Download Serum levels of tumor necrosis factor-alpha and interleukin-6

Serum Levels of Tumor Necrosis Factor-Alpha and
Interleukin-6 in Ocular Cicatricial Pemphigoid
Soon Jin Lee, Zhengzhi Li, Brian Sherman, and C. Stephen Foster
Purpose. These studies examined regulation of the cytokines interleukin-6 and tumor necrosis
factor-alpha in ocular cicatricial pemphigoid (OCP), a systemic autoimmune disease.
Methods. Serum levels of interleukin-6 and tumor necrosis factor-alpha in sera collected from
35 patients with OCP, 29 normal persons and 17 patients with ocular inflammatory diseases
were determined using an enzyme-linked immunosorbent assay.
Results. Levels of interleukin-6 were significantly decreased in sera of patients with OCP (median, 28.9; range, 7.5 to 136.7 pg/ml, P < 0.001) compared with sera from normal subjects
(median, 65.2; range, 21.1 to 303.9 pg/ml). Sera from patients with non-OCP, extraocular
inflammatory diseases and uveitis, showed no such decrease. In contrast, tumor necrosis factor-alpha levels were significantly elevated in OCP patients (median, 22.5; range, 8.3 to 44.4
pg/ml, P < 0.001), whereas no such increase was observed in sera from patients with extraocular inflammatory disease or uveitis, compared to normal sera controls (median, 17.4; range, 5
to 27.2 pg/ml).
Conclusions. These results suggest that elevated serum tumor necrosis factor-alpha levels and
decreased serum interleukin-6 levels can be added to the increasing list of systemic immunologic correlates of active OCP, again emphasizing that OCP is a systemic disease whose primary
manifestation is ocular. Invest Ophthalmol Vis Sci. 1993;34:3522-3525.
1 he term cytokine refers to a class of proteins, produced by a variety of cells, that play critical roles in
many biologic processes including inflammation and
immunity.1 Cytokines play important roles in many
physiological responses and are involved in the pathophysiology of a wide range of diseases. Because of
their regulatory nature, their potential as therapeutic
agents has been explored. The clinical application of
pure recombinant cytokines or their inhibitors in
cancer, infectious disease, and autoimmune disease
has recently met with some success.
Interleukin-6 (IL-6) has a wide variety of activities
on numerous cell types. IL-6 is expressed by tumor
cells, mitogen-stimulated lymphocytes and cytokinestimulated fibroblasts.2 IL-6 enhances immunoglobu-
From the Immunology and Uveitis Unit, Rhodes Molecular Immunology
Laboratory, Department of. Ophthalmology, Massachusetts Eye and Ear Infirmary,
Harvard Medical School, Boston, Massachusetts.
This work was supported by the Mr. & Mrs. Richard Rhoads Fund.
Submitted for publication: October 28, 1992; accepted June 15, 1993.
Proprietary interest category: N.
Reprint requests: C. Stephen Foster, Department of Ophthalmology, Massachusetts
Eye & Ear Infirmary, Harvard Medical School, 243 Charles Street, Boston, MA
02114.
3522
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lin secretion by B lymphocytes and serves as a second
signal in murine T cell activation.2-3 Increased serum
levels of IL-6 have been reported during renal transplant rejection episodes, and IL-6 has been associated
with certain autoimmune diseases.4
Tumor necrosis factor-alpha (TNF-a) also is produced by many different cell types, including macrophages, T cells, and NK cells; it has a variety of physiological properties, including stimulation of fibroblast
growth and neutrophil function.5 TNF-a enhances
both IL-2 receptor and human leukocyte antigen-II
expression on T cells and is an autocrine T cell growth
factor. Elevated TNF-a gene expression has been observed in graft versus host disease, and TNF-a has
been associated with rheumatoid arthritis.6
As a first step in understanding the abnormal regulation of cytokine networks in autoimmune eye diseases, we examined IL-6 and TNF-a levels in the sera
of patients with ocular cicatricial pemphigoid (OCP), a
systemic autoimmune disease, as well as in sera from
patients with other ocular inflammatory eye diseases,
and in sera from normal subjects. We found decreased
levels of IL-6 and increased levels of TNF-a in OCP
sera compared to sera from normal groups. These re-
Investigative Ophthalmology & Visual Science, December 1993, Vol. 34, No. 13
Copyright © Association for Research in Vision and Ophthalmology
Serum TNF-a and IL-6 Levels in Cicatricial Pemphigoid
suits support the notion that OCP is an autoimmune
disease associated with an abnormal cytokine network.
MATERIALS AND METHODS
Sample Collection
Before initiating treatment, informed consent about
the nature and possible consequences of this study was
obtained from all participants. Sera were collected
from patients with OCP (n = 35), uveitis (n = 8), other
extraocular inflammatory diseases (n = 9), and normal
(control) individuals (n = 29) and stored at -70°C
until assayed. The diagnosis of OCP was confirmed by
immunohistochemical demonstration of immunoreactants at the epithelial basement membrane zone. The
protocols used in this study followed the tenets of the
Declaration of Helsinki and were approved by the
Massachusetts Eye and Ear Infirmary Human Experimentation Committee.
Determination of Tumor Necrosis FactorAlpha and Interleukin-6 Levels
Serum TNF-a and IL-6 levels were measured by a twosite sandwich enzyme-linked immunosorbent assay,
using a commercial kit (T-cell Sciences, Inc., Cambridge MA). Briefly, 96-well plates coated with either
an anti-TNF-a or an anti-IL-6 monoclonal antibody
were incubated with sera samples from each group.
After washing the plates to remove unreacted sera
components, enzyme-conjugated monoclonal antibodies directed against either TNF-a or IL-6 were added
and incubated. Unbound enzyme-conjugated antibodies were removed by washing and the substrate, Ophenylenediamine, was added. The reaction was terminated and absorbance at 490 nm was measured. All
samples were tested in duplicate and mean values were
determined. The values for TNF-a and IL-6 were calculated using a standard curve determined using purified TNF-a and IL-6 provided with these kits.
Statistical Method
The Wilcoxon test was used to compare the mean
serum levels of TNF-a and IL-6 between the different
v
groups of patients and control subjects. The level of
significance chosen was P < 0.05.
3523
cluded seven diagnosed with scleritis, one with sarcoidosis-associated chronic conjunctivitis, and one
with episcleritis.
First, we studied TNF-a in the sera of normal subjects grouped by either age or sex. No significant differences were observed between subjects older or
younger than 50 years (P = 0.429) or between male
and female subjects (P = 0.154; Fig. 1). For this reason, samples from patients or control subjects were
not divided into groups before comparison of serum
TNF-a levels.
TNF-a levels were significantly increased in the
sera of patients with OCP (median, 22.5; range, 8.3 to
44.4 pg/ml, P < 0.001) compared to serum levels in
normal subjects (median, 17.4; range, 5 to 27.2 pg/ml;
Table 1). Statistically significant increases in serum
TNF-a levels were not observed in patients with uveitis
(median, 22.5; range, 8.9 to 30.1 pg/ml; P = 0.257) or
extraocular inflammatory diseases (median, 19.5;
range, 14.2 to 22.5 pg/ml; P = 0.057) compared to
normal subjects (Table 1).
Serum IL-6 levels also did not significantly differ
in normal subjects grouped by either age or sex (Fig.
2). Serum IL-6 levels were significantly lower in OCP
patients (median, 28.9; range, 7.5 to 136.7 pg/ml; P <
0.001) compared to normal subjects (median, 65.2;
range, 21.1 to 303.9 pg/ml; Table 1). There was no
significant difference in the mean serum IL-6 levels of
patients with extraocular inflammatory diseases (median, 45.8; range, 0 to 450 pg/ml; P = 0.476) or with
50
~
o
o
40
-
30
-
•
20
-
10
Patients included in this study varied in age from 20 to
60 years. No significant differences were observed between normal subjects grouped on the basis of age or
sex, so normal subjects were not divided into groups
for comparison with patients with ocular inflammation. Extraocular inflammatory disease patients in-
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_l_
—W~
o
-€-
0
8
o
•
-
a
a
i
RESULTS
.
51
:
i
i
>60yr
<50yr
p=0.429
o
.
i
I
8
i
Female
I
i
Male
p-0.154
FIGURE l. TNF-a levels in the sera of normal subjects. Sera
from normal subjects were obtained as described in Methods. Normal subjects were divided into groups on the basis
of either age or gender and their serum TNF-a levels were
compared. P values are presented for the significance of
differences between the means of each group.
Investigative Ophthalmology & Visual Science, December 1993, Vol. 34, No. 13
3524
uveitis (median, 79.6; range, 0 to 435.6 pg/ml; P =
0.200) compared to normal subjects (Table 1). Because the difference in age of the normal group for
IL-6 had borderline significance, we reanalyzed the
data with age-matched group for comparison of serum
IL-6 levels from patients with OCP, extraocular inflammatory disease, and uveitis and those from normal
subjects. The new P values were OCP = 0.01 (n = 9),
uveitis = 0.163 (n = 8) and extraocular inflammatory
diseases = 0.199 (n = 7), all similar to the values obtained without age matching.
400
300
-
200
100
-
DISCUSSION
Elevated serum levels of IL-6 and TNF-a have been
previously reported in autoimmune diseases such as
rheumatoid arthritis, suggesting abnormal regulation
of these two cytokines in the peripheral immune system.4-6 Our studies examined the serum levels of these
two cytokines in patients with OCP, an extraocular
inflammatory problem that we and others have contended represents local expression of a systemic autoimmune disease.7'8 Serum TNF-a levels were substantially elevated in OCP patients, suggesting a potential role for this cytokine in the increased activation
of the peripheral immune system, as judged by elevated serum levels of soluble IL-2 receptor and soluble CD8 (unpublished observation). TNF-a has been
shown to potentiate both CD8 T cell function in vivo
and in vitro and the release of soluble CD8 from synovial fluid mononuclear cells in vitro.9 The observed
increase of serum-soluble CD8 levels in OCP patients
may result from elevated levels of TNF-a, which has
been shown to trigger CD8 T cell activation.9 The elevated serum TNF-a levels observed in OCP patients in
this study suggest an association between expression
of this cytokine and OCP, a finding reported for other
TABLE l.
Comparison of Serum TNF-a
and IL-6 Levels
TNF-a
OCP
Uveitis
Extraocular
Normal
IL-6
OCP
Uveitis
Extraocular
Normal
Median Value
(pg/ml)
No. of
Samples
P Value*
22.5
22.5
19.5
17.4
35
8
9
<0.001
0.257
0.057
28.9
79.6
45.8
65.2
26
8
9
26
29
<0.001
0.2
0.476
—
* P values are relative to normal controls as calculated by the Wilcoxon test.
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FIGURE 2. IL-6 levels in the sera of normal subjects. Sera
from normal subjects were obtained as described in Methods. Normal subjects were divided into groups on the basis
of either age or gender and their serum IL-6 levels were
compared. P values are presented for the significance of
differences between the means of each group.
autoimmune diseases, such as rheumatoid arthritis.5
Increased IL-6 expression has been implicated in
polyclonal B cell activation, which is observed in autoimmune diseases such as rheumatoid arthritis.3 However, IL-6 levels in the sera of our OCP patients were
decreased, suggesting that a different mechanism may
be involved in the pathogenesis of OCP. Although
these studies comprised a relatively small number of
subjects and a large variation in mean values was
noted, the findings obtained from this study warrant
further investigation with a larger study population.
Reduced IL-6 serum levels were reported in cyclosporin-induced nephrotoxicity in human renal allograft recipients.10 Decreased serum levels of IL-6 observed in this study may have resulted from abnormal
function of cells secreting IL-6 or from secondary effects of other cytokines involved in the cytokine regulatory network. More studies are needed to fully establish the potential relationship between serum levels of
IL-6 and TNF-a and their relationship to conjunctival
inflammation.
Key Words
tumor necrosis factor, interleukin-6, cicatricial pemphigoid,
ocular immunology, cytokines
Acknowledgments
The authors thank Dr. George T. Cicila for critically reviewing the manuscript.
Serum TNF-a and IL-6 Levels in Cicatricial Pemphigoid
3525
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