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From the Inflammation Research Unit, School of Pathology, University of New South Wales, Sutherland Centre of Immunology, Sydney, Australia.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. An anti–IL-8 antibody (WS-4) was injected intravitreal 2 hours before, simultaneously with, or 6 hours after endotoxin challenge in rabbits. Eyes were examined for clinical signs of inflammation, and aqueous humor (AH) was sampled to study cellular infiltration and protein content. Leukocyte subset analysis was performed on Giemsa-stained AH cytospins. Histologic grading of inflammation was performed on hematoxylin-eosin–stained sagittal sections of enucleated eyes. In separate experiments, animals received the anti–IL-8 antibody simultaneously with the endotoxin challenge, before repeated anterior chamber paracentesis was performed (at 6, 12, 24, 48, and 72 hours after injection) to estimate the kinetics and durability of changes in total cell count and protein concentration in AH.
RESULTS. Anti–IL-8 therapy caused a decrease in the clinical and histologic grade of inflammation in EIU. The mean cell count in the AH at the peak of inflammation (24 hours) in eyes receiving endotoxin only was 6419 ± 1165/µl (mean ± SE) compared to 2546 ± 573/µl in rabbits treated simultaneously with 250 µg of anti–IL-8 antibody (P < 0.05). The protein concentration in the AH was not significantly altered by anti–IL-8 treatment. Kinetic analysis of the leukocyte count in the AH demonstrated persistent inhibition of leukocyte accumulation (range, 60%–91% compared to control eyes) by the anti–IL-8 antibody administered simultaneously with endotoxin. This inhibition was sustained for up to 72 hours after injection.
CONCLUSIONS. Anti–IL-8 antibody treatment partially blocks EIU in rabbits. A consistent decrease in the recruitment of polymorphonuclear leukocytes into the anterior chamber was obtained when neutralizing antibody was injected simultaneously with endotoxin. These findings suggest that IL-8 contributes to the chemotactic signal for the recruitment of leukocytes in EIU.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(C-X-C subfamily)
chemokine and a potent stimulus for polymorphonuclear (PMN)
leukocyte recruitment and activation. It is produced in
settings of inflammation by a wide range of cells, including
monocyte–macrophages, lymphocytes, vascular endothelium, dermal
fibroblasts, keratinocytes, synovial cells, hepatocytes,1
and the ocular epithelium of cornea and retina.2
3
4
IL-8
production is typically stimulated by endotoxin or proinflammatory
cytokines, such as IL-1 or tumor necrosis factor (TNF)-, and is
associated with a rapid influx of PMN over 4 to 6 hours. IL-8 promotes
PMN leukocyte recruitment by regulating the expression of the integrin
family of leukocyte adhesion receptors,5
resulting in
neutrophil adherence to vascular endothelium, transendothelial
migration, and haptotactic migration through extracellular matrix to
sites of inflammation.6
7 Previous studies have reported the anti-inflammatory effects of neutralizing anti–IL-8 antibody treatment in animal models of neutrophil-dominant inflammation, including a rat model of IgG immune complex–induced lung injury7 and a rabbit model of ischemia–reperfusion lung injury.8 We recently documented significantly increased levels of several chemokines, including IL-8, in the aqueous humor (AH) of patients with acute anterior uveitis (AU).9 In an attempt to evaluate further the role of this chemokine in the regulation of inflammation in acute AU, we have studied the effects of neutralizing anti–IL-8 antibody on the induction of endotoxin-induced uveitis (EIU) in rabbits.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preliminary dose–response studies determined that a dose of 100 ng of endotoxin (lipopolysaccharide [LPS] from Salmonella typhimurium; Difco Laboratories, Detroit, MI) was optimal for the induction of uveitis. The endotoxin was administered by intravitreal injection. Each experimental group consisted of 3 to 4 rabbits (6–8 eyes). To control for animal-to-animal variability in this model, the right eye was always considered as the experimental eye (i.e., endotoxin plus neutralizing antibody), whereas the left eye acted as control (i.e., endotoxin plus control antibody). Preliminary experiments revealed that a contralateral untreated eye remains reliably unaffected after induction of EIU in the other eye.
Animals were anesthetized using 0.4 to 0.8 ml of ketamine (20–40 mg/kg) and 0.1 to 0.2 ml of xylazine (1–2 mg/kg) injected intramuscularly in the hind limb. Amethocaine (0.5%) administered topically to the eyes supplemented the general anesthesia. Before injection, the eye was exposed by retracting the upper lid. A 30-gauge needle was inserted transconjunctivally at the 12 o’clock position, 3 to 4 mm posterior to the limbus, and 20 µl of the endotoxin solution was injected using a disposable syringe. The animals were observed closely until recovery from anesthesia. The eyes subsequently were monitored for redness, photophobia, and lacrimation. Each eye was examined using diffuse and oblique illumination and indirect ophthalmoscopy for signs of uveitis. Clinical scoring of AU was done using Hogan’s classification.10 Animals were killed by intracardiac injection of 2 ml of euthanasia solution (phenobarbital) at a concentration of 350 mg/ml. The eyes were enucleated immediately after death and stored in 10% buffered formalin for 24 to 48 hours before processing. AH was aspirated before enucleation (see AH Sampling).
AH Sampling
Immediately after death, 200 to 250 µl of AH was aspirated from
the anterior chamber using a 30-gauge disposable insulin syringe,
avoiding injury to the iris or lens. To inhibit fibrin clot formation,
freshly prepared plasmin at a final concentration of 50 nM was added to
the AH and incubated at 37°C for 90 to 120 minutes. The plasmin
reaction was stopped using 100 µl of 2 mM aprotinin (Trasylol; Bayer
Australia Ltd., Sydney). The AH cell count was performed in
duplicate using a hemocytometer under 100x magnification. The AH was
then centrifuged, and the supernatant was stored at -70°C, and the
cell pellet was reconstituted in buffer and spread on a glass slide for
Giemsa staining and differential cell count.
Histopathologic Examination
Hematoxylin and eosin–stained sections of 3 to 5 µm thickness
were prepared from paraffin-embedded blocks of the enucleated eyes to
characterize the histopathologic features of inflammation. Sections
were examined for the presence of keratic precipitates, inflammatory
cells in anterior chamber, and ciliary body and retina and for altered
vascularity of the iris and ciliary body. Infiltrating cells were
counted under 200x magnification in contiguous fields across the whole
section and were assigned to a histologic grade on a semi-logarithmic
scale using the following criteria: grade 0, no cells/field; grade 1, 1
to 10 cells/field; grade 2, 11 to 30 cells/field; grade 3,
31 to 100 cells/field; and grade 4, 101 to 300 cells/field. The
saggital section included two fields each of the ciliary body, anterior
chamber angle, and iris and ora serrata and one field each of anterior
chamber, pupillary area, vitreous, and the retina. A differential count
of the infiltrating cells in the experimental eyes was performed by
counting the mononuclear and PMN cell proportions under 400x
magnification in five randomly selected fields. The mean value for each
subset was then calculated and presented as a percentage of the total.
Protein Assays
Protein levels in the AH were measured using a standard
bicinchonic acid microtiter assay protocol (Bio-Rad; Pierce, Rockford,
IL).
IL-8–Induced Ocular Inflammation
Sixty microliters of recombinant human IL-8 (gifted from Dennis
Taub’s laboratory at NIH) in a dose of 1 µg was administered into
the anterior chamber or in four divided doses into four quadrants of
the ciliary body of the rabbit eyes (n = 6 to 8 eyes).
The severity of ocular inflammation was assessed using clinical and
histopathologic grading. AH was aspirated at different time points to
estimate the cell accumulation and the protein content.
Neutralizing Antibody to IL-8
A murine monoclonal antibody against human IL-8 (WS-4) and a
control antibody were prepared in the Department of Pharmacology,
Cancer Research Institute, Kanazawa University, Kanazawa,
Japan. This anti–IL-8 antibody has previously been shown to
neutralize the biological activity of rabbit IL-8 and to abrogate early
neutrophil accumulation in a rabbit model of
arthritis.11
These antibodies were purified and
tested for endotoxin contamination by the Limulus amebocyte
lysate test (Associates of Cape Cod, Falmouth, MA). Endotoxin
contamination of less than 10 pg/ml was considered acceptable, as our
previous dose–response studies of EIU in the rabbit have shown no
significant inflammatory response to intravitreal injection of
endotoxin doses at or below this range.
Neutralizing Experiments
Experiments were conducted to study the effects of anti–IL-8
antibody on the evolution of EIU in the rabbit. Each set of experiments
was performed on a minimum of 6 to 8 eyes (3 to 4 rabbits) and a
maximum of 24 eyes. Varying doses of anti–IL-8 antibody (50, 100, 250,
500, and 1000 µg) and 100 ng of LPS in the volume of 20 µl were
injected intravitreal into the right eye, whereas the left eye was
injected with LPS and control antibody at the same concentrations. All
animals were killed at 24 hours after injection. In other experiments,
the kinetics of EIU over 48 hours was studied after injecting the
chosen dose of anti–IL-8 antibody (250 µg) in groups of animals that
were killed at 6, 12, 24, and 48 hours after LPS administration. In the
subsequent experiments an anti–IL-8 antibody was administered at three
time points: 2 hours before, simultaneously with, or 6 hours after
endotoxin challenge. All three groups of animals were killed at 24
hours after endotoxin challenge, irrespective of the time of anti–IL-8
antibody injection. Inflam-matory activity in the eyes was
documented as described above.
To examine the evolution of EIU using 250-µg dose of anti–IL-8 antibody, the eyes were treated simultaneously with endotoxin injection in a group of three to four animals. Repeated anterior chamber paracentesis was performed at 6, 12, 24, 48, and 72 hours after injection. Animals were killed at the end of the observation period, 72 hours after the endotoxin challenge. In addition to estimating leukocyte exudation and protein content in AH, clinical evaluations were performed to assess the severity of ocular inflammation.
Statistical Analysis
Experimental and control eye pairs formed the basis of the
statistical analyses, using two-tailed paired Student’s
t-tests, 
2 distribution, and the
nonparametric test of Wilcoxon matched pairs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutralizing Experiments
Initial experiments were designed to optimize the dosage and
kinetics of the effect of anti–IL-8 antibody on the inflammatory
response measured at 6 to 48 hours after endotoxin injection in groups
of animals. Subsequent experiments evaluated the effect of anti–IL-8
antibody administered at three time points: 2 hours before,
simultaneously with, or 6 hours after endotoxin challenge. Another
kinetic study was performed to examine the evolution of EIU in a group
of animals (using 250-µg dose of anti–IL-8 antibody), wherein the
inflammatory response was observed at 6, 12, 24, 48, and 72 hours
post-LPS injection to measure leukocyte counts and protein
concentration. Preliminary experiments revealed that repeated aqueous
sampling during the evolution of EIU did not change significantly the
total or differential count of inflammatory cells or protein
concentration in the AH over a time period of 6 to 72 hours.
Dose Response and Time Course of Anti–IL-8 Antibody Inhibition of
EIU
Anti–IL-8 antibody treatment produced a significant decrease in
the AH leukocyte count in EIU when endotoxin and anti–IL-8 antibody
were administered simultaneously. The significant inhibition (63.2% ±
35.6%, mean ± SE) of the cell count in the treated eyes measured
at 24 hours after injection was obtained with the 250-µg dosage of
antibody (P < 0.05) (Fig. 1) . The mean protein level in the AH of control antibody–treated eyes
was 31.4 ± 2.1 mg/ml (mean ± SE), which was not
significantly different from the mean of 38 ± 4.3 mg/ml for the
treated eyes at the peak of inflammatory activity. The histologic
grading of tissue sections obtained at 24 hours after injection showed
a median grade of 3 (31–100 cells/field) in the treated eyes, whereas
for the control eyes, the median grade of inflammation was 4 (101–300
cells/field). Compared to controls, four of six anti–IL-8
antibody–treated eyes showed a decrease in the histologically
evaluated inflammation by at least one grade.
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Evolution of Ocular Inflammation after Anti–IL-8 Treatment
The course of ocular inflammation after anti–IL-8 antibody
treatment was studied in group of animals by examining AH for cellular
accumulation and protein level at time points from 6 to 72 hours, after
simultaneous administration of anti–IL-8 antibody and endotoxin. There
was a reduction in the AH leukocyte count at all time points, with
significant changes at the peak of inflammation and at 48 and 72 hours
after injection. A substantial and consistent inhibition of cell
infiltration (range, 60.3% ± 33% to 91% ± 12.2%, mean ± SE)
was seen from as early as 6 hours to 72 hours after injection (Fig. 3)
. During the evolution of EIU treated with anti–IL-8 antibody, there
was no significant change in the level of protein in AH.
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2
distribution) and 48 hours (P = 0.01) after treatment
with anti–IL-8 antibody in comparison to the group injected with
control antibody. However, at a later stage of EIU (72 hours), the
ratio of PMN leukocytes and mononuclear cells was reversed (11:89),
maintaining a similar proportions in both the treated (anti–IL-8
antibody) and untreated (control antibody) groups, unlike at 24 to 48
hours. The absolute number of cells (monocytes and polymorphs) was not
performed.
Effect of Anti–IL-8 Antibody Treatment on Clinical Uveitis Grade
When compared to the control eyes, the anti–IL-8
antibody–treated eyes showed reduced signs of inflammation in the form
of congestion, lacrimation, keratitis, flare, and aqueous cells in the
anterior chamber. The control eyes had a mean clinical uveitis score of
4 with flare, cellular accumulation, and membrane formation in the AC,
whereas the treated eyes had a mean clinical score of 2 at the peak of
AU.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti–IL-8 antibody partially blocks the development of EIU by neutralizing locally produced IL-8 in the eye. As the animal-to-animal variability in this model is significant, this study adopted a matched (treated/control) eye pair strategy to evaluate the anti–IL-8 antibody treatment. The clinical features and cellular infiltration in the anterior uvea during EIU in the rabbits were significantly reduced after treatment with anti–IL-8 antibody compared to administration of control antibody.
A dose of 250 µg of anti–IL-8 antibody was adequate in inhibiting inflammation throughout the time course of induction of EIU, including at the peak of inflammation (24 hours after injection). There was a significant reduction in total leukocyte accumulation and change in the ratio of PMN leukocytes to monocytes during the evolution of EIU. In experimental arthritis animal model, a higher dose of 500 µg of IL-8 monoclonal antibodies produced a significant inhibition of PMN infiltration into rabbit synovial fluid during the early phase of joint inflammation, rather than at the peak of the disease process.11
Both in vitro and in vivo studies, in endotoxin-induced pleurisy in rabbits, addition of anti–IL-8 antibody or desensitization of the neutrophils to IL-8, significantly decreased PMN migration in response to pleural fluid as the chemoattractant source.14 15 This inhibitory effect has been further corroborated in similar in vitro studies in ocular inflammation and experimental arthritis.11 A study13 reported inhibition of PMN migration by 41% to 79% when the vitreous samples obtained from uveitis- and nonuveitis-affected eyes were treated with a monoclonal antibody to IL-8. It is interesting to note that excessive accumulation of IL-8 itself at the interface of the endothelium and the circulating neutrophil may inhibit accumulation of leukocytes, perhaps by decreasing the chemotactic gradient or desensitization of the neutrophils.7
Disruption of the blood aqueous barrier (BAB) and changes in vascular
permeability lead to the accumulation of cells and protein in the AH.
Recruitment of PMN leukocytes into the extravascular space is regulated
by chemoattractants, including -chemokines, particularly IL-8.
Reduction in the uveal cellular infiltrate after anti–IL-8 antibody
therapy highlights the chemotactic role of IL-8 in leukocyte
trafficking into the anterior chamber. Usually BAB disruption starts
early, 1 to 6 hours after endotoxin injection,16
resulting
in protein accumulation earlier than leukocyte infiltration. This
sequence of events was not affected by treatment with anti–IL-8
antibody, which did not lead to a significant change in protein
exudation. Eyes with IL-8–induced inflammation showed raised AH
proteins in contrast to the previous studies, in which the anterior
chamber injection of CINC (cytokine-induced neutrophil
chemoattractant), which shares similarity with IL-8, did not alter the
protein exudation in AH.17
This divergence in the effect of anti–IL-8 antibody treatment suggests that other factors, including cytokines released in response to LPS, mediate the changes in vascular permeability responsible for protein exudation. These factors may remain unaffected by a reduction in the IL-8 concentration after treatment with neutralizing antibody to IL-8.
Anti–IL-8 antibody administered at different time points during the induction of EIU consistently produced a reduction in inflammation, which was most evident when the anti–IL-8 antibody treatment was given concurrently with endotoxin. Similarly, the cellular infiltration observed from 6 to 72 hours post-LPS injection was consistently inhibited by anti–IL-8 antibody administered simultaneously with the endotoxin. Preliminary studies in our laboratory have demonstrated that in rabbit EIU, the leukocyte infiltration persists until 96 hours post-LPS injection. In this study, the persistent inhibition of leukocyte accumulation indicates that a single administration of anti–IL-8 antibody at the onset of inflammation may be sufficient to neutralize locally produced IL-8 throughout the evolution of the AU.
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Footnotes |
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Commercial relationships policy: N.
1 Present address: Department of Pharmacology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan. 
Corresponding author: Denis Wakefield, Inflammation Research Unit, School of Pathology, University of NSW, Sutherland Centre of Immunology, Sydney, Australia. E-mail: d.wakefield{at}unsw.edu.au
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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