Synthesis of Platelet Activating Factor by Ocular Tissue From Inflamed Eyes James T. Rosenbaum, MD; Richard S. Boney; John R.
\s=b\ Platelet
activating factors (PAFs) are lipids with properties that suggest a major role in inflammation. We have previously implicated a
family
of ether
PAFs in ocular inflammation based on the inhibition of several rabbit models of iritis with a specific PAF receptor antagonist. We have tested ocular tissues for the ability to synthesize PAF. Iris, ciliary body, cornea, and/or retina were carefully dissected from New Zealand white rabbits, and tissue from four eyes was pooled. Tissues were stimulated with calcium ionophore (10 \g=m\mol/L), and supernatants were extracted with chloroformmethanol. Platelet-aggregating activity was found in the chloroform phase in 2 of 9, 1 of 8, 0 of 9, and 3 of 9 studies
involving iris, retina, ciliary body, or corTwenty-four hours afinjection of 125 ng of
nea, respectively. ter the intravitreal
'" he
activating factors family of ether lipids strongly implicated in inflam¬
platelet
(PAFs)
are a
that are mation. The
designation PAF does not
the full biologic activity of this mediator. Platelet activating factor induces vascular permeability far more potently than histamine,1 acts as a chemoattractant for neutrophils,2 stimulates a variety of neutrophil fune-
acknowledge
Accepted for publication October 1, 1990. From the Departments of Medicine (Dr Rosenbaum), Ophthalmology (Drs Rosenbaum and Samples and Mr Boney), and Cell Biology (Dr Rosenbaum), Oregon Health Sciences University, Portland, and the Department of Medicine, University of California, San Francisco (Dr Valone). Reprint requests to Department of Medicine, Oregon Health Sciences University, 3181 SW Sam
Jackson Park Rd,
Rosenbaum).
Portland, OR 97201 (Dr
Samples, MD; Frank H. Valone, MD
endotoxin, aggregating activity was consistently detectable from supernatants of stimulated iris and ciliary body, occasionally present from stimulated retina
but not detectable from cornea. The shape of the aggregation curve resembled that produced by 0.5 to 2.0 ng of authentic PAF. Moreover, the aggregation could be completely inhibited by a PAF receptor antagonist and the aggregating activity chromatographed identically on high-performance liquid chromatography to a PAF standard. These studies indicate that PAF-like activity could be detected from several ocular tissues subsequent to inflammation. Iris, ciliary body, retina, vascular endothelium, and/or leukocytes could each contribute to the presence of this inflammatory mediator.
(Arch Ophthalmol. 1991;109:410-413)
tions,3 induces the synthesis of arachidonic acid metabolites,4 and triggers the synthesis of interleukin 1 and tu¬ mor
necrosis factor.5" Platelet activat¬
ing factor is synthesized by a wide variety of tissues, including neutro¬ phils,' monocytes,7 vascular endotheli¬ um,8 and kidney.9 We have previously reported that a PAF receptor antagonist reduces ocu¬
lar inflammation in several rabbit mod¬ els of iritis.1IM2 Ocular vascular perme¬ ability and/or cellular infiltration can be reduced by a PAF receptor antago¬ nist for inflammation subsequent to intravenous endotoxin, intravitreal in¬ terleukin 1, an anterior chamber para¬ centesis, or an ocular reverse passive Arthus reaction. Platelet activating factor has also been implicated in such
Downloaded From: by a University of Otago Library User on 01/18/2019
diverse disease processes
as
corneal
injury,13 immune-complex deposition glomerulonephritis," asthma,10 shock,"
and ischemie necrosis of the bowel.17 All pharmacologie mediators, howev¬ er, have the potential to act in a non¬ specific fashion. Therefore, to test fur¬ ther the potential role for PAF to contribute to ocular inflammation, we have assessed the ability of rabbit ocu¬ lar tissue to synthesize PAF. MATERIALS AND METHODS Rabbits
Two-kilogram New Zealand white rab¬ bits of either sex were purchased from Western Oregon Rabbit Company (Philo¬ math, Ore) and housed in the animal care facilities of the Oregon Health Sciences University. The handling of rabbits con¬ formed to the Association for Research in Vision and Ophthalmology policy on animal care. A total of 38 rabbits were studied. Eighteen of these received endotoxin as described below. Tissue Dissection were
killed
by
overdose of enucleat¬ ed. A careful dissection was then per¬ formed. The initial incision into the eye was approximately 3 mm posterior to the lim¬ bus. The iris and ciliary body were grasped, removed, and placed into a balanced salt solution. A curved, sharp scissors (Vannas, Storz Instruments, St Louis, Mo) was used to separate the iris and ciliary body. The cornea was excised in a conservative fash¬ ion so that only clear cornea and no limbal or conjunctival tissue remained. Retina epi¬ thelium and pigment epithelium were ob¬ tained by teasing them gently from the adjacent choroidal structures. Dissected tissue was suspended in Hanks' balanced salt solution (GIBCO, Grand Is¬ land, NY) containing 0.25% human serum Rabbits
phénobarbital and the
eyes
an
were
Detection of PAF-like Activity From Ocular Tissue* Uninflamed
Inflamed
Ciliary body
3/9 2/9 0/9
0/4 6/6 5/6
Retina
1/8
1/4
Cornea Iris
*PAF indicates platelet-activating factor. Values are pooled tissue with detectable platelet-aggregat¬ ing activity per number of times different pooled tissues were tested. Inflamed tissue was analyzed 24 hours after 125 ng of endotoxin
was
administered ¡n-
travitreally.
(Alpha Therapeutic Corp. Los An¬ geles, Calif) and stimulated with 10 µ /L of a calcium ionophore (A23187, Sigma Chemical Co, St Louis, Mo) for 15 to 20 minutes. Supernatant from stimulated tis¬ sue was extracted by a modified Bligh-Dyer technique, as previously described.'" Fol¬ lowing extraction, the remaining aqueous phase was reextracted with chloroform. albumin
The combined chloroform extracts were stored overnight at 70°C to promote fur¬ ther separation of the chloroform and wa¬ ter. The chloroform phase of the chloroform-methanol extraction was dried under nitrogen and resuspended in 150 to 200 µL of Hanks' balanced salt solution containing 0.25% human serum albumin for platelet aggregation studies. -
as
retina. Panels F and H indicate that this
pe¬
at 1100 rprn to obtain was
or
ear
artery. Nine parts blood was added to one part sodium citrate (3.8%). Blood was cen-
trifuged plasma. Aggregation platelet-rich plasma
present in the supernatant of extracted iris
aggregating activity is completely inhibited by the PAF receptor antagonist.
Platelet Aggregation Rabbit platelets were isolated from ripheral blood drawn from the central
Fig 1. —Panels A through H show representative rabbit platelet aggregation curves. Each aggregation curve represents the change in light transmission (y-axis) vs time (x-axis). Aggre¬ gation is indicated by an increase in light transmission. Each aggregation curve was recorded continuously for 3 minutes after the addition of the stimulus for aggregation. In panels A through C, progressively increased platelet aggregation is noted with increasing quantities of extracted platelet activating factor (PAF). In panel D, extracted supernatant from corneal tissue failed to produce significant platelet aggregation. Panels E and G show that some aggregatory activity is
platelet-rich performed using previously de¬
scribed11' on an aggregometer (model PAP3, Bio-Data, Hatboro, Pa).
High-Performance Liquid Chromatography (HPLC) The chloroform extracts were reex¬ tracted with chloroform:methanol:water (1.1:1.1:1.0, vol/vol/vol), as previously de¬ scribed."" These extracts were dried in a centrifugal evaporator and resuspended in 40 µL of methanol containing 50 µg each of phosphatidylcholine and lysophosphatidylcholine (Sigma Chemical Co) as internal standards for elution times of known com¬ pounds. The extracts were then injected onto an HPLC column (Ultrasphere, Beckman, Palo Alto, Calif), which was developed at a flow rate of 1.5 mL/min with a linear gradient from 96%:4% to 92%:8
\s=b\ Platelet
activating factors (PAFs) are lipids with properties that suggest a major role in inflammation. We have previously implicated a
family
of ether
PAFs in ocular inflammation based on the inhibition of several rabbit models of iritis with a specific PAF receptor antagonist. We have tested ocular tissues for the ability to synthesize PAF. Iris, ciliary body, cornea, and/or retina were carefully dissected from New Zealand white rabbits, and tissue from four eyes was pooled. Tissues were stimulated with calcium ionophore (10 \g=m\mol/L), and supernatants were extracted with chloroformmethanol. Platelet-aggregating activity was found in the chloroform phase in 2 of 9, 1 of 8, 0 of 9, and 3 of 9 studies
involving iris, retina, ciliary body, or corTwenty-four hours afinjection of 125 ng of
nea, respectively. ter the intravitreal
'" he
activating factors family of ether lipids strongly implicated in inflam¬
platelet
(PAFs)
are a
that are mation. The
designation PAF does not
the full biologic activity of this mediator. Platelet activating factor induces vascular permeability far more potently than histamine,1 acts as a chemoattractant for neutrophils,2 stimulates a variety of neutrophil fune-
acknowledge
Accepted for publication October 1, 1990. From the Departments of Medicine (Dr Rosenbaum), Ophthalmology (Drs Rosenbaum and Samples and Mr Boney), and Cell Biology (Dr Rosenbaum), Oregon Health Sciences University, Portland, and the Department of Medicine, University of California, San Francisco (Dr Valone). Reprint requests to Department of Medicine, Oregon Health Sciences University, 3181 SW Sam
Jackson Park Rd,
Rosenbaum).
Portland, OR 97201 (Dr
Samples, MD; Frank H. Valone, MD
endotoxin, aggregating activity was consistently detectable from supernatants of stimulated iris and ciliary body, occasionally present from stimulated retina
but not detectable from cornea. The shape of the aggregation curve resembled that produced by 0.5 to 2.0 ng of authentic PAF. Moreover, the aggregation could be completely inhibited by a PAF receptor antagonist and the aggregating activity chromatographed identically on high-performance liquid chromatography to a PAF standard. These studies indicate that PAF-like activity could be detected from several ocular tissues subsequent to inflammation. Iris, ciliary body, retina, vascular endothelium, and/or leukocytes could each contribute to the presence of this inflammatory mediator.
(Arch Ophthalmol. 1991;109:410-413)
tions,3 induces the synthesis of arachidonic acid metabolites,4 and triggers the synthesis of interleukin 1 and tu¬ mor
necrosis factor.5" Platelet activat¬
ing factor is synthesized by a wide variety of tissues, including neutro¬ phils,' monocytes,7 vascular endotheli¬ um,8 and kidney.9 We have previously reported that a PAF receptor antagonist reduces ocu¬
lar inflammation in several rabbit mod¬ els of iritis.1IM2 Ocular vascular perme¬ ability and/or cellular infiltration can be reduced by a PAF receptor antago¬ nist for inflammation subsequent to intravenous endotoxin, intravitreal in¬ terleukin 1, an anterior chamber para¬ centesis, or an ocular reverse passive Arthus reaction. Platelet activating factor has also been implicated in such
Downloaded From: by a University of Otago Library User on 01/18/2019
diverse disease processes
as
corneal
injury,13 immune-complex deposition glomerulonephritis," asthma,10 shock,"
and ischemie necrosis of the bowel.17 All pharmacologie mediators, howev¬ er, have the potential to act in a non¬ specific fashion. Therefore, to test fur¬ ther the potential role for PAF to contribute to ocular inflammation, we have assessed the ability of rabbit ocu¬ lar tissue to synthesize PAF. MATERIALS AND METHODS Rabbits
Two-kilogram New Zealand white rab¬ bits of either sex were purchased from Western Oregon Rabbit Company (Philo¬ math, Ore) and housed in the animal care facilities of the Oregon Health Sciences University. The handling of rabbits con¬ formed to the Association for Research in Vision and Ophthalmology policy on animal care. A total of 38 rabbits were studied. Eighteen of these received endotoxin as described below. Tissue Dissection were
killed
by
overdose of enucleat¬ ed. A careful dissection was then per¬ formed. The initial incision into the eye was approximately 3 mm posterior to the lim¬ bus. The iris and ciliary body were grasped, removed, and placed into a balanced salt solution. A curved, sharp scissors (Vannas, Storz Instruments, St Louis, Mo) was used to separate the iris and ciliary body. The cornea was excised in a conservative fash¬ ion so that only clear cornea and no limbal or conjunctival tissue remained. Retina epi¬ thelium and pigment epithelium were ob¬ tained by teasing them gently from the adjacent choroidal structures. Dissected tissue was suspended in Hanks' balanced salt solution (GIBCO, Grand Is¬ land, NY) containing 0.25% human serum Rabbits
phénobarbital and the
eyes
an
were
Detection of PAF-like Activity From Ocular Tissue* Uninflamed
Inflamed
Ciliary body
3/9 2/9 0/9
0/4 6/6 5/6
Retina
1/8
1/4
Cornea Iris
*PAF indicates platelet-activating factor. Values are pooled tissue with detectable platelet-aggregat¬ ing activity per number of times different pooled tissues were tested. Inflamed tissue was analyzed 24 hours after 125 ng of endotoxin
was
administered ¡n-
travitreally.
(Alpha Therapeutic Corp. Los An¬ geles, Calif) and stimulated with 10 µ /L of a calcium ionophore (A23187, Sigma Chemical Co, St Louis, Mo) for 15 to 20 minutes. Supernatant from stimulated tis¬ sue was extracted by a modified Bligh-Dyer technique, as previously described.'" Fol¬ lowing extraction, the remaining aqueous phase was reextracted with chloroform. albumin
The combined chloroform extracts were stored overnight at 70°C to promote fur¬ ther separation of the chloroform and wa¬ ter. The chloroform phase of the chloroform-methanol extraction was dried under nitrogen and resuspended in 150 to 200 µL of Hanks' balanced salt solution containing 0.25% human serum albumin for platelet aggregation studies. -
as
retina. Panels F and H indicate that this
pe¬
at 1100 rprn to obtain was
or
ear
artery. Nine parts blood was added to one part sodium citrate (3.8%). Blood was cen-
trifuged plasma. Aggregation platelet-rich plasma
present in the supernatant of extracted iris
aggregating activity is completely inhibited by the PAF receptor antagonist.
Platelet Aggregation Rabbit platelets were isolated from ripheral blood drawn from the central
Fig 1. —Panels A through H show representative rabbit platelet aggregation curves. Each aggregation curve represents the change in light transmission (y-axis) vs time (x-axis). Aggre¬ gation is indicated by an increase in light transmission. Each aggregation curve was recorded continuously for 3 minutes after the addition of the stimulus for aggregation. In panels A through C, progressively increased platelet aggregation is noted with increasing quantities of extracted platelet activating factor (PAF). In panel D, extracted supernatant from corneal tissue failed to produce significant platelet aggregation. Panels E and G show that some aggregatory activity is
platelet-rich performed using previously de¬
scribed11' on an aggregometer (model PAP3, Bio-Data, Hatboro, Pa).
High-Performance Liquid Chromatography (HPLC) The chloroform extracts were reex¬ tracted with chloroform:methanol:water (1.1:1.1:1.0, vol/vol/vol), as previously de¬ scribed."" These extracts were dried in a centrifugal evaporator and resuspended in 40 µL of methanol containing 50 µg each of phosphatidylcholine and lysophosphatidylcholine (Sigma Chemical Co) as internal standards for elution times of known com¬ pounds. The extracts were then injected onto an HPLC column (Ultrasphere, Beckman, Palo Alto, Calif), which was developed at a flow rate of 1.5 mL/min with a linear gradient from 96%:4% to 92%:8
