical characteristics.
Recent studies have extensively characterized in
situ and in vitro some of the immunologic and biochemical features of HSyMC (4,9). However, except for two
studies (6,7), the pharmacologic modulation of HSyMC
is largely unexplored. Indeed, several fundamental questions concerning the pharmacologic effects of antiinflammatory and immunosuppressive medications on the
activation and modulation of synovial mast cells have
not been addressed or studied in detail (4).
The present study was designed to ascertain
whether various nonsteroidal antiinflammatory drugs
(NSAIDs; nimesulide, acetylsalicylic acid [ASA], piroxicam, and diclofenac) and immunosuppressive drugs
(cyclosporins A and H [CSA and CSH], FK-506 and
rapamycin, and methotrexate [MTX]), which are widely
used in the treatment of patients with rheumatic diseases, modulate the in vitro release of preformed (histamine and tryptase) and de novo-synthesized (prostaglandin D, [PGD,] and leukotriene C, [LTC,])
proinflammatory mediators from HSyMC.
MATERIALS AND METHODS
Reagents. The following were purchased from the
companies indicated: 60% HCIO, (Baker, Deventer, The
Netherlands); adenosine, ASA, bovine serum albumin (BSA),
PIPES, hyaluronidase, chymopapain, diclofenac, elastase type
I, synthetic LTC,, piroxicam, PGD,, and substance P (Sigma,
St. Louis, MO); collagenase (Worthington, Freehold, NJ);
5’-N-ethylcarboxamide adenosine (Byk Gulden Italia, Milan,
Italy); R-phenylisopropyl adenosine (Boehringer Mannheim,
San Diego, CA); Hanks’ balanced salt solution and fetal calf
serum (FCS) (Gibco, Grand Island, NY);DNase I and pronase
(Calbiochem, La Jolla, CA); RPMI 1640 with 25 mM HEPES
buffer and Eagle’s minimum essential medium (Flow Laboratories, Irvine, Scotland); Percoll (Pharmacia, Uppsala, Sweden); 3H-labeled LTC, (39.3 Ci/mmole) and 3H-labeled PGD,
(210 Ci/mmole) (New England Nuclear, Boston, MA); and
nimesulide (LPB Sandoz, Milan, Italy).
Rabbit anti-human FCEantibody was a generous gift
from Drs. Teruko and Kimishige Ishizaka (La Jolla Institute
for Allergy and Immunology, La Jolla, CA). The rabbit antisulfidopeptide LTC, and rabbit anti-PGD, antisera were
kindly donated by Dr. Laurence M. Lichtenstein (Johns Hopkins University, Baltimore, MD). The tryptase radioimmunoassay (RIA) kit (Pharmacia Tryptase RIACT 50) was donated
by Kabi Pharmacia (Milan, Italy). CSA and CSH were generous gifts from Drs. D. Romer and E. Rissi (Sandoz, Basel,
Switzerland).
Buffers. The PIPES buffer used in these experiments
was made up of 25 mM PIPES, pH 7.37 (110 mM NaCI, 5 mM
KCl). Buffer P2CG contained PIPES buffer plus 2 mM CaCI,
and 1 gm/liter dextrose (10); the pH was titrated to 7.4 with
sodium bicarbonate. Buffer PGMD contained PIPES buffer,
pH 7.37, plus 0.25 gm/liter MgC1,*6H,O, 10 mg/liter DNase,
and 1 gm/liter gelatin.
Isolation and partial purification of HSyMC. The
synovial tissue used in this study was obtained from 27-66year-old patients with osteoarthritis or rheumatoid arthritis,
who were undergoing synovectomy at the Division of Orthopedic Surgery, University of Naples Federico II. None of the
synovial tissue donors had been treated with corticosteroids or
immunosuppressive drugs (azathioprine, cyclophosphamide,
MTX, CSA, etc.), gold salts, D-penicillamine, hydroxychloroquine, or sulfasalazine for at least 7 days before surgery. In
addition, the donors did not take any NSAIDs for at least 2
days before surgery.
Resected joint tissue was immediately immersed in
cold (4°C) PIPES buffer and processed within 2 hours of
removal, using a recently described technique (9). Fat, cartilage, and fibrous tissue were removed. The tissue was finely
minced with scissors into 2-5-mm fragments, suspended in
PIPES buffer (10 ml per 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 (I mg/ml) and pronase
(0.5 mg/ml) in 1 ml of Tyrode’s buffer per gram of 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
200p-pore Nytex cloth (Tetko, Elmsford, NY), centrifuged
(200g for 8 minutes at 22”C), and washed twice with PIPES
buffer. Cells were cultured for up to 4 hours in RPMI 1640
containing 25 mM HEPES, 2 mM I-glutamine, 1% gentamicin,
and 10% FCS.
At the end of incubation, the cells were harvested by
centrifugation (200g for 8 minutes at 22”C), washed 3 times in
PIPES, and resuspended in P2CG. Yields with this technique
ranged between 0.1 X lo6 and 0.9 X lo6 mast cells/gm of wet
tissue, and purities ranged between 1% and 8%. HSyMC were
purified further as previously described (9). The viability of
mast cells was >95%. The purity of these populations ranged
from 14% to 64%.
Histamine release from HSyMC. Cells (-3 X lo4 mast
cells/tube) were resuspended in P2CG, and 0.3 ml 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 (11). At the end of this step,
the reaction was stopped by centrifugation (1,000g for 2
minutes at 22”C), and the cell-free supernatants were stored
(at -70°C) for subsequent assay of histamine, tryptase, and
LTC, or PGD, content.
The cell-free supernatants were assayed for histamine
with an automated fluorometric technique (12). Total histamine content was assessed by lysis induced by incubating cells
with 2% HC10,. To calculate histamine release as a percentage of the total cellular histamine, the “spontaneous” release
of histamine from mast cells (4-12% of the total cellular
histamine) was subtracted from both the numerator and the
denominator (13). All values are based on the mean of
duplicate or triplicate determinations. Replicates differed in
histamine content by (10%.
DRUG EFFECTS ON HUMAN SYNOVIAL MAST CELLS
RIA of tryptase. Total tryptase content was assessed by
lysis induced by incubating cells with 100 pl of Triton X-100
(0.1%). Tryptase was analyzed by a solid-phase RIA (Pharmacia Tryptase RIACT 50); duplicate samples or 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 (14). After washing the tubes 3 times with
2 ml of saline at 22"C, bound radioactivity was counted and
gave a linear concentration-response curve within a range of
2-50 pgfliter.
RIA of LTC, and PGD,. In some experiments, 200-pl
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. To minimize degradation of PGD,, the compound was assayed with a previously
described RIA (15) within 24 hours of the experiment (16).
The anti-PGD, antibody is highly selective, with <1% crossreactivity with other eicosanoids (15). The rabbit anti-LTC,
antiserum has been characterized and its cross-reactivity for
heterologous ligand described (17). Supernatants of HSyMC
incubated with buffer or with the drugs we examined were
separated by high-performance liquid chromatography (10,ll)
to evaluate the possibility that these compounds might interfere with the catabolism of LTC, and PGD,. None of the
compounds examined appeared to interfere with peptide leukotrienes and PGD, degradation.
Statistical analysis. The results are expressed as the
mean -t SEM. Comparisons between unpaired groups were
analyzed for significance with the Mann-Whitney U test;
comparisons between pairs of matched groups (e.g., inhibition
of histamine and PGD, release in the same cell populations)
were analyzed with the Wilcoxon matched-pairs test; comparisons between treatments ( e g , CSA versus CSH) were analyzed with the Friedman nonparametric two-way analysis of
variance (18).
RESULTS
Effects of NSAIDs on IgE-mediated histamine
release from HSyMC. NSAIDs are widely prescribed for
patients with rheumatic diseases (19). The NSAIDs that
are currently available come from a variety of chemical
classes with different physicochemical properties
(19,20). The vast majority of these drugs are weakly
acidic, with ionization constants (pKJ that range from 3
to 5 (19). Nimesulide is a sulfonanilide NSAID (19,21)
that is used in the treatment of various inflammatory
diseases (22) and is chemically unrelated to other acidic
NSAIDs, such as ASA, indomethacin, diclofenac, and
piroxicam (19). We compared the effects of nimesulide
with ASA, diclofenac, and piroxicam on the immunologic release of histamine from HSyMC. HSyMC were
preincubated (15 minutes at 37°C) with increasing concentrations (10-9M-10p4M)of nimesulide, ASA, diclofenac, and piroxicam and then challenged with antiIgE (1 pg/ml). The NSAID concentrations used
471
HSyMC
-
0 Nimesulide
- 0 Diclofenac
0 Piroxicarn
ASA
+
H
I
10-~
I
I
10-~
lo-*
10-~
lo-'
Drug Concentration (M)
Figure 1. Effects of acetylsalicylic acid (ASA), piroxicam, diclofenac,
and nimesulide on IgE-mediated release of histamine from human
synovial mast cells (HSyMC). Cells were preincubated for 15 minutes
at 37°C with the indicated concentrations of drugs and then challenged
(30 minutes at 37°C) with anti-IgE (1 pg/ml). Values are the mean ?
SEM of 7 experiments in which anti-IgE induced the release of 10.4 t
1.1% of the total histamine content. * = P < 0.01 and ** = P < 0.001
versus anti-IgE alone.
included those reached in vivo during treatment with
nimesulide (5 X 10-5-10-4M), ASA (-lOP4M), diclofenac (5 X 10p7-10p6M), and piroxicam (10-'-5 X
10p6M) (19,22-24) and are known to inhibit cyclooxygenase activity (22-26). At these concentrations neither
nimesulide, ASA, diclofenac, nor piroxicam affected the
spontaneous release of lactate dehydrogenase or histamine (data not shown).
Nimesulide
(10p7-10p4M)
caused
a
concentration-dependent inhibition (3-90%) of antiIgE-induced histamine release (Figure l) , with maximal
inhibition at 10-4M (mean ? SEM 90.0 ? 8.4%) (P <
0.001). In contrast ASA and piroxicam had no effect on
HSyMC activated by anti-IgE, whereas diclofenac
( z ~ O - ~ M had
)
a modest inhibitory effect. The time
course of the effect of nimesulide on IgE-mediated
histamine release from HSyMC was rapid. Nimesulide
induced a time-dependent decrease in IgE-mediated
histamine release that was maximal at -15 minutes
(data not shown). Therefore, in all experiments, cells
were incubated with nimesulide and other NSAIDs for
15 minutes at 37°C before challenge with different
stimuli. Nimesulide o r other NSAIDs were present
throughout the period of incubation with these stimuli.
DE PAULIS ET AL
472
HSyMC
**
**
0 Nirnesulide
Diclofenac
1
10-~
I
I
IO-~
lo-’
I
I
10-~
I
10-~
Drug Concentration [MI
Figure 2. Effect of acetylsalicylic acid (ASA), piroxicam, diclofenac,
and nimesulide on IgE-mediated release of prostaglandin D, (PGD,)
from human synovial mast cells (HSyMC). Cells were preincubated for
15 minutes at 37°C with the indicated concentrations of drugs and then
challenged (30 minutes at 37°C) with anti-IgE (1 pg,/ml). Values are
the mean + SEM of 4 experiments in which anti-IgE induced the
release of 109.2 2 28.2 ng of PGDd106 cells. * = P < 0.01 and ** =
P < 0.001 versus anti-IgE alone.
Effects of NSAIDs on IgE-mediated release of
PGD, from HSyMC. IgE-mediated activation of HSyMC
results in the de novo synthesis and release of PGD,
(6,9), a proinflammatory mediator implicated in the
pathogenesis of several inflammatory arthritides (4).
The pharmacologic modulation of de novo-synthesized
mediators from human basophils and mast cells does not
always parallel that of preformed mediators (27). Figure
2 compares the effects of ASA, diclofenac, nimesulide,
and piroxicam on the IgE-mediated release of PGD,
from HSyMC.
ASA ( 10-5-10-4A4) caused a concentrationdependent inhibition (3040%) of the IgE-mediated de
novo synthesis of PGD, by HSyMC. Diclofenac and
piroxicam were potent inhibitors of IgE-mediated synthesis of PGD, by HSyMC. The inhibition caused by
diclofenac ranged from 28% at 10p8Mto 96% at lOP5M,
with a mean t SEM concentration producing 50%
inhibition (IC,,J of 3.4 3.3 X 10p7M.The inhibition of
PGD, synthesis caused by piroxicam ranged from 13%
at lO-*M to 94% at lo-”, with an IC,, of 1.9 ? 0.8 x
10p7M. Nimesulide (10p7-10-4M) also caused a
concentration-dependent inhibition of PGD, synthesis.
The inhibition ranged from 36% at 10p7M to 90% at
*
lO-,M, with an IC,, of 2.4 2 0.8 X 10-7M, which is
significantly lower than that calculated for the corresponding inhibition of histamine release (8.0 2 4.4 X
10-6A4) (P < 0.01).
Effects of NSAIDs on IgE-mediated LTC, release
from HSyMC. IgE-mediated activation of HSyMC results in the de novo synthesis and release of LTC, (6,9),
a proinflammatory and vasoactive mediator implicated
in the pathogenesis of inflammatory arthritides (28).
Figure 3 shows that nimesulide (10-7-10-511/1) caused a
concentration-dependent inhibition of the IgE-mediated
de novo synthesis of LTC, by HSyMC from 4 donors.
The inhibition ranged from 53% at 10-6M to 62% at
lOP5M,with an IC5, of 5.0 2 4.5 X 10-%4. Leukotrienes
D, and E, were undetectable in control and in
nimesulide-treated supernatants (data not shown);
therefore, the inhibitory effect presumably was not due
to an increased catabolism of LTC,. Preincubation of
HSyMC with diclofenac and piroxicam (1 0 - 9 - 1 0 - 5 ~
had little or no effect on LTC, release from HSyMC
activated by anti-IgE.
Effects of nimesulide on the release of tryptase
from HSyMC. Mast cell proteases (tryptase, etc.) can
cause significant remodeling of extracellular matrix proteins (4,29). Activation of HSyMC with anti-IgE caused
the release of tryptase in parallel with that of histamine
HSyMC
/
10-9
10-8
10-7
Al
10-6
1o - ~
Drug Concentration [MI
Figure 3. Effects of piroxicam, diclofenac, and nimesulide on IgEmediated release of leukotriene C, (LTC,) from human synovial mast
cells (HSyMC). Cells were preincubated for 15 minutes at 37°C with
the indicated concentrations of drugs and then challenged (30 minutes
at 37°C) with anti-IgE (1 pdml). Values are the mean 2 SEM of 3
experiments in which anti-IgE induced the release of 82.8 5 23.8 ng of
LTCJlO‘ cells. ** = P < 0.001 versus anti-IgE alone.
DRUG EFFECTS ON HUMAN SYNOVIAL MAST CELLS
HSvMC
d
I
IO-~
I
I
lo-?
I
10-~
lo-‘
Nimesulide [MI
Figure 4. Effects of nimesulide on IgE-mediated release of tryptase,
histamine, and prostaglandin D, (PGD,) from human synovial mast
cells (HSyMC). Cells were preincubated for 15 minutes at 37°C with
the indicated concentrations of nimesulide and then challenged (30
minutes at 37°C) with anti-IgE (1 pdml). Values are the mean 2 SEM
of 5 experiments in which anti-IgE induced the release of 12.1 Z 2.3%
of the total histamine content, 12.6 2 1.8 pg of tryptase/lO’ cells, and
86.4 2 19.7 ng of PGD2/106cells. * = P < 0.01 and ** = P < 0.001
versus anti-IgE alone.
(9). Figure 4 shows that nimesulide (10p6-10p4w
caused a concentration-dependent inhibition of IgEmediated release of tryptase and histamine. The inhibition ranged from 5% at 1OP6Mto 80% at 10-4M.
In these experiments, nimesulide also caused a
concentration-dependent inhibition of the IgE-mediated
de novo synthesis of PGD, by HSyMC.
Effects of NSAIDs on non-IgE-mediated histamine release from HSyMC. Substance P induces histamine release from HSyMC by interacting with specific
membrane receptors independent of the IgE receptors
(9). To assess whether the inhibitory effect of nimesulide
and NSAIDs on mediator release from HSyMC was
stimulus specific, we evaluated their effects on the
release of histamine induced by substance P. After
incubation (15 minutes at 37°C) with increasing concentrations ( 10-6-10-4M) of nimesulide, HSyMC were
challenged (30 minutes at 37°C) with substance P
(10-4M). In 3 experiments, nimesulide had no effect on
substance P-induced histamine release from HSyMC.
Similar results were obtained with ASA, diclofenac, and
piroxicam (data not shown).
Effects of CSA and CSH on IgE-mediated histamine release from HSyMC. There is increasing evidence
that low-dose CSA is effective in the treatment of
473
patients with rheumatoid arthritis (30-32) and other
inflammatory arthritides (33). In this series of experiments, we compared the effects of CSA and CSH, which
bind with different affinities to cyclophilin (34), on the
release of inflammatory mediators from HSyMC in
order to evaluate their roles in this process. The concentrations of CSA used in these experiments (2.4-800 “1M)
included those reached in vivo during CSA treatment
(10-100 nM) (35,36) and did not induce the spontaneous
release of lactate dehydrogenase or of histamine from
HSyMC (data not shown).
Preincubation of HSyMC for 5 minutes with CSA
caused a concentration-dependent inhibition of histamine release induced by anti-IgE (Figure 5). The inhibition varied from 5% at 24 nM to -60% at 800 nM. In
contrast, CSH, which has an extremely low affinity for
cyclophilin (34), did not inhibit the anti-IgE-mediated
release of histamine from HSyMC. Neither CSA (24800 nM) nor CSH (24-800 nM) inhibited the release of
histamine from HSyMC induced by substance P (data
not shown).
Effects of FK-506 and rapamycin on IgEmediated histamine release from HSyMC. FK-506, a
carbocyclic lactone-lactane isolated from Streptomyces
tsukubaensis (37), possesses immunosuppressive activi-
HSyMC
0 CsA
++
0 CsH
-
-0
I
I
I
24
80
240
I
800
Drug Concentration [nMJ
Figure 5. Effects of various concentrations of cyclosporin A (CsA)
and cyclosporin H (CsH) on histamine release from human synovial
mast cells (HSyMC). Cells were preincubated for 15 minutes at 37°C
with the indicated concentrations of CsA or CsH and then challenged
(30 minutes at 37°C) with anti-IgE (1 pdml). Values are the mean ?
SEM of 3 experiments in which anti-IgE induced the release of 19.2 2
6.9% of the total histamine content. * = P < 0.01 and ** = P < 0.001
= P < 0.01 versus CsH.
versus anti-IgE alone;
+
DE PAULIS ET AL
474
HSYMC
_I
0 FK-506
-
+*
MRapamycin
c o
c
MM
-I
I
I
I
I
0.3
1
3
10
30
I
/
I
I
100 300 1000
Drug Concentration hMl
Figure 6. Effects of various concentrations of FK-506 and rapamycin
on histamine release from human synovial mast cells (HSyMC). Cells
were preincubated for 15 minutes at 37°C with the indicated concentrations of FK-506 or rapamycin and then challenged (30 minutes at
37°C) with anti-lgE (1 pg/ml). Values are the mean ? SEM of 4
experiments in which anti-IgE induced the release of 19.4 2 1.6% of
the total histamine content. ** P < 0.001 versus anti-IgE alone.
ties with a potency that is 10-100 times greater than that
of CSA (37). Rapamycin, a macrolide isolated from
Streptomyces hygroscopicus, has some immunosuppressive properties (38). Although rapamycin is structurally
related to FK-506 (39), the immunosuppressive activities
of the two drugs are significantly different (40,41). There
is preliminary evidence that FK-506 and rapamycin are
effective in the treatment of experimental arthritis
(42,43). Despite the remarkable immunosuppressive
properties of FK-506 and rapamycin, the effects of these
molecules on human inflammatory synovial cells have
not been characterized. We compared the effects of
increasing concentrations of FK-506 on histamine release from HSyMC challenged in vitro with anti-IgE.
The concentrations of FK-506 (0.3-30 “M) used in these
experiments included those reached in vivo during FK506 treatment (3-8 nM) (44) and did not affect the
spontaneous release of lactate dehydrogenase or histamine from HSyMC (data not shown).
Preincubation of HSyMC for 5 minutes with
FK-506 caused a concentration-dependent inhibition of
histamine release induced by anti-IgE. Figure 6 shows
that maximal inhibition of 72.3 ? 7.3% (mean ? SEM)
was obtained at 30 nM,with IC,, values of 2.9 ? 2.3 nM.
In contrast, rapamycin (30-1,000 nM),which binds with
high affinity to FK-binding proteins (FKBP) (41), which
are the receptors for both macrolides, caused no inhibition of histamine release induced by anti-IgE. We also
assessed the effect of increasing concentrations of FK506 and rapamycin on histamine release from HSyMC
induced by substance P. In these experiments, FK-506
(0.3-30 “M) and rapamycin (30-1,000 nM) had no effect
on the release of histamine from HSyMC caused by
substance P (data not shown). These results suggested
that the inhibition of histamine release from HSyMC
induced by FK-506 was dependent on the nature of the
stimulus.
Effects of MTX and adenosine on IgE-mediated
histamine release from HSyMC. Although low-dose
MTX is considered by most rheumatologists to be the
drug of choice in the treatment of rheumatoid arthritis
(45), its mechanism(s) of action is still unknown. It has
been suggested that because MTX increases extracellular adenosine at sites of inflammation (46), it might exert
antiinflammatory activity, because high concentrations
of adenosine inhibit histamine release from certain types
of human mast cells (47). However, there is no direct
experimental evidence of the effects of MTX on HSyMC
(4). To assess whether the antiinflammatory effect of
MTX on mediator release from HSyMC was mediated
by high concentrations of adenosine, we evaluated their
in vitro effects on the release of histamine induced by
anti-IgE. After incubation for different times (15-60
minutes at 37°C) with increasing concentrations (lop810p4M) of MTX, HSyMC were challenged (30 minutes
at 37°C) with anti-IgE. The results of 3 different experiments indicated that MTX had no effect on the release
of histamine from HSyMC (data not shown).
In parallel experiments, adenosine and its stable
analogs, R-phenylisopropyl adenosine (R-PIA) and 5’N-ethylcarboxamide adenosine (NECA), were incubated (15 minutes at 37°C) with HSyMC before challenge with anti-IgE. Figure 7 shows that adenosine,
R-PIA, and NECA (10-6-10-4M) enhanced the antiIgE-induced release of histamine from HSyMC in a
concentration-dependent manner.
DISCUSSION
In this study, we characterized the pharmacologic
effects of several classes of antiinflammatory and immunosuppressive drugs on the release of preformed (histamine and tryptase) and newly generated (PGD, and
LTC,) mediators from HSyMC activated by immunologic (anti-IgE) and nonimmunologic (substance P)
stimuli. A striking feature of our study was the heterogeneity of the pharmacologic effects exerted by the
DRUG EFFECTS ON HUMAN SYNOVIAL MAST CELLS
HSyMC
**
T
0 Adenosine
0 R-PIA
0 NECA
I
I
10-7
10-6
I
1o - ~
I
10-4
Adenosine/Analogues [Ml
Figure 7. Effects of 5'-N-ethylcarboxamide adenosine (NECA),
R-phenylisopropyl adenosine (R-PIA), and adenosine on IgEmediated histamine release from human synovial mast cells (HSyMC).
Cells were preincubated for 15 minutes at 37°C with the indicated
concentrations of adenosine or analog and then challenged (30 minutes at 37°C) with anti-IgE (1 p g h l ) . Values are the mean 2 SEM of
4 experiments in which anti-IgE induced the release of 18.3 2 2.8% of
the total histamine content. ** = P < 0.001 versus anti-IgE alone.
antiinflammatory and immunosuppressive drugs we examined on the release of mediators from HSyMC. Four
levels of pharmacologic heterogeneity were identified,
the first being among the different NSAIDs examined.
For instance, pharmacologic concentrations of ASA,
diclofenac, and piroxicam had little or no effect on the
immunologic release of preformed mediators from
HSyMC, whereas pharmacologic concentrations of
nimesulide inhibited the release of histamine and
tryptase from HSyMC.
A second level of heterogeneity was found with
respect to the proinflammatory mediators examined. In
fact, although the NSAIDs examined, with the exception
of nimesulide, had little or no effect on the release of
preformed mediators (histamine and tryptase), they
caused concentration-dependent inhibition of PGD, release. Moreover, when the de novo synthesis of LTC, by
HSyMC was examined, only nimesulide, unlike other
NSAIDs, inhibited the immunologic release of LTC,
from HSyMC.
The third level of pharmacologic heterogeneity
became apparent when different immunosuppressive
drugs were evaluated. We examined 2 classes of immunosuppressive drugs: CSA and CSH as well as FK-506
and rapamycin, which bind to a family of cytoplasmic
475
receptors, immunophilins, and MTX. Low doses of
MTX (45) or of CSA (30-32) can be effective treatments
in some patients with arthritis. There is also evidence
that FK-506 and rapamycin exert antiinflammatory effects in some models of arthritis (42,43). Although
pharmacologic concentrations of CSA and FK-506 inhibited the release of proinflammatory mediators from
HSyMC, CSH and rapamycin had no effects. Incubation
of HSyMC for different times with a wide range of
concentrations of MTX had no effect on the release of
mediators from these cells. These results provide evidence of the heterogeneity of the antiinflammatory
activity of immunosuppressive drugs on HSyMC.
A fourth level of heterogeneity concerns the
types of stimuli examined. When cross-linking of IgE
receptors was used as the activating signal, nimesulide,
CSA, and FK-506 were potent inhibitors of mediator
release from HSyMC. In contrast, the engagement of
substance P receptors on HSyMC was unaffected by
preincubation with NSAIDs, CSA, and FK-506. These
results provide evidence that the inhibitory effect of
different classes of the compounds on the activation of
HSyMC has a high degree of selectivity, depending on
the nature of the stimulus. This observation is probably
clinically relevant because there is increasing evidence
that neurogenic inflammation can contribute to the
clinical features of arthritis (48,49).
An interesting finding of this study was the
remarkable difference between nimesulide and the 3
other NSAIDs we examined in the modulation of mediator release from HSyMC. Nimesulide inhibited the
release of histamine, tryptase, LTC,, and PGD, from
HSyMC, whereas ASA, diclofenac, and piroxicam affected only the release of PGD,. These findings extend
previous observations that nimesulide inhibits the immunologic release of proinflammatory mediators from human basophils and mast cells isolated from lung and skin
tissues (50). Nimesulide is effective in the treatment of
some patients with inflammatory arthritides (19,21,22).
Our results suggest that the beneficial effect of nimesulide in these patients could be due, at least in part, to the
inhibitory effect of pharmacologic concentrations of
nimesulide on the release of preformed and de novosynthesized mediators from HSyMC. The possibility that
nimesulide inhibits the release of histamine and eicosanoids from HSyMC by activating/inhibiting signals
other than those shared by other NSAIDs is interesting.
The main recognized mechanism of the antiinflammatory activity of NSAIDs appears to be their ability to
inhibit arachidonic acid metabolism through the cyclo-
476
oxygenase (COX) pathway (19). Our results seem to
imply an additional mechanism of action of nimesulide.
The finding that some NSAIDs inhibit, and that
others do not affect, synovial mast cell-induced inflammation coincides with clinical data. Indeed, some
NSAIDs are ineffective at alleviating the symptoms of
some aspects of synovial inflammation and, perhaps,
may even be deleterious (2031). In addition, the in vivo
administration of NSAIDs causes different pharmacologic effects in experimental synovial mast cell-mediated
arthritis (51). Our results demonstrate the heterogeneity
of the effects of NSAIDs on HSyMC, and suggest that
studies should be conducted to characterize the antiinflammatory activities of nimesulide in vivo and in vitro
and to determine its biochemical mechanism(s) of action. The effect of nimesulide on the 5-lipoxygenase and
on COX-1 and COX-2 activity in HSyMC is presently
under investigation in our laboratory.
Our results demonstrate that CSA, but not CSH,
exerts a potent antiinflammatory effect on the immunologic release of chemical mediators of inflammatory
reactions from HSyMC. Characterization of the molecular site of action of CSA is critical for the rational and
optimal therapeutic use of this agent in various forms of
arthritis (30-33) and for the search for new CSA-like
drugs. Two mechanisms whereby cyclosporins exert their
antiinflammatory activity have been proposed. One is
mediated by binding to cyclophilin (33) and inhibition of
the phosphatase IIB activity of calcineurin (52). Alternatively, CSH could be a competitive antagonist of
formyl peptide receptors on human inflammatory cells
(53). However, HSyMC do not possess FMLP receptors
(9), which seems to discount the latter mechanism. The
former hypothesis is compatible with the inhibitory
effect of FK-506 on mediator release from HSyMC.
FK-506 binds with high affinity to FKBP, and the
FK-506-FKBP complex inhibits the phosphatase IIB
activity of calcineurin (54). This hypothesis is supported
by the lack of antiinflammatory effect of rapamycin,
which binds to FKBP (40,41) but does not inhibit
calcineurin activity (55).
Taken together, the observation that CSA and
FK-506 inhibit mediator release from HSyMC, presumably by inhibiting phosphatase IIB activity, suggests that
drugs binding to this family of proteins, cyclophilin and
FKBP, represent a new class of potent antiinflammatory
agents. Low doses of CSA are effective in the treatment
of some patients with rheumatoid arthritis (30-32) and
other inflammatory arthritides (33). Our results suggest
that the beneficial effects of CSA in these patients are
DE PAULIS ET AL
due, to some extent, to the inhibitory effect of pharmacologic concentrations of CSA on the release of proinflammatory mediators from HSyMC.
Low doses of MTX are effective in the treatment
of rheumatoid arthritis (45). The mechanism(s) of action
of MTX in these patients is unknown, although it has
been suggested that MTX may suppress mast cell function by increasing extracellular adenosine at the site of
inflammation (4,46). Our results indicate that incubation
of HSyMC for different times with a wide range of
concentrations of MTX had no effect on the immunologic
release of histamine from these cells. In addition, high
concentrations of adenosine and its stable analogs potentiated the release of histamine from HSyMC. According to
these findings, it is unlikely that the antiinflammatory
effect of MTX is mediated by the adenosine-synovial mast
cell network.
The enhancing effect of adenosine and its analogs on the activation of HSyMC emphasizes the heterogeneity of the modulation of human mast cells isolated
from different anatomic sites. For instance, adenosine
and its analogs potentiate the release of mediators from
human lung mast cells (47,56), whereas high concentrations of adenosine inhibit histamine release from human
skin mast cells (47). We found that adenosine and its
analogs enhance synovial mast cell degranulation. These
results demonstrate that extracellular adenosine concentrations can exert opposite effects on the activation of
human mast cells at different sites of inflammation.
Very few studies have examined the in vitro and
in vivo effects of different compounds on human synovial mast cells. In one study, in vivo administration of
NSAIDs to patients with rheumatoid arthritis and osteoarthritis had no effect on the release of mediators from
mast cells isolated from synovium (7). In another study,
auranofin inhibited the in vitro release of histamine
from immunologically activated HSyMC (6).
In this study, we examined systematically the
effects of different classes of drugs that are widely used
in the treatment of arthritis on the immunologic and
nonimmunologic release of preformed and de novosynthesized mediators from HSyMC. Our results reveal
the hitherto-unappreciated complex heterogeneity of
the effects of antiinflammatory and immunosuppressive
drugs on mediator release from HSyMC. This experimental model might be useful for identifying pharmacologic agents that could selectively act on these cells and
for exploring the effects of compounds on cytokines
synthesized by HSyMC.
DRUG EFFECTS ON HUMAN SYNOVIAL MAST CELLS
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