Download Involvement of the pineal gland in rats with experimental

October 1983
Vol. 24/10
Investigative Ophthalmology
& Visual Science
A Journal of Dosic ond Clinical Research
Articles
Involvement of the Pineal Glond in Rots with
Experimental Autoimmune Uveitis
Manabu Mochizuki, John Charley, Toichiro Kuwabara, Robert D. Nussenblort, and Igal Gery
Pineal glands of rats with experimentally induced autoimmune uveitis (EAU) were studied histologically.
Inflammatory changes, characterized by mononuclear infiltration, were found in the pineal glands of
one-third of the Lewis rats that developed EAU by active immunization with S-antigen. No changes
in the pineal gland were observed in AVN rats which are "low responders" for EAU and did not
develop ocular disease. Frequency and severity of both pineal gland and ocular involvement clearly
were elevated by intravenous injection of Bordetella pertussis along with the S-antigen immunization;
all B. pertussis-treated rats of both Lewis and AVN strains developed pineal and ocular changes.
Inflammatory changes of the pineal gland also were found in rats in which EAU was induced passively
by transfer of lymphocytes from S-antigen-immunized donors. The frequency of involvement of the
pineal gland was found to be lower than that of the retinas in rats where EAU was induced by active
immunization or by adoptive transfer of lymphocytes. Invest Ophthalmol Vis Sci 24:1333-1338, 1983
The pineal gland of lower vertebrates has anatomic
features, as well as functional capacities, similar to
those of the retina.1'2 Although the pineal gland in
mammals has become a secretory organ, its unique
relationship to the retina is retained partially, as indicated by certain morphologic similarities3 and by the
presumed origin of retinoblastoma-like tumors in the
pineal gland.4 A more direct demonstration of the association between the retina and pineal gland has been
provided by Kalsow and Wacker who showed that the
pineal gland in guinea pigs contains the retinal-specific
S-antigen.5 These authors have shown further that
guinea pigs immunized with S-antigen develop inflammation in both the eye and the pineal gland,6 and that
inflammatory changes in both tissues are induced in
guinea pigs by immunization with pineal gland extract.7
More recently, Wacker et al8 reported that pineal
changes also occur in guinea pigs that are recipients
of cultured peritoneal lymphocytes from donors immunized with S-antigen.
No data have been reported, to the best of our
knowledge, concerning the involvement of the pineal
gland in species other than the guinea pig in which
experimental autoimmune uveitis (EAU) can be induced. We report here our findings with regard to the
histologic changes in the pineal gland of rats immunized with S-antigen. The rats used were of two genetically different strains, with different susceptibility
to EAU.9 In addition, we examined the changes in
pineal glands of rats in which EAU was induced by
transferred lymphocytes from donors immunized with
S-antigen.
Materials and Methods
Animals
Lewis rats were purchased from M.A. Bioproducts,
Walkersville, MD. AVN rats were offsprings of breeding pairs, provided by Dr. David Gasser (University
of Pennsylvania). Male and female rats of both strains
between 7 weeks and 6 months of age were used.
From the Laboratory of Vision Research and Clinical Branch,
National Eye Institute, National Institutes of Health, Department
of Health and Human Services, Bethesda, Maryland.
Supported in part by the US-Japan exchange scientist program
in vision research.
Submitted for publication: February 11, 1983.
Reprint requests: Dr. Manabu Mochizuki, Building 6, Room 232,
National Institutes of Health, Bethesda, MD 20205.
Immunization
Bovine S-antigen was provided by Drs. Magda ElSaied and Hitoshi Shichi (NEI) and Dr. Waldon B.
Wacker (University of Louisville), and prepared as described by Wacker et al.10 The antigen was emulsified
(1:1) in complete Freund's adjuvant (CFA) (GIBCO,
0146-0404/83/1000/1333/$ 1.00 © Association for Research in Vision and Ophthalmology
1333
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / October 1983
Table 1. Involvement of the pineal gland in actively
immunized rats
Pathologic changes
Rat
strain
Lewis
Mode of
immunization
Eye
Pineal gland
S-antigen in CFA
12/12
4/12
S-antigen in CFA
+ B. pertussis
PBS in CFA
MBP* in CFA
+ B. pertussis
S-antigen in CFA
S-antigen in CFA
+ B. pertussis
AVN
5/5
5/5
0/4
0/4
0/4
0/4
0/3
0/3
6/6
6/6
0/6
0/6
MBPt in CFA
+ B. pertussis
* All rats of this group showed severe experimental allergic encephalomyelitis
(EAE).
•f None of these animals developed EAE.
Abbreviations: CFA, complete Freund's adjuvant; PBS, phosphate-buffered
saline; MBP, myelin basic protein.
Vol. 24
Grand Island, NY), enriched with Mycobacterium tuberculosis H37Ra to a concentration of 2.5 mg/ml.
As a control for S-antigen, Dulbecco's phosphate-buffered saline (PBS) and myelin basic protein (MBP) from
guinea pigs were used. The MBP was given by Dr.
Bernard F. Driscoll (National Institute of Mental
Health). A total volume of 0.1 ml/rat, containing 3050 Mg of S-antigen or 50 ng of MBP, was injected into
the hind footpad of rats. In some experiments, an additional bacterial adjuvant, Bordetella pertussis, 1010
bacteria per rat, was injected intravenously along with
aforementioned immunization. The B. pertussis was
of batch C-507F, gifts from Dr. Dale McFarlin (National Institutes of Health) and Dr. Scott Linthicum
(University of Southern California).
Adoptive Transfer of EAU
The spleen and draining lymph node were collected
from donor Lewis rats 12 or 13 days after immuni-
•* V
9
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Fig. 1. Lymphocytic infiltration in the pineal gland of
a Lewis rat immunized with
S-antigen in CFA. The rat
was killed on day 13 after
immunization. A, The cell
infiltration is mild and located mainly at the peripheral
or subcapsular area of the
gland (Hematoxylin and
eosin, XI30). B, The infiltrated cells are mononuclear,
small, and round in shape
(Hematoxylin and eosin,
X800).
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THE PINEAL GLAND IN EAU RATS / Mochizuki er al.
.#
4
•' NT
Fig. 2. Pathologic changes in the eye of a
Lewis rat immunized with S-antigen in CFA.
The rat was killed on day 13 after immunization. Unlike the pineal gland, a heavy involvement of polymorphonuclear cells (P), in
addition to the mononuclear infiltration (M),
was found in the retina, especially in the visual
cell layer (Hematoxylin and eosin, X375).
U
zation with S-antigen. Cell suspensions, prepared by
gentle teasing, were washed and cultured at 2 X 106
cells/ml in RPMI-1640 medium (GIBCO) containing
5% heat-inactivated fetal calf serum, 2-mercaptoethanol (2 X 10~5 M) and either S-antigen (2 fig/ml) or
concanavalin A (Con A, 1 jig/ml; Miles-Yeda, Rehovot,
Israel). Following an incubation period of 3 days, the
cells were harvested, washed twice, and injected intraperitoneally into naive Lewis recipients.
Evaluation of EAU and Pineal Gland Involvement
Clinical signs of EAU were monitored daily, with
or without a slit-lamp microscope. Actively immunized
rats were killed usually between days 14 and 19
postimmunization. Recipient rats were killed between
days 6 and 9 postinjection of the cultured cells. Eyes
and pineal glands were fixed in 2.5% glutaraldehyde
and embedded in glycol methacrylate. Sections cut at
M
3 ^m were stained with toluidine blue or hematoxylin
and eosin, and examined with a light microscope.
Results
Table 1 summarizes the data concerning the involvement of the pineal gland in Lewis or AVN rats
immunized with S-antigen or control antigens. All 12
of the Lewis rats that were tested developed EAU clinically as well as histologically at day 12 to day 19
postimmunization with S-antigen emulsified in CFA.
Four of these 12 Lewis rats showed cell infiltration in
the pineal gland. Among the 12 rats, three rats were
killed on day 12 after immunization when clinical
signs of ocular inflammation first were observed. One
of these rats already had cell infiltration in the pineal
gland. The cell infiltration was moderate and located
mainly at the peripheral or subcapsular area of the
pineal gland (Fig. 1). The infiltrating cells were mono-
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Ocrober 1983
1336
Vol. 24
Fig. 3. Pathologic changes
in the pineal gland of a Lewis
rat immunized with S-antigen in CFA with additional
injection of B. pertussis. The
rat was killed on day 13 after
immunization. The micrograph shows intense lymphocytic infiltration as far as
the central area of the gland
(Hematoxylin and eosin,
XI30).
nuclear, small, and round in shape, and considered to
be mostly lymphocytes. Unlike the pineal glands, a
heavy involvement of polymorphonuclear cells
(PMNs), in addition to the mononuclear infiltration,
was found in the eyes of the same rats (Fig. 2). The
Lewis rats treated with B. pertussis in addition to the
S-antigen immunization (n = 5) developed more severe
EAU as well as an earlier onset of the disease than
rats immunized with S-antigen alone (11-15 days vs.
12-19 days). In addition, all rats in this group showed
lymphocytic infiltration in the pineal gland, with many
cells invading as far as the central area of the gland
(Fig. 3). In contrast, control Lewis rats immunized
with either PBS (n - 4) or MBP (n = 4) along with
B. pertussis did not develop EAU, and no lymphocytic
infiltration was found in their glands. All rats immunized with MBP showed experimental autoimmune
encephalitis (EAE) and lymphocytic infiltration in the
brain tissue.
Table 2. Involvement of the pineal gland of
recipient rats
Pathologic
changes
Origin of
transferred
cells
Stimulus in
culture
Lymph nodes
S-antigen
Spleen
Concanavalin A
Number of celts
transferred
(XJO6 cells)
Eye
5-10
20-60
90-130
3/3
4/4
2/2
Pineal
gland
0/3
3/4
2/2
In AVN rats, none of the three immunized with Santigen in CFA showed EAU or any change in the
pineal gland. On the other hand, all six AVN rats
immunized with S-antigen with the additional injection
of B. pertussis developed EAU and lymphocytic infiltration in their pineal glands. The histologic changes
were similar to those found in the Lewis rats without
B. pertussis treatment (see above). Again, control immunization with MBP in CFA along with B. pertussis
injection did not induce any pathologic changes in
both the eyes and the pineal gland (n = 6).
Table 2 summarizes the results concerning the involvement of the pineal gland in transferred EAU. In
preliminary experiments, recipients of uncultured
lymphocytes from immunized donors showed neither
EAU nor pineal inflammation, even with high numbers
of cells (10 8 -10 9 cells per recipient). However, once
lymphocytes were stimulated in culture with either
Con A or S-antigen, EAU and pineal infiltration were
found in recipients of even lower numbers of cells
(Table 2). As few as 5 X I06 lymph node cells cultured
with S-antigen were reproducibly capable of transferring EAU to naive Lewis rats. EAU developed in recipient rats faster than in actively immunized rats ( 5 9 days vs. 12-19 days), and the clinical and histologic
changes of EAU in recipients resembled those in actively immunized rats. Lymphocytic infiltration of the
pineal gland was observed in three of the four recipients
of 20 to 60 X 106 lymph node cells cultured with Santigen, but in none of the three recipients of a low
number of cells (5 to 10 X 10" cells/rat). The two
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THE PINEAL GLAND IN EAU RATS / Mochizuhi er al.
Fig. 4. The pineal gland of a
Lewis rat with passively
transferred EAU. The rat was
injected with 90 X 106 spleen
cells from a S-antigen-immunized donor that were
stimulated in culture with
concanavalin A. The recipient rat was killed eight days
after cell transfer. Mild lymphocytic infiltration is seen
in the peripheral zone of the
gland (Hematoxylin and
eosin, X80).
* *
*
1&-
recipients of spleen cells cultured with Con A (90 to
130 X 106 cells/rat) also developed EAU and inflammatory changes in the pineal gland. The lymphocytic
infiltration in the recipient pineal gland was mild and
located only in the peripheral area (Fig. 4). The intensity of the pineal infiltration in the recipients of
both.S-antigen- or Con-A-stimuIated lymphocytes was
similar to that of actively immunized Lewis rats without B. pertussis treatment.
Discussion
The present study shows that rats that develop EAU
after immunization with S-antigen also may develop
pathologic changes in their pineal glands. The pineal
changes were found to be antigen-specific, since no
such changes were detected in control rats injected
with CFA alone, or with the encephalitogenic antigen
MBP. The close relationship between the ocular and
pineal involvement was depicted further by the finding
that the frequency and severity of pathologic changes
in the two organs were affected similarly by the genetic
makeup of the immunized rats and by the mode of
immunization. Thus, pineal involvement was found
in Lewis rats that are "high responders" for EAU,9 but
not in the AVN rats that are "low responders"9 and
did not develop EAU in this study following immunization with S-antigen and CFA alone. The ocular
and pineal involvements were enhanced remarkably
^
by treating the immunized rats with B. pertussis: all
S-antigen-immunized rats, of both strains, developed
both pineal and ocular changes following treatment
with these bacteria. The mode of action of B. pertussis
on disease induction is not clear, but accumulating
evidence supports the notion that these bacteria increase the reaginic immunity and the activity of the
mast cell system, and consequently, the permeability
of the blood-organ barrier.9'""15 The close relationship
between the ocular and pineal involvement also was
suggested by the findings that the inflammation in the
pineal gland developed with kinetics similar to that of
the ocular involvement. It is noteworthy that treatment
with B. pertussis did not overcome the inability of the
AVN rats to develop EAE. This inability is attributed
to a genetic (histocompatibility antigen-related) deficiency of these rats to respond to the encephalitogenic
peptide (MBP).16 This immunodeficiency cannot be
overcome by the bacteria. On the other hand, the AVN
rats do respond to S-antigen9 and their "low responsiveness" is a result of factors that are affected by the
B. pertussis.
Cellular infiltration in the pineal gland also was
found in rats in which EAU was induced adoptively
by the transfer of lymphocytes from S-antigen-immunized donors. This finding further supports the notion that the changes in both the retina and pineal
gland are produced by the same immune-mediated
mechanism. Moreover, this finding indicates that spe-
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / October 1983
cifically sensitized lymphocytes, rather than antibodies,
play the major pathogenic role in the disease induction
in both the eye and pineal gland. Indeed, no antibodies
to S-antigen were detected in sera of rats that were
recipients of Con-A-stimulated spleen cells (unpublished data). Thus, it may be suggested that humoral
immunity, whenever developed, plays merely an accessory role in the pathogenesis of the pathologic ocular
or pineal changes.
The cells infiltrating the effected pineal glands were
found to be lymphocytes, both in recipient rats and
those actively immunized with S-antigen (Figs. 1, 3,
4). On the other hand, heavy involvement of PMNs
was found in the eyes of the same rats (Fig. 2). The
difference between the two organs is not clear and may
be attributed to different factors such as (1) the immune
response in the eye may be more intense (resulting
from the presence of more S-antigen) and thus, the
mediated inflammation may be more "acute" and favorable to PMN involvement; or (2) hypothetically,
PMNs may be attracted to the eye by certain ocular
components with selective chemotactic activity for
PMNs.
Our findings differ from those of Wacker et al8 in
that the frequency of pineal involvement in the recipient rats was considerably lower than that of the ocular
involvement; the mentioned authors observed more
pineal than ocular involvement in recipient guinea
pigs. The difference between the two animal species
is not clear, but could be related to anatomic differences
between the two species; unlike the rat, the guinea pig
lacks retinal blood vessels. It is worth noting that less
pineal than ocular involvement also was found in rats
actively immunized with S-antigen (Table 1).
Key words: experimental autoimmune uveitis (EAU), retinal
S-antigen, pineal gland, inflammatory changes, Bordetella
pertussis, adoptive transfer of EAU
Acknowledgments
The authors are grateful to Dr. David Gasser for providing
AVN rats, to Drs. Magda El-Saied, Hitoshi Shichi, and Waldon B. Wacker for S-antigen, and to Drs. Dale McFarlin
and Scott Linthicum for B. pertussis. They also thank Ms.
Mary Alice Crawford for excellent technical help and Ms.
Joyce Mclntyre for typing the manuscript.
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