Alkali-Burned Collagen Produces a Locomotory and
Metabolic Stimulant to Neutrophils
Roswell R. Pfisrer, Jeffrey L. Haddox, Robert W. Dodson, and L. Eroles Harkins
Polymorphonuclear leukocytes (PMNs) invade the cornea following an alkali burn apparently undergoing
a respiratory burst and degranulation, which is thought to lead to corneal ulceration. The supernatant
obtained from burned Sigma collagen (Miller type 1) or from bovine cornea produced a significant
locomotory stimulus to PMNs. Citrate inhibited this locomotory stimulus by 69.5% and 98%, respectively.
PMNs were stimulated to undergo a respiratory burst without the concomitant release of/9-glucuronidase
when exposed to the supernatant from alkali-burned commercial collagens, or from bovine or porcine
corneas. This stimulation is reduced by 72% (Sigma collagen) or 89% (bovine cornea) when the supernatant
is dialyzed against distilled water and reinstated when the osmolality is increased. The degree of the
respiratory burst is partially dependent on the volume of the supernatant, the duration of alkali exposure,
and/or the concentration of NaOH used. The respiratory burst of PMNs stimulated by alkali-burned
Sigma collagen supernatant is inhibited by trifluoperazine but not by citrate or EDTA. Light and electron
microscopy of these stimulated PMNs show many large blebs and hairlike projections. The authors
hypothesize that collagen breakdown product(s) from alkali burning might be the initial, or one of the
initial stimuli, for PMN invasion into the cornea and the subsequent activation of the respiratory burst.
Invest Ophthalmol Vis Sci 28:295-304, 1987
Alkali burns of the eye initiate a complex series of
events in cornea and sclera beginning with destruction
of cellular components (epithelium, keratocytes, and
endothelium), hydrolysis of glycosaminoglycans and
damage to the collagen matrix. Injury to the vascular
system in the external and internal eye induces PMN
margination and then diapedesis through the vascular
walls. Some inflammatory cells may cross the walls of
injured blood vessels in conjunctiva and episclera as a
direct and specific result of the trauma. In addition,
chemotactic agents released from the damaged tissues
may diffuse out to the vascular system, stimulating directed locomotion.
PMNs are the predominant inflammatory cell in the
cornea following alkali burns.1"3 They are the most
abundant and, at times, the only inflammatory cell
associated with ulceration in these corneas. Citrate reduces the incidence of ulceration in alkali burned rabbit
eyes probably by chelating divalent cations, thereby
inhibiting PMNs.4"8 The inflammatory mediators that
attract PMNs into the cornea and cause their metabolic
stimulation are unknown.
These studies investigate the locomotory, stimulatory, and morphological characteristics of PMNs exposed to alkali-burned collagen.
Materials and Methods
Materials
Trisodium citrate, disodium ethylenediamine tetraacetic acid (EDTA), bovine serum albumin (BSA),
dimethyl sulfoxide (DMSO), and N-formyl-methionylleucyl-phenylalanine (fMLP) were purchased from
Sigma Chemical Co. (St. Louis, MO). CaCl 2 , MgCl2,
and sucrose were purchased from Mallinkrodt (St.
Louis, MO), Fisher Scientific Co. (Fair Lawn, NJ), and
Baker Chemical Co. (Phillipsburg, NJ), respectively.
Trifluoperazine dihydrochloride (TFP) was a gift
from Smith Kline and French Laboratories (Philadelphia, PA).
Collagen Preparation
Miller Type 1 collagen was obtained from Sigma
Chemical Co. (Cat. No. C-9879, salt extracted) or Calbiochem-Behring (Cat. No. 234112, acid soluble). Unless otherwise noted, these commercial collagens were
treated with 1 N NaOH (w/v, 1:6). Bovine and porcine
eyes (Pel-Freez Biologicals; Rogers, AR) were enucleated and frozen within 2 hr of death. Whole corneal
buttons were excised while frozen and scraped of endothelium and epithelium. Corneas were subsequently
From the Eye Research Laboratories, Brookwood Medical Center,
Birmingham, Alabama.
Supported by NEI Grant EY-04716, Brookwood Medical Center
and Hoffman LaRoche.
Submitted for publication: February 21, 1986.
Reprint requests: Roswell R. Pfister, MD, 2008 Brookwood Medical
Center Drive, Birmingham, AL 35209.
295
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296
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Februory 1987
rinsed three times in distilled water and dried between
layers of paper towel by applying firm pressure. The
remaining water content of these corneas was determined and an equal volume of 2 N NaOH was added
to give a final concentration of 1 N NaOH. The collagen
weight (65% of dry corneal weight) was 1/12 (w/v) of
the total volume of 1 N NaOH. Alkali treatment of
commercial collagen or corneas was carried out at 35°C
for 20 min (prolonged exposure as specified) followed
by 5-15 min titration at 0-4°C with 1 N or 2 N HC1
to pH 7.4. To obtain the largest volume of supernatant,
corneas were subsequently pressed in a metal syringe
that was tightened in a vise. In all cases, the neutralized
suspension was centrifuged at 15,000 X g for 5 min to
remove paniculate material. The resuspended pellet
was inactive, but the supernatant retained full activity
as measured by PMN respiratory burst. Control samples were prepared using Sigma collagen or bovine corneas treated with 0.5 N or 1.0 N NaCl, respectively,
by following a process identical to that of their burned
counterparts.
Dialysis
Ten milliliters of alkali-treated Sigma collagen or
bovine cornea supernatants were dialyzed (1,000 molecular weight cutoff tubing) against distilled water (2
liters/hour) for 3 hr and 0.9% NaCl for 1 hr.
PMN Isolation
Following the technique of Ferrante and Thong, 9
PMNs were isolated from fresh human whole blood
by centrifugation on Hypaque-Ficoll (density 1.114)
as described in a previous paper.8 Isolated PMNs were
resuspended in Hanks balanced salt solution (HBSS),
containing 500 nM Ca and 600 nM Mg, to a purity of
greater than 75% PMNs (96-99% viability) with the
remaining percentage consisting of red blood cells
(RBCs.) and less than 5% platelets, lymphocytes, and
eosinophils. Previous reports have shown that elimination of RBCs through dextran sedimentation and/
or hypotonic lysis caused significant detrimental effects
on PMNs. 610 " 13 Critical experiments were repeated using PMN suspensions free of RBCs, treated with 0.2%
NaCl for 30 sec followed by 1.6% NaCl, and showed
results consistent with the PMN suspensions containing
RBCs.
PMN Locomotion
PMN locomotion induced by supernatants from
Sigma collagen or bovine cornea was compared to that
of the positive control, fMLP. All stock solutions were
prepared each day to pH 7.3. Trisodium citrate (224
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Vol. 28
mM) was dissolved in distilled water. FMLP (final
concentration = 1 X 10"7 M) was dissolved in DMSO
(final concentration = 0.005%) and diluted with HBSS
containing BSA (final concentration = 2%). One part
(volume) of alkali or salt treated collagen or cornea
supernatant was diluted with 10 parts (volume) of a
distilled water and HBSS mixture (3.4/6.6, v/v) containing Ca2+ and Mg 2+ . Distilled water was substituted
for HBSS to maintain the final osmolalities of both
chambers between 270 mOs/Kg and 320 mOs/Kg. The
final Ca 2+ and Mg 2+ concentrations of each incubation
was 500 pM and 600 nM, respectively. The pH range
for all incubations was 7.2 to 7.6.
Locomotion was determined in modified Boyden
chambers (Neuroprobe, Inc.; Cabin John, MD) containing two nitrocellulose filters. The pore sizes of the
upper and lower filters were 5 /im (Toyo, Neuroprobe)
and 0.45 /xm (Millipore Corp.; Bedford, MA), respectively.
Upper compartments contained 560 /il of PMNs (2.2
X 106 cells/ml) in HBSS containing 500 fiM Ca 2+ , and
600 fiM Mg2+, and 2% BSA. Where appropriate, citrate
(final concentration 12 mM) was added to the PMN
suspension 5 min prior to chamber incubation. An
equal volume of distilled water was added to all other
samples. LDH release was less than 5% of the total for
all incubations.
Lower compartments contained 1.4 ml of one of the
following solutions: fMLP, HBSS with 2.0% BSA and
0.005% DMSO (negative control), burned or salttreated Sigma collagen supernatant (1:10 dilution), or
burned or salt-treated bovine cornea supernatant
(1:10 dilution).
The filled chambers were incubated for 3 hr at 37°C
in humidified air. The filters were subsequently stained
with Lerner-1 Hematoxylin (Lerner Laboratories; New
Haven, CT). The number of PMNs/0.01 mm 2 grid was
determined for the bottom of the upper filter (10 grids)
and the top of the lower filter (10 grids). These numbers
were combined to determine the mean PMN count for
that sample.
PMN Stimulation
EDTA (pH, 8.7-9.0) was prepared in an equal concentration of Tris buffer to compensate for the pH drop
caused by the EDTA-Ca2+ binding reaction. TFP was
made up in HBSS (cation-free) at pH 6.6 by titrating
with NaOH. All other stock solutions were prepared
fresh each day in HBSS or distilled water, both containing 500 fiM Ca2+ and 600 fiM Mg 2+ , at pH 7.4.
The respiratory burst of PMNs (4.5 X 106 cells/ml)
was measured with a Clark-type oxygen monitor (YSI
model 53) in an incubation chamber maintained at
No. 2
ALKALI-BURNED COLLAGEN STIMULATION OF NEUTROPHILS / Pfisrer er ol.
37°C with a pH range of 7.2-7.6. Differing volumes
of each collagen or coraeal supernatant were added to
make a total volume0of 3 ml to stimulate oxygen consumption. Test solutions (citrate, EDTA, or TFP) were
added 5 min prior to stimulation, whereas NaCl or
sucrose was added simultaneously with the supernatants for osmolality enhancement experiments. Oxygen
consumption was measured for 10 min, and samples
were subsequently collected from the incubation
chamber for light microscopy observation or the measurement of /3-glucuronidase14 and lactic dehydrogenase (LDH) 1516 activity. LDH release, a measure of
cell viability, was less than 5% of the total for all incubations.
lOOp
» X
M
01
297
B vs D = Not Significant
DvsE = p<.OOI
30
n = l4
E 25
O
d 20
l4
i
I
2 15
o.
"o to
n-14
C
0)
|
5
n = l4
l4
Control
HBSS
fMLP
NaCl
treated
supernafant
Burned
Supernatant
Burned
Supernatanf
+12 mM
Citrate
lOOr
Electron Microscopy
DvsC = .0Kp<.02
DvsE == .02<p<.05
30
At the conclusion of the respiratory burst incubations, control and burned sigma collagen-stimulated
PMNs were immediately fixed with glutaraldehyde and
prepared by conventional techniques for transmission
(TEM) and scanning (SEM) electron microscopy. TEM
examination was performed with a Phillips 200 (Phillips, Eindhoven, Netherlands) on samples stained for
myeloperoxidase to distinguish between azurophilic
and specific granules.17 SEM samples were examined
with an ISI-SS40 after collecting the suspended PMNs
on Whatman filter paper.
Statistics
Statistical analysis was performed with the student
t-tests. Standard error of the mean is used throughout
the paper.
' 25
-
15
-
n = l2
I
n«l2
10
n»l2
5|—
T
Control
HBSS
B
B
FMLP
n«l2
T
c
D
E
NaCl
Burned
Burned
treated supernatant supernatant
supernatant
+l2mM
Citrate
Fig. 1. PMN locomotion was induced by alkali-burned collagen
supernatants. The concentration of fMLP equals 1 X 10~7 M. The
12-mM citrate was placed only in the upper compartment of the
Boyden chambers. The grid area was determined on oil immersion.
(A) Supernatant from burned Sigma collagen was 88% of fMLP locomotion, citrate inhibition = 69.5%; (B) Supernatant from burned
bovine cornea was 40% of fMLP locomotion, citrate inhibition
Results
When compared to controls, PMNs showed a significant locomotory stimulation to supernatants from
burned Sigma collagen and bovine cornea (Fig. 1 A, B).
Locomotion was 88% and 40% of the positive control
(fMLP) for collagen and cornea, respectively. Locomotion induced by either supernatant was dramatically
inhibited by the addition of 12 mM citrate.
PMNs are readily stimulated to undergo a respiratory
burst when exposed to the supernatant from alkali
burned collagen obtained from Sigma, Calbiochem,
bovine or porcine corneas (Fig. 2A, B, and C). In a
majority of the following studies, parallel comparison
experiments were conducted primarily with Sigma
collagen and bovine cornea. When the supernatant
from alkali-burned Sigma collagen or bovine cornea is
dialyzed (1,000 molecular weight cutoff—MWCO)
against distilled water, there is a 72% or 89% loss of
oxygen utilization, respectively (Fig. 2A and 3B). This
stimulation is reinstated when osmolality is artificially
Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017
increased with NaCl or sucrose (Fig. 3A, B). Furthermore, hypertonic osmolalities enhance the stimulation
of PMNs by low volumes of alkali-burned Sigma and
Calbiochem collagen supernatant (Fig. 3A). However,
stimulation of the respiratory burst is not dependent
on hypertonicity (Fig. 4).
The respiratory burst of PMNs increases with increasing volumes, NaOH concentration, or exposure
to alkali of Sigma collagen (Fig. 5 A, B) or bovine cornea
(Fig. 4). Therefore if Sigma collagen is treated with
high concentrations of alkali or for a prolonged period,
then optimal stimulation can be achieved with lower
supernatant volumes than are required for the 1 N-20min burn (Fig. 5A). In all cases, larger volumes result
in diminished oxygen consumption along with a dramatic rise in the LDH levels indicating lysis of PMNs
(Fig. 5A, C). Light and electron microscopy demonstrates widespread cell death and lysis of these and other
excessively stimulated (30-min exposure to optimal
298
Vol. 28
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / February 1987
1-4 vs5»p<.OOI
50
1-5 -Not Significant
1-5 vs6or7-p<.OOI
6vs7-p<.00l
T
§30
n=8
H 20
T
H8SS
only
HBSS +
burned
supernoton
•
—•
o-— — 0
70 -
Control
PMN
PMN +
NoCI
treated
supernotant
PMN +
burned
collagen
pellet
HBSS
only
PMN+
PMN+
burned
burned
supernatant supernatant
(Dialyzed
1000 MWCO)
T
n-17
HBSS +
burned
supernatant
Control
PMN
n=3
PMN +
PMN +
NaCl
burned
treated
supernatant
supernatant
O2 Consunnption
Osmololit)
1 n-5
60 -
SO -
/
to
30
n»6
-
_ 700 o
n-6
E
20
n»IO
n-5
10
HBSS
only
HBSS+
burned
supernatam
fl.Smls)
n-14
n
p.:6.....
T
500
n-75--"" r " - 8
PMN +
NaCl
treated
supernatant
(l.5mls)
B
Fig. 2. Stimulation of PMN respiratory burst by alkali burned
collagen supernatants. (A) Supernatant (300 ^1) from burned
Sigma collagen; (B) Supernatant (600 fi\) from burned Calbiochem collagen; (C) Supernatant from bovine cornea. The osmolality was held constant by partially substituting distilled water with cations for HBSS with cations in the final PMN suspension (asterisk).
E
600
900
1200
1500
Supernatant from burned bovine cornea
volumes) samples (data not presented). Alkali burns
(1 N-20 min) of Sigma or Calbiochem collagen and
bovine cornea did not cause a significant increase (<5%
of the total) in the LDH levels after optimal stimulation.
The respiratory burst of PMNs stimulated by alkaliburned Sigma collagen supernatant is inhibited by tri-
fluoperazine but not citrate or EDTA (Fig. 6). Trifluoperazine inhibited stimulation over the entire 10-min
period. On the other hand, the respiratory burst is increased by citrate and EDTA during the first 6 min of
incubation and suppressed in the remaining 4 min.
The presence of TFP, EDTA, or citrate in these samples
caused increasing LDH levels, in that order (Fig. 7).
01-
2 vs3 = p<OOI
4or 5 vs3= p<.OOI
£ 70
A-2 vs A-l, A-3,or A-4 = p<.00l
B-lvsB-2 orB-3»p<.OOI
E 60
1
n-8
T
n-4
n-IO
T
40
O 30
n-9
n-9
T
T
88.6 % loss of
stimulation
S.20
V "4-"
n=9
Control Control Control
PMN PMNt PMN +
NaCl sucrose
Dialyzed Dialyzed Dialyzed
+N0CI • sucrose
Low
Low
Low
volume volume volume
+ N0CI • sucrose
B
Control
PMN
Optimal
Dialyzed Oialyzed Oialyzed
+ NaCI +sucrose
Fig. 3. Increased osmolality enhances the respiratory burst of PMN stimulated by dialyzed (1,000 MWCO) or low volume supernatants from
alkali burned collagen. Hypertonicity was accomplished by adding aliquots of 1 N NaCl or 40% sucrose to the supernatants. (A) Burned Sigma
collagen supernatant; optimal (300 nl), dialyzed (300 /A), and low volume (50-100 n\); (B) Burned bovine cornea supernatant, optimal (1 ml)
and dialyzed (1 ml).
Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017
No. 2
ALKALI-BURNED COLLAGEN STIMULATION OF NEUTROPHILS / Pfisrer er ol.
Stimulation of PMNs by bovine cornea supernatant is
not altered by citrate (Fig. 6). The presence of citrate
during stimulation by bovine cornea supernatant had
no effect on LDH release from PMNs (12 mM citrate;
x = 1.3 ± 0.3% of total, N = 6).
The supernatant from alkali-burned Sigma and Calbiochem collagen or bovine cornea did not stimulate
the release of/3-glucuronidase from PMNs (<5% of the
total). However, as previously noted, cell lysis and death
occurs when PMNs are excessively stimulated by
burned Sigma collagen supernatant (Fig. 5A, C). This
process leads to the extracellular release of /3-glucuronidase (Fig. 5C) and probably all granular enzymes.
Viable PMNs in suspension, stimulated with burned
Sigma collagen and examined under a coverslip, did
not adhere to glass slides. Blebs appeared suddenly
(seconds) and retracted slowly (minutes). Retraction
seems to be accompanied by a slight increase in cell
size.
Electron microscopic observation revealed an unusual modification of PMN morphology after stimulation with burned Sigma collagen. Control PMNs are
round cells with small protrusions from an otherwise
smooth cell membrane (Fig. 8A, B). Azurophilic (peroxidase positive) and specific (peroxidase negative)
granules are numerous in the cytoplasmic matrix. A
small golgi apparatus and centriole are located near
100-
« 90-
I
I Sigmo Collagen
H l ^ § Colbiochem Collagen
I
I Bovine Cornea
lOOr-
A-2 vs A-3°Not Significant
B-2 vs B-3 • Not Significant
C-2vsC-3-p<.OOI
T
1.0
£20
•C-2
292 mOs
mOs
C-3
Conirol Hyperrofiic isoionic
PMN
SIO
303
2 9 3 mO>
mOs
mO»
Control Hyperronic Isotonic
PMN
373
293
mOj
Fig. 4. The respiratory burst of PMNs is enhanced by, but not
dependent on, increased osmolality when stimulated by alkali burned
collagen supernatants from Sigma (300 fil), Calbiochem (750 MO> or
bovine cornea (500 fil). Isotonicity was achieved by substituting appropriate volumes of distilled water with cations in the final PMN
suspension. Alkali treatment of bovine cornea was for 24 hours.
the center of the cell partially surrounded by a multi-
lobed nucleus with a wide perinuclear cisterna. PMNs
incubated with supernatant from burned collagen exhibit many large blebs from the cell surface, occasion-
</> l00 r
• IN Na0H-20minufes
•• IN NaOH-25 hours
!
2NNoOH-2Ominutes
Control PMN
Cell Aggregation/Lysis
22
§ 80E
299
C 70
E
2 so
O 70-|
B 60H
|so§40-
S
c 30
0)
0.25
100
200
300
600
Supernatant from Burned Sigma Collagen (pi)
I
70
I
f
B-lvsC-l»p<.OOI
A-2.B-2vsC-2-p<OOI
A-3.B-3 vsC-3-p<.OOI
I
1
900
y°°
I
o
|
S
|
§ 30
U 30
3OOpl
Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017
9 0 0 pi
0.50
0.75
1.0
NoOH Concentration
(20 minutes)
I I I M
Burn Time-Hours
(1.0 N NaOH)
Fig- 5. Stimulation of PMNs by supernatant from alkali burned
Sigma collagen increases with greater supernatant volumes, NaOH
concentration, or duration of alkali treatment. Excessive stimulation
leads to cell death resulting in increased enzyme release and decreased
oxygen consumption. (A) Supernatant volume. (B) NaOH concentration and burn time. Supernatant equals 300 fi\. All values corrected
by subtracting the mean control rate. (C) Excessive stimulation.
30D
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / February 1987
A vs B = p < .001
£
A vsC °.0\<p<02
e
a vsb =p<.OOI
a vs c = Not Significant
Pond d vs oil others = p<OOI
E vs F = Nof Significant
e vsf = Not Significant
I
Burned Citrate
Bovine
Supernatant
Fig. 6. The effects of citrate, EDTA and TFP on stimulation of
the resiratory burst of PMN by supernatants from alkali burned Sigma
collagen (300 fil) and bovine cornea (1.2 ml).
ally appearing to almost separate from the membrane
(Fig. 8C, D). These blebs contain a hyaline core and
frequently terminate into hairlike protrusions. Cytoplasmic granules are retained within the cell proper
but are absent from the blebs in most but not all cases.
Frequently blebs are aggregated into a polarized pattern. The perinuclear cisterna is conspicuously absent.
Discussion
Polymorphonuclear leukocytes are the prime effector
cells of the inflammatory response, ultimately leading
to ulceration in the cornea after an alkali burn.1"3 Direct
and indirect damage to ocular vessels would lead to
nonspecific exudation and PMN diapedesis, but heavy
accumulations in the corneal stroma must occur by
chemotaxis. The initial release of chemotactic agents
from the cornea might originate from damage to epi-
IOO
i-
k
A-3vsA-l,A-2-p<OOI
B-3vsB-l,B2-.O2<p<.O5
C-3vsCI.C-2-.02<p<05
I 30
A-l
PMN
A-2
A-3
supernofonf supernot
B-l
B-2
B-3
Control
Burned
Burned
PMN supernatant supernatant
+l.2BmM
EDTA
C-l
C-2
C-3
Control
Burned
Burneo
PMN supernotont supernot
+ 40JJM
TFP
Fig. 7. The effects of citrate, EDTA and TFP on LDH release of
PMNs stimulated by supernatant from alkali burned Sigma collagen
(300 MO-
Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017
Vol. 28
thelium, endothelium, keratocytes, proteoglycans, or
collagen. Chemotactic agents are so powerful, even in
low concentrations, that any or all of these tissues might
elaborate inflammatory mediators. However, the sudden death and total loss of most corneal cells and rapid
loss of proteoglycans after a burn leave the remaining
collagen a strong potential source for the initial stimulation.
In our experiments the locomotory stimulation of
PMNs by alkali treated Sigma collagen or bovine cornea was 88% and 40%, respectively, of the benchmark
chemoattractant fMLP. BSA is a chemokinetic agent
routinely used to increase the effectiveness of many
chemoattractants.18"19 This effect is thought to be the
result of a decrease in the adhesion to the substratum,
allowing mobility of cells that made contact with the
substratum.20 We have not attempted to determine the
nature of the facillatory effect of BSA the burned supernatants. It is important to note that the purity of
this locomotory stimulant, obtained from Sigma collagen, has not been fully tested.
The chemotactic effect of collagen on PMNs has
been controversial. Chang and Houck showed that soluble collagen and collagen degradation products, obtained from salt extracted rat skin, were chemotactic
to PMNs in in vivo millipore chambers.21 In their experiments, cutaneous collagen digested by collagenase
was considerably more chemotactic than intact soluble
collagen. Other studies by Postlethwaite and Kang
showed that monocytes but not PMNs are chemotactic
to low concentrations of native collagen and peptide
chains in modified Boyden chambers using polycarbonate filters.22 In the latter study, the acid-extracted
type 1 collagen was obtained from lathyritic chicken
or degraded human skin. Unlike our alkali-treated collagen, these specimens were treated with cyanogen
bromide, bacterial collagenase, or pepsin. Any or all
of the differences noted could account for the disparity
between their data and the results presented in this
paper.
A precedent exists for locomotion induced by alkaliburned proteins. Albumin and other proteins apparently do become chemotactic when treated with
alkali.23'24 It is suggested that such treatment exposes
hydrophobic groups of the molecule, which permit
penetration of the phospholipid bilayer of the PMN
cell membrane. It is unknown if locomotion induced
by burned collagen is effected by a similar mechanism.
The activation of PMN locomotion by alkali-burned
collagen was significantly inhibited by the addition of
citrate to the Boyden chamber. Citrate also reduces the
locomotory stimulus of fMLP to PMNs. 8 This is consistent with data showing citrate inhibition of PMN
accumulation in the anterior segment of the eye4'25 and
the reduction of ulceration in alkali-burned eyes treated
No. 2
ALKALI-DURNED COLLAGEN STIMULATION OF NEUTROPHILS / Pfisrer er ol.
301
Fig. 8. Electron microscopic comparison between control PMNs and PMNs stimulated with 300 p] of the supernatant from burned Sigma
collagen. (A) SEM of control PMN. PMNs are collected on filter paper after fixation. (B) TEM of control PMN. (C) SEM of stimulated PMN.
Hairlike protrusions (HP) frequently emanate from the apical portion of cell membrane blebs (MB). (D) TEM of stimulated PMN. Membrane
blebs have a hyaline core (HC) usually surrounded by agranular cytoplasm. Occasionally a bleb appears to almost separate from the cell
membrane (arrow), n: nucleus; a: azurophilic granule; s: specific granule; p: perinuclear cisterna; g: golgi apparatus.
with citrate.4'5 These findings are not surprising, considering that locomotion requires actin microfilament
polymerization and subsequent interaction with the
plasma membrane, a Ca2+ dependent process.26 In vitro
chemotaxis is also known to require the presence of
extracellular Ca 2+ and Mg2+.27 Citrate is a chelator of
these divalent cations.
Metabolic stimulation of PMNs (respiratory burst)
by the supernatant from Sigma and Calbiochem col-
Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017
lagen burned by alkali is a new and important finding
in our studies. Alkali burns of porcine and bovine corneas also yield stimulation of a PMN respiratory burst
similar to commercial collagens. Although the metabolic requirements of chemotaxis are not fully understood, it is known that aerobic respiration is needed to
fuel PMN production of highly reactive oxygen-free
radicals including superoxide species.28 This increased
metabolic activity in the cornea might lead to tissue
002
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / February 1987
destruction or to the formation of other inflammatory
mediators.29'30 The absence of azurophilic granular release, as measured by /3-glucuronidase activity, in the
presence of alkali-burned collagen might explain, in
part, why corneal ulceration does not occur early after
alkali injury. The development of ulceration 2-3 wk
after the burn is probably the result of granular enzyme
and oxygen-free radical release under the influence of
secondary inflammatory mediators. The observation
that collagen exposure to NaOH in higher concentrations or for longer periods of time causes greater metabolic stimulation of PMN, even to the point of cell
death and the subsequent release of granular enzymes,
may correlate to the clinical finding that the more severe the burn, the greater is the likelihood of corneal
ulceration.
One unusual aspect to burned collagen stimulation
of the respiratory burst is its enhancement by hypertonicity. We are unaware of any reports in the literature
where hypertonicity has a similar effect. In fact, hypertonicity is generally known to reduce the stimulation
of PMNs.6'31"34 Although speculative, the increased osmolality might induce alterations in the cell membrane
allowing greater contact by the collagen breakdown
product(s).
The possibility exists that dialysis of the supematants
from burned collagen results in the loss of a collagen
breakdown product(s) (< 1,000 MW), which at least
partially contributes to the stimulatory process. The
results of our dialysis and hypertonic enhancement experiments, however, lead us to believe that most, if not
all, of the lost stimulation after dialysis is caused by a
loss of hypertonicity.
In these studies the presence of citrate or EDTA
caused PMN, exposed to alkali burned collagen, to
show an increased initial stimulation, followed by an
inhibitory response. It is possible that these chelators
induce a loss of Ca 2+ , thus causing membrane perturbation, much like hypertonicity, and allowing the collagen breakdown product(s) greater access to the cell
membrane. This might initially enhance the respiratory
burst, rapidly depleting their limited intracellular Ca 2+
stores. The subsequent drop in oxygen consumption
correlates to widespread cell lysis as evidenced by increased LDH release. This situation is therefore similar
to excessive stimulation by large supernatant volumes,
high alkali concentration, or prolonged exposure to alkali. Complete inhibition of oxygen consumption by
TFP (an activated Ca2+-calmodulin inhibitor) provides
evidence that intracellular Ca 2+ is a key element in this
stimulatory process.
The rapid appearance and slow disappearance of
blebs and hairlike protrusions evaginating from the cell
surface of PMNs, undergoing a respiratory burst from
burned collagen, are similar to those observed by Bad-
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Vol. 28
way et al after cis-unsaturated fatty acid stimulation
and on the plasma membranes of epithelium migrating
over cornea burned by alkali.35'36 This later observation,
as well as our own in this paper may be similar to the
phenomenon of zeiosis occurring during anaphase37 or
induced by various agents38"42 including anesthetics43
and EDTA.44 Dornfeld and Owczarzak proposed that
zeiosis results from a loss of Ca 2+ from the cell surface,
producing weakened areas in the membrane. Alternately these morphological characteristics might be related to cell agony leading to cell death. 45 Massive cell
lysis occurring after excessive stimulation with burned
collagen gives some support to the latter hypothesis.
The purity of the collagen preparations used is of
considerable importance. Preliminary experiments
show that Sigma and Calbiochem collagen is 90% and
98% pure collagen protein, respectively (amino acid
composition analysis based on gas-liquid chromatography performed by Dr. David Whikehart). The Sigma
collagen preparation is, however, contaminated by
lipid. An analysis revealed that 1.5% of the supernatant
is lipid (preliminary data). Separation of lipid from
burned Sigma collagen by sucrose density gradients (530% sucrose, w/v) as well as ultrafiltration (molecular
weight exclusion >25,000) of the supernatants shows
that the respiratory burst stimulant resides in the high
molecular weight collagen fraction and not in the low
molecular weight portion (preliminary data from Dr.
Kwok-Wai Lam). On this basis as well as the finding
that Calbiochem collagen and scraped corneas are virtually lipid-free and that all sources show a similar
stimulation strongly supports the position that the
stimulant producing the respiratory burst is derived
from alkali-burned collagen and not from a lipid contaminant (ie, cis-unsaturated fatty acids).35 These findings have not been confirmed on the locomotory stimulant of PMNs.
The results obtained by dialysis and hypertonic enhancement of alkali-treated Sigma collagen and bovine
corneas and the lack of /3-glucuronidase release by
PMNs exposed to all collagen sources suggest that the
stimulant obtained from the different sources is similar.
The data presented here suggest the following hypothesis: Alkali-burning of the cornea may cause limited breakdown of the collagen, releasing one or more
peptide fragments. The fragment(s) could diffuse away
from the cornea across the surfaces or through the
stroma to the limbal area, where the presence of serum
albumin 46 might facilitate PMN locomotion. This
could provide one of the initial chemotactic stimuli for
PMN invasion into the damaged collagen matrix. Because the stimulus would not release j8-glucuronidase
(azurophilic granules) during their migration, PMNs
might arrive in the corneal target tissue with their full
complement of lytic enzymes. The same collagen frag-
No. 2
ALKALI-BURNED COLLAGEN STIMULATION OF NEUTROPHILS / Pfisrer er ol.
ment(s) at a higher concentration, other long chain
polypeptide fragment(s) from collagen, or other inflammatory mediators might trigger the respiratory
burst in PMNs located in the cornea. When very high
concentrations of these collagen breakdown products
are present, then the PMNs might ultimately lyse
through excessive stimulation, thus releasing their
granular enzymes. The alkali burned collagen fragment(s) might then represent the first of a series of
inflammatory mediators. Other mediators, some directly released from PMNs attracted into the cornea
such as leukotriene B4 (LTB4),47 plasminogen activator
(PA),48 and others produced as a result of the release
of byproducts from these PMNs (superoxide-lipid-albumin complex,30 and C5a49), might trigger further
enzyme release and bring more PMN to the cornea.
The finding of large amounts of LTB 4 in the aqueous
humor of alkali-burned eyes strengthens this point of
view.50 Other potential sources of PMNs chemotactic
agents cannot be ignored, such as PA derived from the
corneal epithelium after an alkali burn. 51
The locomotion of PMNs by alkali-burned collagen
breakdown products should be differentiated from
stimulation to undergo the respiratory burst. Our studies have not extended to the characterization of the
locomotory stimulus. The locomotory and metabolic
stimuli may or may not be the same polypeptide.
The concept of an inflammatory mediator released
from alkali-burned collagen has important implications
for other burned tissues. PMN infiltration leads to necrosis, ulceration, and perforation of tissues and viscera
elsewhere in the body. The development of substances
to abrogate this response, including citrate, could significantly alter an anticipated poor prognosis in these
cases.
Key words: collagen, alkali, polymorphonuclear leukocytes,
locomotion, metabolic stimulation
Acknowledgments
We wish to acknowledge the helpful discussions and editorial contributions of Dr. Kwok-Wai Lam and Dr. David
Whikehart in the preparation of this manuscript. Jonathan
Preble, Daryl Pfister, and Barbara Summerville provided
technical assistance.
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