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The effect of long-term methotrexate therapy on hepatic fibrosis in rheumatoid arthritis.

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Number 12, December 1993, pp 1697-1701
Q 1993, American College of Rheumatology
Objective. To evaluate the progression of hepatic
fibrosis in patients with rheumatoid arthritis (RA)
treated with methotrexate (MTX).
Methods. Fifteen patients receiving MTX for RA
were prospectively studied by electron microscopic analysis of biopsy specimens.
Results. Five of the 15 patients had evidence of
increased hepatic collagen after 2 years of MTX therapy.
Conclusion, Hepatic fibrosis may progress in a
subgroup of RA patients treated with MTX.
Low-dose pulse methotrexate (MTX) is an effective treatment for rheumatoid arthritis (RA). A
recent metaanalysis demonstrated that light microscopic evidence of hepatic fibrosis occurs in 29% of
RA patients receiving MTX (1). Electron microscopic
evidence of fibrosis, manifested as increased sinusoidal collagen, occurs in the majority of patients receiving MTX for RA (2). The clinical significance of this
From the Divisions of Gastroenterology and Rheumatology, University of Utah College of Medicine and Veterans Administration Medical Center, Salt Lake City, Utah.
David J. Bjorkman, MD: Associate Professor, Director of
Endoscopy, Gastroenterology Division, University of Utah; Mark
Boschert, MD: Fellow in Gastroenterology,Gastroenterology Division, University of Utah; Keith G. Tolman, MD: Professor and
Chairman, Gastroenterology Division, University of Utah; Daniel
0. Clegg, MD: Professor, Rheumatology Division, University of
Utah; John R. Ward, MD: Professor, Rheumatology Division,
University of Utah.
Address reprint requests to: David J. Bjorkman, MD,
University of Utah Medical Center, 50 N. Medical Drive, Room
4R118, Salt Lake City, UT 84132.
Submitted for publication February 12, 1993; accepted in
revised form May 6, 1993.
fibrosis is controversial (3,4). In view of the fact that
clinically significant liver failure is rare in these patients, it is unclear whether the fibrosis progresses
with continued MTX therapy (5). Light microscopy, as
used in prior studies, is an insensitive measure of
hepatic fibrosis. Therefore, in the present study, we
investigated the progression of hepatic fibrosis by the
more sensitive method of electron microscopy performed sequentially over 2 years, in patients receiving
MTX for RA.
Fifteen patients (12 men, 3 women) receiving MTX
therapy for active RA were studied. No patient had clinical
evidence of prior or current liver disease. No patient consumed more than 1 alcoholic beverage per week. One patient
was obese and had a history of adult-onset diabetes. At entry
into the study, the duration of MTX therapy was 4.6 2 0.3
years (mean ? SEM). The mean f SEM weekly dosage of
MTX was 9.8 f 0.7 mg, with a cumulative dose of 2.5 f 0.4
gm. The study was approved by the Institutional Review
Board for Human Subjects at both the University of Utah
and the Salt Lake City Veterans Administration Hospital.
A liver biopsy was performed on each patient at
entry into the study and 2 years later. Biopsy specimens
were prepared for light and electron microscopy as previously described ( 2 ) . Electron micrographs were obtained
using a standard magnification of randomly selected hepatic
lipocytes from all hepatic zones, with a JEM-100CX I1
electron microscope (JEOL USA, Peabody, MA).
The amount of sinusoidal collagen was determined in
each biopsy specimen by 2 independent methods. In the first
method, 10 micrographs from each biopsy sample were
coded and evaluated independently by 2 observers who were
blinded to the source of the specimen. The sinusoidal
Figure 1. Grade 0 fibrosis. This electron micrograph shows a central lipocyte without any evidence of
surrounding collagen (original magnification x 15,000).
Figure 2. Grade 1 fibrosis. Two small collagen bundles are seen near a perisinusoidal lipocyte
(arrows); this small amount of collagen is a normal finding (original magnification X 15,000).
collagen in each biopsy specimen was graded on a 4-point
scale (called the fibrosis grade) as illustrated in Figures 1-4.
Grade 0 was defined as no evidence of collagen bundles, and
grade 1 as 1-3 small collagen bundles in the sinusoidal space.
Grade 2 fibrosis was defined as moderate sinusoidal collagen
(>3 small bundles, or the presence of large bundles) and
grade 3 as extensive collagen deposition extending from the
sinusoidal space into surrounding areas. Both grade 0 and
grade 1 fibrosis are normal findings, but fibrosis grades 2 and
3 indicate abnormal increases in hepatic collagen.
In the second method, collagen was quantitated
using a computerized digitizing table (Sigmascan; Jandel
Scientific, Sausalito, CA). Hepatic sinusoidal collagen is
most prominent in the area of collagen-producinglipocytes.
In order to control for variations in sectioning, the area of
collagen in each micrograph was compared with the area of
the adjacent lipocyte. The area of each bundle of collagen
was measured, and the sum of the areas of all collagen
bundles was determined. This value was compared with the
area of the lipocyte in the same micrograph. The collagento-lipocyte area ratio (CLR) was then averaged for 10
micrographs from each specimen. The mean fibrosis grade
and mean CLR were determined at each time point and
compared using a t-test for independent variables.
At entry into the study, 4 patients had a fibrosis
grade of 1 , 10 had a grade of 2, and 1 had a grade of 3.
The mean k SEM fibrosis grade was 1.8 t 0.14. After
2 years of MTX therapy, 3 patients had a lower fibrosis
grade (1 with a grade of 0), 5 had a higher grade, and 7
had no change. The mean fibrosis grade did not change
significantly (1.9 0.18; P > 0.05).
The initial mean -C SEM CLR was 0.16 +- 0.05.
It also did not change significantly over the 2-year
period (0.13 k 0.02; P > 0.05). The CLR increased in
1 1 patients and decreased in 4. There was a significant
positive correlation between the fibrosis grade and the
CLR. Statistical correlations of fibrosis grades 0 and 1
(normal collagen content) compared with grades 2 and
3 (increased collagen) by Mann-Whitney U test
showed a significant increase in the CLR (P= 0.008) in
patients with the higher fibrosis grades. In the 5
patients with fibrosis grades that were increased over
the initial measurements, the CLR also increased,
from a mean of 0.075 0.013 to 0.149 t 0.058. This
did not reach statistical significance, however (P =
0.25), probably because of the small number of patients. Two of the 3 patients with a decreased fibrosis
grade also had a decrease in the CLR. The other
patient had a trivial increase in the CLR. No patient
had light microscopic evidence of hepatic fibrosis at
either time point.
In summary, a subgroup of 5 patients (4 males,
1 female) had progression of fibrosis, while the remain-
ing 10 either had no progression or a decrease in
fibrosis after 2 years of MTX therapy. This increase in
hepatic collagen was identifiable by electron microscopy, but not by standard light microscopy.
The data presented herein demonstrate that
continuous MTX therapy does not uniformly increase
hepatic sinusoidal collagen, as determined by electron
microscopy. When evaluating our 15 patients as a
group, the results show no effect of 2 years of additional MTX therapy on the collagen grade or content.
Close examination of the data, however, indicates that fibrosis does progress in a subgroup (approximately one third) of the patients receiving MTX
for 2 years. This number is similar to that reported for
fibrosis assessed by light microscopy in other studies
(1,6,7). Light microscopy, however, is an insensitive
marker of hepatic fibrosis (2). This is the first evaluation of evidence of the progression of fibrosis by the
more sensitive technique of electron microscopy.
It is unclear why hepatic fibrosis progresses in
only a minority of patients receiving MTX for RA. No
patient in this study had a known predisposition to
liver disease (e.g., use of alcohol or drugs, hepatitis,
etc.) The 1 patient with diabetes and obesity had no
change in the hepatic collagen level. The duration of
MTX therapy (4.6 +- 0.4 years), weekly dosage (9.5 t
0.5 mg), and cumulative dose (2.48 2 0.45 gm) were
not different in these patients than in patients who had
no progression of fibrosis. The incidence of increased
fibrosis was the same in male patients and female
patients. The bimodal results in this study population
raise the possibility that there may be a genetic predisposition to fibrogenesis in a subgroup of patients. A
similar genetic predisposition is seen with other liver
diseases, such as alcoholic cirrhosis.
Increased sinusoidal collagen was seen on the
initial biopsy in 1 1 of 15 patients. These findings
support our previous studies showing electron microscopic evidence of increased collagen in the majority
of patients receiving MTX for RA (2). Because pretreatment biopsies were not available in these patients,
we cannot definitively attribute this fibrosis to MTX.
Rheumatoid arthritis patients not treated with MTX
may also have hepatic fibrosis, albeit at a much lower
incidence than in those treated with MTX (8,9). Other
studies have suggested a relationship between MTX
therapy and sinusoidal collagen in RA (10). While light
microscopy did not identify increased fibrosis in any
Figure 3. Grade 2 fibrosis. A transformed lipocyte shows increased collagen deposition, seen in multiple
bundles surrounding the cell (arrows) (original magnification x 15,000).
Figure 4. Grade 3 fibrosis. Confluent bands of collagen are seen near the lipocyte and extending into
surrounding areas (arrows) (original magnification X 15,000).
patient, electron microscopy identified a subset of
patients in whom sinusoidal collagen was increased.
In conclusion, MTX therapy for RA is associated with electron microscopic evidence of increased
hepatic sinusoidal collagen, even in the absence of
light microscopic evidence of fibrosis. Progression of
collagen deposition occurs in only a minority of patients receiving continued MTX therapy. The factors
responsible for progression of fibrosis are unknown.
The clinical significance of this electron microscopic
finding is unclear. Further study using electron microscopy in a controlled setting may be helpful in addressing this question.
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effect, terms, long, arthritis, hepatica, methotrexate, fibrosis, therapy, rheumatoid
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