Full text of DOI:10.1038/labinvest.3780395

0023-6837/02/8201-061$03.00/0
LABORATORY INVESTIGATION
Vol. 82, No. 1, p. 61, 2002
Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc.
Printed in U.S.A.
Selectin-Mediated Interactions Regulate Cytokine
Networks and Macrophage Heme Oxygenase-1
Induction in Cardiac Allograft Recipients
Ana J. Coito, Gray D. Shaw, Jiye Li, Bibo Ke, Jeffrey Ma, Ronald W. Busuttil, and
Jerzy W. Kupiec-Weglinski
Dumont-UCLA Transplant Center (AJC, JL, BK, JM, RWB, JWK-W), Department of Surgery, UCLA School of
Medicine, Los Angeles, California; and Genetics Institute/Wyeth Research (GDS), Cambridge, Massachusetts
SUMMARY: Host sensitization to major histocompatibility complex (MHC) antigens is among the most critical of problems facing
heart transplantation. Selectins are postulated to mediate the early adhesive events in the recruitment of leukocytes at the
allograft site. We investigated the significance of selectin–P-selectin glycoprotein ligand-1 (PSGL-1)–mediated in vivo interactions
in the immune cascade leading to rejection of cardiac allografts in skin presensitized rats. Infusion of a soluble recombinant form
of PSGL-1 (rPSGL-Ig) during skin graft–mediated sensitization prevented Day 1.0 Ϯ 0.1 “accelerated” rejection in sensitized rat
recipients, and prolonged cardiac allograft survival to Day 3.8 Ϯ 1.0 (p Ͻ 0.001). This therapy significantly depressed serum IgM
levels and decreased intragraft expression of Th1 type cytokines (IL-2 and IFN-␥) as well as of IL-1␤ and MCP-1, as compared
with controls, without affecting the initial number of infiltrating mononuclear cells (MNC). A profound decrease in graft-infiltrating
MNC was recorded at 24 hours in rPSGL-Ig–treated rats. The expression of heme oxygenase-1 (HO-1), an inducible heat shock
protein 32 that protects against oxidative cell/tissue injury, was found in approximately 14-fold higher levels in the rPSGL-Ig–
treated recipients as compared with controls. The HO-1 overexpression in rPSGL-Ig–treated hosts, primarily by infiltrating
macrophages, was accompanied by virtual absence of myocardial infarcts and decreased frequency of TUNEL ϩ cells at the graft
site. Moreover, down-regulation of HO-1 expression by zinc protoporphyrin, an HO-1 antagonist, decreased expression of
antiapoptotic Bag-1 molecule in recipients conditioned with rPSGL-Ig. Thus, the blockade of selectin–PSGL-1 interactions
depresses intracardiac allograft expression of Th1 type cytokines, and might inhibit the differentiation of Th1 type cells. In
addition, it up-regulates HO-1 expression and protects against myocardial infarction and apoptosis. Hence, this study reports on
a previously unrecognized role of selectin–PSGL-1–mediated interactions after in vivo alloantigenic challenge. (Lab Invest 2002,
82:61–70).
multistep series of adhesive and signaling events
regulates the recruitment of leukocytes to major
with relatively high affinity to only a few glycoproteins on
leukocytes or endothelial cells (McEver and Cummings,
1997). P-selectin glycoprotein ligand-1 (PSGL-1), a
disulfide-bonded homodimeric mucin-like glycoprotein
expressed virtually on all leukocytes, is the best-defined
ligand of the selectin family. Although PSGL-1 was
originally discovered as the principal ligand for
P-selectin, it was subsequently demonstrated that this
glycoprotein ligand binds to all three P-, E-, and
L-selectin members (McEver and Cummings, 1997).
We have shown that the blockade of PSGL-1–medi-
ated interactions with a soluble recombinant form of
PSGL-1 (sPSGL-1) inhibits recruitment of polymorpho-
nuclear leukocytes (PMN) and mononuclear cells (MNC)
in rat models of kidney (Takada et al, 1997) and liver
ischemia/reperfusion (I/R) injury (Dulkanchainun et al,
1998), resulting in significantly less organ dysfunction.
There is evidence that functional selectin ligands are
predominantly expressed on Th1 rather than Th2 cells
(Lim et al, 1999). In organ transplantation, host immune
responses seem to be qualitatively distinct depending
on whether there is predominance of Th1 or Th2 cells at
the graft site (Zhai and Kupiec-Weglinski, 1999).
A
histocompatibility complex (MHC)-incompatible organ
allograft (Coito et al, 1994). The selectin family of
adhesion receptors, L-, E-, and P-selectin, and their
carbohydrate ligands, are considered the primary me-
diators of leukocyte tethering and rolling on the sur-
face of endothelial cells (McEver and Cummings,
1997). Indeed, studies in selectin-deficient mice have
not only confirmed the role of these membrane-
anchored C-type lectins in leukocyte rolling, but also
provided evidence indicating an overlapping function of
the selectins in the regulation of inflammatory processes
(Frenette et al, 1996; Homeister et al, 1998). L-selectin is
expressed on most leukocytes. E- and P-selectins are
expressed on activated endothelial cells, and P-selectin
is also expressed on activated platelets. Selectins bind
Received September 27, 2001.
This work was supported by American Heart Association Grant 0030006N
(AJC), NIH Grants RO1 AI23847, RO1 AI42223 (JWK-W), and The
Dumont Research Foundation.
Address reprint requests to: Dr. Ana J. Coito, The Dumont-UCLA Trans-
plant Center, Rm. 77-120 CHS, Box 957054, Los Angeles, CA 90095.
E-mail: acoito@mednet.ucla.edu
The present study is the first to elucidate the signifi-
cance of selectin–PSGL-1–mediated in vivo interactions
Laboratory Investigation • January 2002 • Volume 82 • Number 1 61

Coito et al
in sensitized cardiac allograft recipients. The blockade of
selectin-mediated interactions, with a soluble recombi-
nant form of the PSGL-1 (rPSGL-Ig), favored inhibition of
Th1 differentiation and decreased expression of the
monocyte chemoattractant protein (MCP-1) in a model
of accelerated cardiac allograft rejection in presensitized
rats. Moreover, rPSGL-Ig therapy induced intragraft ex-
pression of heme oxygenase-1 (HO-1), a heat shock
protein (hsp32) that protects against oxidative tissue
injury, prevented the development of myocardial infarcts,
and protected against myocardial apoptosis.
allograft rejection (Fig. 1A). At both Day 0 and ϩ1 the
IgM allo-Ab levels were significantly decreased in the
rPSGL-Ig–treated group as compared with controls (p
Ͻ 0.03 and p Ͻ0.05, respectively). No significant
differences in anti-donor IgG allo-Ab levels between
treated and control groups were detected (Fig. 1B).
rPSGL-Ig Therapy Arrests Intramyocardial Leukocyte
Infiltration Without Affecting P-Selectin Expression
rPSGL-Ig treatment during the sensitization phase ex-
tended cardiac allograft survival from less than 24 hours
to more than 3 days. We analyzed intragraft cellular
infiltration at 6 hours and 24 hours post-transplant (n ϭ
3–5/group). At 6 hours, cardiac allografts from rPSGL-
Ig–treated and control recipients showed relatively nor-
mal histology with only slight interstitial edema and
sparse intramyocardial MNC infiltration (Table 2). By 24
hours, control grafts exhibited severe interstitial edema,
large areas of myocardial infarction, and dense MNC
infiltration. The infiltrating cells consisted predominantly
of T lymphocytes, CD4ϩ cells, CD8ϩ cells, macro-
phages, and IL-2Rϩ cells (Table 2; Fig. 2, A to C). In
contrast, the 24-hour grafts from rPSGL-Ig–treated re-
cipients revealed good preservation of myocardial archi-
tecture, with significantly less intramyocardial T cell (p Ͻ
0.001), CD4ϩ cell (p Ͻ 0.04), CD8ϩ cell (p Ͻ 0.0001),
macrophage (p Ͻ 0.001), and IL-2Rϩ cell (p Ͻ 0.01)
infiltration, as compared with respective controls (Table
2; Fig. 2, F to H). As P-selectin represents the principal
ligand for PSGL-1, we then investigated whether admin-
istration of rPSGL-Ig depressed P-selectin expression.
The expression of P-selectin by vascular graft endothe-
lium was comparable at 6 hours (Ϯ/ϩ) and 24 hours
(ϩϩ/ϩϩϩ) after transplantation in both treated and
untreated groups, suggesting that rPSGL-Ig blocks the
interaction rather than regulates P-selectin molecule
expression. Moreover, rPSGL-Ig therapy failed to affect
the expression of other vascular adhesion molecules,
with both control and treated 24-hour grafts showing
moderate to high-level expression of VCAM-1 (ϩ/ϩϩ)
and ICAM-1 (ϩϩ/ϩϩϩ).
Results
Treatment with rPSGL Ig Prolongs Cardiac Allograft
Survival in Sensitized Rats
(LEWϫBN)F1 cardiac allografts are rejected in less
than 24 hours in Lewis (LEW) rats sensitized with
Brown Norway (BN) skin grafts 7 days earlier (Day Ϫ7)
(Kupiec-Weglinski et al, 1991). Treatment with
rPSGL-Ig during the effector phase (postcardiac en-
graftment) failed to affect the 24-hour rejection re-
sponse (Table 1). In contrast, cardiac allograft survival
in rats that received a course of rPSGL-Ig during the
sensitization phase (between skin and cardiac engraft-
ment) was significantly increased to Day 3.8 Ϯ 1.0, as
compared with controls (Day 1.0 Ϯ 0.1, p Ͻ 0.001; Table
1). Extended pre- and post-transplant rPSGL-Ig treat-
ment did not improve further allograft survival. Animals
conditioned with rPSGL-Ig during the sensitization
phase (Day Ϫ7 to Ϫ1) were used in these studies.
Treatment with rPSGL-Ig Depresses Serum
IgM Allo-Ab Levels
To define the donor-specific allo-Ab responses elic-
ited by (LEWϫBN)F1 cardiac allografts in LEW rats
sensitized with BN skin grafts, serum was harvested
from recipients preconditioned with rPSGL-Ig or PBS
at Day Ϫ5, Ϫ3, 0 (the day of cardiac engraftment), and
at Day ϩ1, ϩ4, ϩ7, ϩ14, and ϩ21 thereafter. Serum
samples were then tested for IgM and IgG binding to
BN lymph node target cells by flow cytometry. In
control recipients, IgM allo-Ab levels increased con-
siderably by Day 0 and remained elevated until Day 7
post-transplant (Fig. 1A). In contrast, in recipients
treated with rPSGL-Ig, the anti-donor IgM response
peaked at Day 4, that is, around the time of ultimate
rPSGL-Ig Therapy Modifies Intragraft Cytokine Networks
We have employed competitive RT-PCR and immu-
nohistology to analyze cytokine gene and protein
expression patterns in cardiac allografts. Interestingly,
despite comparable numbers of MNC infiltrating car-
diac allografts at 6 hours in both rPSGL-Ig and control
recipients, the cytokine networks were highly affected.
At 6 hours, the mRNA levels coding for IL-1␤ (p Ͻ
0.04), IL-2 (p Ͻ 0.02), IFN-␥ (p Ͻ 0.005), and MCP-1 (p
Ͻ 0.05) were significantly depressed in treated recip-
ients as compared with the respective controls (Fig. 3).
At 24 hours, intragraft expression of IL-2 mRNA re-
mained depressed in rPSGL-Ig–treated hosts, as
compared with controls (p Ͻ 0.004; Fig. 3). The
immunohistochemical analysis confirmed the transla-
tion of mRNA into the relevant proteins (Fig. 2).
Table 1. The Effects of rPSGL-Ig Therapy on the Survival
of LBNF1 Cardiac Allografts in Skin Presensitized
LEW Rats
Cardiac
allograft
survival (days)
Significance
Group
(mean Ϯ SD) n
(pϽ)
Control
1.0 Ϯ 0.1
1.0 Ϯ 0.3
3.8 Ϯ 1.0
3.3 Ϯ 0.5
6
5
6
3
rPSGL-Ig (Day 0 to ϩ1)
rPSGL-Ig (Day Ϫ7 to Ϫ1)
rPSGL-Ig (Day Ϫ7 to ϩ2)
0.37
0.001
0.007
Figure 3 also shows that at 6 hours, the CD3 and
CD25 mRNA levels were comparable in both recipient
62
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PSGL-1–Mediated Interactions in Heart Transplants
Figure 1.
Flow cytometry analysis of circulating IgM and IgG allo-Ab levels in untreated (no Rx) and rPSGL-Ig-treated (rPSGL-Ig Rx) presensitized cardiac allograft recipients.
In control recipients, IgM allo-Ab levels increased at Day 0 to 1 and remained elevated until Day 7 post-transplant, contrasting with rPSGL-Ig–treated recipients in
which IgM responses peaked at Day 4, ie, around the time of ultimate allograft rejection (A). No significant difference was found in the IgG allo-Ab response between
control and treated recipients (B).
Table 2. Immunohistochemical Analysis of Intramyocardial Infiltrating MNC in Cardiac Allografts
No Rx.
rPSGL-Ig Rx.
rPSGL-Ig ϩ ZnPP Rx.
Feature
T cells
CD4ϩ cells
CD8ϩ cells
Mono/Mac
6 hours
24 hours
6 hours
24 hours
24 hours
5.0 Ϯ 2.3
33.0 Ϯ 2.0
1–2
24.8 Ϯ 3.2
1.0 Ϯ 1.0
67.0 Ϯ 3.0
40.5 Ϯ 11.0
Ͼ200
Ͼ200
42.5 Ϯ 4.5
3.3 Ϯ 2.1
24.5 Ϯ 7.5
1–2
22.0 Ϯ 0.6
0.6 Ϯ 0.6
18.2 Ϯ 10.9
20.6 Ϯ 5.5
34.2 Ϯ 18.5
56.5 Ϯ 32.0
13.7 Ϯ 6.6
35.0 Ϯ 4.2
26.0 Ϯ 1.4
85.0 Ϯ 16.9
96.5 Ϯ 2.1
32.5 Ϯ 7.0
IL-2Rϩ cells
groups. At 24 hours, in agreement with our intragraft
MNC infiltration analysis (Table 2; Fig. 2), mRNA levels
coding for CD3 (p Ͻ 0.031) and CD25 (p Ͻ 0.05) were
profoundly depressed in the rPSGL-Ig–treated recipi-
ents, compared with controls (Fig. 3).
a few vessels were sparsely positive for HO-1 at 24
hours after transplantation. We identify the infiltrating
MNC cells as the primary source of HO-1 in the grafts. In
rPSGL-Ig–treated grafts the infiltrating MNC were
strongly HO-1 positive, whereas in control grafts MNC
were barely HO-1 positive (Fig. 4, B and C). The HO-1–
positive cells usually had the convoluted shape typical of
macrophages. We also performed a comparative study
in sequential section stained for ED1ϩ and HO-1ϩ cells,
and we could identify macrophages as a predominant
source of HO-1.
HO-1 is Induced by Intragraft Macrophages in
rPSGL-Ig–Conditioned Recipients
The expression of HO-1 at 6 hours post-transplant was
negligible in both rPSGL-Ig and control groups. By 24
hours, however, cardiac allografts from treated recipi-
ents showed up-regulation of HO-1 expression as com-
pared with controls (Fig. 4A). Indeed, Western blot–
assisted densitometry measurements indicated 14-fold
higher levels of HO-1 in rPSGL-Ig–treated recipients (p Ͻ
0.001). To identify the source of HO-1 in cardiac allo-
grafts, we performed immunocytochemistry studies. In
cardiac grafts of both treated and untreated groups, only
rPSGL-Ig Therapy Protects Against Apoptosis in Infarcted
Myocardial Areas
We then performed the terminal deoxynucleotidyl trans-
ferase (TdT) nick-end labeling (TUNEL) assay to assess
whether rPSGL-Ig therapy protected against apoptosis.
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Coito et al
Figure 2.
Immunohistochemical analysis of infiltrating cells/cytokines in rat cardiac allografts (24 hours). Representative analysis of cardiac allografts in skin sensitized,
otherwise untreated, recipients (A to E), and after rPSGL-Ig treatment (F to J). Rejecting grafts were characterized by an elevated number of infiltrating T cells (A)
and macrophages (B), contrasting with their decreased number in allografts from rPSGL-Ig–treated hosts (F and G, respectively). In addition, rPSGL-Ig therapy
virtually abolished intragraft cell activation markers, such as IL-2Rϩ cells (H), IL-2 (I), and IFN-␥ (J). Moderate to high numbers of IL-2 Rϩ cells (C), IL-2 (D), and
IFN-␥ (E) were found in control cardiac allografts. Original magnification, ϫ200.
No TUNEL–positive cells were observed in cardiac grafts
of rPSGL-Ig–treated or control recipients at 6 hours
post-transplant. However, unlike in the rPSGL-Ig–
treated grafts that remained negative for TUNEL staining
(Fig. 4E), increased numbers of TUNEL–positive myo-
cytes were readily detectable by 24 hours in infarcted
areas and in some vessels of control grafts (Fig. 4D).
Zinc Protoporphyrin (ZnPP) Increases T Cell
Infiltration/Activation and Down-Regulates Bag-1
Expression in rPSGL-Ig–Conditioned Hosts
To determine whether the beneficial effect of rPSGL-Ig
therapy was HO-1–mediated, we administrated ZnPP, a
competitive inhibitor of HO-1 activity, to rPSGL-Ig–
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PSGL-1–Mediated Interactions in Heart Transplants
Figure 3.
Competitive template RT-PCR–assisted analysis of intragraft cytokines in untreated (no Rx) and rPSGL-Ig-treated (rPSGL-Ig Rx) cardiac allograft recipients. The levels
of CD3 and CD25 mRNA were comparable in both recipient groups at 6 hours post-transplant. In contrast, rPSGL-Ig therapy significantly decreased the expression
of mRNA coding for IL-1␤ (p Ͻ 0.04), IL-2 (p Ͻ 0.02), IFN-␥ (p Ͻ 0.005), and MCP-1 (p Ͻ 0.05). IL-10 was almost absent in all cardiac grafts at this initial time
point. At 24 hours, control allografts were characterized by elevated CD3, CD25, and IL-2 mRNA levels, as compared with those in the rPSGL-Ig–treated group (p
Ͻ 0.03, p Ͻ 0.05, p Ͻ 0.004, respectively).
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Coito et al
Figure 4.
Western blot–assisted detection of HO-1 and of TUNEL-assisted staining of apoptotic cells in rat cardiac allografts (24 hours). A, rPSGL-Ig therapy up-regulated
detection of HO-1 expression (Lanes 3 and 4), as compared with control grafts (Lanes 1 and 2). B, Despite dense infiltration of control grafts by MNC, including
macrophages, cells are faintly positive for HO-1 staining. C, An intense staining for HO-1 by infiltrating macrophages in cardiac allografts from rPSGL-Ig–treated rats.
Enlargements of cellular staining are provided in the lower left inserts. D, TUNELϩ staining in both myocardium and vessels of control grafts. Areas of positive labeling
are identified by asterisk (*), and enlargement of cellular staining is provided in lower left insert. E, In contrast, no TUNELϩ cells were detected in the grafts of
rPSGL-Ig–treated recipients. Original magnification, ϫ200.
treated recipients. We have shown that ZnPP exerts its
biological effects quickly by down-regulating HO-1 pro-
tein/activity in cardiac grafts (Katori et al, in press).
Adjunctive administration of ZnPP to rPSGL-Ig–treated
recipients resulted in a loss of myocardial preservation
and a significant increase of T cell (p Ͻ 0.03), CD8ϩ cell
(p Ͻ 0.04), and IL-2Rϩ cell (p Ͻ 0.05) infiltration as
compared with recipients treated with rPSGL-Ig alone
(Table 2). No significant effect was observed in CD4ϩ
cell and macrophage infiltration after ZnPP administra-
tion (Table 2). In addition, to decreased T cell infiltration/
activation, adjunctive ZnPP therapy markedly decreased
the expression of antiapoptotic Bag-1 protein (mean
densitometric ratio rPSGL-Igϩ ZnPP : rPSGL-Ig ϭ 0.6; n
ϭ 3). Moreover, intragraft expression of proapoptotic
caspase-3 (p20), undetectable otherwise in recipients
treated with PSGL-Ig alone, was moderately increased
by densitometry (net intensity: 1567 Ϯ 343) after adjunc-
tive ZnPP therapy.
number of infiltrating MNC; (d) induced intragraft over-
expression of HO-1; and (e) protected against the
development of myocardial infarcts and apoptosis.
Administration of rPSGL-Ig during the sensitization
phase ameliorated 24-hour rejection and significantly
prolonged the survival of cardiac allografts to over 3
days. Very few therapeutic modalities are effective in
this stringent sensitized rat cardiac transplant model,
which makes rPSGL-Ig–induced prolongation of allo-
graft survival of particular significance.
A growing number of reports have been showing the
crucial role for selectin-mediated interactions on leu-
kocyte tethering and rolling on endothelial cells
(Frenette et al, 1996). We observed that administration
of rPSGL-Ig to cardiac recipients significantly de-
creased the expression of type-1 cytokines, com-
pared with controls, consistent with their role in the
rejection cascade (Zhai and Kupiec-Weglinski, 1999).
Interestingly, at 6 hours post-transplantation, despite
differences in cytokine expression in cardiac grafts of
treated versus control recipients, grafts from both
groups showed comparable T cell infiltrate and CD3
mRNA levels. These results might favor the notion that
rPSGL-Ig preferentially inhibited the recruitment of
Th1-like cells to the graft site. Indeed, the selectin
ligands are preferentially expressed on Th1 cells and
play a key role in their recruitment to sites of inflam-
mation (Austrup et al, 1997; Lim et al, 1999). Support-
ing this concept is our observation of up-regulated
Discussion
We report here the results of our studies on the role of
selectin–PSGL-1–mediated interactions in
a well-
defined model of cardiac transplantation in sensitized
rat recipients. Treatment with rPSGL-Ig before cardiac
engraftment (a) significantly prolonged cardiac allo-
graft survival; (b) profoundly depressed intragraft ex-
pression of type-1 cytokines (IL-2 and IFN-␥), as well
as of IL-1␤, and MCP-1; (c) significantly depressed the
66
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PSGL-1–Mediated Interactions in Heart Transplants
P-selectin expression on the vascular endothelium in
the first few hours post-transplantation. If the inhibi-
tion of lymphocyte rolling by rPSGL-Ig can explain, at
least in part, our results, it does not answer the
question of why treatment of sensitized recipients with
rPSGL-Ig failed when given immediately after heart
transplantation. It seems logical to raise the assump-
tion that selectin-mediated interactions may play a
role in the T cell costimulatory pathway favoring the
generation of effector Th1 cells. The immunosuppres-
sive properties of rPSGL-Ig might, to a certain extend,
contribute to the inhibition of Th1 cell differentiation
during the sensitization phase. The expression of P-
and E-selectin ligands is controlled by the activity of
␣(1, 3)-fucosyltransferases that modify carbohydrates
on PSGL-1 and other surface receptors (Fuhlbrigge et
al, 1997). Moreover, the expression of one enzyme of
the ␣(1, 3)-fucosyltransferase family, FucTVII, is highly
linked to Th1 cell differentiation (Wagers et al, 1998).
The hypothesis that a primary role of the rPSGL-Ig–
mediated therapy is to inhibit Th1 differentiation pro-
vides a better justification of why postcardiac trans-
plant therapeutic approach failed to affect the under
24-hour rejection in our model. At that time, the
effector T cells triggered by skin graft–driven host
immune response are already differentiated and ready
to reject cardiac transplants in an accelerated manner.
In support of our theory that the rPSGL-Ig–mediated
blockade of selectin interactions favors inhibition of T
cell activation/differentiation rather than lymphocyte
rolling are our recent observations in the ischemic
livers in which rPSGL-Ig therapy failed to affect the
initial recruitment of T lymphocytes into the liver grafts
(AJ Coito, unpublished data). Indeed, a growing num-
ber of studies illustrate that other adhesion molecules
can mediate leukocyte rolling. For example, the ␣4␤1
integrin also supports leukocyte tethering and rolling
in flow on VCAM-1 (Alon et al, 1995). Moreover, the ␣4
integrin can initiate leukocyte–endothelial cell interac-
tions in the absence of selectins in vivo and, in
IL-4–treated mice, the absence of selectins does not
affect the recruitment of MNC cells in cremasteric
postcapillary venules (Hickey et al, 1999). In our stud-
ies, VCAM-1 was up-regulated on the vascular cardiac
endothelium independent of the rPSGL-Ig therapy.
Furthermore, IL-4 was predominantly expressed in the
rPSGL-Ig–treated grafts at 24 hours (data not shown).
Interestingly, at 6 hours, MCP-1, a chemokine that
specifically stimulates the directional migration of T
cells as well as monocytes, was found to be nearly
absent in the cardiac grafts of rPSGL-Ig–treated rats.
It has been postulated that MCP-1, a member of the
C-C chemokines, may play an important role in im-
mune cell recruitment into sites of the antigenic chal-
lenge (Russell et al, 1993). The initial lack of MCP-1
observed in the rPSGL-Ig–treated grafts might also
represent one of the mechanisms by which this ther-
apeutic approach effectively reduced the number of
leukocytes at the graft site.
morphology of those grafts was largely preserved.
There is a growing body of evidence that oxidative
stress causes cardiac injury (Aikawa et al, 1997).
Heme oxygenases, rate-limiting enzymes in the deg-
radation of heme, catalyze the conversion of heme
into biliverdin, carbon monoxide (CO), and free iron
(Soares et al, 1998). CO increases cellular levels of
cGMP, which regulates vascular tone and smooth
muscle development and, as recently shown, has
antiinflammatory effects (Otterbein et al, 2000). Biliru-
bin is known to be a potent antioxidant (Stocker et al,
1987). HO-1, also known as hsp32, is induced by a
variety of cellular stresses, including oxygen depriva-
tion and free radical–mediated stress. The overex-
pression of this enzyme exerts potent cytoprotective
effects (Soares et al, 1998), consistent with our recent
studies in steatotic rat liver models of I/R injury (Amersi
et al, 1999). Moreover, it has been reported that HO-1
has a critical role in survival of concordant mouse to
rat cardiac transplants (Lin et al, 1999; Soares et al,
1998). We analyzed HO-1 induction in cardiac grafts
on rPSGL-Ig therapy. This stress protein was almost
absent at 6 hours and highly up-regulated by infiltrat-
ing MNC, primarily macrophages, at 24 hours after
transplant. In contrast, HO-1 expression was minimal
in control grafts at both 6 hours and 24 hours. The
virtual absence of HO-1 expression in 24-hour control
grafts might relate to the myocardial apoptosis ob-
served in the graft infarcted areas. In an attempt to
elucidate the role mediated by HO-1 in our experimen-
tal settings we administered ZnPP, a known HO-1
antagonist, to recipients pretreated with rPSGL-Ig.
Interestingly, ZnPP administration resulted in signifi-
cant increased MNC infiltration and activation. The
numbers of intragraft T cells, particularly CD8ϩ cells,
in the ZnPP-treated recipients were significantly
higher than in recipients treated with rPSGL-Ig alone,
whereas the levels of IL-2Rϩ cells in the ZnPP-treated
recipients were comparable with those observed in
untreated controls. These findings support the con-
cept that HO-1 and/or its downstream mediators,
such as CO, are powerful antiinflammatory agents
(Otterbein et al, 2000). Interestingly, we did not find
significant differences in CD4ϩ cells and macro-
phages infiltrating the cardiac allografts treated with
rPSGL-Ig alone or in combination with ZnPP. These
observations suggest that HO-1 may account for
some but not all of the effects of the rPSGL-1 therapy
on leukocyte infiltration. Although it is important that in
our experimental settings ZnPP is a potent inhibitor of
HO-1 activity, it never completely abolishes HO-1
expression (Amersi et al, 1999; Katori et al, in press).
Administration of ZnPP to rPSGL-Ig–treated recipients
has also led to a decrease in antiapoptotic Bag-1
protein expression. In addition, caspase-3 (p20) a
proapoptotic protein that was absent in recipients
conditioned with rPSGL-Ig, moderately increased at
the graft site in recipients that received adjunctive
ZnPP. This evidence for the role of HO-1 in the
regulation of apoptotic protein networks is consistent
with our recent data in the ischemic rat livers in which
gene therapy–induced HO-1 overexpression was as-
One of the most striking effects of rPSGL-Ig therapy
was the apparent absence of myocardial infarcts in
the treated hosts. Despite some MNC infiltration, the
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Coito et al
sociated with up-regulation of antiapoptotic proteins
(Coito, unpublished). In supporting our interpretation
are recent findings that myocardial apoptosis occurs
during myocardial infarction and that HO-1 null mice
are more prone to develop myocardial infarcts than
their wild-type counterparts (Yet et al, 1999; Bialik et
al, 1997).
heart I/R, a single dose of ZnPP given at reperfusion to
CoPP-treated grafts reduces HO-1 enzymatic activity
to control levels as earlier as 4 minutes after trans-
plantation (Katori et al, in press).
Serum IgM/IgG Allo-Ab Responses
In conclusion, the blockade of selectin–PSGL-1
interactions depresses intragraft expression of Th1
type cytokines and might inhibit the differentiation of
Th1 type cells. In addition, it up-regulates HO-1 ex-
pression and protects against myocardial infarction
and apoptosis. Hence, this study reports on the pre-
viously unrecognized role and mechanisms of selec-
tin–PSGL-1–mediated interactions after in vivo alloan-
tigenic challenge.
The circulating donor–specific allo-Ab responses were
screened in the recipient’s serum as described (Wa-
sowska et al, 1996). Briefly, cervical BN lymph node
target cells were first incubated with serially diluted
(1/4 to 1/128) heat-inactivated experimental serum
samples for 45 minutes at 4° C. To stain for IgG and
IgM, the cells were reacted with a mixture of FITC-
conjugated goat anti-rat IgG (Fc specific) and PE-
conjugated goat anti-rat IgM (␮-chain) (Jackson Im-
munoresearch, West Grove, Pennsylvania) for 30
minutes at 4° C. The cells were then washed, fixed in
1% neutral buffered formalin, and analyzed on an
EPICS C cell sorter (Coulter, Hialeah, Florida).
Materials and Methods
Animals and Grafting Techniques
Male inbred adult rats (Harlan Sprague-Dawley, Indi-
anapolis, Indiana) were used. Lewis (LEW; RTl) rats
were recipients of cardiac allografts from Lewis ϫ
Brown Norway F1 hybrids (LBNF1; RT1l/n) and Brown
Norway (BN; RT1n) rats served as a source of skin
grafts (Kupiec-Weglinski et al, 1991). Orthotopic skin
(3 ϫ 3 cm) grafts were placed bilaterally to defects on
the dorsal thorax of prospective recipients (Day Ϫ7).
Hearts were transplanted to the abdominal great ves-
sels of the skin graft–sensitized recipients, and their
function was monitored by palpation. Rejection was
defined as the day of cessation of heartbeat and was
confirmed by histology. All the surgical procedures
were done under the guidelines of the UCLA Animal
Research Committee.
Intragraft Cell Infiltration
Hematoxylin/eosin and immunoperoxidase staining
were used for serial assessment of myocardial/vascu-
lar preservation and detection of inflammatory cell
infiltration/activation, adhesion molecules, and HO-1
expression (Coito et al, 1995). The presence of cyto-
kine gene products was demonstrated by the
saponin-immunoperoxidase method as previously de-
scribed (Coito et al, 2000). Antibodies against human
P-selectin (CD62P, cross reacts with rat P-selectin;
Pharmingen, San Diego, California), and rat VCAM-1
(CD106; courtesy of Dr. R. Lobb, Biogen, Cambridge,
Massachusetts), ICAM-1 (CD54; Harlan Bioproducts
for Science, Indianapolis, Indiana), T cells (TCR-␣/␤
chains, R73; Harlan), monocytes/macrophages (ED1;
Harlan), CD25 (IL-2R; ART-18; courtesy of Dr. T.
Diamantstein, Berlin, Germany), HO-1 (does not react
with HO-2; StressGen Biotechnologies, Victoria, B.C.,
Canada), IL-2 (R&D Systems, Minneapolis, Minneso-
ta), IFN-␥ (DB-1; Harlan), RANTES and MCP1 (Pepro
Tech, Rocky Hill, New Jersey) were used. The sections
were evaluated blindly by counting the labeled cells in
triplicates within 10 high-power fields (HPF)/section.
Some antigens were analyzed in a semiquantitative
fashion, and the relative abundance of each was
judged as (Ϫ) negative, (Ϯ) minimal, (ϩ) little, (ϩϩ)
moderately abundant, or (ϩϩϩ) very abundant.
Treatment with rPSGL-Ig
rPSGL-Ig (Genetics Institute, Cambridge, Massachu-
setts) is a recombinant soluble form of PSGL-1 (Sako
et al, 1995). This dimeric molecule consists of the
amino terminal 47 a.a. and carbohydrate portion of
mature PSGL-1 fused to the Fc portion of human IgG1
(Sako et al, 1995). rPSGL-Ig, free of endotoxin, was
administered intravenously to skin-presensitized car-
diac allograft recipients (100 ␮g/day/rat) according to
three protocols: (a) during the effector phase, that is,
at Day 0 and Day ϩ1 postcardiac engraftment (n ϭ 5);
(b) during the skin graft–mediated sensitization phase,
that is, between Day Ϫ7 and Day Ϫ1 (n ϭ 11); and (c)
during both sensitization and effector phases, that is,
between Day Ϫ7 and Day ϩ2 (n ϭ 6). The control
group received PBS only (n ϭ 10).
Intragraft Cytokine Gene Expression
RNA from cardiac grafts was prepared by the guani-
dinium thiocyanate–cesium chloride method and
competitive template (CT) RT-PCR was performed, as
described (Siegling et al, 1994). Quantification of cyto-
kine cDNA derived from this RNA was carried out using
multispecific control fragment as an internal standard for
competitive PCR. The sense and antisense primer se-
quences derived from rat CD3, CD25, IL-2, IFN-␥, IL-10,
RANTES, MCP-1, and ␤-actin cDNA were used (Nadeau
et al, 1995; Siegling et al, 1994).
Treatment with ZnPPn
ZnPP was given at Day 0 (1.5 mg/kg iv) to recipients
previously treated with rPSGL-Ig during the sensitiza-
tion phase (Day Ϫ7 to Day Ϫ1). This therapy was
based on our previous findings that ZnPP is a potent
and fast inhibitor of HO-1 expression (Amersi et al,
1999; Katori et al, in press). For instance, in a model of
68
Laboratory Investigation • January 2002 • Volume 82 • Number 1

PSGL-1–Mediated Interactions in Heart Transplants
Coito AJ, Binder J, Brown LF, de Sousa M, Van de Water L,
and Kupiec-Weglinski JW (1995). Anti-TNF-alpha treatment
down-regulates the expression of fibronectin and decreases
cellular infiltration of cardiac allografts in rats. J Immunol
154:2949–2958.
Intragraft HO-1 and Bag-1 Protein Expression
Proteins extracted from cardiac samples (60 ␮g/sam-
ple) were subjected to 12% SDS-PAGE and trans-
ferred to nitrocellulose membranes (Bio-Rad Labora-
tories, Hercules, California), as described (Ke et al,
2000). Gels were then stained with Coomassie blue to
document equal protein loading. Membranes were
blocked (Ͼ2 hours) in 5% defatted dry milk/PBS/0.1%
Tween-20 and incubated with anti-HO-1 (Sangstat,
San Francisco, California), anti-Bag-1 (C-16), anti-
caspase-3 (H-277), or the control anti-actin (I-19)
(Santa Cruz Biotechnology, Santa Cruz, California)
antibodies. After 1 hour of incubation with the primary
Ab, blots were washed three times (PBS/0.1%
Tween), and the secondary, peroxidase-conjugated
Ab (Pierce, Rockford, Illinois) was added for another
hour. Finally, the blots were washed three times (PBS/
0.1% Tween) and immunoreactive proteins were visu-
alized using Western blot chemiluminescent detection
(Amersham Pharmacia Biotech, Piscataway, New Jer-
sey). Relative quantities of proteins were determined
using a densitometer (Kodak Digital Science 1D Anal-
ysis Software, Rochester, New York).
Coito AJ, de Sousa M, and Kupiec-Weglinski JW (1994). The
role of cellular and extracellular matrix adhesion proteins in
organ transplantation. Cell Adhes Commun 2:249–255
Coito AJ, Onodera K, Kato H, Busuttil RW, and Kupiec-
Weglinski JW (2000). Fibronectin-mononuclear cell interac-
tions regulate type 1 helper T cell cytokine network in tolerant
transplant recipients. Am J Pathol 157:1207–1218.
Dulkanchainun TS, Goss JA, Imagawa DK, Shaw GD,
Anselmo DM, Kaldas F, Wang T, Zhao D, Busuttil AA, Kato H,
Murray NG, Kupiec-Weglinski JW, and Busuttil RW (1998).
Reduction of hepatic ischemia/reperfusion injury by a soluble
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Frenette PS, Mayadas TN, Rayburn H, Hynes RO, and
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Fuhlbrigge RC, Kieffer JD, Armerding D, and Kupper TS
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Intragraft Apoptosis
Gold R, Schmied M, Giegerich G, Breitschopf H, Hartung HP,
Toyka KV, and Lassmann H (1994). Differentiation between
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out on 5-␮m cryostat sections using the TACS 2
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