Transcript Expression of Intracellular Cytokines TNF and IL-4

Effects of Brimonidine, Timolol, and
Fixed–Combination Brimonidine-Timolol
on FcεR1–Stimulated Human
Mast Cells
G. Galatowicz,1 V. Chan,1 M.E. Stern,2
R.M. Schiffman,2 V.L. Calder1
1. UCL Institute of Ophthalmology, London UK; 2. Allergan Inc., Irvine, CA
This study was funded by an unrestricted grant from Allergan, Inc.
G. Galatowicz and V. Chan have no financial relationships to disclose;
M.E. Stern and R.M. Schiffman are employees of Allergan, Inc.;
V.L. Calder is a consultant to Allergan, Inc.
Abstract
Purpose: The IOP-lowering fixed combination of brimonidine/timolol has been associated
with a lower rate of ocular allergy as compared with brimonidine alone. The aim of this
study was to examine the effects of brimonidine, timolol or a combination on resting and
FcεR1- activated human mast cells in vitro.
Methods: Human cord blood CD34+stem-cells (CBMC; 105) were expanded in
Stemspan™ medium with SCF [100ng/ml], IL-6 [50ng/ml] and IL-3 [1ng/ml] during the first
14-days. At week 11, differentiation was confirmed by CD117 (c-kit) and FcεR1
expression. Unstimulated-CBMC (106 cells/mL) were cultured alone or in presence of
brimonidine, timolol, or combination (dilutions of 1:20 – 1:2000). For activation, CBMC
were incubated with 4μg/ml IgE for 16hrs, with drug combinations given 30-min before
adding 25μg/ml anti-IgE-Ab. Culture supernatants were harvested (1hr) for histamine
determination (ELISA). After 24 hr, cells were stained for intracellular expression of TNFa
and IL-4, and analyzed by flow cytometry.
Results: Histamine levels were low in unstimulated cells [9.9 ± 0.7 ng/mL] and in the
presence of drugs. After FceR1 stimulation, histamine levels were increased [36.6 ± 2.1
ng/mL; P < .02] relative to control. In stimulated CBMC, timolol significantly decreased
histamine production (P < .05) at both high and low drug concentrations. The histamine
upregulation seen with brimonidine (P < .05) was reduced in combination with timolol at
low concentrations although this failed to reach statistical significance. Brimonidine,
timolol and the combination of brimonidine and timolol had little or no effect on IL-4 or
TNFa expression at 24 hr in resting or activated CBMC.
Conclusions: In activated CBMC, timolol was selectively inhibitory for histamine
secretion. The brimonidine-mediated upregulation of histamine also appears to be
reduced in the presence of timolol.
Introduction
 The IOP-lowering drugs brimonidine and timolol have been
reported to cause hyper-responsiveness at the ocular surface
when given long term in some cases.
 The fixed combination of 0.2% brimonidine/0.5% timolol is
thought to be associated with a lower rate of ocular surface
involvement when compared with 0.2% brimonidine alone.1
1. Sherwood MB. Arch Ophthalmol. 2006, 124:1230-1238
Purpose
 The aim of this study was to examine the effects of
brimonidine, timolol, and the 2 agents in combination on resting
and FcεR1–activated human mast cells in vitro.
Methods
 Human cord blood CD34+ stem cells (CBMC; 105) were expanded in
Stemspan® medium with SCF (100 ng/mL), IL-6 (50 ng/mL) and IL-3
(1 ng/mL) during the first 14 days.
– At week 11, differentiation was confirmed by CD117 (c-kit) and FcεR1
expression.
 Unstimulated CBMC (106 cells/mL) were cultured alone or in the
presence of brimonidine, timolol, or combination.
– Dilutions of 1:20 to 1:2000 were used.
 For activation, CBMC were incubated with 4 μg/mL IgE for 16 h,
with drug combinations given 30 minutes before adding 25 μg/mL
anti-IgE-Ab.
 Culture supernatants were harvested 1 hour later for histamine
determination by ELISA.
 After 24 hours, cells were stained for intracellular expression of TNFα
and IL-4, and analyzed by flow cytometry.
Mast Cell Stimulation
Wash CBMC and resuspend in Stemspan® + 10% FCS + 100ng/mL SCF
2x105 CBMC/200µl well
Add 4µg/mL IgE
16 hours
Add drugs (T, B, T+B)
30 minutes
Add 25µg/mL anti IgE
1 hour
24 hours
Harvest 70µl cell-free medium for histamine-release assay
±10g/mL BFA 6h before staining
Stain cells for surface markers
(CD54, HLA-DR, CCR5)
T = timolol; B = brimonidine; T+B = timolol and brimonidine combined
Harvest cells for intracellular
cytokine determination
Timolol Blocks the Mast Cell
Response
80.0
Resting cells
Activated cells
Histamine (ng/mL)
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
T+B 1:20
T+B 1:200
T+B 1:2000
B 1:20
B 1:200
B 1:2000
T 1:20
T 1:200
T 1:2000
aIgE
T+B 1:20
T+B 1:200
T+B 1:2000
B 1:20
B 1:200
B 1:2000
T 1:20
T 1:200
T 1:2000
cont
 Histamine levels were low in unstimulated CBMC (9.9  0.7 ng/mL) and in presence of drugs.
 After FceR1 stimulation, histamine levels increased (36.6  2.1 ng/mL; P < 0.02) relative to control.
 In stimulated CBMC, timolol significantly decreased histamine levels (P < 0.05) at both high and
low drug concentrations.
 Histamine upregulation seen with brimonidine (P < 0.05) was reduced in combination with timolol
at low concentrations, although failed to reach statistical significance.
T = timolol; B = brimonidine; T+B = timolol and brimonidine combined
Expression of Cell Surface Markers
Resting
95
Activated
Resting
75
CD54 (ICAM-1)
90
85
80
Activated
CCR5
70
% expression
% expression
100
65
60
55
75
50
T
B
T+B
T
B
T
T+B
B
T+B
T
B
4
47
3
53
3
2
10
CD54 PE
2
10
CD54 PE
15
43
10
9
1
1
10
10
10
% expression
10
44
3
HLA-DR
10
20
Activated
10
Resting
25
Activated
4
Resting
T+B
5
10
0
T
B
T+B
T
B
T+B
1
10
2
10
3
CCR5 FITC
10
4
10
1
10
2
10
3
10
4
CCR5 FITC
Cell Surface Marker Expression Graphs: Open bars – untreated, resting; Closed bars – untreated, activated;
Drug concentrations: (Lighter – dark) 3.4, 4.5, 6.8, 13.6, 34.0 g/ml T; 1.0, 1.3, 2.0, 4.0, 10.0 g/ml B;
T = timolol; B = brimonidine; T+B = timolol and brimonidine combined
 Activation of the mast cells partially increased the level of CD54 expression whereas
HLA-DR did not increase. CCR5 expression was decreased after stimulation. Addition
of any of the drugs had little, if any, effect on these surface molecules.
 Activation of CBMC induces a small upregulation of CD54+ CCR5+ cells. Addition of the
drugs had no affect on CD54 expression either in resting or stimulated cultures.
Expression of Intracellular
Cytokines TNFa and IL-4
2
10
3
10
4
1
10
2
10
3
10
4
IL-4 FITC
4
3
1
10
2
IL-4 FITC
10
3
10
4
2
10
87
1
10
10
1
10
2
IL-4 FITC
10
3
10
4
10
1
10
2
10
IL-4 FITC
 Brimonidine, timolol, and the 2 agents in combination had little
or no effect on IL-4 or TNFa expression at 24 hours in resting
or activated CBMC.
T = timolol; B = brimonidine; T+B = timolol and brimonidine combined
12
10
TNFa PE
2
TNFa PE
10
76
10
10
0.4
10
21
3
10
1
1
1
10
10
Activated
4
4
3
84
2
TNFa PE
2
1
10
IL-4 FITC
14
10
76
10
1
1
10
21
10
TNFa PE
2
10
10
10
Resting + T+B
10
10
65
3
10
31
3
4
4
10
3
10
2
1
TNFa PE
Resting + B
10
Resting + T
Resting
3
10
4
Conclusions
 In activated CBMC, timolol was selectively inhibitory for
histamine secretion.
 Brimonidine-mediated upregulation of histamine secretion also
appears to be reduced in the presence of timolol.