Use of C4d as a Diagnostic Adjunct in Lung Allograft Biopsies

Copyright
American Journal of Transplantation 2003; 3: 1143–1154
Blackwell Munksgaard
#
Blackwell Munksgaard 2003
ISSN 1600-6135
Use of C4d as a Diagnostic Adjunct in Lung Allograft
Biopsies
Cynthia M. Magroa, Amy Pope Harmanc, Dana
Klingerb, Charles Oroszd, Patrick Adamsd,
James Waldmana, Deborah Knighta, Moira
Kelseyb and Patrick Ross Jr.b,*
a
Department of Pathology, bDepartment of Cardiothoracic
Surgery, cDepartment of Medicine, dClinical Histocompatibility Laboratory, Department of Surgery, The Ohio State
University, Columbus, OH, USA
*Corresponding author: Patrick Ross Jr., ross-3@medctr.
osu.edu
Purpose: Humoral allograft rejection is a defined
mechanism for cardiac and renal graft dysfunction;
C4d deposition, a stable component of complement
activation, inversely correlates with graft survival.
With the recent recognition of humoral rejection in
lung grafts, we examined C4d’s role as a prognostic
adjunct in lung allografts. Material and Methods:
Twenty-three lung recipients underwent biopsies for
deterioration in clinical status or routine surveillance.
Clinically unwell patients possessed acute rejection or
bronchiolitis obliterans syndrome (BOS). Biopsies
attributable to infection were excluded from the
study. In addition to routine light microscopy, an
attempt was made to correlate the clinical status
and morphologic findings with the pattern of C4d
deposition and also to compare these clinical and
morphologic parameters with the other assessed
immunoreactants. Panel reactive antibody testing
was also carried out at various points in their post
transplantation course whereby in 6 of the cases the
samples were procured at exactly the same time as
the tissue samples. Results: The patients were segregated into two groups: those patients with recurrent
acute rejection and those with BOS. In those patients
with symptomatic acute rejection, all biopsies
showed light microscopic and immunofluorescent
evidence of humoral allograft rejection. The level of
C4d was positively correlated with the degree of parenchymal injury, the hallmark being one of septal
capillary necrosis. In addition, high and intermediate
levels of C4d correlated with a clinical diagnosis of
acute rejection. C4d was the strongest predictor of
parenchymal injury and of the clinical status
(p < .0001) compared to other the immunoreactants
C1q, C5b-9 and immunoglobulin. There was no
specific correlation between C4d deposition and the
presence of acute cellular rejection. In those patients
fulfilling clinical criteria of BOS, deposits of C4d as
well as other immunoreactants were found in the
bronchial wall as opposed to the rarity of this finding
in bon-BOS patients. However the only statistically
significant predictor of BOS was bronchial wall
deposition of C1q. In no case were panel reactive
antibodies at significant levels discovered post
transplantation. Conclusions: In the context of acute
rejection, C4d deposition correlates with clinical evidence of rejection and the degree of humoral rejection
assessed pathologically; there is no association with
the presence of histocompatibility related antibodies.
It is a more specific predictor of allograft status compared to other immunoreactants. C4d deposition
within the bronchial wall is a feature of BOS and
hence may be used as a marker of chronic graft
dysfunction. The antigenic target resulting in C4d
deposition may not be histocompatibility related.
Key words: Antiendothelial cell antibody, C4d deposition, humoral allograft rejection, lung transplantation
Abbreviations: BOS, bronchiolitis obliterans syndrome;
MAC, membranolytic attack complex.
Received 12 February 2003, revised 22 January and 19
February 2003, and accepted for publication 4 March
2003
Introduction
Lung transplantation constitutes an important therapeutic
option in patients with endstage lung disease. Nevertheless,
the 5-year survival for lung transplantation is significantly
less compared with other solid organ transplants (1). The
rate-limiting factor is chronic graft failure, the hallmark of
which is progressive scarring of the bronchial tree for
which the designation of bronchiolitis obliterans syndrome
(BOS) is used (1,2). The commonly regarded pathway in
acute and chronic lung allograft dysfunction has always
been espoused to be one of type IV immunity to donor
alloantigens (2). This is at variance with other solid organ
transplants, namely the kidney and heart. Although at one
time complement-binding recipient antibodies were held to
play a dominant role in the context of hyperacute rejection, it
is now becoming increasingly apparent antidonor humoral
responses may play a role in other forms of cardiac and renal
allograft rejection (3–5). In the context of lung transplantation, until recently the concept of humoral allograft rejection,
was largely limited to acute lung graft dysfunction within
hours following transplantation. There was a specific correlation between the presence of preformed donor-specific
antibodies as detected by flow cytometry and the development of hyperacute rejection within hours following transplantation (6). Recently a post transplant septal injury
proffered to represent humoral allograft rejection has been
1143
Magro et al.
described in the lung (7). The defining hallmarks of the lung
humoral allograft syndrome are clinical features to suggest
rejection/graft dysfunction in concert with septal capillary
injury accompanied by significant immunoglobulin, C1q,
C4d, and/or MAC deposition in the septal capillaries. C4d, a
stable component of classic complement activation and
representing a degradation product of the complement factor C4, is a marker of humoral allograft rejection. It has been
specifically correlated with cardiac and renal graft dysfunction, be it in the context of clinical rejection, elevated panel
reactive antibodies, long-term poor graft survival, and/or histologic evidence of humoral allograft rejection in the kidney
(8–13) and heart (13). To date, we are only aware of our earlier
study, which describes C4d deposition in lung allografted
tissue (7). In addition, despite substantial literature on C4d
deposition in the heart and kidney and its correlation with graft
outcome, there are no reports addressing C4d expression in
the context of being a significant prognostic determinant in
lung allograft function. This is in the context of either
morphologic evidence of parenchymal injury and or clinical
features indicative of rejection. The purpose of our study was
to define the significance of C4d deposition as a correlative
diagnostic adjunct in lung graft dysfunction.
Materials and Methods
The patient population was defined by 23 recipients of unilateral lung transplants who underwent transbronchial biopsies prompted by either a deterioration in their clinical status or who were well clinically and in whom the biopsy
was part of routine surveillance. Biopsies in which an infectious-based etiology was likely were excluded as part of the analysis. An attempt was made to
correlate the clinical status and morphologic findings with the pattern of C4d
deposition and also to compare these clinical and morphologic parameters
with the other assessed immunoreactants. Patients were categorized into
those manifesting acute rejection, BOS (i.e. chronic graft dysfunction), and/or
who were clinically well. Features of acute rejection included dyspnea, cough,
fever, and chest roentgenogram showing infiltrates and/or effusions and
decreased in forced expiratory volume (FEV1). The revised scoring system
for BOS was used to determine which patients fulfilled the clinical criteria to
warrant the designation of BOS. According to this new system, a FEV1 of
66% to 80% of the baseline value was categorized as stage I BOS (BOS 1),
51% to 65% as stage 2 (BOS 2), and 50% or less as stage 3 (BOS 3). Also, a
potential BOS stage (BOS 0-p) was assigned to those cases showing a 10%
to 19% decrease in FEV1 and/or a 25% decrease in FEF2575.
Assessment of alloantibodies through flow cytometric alloantibody
detection
As outlined by Pelletier et al. (7), a commercially available pool of microparticle beads coated with various purified major histocompatibility complex (MHC) antigens of known specificity were used according to
manufacturer’s instructions (FlowPRA, OneLambda, Canoga Park, CA). A
total of 16 patients were examined. In six of the cases, the samples were
obtained simultaneously with the biopsy, while in the remaining 10 cases
the serum samples were obtained 1–3 months after the procurement of
the biopsy. Briefly, 20 mL of recipient sera was incubated with 5 mL of MHC
class I plus 5 mL of MHC class II microparticle beads for 30 min at room
temperature (RT). The beads were washed twice with buffer and centrifuged at 10000 r.p.m. for 2 min. The beads were resuspended in 100 mL of
solution containing FITC-conjugated goat antihuman IgG and incubated for
1144
30 min at RT. The wash step was repeated and the beads were resuspended in 500 mL of wash buffer. Negative control serum using pooled sera
from nontransfused males was similarly prepared. Samples were read with
the aid of a Beckman Coulter XL2 flow cytometer. The fluorescence profile
obtained with negative control sera was used as the baseline fluorescence.
Major histocompatibility complex class I and class II beads were readily
distinguishable, as they are fluorescent (excited at 488 nm and maximum
emission at 580 nm) and have unique emission spectra. The positive/negative cutoff was empirically determined for each assay by setting a histogram region that excluded 98% of the peak obtained with the negative
control serum. The median channel associated with this cut-off point was
recorded for each assay. A test was deemed positive for alloantibody if a
distinct peak was noted or if there was a shift to the right in bead fluorescence of 6% to the right of the cut-off point.
Light microscopic evaluation
Routine light microscopic studies were performed on a total of 52 biopsies.
The biopsies were assessed for foci of frank septal capillary necrosis, and a
grading score was assigned as follows: 0 (no discernible capillary necrosis),
5–15%: mild (grade I), 20–30%, moderate (grade II), and > 30% severe
(grade III). The demonstration of this light microscopic finding in the absence
of discernible infection and of a reperfusion ischemia injury was considered
morphologic evidence of humoral allograft rejection whereby the extent of
necrosis determined the severity of this type of rejection. The presence or
absence of intra-alveolar fibrin (present or absent), hemosiderin (present or
absent), septal neutrophilia, septal fibroplasia, concomitant acute cellular
rejection, and chronic cellular rejection was also determined. In addition
bronchial wall changes observed with BOS were evaluated such as epithelial
thinning, bronchial wall fibrosis, and vasculitic changes of the bronchial
wall.
Direct immunofluorescent analysis
One piece of tissue was frozen and cryostat sections of the tissue samples
were examined for a conventional battery of antibodies directed against
C3, and C1q and immunoglobulin (IgG, IgM, and IgA; all rabbit antihuman
FITC-conjugated monoclonal antibodies were from DAKO A/S, Glostrup,
Denmark). In addition, the cases were assessed for C4d and C5b-9 deposition. The methodologies for the C4d and C5b-9 assay are described in
detail later (9,10). The grading of immunoreactant deposition is one based
on a subjective quantitative assessment of deposition intensity with the
four standard assigned grades being absent (0), mild (1), moderate (2), and
marked (3).
C4d assay: The tissue was cut at 2–4 mm in thickness and placed on
Fisher/Superfrost Plus slides; they were allowed to air dry for 30 min
followed by a rinse in phosphate buffered saline (PBS). Avidin D 150 mL
(100 g/mL in PBS/1% BSA) was applied as a preblock for 20 min followed
by a PBS rinse. Subsequently 150 mL (10 g/mL in PBS) of d-biotin preblock
for 20 min was applied to the slide followed again by a PBS rinse. The
monoclonal antibody to C4d (clone 10–11) at a dilution of 1 : 100 (10 g/mL)
was applied for 30 min followed by a PBS wash for 2–3 min. An antimouse
IgG (H & L) diluted to a concentration of 1 : 100 was applied for 30 min
followed again by a PBS wash. FITC-streptavidin 1 : 50 for 30 min followed
by a 2–3 min of PBS.
C5b-9 assay: Mouse anti-C5b-9 (DAKO #M777) diluted at 1 : 25 in PBS
was applied to cryostat sections for 45 min and then washed twice in PBS
for 5 min, following which a fluorescein conjugated antimouse immunoglobulin (DAKO F232) diluted in PBS at 1 : 25 (DAKO) was applied and incubated for 30 min. Two additional 5-min PBS washes were performed.
American Journal of Transplantation 2003; 3: 1143–1154
C4d in Lung Allograft Biopsies
Table 1: Patients with recurrent acute rejection
Date of
birth
Date of
biopsy
Date of
transplant
Type of
transplant
Reason
for transplant
1
08-09-46
05-01-02
06-04-02
10-08-02
03-27-02
Single
Emphysema
65
99
225
2
04-11-42
07-23-02
08-27-02
10-22-02
07-01-02
Single
Emphysema
22
57
113
3
02-26-61
11-19-02
07-02-02
Single
Emphysema/alpha 1
4
08-25-49
09-12-02
07-21-01
Single
IPF
5
06-09-37
03-26-02
04-15-99
Single
Emphysema
1075
6
01-12-41
02-19-02
03-25-02
05-21-02
01-11-02
Single
Emphysema
39
73
130
X
X
7
07-16-47
02-12-02
03-12-02
04-24-02
09-16-02
10-01-02
01-05-02
Single
Emphysema
38
66
109
254
269
X
02-19-02
07-10-02
10-01-02
06-12-01
252
393
476
X
02-14-02
06-28-02
07-10-02
07-25-01
204
338
350
X
04-19-02
05-21-02
08-23-02
03-18-02
32
64
158
X
8
9
10
06-27-35
03-27-40
06-11-39
Single
Single
Single
Emphysema
Emphysema
Emphysema
Days post
transplant
Acute
rejection
No
rejection
X
X
X
X
X
X
29
X
418
X
X
X
X
X
X
X
X
X
X
X
X
X
11
07-15-35
01-29-02
04-23-02
10-28-02
10-19-01
Single
Emphysema
102
186
374
X
X
X
12
08-22-38
07-23-02
06-08-02
Single
Emphysema
45
13
07-21-35
10-08-02
08-11-02
Single
Emphysema
58
X
14
09-07-72
03-19-02
07-01-02
02-23-01
Single
IPF
330
420
X
X
15
01-15-41
01-15-02
09-07-00
Single
IPF
495
X
16
10-19-51
09-19-02
07-01-02
Single
IPF
80
X
X
IPF ¼ idiopathic pulmonary fibrosis, alpha 1 ¼ alpha-1 antitrypsin.
Scoring of C4d deposition
A correlation was sought between the degree of C4d deposition and the
clinical status and degree of graft dysfunction as determined by morphologic assessment be it in the context of acute cellular rejection, humoral
allograft rejection, and or morphologic features of BOS.
The percentage of terminal lung parenchyma (i.e. interalveolar septae)
manifesting C4d deposition was assessed. There were three categories:
1, low when less than 30% of the sample showed some deposition; and 2,
intermediate when 30–50% of the sample showed deposition and high
when greater than 50% of the lung parenchyma exhibited deposition. The
staining intensity was graded as 0/3, 1/3, 2/3, and 3/3.
reactant deposition (i.e. C4d, C1q, C5b-9, IgG, IgM, IgA) and the degree of
humoral allograft rejection. By grouping the moderate and severe deposition
as a predictor of the clinical status of the patient, we estimated it as a risk with
Odds ratios. The confidence interval for the Odds ratio was set at 95%.
To assess the presence and type of immunoreactant deposition within the
bronchial wall as a potential predictor of BOS, a chi-square analysis with
estimates of Odds ratios and a 95% confidence interval was used. Multiple
logistic regression modeling was also carried out to find a combination of
independent factors/predictors.
Results (see Tables 1–4)
Statistical analysis
For non-BOS patients with recurrent acute rejection, a Spearman rank correlation was used to assess the correlation between the degree of immuno-
American Journal of Transplantation 2003; 3: 1143–1154
Sixteen patients had recurrent episodes of acute rejection
with interposed periods of being clinically well, while
1145
1146
73
130
02-19-02 01-11-02
03-25-02
05-21-02
6
9
8
7
39
03-26-02 04-15-99
5
204
338
350
02-14-02 07-25-01
06-28-02
07-10-02
393
476
269
10-01-02
07-10-02
254
10-01-02
109
04-24-02
09-16-02
252
66
03-12-02
02-19-02 06-12-01
38
02-12-02 01-05-02
1075
418
09-12-02 07-21-01
4
445
05-07-02 02-16-01
57
113
08-27-02
10-22-02
10-08-02
22
99
225
06-04-02
07-23-02 07-01-02
65
0
0
0
0
0
2/3(20sc)
0
0
0
0
0
2/3bmz
3bmz
1(>50%sc)
transplant transplant Low C4d Int C4d
05-01-02 03-27-02
biopsy
3
2
1
Days Post
Date of
Date of
IgG
0
0
0
0
2/3(<5sc)
2/3(40sc)
3/3(50sc)
3/3(90sc)
0
3/3(90sc)
2/3(90sc)
3/390sc
<5sc
<5sc
2/3(50sc)
0
0
2/3bmv
0
3/3(<5sc)
0
3/3 > 50sc 1/3(<5sc)
3(90sc)
3/3(90sc)
3/3(50sc)
0
3/3bmz
2(70sc;be) 2/3(<5sc)
2(90sc)
High C4d
IgA
2/3(<5sc)
2/3(15sc)
2/3(20sc)
0
1/3(15sc)
2/3(<5sc)
C1q
2/3(50sc)
0
2/3(30sc;2ch)
2/3(20sc)
1/3(60sc)
2/3(90sc)
C3
2/3(30sc)
0
0
nd
0
1/3(<5sc)
0
1/3(<5sc)
0
0
0
1/3(10sc)
0
2/3 < 5sc
3/3 90sc
0
3/3 < 10sc
2/3(30sc)
0
2/3(30sc)
0
0
nd
0
2/3(50sc)
0
1/3(<5sc)
1/3(<10sc)
0
2/3(<5sc)
3/3(<5sc)
3/3(<10sc)
3/3(<15sc)
0
<5sc
2/3(20sc)
0
0
ch
2/3 ch
1/3(10sc)
1/3(20sc)
1/3(>50sc)
1/3(>50sc)
0
2/3(<5sc)
3/3(90sc)
1/3(<5sc)
2/3(70sc)
0
2/3(20sc)
2/3(50sc)
0
0
C5b-9
1/3(<5sc)
2/3(60sc)
2/3(30sc)
2/3(20sc)
2/3(<5sc)
3/3(<5sc)
2/3(<5sc)
2/3(<5sc)
3/3(90sc)
0
0
0
0
1/3(10sc)
2/3(>50sc)
2/3(>50sc)
2/3(20sc)
2/3bmz
2/3(90sc)
0
1/3(<10sc) 0
2/3(<5sc)
moderate HR
no HR;grade a2b
neg HR; A2a; bronchial wall vasculitis
moderate HR
neg
moderate HR; A1d
grade of cellular rejection
Humoral rejection;
0
0
(mild HR)
mild HR
severe HR
no HR; grade A2b
mild HR
severe HR
changes suggestive of BOS
mild HR; A2-3b
mild HR; A2b
residual mild HR
severe HR
mild HR; A2c
mild HR; A2d
moderate HR; A2a
moderate HR
neg HR
0
0
0
0
0
4
nd
1
0
0
0
12
2
nd
5
0
0
CL. I PRA CL. II PRA
% Reactive antibody
3/3bmz;2/3(<5sc) neg HR; changes suggestive of early BOS 5
0%
2/3(<5sc)
2/3ch;2/3bmz
2/3(50)
2/3(<5)
nd
3/3(<10sc) 3/3(90sc)
1/3(<5sc)
1/3(<5sc)
2/3(10sc)
2/3(20sc)
0
1/3ch/1/3 < 5sc 3/3bmz;2/3(40sc) 1/3 ch;2/3(<5sc) 2/3bmv
0
0
2/3(<20sc)
2/3(<5sc)
2/3(15sc)
2/3(30sc)
IgM
Table 2: C4d as a prognostic variable in patients with acute rejection
Magro et al.
American Journal of Transplantation 2003; 3: 1143–1154
American Journal of Transplantation 2003; 3: 1143–1154
330
14 03-19-02 02-23-01
80
16 09-19-02 07-01-02
0
+1/3 (<5)
0
30
2/3(90sc)
3/3(>90sc)
0
0
0
0
0
0
<5sc
2/3(<5sc)
3/3(90sc)
3/3(40sc)
2/3(20sc)
0
0
0
3/3(40sc)
0
2/3(<5sc)
<5sc
2/3(<15sc)
3/3(30sc)
0
2/3(<5sc)
2/3bmz
2/3(30sc)
1/3(<5sc)
0
0
0
2/3(>90sc)
3/3(30sc)
0
2/3(20sc)
2/3(<5sc)
2/3(20sc)
2/3(50sc)
3/3(10sc)
0
2/3(50sc)
0
0
2/3(<5sc)
nd
3/3(40sc)
3/3(30sc)
0
0
0
1/3(<5sc)
1/3(50sc)
0
2/3(<5sc)
2/3(20sc)
3/3(40sc)
3/3(90sc)
2/3(50sc)
2/3(90sc);2bmz
3/3(90sc)
3/3(90sc)
3/3(90C5b-9)
1/2(<20sc)
0
1/3(>90sc) 0
0
(mild HR; A3b)
neg
neg
(severe HR; A1d/3)
(moderate HR)
moderate HR
neg
neg; neg
(severe HR; A1a)
moderate HR
mild HR
neg
0
0
0
4
4
0
1
1
0
1
0
0
0
1
neg ¼ negative, % ¼ percentage of lung parenchyma showing staining, pos ¼ positive, SC ¼ septal capillary, H ¼ homogeneous, CH ¼ chondrocytes, SCN ¼ septal capillary necrosis, BE ¼ bronchial epithelium, PRA ¼ panel reactive antibodies.
BOS ¼ bronchiolitis obliterans syndrome, HR ¼ humoral rejection, int ¼ intermediate, BMZ ¼ basement membrane zone, bmv ¼ basement membrane vessels, nd ¼ not done, BW ¼ bronchial wall, hemos ¼ hemosiderin, CL. ¼ class,
495
15 01-15-02 09-07-00
420
58
13 10-08-02 08-11-02
07-01-02
45
0
0
0
374
3/3(40sc)
3/3(30sc)
3/3 h linear 0
3/3 90(sc) 0
3/3(40sc)
2/3(90sc)
186
0
0
158
102
1/3(<5sc)
32
64
12 07-23-02 06-08-02
10-28-02
04-23-02
11 01-29-02 10-19-01
08-23-02
05-21-02
10 04-19-02 03-18-02
C4d in Lung Allograft Biopsies
1147
Magro et al.
Table 3: Clinical features of BOS Patients
Current Transplant Type of
Transplant
Sex age
date
transplant indication
1 M
67
10-30-99
Single
2 F
66
04-16-99
Single
3 M
42
02-16-01
Single
4 M
5 M
6 M
7 M
63
48
66
60
10-30-99
11-08-99
10-05-01
11-08-99
Single
Single
Single
Single
Date of
Days Post
FEV1
FEF25–75 New BOS
pulmonary studies transplant FEV1 % baseline % baseline recommend
Emphysema 01-14-00
02-29-00
03-20-00
02-28-02
Emphysema 10-22-99
05-21-02
Emphysema 03-29-01
05-08-01
09-14-01
10-09-01
01-02-02
05-06-02
Emphysema 09-29-98
09-29-98
04-12-99
06-20-00
05-16-02
Emphysema 04-11-00
05-30-00
02-19-02
Emphysema 12-21-01
02-05-02
03-05-02
04-08-02
Emphysema 02-16-00
10-08-01
11-15-01
12-06-01
01-15-02
05-07-02
76
36585
36605
852
189
1131
41
81
210
235
320
444
234
929
155
204
834
77
37292
37320
185
100
700
738
759
799
911
1.32
1.75
1.57
1.12
1.39
0.85
1.51
1.48
1.43
1.57
1.87
1.72
0.48
0.53
0.5
1.88
1.12
0.77
0.90
0.81
1.68
1.93
1.61
1.61
1.19
0.57
0.61
0.65
0.52
0.54
base
89.71
64.00
base
68.25
base
92.31
base
28.57
base
46.43
base
80.65
64.52
48.39
100.00
80.65
base
59.57
base
58.33
2
base
90.00
base
66.67
0-p
base
83.42
83.42
base
48.71
53.85
56.41
46.15
46.15
base
52.94
43.14
base
56.25
37.50
43.75
18.75
37.50
2
2
0
0-p
0-p
3
2
2
3
3
base ¼ baseline, FEF25–75 ¼ mid expiratory flow rate, FEV1 ¼ forced expiratory flow volume.
seven patients fulfilled the clinical criteria for BOS. Each
group is considered separately.
Non-BOS patients with recurrent acute rejection
(Table 1): Clinical features
This group was defined by 16 patients, representing six
women and 10 men, ranging in age from 29 to 68 years.
They developed evidence of acute rejection on one or
more occasions within 22–1075 days post transplantation.
With each episode of clinical deterioration, an infectiousbased etiology had been excluded both with cultures and
special stains on pathologic material. Further clinical
details of this group are provided in Table 1.
Pathological findings (Table 2)
All 16 patients had one or more biopsies manifesting
changes compatible with acute humoral allograft rejection.
The hallmark was one of septal capillary necrosis as characterized by septal and intra-alveolar fibrin deposition
accompanied by extensive hemorrhage. The severity of
the reaction was based on the degree of septal capillary
1148
necrosis (Figure 1). Five, eight and 10 biopsies showed
severe, moderate and mild humoral allograft rejection,
respectively. Twelve biopsies showed concomitant acute
cellular rejection. However in each patient there were
biopsies performed during periods of clinical quiescence
in which there were no remarkable features to suggest
humoral allograft rejection, representing a total of 10
biopsies.
Correlation of C4d with morphologic evidence of
humoral allograft rejection
Twelve of the biopsies exhibited high C4d deposition patterns within the septal capillaries. The staining intensity
was either moderate or marked (Figures 2 and 3). This
deposition pattern corresponded to moderate or severe
humoral allograft rejection as determined by the degree
of septal capillary necrosis in nine biopsies, while in three
the humoral allograft rejection was only mild. An intermediate/moderate degree of C4d deposition within the
interalveolar septae was observed in four cases and corresponded to moderate humoral allograft rejection in three
and a severe pattern in one. A mild degree of C4d within
American Journal of Transplantation 2003; 3: 1143–1154
C4d in Lung Allograft Biopsies
Figure 1: Light microscopic features of the lung humoral
allograft rejection phenomenon. There is fibrinoid necrosis of
the interalveolar septae (arrow 1) with associated intra-alveolar
fibrin deposition with admixed erythrocytes (arrow 2) compatible
with humoral allograft rejection.
the interalveolar septae was observed in three cases corresponding to no rejection in two and mild rejection in one.
In 14 cases there was no detectable C4d deposition and in
these cases the biopsies showed no humoral allograft
rejection in 10 cases, while in four cases the degree of
septal injury was mild. These patients however, had
other biopsies that showed significant deposits of C4d
and morphologic evidence of humoral allograft rejection
(Figures 4). Overall in 30 of the 33 cases the deposition of
C4d could be correlated to the degree of septal capillary
necrosis with higher deposition patterns corresponding
to moderate or severe degrees of septal capillary necrosis
and mild or absent deposition of C4d being found in
biopsies showing either minimal necrosis and/or no
necrosis.
Correlation of C4d with acute cellular rejection
Five of the 12 biopsies showing high C4d deposition
demonstrated perivascular and interstitial lymphocytic
infiltration consistent with acute cellular rejection. Seven
of the 17 biopsies showing either no or weak deposits of
C4d demonstrated acute cellular rejection. None of the
biopsies showing an intermediate pattern of C4d deposition within the septal capillaries demonstrated acute cellular rejection. There was no definite correlation between
the level of C4d deposition and the presence or absence
of acute cellular rejection.
Correlation of other immunoreactants with the degree of
humoral allograft rejection
There was no significant correlation between the degree
and extent of immunoglobulin deposition and the presence of humoral allograft rejection/septal capillary necrosis. With respect to C1q deposition a high deposition
American Journal of Transplantation 2003; 3: 1143–1154
Figures 2 and 3: C4d deposition as a marker of acute humoral
allograft rejection. There is granular C4d deposition within the
alveolar septae with capillary and endothelial cell localization (!).
Concomitant features of humoral allograft rejection along with
clinical evidence of rejection were observed.
pattern was observed in one case, and corresponded to
severe rejection in two cases, moderate rejection in four
cases, and mild or no rejection in five cases. A moderate
pattern of deposition was seen in one case and there was
no evidence of humoral allograft rejection. In 20 cases the
deposition of C1q was absent or weak, corresponding to
no rejection in nine cases, mild rejection in six cases,
moderate rejection in three cases, and severe rejection
in two cases. With respect to C5b-9 deposition in seven
cases the deposition pattern was weak or negative, corresponding to septal capillary necrosis/humoral allograft
rejection, which was absent in nine cases, weak in six
cases, moderate in one case, and severe in one case.
Moderate deposits were observed in four cases, corresponding with moderate humoral allograft rejection/
moderate septal necrosis in three cases and severe rejection in one case. A high pattern of deposition was
observed in nine cases and corresponded to moderate
1149
Magro et al.
Patients with BOS (Table 3): Clinical features
This patient population comprised six males and one
female, ranging in age from 48 to 67 years. All had undergone unilateral lung transplantation for emphysema, on
average, 2.5 years before the assessment of their biopsies
for C4d deposition. They had all experienced a progressive
decrement in their forced expiratory volumes and mid
forced expiratory flow rates, with BOS scores ranging
from 1 to 3 in five cases (see Table 3); their average
FEV1s ranged from 22 to 48% of predicted, while their
mid expiratory flow rates varied from 8 to 33%. They
developed the clinical syndrome of BOS within 2 years
of transplantation.
Figure 4: Morphologic and direct immunofluorescent C4d
differences between biopsies procured from a patient
during acute rejection versus a time when clinically well.
Patient 2 of the recurrent acute allograft rejection group
manifesting deposits of C4d day 113 post transplantation in
association with morphologic evidence of humoral allograft
rejection; the patient was clinically unwell. A biopsy performed
at day 57 following transplantation did not demonstrate C4d
deposition and neither was there morphologic evidence of
rejection; the patient had no clinical evidence of rejection.
rejection necrosis in three cases, weak rejection/mild
necrosis in two cases, and no rejection/necrosis in two
cases. There was no specific correlation between the
degree and type of immunoreactant deposited and the
presence or absence of cellular rejection. Regarding the
C3 deposition, in those cases showing a weak or negative
pattern of deposition, nine cases corresponded to weak
septal capillary necrosis/weak humoral allograft rejection,
three cases corresponded to moderate humoral allograft
rejection, and two cases corresponded to severe humoral
allograft rejection. A moderate degree of deposition was
seen corresponding to moderate septal capillary necrosis
in three cases and marked vascular injury in one case. A
marked degree of deposition seen in two cases, corresponding to mild humoral allograft rejection in one and
severe in the other.
Statistical analysis
With respect to those patients with recurrent acute rejection, the degree of C4d deposition was positively correlated with the degree of humoral allograft rejection
(p < 0.0001). A significant positive correlation was also
found with C1q (p ¼ 0.0053). There was no significant
correlation of the levels of C3, C5b-9, and immunoglobulin
with the degree of humoral allograft rejection. C4d was a
predictor of the clinical status of the patient. Specifically
intermediate and or marked degrees of C4d deposition in
the interalveolar septae was a predictor of rejection
(OR ¼ 17.3; p < 0.0001) with 76.92% sensitivity and 83.87%
specificity. Conversely, absent and/or minimal deposits of
C4d correlated with a state of clinical well being.
1150
Light microscopic and immunofluorescent findings of
patients with BOS including the pattern of C4d
deposition (Table 4)
All patients with BOS had light microscopic findings on
one or more biopsies suggestive of BOS. Specifically the
biopsies showed variable epithelial attenuation, bronchial
wall fibrosis, and neutrophilic infiltration accompanied by
vasculitic changes of the bronchial wall (Figure 5). Immunofluorescent studies revealed deposition of C4d along the
BMZ of the bronchial epithelium, as well as deposition of
C4d within the bronchial epithelium and or bronchial
mucosal vessels in one or more biopsies in all patients
excluding patient 3 in whom deposits of C4d were not
discernible (Figure 6). A similar deposition pattern was
observed for C5b-9 and C1q, IgM, and IgA. The deposition
pattern for C1q and C5b-9 exceeded that observed for
C4d, and in every case both immunoreactants demonstrated BMZ and/or bronchial epithelial localization. All
biopsies excluding one showed concomitant septal
Figure 5: Morphologic features of bronchiolitis obliterans
syndrome. There is prominent basement membrane zone
thickening of the bronchial epithelium (!). Also, the subjacent
mucosa appears fibrotic with vascular drop out. The morphologic
findings are most compatible with bronchiolitis obliterans syndrome.
American Journal of Transplantation 2003; 3: 1143–1154
American Journal of Transplantation 2003; 3: 1143–1154
215
05-07-02
880
3/3 bmz
0
0
<5sc
0-1 (<10)
0
1/3(<5sc)
0
2/3(30sc;be;bmz)
3 bmz;<5% sc
3be,3 bmv
2/3(50% SC);
IgG
IgM
2/3(<10sc)
2/3(90sc)
0
2/3(<5%sc)
IgA
2/3(90sc)
0
(single vessel)
2/3 bmv
2/3(10sc)
0
3 bmz
0
1/3(5sc)
1(<5%sc)
0
2/3(90sc)
2/3(90sc)
0
1/3(<10% sc)
2/3(90sc)
0
3/3(<5%)
2/3(<10sc)
2/3 < 5sc
2/3 grbmz
2/3(<10sc)
1/3(30sc)
C3
C1q
C5b-9
1/3(20%sc)
1/3(<5sc)
3/3 bmz
2/3(20sc)
1/3(<10sc)
2/3(be;bmz)
2/3(90sc)
3/3(30sc)
3/3(90sc)
2/3 bmz;be
3/3(30sc)
0
2/3(80%sc)
Chronic rejection
pos (moderate HR); BOS
0 cellular rejection
mild humoral allograft;
of BOS
mild HR; features suggestive
pos (moderate HR; BOS
pos (moderate HR)
moderate HR; 1c; BOS
mild HR
moderateHR; BOS
mild HR; BOS
pos (moderate HR)
(neg; A2d)
neg; BOS
moderate HR; A2c
moderate HR; A2b
bronchial wall sampled)
2/3(90%sc;bmz;bmv) pos (moderate HR); BOS
2/3(30%sc)
3/3(90sc)
3/3 bmz;3be
3/3(90sc)
3/3(90sc)
3/3 bmz;2/3 (15%sc) pos (chronic); BOS (only
2/3(90 sc);2bmv
1/3(<5sc)
2/3(30sc)
3/3(90sc);1/3bmz
2/3(40sc)
2/3(40%sc)
2/3(>90%)
2/3 (90 sc;be) 2/3(90 sc;bmv)
0
2/3(<5%sc;be) 1/3 sc end
1/3 < 5sc
0
2/3(90sc)
1/3(20sc)
2/3(30%sc)
2/3(90sc)
1-2/3 30sc
2/3(90sc)
1/3(90sc);2be
1/3(20sc)
0
nd
2/3 (50sc)
1/3(<5%sc)
3/3(<10% sc);be 2/3(<5%sc;be) 2/3(<5%;be)
0
2/3(10sc)
1/3gr bmz
1/3(<10sc)
1/3(<5sc)
2/3(<sc;be;bmz) 1/3(<5%sc)
2/3(30sc)
1-2/3(30sc)
2/3(<5sc)
3/3be(90sc) 2/3be,bmv,90sc 2/3be;bmv;90sc
nd
0
0
focal 3 bmz 0
0
3/3(60% sc) 0
1/3(50sc)
2/3(90)
3/3(50sc)
3/3(90sc)
+3 (bmz);
High C4d
Humoral/Cellular/
0
1
0
nd
0
0
0
0
1
4
6
nd
0
0
2
1
CL. I PRA CL. II PRA
% Reactive antibody
% ¼ percentage of lung parenchyma showing staining, bmv ¼ basement membrane vessels, end ¼ endothelium, PRA ¼ panel reactive antibody.
be ¼ bronchial epithelium, bmz ¼ basement membrane zone, BOS ¼ bronchiolitis obliterans syndrome, HR ¼ humoral rejection, int ¼ intermediate, nd ¼ not done, CL. ¼ Class, neg ¼ negative, pos ¼ positive, sc ¼ septal capillary necrosis,
05-07-02
768
179
04-01-02
7 01-15-02 12-08-99
152
124
6 02-05-02 10-04-01
03-05-02
920
803
620
10-29-02
4 05-07-02 10-29-99
550
5 02-19-02 12-08-99
445
08-20-02
333
1131
996
1087
937
05-07-02
3 01-15-02 02-16-01
05-21-02
Days Post
transplant transplant Low C4d Int C4d
Date of
2 01-06-02 04-15-99
10-22-02
1 05-25-02
biopsy
Date of
Table 4: IF and histologic assessment in patients with BOS
C4d in Lung Allograft Biopsies
1151
Magro et al.
Discussion
Figure 6: Direct immunofluorescent features of bronchiolitis
obliterans syndrome. There is significant deposition of C4d
along the basement membrane zone of the bronchial epithelium.
The patient has advanced BOS morphologically defined by
basement membrane zone thickening, epithelial atrophy and
fibrosis of subsegmental bronchial walls.
capillary necrosis accompanied by septal capillary deposition of complement and immunoglobulin, hence compatible with humoral allograft rejection.
Statistical analysis
The presence of deposition of C4d, immunoglobulin, C1q,
and C5b-9 within the bronchial epithelium, basement
membrane zone, and bronchial mucosal blood vessels
was assessed as a potential predictor of BOS. The deposition of C1q within the aforesaid sites of the bronchus was
the only independent predictor of BOS based on logistic
regression. The Odds ratios, 95% confidence intervals,
and p-values obtained with C4d were 4.85 (0.99, 23.7,
p ¼ 0.04), IgG 2.36 (0.30, 18.4, p ¼ 0.4), IgA 5.5 (0.89,
34.0, p ¼ 0.04), C5b-9 4.65 (1.09, 19.87, p ¼ 0.03), and
C1q 15.4 (1.63, 146.9, p ¼ 0.0038). While most of the
deposition of bronchial wall immunoreactants tend to be
more specific than sensitive, with specificity ranging from
88.6 to 100%, there was a trend to suggest that deposition within the bronchial wall was a marker of BOS.
Histocompatibility alloantibody status
As shown in Table 1, PRA testing demonstrated a remarkable absence of both MHC Class I and Class II circulating
alloantibodies in this patient population at all of the time
points tested during the post transplant period. Panel reactive antibodies (PRA) values were less than the 6% value
our program employs as an indicator of sensitization in all
cases with one exception. No MHC Class II specificity could
be assigned when the one sample that demonstrated Class
II sensitization (PRA ¼ 12%) was further tested for MHC
Class II specificity.
1152
We have shown a significant correlation between the
degree of C4d deposition with respect to both the clinical
evidence of acute rejection and the degree of humoral
allograft rejection assessed morphologically. Hallmarks of
the latter are defined by septal capillary injury, including
septal and intra-alveolar fibrin and hemorrhage. There was
no specific correlation between the findings of morphologic features of cellular rejection and the degree of C4d
deposition. Compared with the other immunoreactants
IgG, IgM, IgA, C3, C1q and C5b-9, the C4d deposition
pattern was a more superior predictor of recurrent acute
graft dysfunction, both in the context of the clinical features and based on the morphologic assessment whereby
the deposition pattern was primarily interalveolar septal
with capillary accentuation. In those patients with BOS
however, significant deposition of all immunoreactants
including C4d was noted in the bronchial wall; only C1q
was a statistically valid predictor in this respect.
There is considerable precedent in the literature regarding
the significance of C4d deposition as a prognostic and
diagnostic marker of humoral rejection in the kidney and
heart. Although earlier studies emphasized its importance
as a positive correlate to a state of acute rejection, recent
studies outline its value as a predictor of long-term graft
survival (8–13). For instance, in one multivariate analysis
study, C4d positivity was observed to be an independent
predictor for inferior 12-month renal graft survival (8). In a
second study, acute humoral rejection was recognized in
45% of biopsies showing C4d positivity, while acute cellular rejection unaccompanied by acute humoral rejection
was seen in 15% of cases. The cardinal distinguishing
features separating the C4d+ cases from the C4d– cases
included neutrophils in peritubular capillaries, neutrophilic
glomerulitis, and neutrophilic tubulitis. It was further discovered that the 1-year graft failure rate was significantly
higher in those CD4d+ cases in which there was fibrinoid
necrosis of vessel walls compared with those cases in
which there were just neutrophils in the vascular lumens
unaccompanied by C4d deposition (5). They concluded
that a distinct and common type of acute renal allograft
rejection is mediated by specific antidonor antibodies that
react with graft endothelium, leading to the deposition of
C4d. A third study correlated C4d deposits in peritubular
capillaries with chronic allograft dysfunction specifically in
the context of being associated with chronic transplant
glomerulopathy and basement membrane multilayering
and mononuclear cell infiltration (14). A further study
found that those kidney grafts demonstrating capillary
deposition of C4d had a markedly shorter survival, and
there was a correlation of C4d expression and the presence of panel reactive antibodies as well (15). In the
kidney and heart, the correlation between C4d and longterm graft survival emphasizes the critical role for humoral
immunity in contributing to allograft dysfunction. The
American Journal of Transplantation 2003; 3: 1143–1154
C4d in Lung Allograft Biopsies
question arises as to why C4d deposition is a reliable marker
of organ graft tissue injury and ultimately a predictor of
graft survival.
According to our work, the brunt of injury in the lung
humoral allograft rejection syndrome is directed at pulmonary septal capillary endothelium (7). With respect to
the patients with chronic graft dysfunction and BOS,
potential antigenic targets include the bronchial epithelium, chondrocyte, and mucosal microvasculature. The
immunofluorescent profile, being one of the significant
deposits within the interalveolar septae and the bronchial
epithelium, chondrocytes, and blood vessels, is corroborative of this hypothesis. In humoral allograft rejection of the
heart and kidney, a microvascular target has been proposed (3,4,13,16). Antibody binding to a cellular target,
which in the context of humoral allograft rejection is
donor endothelium, induces the assembly of C1q
complexes, resulting in the cleavage of C4 and C2 to
generate C4b. C4b then binds to nearby proteins on
endothelial cell surfaces and associates with C2a to form
C3 convertase; designated C4b2a (17). Formation of the
C3 convertase on the surface of the target cell amplifies
further activation of the complement cascade sequence.
Specifically, C4b2a activates C5 to C5b, which then binds
C6, C7, C8, and C9 to form the membranolytic attack
complex C5b-9; the effector mechanism of complementmediated cell lysis (18). Because complement activation
can damage cells via the formation of MAC, it is critical to
control C3 convertase by some regulatory mechanism.
There are certain plasma-based factors that will cleave
C4b into C4d; the latter being catalytically inactive (17–20),
representing the alpha 2 domain of C4. Immediately after
repair of the endothelial injury, C1q, C3, C4c, and MAC
disappear but C3d and C4d remain attached to the activated surface (20) via a proteolytically exposed thioester
group (10). Hence while the other components of the
complement cascade sequence are no longer bound
to the tissue, C4d does remain covalently attached
to endothelium, establishing it as a reliable marker of
activation of the classic complement cascade sequence
(20).
The presence of antibodies to MHC type I and type II has
been held to be a sign of humoral rejection based on the
emergence of positive anti-HLA profiles post transplantation and their association with clinical features of rejection
(21–25). We found no evidence of panel reactive antibodies to class I and class II antigens either prior to or
subsequent to transplantation. As shown in Table 1, PRA
testing demonstrated a remarkable absence of both MHC
Class I and Class II circulating alloantibodies in this patient
population at all of the time points tested during the post
transplant period. PRA values were less than the 6% value
our program employs as an indicator of sensitization in all
cases with one exception. No MHC Class II specificity
could be assigned when the one sample that demonstrated Class II sensitization (PRA ¼ 12%) was further
American Journal of Transplantation 2003; 3: 1143–1154
tested for MHC Class II specificity. This concords with
other studies where humoral allograft rejection and C4d
deposition may not be correlated with panel reactive antibodies (9), suggesting a role for non-HLA antigenic sites
(26).
We are currently trying to elucidate more fully the nature
of the antigenic targets involved in the lung humoral allograft phenomenon. As previously mentioned it is our belief
that antiendothelial cell antibodies may play an important
role (7). The concept of antiendothelial cell antibodies was
first introduced in the context of autoimmune disease,
including scleroderma, antiphospholipid antibody, Behçet’s disease, lupus erythematosus, and Henoch Schönlein purpura (27–35). With respect to antiendothelial cell
antibodies as an inciting trigger to graft dysfunction, one
study found a direct correlation between antiendothelial
cell antibodies with myocardial damage, suggesting that
the presence of these antibodies may predict the future
development of coronary artery disease in patients undergoing heart transplant (36–38).
In summary, this pilot study does suggest that C4d
deposition in the lung allograft may be a prognostic and
diagnostic adjunct comparable to that described in renal
and cardiac allografts. However, longer term follow up is
needed, and additionally, other centers need to assess the
role of C4d as a predictor of acute and chronic lung allograft dysfunction and ultimately lung allograft survival
before C4d expression will emerge as an accepted marker
of graft dysfunction.
Acknowledgment
We would like to thank Mary Chaeng from the Department of Biomedical
Statistics, University of Manitoba, for her statistical analysis of the data,
Aimee Sisinger and Lynn Lepper for outstanding technical, editorial and
adata assistance in the preparation of this manuscript and Mary Marin
from Core Histology Laboratory, Ohio State University for her superb
technical assistance in all of the immunofluorescent studies.
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