Transplantation

     Issue: Volume 62(10), 27 November 1996, pp 1441-1450
     Copyright: (C) Williams & Wilkins 1996.  All Rights Reserved.
     Publication Type: [Clinical Transplantation]
     ISSN: 0041-1337
     Accession: 00007890-199611270-00011    

 [Clinical Transplantation]

CYTOKINE PATTERN DURING REJECTION AND INFECTION AFTER LIVER TRANSPLANTATION-IMPROVEMENTS
IN POSTOPERATIVE MONITORING?

Platz, Klaus-Peter1; Mueller, Andrea R.2; Rossaint, Rolf3; Steinmuller, Thomas2;
Lemmens, H.-Peter2; Lobeck, Hartmut4; Neuhaus, Peter2

Author Information
Departments of Surgery, Anesthesiology and Pathology, Humboldt University of
Berlin, Virchow Klinikum, Berlin, Germany

1 Address correspondence to Dr. Klaus-Peter Platz, Department of Surgery,
Virchow Klinikum, Humboldt University of Berlin, Augustenburger Platz 1, 13353
Berlin, Germany.

2 Department of Surgery.

3 Department of Anesthesiology.

4 Department of Pathology.

Received 16 February 1996.

Accepted 23 July 1996.

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  Outline

    Abstract

    MATERIALS AND METHODS

    RESULTS

    DISCUSSION

    REFERENCES

Abstract

Despite improvements in immunosuppression, rejection occurs in 50% of liver
transplant patients and may cause significant morbidity. The most frequent cause
of death after liver transplantation is severe infection. Determination of the
cytokine network may lead to earlier detection of patients at risk for severe
rejection and infection. For this purpose, 81 patients with 85 liver transplants
were monitored for cytokines and neopterin on a daily basis. During the first
postoperative month, 28 patients (34.6%) developed acute rejection; 14 patients
were successfully treated with methylprednisolone (steroid-sensitive rejection),
while 14 patients required additional treatment with FK506 and OKT3 (steroid-resistant
rejection). Ten patients developed severe infections, and 11 patients experienced
asymptomatic cholangitis. Patients with an uneventful postoperative course
(n=37) were the control group. One-year patient survival was 88.9%: 1 patient
died because of chronic rejection and Pseudomonas urosepsis; a further 4
patients died of aspergillus pneumonia and bacterial sepsis. Soluble TNF-RII,
sIL-2R-, and IL-10 levels were significantly elevated 3 days prior to or at the
onset of acute steroid-resistant rejection(PP.

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In recent years liver transplantation has become a highly successful treatment
for endstage liver disease and fulminant acute liver failure. However, despite
improvements in immunosuppression, approximately 50% of liver transplanted
patients will develop one or more acute rejection episodes (1-5). While some
rejection episodes are mild and will be successfully treated with a short course
of methylprednisolone, others are more dangerous with regard to morbidity and
possible graft loss. Furthermore, overimmunosuppression-associated atypical
bacterial and fungal infections are one of the most frequent causes of death
after liver transplantation (6-10).

New parameters such as neopterin and cytokines may differentiate between mild
rejection and the risk of serious rejection after liver transplantation.
Furthermore, the different patterns of these parameters may differentiate
between acute rejection and infection. For this purpose, a prospective study was
conducted to measure neopterin and various cytokines daily after liver
transplantation, and it focused on the clinically most important parameters.
 MATERIALS AND METHODS

Patients. A total of 81 patients receiving 85 orthotopic liver transplants were
prospectively monitored for neopterin and cytokines between August 1993 and July
1994. Indications for liver transplantation included 5 patients with acute liver
failure due to fulminant hepatitis B (HBV)* and hepatitis C (HCV) infections or
intoxication, 23 with HBV and HCV-cirrhosis, 16 with alcoholic cirrhosis, 11
with primary biliary cirrhosis (PBC), 8 with cryptogenic cirrhosis, 4 with
autoimmune cirrhosis, and 15 with various other indications. Six patients who
were undergoing retransplantation were included: 1 initial nonfunction (INF), 1
refractory acute rejection, 1 chronic rejection, and 1 early graft failure
following rt-PA (recombinant tissue-type plasminogen activator) lysis therapy
and DIC (disseminated intravascular coagulopathy) due to lung artery embolism; 2
patients first entered the study at the time of retransplantation: 1 patient
with chronic rejection and 1 with HBV recurrence. The study was approved by the
Ethics Committee of the Humboldt University of Berlin and informed consent was
received from each patient prior to participation in the study. The surgical
procedure as well as antibiotic and other prophylaxes were performed perioperatively
as previously reported (2, 3).

Immunosuppression. Immunosuppression was commenced as quadruple therapy
including cyclosporine (CsA), azathioprine, prednisolone, and ALG(antilymphocyte
immunoglobulin, Merrieur, France; n=24) or IL-2 receptor antagonist BT563
(Biotest, Dreieich, Germany; n=47) for the first 7 to 12 postoperative days,
respectively, and continued as triple therapy thereafter (2); 12 patients
received no induction therapy (primary triple therapy). Two patients undergoing
retransplantation who had been previously converted to FK506 because of
steroid-resistant and chronic rejection received FK506 (Fujisawa, Osaka, Japan)
in conjunction with prednisolone.

Management of rejection. Diagnosis of acute rejection was based on clinical
(fever, change of color, and amount of bile production) and laboratory (AST,
ALT, bilirubin, [gamma]GT, and alkaline phosphatase) findings and was confirmed
by histological evaluation of graft biopsies. Liver biopsies were performed
routinely on POD 7 and whenever rejection was suspected. The histological
classification of acute rejection used was previously reported (3, 11, 12).
Patients received methylprednisolone for treatment of acute rejection at a
dosage of 500 mg/day for 3 days and FK506 (n=8) or a combination of FK506 and
OKT3 monoclonal antibody (n=6) (Cilag GmbH, Sulzbach, Germany) simultaneously
for steroid-resistant or severe recurrent rejection. Acute rejection was defined
as steroid-resistant by a missing response or second increase in liver enzymes
after one course of methylprednisolone in combination with histological signs of
ongoing rejection in repeated liver biopsies prior to initiation of rescue
therapy. Steroid recycles have never been used at our center-in more than 600
liver transplants. For treatment of early chronic rejection, high-dose FK506
rescue therapy was initiated.

Management of infection. CMV infection was diagnosed by routinely performed
polymerase chain reaction (PCR) techniques and treated with gancyclovir. If
pneumonia was suspected following clinical examination or chest oentgenogram,
diagnostic bronchoscopy was performed to confirm diagnosis and allow appropriate
antibiotic, antiviral, or antifungal treatment. Infections were defined as
serious when more than two secondary organ failures or dysfunctions-including
acute renal failure (ARF; serum creatinine >=3.5 mg/dl, or hemodialysis), acute
respiratory insufficiency (ARI; requirement of ventilatory support), severe
liver dysfunction, DIC, sepsis, or SIRS (systemic inflammatory response
syndrome), a requirement for cathecholamines, neurologic disorders, or acute or
chronic rejection-were present (13). Asymptomatic cholangitis was defined as
microbiological proof of bacteria in bile, increase in cholestatic enzymes(bilirubin,
[gamma]GT, alkaline phosphatase)- with or without histological signs of
cholestasis and cholangitis but without clinical signs of infection(fever,
leukocytosis, critical illness). Asymptomatic cholangitis was treated with
appropriate antibiotics according to test results.

Experimental monitoring. Experimental parameters were determined prior to and
after transplantation on a daily basis for the first postoperative month or
until complications were resolved. Heparinized blood was immediately placed on
ice and centrifuged at 4 [degrees]C for 10 min within 30 min of retrieval.
Plasma was stored at -70 [degrees]C until measured. Commercial immunoassays with
96-well microtiter plates were used in all cases: neopterin(ELItest neopterin,
B.R.A.H.M.S. Diagnostica, Berlin); IL-1[beta], IL-4, IL-6, IL-10, and IFN-[gamma]
(Biozol, Eching, Germany); and sIL-2R, sTNF-RII, and IL-8 (DPC Biermann, Bad
Nauheim, FRG). The normal ranges (n=45) of various plasma levels were: 6.2+/-0.3
nmol/L for neopterin; 0.0 pg/ml for IL-1[beta]; 0.2+/-0.1 pg/ml for IL-4;
28.8+/-0.9 pg/ml for IL-6; 4.7+/-0.3 pg/ml for IL-10, 2.1+/-0.8 pg/ml for IFN;
380+/-20.6 U/L for sIL-2R; 1,450+/-52.7 pg/ml for sTNF-RII; and 29.3+/-1.7 pg/ml
for IL-8.

Statistical analysis. Kaplan Meier estimates, Wilcoxon, chisquare and Kruskal-Wallis
tests and analysis of variance (one-way ANOVA and multivariate analysis) were
used as indicated. Linear regression analysis was used to determine if changes
of various parameters correlated with the incidence and severity of early
postoperative rejection or infection and if parameters correlated with the
ultimate outcome of infection. Multivariate analysis was also used to test for
the significant impact of individual parameters for rejection, infection, and
the differentiation of both adverse events. Results were expressed as means+/-SEM.
 RESULTS

Survival and incidence of rejection. Actuarial 1-month and 1-year patient and
graft survivals were 97.5% (79/81) and 88.9% (72/81) for patients and 94.1%
(80/85) and 84.7% (72/81) for grafts, respectively. During the first year after
transplantation, 9 patients died; 5 patients died of severe infection (one of
these patients additionally suffered from chronic rejection); 3 patients died of
HBV or HCV recurrence, and one patient died of tumor recurrence.

A total of 28 patients (34.7%) developed acute rejection during the first month
after transplantation; 14 were successfully treated with methylprednisolone
(steroid-sensitive rejection). In 14 cases rejection was steroid-resistant and
required treatment with FK506 alone (8 patients) or a combination of FK506 and
OKT3 simultaneously (6 patients)(Table 1). Eleven patients with steroid-resistant
rejection recovered completely, while 3 patients subsequently developed early
chronic rejection; 1 patient recovered under highdose FK506-rescue therapy, 1
patient was successfully retransplanted, and 1 patient died of chronic rejection
and Pseudomonas sepsis. Only one patient developed chronic rejection without
previous signs of acute rejection. The pattern of cytokines of the 14 patients
with steroid-resistant rejection was compared with the pattern of the 14
patients with steroid-sensitive rejection, and with the pattern of 37 patients
with an uneventful postoperative course (no rejection, no infection). Day 0 for
the control group was POD 7, because complications reported occurred during the
early postoperative period, and routine liver biopsy on POD 7 in conjunction
with clinical and laboratory investigations excluded acute rejection.

Incidence of infection. Ten patients developed serious infections according to
the criteria described in Materials and Methods; 5 subsequently died (Table 2).
Since cholangitis is the most liver-specific infection that also includes the
effect of cholestasis, 11 patients with asymptomatic cholangitis, but no sign of
concomitant acute allograft rejection (as confimed by routine liver biopsies)
and no more than 2 secondary organ dysfunctions, acted as the control group.

Routine laboratory investigations. At the onset of acute rejection, bilirubin
levels increased significantly in all patients. A persistent increase in
bilirubin levels for a duration of 14 days was observed in patients with acute
steroid-resistant rejection, while bilirubin levels in patients with steroid-sensitive
rejection subsequently normalized (Table 3). AST levels increased at onset of
acute rejection but remained elevated throughout the observation period of 2
weeks in patients with steroid-resistant rejection, while AST levels in patients
with steroid-sensitive rejection normalized within 1 week. A similar picture was
seen for ALT, with the exception of a slower rise and prolonged decrease in
patients with steroid-sensitive rejection (Table 3). Of various laboratory
investigations performed on a routine basis, slight differences were observed
for [gamma]GT and alkaline phosphatase levels between patients with steroid-resistant
versus steroid-sensitive rejection and patients with an uneventful postoperative
course (P=n.s.). Furthermore, a significant decrease in prothrombin time and
serum cholesterol occurred in patients with steroid-resistant rejection (P

During the entire infectious period, mean bilirubin levels were significantly
higher in patients with serious infections compared with patients with
asymptomatic cholangitis and patients with an uneventful postoperative course
(Table 3). Higher mean bilirubin levels were observed in patients who subsequently
died (15.9+/-1.7 mg/dl versus 11.6+/-2.2 mg/dl in surviving patients). Mean AST
levels increased during the late course of severe infection coinciding with
impaired graft function in patients with a fatal outcome (98.7+/-21.7 U/L versus
43.6+/-8.6 U/L in surviving patients). Mean ALT levels revealed no significant
differences between either group except for an increase during the late course
of infection in patients who subsequently died (97+/-42.3 U/L versus 63.4+/-16.5
U/L for surviving patients). A significant rise was also observed for mean CRP
(C-reactive protein) levels in patients with serious infections compared with
patients with cholangitis and with patients with an uneventful postoperative
course. No differences in laboratory investigations were observed in any of the
groups except for a significant decrease in prothrombin time and serum
cholesterol, and an increase in BUN and serum creatinine, because all patients
with serious infections suffered from ARF. Patients who died because of severe
infections developed a significant decrease in thrombocyte counts and prothrombin
time (P

Soluble IL-2R. The TH1 cytokine IL-2 and its receptors are well known to trigger
acute allograft rejection. As expected, sIL-2R levels significantly increased at
onset of acute rejection and were higher in patients with steroid-resistant
rejection (mean sIL-2R levels at days -3 -14 after onset of rejection:
3627+/-678 U/L; PPP

Soluble TNF-RII. A significant rise in mean sTNF-RII, which is released by
various cell types upon stimulation of TH1 lymphocytes, occurred in patients
with steroid-resistant rejection 3 days prior to onset of rejection (19,110+/-3921
pg/ml; P

Since TNF and its receptors are also well known as important mediators of
sepsis, as expected, a significant rise in mean sTNF-RII occurred in patients
with serious infections more than 3 days prior to onset of first symptoms of
infection (25,522+/-6744 pg/ml; PMaterials and Methods).

IL-10. The counter-regulatory TH2 cytokine IL-10, known to down-regulate TH1
lymphocyte function and IL-2-production, was expected to increase during the
late course of rejection. However, IL-10 levels significantly increased at onset
of acute rejection, simultaneously with the IL-2 increase, and mean IL-10 levels
were higher in patients with steroid-resistant rejection (173+/-25.1 pg/ml; P

IL-10 down-regulates also inflammatory cytokines such as IL-1[beta], IL-8, and
TNF-[alpha]-and mean levels of IL-10 were significantly elevated in patients
with serious infections (114+/-25.1 pg/ml; PP

IL-8. The inflammatory cytokine IL-8, which is released by various cell types,
increased slightly at the onset of rejection and significantly during the second
week in patients with steroid-resistant rejection (316+/-99 pg/ml; P

IL-8 levels increased significantly 3 days prior to onset of earliest symptoms
of infection in patients with severe infections (514+/-103 pg/ml; PP

Neopterin. Neopterin, an intermediate of tetrahydrobiopterin synthesis, is
produced by INF-[gamma]-activated macrophages. Neopterin levels increased at the
onset of rejection in patients experiencing steroid-resistant rejection
(71.6+/-9.8 nmol/L) and normalized within one week. No significant increase was
observed in patients with steroid-sensitive rejection and with an uneventful
postoperative course (55.5+/-17.1 nmol/L and 43.1+/-13.5 nmol/L, respectively)
(Fig. 5A). Neopterin levels increased in 9 of 14 patients with steroid-resistant
rejection but the rise in neopterin was significantly lower than that seen
during severe infection (P

Mean neopterin levels increased significantly in patients with serious
infections more than 3 days prior to the onset of the first symptoms of
infection (192+/-48.2 nmol/L; P

Other cytokines. IFN-[gamma], which is produced by IL-2 induced NK cells,
increased significantly during the second week of steroid-resistant rejection
and during lethal infection to a similar extent, and it remained within the
normal range in all control groups (Fig. 6; Table 4). Similar to IFN-[gamma],
the proinflammatory cytokine IL-1[beta] and the counter-regulatory cytokine IL-6
also increased during the late course of steroid-resistant rejection and lethal
infection, while the increase in IL-4 occurred in both events only in 50% of the
patients. Due to the late increase and the similar timing, the latter 4
cytokines failed to be early indicators for both adverse events, and failed to
differentiate between rejection and infection (Table 5).

Impact of various parameters on severity of rejection. Linear regression
analysis showed that the combined parameters correlated significantly with the
incidence and severity of rejection(uneventful-steroid-sensitive-steroid-resistant)
with an R2 of 53.9%(P2=33.9%; P2=23.1%; P2=16.9%; PPPP

Impact of various parameters on severity of infection. Linear regression
analysis showed that the combined parameters correlated significantly with
incidence and severity of infection(uneventful-cholangitis-serious infection)
with an R2 of 57.8%(P2=33.8%;P2=29.1%;P2=20.6%; P2=16.4%; P2=12.8%;P2=11.7%;
P2=10.4%; PPP

Impact of various parameters on fatal outcome. By linear regression analysis,
the combined parameters correlated significantly with the ultimate outcome of
infections (dead versus alive) with an R2 of 63.1%(P2=31.8%; P2=27.2%; P2=22.6%;
P2=17.1%;P2=14.9%; P2=12.8%; P2=12.1%;P2=10.4%;P

Differentiation between rejection and infection. At its onset as well as during
the entire period of rejection and infection, multivariate analysis revealed
significant differences in cytokine pattern between rejection and infection for
neopterin, IL-8 (PPP
 DISCUSSION

Improvements in organ preservation, surgical techniques and last but not least
in immunosuppression, have resulted in higher success rates in liver transplantation
with 1-year graft and patient survival figures of 90%(2-5). New approaches in
liver transplantation may now concentrate on patients at risk, including
patients with severe acute steroid-resistant rejection and serious infections.
Some early acute rejection episodes are mild and recover immediately after
methylprednisolone therapy. Severe acute rejection episodes, however, may cause
significant morbidity, or may result in graft failure due to hyperacute or
subsequent chronic rejection (14, 15). While histology is still the gold
standard for diagnosis of rejection (16), the initial severity of histological
changes failed to correlate with response or nonresponse to high-dose steroid
therapy-i.e., initial histological signs of rejection can be mild but will
progress despite high-dose steroid bolus therapy. In order to differentiate mild
from clinically severe rejection at the onset of acute rejection and to detect
patients at risk for severe or lethal infection, neopterin and various cytokines
were measured daily in 81 patients.

AST and ALT levels significantly increased with the onset of acute rejection,
and mean levels of both parameters correlated with severity of rejection.
However, differences in levels between steroid-resistant and steroid-sensitive
rejection were minor, and AST and ALT levels of individual patients may not
predict which type of rejection is present. Bilirubin levels were best
predictive of severity of rejection. Serum cholesterol and prothrombin time
decreased with impairment of graft function during the late course of severe
steroid-resistant rejection. Bilirubin and AST were also increased during severe
infection; bilirubin levels were higher during infection than rejection, while
AST levels were in a similar range and ALT levels were not increased during
infection. Levels of [gamma]GT and alkaline phosphatase failed to correlate with
either complication or to differentiate between the two events. Most significant
for severity of infection and indicative of lethal outcome were the decrease in
platelet count and prothrombin time, especially during the late course of lethal
infection.

Previous reports have shown an increase in IL-2 or sIL-2R levels in liver and
kidney allograft rejection (17-19). An increase in immunostimulatory TH1
cytokines was observed not only during rejection but also during infection.
There were no differences in the extent of increase in sIL-2R but there was a
difference in the timing. Soluble IL-2R increased rapidly 3-5-fold in all 14
patients with steroid-resistant rejection, while only a slight increase was
observed in patients with steroid-sensitive rejection. A more constant, slower
increase in sIL-2R was observed in patients with lethal infection. However,
markedly increased sIL-2R levels also occurred during the late course of
asymptomatic cholangitis in patients with no signs of rejection and no critical
illness. Furthermore, significant differences in sIL-2R levels also have to be
considered for different induction therapies. Soluble IL-2R levels were
significantly reduced by IL-2 receptor antagonist (BT563), but increased at the
onset of steroid-resistant rejection and after discontinuation. IL-2 induced NK
cells are probably the most important source of IFN-[gamma]-production (20).
Accordingly, the increase in IFN-[gamma] was observed one week after sIL-2R-increase.
Most patients with steroid-resistant rejection and high sIL-2R levels showed
this increase in IFN-[gamma].

Elevation of sIL-2R and IFN-[gamma] in patients with lethal infection may result
from general activation of the immune response. The current management of severe
infection commonly resulted in reduction of immunosuppression. However, our data
support the hypothesis that stimulation of the immune response and rejection may
very well occur during lethal infection. This complicates the dilemma of how to
treat patients with severe infection and signs of activated immune response and
makes specific inhibition of the respective cytokines or their receptors
desirable in the future.

In accordance with previous reports which have shown an increase in TNF-[alpha]
prior to acute rejection of human liver allografts (21), we observed a
significant rise of sTNF-RII in patients with steroid-resistant rejection prior
to onset of acute rejection. Soluble TNF-RII is not specific for rejection. A
variety of studies have shown an increase in proinflammatory and inflammatory
cytokines including IL-1[beta], TNF-[alpha], and IL-8 in nontransplanted
patients with serious infection, sepsis, and SIRS. In some studies, these
cytokines-and especially the counter-regulatory cytokine IL-6-correlated with
severity of disease and the ultimate outcome of infection (22-25). In response
to the activation of proinflammatory cytokines, sTNF-RII increased significantly
prior to onset of serious infection to an even greater extent than seen during
steroid-resistant rejection. A significant rise was observed in 4 of the 5
nonsurviving patients, indicating a positive correlation of sTNF-RII-increase
with severity of inflammation and also with ultimate outcome. Soluble TNF-RII
has been shown to correlate with TNF-[alpha]-production (26). Because of its
longer half-life, determination of sTNF-RII may more reliably reflect TNF-[alpha]-production
than the cytokine itself (27, 28). Soluble TNF-RII levels were generally higher
in all liver transplanted patients than in normal healthy individuals (see
Materials and Methods), but increased further during infection, and acute and
chronic rejection. However, differentiation between rejection and infection may
be difficult because of the similar timing in sTNF-RII increase during both
complications.

The counter-regulatory cytokine IL-10, which is produced by monocytes and CD4+
TH2 cells has been shown to down-regulate TH1 lymphocyte function and the
release of IL-2, IL-2 receptor and IFN-[gamma](29-34). Furthermore, IL-10 is
thought to have a strong anti-inflammatory effect by suppressing macrophage
activity, as well as the release of reactive oxygen intermediates and reactive
nitrogen intermediates (35-37). IL-10 has been also shown to down-regulate the
activity of proinflammatory cytokines, including TNF-[alpha], IL-1[beta], and
IL-8 (30, 34). IL-10 levels increased significantly with the onset of steroid-resistant
but not steroid-sensitive rejection. This may reflect the more prominent
activation of the immune response in the first-mentioned group of patients.
Similar to IL-10, IL-4 has been shown to down-regulate IL-1, IL-2 receptor and
IFN-[gamma]-production (38). However, in contrast to IL-10 which increased in
almost all patients with steroid-resistant rejection, IL-4 increased only in 50%
of patients with rejection irrespective the severity. While IL-4 in general
failed to correlate with severity of rejection, IL-10 only failed to increase in
2 patients: one patient subsequently developed chronic rejection and the other
patient subsequently suffered from HCV recurrence. A possible explanation may be
the disability of a protective immune response in these two patients.

The most important feature of IL-10 determination in the context of infection is
the marked increase prior to onset of earliest symptoms of infection exclusively
in patients with a lethal outcome. Despite high levels of IL-10 in all 5
patients who died, proinflammatory and immunostimulatory cytokines showed a
progressive rise during the late course of lethal infections. This is in
accordance with the view that an overwhelming immune response takes place in
this group of patients. An inflammatory and immunostimulatory effect generally
occurring after transplantation may result in significantly higher IL-10 levels
in all liver transplanted patients than in normal individuals, possibly in a
counter-regulatory manner.

The inflammatory cytokine IL-8 is released by various cell types, including the
vascular endothelium, neutrophils, macrophages, monocytes, Kupffer cells, and
fibroblasts in response to IL-1[beta] and TNF-[alpha]-stimulation. IL-8 has been
shown to have potent attraction for neutrophils adhering to the endothelial
surface, and to evoke infiltration of neutrophils, the release of enzymes, and
reactive oxygen intermediates, leading to ultimate tissue damage (25, 39).
Although there were baseline levels detectable in all patients after liver
transplantation that were higher than in normal individuals, a significant rise
of IL-8 was observed during the second week of steroid-resistant rejection. The
increase in IL-8 and IL-1[beta] indicates that severe inflammation takes place
relatively early(during the second week) of severe rejection. Furthermore, as
expected, IL-8 increased prior to onset of severe infection and a tremendous
increase in IL-8 levels occurred only in patients who subsequently died. All 5
non-surviving patients showed this increase in IL-8 as early as 3 days prior to
the onset of first symptoms of infection.

Other cytokines-including IFN-[gamma], IL-1[beta], IL-4, and IL-6- were also
increased during severe rejection and infection. However, these cytokines
increased after the onset of either event and were, therefore, late indicators,
which are of less clinical value in terms of early or specific monitors.
However, they may be of value when analyzing mechanisms of rejection and
infection or when developing new therapeutic strategies for treatment of
rejection and infection in terms of immunomodulation.

Neopterin has been shown to increase with stimulation and alteration of the
immune response caused by rejection, infection, and tumor diseases (40-42).
Neopterin production is mediated by IFN-[gamma]- and other cytokine-activated
macrophages (43). Indeed, we observed an increase in neopterin levels during the
early course of steroid-resistant rejection. However, this increase was much
lower than that seen in patients with severe infection. A marked increase
occurred only in 2 patients with steroid-resistant rejection and in patients who
developed chronic rejection. Of clinical interest is the observation that
neopterin increased in all patients with severe infection prior to the onset of
the earliest symptoms. The highest neopterin levels were observed in the
patients who died, while patients who recovered showed a decrease in neopterin
in relation to recovery of clinical signs of infection. Besides retransplantation
and chronic rejection, which also showed a rise in neopterin, neopterin was
highly specific for severe infections, which is in accordance with observations
by others (44).

Neopterin and sTNF-RII are eliminated by the kidneys, and acute renal failure
may result in an increase in circulating plasma levels of both parameters (41,
45). There was indeed a positive correlation of these two parameters with BUN
and serum creatinine. However, there was no increase in neopterin and sTNF-RII
levels in patients with acute renal failure related to CsA toxicity or origins
other than infection, indicating that the most prominent source of neopterin
increase during infection was an increased production.

By linear regression analysis, most cytokines and neopterin correlated weakly
but significantly with bilirubin levels (R2=26%;P

None of these parameters specifically indicated an acute rejection episode in
advance. However, when rejection was diagnosed there were several parameters
together that reliably indicated the necessity of augmented immunosuppression in
patients who will subsequently develop steroid-resistant rejection. This means
that a significant increase in sTNF-RII and IL-10 in combination with a rapid
rise in sIL-2R at the time of diagnosis of rejection should prompt us to
initiate immediate rescue therapy simultaneously with steroid therapy. By this
means, a delay in effective antirejection therapy may be avoided, and this may
result in decreased subsequent chronic rejection and graft loss. While histology
is still the gold standard for diagnosis of rejection (16), the initial severity
of histological changes failed to correlate with response or nonresponse to
steroid therapy.

A marked increase in IL-8 and neopterin in otherwise healthy transplanted
patients was highly indicative of subsequent development of severe infection,
and an intensified infectious screening should be initiated immediately. If
IL-10 was also markedly increased at the same time, antiinfectious therapy
should be given more aggressively, because these patients are at high risk for a
lethal outcome. Since all 3 parameters (neopterin, IL-8, and IL-10) increased as
early as 3 days prior to the first symptoms of infection, there may be a chance
for improved management by forcing an earlier diagnosis and giving more
aggressive therapy than would have indicated by conventional management.

If acute rejection and infection will occur simultaneously, all parameters may
be highly increased but none of these parameters is likely to be able to
differentiate between these complications. Further studies must be considered to
answer this question, since, at least in our hands, this is a rare event.
Although most cytokines and neopterin are not specific for any complication, if
used in combination, according to their timing and pattern, they may improve
currently established monitoring procedures after liver transplantation.
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