Download and Progression of Acute Allograft Rejection Cardiac Grafts

Early Chemokine Cascades in Murine
Cardiac Grafts Regulate T Cell Recruitment
and Progression of Acute Allograft Rejection
This information is current as
of August 9, 2017.
Ken Morita, Masayoshi Miura, David R. Paolone, Tara M.
Engeman, Anil Kapoor, Daniel G. Remick and Robert L.
Fairchild
J Immunol 2001; 167:2979-2984; ;
doi: 10.4049/jimmunol.167.5.2979
http://www.jimmunol.org/content/167/5/2979
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References
Early Chemokine Cascades in Murine Cardiac Grafts Regulate
T Cell Recruitment and Progression of Acute Allograft
Rejection1
Ken Morita,* Masayoshi Miura,*† David R. Paolone,* Tara M. Engeman,† Anil Kapoor,*
Daniel G. Remick,‡ and Robert L. Fairchild2*‡§
A
llogeneic cardiac transplantation is an increasingly used
approach for therapy of end-stage heart disease. The loss
of cardiac allografts due to T cell-mediated acute rejection has been substantially decreased by current immunosuppressive strategies. Despite this improvement, acute rejection episodes
remain a significant problem in allograft function and loss (1).
Furthermore, recent clinical studies have clearly indicated that
acute rejection episodes are a critical risk factor for the subsequent
development of chronic rejection, the leading cause of cardiac allograft loss (2, 3). The key factors directing alloantigen-primed T
cell infiltration into allografts during acute rejection remain largely
unidentified. A critical factor facilitating leukocyte infiltration of
tissue is the presence of inflammation. Inflammation in vascularized organ grafts induces up-regulated adhesion molecule expression on vascular endothelium and other cells (4 –7). Antagonism of
adhesion molecules has inhibited acute rejection and prolonged
graft survival in some clinical studies and animal models but not in
Departments of *Urology and †Immunology, Cleveland Clinic Foundation, Cleveland, OH 44195; ‡Department of Pathology, University of Michigan Medical School,
Ann Arbor, MI 48109; and §Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106
Received for publication August 16, 2000. Accepted for publication June 20, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the National Institutes of Health (AI 40459)
and the American Heart Association (0050538N) and a generous gift from the State
of Qatar to the Renal Transplant Research Program at the Cleveland Clinic
Foundation.
2
Address correspondence and reprint requests to Dr. Robert L. Fairchild, NB3-79,
Department of Immunology, Cleveland Clinic Foundation, 9500 Euclid Avenue,
Cleveland, OH 44195-0001. E-mail address: [email protected]
Copyright © 2001 by The American Association of Immunologists
others (7–10), suggesting that other factors may also be important
in directing T cell recruitment into allografts.
Another important component of inflammation is the production
of cytokines with chemoattractant properties for leukocyte populations, chemokines (11, 12). Chemokines are grouped into families (CXC, CC, C, and CX3C) based on cysteine motifs. The CXC
family includes the neutrophil chemoattractants IL-8 and growthrelated oncogene ␣, of which KC is the murine homologue, and
two potent chemoattractants for Ag-activated T cells, IFN-␥-inducible protein-10 (IP-10)3 and monokine induced by IFN-␥
(Mig). The CC family includes the monocyte/macrophage chemoattractant protein-1 (MCP-1), of which JE is the murine homologue, as well as macrophage inflammatory proteins (MIP)-1␣ and
1␤. Studies from this and several other laboratories have indicated
the presence of chemokine mRNA and/or protein during acute rejection of allografts in experimental and clinical transplantation
(13–17). IP-10 and Mig expression was observed in heterotopically transplanted cardiac allografts but not isografts at day 3 after
transplant, whereas expression of MIP-1␣, MIP-1␤, and KC was
absent in iso- and allografts (14, 18). The production of IP-10 and
Mig may be an important step in the acute rejection process due to
their strong chemoattractive properties for Ag-primed T cells (19).
The induction and role of specific chemokines in the rejection of
solid organ allografts remains unclear. In the current study, we
have tested the expression of four chemokines 1.5– 48 h after transplant in murine cardiac iso- and allografts. The results indicate the
induction of organized cascades of chemokine RNA expression
initiated by KC and followed by JE, MIP-1␤, and MIP-1␣ at early
3
Abbreviations used in this paper: IP-10, IFN-␥-inducible protein-10; MCP-1, monocyte chemotactic protein-1; Mig, monokine induced by IFN-␥; MIP, macrophage
inflammatory protein; NRS, normal rabbit serum.
0022-1767/01/$02.00
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The identification of early inflammatory events after transplant in solid tissue organ grafts that may direct T cell recruitment and
promote acute allograft rejection remain largely unknown. To better understand temporal aspects of early inflammatory events
in vascularized organ grafts, we tested the intragraft expression of four different chemokines in heterotopically transplanted A/J
(H-2a) and syngeneic heart grafts in C57BL/6 (H-2b) recipient mice from 1.5 to 48 h after transplant. Similar temporal expression
patterns and equivalent levels of chemokine expression were observed in both syngeneic and allogeneic cardiac allografts during
this time period. Expression of the neutrophil chemoattractant growth-related oncogene ␣ (KC) was observed first and reached
peak levels by 6 h after transplant and was followed by the monocyte/macrophage chemoattractant protein-1 (JE) and then
macrophage inflammatory proteins 1␤ and 1␣. Administration of rabbit KC antiserum to allograft recipients within 30 min of
cardiac transplantation attenuated downstream events including intra-allograft expression of the T cell chemoattractants IFN␥-inducible protein-10 and monokine induced by IFN-␥, cellular infiltration into the allograft, and graft rejection. Similarly,
depletion of recipient neutrophils at the time of transplantation significantly extended allograft survival from day 8 to 10 in
control-treated recipients up to day 21 after transplant. These results indicate the induction of highly organized cascades of
neutrophil and macrophage chemoattractants in cardiac grafts and support the proposal that early inflammatory events are
required for optimal recruitment of T cells into allografts during the progression of acute rejection of cardiac allografts. The
Journal of Immunology, 2001, 167: 2979 –2984.
2980
times after transplant in both iso- and allografts. Administration of
a single dose of rabbit antiserum to KC results in a substantial
increase in allogeneic heart graft survival. The prolonged survival
is associated with the decreased expression of Mig and IP-10, as
well as decreased T cell infiltration into the allograft. These results
indicate the importance of early inflammatory events on the subsequent production of T cell chemoattractants and T cell recruitment into the allograft during the acute rejection process.
Materials and Methods
Animals
A/J (H-2a) and C57BL/6 (H-2b) mice were obtained through Dr. C. Reeder
at the National Cancer Institute (Frederick, MD). Adult males of 8 –12 wk
of age were used throughout this study.
Abs and antiserum
Immunohistology
Heart grafts were retrieved at day 7 after transplant, embedded in OCT
compound (Sakura Finetek, Torrence, CA), and frozen at ⫺80°C. Sections
were cut at 8 ␮m and mounted onto slides. For immunohistochemistry,
sections were fixed in acetone for 10 min and air dried. Slides were immersed in PBS for 10 min and then in 0.03% H2O2 for 10 min to eliminate
endogenous peroxidase activity. The slides were then stained for 1 h with
5 ␮g/ml anti-CD4 mAb (GK1.5) or anti-CD8 mAb (53-6.7) in 0.05 TrisHCl with 1% BSA. Control slides were incubated with rat IgG as the
primary Ab. After three washes in PBS for 5 min each, slides were incubated for 20 min with biotinylated goat anti-rat IgG diluted 1/300 in PBS.
After three washes in PBS, slides were incubated with streptavidin-HRP
(DAKO) for 20 min and washed another three times. To prepare the substrate-chromagen solution, a 10-mg tablet of 3,3⬘-diaminobenzidine (Sigma, St. Louis, MO) was dissolved in 15 ml of PBS plus 12 ␮l of 30%
H2O2. The solution was applied to the slides, which were incubated for 3–7
min and then rinsed in dH2O to stop the reaction. The slides were counterstained with hematoxylin for 3 min, rinsed with tap water, and immersed
in 37 nM NH4OH for 10 s. Finally, the slides were dehydrated, viewed
under light microscopy, and the images were captured using ImagePro Plus
(Media Cybernetics, Silver Spring, MD). Numbers of cells staining positive were counted in eight random fields from three different tissue sections
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Rabbit immune serum to a KC-specific peptide (sequence QTMAGIHLKNIQS) was made at BioSynthesis (Louisville, TX). This antiserum reacts with KC and not with other CXC chemokines including IP-10
and Mig in Western blot analyses and inhibits recombinant KC-mediated
chemotaxis of thioglycolate-induced peritoneal neutrophils in in vitro chemotaxis assays (Ref. 20 and data not shown). Normal rabbit serum (NRS)
was used as control Ig for in vivo Ab treatment experiments. Mice were
depleted of neutrophils by giving 100-␮g aliquots of the rat anti-Ly6G
mAb, RB6.8C5 (21), on two consecutive days. This treatment resulted in
⬍5% neutrophils in the peritoneal wash of mice 4 h after thioglycolate
injection as assessed by staining the peritoneal cells with Wright’s stain.
Previous studies have shown that treatment with either the KC antiserum or
RB6.8C5 does not affect the viability of circulating or lymphoid T cell
populations (20). For use in immunocytochemistry, GK1.5, rat anti-mouse
CD4 mAb, was obtained from BD PharMingen (San Diego, CA); 53-6.7,
rat anti-mouse CD8 mAb, and biotinylated goat anti-rat polyclonal Ab
were purchased from DAKO (Carpinteria, CA); and control rat IgG was
purchased from R&D Systems (Minneapolis, MN).
EARLY CHEMOKINES IN CARDIAC ALLOGRAFTS
Heterotopic cardiac transplant
Cardiac transplants were performed according to the method of Corry and
coworkers (22). Briefly, donor and recipient mice were anesthetized with
phenobarbital. Donor hearts were harvested and placed in chilled lactated
Ringer’s solution while the recipient mice were prepared. The donor heart
was anastomosed to the recipient abdominal aorta and vena cava using
microsurgical techniques. Upon completion of the anastomosis and organ
perfusion, the transplanted hearts resumed spontaneous contraction. The
strength and quality of cardiac impulses were graded each day, as described
previously (14, 18). Rejection of cardiac grafts was considered complete by
the cessation of impulse and was confirmed visually for each graft by
laparotomy. In C57BL/6 recipients, complete rejection of A/J cardiac
grafts occurs between 8 and 10 days after transplantation. Cardiac isografts
in the C57BL/6 recipients functioned for ⬎300 days. Significance in allograft survival between recipient treatment groups was analyzed by log rank
test, and p ⬍ 0.01 was considered a significant difference between groups.
Northern blot analysis
Whole-cell RNA was isolated from transplanted and native heart tissue
using TRIzol (Life Technologies, Grand Island, NY). Briefly, transplanted
and naive hearts were excised from recipients, snap frozen, and homogenized in TRIzol. After extraction, precipitation, and resuspension in diethylpyrocarbonate-treated dH2O, 10-␮g aliquots of RNA were electrophoresed in 1% agarose formaldehyde-denaturing gels and analyzed by
Northern blot analysis, as described previously (14). Blots were hybridized
with 32P-labeled oligonucleotide probes specific for KC, MIP-1␣, MIP-1␤,
JE, IP-10, and Mig. After hybridization and exposure with one cytokine
oligonucleotide probe, the filter was stripped of the probe by washing the
blot three times in 0.4% SDS at 90°C and then hybridized with the next test
cytokine probe. After hybridization with the test cytokine probes was completed, the blot was stripped and probed with a rat GAPDH cDNA (23).
Densitometry using Storage Phosphor Screen analyzer (Molecular Dynamics, Sunnyvale, CA) was used to comparatively measure the cytokine signal and the GAPDH signal for each sample of the blot. The cytokine signals for each sample of the blot were then normalized by expressing the
density of the cytokine signal as a ratio to the signal of the GAPDH signal
for each RNA sample. The mean ratio for each group (i.e., time point or
treatment) was determined, and differences between means were tested
using Welch’s t test. A p ⬍ 0.05 was considered a significant difference.
FIGURE 1. Expression of chemokine genes following reperfusion of
heart grafts. Groups of five cardiac isografts and A/J allografts were harvested from C57BL/6 recipients at 1.5, 3, 6, 9, 12, 18, 24, and 48 h after
reperfusion, and total cellular RNA was prepared and analyzed by Northern
blot for expression of the indicated genes. Intensity of the chemokine RNA
signals were plotted as a ratio to the GAPDH signal, and the mean ratio ⫾
SD of the five grafts in each group is shown
The Journal of Immunology
2981
from three different grafts, and significance between mean numbers of
positive cells per field in different treatment groups was tested using MannWhitney U test.
Results
FIGURE 3. Histological analyses of A/J cardiac allografts from C57BL/6 recipients treated with NRS vs KC antiserum. C57BL/6 recipients were given
200 ␮l KC antiserum (A and C) or NRS (B and D) within 30 min of cardiac allograft reperfusion. Allografts were harvested at day 7 after transplant and
fixed with 10% buffered formalin. Sections were prepared and stained by H&E. Magnification: A and B, ⫻100; C and D, ⫻400.
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FIGURE 2. Effect of KC antiserum on heart allograft rejection.
Groups of C57BL/6 recipients were given 200 ␮l KC antiserum (dotted
line) or NRS (solid line) within 30 min of A/J cardiac allograft reperfusion. Survival of heart allografts in KC antiserum-treated C57BL/6
recipients (n ⫽ 6) was compared with survival of allografts in NRStreated recipients (n ⫽ 8). Allograft rejection in all recipient mice was
visually confirmed by laparotomy. Isografts from recipients treated with
KC antiserum or NRS were maintained ⬎300 days. Recipient treatment
with KC antiserum significantly prolonged heart allograft survival (p ⬍
0.01).
In a previous study, expression of neutrophil and macrophage chemoattractant chemokines were absent from allogeneic and syngeneic vascularized cardiac grafts at day 3 after transplant (14). To
begin to test if neutrophil and monocyte/macrophage chemoattractants were expressed at earlier time points in cardiac iso- and allografts, C57BL/6 (H-2b) mice received syngeneic or A/J (H-2a)
heart grafts and at various times from 1.5 to 48 h after transplant
groups of five grafts were harvested at each time point. RNA was
then prepared and analyzed by Northern blot hybridization for expression of KC, JE, MIP-1␣, and MIP-1␤. KC and JE expression
was first apparent at 3 h after transplant and peaked at 6 h after
transplant in both iso- and allografts (Fig. 1). These levels dropped
quickly by 9 h after ransplant, and by 48 h after transplant, levels
of KC and JE expression in iso- and allografts had receded to
background levels.
Expression of MIP-1␤ in both syngeneic and allogeneic heart
grafts also began to appear at low levels at 3 h after transplant. In
contrast to KC and JE, expression levels of MIP-1␤ dipped slightly
after 6 h after transplant and rose to a second peak at 24 h after
transplant before beginning to fall to background levels 48 h after
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begins at day 2 after transplant and increases to the time of acute
graft rejection (18). We tested the impact of KC on expression of
these chemokines at day 2 after transplant by Northern blot hybridization. NRS or rabbit antiserum to KC was given i.v. to
groups of allogeneic heart graft recipients 30 min after graft reperfusion. Two days later, the hearts were retrieved, and RNA was
prepared and tested for expression of IP-10 and Mig. Recipient
treatment with KC antiserum reduced intraallograft expression of
both IP-10 and Mig ⬎50% (Fig. 6).
The ability of Abs to the neutrophil chemoattractant KC to inhibit T cell infiltration into cardiac allografts and prolong survival
suggested a role for neutrophils in the acute rejection process of
these allografts. To examine this role more closely, we tested the
effect of recipient neutrophil depletion on the survival of cardiac
allografts. C57BL/6 mice were treated on two consecutive days
with the neutrophil-depleting Ab RB6.8C5 or rat IgG as a control.
On the day of the second treatment, groups of the mice received
either syngeneic C57BL/6 or MHC-mismatched A/J cardiac allografts. Treatment with the control rat IgG did not prolong allograft
survival when compared with nontreated allograft recipients (Fig.
7). However, depletion of recipient neutrophils at the time of transplantation extended the survival of the allografts up to day 21 after
transplantation. In conjunction with the extended survival in recipients treated with the KC antiserum, these results indicate the
importance of neutrophils in acute rejection of vascularized heart
allografts.
Discussion
The introduction of wounds and ischemia/reperfusion injury initiates a tissue inflammatory response that includes production of
TNF-␣, IL-1, and chemokines and the up-regulation of adhesion
molecules (4 –7, 24, 25). Although chemokines play a critical role
FIGURE 4. CD8⫹ T cell infiltration into heart allografts from KC antiserum- and NRS-treated recipients. C57BL/6 recipients were given 200 ␮l KC
antiserum (A) or NRS (B) within 30 min of cardiac allograft reperfusion. Allografts were harvested at day 7 after transplant, and frozen sections were
prepared and stained with anti-CD8 mAb for immunohistochemistry. Magnification, ⫻200.
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transplant. Expression of MIP-1␣ did not begin to become apparent until 12–18 h after transplant, reached peak levels at 24- 48 h
after transplant, and then decreased.
Because KC was the first chemokine we observed expressed in
the cardiac grafts, we tested the effect of KC antagonism on the
survival of the A/J cardiac allografts in C57BL/6 recipients.
Groups of allograft recipients were given 200 ␮l NRS or rabbit KC
antiserum i.v. within 30 min after graft reperfusion. Heart graft
beating in recipients was monitored daily by palpation and cessation of beating was confirmed by laparotomy. With one exception,
heart allografts in recipients treated with NRS ceased beating between days 7 and 10 after transplant (Fig. 2). In contrast, significant prolongation of allograft survival up to day 23 after transplant
was observed in recipients treated with the KC antiserum. This
enhanced survival was accompanied by a visible decrease in cellular infiltration of the heart allografts from the KC antiserumtreated recipients when compared with allografts from NRStreated recipients at day 7 after transplant (Fig. 3, A vs B). The
decrease in cellular infiltration was particularly evident around
vessels and within the graft interstitium (Fig. 3, C vs D). Furthermore, vessels in allografts from NRS-treated recipients had signs
of endothelial degeneration at day 7 after transplant that were absent in allografts from the KC antiserum-treated recipients. To
compare T cell infiltration into allografts from control and KC
antiserum-treated recipients, graft tissue sections were also prepared and stained with anti-CD8 or anti-CD4 mAb. KC antiserum
treatment resulted in a clear decrease in CD8⫹ and CD4⫹ T cell
infiltration into the allograft interstitium (Figs. 4, A vs B, and 5).
There was little to no T cell infiltration evident in sections prepared
from isografts at day 7 after transplant (data not shown).
Recent results from this laboratory have shown that expression
of the T cell chemoattractants IP-10 and Mig in cardiac allografts
EARLY CHEMOKINES IN CARDIAC ALLOGRAFTS
The Journal of Immunology
in leukocyte recruitment to peripheral tissues, few studies have
tested the induction and role of chemokines during acute rejection
of solid organ allografts. The results of the current report indicate
coordinated cascades of chemokine gene expression in transplanted cardiac grafts within hours after graft revascularization.
Northern blot analysis of RNA isolated from cardiac grafts 1.5– 48
h after revascularization indicated no difference between isografts
and allografts with respect to patterns and levels of chemokine
expression. The equivalent expression in allografts and isografts
suggests that the early cascades occur without the influence of
adaptive immune mechanisms.
The chemokines expressed in cardiac grafts during the early
time periods following transplant are those directing the recruitment of neutrophils and macrophages during wound healing.
Whether the expression of these early chemokines is induced directly by the ischemia and trauma of the transplant surgery or
induced indirectly by other cytokines produced during the initial
inflammation is unclear at this time. Several investigators have
reported the rapid after transplant induction of TNF-␣ in allo- and
isografts (26, 27). Because TNF-␣ also induces expression and
FIGURE 6. Expression of T cell chemoattractants in allografts at day 2
after transplant. KC antiserum or NRS was given, 200 ␮l i.v., to groups of
four C57BL/6 recipient mice within 30 min after A/J cardiac allograft
reperfusion. At day 2 after transplant, allografts were retrieved, and RNA
was prepared and analyzed for levels of IP-10 and Mig RNA by Northern
blot hybridization. Intensity of intra-allograft IP-10 and Mig RNA signals
was plotted as a ratio to the GAPDH signal, and the mean ratio ⫾ SD of
the five grafts in each group is shown. ⴱ, p ⬍ 0.04.
FIGURE 7. Depletion of recipient neutrophils extends cardiac allograft
survival. C57BL/6 mice were given 100 mg RB6.8C or control rat IgG on
two consecutive days. On the second day of treatment, the treated mice
received A/J heart allografts. Survival of heart allografts in neutrophildepleted C57BL/6 recipients (n ⫽ 5) was compared with survival of allografts in control rat IgG-treated recipients (n ⫽ 8). Allograft rejection in all
recipient mice was visually confirmed by laparotomy. Isografts from recipients treated with RB6.8C5 were maintained ⬎100 days. Recipient
treatment with RB6.8C5 significantly prolonged heart allograft survival
(p ⫽ 0.001).
production of IL-8 and MIP-1␣ (28, 29), this cytokine may initiate
the early chemokine cascades observed in the current studies.
The results of the current report clearly indicate that induction of
early inflammatory events in the cardiac grafts influences the progression of downstream events involving adaptive immune responses to the allograft. Recent results from this laboratory have
demonstrated that Mig and IP-10 expression is observed as early as
day 2 after transplant in cardiac allografts but not in isografts (18),
⬃1 day after intragraft expression of KC has subsided. Treatment
of recipients with KC antiserum at the time of transplantation decreased intraallograft expression of the T cell chemoattractants
IP-10 and Mig. Ab depletion studies and the use of recipients
deficient in CD4⫹ vs CD8⫹ T cells have indicated that day 2
expression of IP-10 and Mig is mediated by IFN-␥-producing
CD8⫹ T cells (18). On the basis of these results, we have proposed
that circulating CD8⫹ T cells interact with inflammatory sites in
the vascular endothelium and those T cells with allogeneic class I
MHC specificity are stimulated to produce IFN-␥ that in turn stimulates endothelial cell production of Mig and IP-10. The importance of Mig production in the rejection of the heart allografts has
been shown in our laboratory by the ability of Mig-specific Abs to
inhibit T cell infiltration into the cardiac allograft and prolong
survival from day 8 –9 to day 18 –20 after transplant.4 In light of
these results, the effects of KC antagonism on T cell graft infiltration and survival observed in the current report may be indirectly mediated through decreases in Mig. The early chemokine
cascades in cardiac grafts may be coordinated so that optimal induction of each cascade is dependent on induction of the preceding
chemokine cascade.
The mechanism by which treatment with the KC antiserum attenuates the expression of IP-10 and Mig and T cell infiltration into
the graft remains unclear at this time. The chemoattractive properties of KC for neutrophils is suggestive of a potential role of
these cells in initiating downstream events in the graft rejection
process. Alternatively, recipient treatment with KC antiserum may
inhibit a critical neutrophil-mediated activity without affecting
M. Miura. Monokine induced by IFN-␥ is a dominant factor directing T cells into
murine cardiac allografts during acute rejection. Submitted for publication.
4
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FIGURE 5. CD4⫹ and CD8⫹ T cell infiltration into allogeneic cardiac
grafts from KC antiserum- and NRS-treated recipients. Cardiac allografts
harvested from KC antiserum- and NRS-treated recipients were used to
prepare frozen sections that were stained with anti-CD4 or anti-CD8 mAb
for immunohistochemistry. The slides were viewed at ⫻200 magnification,
and the number of positively staining cells in eight random fields from
three different sections from three different grafts was counted. Each circle
represents the number of positive cells per random field for the indicated
stain, with the horizontal bar indicating the mean number of positive cells
per field. Treatment with the KC antiserum significantly (ⴱ, p ⬍ 0.0001)
inhibited infiltration of both CD4⫹ and CD8⫹ T cells into the allograft.
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neutrophil infiltration into the allograft. Further evidence for a neutrophil role in acute rejection of the heart allografts is indicated by
the ability of recipient neutrophil depletion to extend heart allograft survival. Although Ab-mediated neutrophil depletion results
in ⬃50% mortality of the heart allograft recipients from decreased
resistance to infections, rejection of allografts in the surviving recipients is delayed until 18 –21 days after transplant, which is similar to that observed in the KC antiserum-treated recipients. Such
observations support a role for neutrophils in the establishment of
inflammatory foci in the allograft that facilitate T cell recruitment
and graft infiltration. Several laboratories have documented the
critical role of neutrophils in ischemia/reperufsion injury (30 –32).
Antagonism of neutrophil adhesion to the endothelium effectively
attenuates this injury in many models. Recent results from this
laboratory have indicated marked inhibition of neutrophil infiltration into renal tissue and decreased tissue pathology by treating
mice with KC-specific antiserum following reperfusion of kidneys
subjected to 1 h of warm ischemia (M. Miura, manuscript in preparation). In addition to their role in ischemia/reperfusion injury,
activation of neutrophils with solid-phase IL-8 stimulates neutrophils to produce or release many different chemoattractants for T
cells (33, 34). Such events could certainly play a role in recruiting
and/or focusing T cells to the sites of inflammation in the vascular
endothelium of cardiac allografts during progression of acute allograft rejection.
In summary, the results in the current report have indicated the
induction of a programmed cascade of chemokines in both
isografts and allografts at early times following the transplantation
surgery. Treatment of recipients with Abs to one of the first components in these cascades attenuates downstream events including
the expression of chemokines with chemoattractive properties for
Ag-primed T cells and cellular infiltration into the graft. These data
are the first to expose the induction of such cascades in transplanted organ allografts. Furthermore, these data indicate the profound effects of innate immune components on alloantigen-specific
rejection responses and suggest that strategies aimed at antagonizing early inflammatory events in heart allografts will have substantial benefit in attenuating acute rejection of cardiac allografts.
EARLY CHEMOKINES IN CARDIAC ALLOGRAFTS