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Receptors in Antibody-Dependent Corneal Inflammation1
Departments of Medicine and Ophthalmology, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH 44106
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R
interactions in triggering immune complex-mediated corneal disease.
FcR-/- mice, C3-/- mice, and
immunocompetent control (B6/129Sj) mice were immunized s.c. and
injected intrastromally with Onchocerca volvulus Ags.
Slit lamp examination showed that control mice, C3-/-
mice, and control mice injected with cobra venom factor developed
pronounced corneal opacification, whereas corneas of
FcR-/- mice remained completely clear. Furthermore,
recruitment of neutrophils and eosinophils to the corneal stroma was
significantly impaired in FcR-/- mice, but not in
C3-/- mice or cobra venom factor-treated mice. We
therefore conclude that FcR-mediated cell activation, rather than
complement activation, is the dominant pathway of immune complex
disease in the cornea. These findings demonstrate a novel role for
FcR interactions in mediating ocular
inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Taken together, these observations imply that: 1) individuals with chronic infections are likely to have pathogen-specific Ab in the corneal stroma; and 2) infiltration of pathogens into the cornea and release of Ags would trigger immune complex formation and an Arthus-type response at this site. As the transparent nature of the cornea is tightly regulated by the parallel arrangement of collagen fibrils and by a strict level of hydration, development of an inflammatory response in this tissue will disrupt the normal physiology of the cornea, resulting in loss of corneal clarity, visual impairment, and possibly blindness.
Two pathways are involved in immune complex-mediated inflammatory
responses. First, immune complexes can activate the complement cascade
and generate peptide fragments C3a and C5a, which are highly
chemotactic for neutrophils and eosinophils (6, 7).
Second, immune complexes can bind to FcR on the cell surface and
trigger cellular activation (2, 3). Ravetch and coworkers
(8, 9) demonstrated that FcR interactions are essential
for development of the reverse passive Arthus reaction in the skin,
whereas the absence of complement components C3 or C4 had no effect.
However, the relative contribution of FcR interactions and complement
in development of immune complex disease depends on the genetic
background of the host, and on the tissue that is involved (3, 10, 11, 12). For example, C5a receptor expression is important in
immune complex-mediated alveolitis, but has less of a contribution in
peritonitis and skin inflammation (12).
Our studies have focused on river blindness, which is the second leading cause of infectious blindness worldwide (13, 14, 15, 16).The parasitic nematode that causes this disease, Onchocerca volvulus, survives in the host for over 10 years in the presence of an ongoing T cell response and high titers of parasite-specific IgG1, IgG4, and IgE (16). The larval form of the parasite invades the cornea and posterior ocular tissue, causing severe visual impairment and eventual blindness. Epidemiological studies indicate that ocular inflammation and subsequent tissue damage develop only after parasite death and release of somatic Ags into the immediate environment (16, 17). Although immune complexes have been detected in the serum of O. volvulus-infected individuals, their role in the pathogenesis of this disease is not known (18, 19, 20). In the current study, we explore the possibility that the presence of O. volvulus Ags and parasite-specific Ab in the cornea triggers an Arthus-type reaction that drives the inflammatory response in ocular onchocerciasis.
We have developed a murine model of river blindness in which mice are immunized s.c. and then injected into the corneal stroma with O. volvulus Ags (15, 21). Immunized mice produce high titers of anti-parasite IgG, and develop pronounced corneal opacification and neovascularization (15, 22). In this model, neutrophil recruitment to the cornea peaks at 24 h after intracorneal injection, whereas eosinophil recruitment peaks after 72 h, completely replacing neutrophils as the predominant inflammatory cell type in the cornea (15, 22). Although neutrophils and eosinophils mediate corneal pathology that is similar in appearance, these cell types differ both in the molecular basis of recruitment to the cornea and in the cytotoxic mediators produced. For example, neutrophil infiltration is dependent on expression of platelet endothelial cell adhesion molecule-1 and expression of CXCR2, whereas eosinophil recruitment to the cornea is dependent on expression of P-selectin and ICAM-1, and is regulated by CD4+ cells (23, 24, 25, 26) and by eotaxin (53). These studies also demonstrated that complete inhibition of corneal pathology depends on blocking infiltration of both cell types to the cornea. Furthermore, whereas the primary cytotoxic activity of neutrophils is by generating reactive oxygen species, eosinophils also secrete cationic granule proteins such as eosinophil major basic protein, which are directly cytotoxic for resident corneal cells (27).
Results from several sets of experiments led us to conclude that
formation of immune complexes is essential for development of O.
volvulus keratitis, as corneal pathology is detected only when
parasite Ags are injected in the presence of O.
volvulus-specific Ab. For example, keratitis is not induced when
parasite Ags are injected into unimmunized, immunocompetent mice
(21), immunized Ab-deficient (µMT) mice
(28), or immunized µMT mice together with normal mouse
sera (28). Keratitis is induced in µMT mice only when
parasite Ags are injected into the corneal stroma together with sera
from immunized mice (28). Results from the current study
provide further evidence for an essential role for immune complexes in
this disease, as FcR-deficient mice fail to develop keratitis
despite the presence of Ag in the cornea and high titer
parasite-specific serum Ab. Furthermore, we found that O.
volvulus-induced immune complex disease in the cornea is
independent of the pivotal complement component C3.
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Materials and Methods |
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O. volvulus worms were obtained from nodules that had been surgically removed from patients in Cameroon, and were kindly provided by J. Bradley (University of Nottingham, Nottingham, U.K.). Soluble O. volvulus Ags were prepared as described previously (21). Briefly, worms were isolated from s.c. nodules after digestion with collagenase (Sigma, St. Louis, MO). Parasites were homogenized in HBSS, sonicated, and centrifuged to remove insoluble material. The soluble O. volvulus Ag preparation was adjusted to 1 mg/ml, filter sterilized, and stored at -70°C.
Animals
Mice with a targeted deletion in the common FcR chain (B6,
129-Fc
r1gtm1Rav) were derived by Ravetch and
coworkers (29), and were obtained from The Jackson
Laboratory (Bar Harbor, ME). Control F2
generation mice were on same genetic background (B6/129SJ) and were
also obtained from The Jackson Laboratory. C3-deficient mice on the
same genetic background were derived by Carroll et al.
(30), and were a kind gift from his laboratory. In some
experiments, FcR mice backcrossed to C57BL/6 background were
obtained from Taconic Laboratories (Germantown, NY).
Immunization and intrastromal injection
Mice received three weekly s.c. immunizations with 10 µg O. volvulus Ags in a 1:1 ratio with adjuvant containing squalene (Aldrich Chemical, Milwaukee, WI), Tween 80 (Fisher, Fair Lawn, NJ), and pluronic acid (BASF, Parsippany, NJ). For intrastromal injections, the cornea was scarified with a 30-gauge needle, and 5 µg O. volvulus Ags were injected into the corneal stroma using a 33-gauge needle attached to a Hamilton syringe (Hamilton, Reno, NV). Corneal opacification was monitored daily by slit lamp examination and evaluated as previously described (21).
Depletion of C3 by treatment with cobra venom factor
Cobra venom factor (CVF)3 was obtained from Sigma (St. Louis, MO) and prepared at 20 µg/ml HBSS. A total of 0.5 ml was injected i.p. 1 day before intrastromal injection, the day of intrastromal injection, and each day before sacrifice. Control mice were injected with HBSS.
Immunohistochemistry
Eyes were removed, fixed for 18 h in 10% formaldehyde (Sigma), processed, and embedded in paraffin by standard methods. To detect neutrophils, 5-µm sections were immunostained with mAb NIMP-R14, followed by biotinylated rabbit anti-rat Ig (BioGenex, San Ramon, CA). Eosinophils were detected using rabbit antisera to major basic protein diluted 1/5000 for 2 h, as described (22, 28). After incubation with a secondary Ab, sections were incubated with alkaline phosphatase-conjugated streptavidin (BioGenex). Positive reactivity was visualized using Vector Red Substrate (Vector Laboratories, Burlingame, CA) containing Levamisole (Sigma), and counterstained with modified Harris’ hematoxylin (Richard-Allen, Kalamazoo, MI). Cells were visualized by bright field and fluorescent microscopy.
Measurement of serum IgG and C3 levels by ELISA
Sera were collected at the time of sacrifice and assayed for Ab by ELISA. Immulon-4 ELISA plates (Dynatech, Chantilly, VA) were coated with 50 µl of 1 µg/ml parasite Ags and incubated overnight at 4°C. After blocking with 1% FBS, dilutions of mouse sera were incubated 2 h at room temperature, washed, and incubated with biotinylated goat anti-mouse isotype-specific Abs (Southern Biotechnology, Birmingham, AL). Reactivity was determined after incubation with peroxidase-labeled anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA), and tetramethyl benzidine (Zymed, San Francisco, CA) was used as a substrate. Reaction was stopped after 10 min with 1 N HCl. Absorbance was measured at 450 nm on a kinetic microplate reader (Molecular Devices, Sunnyvale, CA).
Serum C3 levels were detected by ELISA using unconjugated anti-C3 to coat the wells, and HRP-conjugated anti-C3 as detecting Ab (Cappell, Aurora, OH). Serum C3 levels in mice treated with CVF were reduced >90%.
Statistics
p Values were determined by unpaired t test using Prism software (GraphPad, San Diego, CA). A value of <0.05 was considered to be statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R-/- expression is essential for development of
O. volvulus-induced corneal disease
To determine the role of FcR expression on O.
volvulus keratitis, FcR-/- and control,
129Sj/C57BL/6 mice were immunized s.c. and injected into the central
cornea with O. volvulus Ags, and the severity of corneal
opacification was determined by slit lamp examination. As shown in Fig. 1
, control mice developed pronounced corneal opacification within 24
h after intrastromal injection, and corneas remained opaque throughout
the course of the experiment. In marked contrast to control mice,
corneas of FcR-/- mice failed to develop
opacities and remained transparent throughout the study (Fig. 1, lower right panel).
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R-/- mice
Previous studies demonstrated that corneal opacification is
associated with a biphasic recruitment of neutrophils and eosinophils
to the corneal stroma, with neutrophils at maximal numbers in the first
24 h after intrastromal injection, and eosinophils the predominant
inflammatory cell type after 72 h (15, 22, 28). To
determine the role of FcR in extravasation of neutrophils from the
limbus to the peripheral cornea and migration to the central cornea,
control and FcR-/- mice were immunized and
injected intrastromally with worm Ags, as described above. Neutrophils
in the peripheral, paracentral, and central regions of the cornea were
counted after immunostaining with Ab to neutrophil surface marker that
does not react with eosinophils.
As shown previously for C57BL/6 mice (22, 28), neutrophils
infiltrate into each region of the corneal stroma of control,
immunocompetent mice within 24 h of intrastromal injection. The
highest numbers were in the peripheral region of the corneal stroma and
the fewest in the center, reflecting migration of these cells from
limbal vessels to the site of trauma in the central cornea. However,
neutrophil recruitment to the corneas of
FcR-/- mice was significantly impaired in
the peripheral cornea and in the paracentral and central regions of the
cornea (Figs. 2 and 3). This deficiency was not a result of delayed recruitment, as
neutrophils were not detected in corneas of either strain of mice after
72 h (data not shown).
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R-/- mice
To determine the effect of FcR deficiency on eosinophil
infiltration of the cornea, FcR-/- and
control, 129Sj/C57BL/6 mice were immunized s.c. and injected
intrastromally, as described above. Mice were sacrificed 72 h
later, and eosinophils were identified by immunostaining with Ab to
eosinophil major basic protein. As shown for neutrophils, eosinophils
were also recruited to peripheral, paracentral, and central regions of
the corneas of immunocompetent, control mice (Figs. 4 and 5). In contrast, eosinophil recruitment to the cornea was significantly
impaired in FcR-/- mice compared with
control, 129Sj/C57BL/6 mice. As shown previously for µMT mice
(28), eosinophils’ recruitment from peripheral vessels to
the peripheral cornea of FcR-/- mice was
only partially impaired, whereas infiltration from the peripheral to
the central cornea was completely inhibited in the absence of FcR
expression.
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R-/-
mice
To determine whether the severity of corneal disease and
recruitment of neutrophils and eosinophils are related to differences
in Ag-specific isotype production, we measured IgG1 and IgG2a responses
in FcR-/- mice. As shown in Fig. 6, parasite-specific IgG1 production was elevated compared with IgG2a in
FcR-/- mice, consistent with a predominant
Th2-associated response shown previously for immunocompetent mice
(21). However, there was no significant difference between
parasite-specific Ab titers in FcR-/- mice
compared with control mice. These data indicate that FcR expression
is not essential for development of Th2 responses, and also suggest
that impaired neutrophil and eosinophil recruitment to the cornea is
not due to a deficiency in Ab production or a switch in isotype of
parasite-specific Ab.
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Two approaches were taken to determine the effect of complement on
development of O. volvulus keratitis. First,
C3-/- mice were immunized and injected using
the same experimental protocol as FcR-/-
mice, and second, mice were depleted of C3 by i.p. injection of CVF.
Systemic administration of CVF depletes complement not only from the
serum, but also from the cornea (31). Serum complement
levels after i.p. injection of CVF were reduced by >90%
(OD450 0.84 +/- 0.06 for control,
saline-injected mice; 0.07 +/- 0.01 in CVF-treated mice).
As shown in Fig. 7, the total number of neutrophils and eosinophils in the corneal stroma
of C3-/- mice and CVF-treated mice was not
significantly different from control animals, and there was no
difference in distribution of these cells within the corneal stroma.
Furthermore, there were no significant differences in corneal
opacification scores between control mice and either
C3-/- mice or C3-depleted mice at any time
point (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R-mediated responses that is
independent of C3. Our findings differ from other models of infectious
keratitis and of helminth-induced immunopathology. In Pseudomonas
aeruginosa keratitis, for example, depletion of C3 with CVF
abrogates development of stromal disease, indicating an essential
role for complement (32, 33). Also, our findings
that FcR interactions have a proinflammatory role in ocular
onchocerciasis differ from the antiinflammatory role of FcR in
granuloma formation in mice infected with the parasitic helminth
Schistosoma mansoni, in which
FcR-/- mice failed to down-regulate liver
granulomas (34).
The dominant role for FcR-mediated interactions over complement in
the current study is consistent with findings by Ravetch and coworkers
(8, 35) using a murine model of reverse Arthus reaction.
In those studies, FcR-/- mice had reduced
neutrophil infiltration, edema, and hemorrhage compared with control
animals, whereas Arthus responses developed normally in
C3-/- and C4-/- mice,
and in animals depleted of C3 using CVF (8, 9). However,
studies in which C5a receptor interactions were blocked using either
receptor antagonists or C5aR-deficient mice indicate an important
contribution of C5a receptors in immune complex disease, although the
relative contribution depends on the tissue involved (10, 12, 36). The apparent paradox that C5a contributes to the
inflammatory response in the absence of C3 may be explained by local
production of complement components in affected tissues
(10). Various cell types, including mononuclear
phagocytes, endothelial cells, and corneal fibroblasts, can produce C3
and C5, and neutrophils and macrophages can secrete the proteases that
cleave them (10, 37).
The relative contribution of complement to immune complex disease also has a genetic component, with C57BL/6 mice less susceptible to complement-mediated immune complex disease than BALB/c mice, which have a codominant role together with FcR (11, 36). In the present study, we found that CVF treatment did not inhibit development of keratitis B6/129Sj mice; however, we also found similar results in CVF-treated BALB/c and C57BL/6 mice, indicating that there are no apparent strain differences in response to CVF (L. R. Hall, unpublished observations). Together with our findings that C3-/- mice develop O. volvulus keratitis with the same intensity as immunocompetent control animals, we conclude that C3 has no essential role in this model of immune complex disease. However, as resident stromal fibroblasts can synthesize complement components (37), future studies will examine whether C5a receptor interactions function independently from C3 in O. volvulus keratitis.
Resident cells in the cornea, including epithelial cells and stromal
fibroblasts, can also be induced to express FcR in vitro (38, 39). However, it is likely that FcR-dependent keratitis is
mediated primarily by infiltrating neutrophils and eosinophils, with
resident cells involved only in initial recruitment of neutrophils by
an FcR-independent pathway. Although this has yet to be verified,
our recent studies demonstrate that CXCR2 mediates neutrophil
recruitment to the corneal stroma, and that neutrophil chemokines
macrophage-inflammatory protein (MIP)-2 and KC are produced in
the corneal stroma rather than the epithelium (24). Taken
together with in vitro studies showing that human keratocytes can
secrete IL-8 (40), it is feasible that stromal keratocytes
mediate early recruitment of neutrophils by producing MIP-2 and KC.
Once these cells infiltrate the cornea, we speculate that binding of
immune complexes to FcR on the neutrophil surface stimulates
production of chemotactic and immunoregulatory cytokines such as IL-12
and MIP-1
(41, 42).
The high affinity FcRI and the low affinity FcRIII, which are
nonfunctional in FcR-/- mice, are multimeric
receptors with ligand binding and signaling subunits. The signaling
subunit for FcRI and FcRIII is the immunoreceptor tyrosine-based
activation motif (ITAM), which mediates cell activation signals such as
production of proinflammatory cytokines (3, 43). In
contrast, FcRII is a single subunit receptor that signals through
the immunoreceptor tyrosine-based inhibitor motif, which can inhibit
ITAM-mediated cell activation (44, 45). As neutrophils and
eosinophils express more than one type of FcR on their surface,
interactions with IgG-containing immune complexes may result in
coaggregation of different FcR, and the balance of activating and
inhibitory FcR signals most likely determines the function of these
effector cells in the cornea. In the current study, the finding that
ocular inflammation is dependent on FcRI and FcRIII expression
indicates that inflammation is dependent on ITAM signaling. Future
studies will examine the interplay of ITAM and immunoreceptor
tyrosine-based inhibitor motif-mediated interactions in ocular
disease.
The common -chain of the FcR is an integral part not only of FcRI
and FcRIII, but also the high affinity IgE receptor FcRI
(2). However, although IgE levels are elevated in infected
individuals and immunized animals (16, 21), it is unlikely
that FcRI is involved in O. volvulus keratitis, as this
receptor is not expressed by neutrophils or by murine eosinophils
(2, 46). Mast cells, which mediate the reverse Arthus
response (35), have not been detected in O.
volvulus keratitis.
In addition to chemotactic and immunoregulatory cytokines produced by
neutrophils, ITAM-mediated interactions may trigger release of
cytotoxic mediators from neutrophils, including oxygen radicals and
proteolytic enzymes (47). Similarly, FcR stimulation of
eosinophils could stimulate production of chemotactic and
immunoregulatory cytokines such as eotaxin, IL-4, and IFN-
(48, 49, 50), and release of granule proteins such as
eosinophil major basic protein, which has a direct cytotoxic effect on
corneal epithelial cells (27, 51, 52). The presence of
these mediators in the tightly regulated environment of the corneal
stroma could then disrupt the function of epithelial cells and corneal
endothelial cells, which maintain the critical hydration level of the
stroma, resulting in stromal edema, and opacification of normally
transparent cornea. Similarly, a cytotoxic effect on stromal
keratocytes would adversely affect the structural integrity and spacing
of the collagen fibrils.
Our previous studies demonstrated that O. volvulus keratitis
is dependent on development of a CD4+,
Th2-associated response, consistent with a type IV response
(21). However, it appears that the primary role for the
Th2 response is to generate parasite-specific IgG1 and IgE by
production of IL-4, and to induce differentiation and maturation of
eosinophils by production of IL-5. Although CD4+
cells also regulate eosinophil recruitment to the cornea at the later
stage of disease (23), results of the present study
indicate that immune complex formation is essential for mediating the
inflammatory response in the cornea. In the absence of specific Ab,
neutrophil and eosinophil recruitment to the central cornea is
significantly impaired, and corneas remain transparent
(28). However, the current study shows that even in the
presence of high titer anti-parasite IgG, FcR expression is
essential for development of keratitis.
In conclusion, the findings presented in this work represent a novel
role for FcR interactions in mediating ocular inflammation and loss
of vision.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eric Pearlman, Division of Geographic Medicine, Case Western Reserve University School of Medicine, W137, 2109 Adelbert Road, Cleveland, OH 44106. E-mail address: exp2{at}po.cwru.edu
3 Abbreviations used in this paper: CVF, cobra venom factor; ITAM, immunoreceptor tyrosine-based activation motif; MIP, macrophage-inflammatory protein.
Received for publication March 6, 2001. Accepted for publication May 4, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI/III and C5aR in the reverse Arthus reaction. J. Immunol. 164:1065. chain deletion results in pleiotrophic effector cell defects. Cell 76:519.[Medline]
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receptors on murine eosinophils: implications for the functional significance of elevated IgE and eosinophils in parasitic infections. Blood 89:3826.): inhibition by immunoglobulin A complexes. J. Exp. Med. 190:487.This article has been cited by other articles:
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I. Gillette-Ferguson, A. G. Hise, Y. Sun, E. Diaconu, H. F. McGarry, M. J. Taylor, and E. Pearlman Wolbachia- and Onchocerca volvulus-Induced Keratitis (River Blindness) Is Dependent on Myeloid Differentiation Factor 88 Infect. Immun., April 1, 2006; 74(4): 2442 - 2445. [Abstract] [Full Text] [PDF]
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A. Casadevall and L.-a. Pirofski New Concepts in Antibody-Mediated Immunity Infect. Immun., November 1, 2004; 72(11): 6191 - 6196. [Full Text] [PDF]
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