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Use of sensitive assays to detect soluble Fas in patients with systemic lupus erythematosusComment on the article by Knipping et al and the article by Goel et al.

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LETTERS
Therefore, we think that we can detect a very low
number of copies of the HTLV-I pX gene in 25% of salivary
glands of French SS patients. Sequencing of a long fragment in
a polymorphic region of pX from all the positive samples will
allow us to definitely rule out contamination and to determine
precisely whether the detected sequence belong to HTLV-I
itself or to another retrovirus.
X. Mariette, MD, PhD
P. Cherot, PhD
F. Agbalika, MD
F. Morinet, MD, PhD
Hbpital SaintLouis
Paris, France
1. Kwok S, Ehrlich G, Poiesz B, Kalish R, Sninsky JJ: Enzymatic
amplification of HTLV-I viral sequences from peripheral blood
mononuclear cells and infected tissues. Blood 72:1117-1123, 1988
2. Mariette X, Agbalika F, Daniel M-T, Bisson M, Lagrange P, Brouet
J-C, Morinet F: Detection of human T lymphotropic virus type I tax
gene in salivary gland epithelium from two patients with Sjogren's
syndrome. Arthritis Rheum 36:1423-1428, 1993
3. Mariette X, Cherot P, Cazals D, Brocheriou C, Brouet JC, Agbalika
F: Antibodies to HTLV-I in Sjogren's syndrome (letter). Lancet
34571, 1995
Reply
To the Editor:
We thank Dr. Rigby and colleagues for their interest in
our previous paper on HTLV-I tax expression in labial salivary
glands from seronegative Japanese patients with SS (1). Using
PCR, they found that HTLV-I tax DNA (159 bp) was not
present in salivary gland biopsies from 49 British patients;
however, in a parallel PCR, ERV-3 (403 bp), an endogenous
human retrovirus used as a control, was present in all 49
patients. Their PCR assay was sufficiently sensitive to detect 30
molecules of HTLV-I tax gene in a single PCR. Judging from
these data, they concluded that the presence of HTLV-I tax
gene in the salivary glands of Japanese and French SS patients
can be explained by geographic variation, although they suggest the possibility of PCR contamination.
Concerning the sensitivity of the PCR method, we used
PCR-Southern blot analysis to detect am lified DNA, since
the efficiency of Southern blot analysis is 10 to lo4 times better
than ethidium bromide staining. In our system, lo-' pg of a
188-bp tax gene for PCR was the minimum detectable by
PCR-Southern blot analysis, as shown in our previous study
(1). This amount of double-stranded DNA (dsDNA) is equivalent to about 48 molecules. In contrast, Venables described
that 30 molecules of the 159 bp of HTLV-I tax gene was
detectable by ethidium bromide staining. We estimate that 30
molecules of 159-bp dsDNA is about 5.3 X lo-" pg and predict
that these molecules are not detectable by ethidium bromide
staining after a single PCR. Therefore, further experiments on
the PCR-Southern blot analysis in their study are necessary
before they conclude that no HTLV-I tax gene can be detected
in the salivary glands of British patients with SS.
Regarding the possibility of contamination of the
HTLV-I gene in the PCR process, we believe that the expression and detection of the HTLV-I tax gene in the labial salivary
P
glands of SS patients is not due to artifactual contamination of
HTLV-I provirus, for the following 3 reasons. First, the
efficiency of primers for HTLV-I tax is the same as that of
primers for env and pol genes, as shown previously (1).
However, only the HTLV-I tax gene can be detected, while
neither the env nor the pol gene was found, even with longer
exposures. This strongly supports the notion that the HTLV-I
tax gene is expressed selectively in the labial salivary glands of
SS patients. Second, the HTLV-I tax gene was detected in
different samples prepared individually, such as RNA and
DNA, from the same patients. Third, the tax gene is expressed
in the labial salivary glands of only SS patients (4 of 14, or
29%), not healthy subjects (0 of 14), suggesting the diseasespecific expression of the tax gene. For these reasons, we
conclude that the finding of tax gene is not simple contamination of positive DNA.
Mariette et a1 (2) reported that HTLV-I tax DNA was
found by in situ hybridization in the salivary glands from 2 of
9 (22%) French patients with SS. Their subsequent PCR
analysis demonstrated the HTLV-I tax gene in 9 of 40 (23%)
patients (3). Taken together, their 2 studies and our previous
work clearly show the presence of HTLV-I tax DNA and the
expression of HTLV-I transcripts in the labial salivary glands
of 22-29% of French and Japanese SS patients, using 2
different strategies, in situ hybridization and the PCR method.
The origin of this HTLV-I tax gene is unknown; however, it
may be defective HTLV-I virus, other exogenous retroviral
infection, or a part of an endogenous retrovirus. Further
examination of the DNA sequence of the HTLV-I tax gene
should shed light on the genetic origin of this gene and its role
in the pathogenesis of SS.
Takayuki Sumida, MD
Toshiro Maeda, MD
Institute of Medical Science
St. Marianna University School of Medicine
Kawasaki, Japan
1. Sumida T, Yonaha F, Maeda T, Kita Y, Iwamoto I, Koike T,
Yoshida S : Expression of sequences homologous to HTLV-I tax
gene in the labial salivary glands of Japanese patients with Sjogren's
.
syndrome. Arthritis Rheum 37545-550, 1994
2. Mariette X,Agbalika F, Daniel M-T, Bisson M, Lagrange P, Brouet
J-C, Morinet F Detection of human T lymphotropic virus type I tax
gene in salivary gland epithelium from two patients with Sjogren's
syndrome. Arthritis Rheum 36:1423-1428, 1993
3. Mariette X, Cherot P, Cazals D, Brocheriou C, Brouet JC, Agbalika
F: Antibodies to HTLV-I in Sjogren's syndrome (letter). Lancet
345:71. 1995
Use of sensitive assays to detect soluble Fas in
patients with systemic lupus erythematosus: comment
on the article by h i p p i n g et a1 and the article by
Goel et al
To the Editor:
h i p p i n g et a1 (1) and Goel et a1 (2) recently reported
a failure to detect elevated levels of soluble Fas (sFas) in
patients with human autoimmune disease. We believe the
1612
likely explanation for the discrepancy between these results
and the original results published by us 2 years ago (3) is our
original overestimation of the levels of sFas in humans. Using
a more highly purified sFas recombinant protein to study
serum samples from 260 normal subjects, we found the
mean t SEM normal level of sFas to be 1.3 ? 0.3 ng/ml,
whereas a significant number of patients with autoimmune
disease expressed serum sFas levels >2 ng/ml (43). Patients
with active systemic lupus erythematosus (SLE) had elevated
levels of sFas (6.1 2 2.3 ng/ml). Patients with osteoarthritis and
those with chronic fatigue syndrome had lower levels of sFas
(0.86 2 0.2 and 0.51 ? 0.3, respectively). Since the assay used
by h i p p i n g et a1 has a detection limit of 2 ng/ml, which also
appears to be the detection limit in the assay used by Goel et
al, it is likely that these assays are not sensitive enough to
detect elevated serum levels of sFas. It is unfortunate that the
2 groups of investigators failed to mention our revised lower
limit for normal levels of sFas, which we have made known by
personal communication, meetings, and our further publication (4).
Despite our extensive experience with measurement of
sFas, there are several unanswered questions and special
points about the assay. One technical problem we have observed is that failure to absorb human rheumatoid factor (RF)
from serum samples can result in binding of the capture
antibody to the revealing antibody, resulting in a falsely
elevated sFas reading. In our original assay, we found it
necessary to absorb R F from the serum samples. A second
technique to decrease background is the use of an isotype
control mouse monoclonal capture antibody with each serum
sample. The sFas level is calculated using the difference
between enzyme-linked immunsorbent assay (ELISA) plate
readings with the specific and nonspecific capture antibodies.
Third, the sensitivity varies with different anti-human Fas
extracellular domain capture and revealing antibodies, possibly
due to competition for a close or overlapping antigenic site by
different antibodies. We are currently developing an improved
ELISA using an anti-human Fas extracellular domain capture
antibody along with an anti-human Fas intracellular domain
revealing antibody to prevent such competition and also to
distinguish between full-length alternatively spliced sFas and
sFas consisting of the extracellular domain that is cleaved from
the surface. ELISA using a polyclonal antibody, as reported by
Goel et al, is not reliable due to the high probability of
overlapping antigenic determinants, resulting in a low sensitivity. Interestingly, in accordance with the report by Goel and
colleagues, we occasionally find that an apparently normal,
young, healthy volunteer reproducibly expresses very high
levels of sFas detected by our assay.
A finding of low serum levels of sFas is consistent with
our original described role of sFas as functioning at the cellular
level to regulate apoptosis (3). Since our original report of the
existence of RNA encoding a soluble form of Fas produced by
alternative splicing, and the capability of a mouse and human
form of sFas or Fas Fc to inhibit apoptosis, this structure and
function have been verified by numerous investigators (5-13).
The production of an alternatively spliced form of sFas by a
subpopulation of T cells during an immune response is supported by a recent finding by Kovacs et a1 (5) of defective
activation-induced cell death in a T cell line isolated from a
patient with SLE and in patients with certain malignancies
LETTERS
(1,8,13,14). Future studies using sensitive assays for measurement of sFas will be required, in order to determine whether
the presence of sFas occurs in the early acute stage of
autoimmune disease, the cell type capable of production of
sFas, and the protein structure of active sFas.
John D. Mountz, MD, PhD
Tong Zhou, M D
Jianhua Cheng, M D
The University of Alabama at Birmingham
1. Knipping E, Krammer PH, Onel KB, Lehman TJA, Myler E,
Elkon KB: Levels of soluble Fas/APO-l/CD95 in systemic lupus
erythematosus and juvenile rheumatoid arthritis. Arthritis Rheum
38:1735-1737, 1995
2. Goel N, Ulrich DT, St. Clair EW, Fleming JA, Lynch DH, Seldin
MF: Lack of correlation between serum soluble Fas/APO-1 levels
and autoimmune disease. Arthritis Rheum 38:1738-1743, 1995
3. Cheng J, Zhou T, Liu C, Shapiro JP, Brauer MJ, Kiefer MC, Barr
PJ, Mountz JD: Protection from Fas-mediated apoptosis by a
soluble form of the Fas molecule. Science 263:1759-1762, 1994
4. Mountz JD, Pierson MC, Zhou T, Cheng J, Elkon KB. Hasunuma
T, Nishioka K, Okumura K, Lin A, Song GG, Dale JK, Gorley M,
Straus SE: sFas expression in patients with autoimmune disease
(abstract). Arthritis Rheum 38 (suppl 9):S174, 1995
5. Kovacs B, Szentendrei T, Bednarek JM, Pierson MC, Mountz JD,
Vogelgesang S, Tsokos GC: Persistent expression of a soluble form
of Fas/APOl in continuously activated T cells from a patient with
SLE. Submitted for publication
6. Cascino I, Fiucci G, Papoff G. Ruberti G: Three functional soluble
forms of the human apoptosis-inducing Fas molecule are produced
by alternative splicing. J Immunol 154:2706-2713, 1995
7. Hughes DPM, Crispe IN: A naturally occurring soluble isoform of
murine Fas generated by alternative splicing. J Exp Med 182:13951401, 1995
8. Owen-Schaub LB. Angelo LS,Radinsky R, Ware CF, Gesner TG,
Bartos DP: Soluble Fas/APO-1 in tumor cells: a potential regulator of apoptosis? Cancer Lett 94:l-8, 1995
9. Liu C, Cheng J, Mountz JD: Differential expression of human Fas
mRNA species upon peripheral blood mononuclear cell activation. Biochern J 310:957-963, 1995
10. Cheng J, Liu C, Koopman WJ, Mountz JD: Characterization of
human Fas gene: exon/intron organization and promoter region.
J Immunol 154:1239-1245, 1995
11. Behrmann I, Walczak H, Krammer PH: Structure of the human
APO-1 eene. Eur J Immunol 24:3057-3062. 1994
19
I L . MountpJD, Wu J, Cheng J, Zhou T: Autoimmune disease: a
problem of defective apoptosis. Arthritis Rheum 37:1415-1420,
1994
13. Natoli G, Ianni A, Costanzo A, De Petrillo G, IIari I, Chirillo P,
Balsano C, Levrero M: Resistance to Fas-mediated apoptosis in
human hepatoma cells. Oncogene 11:1157-1164, 1995
14. Knipping E, Debatin K-M, Stricker y Heilig B, Eder A, Krammer
PH: Identification of soluble APO-1 in supernatants of human B
and T cell lines and increased serum levels in B and T cell
leukemias. Blood 85:1562-1569. 1995
Elevated levels of soluble Fas in systemic lupus
erythematosus: comment on the article by Knipping
et al
To the Editor:
We were interested in the article by h i p p i n g et al
(Knipping E, Krammer PH, Onel KB, Lehman TJA, Mysler E,
Elkon KB: Levels of soluble Fas/APO-I/CD95 in systemic
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