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Acts Directly on Rejecting Renal Allografts to Prevent Graft Necrosis
From the Departments of Medicine,*
Surgery,
and Laboratory Medicine and
Pathology,
University of Alberta, Edmonton,
Alberta, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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regulates the
recipient immune response but also acts directly on IFN- receptors
in the graft. We investigated these direct actions by comparing
rejecting kidneys from donors lacking IFN- receptors (GRKO mice) or
control donors (129Sv/J) in CBA recipients. Beginning day 5,
129Sv/J kidneys displayed high major histocompatibility complex (MHC)
expression, progressive infiltration by inflammatory
cells, but no thrombosis and little necrosis, even at
day 21. GRKO kidneys showed increasing fibrin thrombi in small
veins, peritubular capillary congestion, hyaline
casts, and patchy parenchymal necrosis, progressing to
near total necrosis at day 10. Terminal dUTP nick-end labeling assays
were positive only in the interstitial infiltrate, confirming
that massive cell death in GRKO transplants was not apoptotic.
Paradoxically, GRKO kidneys showed little donor MHC induction
and less inflammatory infiltration. Both GRKO and 129Sv/J allografts
evoked vigorous host immune responses including alloantibody and mRNA
for cytotoxic T cell genes (perforin, granzyme B, Fas
ligand), and displayed similar expression of complement
inhibitors (CD46, CD55, CD59). GRKO kidneys displayed
less mRNA for inducible nitric oxide synthase and monokine inducible by
IFN- but increased heme oxygenase-1 mRNA. Thus IFN- acting on
IFN- receptors in allografts promotes infiltration and MHC induction
but prevents early thrombosis, congestion, and
necrosis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a major player in host defense, graft
rejection, and autoimmunity.1-6
IFN- acts through the
IFN- receptor (IFN-R), composed of a ligand-binding chain R1 and
an accessory chain R2,7
members of the same family as
IFN-
/ßR and IL-10R.8-12
IFN- generally promotes
host defense and its absence creates a mild immunodeficiency. Humans
with defects in IFN-R display increased susceptibility to
mycobacterial infection.13,14
Mice with targeted
disruption of the genes for IFN- (GKO mice),15
IFN-R1 (GRKO),16
or IFN-R217
have
multiple defects in immune function,18,19
including
decreased nitric oxide (NO) and IgG2a production and increased
susceptibility to mycobacteria,14
Leishmania,20
and certain viruses.21
In
addition to its role in immune regulation, IFN- differs from other
cytokines in its extensive effect on nonmarrow derived cells, as
evident in the unique role of IFN- in inducing major
histocompatibility complex (MHC) expression in epithelial and
endothelial cells. Mice lacking IFN- or IFN-R1 show decreased
class I expression in the basal state, much less induction of MHC
expression in response to stimuli, and decreased class I and II
induction after local injury.22,23
Thus, defects in IFN- or IFN-R genes
were expected to impair allograft rejection. For example, IFN-
should increase antigenicity because it increases the expression of MHC
products, which are potent alloantigens and are massively increased in
rejecting grafts24,25
and in the recipient
tissues,26,27
in large part because of IFN-. IFN-
promotes chronic immune injury to transplant vessels.28,29
In contrast, acute rejection is brisk in IFN--deficient
hosts,30
associated with increased T cell proliferation
and cytotoxic T lymphocyte generation.30-32
We have found
that mice lacking IFN- reject with altered pathology in kidney or
heart allografts, including thrombosis, congestion, and infarction in
the rejecting organ despite having little MHC induction (P. F.
Halloran, L. W. Miller, A. Battocchio, J. Urmson, L.-F. Zhu,
N. M. Kneteman, and K. Solez: Unique roles of IFN- in graft
rejection: induction of MHC expression and protection from early
vascular injury. Submitted for publication.) The rejection is immune
because it is absent in isografts and is blocked by immunosuppressive
drugs. The ability of IFN- to restrain early tissue destruction
could be relevant to the essential role of IFN- in some tolerance
models.32
IFN- also plays a role in xenografts by
preventing vascular injury.33
These observations highlight
the existence of an unexpected tendency of IFN- to protect
allografts and concordant xenografts during the early posttransplant
period. In later periods IFN- may promote graft
injury.28,29
While the best-studied effects of IFN- are on the immune response,
IFN- can act directly on allografts independently of its effect on
the immune system. To explore how such direct effects alter rejection,
we examined the pathology of rejecting kidney allografts lacking
IFN-Rs. We transplanted H-2b kidneys from
donors lacking or expressing IFN-R into allogeneic
H-2k recipients with normal IFN- production.
Grafts with IFN-R showed typical interstitial infiltration and MHC
induction at days 5, 7, and 10, but remained viable despite severe
rejection at day 21. Grafts lacking IFN-R showed little MHC
induction and reduced cellular infiltration but increased vascular
injury as manifest by venous thrombosis, congestion of peritubular
capillaries, and massive necrosis of parenchymal cells by days 7 to 10.
Thus a major early effect of IFN- in acute rejection is a direct
action on the IFN-Rs of graft that maintains graft viability during
the intense inflammation of acute rejection by preventing thrombosis,
congestion, and necrosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The original GRKO (129/Sv/Ev) mouse strain with disrupted
IFN-R1-chain genes was generated by gene targeting in murine
embryonic stem cells.16
The gene was disrupted by
inserting the neomycin-resistance gene
(neor) into exon V, which encodes an
extracellular domain. Homozygous 129/Sv/Ev mice were provided to us
though Dr. Michel Aguet (University of Zurich, Switzerland).
As controls with wild-type IFN-R1 genes, we obtained 129Sv/J mice
from Jackson Laboratories (Bar Harbor, ME). The mice were maintained in
the Health Sciences Laboratory Animal Services at the University of
Alberta and were kept on acidified water. All experiments conformed to
approved animal care protocols. As in many other transplant experiments
with knockout mice,34
the GRKO and 129Sv/J are H-2
identical but may differ at minor loci. Nevertheless the short time
frame and the complete H-2 disparity in the present experiments make it
highly unlikely that any minor differences influence this experiment.
Kidney Transplants
In anesthetized 129Sv/J and GRKO donor mice, the abdomen was opened by a midline incision and the left kidney was excised and preserved in cold lactate Ringers solution. The recipient CBA mice were similarly anesthetized and the right native kidney was excised. The donor kidney was then anastomosed heterotopically to the abdominal aorta and vena cava, without removing the recipient left kidney. The mice were allowed to recover and were killed at days 5, 7, 10, and 21 by cervical dislocation under anesthesia. Tissue samples were obtained for histological staining [hematoxylin and eosin (H&E), periodic acid Schiff , and Martius scarlet blue], as well as indirect immunoperoxidase staining for class I and II antigen expression.
Banff Scoring System
Using a modified version of the Banff scoring system35 two pathologists assigned scores for the lesions observed in whole kidney sections including cortex and outer medulla.35 Additional findings not included in the Banff scoring system such as the extent of necrosis, peritubular capillary congestion, and cast formation were scored from 0 to 3 based on the percentage of parenchymal involvement (0, no changes; 1, <25% of the total parenchyma involved, 2, 25 to 75% of total parenchyma involved; 3, >75% of the total parenchyma involved). Venous thrombosis was first assessed by H&E stain and the presence of fibrin in the thrombus was confirmed by Martius scarlet blue stain for fibrin. For scores on venous thrombosis the number of veins showing venous thrombosis was counted in each specimen.
Antibodies
Monoclonal antibodies (mAb) were purified in our laboratory from supernatants of hybridoma cell lines, AF 6-120.1.2 (mouse IgG against mouse I-Ab), 20-8-4S (mouse IgG against mouse H-2KbDb), 11-4.1 (mouse IgG against mouse H-2K,) 11-5.2.1.9 (mouse IgG against mouse I-Ak), M1/42.3.9.8 (rat IgG2a against all mouse H-2 haplotypes), and M5/114.15.2 (rat IgG2b against mouse I-Ab,d,q and I-Ed,k), obtained from American Type Culture Collection (Rockville, MD). Briefly, the hybridoma cell lines were maintained in tissue culture, and the supernatants containing AF 6-120.1.2 (anti-I-Ab) and 20-8-4S (anti-H-2KbDb), 11-4.1 (anti-H-2K), and 11-5.2.1.9 (anti-I-Ak) were purified by protein A chromatography. The supernatants containing M1/42.3.9.8 (anti–2 antigens all haplotypes) and M5/114.15.2 (anti-I-Ab,d,q and I-Ed,k) were ammonium-sulfate precipitated, and then the antibodies were obtained by purification with a DE52 anion exchanger column (Whatman, Hillsboro, OR) and by concentration with Amicon ultrafiltration. The protein concentration was adjusted to 1 mg/ml by a modified Lowry method and maintained at -70°C. Radioiodination was performed by the Iodogen method (Pierce Chemical Co., Rockford, IL).36
Radiolabeled Antibody-Binding Assay
Anti-H-2KbDb mAb and
anti-I-Ab mAb were radiolabeled with
[125I]iodide.36
Tissues of
individual mice were prepared as described
previously.37-39
The tissue concentration was adjusted to
20 mg/ml and 5 mg of kidney tissue was aliquoted in triplicate and
spun. The pellets were incubated on ice with
125I-labeled mAb in 10% normal mouse serum
(100,000 cpm per 100 µl) with agitation for 1 hour. After washing,
the pellets were counted in a counter and the nonspecific binding
of a negative tissue was subtracted. Based on standard curves, the
change in specific cpm bound in this assay corresponds to approximately
a sevenfold change in antigen output.39
Indirect Immunoperoxidase Staining of Tissue Sections
Fresh frozen cryostat sections were fixed in acetone, then incubated with normal goat serum. The slides were incubated with rat mAb against class I (M1) and class II (M5) or with phosphate-buffered saline (PBS) as a control. The slides were then incubated with affinity purified peroxidase-conjugated goat anti-rat IgG F (ab')2 fragment (ICN, Costa Mesa, CA). Immune complexes were visualized by the use of 3'3 diaminobenzidine tetrahydrochloride and hydrogen peroxide for the color reaction and then counterstained with hematoxylin. To count the number of infiltrating cells stained for various markers, 10 fields were counted and the results were expressed as the mean number per high-power field.
Assessment of Gene Expression
Total RNA was extracted and pooled from three kidneys that were
harvested on day 7. RNA was transcribed into cDNA using Superscript
reverse transcriptase (BRL, Burlington, Ontario) and amplified in a
Perkin Elmer Cetus thermal cycler (Perkin Elmer Cetus, Emeryville, CA)
using Taq DNA polymerase. The sequences of the polymerase
chain reaction primers are shown in Table 1
. The polymerase chain reaction products
were Southern blotted and probed with radiolabeled oligonucleotide
probes. Quantitation was performed by phosphoimaging of blots and
analysis by ImageGuage Software (Fuji, CT).
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To stain apoptotic cells, we performed TUNEL of fragmented DNA on 3-µm sections of paraffin-embedded tissue.40,41 Briefly, sections were deparaffinized in xylene for 5 minutes twice and then hydrated through a series of alcohols. To inactivate endogenous peroxidase, the sections were immersed in 1% H2O2 for 8 minutes at room temperature and then rinsed twice in distilled water. The sections were then treated with proteinase K (20 µg/ml in PBS) for 10 minutes at room temperature and then rinsed in PBS three times. The sections were air dried and then flooded with terminal transferase (TDT) buffer (30 mmol/L Tris-HCl, pH 7.2, 1 mmol/L CoCl2, 140 mmol/L sodium cacodylate) for 30 minutes at room temperature. To begin labeling the TDT buffer was replaced with TDT buffer containing 0.25 nmol/µl biotin-16-dUTP and 0.25 U/µl TDT (Hoffmann-La Roche, Quebec). After a 1-hour incubation at 37°C in a humidified chamber the slides were rinsed twice in PBS for 5 minutes. Nonspecific staining was blocked by immersing the slides in 2% bovine serum albumin in PBS for 20 minutes at room temperature, followed by two rinses in PBS for 5 minutes. The slides were then incubated with the avidin-biotin complex (Vector Laboratories, Burlingame, CA) for 30 minutes and then rinsed in PBS two times for 5 minutes. The reaction was visualized using the diaminobenzidine substrate kit (Vector Laboratories). The slides were counterstained with methyl green, dehydrated through a series of alcohols and xylene, and mounted with Permount (Fisher, Nepean, ON). For negative controls TDT was omitted. Thymus from young mice or kidney sections treated with 10 to 1,000 ng/ml DNase I (Sigma Chemical Co., St. Louis, MO) were used as positive controls. Apoptotic cells were counted in 10 fields (x200) in one kidney in each group.
Assessment of Cytotoxic Alloantibody
129Sv/J and GRKO spleen cells (10 x 106) were injected intraperitoneally into CBA mice, bled at day 7, boosted with 10 x 106 spleen cells and bled at day 14. Transplanted mice (CBA) with 129Sv/J or GRKO kidneys were bled at the time of harvest and their sera were assessed in a microcytotoxicity assay. Serum (2 µl) and spleen cells (2 µl) (2 x 106/ml) were incubated in a 37°C CO2 incubator for 30 minutes. Rabbit complement (2 µl at 1:3 dilution) was added and incubated at room temperature for 90 minutes. Two µl of 5% eosin and one drop of 10% buffered formalin were added to each well. The plates were flooded with formalin and examined under an inverted microscope to determine the percentage of dead cells.
Statistical Analysis
Values are given as mean ± SE in Table 2
and statistical analysis was performed
using the Mann Whitney U test. Analysis of variance was
performed in Table 3
. A P
value of 0.05 was considered to show a significant difference between
the two groups.
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Results |
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Pathology
Syngeneic control kidney transplants were performed with 129/J into 129/J, GRKO into GRKO, and GRKO into 129Sv/J mice. None of these transplants showed rejection or thrombosis at day 7. The left native kidneys were normal throughout and served as controls.
All 129Sv/J and GRKO kidney allografts showed rejection by day 5,
progressing at the later times. The rejecting 129Sv/J and GRKO
allografts were enlarged compared to the recipient left kidneys but
GRKO transplants were more enlarged than 129Sv/J, particularly at day 7
(Table 2)
. The 129Sv/J transplants showed more mononuclear interstitial
infiltrate but little necrosis, peritubular capillary congestion, or
cast formation (Figure 1A)
. The infiltrate
was present at day 5 and increased at day 7. There was mild tubulitis
and no arteritis at days 5 and 7, although venulitis was common.
Despite the mononuclear infiltrate, the 129Sv/J’s at days 5, 7, and 10
displayed little parenchymal cell death (Table 2
and Figure 1A
). By day
21 all 129Sv/J kidneys showed severe tubulitis and heavy infiltrate
(Figure 1B)
. No 129Sv/J transplants showed venous thrombosis at days 5,
7, 10, or 21.
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. However, beginning at day 5 and increasing at day 7
GRKO kidneys showed mural fibrin thrombi in small veins, peritubular
capillary congestion, and cast formation in tubules (Figure 1, C and D)
. Inflammation in small veins (venulitis) was common in GRKO and
129Sv/J kidneys (Figure 1E)
. Venous thrombosis was found in two of five
GRKO transplants at day 5 (Figure 1E)
and in all eight GRKO transplants
at day 7 (Table 2)
(Figure 1F)
. By day 7 GRKO transplants showed
extensive parenchymal cell necrosis (Figure 1D)
, which was massive at
days 10 and 21 (not shown). There was little tubulitis and no arteritis
in GRKO kidneys at days 5 and 7 (Table 2)
. Electron microscopy of 129Sv/J and GRKO transplants at days 5 and day 7 confirmed that the peritubular capillaries were congested in the GRKO transplants, but neither 129Sv/J nor GRKO kidneys displayed interstitial hemorrhage. In both 129Sv/J and GRKO transplants interstitial inflammatory cells were shown to be outside of peritubular capillaries. The electron microscopic assessment of renal tubular epithelium showed changes compatible with ischemic necrosis (data not shown).
TUNEL Assay
We used the TUNEL assay to clarify the character of cell death in
129Sv/J and GRKO transplants. Apoptosis was frequent among the
inflammatory cells located in the interstitium and the peritubular
capillaries at day 5 (Figure 1G)
and at day 7 (Figure 1H)
but was not
seen in the parenchymal cells, even in areas of extensive cell death.
Thus by TUNEL assay the loss of parenchymal viability was not because
of apoptosis.
Immunostaining of the Infiltrate
We counted the infiltrating mononuclear cells by indirect
immunoperoxidase staining at days 5 and 7 (Table 3)
. The rejecting GRKO
kidneys contained fewer cells staining for CD3, CD8, CD4, and CD45 than
did the rejecting 129Sv/J kidneys, particularly at day 7. In contrast
class II-positive interstitial cells tended to be increased in GRKO
kidneys.
Assessment of MHC Expression by Immunostaining and Radiolabeled Antibody-Binding Assay
The pattern of MHC expression was determined by indirect
immunoperoxidase staining. We used rat monoclonal anti-class I (M1) and
anti-class II (M5), plus peroxidase-labeled goat anti-rat IgG (Figure 2)
. Class I and II was strongly expressed in
the tubules, glomeruli, and arterial endothelium of rejecting 129Sv/J
kidneys. The tubule staining was localized to the basolateral aspect
(Figure 2, A and B)
. The intensity and extent of MHC expression in
nonnecrotic tubules was much less in GRKO kidneys (Figure 2, C and D)
,
and the expression was mainly confined to the cellular infiltrate. M5
stained some tubules of the GRKO kidneys, predominantly those that were
dilated or damaged. The MHC expression patterns were also confirmed
with mouse monoclonals against donor and recipient class I and class
II, plus goat anti-mouse IgG (data not shown).
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) or
H-2k (recipient type, Figure 3B
). The extent of
MHC induction was calculated by a standard curve, and is shown as a
number above the bar indicating fold induction over normal kidney
controls. Donor (H-2b) class I and II expression
was strongly induced in 129Sv/J transplants but was much less increased
in GRKO transplants (Figure 3A)
. The MHC induction in 129Sv/J
transplants was 10 times greater for class I and 4.5 times greater for
class II than in GRKO transplants. Donor class I or class II was not
induced in the native kidney of the recipient, as expected.
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,
because of the infiltrating recipient cells. Recipient MHC was also
strongly induced in the native kidney of CBA recipients rejecting
either 129Sv/J or GRKO transplants (Figure 3B)
. We have previously
established that systemic MHC induction in allogeneic responses is
highly dependent on IFN-.22,26
Thus the MHC induction
in the native kidney confirms that both 129Sv/J and GRKO allografts
evoked IFN- production. Alloantibody Responses of CBA Mice Against 129Sv/J or GRKO
Because vascular injury and necrosis in renal transplants suggests
alloantibody-mediated damage,42
we investigated the
alloantibody response, either to spleen cell immunizations or to kidney
allografts. CBA mice were immunized intraperitoneally with
107
129Sv/J or GRKO spleen cells, and boosted
with a second injection at day 7 (Figure 4A)
.
The first injection of GRKO or 129Sv/J spleen cells evoked equivalent
cytotoxic alloantibody production in CBA mice. The second injection
increased the cytotoxic activity more in 129Sv/J than in GRKO. Thus the
absence of IFN-R in the immunizing cells did not prevent the initial
alloantibody response but may reduce the secondary response.
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. CBA recipients had 129Sv/J- or GRKO-specific alloantibody
demonstrable at day 7. Antibody titers were lower in mice rejecting
transplants than in mice immunized with spleen cells. The cytotoxic
antibody titer was higher in hosts rejecting GRKO transplants compared
to 129Sv/J transplants. Gene Expression in Rejecting GRKO versus 129Sv/J Allografts
We studied the mRNA levels for cytotoxic T-cell effector
molecules, complement inhibitory proteins, and the endothelial
protective gene heme oxygenase-1 (HO-1) in the
transplant.43,44
Rejecting 129Sv/J and GRKO kidneys both
showed increased expression of FasL, perforin, and granzyme B mRNA
compared to control kidneys, with no obvious differences between GRKO
kidneys and 129Sv/J kidneys (Figure 5A)
. All
kidneys had abundant mRNA for complement inhibitory proteins CD46,
CD55, and CD59, with no differences between rejecting and control
kidneys or between GRKO and 129Sv/J transplants (Figure 5B)
. HO-1 mRNA
was increased in rejecting 129Sv/J and GRKO transplants, but the
increase was greater in the GRKO than in the 129Sv/J.
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-inducible
chemokines—monokine inducible by IFN- (MIG) and IFN--inducible
protein (IP-10)—and for nitric oxide synthetase (NOS2). MIG and IP-10
mRNA levels were strongly induced in rejecting transplants, but the
levels were lower in GRKO transplants than in 129Sv/J transplants
(Figure 5B)
. Rejecting GRKO kidneys also displayed lower levels of mRNA
for NOS2. Phosphoimaging analysis was used to estimate the relative intensity of hybridization between the 129Sv/J and GRKO transplants at day 7. The expression of NOS2, IP-10, and MIG was twofold, 2.6-fold, and 3.3-fold lower, respectively, and HO-1 expression was 2.7-fold higher in rejecting GRKO kidney allografts compared to 129Sv/J allografts.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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strikingly affects the
evolution of tissue injury in a rejecting allograft by a direct action
on the graft that prevents thrombosis, congestion, and necrosis during
acute rejection. From day 5 allografts lacking IFN-Rs in hosts with
intact IFN--IFN-R systems showed increased venous thrombosis,
peritubular congestion, casts, and massive cell death by necrosis.
There was decreased T-cell interstitial infiltrate; reduced MHC class I
and II induction in the transplant; decreased expression of MIG, IP-10,
and NOS2; and increased expression of HO-1. The host MHC expression in
the host tissues provided evidence for abundant IFN- production and
effect in the hosts, making it unlikely that a lack of host IFN-
production could contribute to these effects. Cytotoxic T-cell gene
expression and alloantibody responses were strong in both GRKO and
129Sv/J transplants. The findings indicate that IFN- acts on
IFN-Rs in the grafted tissue to maintain the circulation of the
allograft during acute rejection. The IFN- may act by modulating
graft susceptibility to immune injury or by altering the host immune
response.
Although it is possible that grafts lacking IFN-Rs evoke an altered
host immune response, there was no obvious difference in the immune
response evoked by allografts with and without IFN-Rs to explain the
differences in tissue injury. These CBA hosts produce IFN- and have
normal IFN-Rs, and produce abundant IFN- as witnessed by the
induction of host MHC expression in the normal native kidney, an effect
that is highly dependent on IFN-.22,26
The expression
of the CTL effector genes perforin, granzyme B, and FasL was increased
both in 129Sv/J and GRKO grafts at day 7. Alloantibody responses were
readily induced by immunization with either both GRKO or 129Sv/J spleen
cells, and during rejection of both GRKO or 129Sv/J grafts with and
without IFN-Rs. Hosts rejecting GRKO kidneys showed higher
alloantibody levels than hosts rejecting 129Sv/J transplants, but this
could reflect increased absorption to the 129Sv/J kidneys because of
their high MHC expression, rather than increased production. In studies
currently in progress we have shown that IgG is deposited on the
basolateral membrane of the tubules of wild-type rejecting
kidneys but not GRKO kidneys, supporting the possibility of absorption
of anti-MHC antibody to host MHC induced by IFN-. However, we have
not yet proven by elution that this IgG is indeed anti-MHC. Moreover,
the modest difference in titer of antibody between hosts rejecting
wild-type versus GRKO kidneys is probably insufficient to
explain the extreme differences in pathology, although we cannot
exclude the possibility that different rates and sites of alloantibody
deposition contribute to these differences.
A working hypothesis for the excessive necrosis in GRKO allografts is
that during acute rejection the lack of IFN-Rs renders the grafts
susceptible to early thrombosis, congestion, and ischemic necrosis. The
TUNEL assays showed little apoptosis of epithelial cells, but do not
exclude apoptosis of endothelial cells in small vessels as a mechanism
of the venous thrombosis, because TUNEL assays may miss such changes.
One mechanism by which IFN- could modulate the pathology of
rejection is induction of NOS2 and nitric oxide production. The grafts
lacking IFN-Rs displayed lower levels of mRNA for NOS2. Host
inflammatory cells presumably contribute to NOS2 expression and NO
production, but the donor vascular cells are unable to respond to
IFN- and thus should have less NOS2 expression and impaired NO
production. Nitric oxide has potential for protective or aggressive
effects in graft rejection. NO limits inflammation at endothelial
surfaces,45
and inhibits apoptosis of some cell
types,46
including endothelial cells,47
despite promoting apoptosis in other cell types.48
Inhibitors of NOS tend to make rejection worse, suggesting a protective
role.49
On the other hand, acute graft rejection is
actually attenuated rather than accelerated in hosts lacking NOS2, and
thrombosis is not increased,50
indicating that NOS2 alone
is probably not the critical mediator of the protective effects of
IFN-, although it may protect against transplant
arteriosclerosis.51
The complex and contradictory effects
of NOS2 and NO in graft rejection models invites us to leave the issue
of the role of NOS2 unresolved at present.52
Moreover,
some effects of NOS2 and NO may be dependent on the microenvironment of
the organ transplanted.46,47
No direct anticoagulant effect of IFN- on platelets or on
coagulation is established, and the hosts in this model should regulate
host platelet and coagulation functions normally. Moreover, there is no
excess of thrombosis in mice lacking IFN- or IFN-Rs or in our
syngeneic control transplants lacking IFN-Rs.
The reduction in expression of IFN--inducible chemokines such as MIG
and IP-10 may have either protective or injurious effects. Reduced
induction of IP-10 and MIG in grafts lacking IFN-Rs could alter the
rejection process, eg, by reducing the mononuclear infiltrate of cells
with CXCR3 receptors. At least one model of rejection shows that
IFN- participates in graft destruction via the induction of
MIG.53
Thus the lack of MIG and IP-10 induction in GRKO
should have protected rather than injured the graft. We also considered
the possibility that the damage and thrombosis in the vasculature
reflect disregulated diapedesis because of a lack of appropriate
chemokine milieu, but the pathology showed that the leukocytes in the
allograft had crossed into the interstitium and did not show excessive
endothelialitis.
Given the potency of antibody to damage endothelium in both
allografts and xenografts, we considered the possibility that the
thrombosis in GRKO mice reflected antibody injury because of disturbed
expression of complement regulatory proteins or HO-1. Wang et
al33
concluded that IFN- protects concordant rat
xenotransplants against vascular injury by antibody, supporting the
concept that IFN- may limit antibody injury to vessels. In our
experiments we found no changes in expression of complement regulatory
proteins in GRKO kidneys, confirming previous conclusions that these
proteins are not regulated by IFN-.54-57
Deficiency in
the endothelial protective effect of HO-1 could not be incriminated in
the thrombosis of GRKO kidneys because the GRKO actually had higher
levels of HO-1, perhaps as a response to the injury to the
tissue.58
Thus antibody injury remains a potential cause
of the excessive thrombosis, but by an undefined mechanism.
Of the genes in the graft that may mediate the effect of
IFN--IFN-R on the pattern of graft rejection, one candidate is
the MHC genes themselves. Induction of intense MHC expression during
graft rejection is often assumed to favor rejection because MHC
products are powerful alloantigens and key antigen presenting
structures. However, allogeneic MHC can also lead to tolerance or
anergy, and MHC negatively regulates natural killer receptors, at least
in vitro. High MHC expression in less critical tissue
components such as tubule epithelium could temporarily divert immune
effectors such as alloantibody or CTL from engaging the critical
endothelial cells of the vasculature. Such effects could be mediated
either by membrane-bound MHC or by soluble MHC released from the graft.
This effect would be temporary, because high MHC expression could
facilitate later invasive lesions such as tubulitis and
endothelialitis. The fact that alloantibody levels are lower in hosts
rejecting 129Sv/J grafts than in hosts rejecting GRKO grafts (which
have much less MHC expression) could be used to support this
explanation. Thus low MHC expression in rejecting GRKO transplants
might favor vascular injury either by a reduced immune-modulating role
on antibody and CTL mechanisms, or by reduced negative signaling to
natural killer cell inhibitory receptors.
There are some parallels between the present results and those
described by Pober et al,59
in that both reflect direct
effects of IFN- on graft vessels. The latter system demonstrated
throughout a longer time frame that IFN- could directly lead to
arteriosclerosis. Other observations also suggest a similar adverse
long-term effect of IFN- on arteries in grafts.28,29
Thus, the early benefits of IFN- actions during acute rejection may
be followed by direct deleterious effects throughout a longer time
frame.
The ability of IFN- to act directly on the target tissue to prevent
vascular thrombosis and maintain tissue viability invites the question
of whether this mechanism operates in other inflammatory diseases.
Because a similar effect operates in concordant
xenografts,33
it is possible that one reason for severe
vascular injury in discordant xenografts is the species specificity of
IFN-Rs, making discordant xenografts comparable to GRKO grafts.
Moreover, in models of autoimmune disease, hosts lacking IFN- or
IFN-Rs sometimes display excessive tissue injury that is usually
attributed to lack of immunoregulatory effects of IFN- on
lymphocytes, eg, in experimental arthritis,60,61
encephalitis,62
and uveitis.63
Thus the
principle is that IFN- can directly affect the target tissue in a
disease process independent of its effects on the host immune response,
reminding us to avoid attributing IFN- effects in disease states
exclusively to its direct effects on inflammatory cells.
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Acknowledgements |
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Supported by the Medical Research Council of Canada, Roche Organ Transplant Research Foundation, Kidney Foundation of Canada, Novartis Pharmaceuticals Canada, Inc., Hoffmann-La Roche Canada, Inc., the Muttart Foundation, Royal Canadian Legion.
Accepted for publication September 18, 2000.
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