Abstract
purpose. To evaluate the differential gene expression of chemokines after
corneal transplantation and to determine the chemokines associated with
allograft rejection.
methods. Orthotopic mouse corneal transplantation was performed in two fully
mismatched-strain combinations using C57BL/6 (H-2b) and
BALB/c (H-2d) mice as recipients and BALB/c and C57BL/6
mice as donors. Normal nonsurgical eyes served as negative control
specimens and syngeneic transplants (isografts) as control specimens
for the alloimmune response. Chemokine gene expression in accepted and
rejected allografts and appropriate control specimens was determined by
a multiprobe RNase protection assay system.
results. In eyes with rejected allografts, there was overexpression of regulated
on activation normal T-cell expressed and secreted (RANTES), macrophage
inflammatory protein (MIP)-1α, MIP-1β, MIP-2, and monocyte
chemotactic protein (MCP)-1 in both C57BL/6 and BALB/c recipients. In
addition, C57BL/6 eyes with rejected allografts expressed very high
levels of interferon-γ–inducible protein of 10 kDa (IP-10) mRNA, in
contrast to BALB/c eyes with rejected allografts, in which IP-10
expression remained very low. In contrast, lymphotactin gene expression
increased only slightly in rejected allografts, and eotaxin mRNA, which
was also detected in normal eyes, remained unchanged among isograft and
allograft groups. T-cell activation gene (TCA)-3 mRNA was not detected
in any of the assayed eyes.
conclusions. Increased expression of mRNA for select chemokines of the CXC (α) and
CC (β) families is associated with corneal allograft rejection.
Significantly elevated IP-10 gene expression in high-rejector C57BL/6,
but not in low-rejector BALB/c, hosts suggests that differential
activation of chemokines may be related to differences in alloimmune
reactivity observed among different murine
strains.
Chemokines are low-molecular-weight proteins that, along with
adhesion molecules, play a critical role in immune and inflammatory
responses by virtue of regulating the trafficking of leukocytes in a
multistep process involving arrest of rolling and firm adhesion to the
vascular endothelium
1 2 and leukocyte migration to target
tissues through a chemotactic gradient.
3 In addition,
chemokines are involved in homeostatic noninflammatory processes such
as lymphocyte homing and recirculation through secondary lymphoid
organs.
4 5 Chemokines have been subdivided into families
on the basis of the relative position of their cysteine
residues.
3 The CXC (α) chemokine family, which includes
interferon-γ–inducible protein (IP)-10, interleukin (IL)-8, and
macrophage inflammatory protein (MIP)-2, has the first two cysteines
separated by one amino acid residue. In the CC (β) chemokine group,
the first two cysteine residues are adjacent to each other. Regulated
on activation normal T-cell expressed and secreted (RANTES), eotaxin,
MIP-1α, MIP-1β, monocyte chemoattractant protein (MCP)-1, and
T-cell activation gene (TCA)-3 are included in the CC chemokine family.
Lymphotactin (Ltn) has only one cysteine and is classified as a C
chemokine.
6 7 These chemokines form a complex functional
network locally and systemically in a variety of inflammatory,
infectious, and immune diseases in which many CXC chemokines (e.g.,
IL-8 and Gro-α) mediate recruitment of neutrophils, whereas CC
chemokines are primarily involved in recruitment of immune cells such
as antigen-presenting cells and T cells.
3 8
A number of molecular and anatomic features of the cornea and anterior
segment are believed to contribute to the immune privilege enjoyed by
orthotopic corneal transplants.
9 In spite of this
privilege, corneal allografts are frequently rejected by a process
characterized by leukocyte infiltration of the graft stroma and
adherence of mononuclear cells to the donor corneal endothelium.
Therefore, chemotactic mechanisms involved in leukocyte trafficking
probably play a critical role in the alloimmune response to corneal
transplants.
Upregulation in chemokine transcription or protein expression has been
related to allograft rejection in a number of vascularized organ
transplants.
10 11 12 13 14 However, to date, chemokine expression
in corneal transplantation has not been characterized. We investigated
the gene expression of a panel of chemokines by assaying for their mRNA
using the RNase protection assay (RPA) system. We hypothesized that
corneal graft rejection is associated with differential overexpression
of chemokines. Specifically, because the alloreactive T-cell response
to corneal grafts has been primarily associated with a T-helper (Th) 1
type phenotype,
15 16 17 and specific chemokines and
chemokine receptors are associated with polarized Th1 and Th2
responses,
18 19 20 21 22 23 24 25 we hypothesized that chemokines
associated with receptors CCR1 (e.g., MIP-1α), CCR2 (MCP-1), CCR5
(e.g., RANTES), and CXCR3 (e.g., IP-10), but not CCR3 (eotaxin), would
be selectively upregulated in the process of rejection of corneal
allografts because they have been associated with Th1 type immune
responses. Moreover, because appreciable differences in corneal graft
survival rates have been observed among high-rejecting Th1-biased
C57BL/6 mice compared with low-rejecting Th2-biased BALB/c
recipients,
26 we hypothesized that differential expression
of chemokines in the two strains may partially account for differences
in graft rejection rates in the two strains. In the aggregate, our
results suggest that there is selective chemokine gene expression
associated with the effector phase of corneal transplant allorejection.
Total RNA was extracted by the single-step method (RNA-STAT-60;
Tel-Test, Friendswood, TX). Eyes were homogenized and centrifuged to
remove cellular debris. The RNA pellet obtained from five eyes was
resuspended in nuclease-free water and processed together as a group.
Detection and quantification of murine chemokine mRNAs were
accomplished with a multiprobe RPA system (PharMingen, San Diego, CA),
as recommended by the supplier. Briefly, a mixture of[α
-32P] uridine triphosphate–labeled
antisense riboprobes was generated from the chemokine template set
mCK-5 (PharMingen). Twenty micrograms total RNA was used in each
sample. Total RNA was hybridized overnight at 56°C with 300 pg of the 32P antisense riboprobe mixture.
Nuclease-protected RNA fragments were purified by ethanol
precipitation. After purification, the samples were resolved on 5%
polyacrylamide sequencing gels. The gels were dried and subjected to
autoradiography.
Protected bands were observed after exposure of gels to x-ray film.
Specific bands were identified on the basis of their individual
migration patterns in comparison with the undigested probes. The bands
were quantitated by densitometric analysis (Image; National Institutes
of Health, Bethesda, MD) and were normalized to
glyceraldehide-3-phosphate dehydrogenase (GAPDH).
We used a multiprobe RPA system to quantify a panel of nine
chemokines’ mRNA levels from a single sample of total RNA. This method
is highly sensitive, and allows for comparative analysis of different
mRNA species from a given RNA sample. In the aggregate, we conclude
from our data that there is increased expression of select chemokines,
in particular RANTES, and to a lesser extent MIP-1α, MIP-1β, and
MCP-1 after corneal allotransplantation regardless of the recipient
host; there is marked overexpression of the Th-1-associated, interferon
(IFN)-γ–induced CXC chemokine IP-10 in high-rejecting C57BL/6, but
not in BALB/c, recipients. Eotaxin is constitutively expressed in
normal control eyes, and its mRNA level is not appreciably affected by
the alloimmune response to corneal transplantation.
RANTES and MIP-1β are known to serve as chemoattractants for
activated CD4+ T lymphocytes, and MIP-1α is chemotactic for activated
CD8+ T lymphocytes.
29 30 MCP-1 attracts memory T
lymphocytes and monocytes.
31 Moreover, these CC chemokines
not only attract natural killer (NK) cells but also enhance their
cytolytic responses.
32 Because the role of NK cell
activity in corneal alloimmunity remains unknown, we speculate that
RANTES, MIP-1α, MIP-1β, and MCP-1 are primarily involved in corneal
transplant immunity by mediating recruitment of alloreactive T cells to
the anterior segment microenvironment. Ltn, known for its function as a
lymphocyte-specific chemoattractant, is thought to play an important
role in trafficking of resting T cells and in activated peripheral CD8+
T cells.
6 7 Our data demonstrate that Ltn mRNA expression
level in eyes with rejected allografts is higher than that of accepted
allografts or the undetectable levels in isografts and naive controls.
However the overall Ltn mRNA level, even in rejected hosts, was
uniformly low, regardless of the strain tested. This could be either a
reflection of the dominant role of the CD4 compartment in corneal
alloimmunity,
15 16 17 or because in corneal transplantation,
CD8+ T cell responses may occur in the later, rather than acute, phase
of allorejection,
15 33 and therefore our assay may have
missed the peak level of Ltn expression.
There are significant differences in the expression of specific
chemokine receptors in leukocyte subsets that are thought to serve as
an important level of regulation for selective recruitment of
lymphocyte subsets in different disease states. For example, the
receptors CXCR3 (for IP-10), CCR1, and CCR5 (for MIP-1α, MIP-1β,
and RANTES) are preferentially expressed on Th1
cells.
20 21 22 24 Conversely, expression of CCR4 (for TARC)
and CCR3 (for eotaxin) have been linked to Th2 type activation and
recruitment.
21 22 24 In this study, levels of mRNA for
eotaxin, which preferentially binds CCR3 expressed on Th2
cells,
34 did not increase in rejected corneal allograft
samples. These results are in accordance with previous observations
suggesting Th1-, but not Th2-, dominant responses in mediating corneal
allograft rejection.
15 16 17
We have been interested by recent observations that fully mismatched
corneal grafts are rejected more swiftly and at a higher overall rate
in C57BL/6 (∼90%) compared with BALB/c (∼50%)
recipients.
26 We were therefore intrigued by the finding
that there was very high ocular mRNA expression for IP-10 in
allografted C57BL/6 hosts, compared with levels in the BALB/c host
group. Moreover, because draining lymph nodes are regarded as important
sites for lymphocyte homing and activation after
transplantation,
35 we have recently examined chemokine
gene expression in these sites. Compared with that in draining lymph
nodes of naive animals, high IP-10 mRNA expression has been detected in
C57BL/6, but not BALB/c, hosts that rejected allografts (unpublished
observations). IP-10 may very well be instrumental in corneal allograft
rejection, because its receptor CXCR3 is expressed almost exclusively
on T cells of the Th1 phenotype,
21 24 and its expression by
interferon-treated monocytes has been shown to regulate the migration
of activated CD4+ T lymphocytes.
36 37 Because C57BL/6 and
BALB/c mice are thought to have preferential Th1- or Th2-polarized
responses, respectively,
23 26 38 our data suggest that
selective high IP-10 expression by C57BL/6 mice may be associated with
the more potent alloreactivity seen in this recipient
strain.
26
It is important to address the potential limitations of this study.
First, we selected for study a group of chemokines from the C, CC, and
CXC families (from among the more than 40 chemokines identified to
date) that are believed to be primarily involved in the recruitment of
immune cells rather than neutrophils. We did not concentrate on CXC
chemokines containing the NH2 terminal sequence glutamic
acid-leucine-arginine that are critically relevant to recruitment of
neutrophils
3 and may therefore play a significant role in
the recruitment of inflammatory cells in corneal transplants. However,
because we detected increased MIP-2 mRNA (MIP-2 binds the murine
homologue of the IL-8 receptor), particularly in the high-rejecting
C57BL/6 recipients, we cannot rule out contribution of CXC neutrophil
chemoattractant chemokines to corneal transplant alloimmunity. This is
especially true of the high-risk corneal transplantation setting in
which we have observed neutrophilic infiltration before migration of
antigen-presenting cells (unpublished data). We believe therefore that
the functional role of CXC chemokines deserves further study in the
high-risk corneal graft setting, particularly in the early induction
phase of alloimmunity.
Second, we primarily used whole-eye homogenates for analysis of
chemokine mRNA to circumvent the problems faced with the very small
quantities of RNA extracted from the murine cornea, which would
translate into significant increases in the number of animals used.
Although admittedly this method does not allow localization of the
chemokine mRNA expression (to the cornea), as may be obtained by in
situ hybridization, it has the benefit of allowing simultaneous
quantification of different RNA species. In addition, whereas leukocyte
infiltration into the posterior compartments of the eye is not observed
after corneal transplantation, effector cells involved in mediating
graft rejection are commonly seen in noncorneal structures of the
anterior segment such as the anterior chamber and iris, most likely a
result of extravasation and recruitment at the level of the ciliary
body and iris root. It is therefore very likely that noncorneal
structures of the anterior segment actively contribute to leukocyte
recruitment by expressing chemokines. Therefore, although analysis of
whole eyes has the disadvantage of not limiting the assay to the cornea
alone, it has the advantage of assaying chemokines expressed by other
structures in the anterior segment that probably play a functionally
relevant role in leukocyte recruitment after corneal transplantation.
To confirm that the expression of specific chemokine mRNA after
allograft rejection reflected in the whole-eye data are also operative
in the corneal microenvironment, we analyzed C57BL/6 control and
rejected corneas (
n = 12) and were able to reproduce
the whole-eye data with the exception that eotaxin, detectable in the
normal whole eye, was not expressed in normal corneas
(Fig. 1C) .
Third, it is important to emphasize that we analyzed chemokine
expression in the effector phase of the alloimmune response. The time
course of chemokine expression may vary significantly from one
chemokine to another. Therefore, detecting low mRNA levels for a
specific chemokine (e.g., Ltn) several weeks after corneal
transplantation does not mean that the chemokine is similarly minimally
expressed early after transplantation in the induction phase of the
alloimmune response. Fourth, because we evaluated only mRNA levels, and
the biologic function of these chemotactic cytokines is dependent on
ligand binding of chemokine receptors, differential levels of genetic
message should not be equated with similar variations in protein
expression. Finally, we emphasize that in these studies we did not
evaluate the functional relevance of chemokines in corneal
transplantation. Further studies, such as those involving knockout
strains or specific antibodies, would be helpful in establishing the
functional relevance of a chemokine or chemokine receptor system in
corneal allograft survival.
Corneal transplant rejection shares with all other immune responses the
fundamental process of leukocyte recruitment to the antigenic site. As
such, chemokines may play a critical role in regulating not only the
migration of inflammatory cells from the intravascular compartment to
the graft site, but also in amplifying the alloimmune response by
selectively activating and recruiting polarized Th1 phenotypic cells.
In addition to demonstrating significant overexpression of RANTES,
MIP-1α, MIP-1β, MIP-2, and MCP-1 mRNA in rejected corneal
allografts of both C57BL/6 and BALB/c host groups, our data suggest
that the extremely high levels of IP-10 mRNA detected in the rejected
allograft of C57BL/6 mice may explain the high rejection rate of
corneal allografts in this strain. Further studies are required to
evaluate the contribution of specific chemokines to corneal
transplantation immunobiology.
Presented in part at the annual meeting of the Association for Research
in Vision and Ophthalmology, Fort Lauderdale, Florida, May 9–14, 1999.
Supported by Grants EY00363 (MRD), GM49661 (SJO), and EY1901 (SJO) from
the National Institutes of Health and by grants from Fight For Sight
(MRD, SJO), Eye Bank Association of America (MRD), the Lucille P.
Markey Foundation (SJO), and Fellowships from Bausch & Lomb (SY, DM).
Submitted for publication February 25, 1999; revised July 6, 1999; accepted July 14, 1999.
Commercial relationships policy: N.
Corresponding author: M. Reza Dana, Laboratory of Immunology, Schepens
Eye Research Institute, Harvard Medical School, 20 Staniford Street,
Boston, MA 02114. E-mail:
[email protected]
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