Download The presence of cytotoxic autoantibody to lacrimal gland cells

The Presence of Cytotoxic Autoontibody to Locrimol
Gland Cells in NZB/W Mice
Yuichi Ohashi,* 5impson K. So, Petros N. Minosi, and Khalid F. Tobboro
New Zealand Black and White F, hybrid mice (NZB/W mice) spontaneously develop an autoimmune
disease which provides us with a suitable animal model for Sjogren's syndrome. With increasing age,
these mice develop foci of mononuclear cell infiltration in the lacrimal and salivary glands, which
closely resemble the lesions seen in patients with Sjogren's syndrome. We studied the cell-mediated
and antibody-mediated immune responses of NZB/W mice to lacrimal gland cells. Lacrimal gland
acinar cells were isolated from 2-month-old NZB/W or BALB/c mice for the target of 5>Cr-release
assay. There was no statistically significant difference in the spleen cell-mediated cytotoxicity to
lacrimal gland cells among NZB/W mice of different ages (2, 5, and 8 months old). With increasing
age, on the other hand, we found a statistically significant increase in the titers of autoantibodies to
lacrimal gland cells in NZB/W mice, while aged BALB/c mice did not develop such antibodies.
Fractionation of pooled positive sera by gel filtration revealed that this cytotoxic activity was mostly
recovered in the IgM fraction. The tissue absorption study showed that these antibodies cross-reacted
with salivary gland and kidney. Invest Ophthalmol Vis Sci 26:214-219, 1985
gland and showed that the changes started as early as
4 months of age.10 However, the exact etiology of the
infiltration to these exocrine glands remains unknown.
In terms of tissue injury, one possible hypothesis may
be that the lacrimal gland cells could be the target of
the host immune system. This study, using 51Crrelease assay, was undertaken to investigate whether
there was any autoimmune response to lacrimal gland
cells in NZB/W mice.
Sjogren's syndrome is a chronic, relatively common
autoimmune disorder characterized by the mononuclear cell infiltration of the lacrimal and salivary
glands. This infiltrative process causes the progressive
destruction of exocrine glands and eventually brings
in intractable symptoms of keratoconjunctivitis sicca
and xerostomia.1"3 The etiology of Sjogren's syndrome
is still unknown, however it has been suggested that
autoimmune mechanisms are involved in the pathogenesis of this disease.
New Zealand Black/White F, hybrid mice (NZB/
W mice) have been found to develop spontaneously
an autoimmune disease with aging. 45 They display a
variety of autoantibodies and usually die from severe
renal failure, which has been caused by immune
complex-mediated glomerulonephritis. 67 Kessler et al
showed that aged NZB/W mice exhibited a characteristic mononuclear cell infiltration in the lacrimal
gland. 89 Recently, deLuise et al, in a chronologic
study, investigated the histopathology of the lacrimal
Materials and Methods
Animals
Female, 2-3-month-old NZB/W mice were purchased from the Jackson Laboratory (Bar Harbor,
Maine). Also, 2- and 6-month-old female BALB/c
mice were obtained from the Simonsen Laboratory
(Gilroy, CA). Most of the NZB/W mice and 2-monthold BALB/c mice were used for the source of target
cells, but some of the NZB/W mice were housed in
the vivarium for a desired period. About 30% of the
NZB/W mice died before reaching 8 months of age.
Studies using these animals were performed in conformance with the ARVO Resolution on the Use of
Animals in Research.
From the Heintz Laboratory, the Francis I. Proctor Foundation
for Research in Ophthalmology and the Department of Ophthalmology, University of California, San Francisco, and the King
Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia.
Supported in part by research grant EY-03436-03 from the
National Eye Institute and in part by a grant from the King Khaled
Eye Specialist Hospital.
* Yuichi Ohashi, MD, is the recipient of the Cecilia Vaughan
Memorial Fellowship during the years 1982-1984.
Submitted for publication: January 30, 1984.
Reprint requests: Khalid F. Tabbara, MD, Francis I. Proctor
Foundation, S-315, University of California, San Francisco, San
Francisco, CA 94143.
Chemicals and Reagents
Collagenase (type 2; 3.4.24.3 156 U/mg), hyaluronidase (HSEP; 3.2.1.35 9134 U/mg), and soybean
trypsin inhibitor were purchased from Worthington
Biochemical Inc. (Freehold, NJ). Ethylenediamine
tetra-acetic acid (EDTA), carbamylcholine chloride,
214
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No. 2
ANTI-LACRIMAL GLAND AUTOANTIDODY IN NZD/W MICE / Ohoshi er ol.
and saponin were obtained from Sigma Chemicals
(St. Louis, MO). Guinea pig complement was purchased from Gibco Laboratory (Grand Island, NY).
Culture Medium
Ham's F-12 medium (F-12), heat-inactivated fetal
calf serum (HIFCS), and Ca ++ , Mg++-free, Hanks'
balanced salt solution (HBSS), were supplied by the
Cell Culture Facility of University of California (San
Francisco, CA). The isolation medium contained 0.1
mg/ml of soybean trypsin inhibitor and the cytotoxicity medium was supplemented additionally with
10% HIFCS and 10~6 M of carbamylcholine as a
secretagogue.
Isolation of Lacrimal Gland Cells
Lacrimal gland cells were isolated from either 2month-old NZB/W or BALB/c mice, based on the
method of Oliver.'' Each animal was killed by cervical
dislocation and was perfused immediately with 10 ml
of F-12 through the left ventricle in order to flush
out the blood from the lacrimal gland. Following
perfusion, both exorbital lacrimal glands were excised
and rinsed in the isolation medium. After several
cycles of washing, the glands were minced into fragments and rinsed in HBSS for 5 min. Fragmented
glands then were transferred into Erlenmeyer's flask
(25 ml) with 5 ml of HBSS containing 0.76 mg/ml
of EDTA, and the chelation was continued for 15
min at 37 °C with continuous stirring. Chelated tissues
were rinsed in the isolation medium for 5 min and
transferred into another flask with 5 ml of enzyme
solution. The enzyme solution consisted of F-12
medium containing 195 U/ml of collagenase and 300
U/ml of hyaluronidase. After the digestion for 15
min at 37 °C, the tissues again were rinsed in HBSS
for 5 min for the next chelation. This chelationdigestion cycle was repeated until the larimal gland
tissues were dispersed considerably. The dispersed
cells were washed twice with the isolation medium
supplemented with 10% HIFCS and were forced
gently through 500 urn and 25 ^m Nytex screens
(Tekto Inc.; Elsford, NY) for further dispersion. The
cells were lightly centrifuged at 50 g for 5 min and
suspended in the cytotoxicity medium for labeling.
A part of the cell suspension was cytocentrifuged and
stained with Diff-Quik (Dade Diagnostics, Aguada
Puerto Rico, 00602) to check the population of the
isolated cells.
Preparation of Target Cells
The isolated lacrimal gland cells were immediately
labelled with 0.1 mCi of 5l Cr (sodium chromate:
specific activity 250-500 mCi/mg, Amersham; Ar-
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215
lington, IL) for 1 hr at 37°C. The labelled cells then
were washed three times with a large volume of the
cytotoxicity medium and suspended in this medium
for 2 hr of preincubation. The lacrimal cells finally
were suspended in the cytotoxicity medium at the
concentration of 1 X 105 cells/ml. The viability of
the target cells after the labelling usually was
around 80%.
Spleen Cell-Mediated Cytotoxicity
The animals examined were composed of four
groups: 2-, 5-, and 8-month-old NZB/W mice and 6month-old BALB/c mice. They were killed by cervical
dislocation and their spleens were excised aseptically.
Minced spleens were wrapped up in a cotton gauze
and squeezed in F-12 medium. After lysing red blood
cells by hypotonic treatment, spleen cells were washed
twice and suspended in the cytotoxicity medium at
the concentration of 1 X 107 cells/ml. The viability
of spleen cells was almost 100% as determined by
trypan blue dye exclusion test. One-tenth ml of target
cell suspension prepared as described above, (104
cells/well) and 0.1 ml of effector cell suspension (106
cells/ml) were mixed at the effector to target cell ratio
of 100/1 in the wells of microtitration plate. After
incubation for 4 hr at 37°C, the plate was centrifuged
to spin down the cells, and 0.1 ml of the supernatant
medium was collected and counted for the released radioactivity, using gamma-counter (Beckman,
Gamma-4000). The maximum release of radioactivity
was determined by incubating the lacrimal gland cells
with the same volume of saponin solution (0.25% in
water) with 1 mM EDTA, and the spontaneous
release was determined by incubating the target cells
with medium alone. The specific cytotoxicity was
calculated according to the following formula: specific
cytotoxicity (%) = cpm (sample) - cpm (spontaneous)/cpm (maximum) — cpm (spontaneous) X 100.
Complement-Dependent Antibody-Mediated
Cytotoxicity (CDAC) Test
When the animals were killed, blood was collected
by cardiac puncture. The sera obtained were heatinactivated for 30 min at 56°C and serial twofold
diluents were made with the cytotoxicity medium,
ranging from 8- to 128-folds. Then, 0.05 ml of the
diluent of each serum was mixed with an equal
volume of 5lCr-labeled target cell suspension (104
cells/well), prepared as described, and incubated for
30 min at 37°C. One-tenth ml of guinea pig complement (1:10 diluted with the cytotoxicity medium)
was added to the wells, and the plates were incubated
for another 30 min at 37°C. For the controls, target
cells were incubated with either eightfold diluted sera
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / February 1985
216
Table 1. Spleen cell-mediated cytotoxicity to
lacrimal gland acinar cells
± 0.5*
± 0.4
±0.5
± 1.0
7)t
13)
9)
9)
II
4.4
6.1
6.1
5.2
II
6
2
5
8
II
BALB/c
NZB/W
Specific cytotoxicity (c,V
II
Age
(months)
z z z z
Mice
examined
* The values represent the mean of specific cytotoxicity ± standard error
of means.
t Number of mice examined.
or complement alone. Finally, the plate was centrifuged at 100 g for 10 minutes to sediment the target
cells and 0.1 ml of the supernatant medium was
collected for counting radioactivity. The specific cytotoxicity was calculated in the same way as already
described.
Absorption of Pooled Positive Sera by
Various Organ and Tissues
The sera of aged NZB/W mice, positive for cytotoxic antibody to lacrimal gland cells, were pooled
and used for the absorption study. The absorption of
the sera was done according to the method of Harbeck
et al.12 Brain, lacrimal glands, salivary glands, liver,
and kidney were obtained from 2-month-old NZB/
W mice and were homogenized separately in F-12
medium. The homogenate was washed twice with the
cytotoxicity medium, and the pellet was used for the
following absorption study. Spleen and thymus cell
suspension were prepared by being minced and
squeezed in F-12 medium and red blood cell suspension was obtained from heparinized cardiac blood.
One hundred microliters of undiluted pooled serum
was mixed with an equal volume of pelleted tissues
or cells and incubated at 4°C for 1 hour. These
absorbed sera were subjected to complement-dependent antibody-mediated cytotoxicity assay at the dilution of 1:4.
Vol. 26
(Cappel Laboratory; Cochranville, PA). IgM was present mainly in fraction 5 and scarcely in fraction 6.
On the other hand, IgG was found largely in fractions
6-9 with a trace amount detectable in fraction 5.
Consequently, fractions 5-9 were dialyzed against the
cytotoxicity medium overnight at 4°C and used for
CDAC test at the dilution of 1:4 against the original
volume of the sera.
Histopathology of Lacrimal Glands
of NZB/W Mice
Both exorbital lacrimal glands were excised when
the animals were killed and fixed in the buffered
formalin. The specimens were embedded in paraffin,
cut in 7 nm thickness, and stained with hematoxylin
and eosin (H & E). The section of both lacrimal
glands was checked for the number of the infiltrative
foci per five fields at X100 magnification. The cluster
consisting of more than 50 lymphocytes was considered as one focus.
Results
Isolation of Lacrimal Gland Cells
Lacrimal gland cells were successfully isolated from
exorbital lacrimal glands by a combination of chelation with EDTA, and digestion with collagenase and
hyaluronidase. The cytocentrifuged specimen of isolated lacrimal cells consisted predominantly of lacrimal acinar cells. The viability of the isolated cells
was between 80-90% and the yield of the cells was
1-2 X 105 cells per gland. The isolated lacrimal gland
cells could not be maintained for long in tissue
culture even in the presence of secretagogue. In fact,
the cell viability dropped to around 40% the next
day, and the cells became degenerated totally within
3 days. Therefore, we decided to use the lacrimal
cells as a target cell immediately after the isolation.
In this way, the spontaneous release of the target cell
after 4 hr of incubation ranged between 10-15%.
Cytotoxicity Test to Lacrimal Gland Cells
Fractionation of Pooled Positive Sera
by Gel Filtration
A Sephadex G-150 column (1.6X90 cm) was
equilibrated with 0.1 M phosphate-buffered saline
(PBS, pH 7.2). One milliliter of pooled sera from
aged NZB/W mice, which were found to be cytotoxic
to lacrimal gland cells, was applied to this column
and fractionated at the flow rate of 7 ml/hr. Three
peaks were obtained in a total of 15 fractions, each
of which consisted of 5 ml. Ouchterlony's double
immunodiffusion test was done for all the fractions,
using rabbit serum anti-mouse IgG as well as IgM
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The results of spleen cell-mediated cytotoxicity test
against lacrimal gland cells is shown in Table 1.
There was no statistically significant difference in the
cytotoxicity among the NZB/W mice with the age of
2, 5, and 8 months, and the values ranged between
4-9%. The spleen cells from aged BALB/c mice, an
H-2 identical allogeneic control mouse, also were
found to have no significant cytotoxicity (Table 1).
There was mild splenomegaly in 5- and 8-month-old
NZB/W mice.
The presence of serum cytotoxic antibody against
lacrimal gland cells was investigated in the same
217
ANTI-LACRIMAL GLAND AUTOANTIDODY IN NZO/W MICE / Ohoshi er ol.
No. 2
Table 2. Complement-dependent antibody-mediated
cytotoxicity to lacrimal gland acinar cells
1.0
Specific cytotoxicity (%) to
Mice
examined
Age
(months)
BALB/c
NZB/W
6
2
5
8
NZB/W lacrimal cells
BALB/c lai rimal cells
0.5
6.6 ± 1.1*1
6.9+ 1.9
18.1 ± 4.7
35.8 ± 10.7£:
(N
(N
(N
(N
=
=
=
=
6)f
13)
9)|
9)
Not done
0.4 ± 1.3
6.0 ± 1.4"
13.2 ± 3.7||
(N == 5)
(N == 9)
(N == 9)
* Tie values represent the mean of specific cytotoxicity ± standard error
of means.
t The number of mice examined.
% Significant from 2-month-old NZB/W mice (P < 0.025); &P < 0.005);
\P < 0.025); \\(P < 0.01), by Student's t-test.
animals that had been evaluated for spleen cellmediated cytotoxicity. Serum was taken from both
NZB/W and BALB/c mice. As shown in Table 2,
cytotoxic effects on the target cells was detectable
with the serum of aged NZB/W mice but not with
the serum of aged BALB/c mice. As far as NZB/W
mice are concerned, the mean level of cytotoxicity
against syngeneic targets increased significantly with
aging.
The incidence of the number of animals, which
showed the cytotoxicity higher than 10%, also was
increased. The incidence was two out of thirteen
(15%), seven out of nine (78%), and six out of nine
(67%), for 2-, 5-, and 8-month-old animals, respectively. There was a statistically significant difference
in the level of specific cytotoxicity between 2- and 5month-old (P < 0.025) as well as 2- and 8-monthold NZB/W mice (P < 0.005). On the other hand,
the sera from aged BALB/c did not react with these
targets. There was no significant level of the cytotoxicity when the target cells were incubated with either
serum or complement alone. The positive sera from
NZB/W mice similarly lysed the lacrimal gland cells
isolated from BALB/c mice. Again, the cytotoxicity
of sera from 5- and 8-month-old NZB/W mice was
increased significantly as compared with 2-month-old
animals (P < 0.01 and P < 0.025, respectively).
Histopathology of the lacrimal glands of the same
animals was checked for infiltration. No infiltration
was observed in 2-month-old NZB/W mice; however,
eight out of nine (5-month-old animals) and nine out
of nine animals (8 months old) showed infiltrative
foci.
I1TT12I13T14I15
FRACTION NUMBER
Fig. 1. Complement dependent antibody-mediated cytotoxicity
of fractionated NZB/W sera to lacrimal gland cells. The solid bar
represents the specific cytotoxicity (%). The curved line shows the
eluation profile of applied sera.
7-9) as demonstrated by Ouchterloney's double immunodiffusion test. As Figure 1 shows, the high
cytotoxic activity was recovered in the fraction 5,
strongly indicating that these antibodies were of IgM
class.
The tissue absorption test was performed to check
the cross-reactivity of these autoantibodies with other
organs. The results showed that the cytotoxic activity
was abolished markedly by lacrimal glands and salivary glands, and moderately eliminated by the kidney.
Any other organs or cells such as brain, liver, spleen,
thymus, or erythrocytes could not affect the cytotoxicity (Fig. 2).
Discussion
New Zealand mice (NZB and NZB/W) are the
strains most commonly studied for autoimmune dis-
SPECIFIC CYTOTOXICITY (%)
14.6
Characterization of Cytotoxic Autoantibody
To determine the class of these autoantibodies, we
fractionated the pooled positive sera from NZB/W
mice by gel filtration with Sephadex G-150. Of three
peaks obtained, the first one was enriched with IgM
(fractions 5, 6), and the second one with IgG (fractions
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20%
Fig. 2. Effect of tissue absorption on complement dependent
antibody-mediated cytotoxicity of pooled NZB/W sera to lacrimal
gland cells. The solid bar indicates the specific cytotoxicity without
absorption, and the hatched bar the cytotoxicity after the tissue
absorption.
218
INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / February 1985
ease. They produce a wide spectrum of autoantibodies
spontaneously and develop diseases which mimic
systemic lupus erythematosus and Sjogren's syndrome
in humans.4-5'8'9 The predominant immunologic feature of NZB/W mice is the presence of hyperactive
B-cells early in life. This results in the abundant
production of various autoantibodies such as antinuclear antibody6'7 and antithymocyte antibody. 1314
Besides these well-known autoantibodies, three kinds
of organ- or tissue-specific autoantibodies have been
found in NZB mice. These include anticerebellar,12
anti-liver,15 and anti-fibroblast antibodies.16 However,
the pathologic significance of these autoantibodies is
not known.
We have shown in the present study that antilacrimal gland cytotoxic autoantibodies of the IgM
class were increased in the sera of aged NZB/W mice.
The level and incidence of such antibodies increased
significantly with age. Even at the age of 2 months,
a considerable level of cytotoxic antibodies was detectable in a few animals. This is not surprising,
however, considering the fact that abnormal early
maturation of B-cells occurs immediately after birth
in these animals. 1718 These antibodies were also capable of lysing the lacrimal gland cells obtained from
BALB/c mice, an H-2 identical allogeneic strain,
suggesting that tissue-associated antigen might be
involved as a target. The result of the tissue absorption
study was of much more interest, since the salivary
gland, in which a mononuclear cell infiltration occurs
in somewhat the same manner as it does in the
lacrimal gland, could almost abrogate the cytotoxic
activity of the sera. This would indicate that a certain
common antigen did exist in both glands, and that
this particular autoantibody may be responsible for
the salivary gland involvement. Also, there is a possibility that the corresponding antigen might be the
product of a virus that is harbored by both glands.19
At present, the role of anti-lacrimal gland autoantibodies in the pathogenesis of cellular infiltration of
the lacrimal gland is unclear. One possible assumption
is that these antibodies, bound to an antigen in the
lacrimal gland cells, could attract inflammatory cells
to the site of the reaction with the help of complement.
In fact, the finding of deLuise et al10 that immunoglobulins as well as C3 components were deposited
in the periductal areas of the lacrimal gland might be
of interest, although we cannot stipulate the specific
antigens against which such deposited immunoglobulins were directed. Although some reports have
suggested the possibility that cell-mediated immunity
may play a role in the pathogenesis of the cellular
infiltration21"23 of lacrimal and salivary glands, there
was no significant level of spleen cell-mediated cyto-
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Vol. 26
toxicity to lacrimal gland cells in any animals of any
age in our study. Although it may be possible that
suppressor cells masked the cytotoxicity in vitro, cellmediated immunity would not appear to be active in
mediating injury. A similar attempt to demonstrate
the cellular cytotoxicity to the cultured salivary gland
cells in patients with Sjogren's syndrome has failed.24
Thus, anti-lacrimal gland antibodies may be responsible for the tissue injury to the lacrimal gland.
However, their presence may simply be the consequence of antibody production in response to damaged lacrimal gland cells. It is possible to obtain
monoclonal antibodies to lacrimal gland cells by
producing a hybridoma with the spleen cells of aged
NZB/W mice, since Eliat et al have succeeded in
producing monoclonal antibody to mRNA. 20 In subsequent experiments, therefore, passive transfer of
antilacrimal gland antibody would be an ideal means
of resolving this crucial question.
Key words: Sjogren's syndrome, autoimmune disease, NZB/
W mice, autoantibody, cytotoxicity, lacrimal gland
Acknowledgments
The authors thank Dr. G. Richard O'Connor for critical
reading of the manuscript, Mr. King Kryger for his editorial
assistance, and Miss Sue Karg for technical assistance.
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