CLINICAL
IMMUNOLOGY
AND
Vol. 65,No. 3,December,
IMMUNOPATHOLOGY
pp. 294-299,
1992
Association between Mast Cells and the Development of Experimental Autoimmune Uveitis in Different Rat Strains QIANLI,YUJIROFUJINO,RACHEL Laboratory
R. CASPI,FATEMEHNAJAFIAN,ROBERT
of Immunology,
National
Eye Institute,
National
To study the role of anterior uveal mast cells in experimental autoimmune uveitis (EAU), the mast cells in the iris and ciliary body of Lewis rats, Brown Norway (BN) rats, and their Fl hybrids (LBNFl) were quantitated in normal rats and during the induction period of EAU. The mean baseline mast cell number was 68.9 f 10.8 per anterior uvea for Lewis rats, 0.3 + 0.2 for BN rats, and 4.6 + 0.6 for LBNFl rats. Detectable mast cells in the anterior uvea of S-Ag-immunized Lewis rats decreasedto 60%of control at 6 days postimmunization, recovered to 80% at 10 days, and dropped again to 16%at 13 days, with diseaseonset around 14days. in Lewis rats that were adoptively transferred with a uveitogenic T-lymphocyte line, a profound drop in anterior uveal mast cell numbers occurred in the eyes with early signs of EAU, 3 days after the transfer. The decrease in detectable mast cells is consistent with mast cell degranulation. The data suggest that anterior mast cells participate in the immunopathogenesis of EAU and may influence the genetic susceptibility to EAU. D 1st~ Academic PM+, IM.
INTRODUCTION
Experimental autoimmune uveitis (EAU) is an organ-specific, T-cell-mediated autoimmune eye disease that has been used as an animal model for some forms of human uveitis (1). EAU can be induced by active immunization with retinal antigens, such as S-antigen retinoid-binding (S-Ag) and th e interphotoreceptor protein (IRBP), and can also be induced by adoptive transfer of retinal antigen-specific T lymphocytes, including long-term T-cell lines specific to uveitogenic peptide (l-3). The first histopathological findings of EAU in the rat are inflammatory cells in the anterior uvea and retinal vessels (4). Later the major part of the damage to the retina is caused by recruited leukocytes attracted to the eye through an amplification cascade initiated by the antigen-specific T cells. The mechanisms involved in this amplification have not been well defined, but are thought to induce cytokine-mediated as well as vascular effects. The relation between ocular tissue mast cells and EAU has been the subject of a number of studies in
of Health,
Bethesda,
AND
Maryland
CHI-CHAOCHAN 20892
recent years (5-8). They have shown that there is a correlation between the number of choroidal mast cells and the susceptibility to EAU in different rat strains (5, 6), and that EAU can be delayed or suppressed by drugs that inhibit the release of mast cell mediators (7, 8). These findings suggest that local release of mast cell products is involved in the induction of EAU. Mast cell degranulation can be caused by receptor crosslinking via cytophilic antibodies and antigen, as well as by mediators released from activated T cells (9-12). Both mechanisms could be operative in EAU, since both cytophilic antibodies and autoimmune T lymphocytes are induced during the process of immunization cl,@. Mediators released from mast cells have been shown to directly increase vascular permeability (9). This effect could be particularly important in inflammatory processes affecting closed organs such as the eye, where leukocytic infiltration must be preceded by a breakdown of the blood-organ barrier. In addition, mast cell mediators can induce expression of adhesion molecules on vascular endothelium and attract leukocytes to sites of inflammation (13-151, thus directly participating in the amplification cascade. We therefore decided to evaluate the number and the distribution of mast cells in the anterior uvea (the iris and ciliary body) of two inbred rat strains, EAUsusceptible Lewis rats and EAU-resistant BN rats, and their Fl hybrids. The present study examined the number of detectable mast cells in normal unmanipulated animals, and investigated the dynamic changes in mast cell numbers during the induction period of EAU. The results are consistent with the interpretation that mast cells located in the anterior portion of the eye have a role in determining susceptibility to EAU and participate in the development of the disease. MATERIALSANDMETHODS Animals. A total of 184 female Lewis rats, BN rats, and their Fl hybrids (LBNFl) weighing 200 g (6-8 weeks of age) were obtained from Harlan SpragueDawley, Frederick, Maryland, and Charles River Raleigh, Raleigh, North Carolina. All animals were
294 0090-1229192 $4.00 C opyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Institutes
B. NUSSENBLA~,
MAST CELLS IN EXPERIMENTAL
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UVEITIS
295
housed in environmentally controlled rooms with a 12hr light and dark cycle. They were provided food and water ad libitum.
Springfield, MA), and the total mast cell numbers on each flat-mounted iris and ciliary body were counted under a microscope by two independent observers.
S-antigen was prepared Antigens and reagents. from bovine retinas according to the method of Dorey et al. (16). R16 (a 15-mer synthetic peptide containing the major pathogenic epitope of interphotoreceptor retinoid-binding protein) was synthesized and purified on a peptide synthesizer (3).
EAU occurrence and presentation of results. Animals were followed daily for signs of EAU, starting 10 days after immunization. Iris hyperemia, exudation and hypopyon were taken as clinical evidence of EAU onset. Incidence is shown as the percent of positive animals, or the number of positive out of total animals in each group. All experiments were repeated at least twice. Statistics on the data were analyzed using students’ t-test.
T-cell lines. An RlG-specific helper-T-cell line (LR16) was established from draining lymph nodes of RlG-immunized Lewis rats, essentially as previously described (2, 3). The cells were maintained in culture by a weekly regimen of stimulation with R16 on antigen presenting cells, followed by expansion in interleukin-a-containing medium. The LR16 lymphocyte line is highly uveitogenic, and transfers EAU to otherwise unmanipulated syngeneic recipients. Disease onset in the recipient usually occurs on the fourth day after injection of 1 to 2 million LR16 cells. Znduction of EAU. Rats were immunized with purified bovine S-Ag emulsified (l:l, v/v) in complete Freund’s adjuvant (CFA, Difco) that had been enriched with Mycobacterium tuberculosis strain H37Ra (Difco) to a concentration of 2.5 mg/ml. Each rat was immunized by a single injection of 30 pg of S-Ag in a total volume of 0.1 ml into one hind footpad. Adoptive transfer was performed as follows: 3 x lo6 LR16 cells and 1 x lo8 thymocytes (irradiated 3000 R) were cocultured with R16 (3 kg/ml) in a volume of 10 ml supplemented RPM1 1640 medium in lo-cm Petri dishes for 48 hr at 37°C. The activated cells were washed twice and injected intraperitoneally into naive Lewis recipients (1.5 x lo6 cells into each animal).
RESULTS
Anterior mast cells in normal rats of different strains. Total mast cells in the anterior uvea of unmanipulated Lewis, BN, and LBNFl rats were enumerated as described under Materials and Methods. Mast cells were located predominantly in the ciliary body and less commonly in the iris, where they were often distributed along vessels (Fig. 1). The number of mast cells in the Lewis strain was strikingly higher than in the BN strain, with the LBNFl hybrids falling closer to the BN parent (Table 1 and Fig. 2). In addition, variation was considerable among individuals within the Lewis strain and even between the two eyes of single animals (Fig. 3). In Lewis rats an average of 52 mast cells were observed in the ciliary body and 16 in the iris. In BN rats, mast cells were recognized only in the ciliary body and not in the iris (only one or two mast cells per eye were found in 4 of the 25 eyes examined). An average of five mast cells per eye were found in LBNFl eyes.
Tissue preparation. Animals were sacrificed by COz inhalation. Enucleated eyes were immediately immersed in 10% neutral buffered formalin for 15 min. Under a dissecting microscope, the globes were opened by an equatorial incision at the limbus. The iris and ciliary body were isolated carefully with a dissecting needle, then floated in distilled water and flat-mounted on poly-L-lysine-coated slides. Slides were air-dried and stored at 4°C for 2 days before staining.
Onset and incidence of EAU in different rat strains. The incidence of EAU was lower, and the time of disease onset was later, in BN and LBNFl rats than in Lewis rats. All 12 Lewis rats developed EAU, whereas only 4 of 12 BN rats developed EAU and 7 of 12 LBNFl rats developed EAU. The difference in EAU incidence in Lewis compared with BN and LBNFl rats was statistically significant at P < 0.0001 and P < 0.009, respectively. In Lewis rats, EAU onset date was between Days 12 and 14 after immunization but was delayed until Days 18 to 23 in BN rats and until Days 14 to 17 in LBNFl rats.
Staining. The slides were depigmented in aqueous potassium permanganate solution for 60 min. A 1% concentration was used for the BN and LBNFl rats, and a 0.25% concentration was used for the Lewis rats, which have no uveal pigmentation. Slides were washed three times in distilled water and bleached with 5% aqueous oxalic acid solution for approximately 10 min until the tissue became clear. The slides were again washed in distilled water, and 0.15% toluidine blue solution was applied for 3 min. The slides were covered with gelvatol (20/30 Resin, Indian Orchard Plant,
Changes in mast cell numbers after EAU induction by immunization with S-Ag. In control, shamimmunized rats (PBS in CFA) mast cell numbers did not change significantly, and there were no signs of ocular inflammation throughout the experiment. In S-Ag-immunized Lewis rats, the number of mast cells decreased to 60% of control on Day 6, recovered to 80% of control on Day 10, and dropped again to 16% of control on Day 13, with an onset of uveitis around Day 14 (Fig. 4). After EAU onset further changes in mast cell numbers could no longer be followed, as it is techni-
LI ET AL
296
FIG. 1. Photomicrograph of mast cells in the iris of a Lewis rat. The detail morphologies the uvea (toluidine blue, x200; inset, x400).
tally difficult to obtain adequate tissue preparations from severely inflamed eyes. It was impossible to reliably evaluate changes in mast cell number in LBNFl and BN rats after S-Ag immunization, because there were very few mast cells present at baseline (Table 2).
.
l
MeanGE: Lewis 68.9f10.8 LBNFl 4.6fO 6 EN 0.3f0.2
z L f :
40
:
20
:
0
.
Changes in mast cell numbers after EAU inductton by adoptive transfer of uveitogenic T cells. Table 3
shows the mast cell numbers in the anterior uvea of Lewis rats injected with the pathogenic LR16 lymphocyte line. No statistical difference relative to baseline was found on Day 1 or 2 after uveitogenic cell challenge, and no eyes had either clinical or histopathological evidence of EAU. On Day 3, however, 5 of 16 eyes had early clinical signs of EAU (i.e., miosis, iris congestion, and edema). In these 5 eyes only a single mast cell in one eye could be identified, constituting a highly significant decrease in mast cell number. In the 11 of 16 eyes without clinical signs of EAU, there was a mild
Numbers
.
.
’
TABLE 1 and Distribution of Mast Cells in Anterior of Normal Rats
t 8
l
of mast cells are poor due to the thickness of
.
l
4t
Lewis
m
LBNFl Rat
EN
Strains
FIG. 2. Total mast cells in the anterior uvea (iris plus ciliary body) of individual eyes of various rat strains. Each symbol represents a single eye. Note the high variability in the counts for mast cells.
Strain __-..-~~~ Lewis LBNFl BN
Number of rats -~-~ 15 9 13
Uvea
Number of mast cells” .-
Iris 16.1 t 3.3* 0.3 t 0.2 0
a Values are means t SE. * Significantly different from BN and LBNFl
~..
Ciliary body --. _. -~ 51.5 f 11.3* 4.3 f 0.8 0.3 i- 0.2
(P < 0.0001).
MAST CELLS IN EXPERIMENTAL I 5
: 0
1
.
l-
I-
4
10
Individual
FIG. 3. Variation single animals.
11
12
13
I14
Rat
in mast cell numbers between the two eyes in
reduction in mast cell number tistical significance compared all eyes had clinical evidence making quantitation of mast flat-mount method.
which did not reach stawith baseline. By Day 4, of severe inflammation, cells impossible by this
DISCUSSION
The present study demonstrates that the number of mast cells present in the anterior uveal tract is much higher in Lewis rats than in BN or LBNFl rats, and is correlated with a higher incidence of EAU and an ear-
.-& ; E a
607060 -
iii tn K
50
D =3
40
r
30-
: Z
20 -
ii !-
10 -
”
.
da;
day
0
Days
6
after
day
10
day
13
lmmunlzatlon
FIG. 4. Kinetics of change in mast cell numbers (*SE) in S-Ag immunized Lewis rats during the induction period of EAU. Asterisks denote statistically significant differences from controls (PBS in CFA).
AUTOIMMUNE
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lier onset of disease. These results are in agreement with a previous study, in which the number of mast cells in the choroid of various rat strains was associated with their respective susceptibility to EAU (6). Our study also points to a similarity in the number of mast cells in the anterior and posterior portions of the eye within the strains studied: high in the Lewis, intermediate in LBNFl, and low in BN. We hypothesize that in the EAU-resistant BN strain, the overall small number of local mast cells may be insufficient to provide efficient amplification of the inflammatory cascade, resulting in a delay or absence of disease induction. The considerable variation in mast cell number between the two eyes of most Lewis rats is of interest. The severity and the onset of EAU sometimes vary between the two eyes of the same animal, and in some cases the disease is unilateral. The unequal distribution of mast cells between the two eyes could explain this phenomenon. Although the target antigen (S-Ag or IRBP) is located in the posterior portion of the eye, the clinical onset of EAU is almost invariably accompanied by acute inflammation in the anterior segment. In fact, anterior inflammation may precede posterior involvement, since the first inflammatory cells entering the eye are frequently observed around the ciliary body (Ghan and Caspi, unpublished data). One possible explanation for this phenomenon is that the mast cells in the anterior segment may be involved in the early stage of anterior uveitis. In the Lewis strain, there was a biphasic drop in detectable mast cell numbers during the induction period: a decrease to 60% on Day 6, with an apparent recovery to 80% on Day 10, and a second profound decrease to 16% on Day 13 just prior to EAU induction which occurred around Day 14 (17, IS>. The apparent drop in mast cell numbers is most consistent with degranulation, which would decrease their detectability for about 24 hr, while regeneration of the granules occurs (9,19). The striking drop in mast cell number 24 hr before EAU onset could be directly involved in facilitating the subsequent infiltration of inflammatory cells. A similar observation was made earlier in experimental allergic encephalomyelitis (EAE), where a decrease in number and functional activity of mast cells was noted at the onset of EAE, just before and during the development of clinical signs in the Lewis rat (2022). Mast cell involvement and subsequent release of vasoactive amines to alter the integrity of the bloodbrain barrier were proposed to play a role in the development of actively induced EAE (23, 24). Recent studies of adhesion molecules in EAE demonstrated their expression in a stage-specific manner within the central nervous system (CNS) and suggested specific homing and attachment of lymphocytes to the CNS vasculature as being the earliest features of
298
LI ET AL.
TABLE 2 Numbers of Mast Cells in Anterior Uvea in Different Strains of Rats during ~- -.
.-.
Lewis
Days after immunization
S-AG + CFA
Baseline 6 10 13
33.2 t- 6.7 (12)* 48.9 2 11.1 (8) 10.0 ” 2.5 (6)*
Induction -. -.
Period
of EAU
PBS + CFA
LBNFl S-Ag t CFA
BN S-Ag -+ CFA
54.8 t 13.4 (8) 61.7 + 15.2 (5) 61.8 + 15.7 (6)
4.6 r 0.6 (9) 3.3 t 0.5 (8) 1.6 I+_0.5 ill) ND
0.3 f 0.2 (13) I 3 ‘- 0.3 i8) 0.6 t 0.3 16) ND
68.9 t 10.8 (15)”
a Values are mean counts t SE, with number of rats in parentheses. ND, not done. * Statistically significant reduction (P < 0.003) in comparison to control.
the disease process (25-27). Thus, lymphocytes may induce local changes at the blood-brain barrier favoring further influx of inflammatory cells. We speculate that the initial moderate decrease in mast cells observed on Day 6 in the present study may be involved in the elevated expression of adhesion molecules occurring in the eyes of Lewis rats 7 days after S-Ag immunization (28). Our results for mast cellular kinetics in EAU differs from those reported by de Kozak et al. (8) for choroidal mast cells, where the numbers were doubled on Day 6 and were lowest on Day 10. Two nonmutually exclusive hypotheses can be proposed to reconcile this apparent discrepancy: either the number of mast cells in the eye is constant and the reciprocal shifts in their numbers represent migration of mast cells between the anterior and posterior compartments, or the changes represent different kinetics of degranulation in the anterior and posterior portions of the eye. We next examined changes in detectable mast cell numbers during the course of EAU induction by adoptive transfer of uveitogenic T lymphocytes in the Lewis strain (uveitogenic T-cell lines are not available in the other strains). It should be noted that EAU induced by adoptive transfer circumvents the early steps of antigen presentation and recognition, as well as induction of high titers of antibodies, and is considered to represent only the cell-mediated, efferent mechanisms of disease (2, 29). In contrast to the situation in active TABLE 3 Number of Mast Cells in Iris and Ciliary Body of Lewis Rats after Adoptive Transfer of LR16 Cell Line Days after adoptive transfer 1
2 3
Rats without
EAU
Rats with EAU
Number of eyes/total
Number of mast cells”
Number of eyes/total
Number of mast cells”
14/14 16116 11/16
54.8 + 6.9 54.1 * 10.3 44.4 t 7.7
o/14 0116 5116 0.2 + 0.3* (a total of only one mast cell was identified)
a Values are means * SE. * Statistically significant reduction (P < 0.0001) in comparison to animals that did not exhibit signs of EAU.
immunization, in the adoptively transferred animals we did not observe a biphasic change in mast cell number. Mast cells became undetectable only in eyes that were already showing early clinical signs of disease. This suggests that the early drop in mast cell counts in S-Ag-immunized animals is connected to an event(s) not represented in the adoptive transfer model, such as induction of serum antibodies. Although there are other genetic differences between Lewis and BN strains, our study points to an association between the genetically determined number of mast cells in the anterior uvea of rats and their susceptibility to EAU. These results strongly suggest participation of anterior uveal mast cells in the immunopathogenesis of ocular autoimmunity, particularly in the early stages of EAU. ACKNOWLEDGMENT
The authors thank Dr. Louis Kasner for his excellent editorial assistance. REFERENCES
1. Nussenblatt, R. B., and Palestine, A. G. (Eds.1, Ocular autoimmunity. In “UVEITIS Fundamentals and Clinical Practice,” pp. 38-46. Year Book Medical, Chicago, 1989. 2. Caspi, R. R., Roberge, F. G., Mcallister, C. G., El-Saied, M., Kuwabara, T., Gery, I., Hanna, E., and Nussenblatt, R. B., T cell lines mediating experimental autoimmune uveoretinitis (EAU) in the rat. J. Zmmunol. 136, 928-933, 1986. 3. Sanui, H., Redmond, T. M., Kotakem, S., Wiggert, B., Hu, L-H., Margalit, H., Berzofsky, J. A., Chader, G. J., and Gery, I., Identification of an immunodominant and highly immunopathogenic determinant in the retinal interphotoreceptor retinoid-binding protein (IRBP). J. Exp. Med. 169, 1947-1960, 1989. 4. de Kozak, Y., Sakai, J., Thillaye, B., and Faure, J-P., S antigeninduced experimental autoimmune uveo-retinitis in rats. Curr. Eye Res. 1, 327-337,
1981.
5. Gery, I., Robinson, W. G., Jr., Shichi, H., El-Saied, M., Mochizuki, M., Nussenblatt, R. B., and Williams, R. M., Differences in susceptibility to experimental autoimmune uveitis among rats of various strains In “Advances in Immunology and Immunopathology of the Eye” (G. R. O’Connor and J. W. Chandler, Eds.), pp. 242-245, Masson, New York, 1982. 6. Mochizuki, M., Kuwabara, T., Chan, C C., Nussenblatt, R. B., Metcalfe, D. D., and Gery, I., An association between suscepti-
MAST CELLS IN EXPERIMENTAL
7.
8.
9. 10.
11. 12. 13.
14.
15.
16. 17.
18.
bility to experimental autoimmune uveitis and choroidal mast cell numbers. J. Zmmunol. 133, 1699-1701, 1984. de Kozak, Y., Sakai, J., Sainte-Laudy, J., Faure, J-P., and Benveniste, J., Pharmacological modulation of IgE-dependent mast cell degranulation in experimental autoimmune uveoretinitis. Jpn. J. Ophthnlmol. 27, 598-608, 1983. de Kozak, Y., Sainte-Laudy, J., Benveniste, J., Faure, J-P., Evidence for immediate hypersensitivity phenomena in experimental autoimmune uveoretinitis. Eur. J. Zmmunol. 11, 612 617,198l. Galli, S. J., Dvorak, A. M., and Dvorak, H. F., Basophils and mast cells: Morphologic insights into their biology, secretory patterns, and function. Prog. Allergy 34, l-84, 1984. Plaut, M., Pierce, J. H., Watson, C. J., Hanley-Hyde, J., Nordan, R. P., and Paul, W. E., Mast cell lines produce lymphokines in response to cross-linkage of FceRI or to calcium ionophores. Nuture 339, 64-67, 1989. Marshall, J. S., and Bienenstock, J., Mast cells. Springer Semin. Zmmunopathol. 12, 191-202. Askenase, P. W., Rosenstein, R. W., and Ptak, W., T cells produce an antigen-binding factor with in vivo activity antalogous to IgE antibody. J. Exp. Med. 157, 862-873, 1983. Maggiano, N., Colotta, F., Castellino, F., Ricci, R., Valitutti, S., Larocca, L. M., and Musiani, P., Interleukin-2 receptor expression in human mast cells and basophils. Znt. Arch. Allergy Appl. Zmmunol. 91, 8-14, 1990. Valent, P., Bevec, D., Maurer, D., Besemer, J., Di Padova, F., Butterfield, J. H., Speiser, W., Majdic, O., Lechner, K., and Bettelheim, P., Interleukin 4 promotes expression of mast cell ICAMantigen. Proc. Natl. Acad. Sci. USA 88, 3339-3342, 1991. Klein, L. M., Lavker, R. M., Matis, W. L., and Murphy, F., Degranulation of human mast cells induces an endothelial antigen central to leukocvte adhesion. Proc. N&l. Acud. Sci. USA 86. 8972-8976, 1989.” Dorey, C., Cozette, J., and Faure, J-P., A simple and rapid method for isolation of retina S antigen. Ophthalmic Res. 14, 249,1982. Chan, C-C., Hooks, J. J., Nussenblatt, R. B., and Detrick, B., Expression of Ia antigen on retinal pigment epithelium in experimental autoimmune uveoretinitis. CUFF. Eye Res. 5, 325330, 1986. Fujikawa, L. S., Chan, C C., McAllister, C., Gery, I., Hooks, J. J., Betrick, B., and Nussenblatt, R. B., Class II histocompatibility
Received April 17, 1992; accepted with revision August 19, 1992
AUTOIMMUNE
19.
20. 21.
22. 23. 24. 25.
26.
27.
28.
29.
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complex antigens (I-A and I-E) in experimental autoimmune uveitis. In “Modern Trends in Immunology and Immunopathology of the Eye” (A. G. Secchi and I. A. Fregona, Eds.), pp. 69-75, Masson, Milan, 1989. Allansmith, M. R., Baird, R. S., Henriquez, A. S., and Bloch, K. J., Regeneration of mast cells after ocular anaphylaxis In “Advances in Immunology and Immunopathology of the Eye” (G. R. O’Connor and J. W. Chandler, Eds.), pp. 173-175, Masson, New York, 1982. Stanley, N. C., Jackson, F. L., and Orr, E. L., Attenuation of experimental autoimmune encephalomyelitis by compound 48/80 in Lewis rats. J. Neuroimmunol. 29,223-228, 1990. Be, L., Olsson, T., Nyland, H., Kruger, P. G., Taule, A., and Merk, S., Mast cells in brains during experimental allergic encephalomyelitis in Lewis rats. J. Neural. Sci. 105, 135-142, 1991. Levi-Schaffer, F., Riesel, N., Soffer, D., Abramsky, O., and Brenner, T., Mast cell activity in experimental allergic encephalomyelitis. Mol. Chem. Neuropathol. 15, 173-184, 1991. Linthicum, D. S., Development of acute autoimmune encephalomyelitis in mice: Factors regulating the effector phase of the disease. Zmmunobiology 162, 211-220, 1982. Dietsch, G. N., and Hinrichs, D. J., The role of mast cells in the elicitation of experimental allergic encephalomyelitis. J. Zmmunol. 142, 14761481, 1989. Cannelia, B., Cross, A. H., and Raine, C. S., Adhesion-related molecules in the central nervous system. Upregulation correlates with inflammatory cell influx during relapsing experimental autoimmune encephalomyelitis. Lab. Invest. 65,23-31,1991. O’Neill, J. K., Butter, C., Baker, D., Gschmeissner, S. E., Kraal, G., Butcher, E. C., and Turk, J. L., Expression of vascular addressins and ICAM- by endothelial cells in the spinal cord during chronic relapsing experimental allergic encephalomyelitis in the Biozzi AB/H mouse. Immunology 72, 520-525, 1991. Yednock, T. A., Cannon, C., Fritz, L. C., Sanchez-Madrid, F., Steinman, L., and Karin, N., Prevention of experimental autoimmune encephalomyelitis by antibodies against a431 integrin. Nature 356, 63-66, 1992. DeBarge, L. R., Chan, C-C., Caspi, R. R., Nussenblatt, R. B., and Whitcup, S., Expression of cell adhesion molecules in mice with experimental autoimmune uveitis. Invest. Ophthulmol. Vis. Sci. 33 (Suppl.), 796, 1992. Caspi, R. R., Basic mechanisms in immune mediated uveitic disease. In “Immunology of Eye Diseases” (S. Lightman, Ed.), pp. 61-86, Kluwer Academic, Boston, 1989.
IMMUNOLOGY
AND
Vol. 65,No. 3,December,
IMMUNOPATHOLOGY
pp. 294-299,
1992
Association between Mast Cells and the Development of Experimental Autoimmune Uveitis in Different Rat Strains QIANLI,YUJIROFUJINO,RACHEL Laboratory
R. CASPI,FATEMEHNAJAFIAN,ROBERT
of Immunology,
National
Eye Institute,
National
To study the role of anterior uveal mast cells in experimental autoimmune uveitis (EAU), the mast cells in the iris and ciliary body of Lewis rats, Brown Norway (BN) rats, and their Fl hybrids (LBNFl) were quantitated in normal rats and during the induction period of EAU. The mean baseline mast cell number was 68.9 f 10.8 per anterior uvea for Lewis rats, 0.3 + 0.2 for BN rats, and 4.6 + 0.6 for LBNFl rats. Detectable mast cells in the anterior uvea of S-Ag-immunized Lewis rats decreasedto 60%of control at 6 days postimmunization, recovered to 80% at 10 days, and dropped again to 16%at 13 days, with diseaseonset around 14days. in Lewis rats that were adoptively transferred with a uveitogenic T-lymphocyte line, a profound drop in anterior uveal mast cell numbers occurred in the eyes with early signs of EAU, 3 days after the transfer. The decrease in detectable mast cells is consistent with mast cell degranulation. The data suggest that anterior mast cells participate in the immunopathogenesis of EAU and may influence the genetic susceptibility to EAU. D 1st~ Academic PM+, IM.
INTRODUCTION
Experimental autoimmune uveitis (EAU) is an organ-specific, T-cell-mediated autoimmune eye disease that has been used as an animal model for some forms of human uveitis (1). EAU can be induced by active immunization with retinal antigens, such as S-antigen retinoid-binding (S-Ag) and th e interphotoreceptor protein (IRBP), and can also be induced by adoptive transfer of retinal antigen-specific T lymphocytes, including long-term T-cell lines specific to uveitogenic peptide (l-3). The first histopathological findings of EAU in the rat are inflammatory cells in the anterior uvea and retinal vessels (4). Later the major part of the damage to the retina is caused by recruited leukocytes attracted to the eye through an amplification cascade initiated by the antigen-specific T cells. The mechanisms involved in this amplification have not been well defined, but are thought to induce cytokine-mediated as well as vascular effects. The relation between ocular tissue mast cells and EAU has been the subject of a number of studies in
of Health,
Bethesda,
AND
Maryland
CHI-CHAOCHAN 20892
recent years (5-8). They have shown that there is a correlation between the number of choroidal mast cells and the susceptibility to EAU in different rat strains (5, 6), and that EAU can be delayed or suppressed by drugs that inhibit the release of mast cell mediators (7, 8). These findings suggest that local release of mast cell products is involved in the induction of EAU. Mast cell degranulation can be caused by receptor crosslinking via cytophilic antibodies and antigen, as well as by mediators released from activated T cells (9-12). Both mechanisms could be operative in EAU, since both cytophilic antibodies and autoimmune T lymphocytes are induced during the process of immunization cl,@. Mediators released from mast cells have been shown to directly increase vascular permeability (9). This effect could be particularly important in inflammatory processes affecting closed organs such as the eye, where leukocytic infiltration must be preceded by a breakdown of the blood-organ barrier. In addition, mast cell mediators can induce expression of adhesion molecules on vascular endothelium and attract leukocytes to sites of inflammation (13-151, thus directly participating in the amplification cascade. We therefore decided to evaluate the number and the distribution of mast cells in the anterior uvea (the iris and ciliary body) of two inbred rat strains, EAUsusceptible Lewis rats and EAU-resistant BN rats, and their Fl hybrids. The present study examined the number of detectable mast cells in normal unmanipulated animals, and investigated the dynamic changes in mast cell numbers during the induction period of EAU. The results are consistent with the interpretation that mast cells located in the anterior portion of the eye have a role in determining susceptibility to EAU and participate in the development of the disease. MATERIALSANDMETHODS Animals. A total of 184 female Lewis rats, BN rats, and their Fl hybrids (LBNFl) weighing 200 g (6-8 weeks of age) were obtained from Harlan SpragueDawley, Frederick, Maryland, and Charles River Raleigh, Raleigh, North Carolina. All animals were
294 0090-1229192 $4.00 C opyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Institutes
B. NUSSENBLA~,
MAST CELLS IN EXPERIMENTAL
AUTOIMMUNE
UVEITIS
295
housed in environmentally controlled rooms with a 12hr light and dark cycle. They were provided food and water ad libitum.
Springfield, MA), and the total mast cell numbers on each flat-mounted iris and ciliary body were counted under a microscope by two independent observers.
S-antigen was prepared Antigens and reagents. from bovine retinas according to the method of Dorey et al. (16). R16 (a 15-mer synthetic peptide containing the major pathogenic epitope of interphotoreceptor retinoid-binding protein) was synthesized and purified on a peptide synthesizer (3).
EAU occurrence and presentation of results. Animals were followed daily for signs of EAU, starting 10 days after immunization. Iris hyperemia, exudation and hypopyon were taken as clinical evidence of EAU onset. Incidence is shown as the percent of positive animals, or the number of positive out of total animals in each group. All experiments were repeated at least twice. Statistics on the data were analyzed using students’ t-test.
T-cell lines. An RlG-specific helper-T-cell line (LR16) was established from draining lymph nodes of RlG-immunized Lewis rats, essentially as previously described (2, 3). The cells were maintained in culture by a weekly regimen of stimulation with R16 on antigen presenting cells, followed by expansion in interleukin-a-containing medium. The LR16 lymphocyte line is highly uveitogenic, and transfers EAU to otherwise unmanipulated syngeneic recipients. Disease onset in the recipient usually occurs on the fourth day after injection of 1 to 2 million LR16 cells. Znduction of EAU. Rats were immunized with purified bovine S-Ag emulsified (l:l, v/v) in complete Freund’s adjuvant (CFA, Difco) that had been enriched with Mycobacterium tuberculosis strain H37Ra (Difco) to a concentration of 2.5 mg/ml. Each rat was immunized by a single injection of 30 pg of S-Ag in a total volume of 0.1 ml into one hind footpad. Adoptive transfer was performed as follows: 3 x lo6 LR16 cells and 1 x lo8 thymocytes (irradiated 3000 R) were cocultured with R16 (3 kg/ml) in a volume of 10 ml supplemented RPM1 1640 medium in lo-cm Petri dishes for 48 hr at 37°C. The activated cells were washed twice and injected intraperitoneally into naive Lewis recipients (1.5 x lo6 cells into each animal).
RESULTS
Anterior mast cells in normal rats of different strains. Total mast cells in the anterior uvea of unmanipulated Lewis, BN, and LBNFl rats were enumerated as described under Materials and Methods. Mast cells were located predominantly in the ciliary body and less commonly in the iris, where they were often distributed along vessels (Fig. 1). The number of mast cells in the Lewis strain was strikingly higher than in the BN strain, with the LBNFl hybrids falling closer to the BN parent (Table 1 and Fig. 2). In addition, variation was considerable among individuals within the Lewis strain and even between the two eyes of single animals (Fig. 3). In Lewis rats an average of 52 mast cells were observed in the ciliary body and 16 in the iris. In BN rats, mast cells were recognized only in the ciliary body and not in the iris (only one or two mast cells per eye were found in 4 of the 25 eyes examined). An average of five mast cells per eye were found in LBNFl eyes.
Tissue preparation. Animals were sacrificed by COz inhalation. Enucleated eyes were immediately immersed in 10% neutral buffered formalin for 15 min. Under a dissecting microscope, the globes were opened by an equatorial incision at the limbus. The iris and ciliary body were isolated carefully with a dissecting needle, then floated in distilled water and flat-mounted on poly-L-lysine-coated slides. Slides were air-dried and stored at 4°C for 2 days before staining.
Onset and incidence of EAU in different rat strains. The incidence of EAU was lower, and the time of disease onset was later, in BN and LBNFl rats than in Lewis rats. All 12 Lewis rats developed EAU, whereas only 4 of 12 BN rats developed EAU and 7 of 12 LBNFl rats developed EAU. The difference in EAU incidence in Lewis compared with BN and LBNFl rats was statistically significant at P < 0.0001 and P < 0.009, respectively. In Lewis rats, EAU onset date was between Days 12 and 14 after immunization but was delayed until Days 18 to 23 in BN rats and until Days 14 to 17 in LBNFl rats.
Staining. The slides were depigmented in aqueous potassium permanganate solution for 60 min. A 1% concentration was used for the BN and LBNFl rats, and a 0.25% concentration was used for the Lewis rats, which have no uveal pigmentation. Slides were washed three times in distilled water and bleached with 5% aqueous oxalic acid solution for approximately 10 min until the tissue became clear. The slides were again washed in distilled water, and 0.15% toluidine blue solution was applied for 3 min. The slides were covered with gelvatol (20/30 Resin, Indian Orchard Plant,
Changes in mast cell numbers after EAU induction by immunization with S-Ag. In control, shamimmunized rats (PBS in CFA) mast cell numbers did not change significantly, and there were no signs of ocular inflammation throughout the experiment. In S-Ag-immunized Lewis rats, the number of mast cells decreased to 60% of control on Day 6, recovered to 80% of control on Day 10, and dropped again to 16% of control on Day 13, with an onset of uveitis around Day 14 (Fig. 4). After EAU onset further changes in mast cell numbers could no longer be followed, as it is techni-
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296
FIG. 1. Photomicrograph of mast cells in the iris of a Lewis rat. The detail morphologies the uvea (toluidine blue, x200; inset, x400).
tally difficult to obtain adequate tissue preparations from severely inflamed eyes. It was impossible to reliably evaluate changes in mast cell number in LBNFl and BN rats after S-Ag immunization, because there were very few mast cells present at baseline (Table 2).
.
l
MeanGE: Lewis 68.9f10.8 LBNFl 4.6fO 6 EN 0.3f0.2
z L f :
40
:
20
:
0
.
Changes in mast cell numbers after EAU inductton by adoptive transfer of uveitogenic T cells. Table 3
shows the mast cell numbers in the anterior uvea of Lewis rats injected with the pathogenic LR16 lymphocyte line. No statistical difference relative to baseline was found on Day 1 or 2 after uveitogenic cell challenge, and no eyes had either clinical or histopathological evidence of EAU. On Day 3, however, 5 of 16 eyes had early clinical signs of EAU (i.e., miosis, iris congestion, and edema). In these 5 eyes only a single mast cell in one eye could be identified, constituting a highly significant decrease in mast cell number. In the 11 of 16 eyes without clinical signs of EAU, there was a mild
Numbers
.
.
’
TABLE 1 and Distribution of Mast Cells in Anterior of Normal Rats
t 8
l
of mast cells are poor due to the thickness of
.
l
4t
Lewis
m
LBNFl Rat
EN
Strains
FIG. 2. Total mast cells in the anterior uvea (iris plus ciliary body) of individual eyes of various rat strains. Each symbol represents a single eye. Note the high variability in the counts for mast cells.
Strain __-..-~~~ Lewis LBNFl BN
Number of rats -~-~ 15 9 13
Uvea
Number of mast cells” .-
Iris 16.1 t 3.3* 0.3 t 0.2 0
a Values are means t SE. * Significantly different from BN and LBNFl
~..
Ciliary body --. _. -~ 51.5 f 11.3* 4.3 f 0.8 0.3 i- 0.2
(P < 0.0001).
MAST CELLS IN EXPERIMENTAL I 5
: 0
1
.
l-
I-
4
10
Individual
FIG. 3. Variation single animals.
11
12
13
I14
Rat
in mast cell numbers between the two eyes in
reduction in mast cell number tistical significance compared all eyes had clinical evidence making quantitation of mast flat-mount method.
which did not reach stawith baseline. By Day 4, of severe inflammation, cells impossible by this
DISCUSSION
The present study demonstrates that the number of mast cells present in the anterior uveal tract is much higher in Lewis rats than in BN or LBNFl rats, and is correlated with a higher incidence of EAU and an ear-
.-& ; E a
607060 -
iii tn K
50
D =3
40
r
30-
: Z
20 -
ii !-
10 -
”
.
da;
day
0
Days
6
after
day
10
day
13
lmmunlzatlon
FIG. 4. Kinetics of change in mast cell numbers (*SE) in S-Ag immunized Lewis rats during the induction period of EAU. Asterisks denote statistically significant differences from controls (PBS in CFA).
AUTOIMMUNE
UVEITIS
297
lier onset of disease. These results are in agreement with a previous study, in which the number of mast cells in the choroid of various rat strains was associated with their respective susceptibility to EAU (6). Our study also points to a similarity in the number of mast cells in the anterior and posterior portions of the eye within the strains studied: high in the Lewis, intermediate in LBNFl, and low in BN. We hypothesize that in the EAU-resistant BN strain, the overall small number of local mast cells may be insufficient to provide efficient amplification of the inflammatory cascade, resulting in a delay or absence of disease induction. The considerable variation in mast cell number between the two eyes of most Lewis rats is of interest. The severity and the onset of EAU sometimes vary between the two eyes of the same animal, and in some cases the disease is unilateral. The unequal distribution of mast cells between the two eyes could explain this phenomenon. Although the target antigen (S-Ag or IRBP) is located in the posterior portion of the eye, the clinical onset of EAU is almost invariably accompanied by acute inflammation in the anterior segment. In fact, anterior inflammation may precede posterior involvement, since the first inflammatory cells entering the eye are frequently observed around the ciliary body (Ghan and Caspi, unpublished data). One possible explanation for this phenomenon is that the mast cells in the anterior segment may be involved in the early stage of anterior uveitis. In the Lewis strain, there was a biphasic drop in detectable mast cell numbers during the induction period: a decrease to 60% on Day 6, with an apparent recovery to 80% on Day 10, and a second profound decrease to 16% on Day 13 just prior to EAU induction which occurred around Day 14 (17, IS>. The apparent drop in mast cell numbers is most consistent with degranulation, which would decrease their detectability for about 24 hr, while regeneration of the granules occurs (9,19). The striking drop in mast cell number 24 hr before EAU onset could be directly involved in facilitating the subsequent infiltration of inflammatory cells. A similar observation was made earlier in experimental allergic encephalomyelitis (EAE), where a decrease in number and functional activity of mast cells was noted at the onset of EAE, just before and during the development of clinical signs in the Lewis rat (2022). Mast cell involvement and subsequent release of vasoactive amines to alter the integrity of the bloodbrain barrier were proposed to play a role in the development of actively induced EAE (23, 24). Recent studies of adhesion molecules in EAE demonstrated their expression in a stage-specific manner within the central nervous system (CNS) and suggested specific homing and attachment of lymphocytes to the CNS vasculature as being the earliest features of
298
LI ET AL.
TABLE 2 Numbers of Mast Cells in Anterior Uvea in Different Strains of Rats during ~- -.
.-.
Lewis
Days after immunization
S-AG + CFA
Baseline 6 10 13
33.2 t- 6.7 (12)* 48.9 2 11.1 (8) 10.0 ” 2.5 (6)*
Induction -. -.
Period
of EAU
PBS + CFA
LBNFl S-Ag t CFA
BN S-Ag -+ CFA
54.8 t 13.4 (8) 61.7 + 15.2 (5) 61.8 + 15.7 (6)
4.6 r 0.6 (9) 3.3 t 0.5 (8) 1.6 I+_0.5 ill) ND
0.3 f 0.2 (13) I 3 ‘- 0.3 i8) 0.6 t 0.3 16) ND
68.9 t 10.8 (15)”
a Values are mean counts t SE, with number of rats in parentheses. ND, not done. * Statistically significant reduction (P < 0.003) in comparison to control.
the disease process (25-27). Thus, lymphocytes may induce local changes at the blood-brain barrier favoring further influx of inflammatory cells. We speculate that the initial moderate decrease in mast cells observed on Day 6 in the present study may be involved in the elevated expression of adhesion molecules occurring in the eyes of Lewis rats 7 days after S-Ag immunization (28). Our results for mast cellular kinetics in EAU differs from those reported by de Kozak et al. (8) for choroidal mast cells, where the numbers were doubled on Day 6 and were lowest on Day 10. Two nonmutually exclusive hypotheses can be proposed to reconcile this apparent discrepancy: either the number of mast cells in the eye is constant and the reciprocal shifts in their numbers represent migration of mast cells between the anterior and posterior compartments, or the changes represent different kinetics of degranulation in the anterior and posterior portions of the eye. We next examined changes in detectable mast cell numbers during the course of EAU induction by adoptive transfer of uveitogenic T lymphocytes in the Lewis strain (uveitogenic T-cell lines are not available in the other strains). It should be noted that EAU induced by adoptive transfer circumvents the early steps of antigen presentation and recognition, as well as induction of high titers of antibodies, and is considered to represent only the cell-mediated, efferent mechanisms of disease (2, 29). In contrast to the situation in active TABLE 3 Number of Mast Cells in Iris and Ciliary Body of Lewis Rats after Adoptive Transfer of LR16 Cell Line Days after adoptive transfer 1
2 3
Rats without
EAU
Rats with EAU
Number of eyes/total
Number of mast cells”
Number of eyes/total
Number of mast cells”
14/14 16116 11/16
54.8 + 6.9 54.1 * 10.3 44.4 t 7.7
o/14 0116 5116 0.2 + 0.3* (a total of only one mast cell was identified)
a Values are means * SE. * Statistically significant reduction (P < 0.0001) in comparison to animals that did not exhibit signs of EAU.
immunization, in the adoptively transferred animals we did not observe a biphasic change in mast cell number. Mast cells became undetectable only in eyes that were already showing early clinical signs of disease. This suggests that the early drop in mast cell counts in S-Ag-immunized animals is connected to an event(s) not represented in the adoptive transfer model, such as induction of serum antibodies. Although there are other genetic differences between Lewis and BN strains, our study points to an association between the genetically determined number of mast cells in the anterior uvea of rats and their susceptibility to EAU. These results strongly suggest participation of anterior uveal mast cells in the immunopathogenesis of ocular autoimmunity, particularly in the early stages of EAU. ACKNOWLEDGMENT
The authors thank Dr. Louis Kasner for his excellent editorial assistance. REFERENCES
1. Nussenblatt, R. B., and Palestine, A. G. (Eds.1, Ocular autoimmunity. In “UVEITIS Fundamentals and Clinical Practice,” pp. 38-46. Year Book Medical, Chicago, 1989. 2. Caspi, R. R., Roberge, F. G., Mcallister, C. G., El-Saied, M., Kuwabara, T., Gery, I., Hanna, E., and Nussenblatt, R. B., T cell lines mediating experimental autoimmune uveoretinitis (EAU) in the rat. J. Zmmunol. 136, 928-933, 1986. 3. Sanui, H., Redmond, T. M., Kotakem, S., Wiggert, B., Hu, L-H., Margalit, H., Berzofsky, J. A., Chader, G. J., and Gery, I., Identification of an immunodominant and highly immunopathogenic determinant in the retinal interphotoreceptor retinoid-binding protein (IRBP). J. Exp. Med. 169, 1947-1960, 1989. 4. de Kozak, Y., Sakai, J., Thillaye, B., and Faure, J-P., S antigeninduced experimental autoimmune uveo-retinitis in rats. Curr. Eye Res. 1, 327-337,
1981.
5. Gery, I., Robinson, W. G., Jr., Shichi, H., El-Saied, M., Mochizuki, M., Nussenblatt, R. B., and Williams, R. M., Differences in susceptibility to experimental autoimmune uveitis among rats of various strains In “Advances in Immunology and Immunopathology of the Eye” (G. R. O’Connor and J. W. Chandler, Eds.), pp. 242-245, Masson, New York, 1982. 6. Mochizuki, M., Kuwabara, T., Chan, C C., Nussenblatt, R. B., Metcalfe, D. D., and Gery, I., An association between suscepti-
MAST CELLS IN EXPERIMENTAL
7.
8.
9. 10.
11. 12. 13.
14.
15.
16. 17.
18.
bility to experimental autoimmune uveitis and choroidal mast cell numbers. J. Zmmunol. 133, 1699-1701, 1984. de Kozak, Y., Sakai, J., Sainte-Laudy, J., Faure, J-P., and Benveniste, J., Pharmacological modulation of IgE-dependent mast cell degranulation in experimental autoimmune uveoretinitis. Jpn. J. Ophthnlmol. 27, 598-608, 1983. de Kozak, Y., Sainte-Laudy, J., Benveniste, J., Faure, J-P., Evidence for immediate hypersensitivity phenomena in experimental autoimmune uveoretinitis. Eur. J. Zmmunol. 11, 612 617,198l. Galli, S. J., Dvorak, A. M., and Dvorak, H. F., Basophils and mast cells: Morphologic insights into their biology, secretory patterns, and function. Prog. Allergy 34, l-84, 1984. Plaut, M., Pierce, J. H., Watson, C. J., Hanley-Hyde, J., Nordan, R. P., and Paul, W. E., Mast cell lines produce lymphokines in response to cross-linkage of FceRI or to calcium ionophores. Nuture 339, 64-67, 1989. Marshall, J. S., and Bienenstock, J., Mast cells. Springer Semin. Zmmunopathol. 12, 191-202. Askenase, P. W., Rosenstein, R. W., and Ptak, W., T cells produce an antigen-binding factor with in vivo activity antalogous to IgE antibody. J. Exp. Med. 157, 862-873, 1983. Maggiano, N., Colotta, F., Castellino, F., Ricci, R., Valitutti, S., Larocca, L. M., and Musiani, P., Interleukin-2 receptor expression in human mast cells and basophils. Znt. Arch. Allergy Appl. Zmmunol. 91, 8-14, 1990. Valent, P., Bevec, D., Maurer, D., Besemer, J., Di Padova, F., Butterfield, J. H., Speiser, W., Majdic, O., Lechner, K., and Bettelheim, P., Interleukin 4 promotes expression of mast cell ICAMantigen. Proc. Natl. Acad. Sci. USA 88, 3339-3342, 1991. Klein, L. M., Lavker, R. M., Matis, W. L., and Murphy, F., Degranulation of human mast cells induces an endothelial antigen central to leukocvte adhesion. Proc. N&l. Acud. Sci. USA 86. 8972-8976, 1989.” Dorey, C., Cozette, J., and Faure, J-P., A simple and rapid method for isolation of retina S antigen. Ophthalmic Res. 14, 249,1982. Chan, C-C., Hooks, J. J., Nussenblatt, R. B., and Detrick, B., Expression of Ia antigen on retinal pigment epithelium in experimental autoimmune uveoretinitis. CUFF. Eye Res. 5, 325330, 1986. Fujikawa, L. S., Chan, C C., McAllister, C., Gery, I., Hooks, J. J., Betrick, B., and Nussenblatt, R. B., Class II histocompatibility
Received April 17, 1992; accepted with revision August 19, 1992
AUTOIMMUNE
19.
20. 21.
22. 23. 24. 25.
26.
27.
28.
29.
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complex antigens (I-A and I-E) in experimental autoimmune uveitis. In “Modern Trends in Immunology and Immunopathology of the Eye” (A. G. Secchi and I. A. Fregona, Eds.), pp. 69-75, Masson, Milan, 1989. Allansmith, M. R., Baird, R. S., Henriquez, A. S., and Bloch, K. J., Regeneration of mast cells after ocular anaphylaxis In “Advances in Immunology and Immunopathology of the Eye” (G. R. O’Connor and J. W. Chandler, Eds.), pp. 173-175, Masson, New York, 1982. Stanley, N. C., Jackson, F. L., and Orr, E. L., Attenuation of experimental autoimmune encephalomyelitis by compound 48/80 in Lewis rats. J. Neuroimmunol. 29,223-228, 1990. Be, L., Olsson, T., Nyland, H., Kruger, P. G., Taule, A., and Merk, S., Mast cells in brains during experimental allergic encephalomyelitis in Lewis rats. J. Neural. Sci. 105, 135-142, 1991. Levi-Schaffer, F., Riesel, N., Soffer, D., Abramsky, O., and Brenner, T., Mast cell activity in experimental allergic encephalomyelitis. Mol. Chem. Neuropathol. 15, 173-184, 1991. Linthicum, D. S., Development of acute autoimmune encephalomyelitis in mice: Factors regulating the effector phase of the disease. Zmmunobiology 162, 211-220, 1982. Dietsch, G. N., and Hinrichs, D. J., The role of mast cells in the elicitation of experimental allergic encephalomyelitis. J. Zmmunol. 142, 14761481, 1989. Cannelia, B., Cross, A. H., and Raine, C. S., Adhesion-related molecules in the central nervous system. Upregulation correlates with inflammatory cell influx during relapsing experimental autoimmune encephalomyelitis. Lab. Invest. 65,23-31,1991. O’Neill, J. K., Butter, C., Baker, D., Gschmeissner, S. E., Kraal, G., Butcher, E. C., and Turk, J. L., Expression of vascular addressins and ICAM- by endothelial cells in the spinal cord during chronic relapsing experimental allergic encephalomyelitis in the Biozzi AB/H mouse. Immunology 72, 520-525, 1991. Yednock, T. A., Cannon, C., Fritz, L. C., Sanchez-Madrid, F., Steinman, L., and Karin, N., Prevention of experimental autoimmune encephalomyelitis by antibodies against a431 integrin. Nature 356, 63-66, 1992. DeBarge, L. R., Chan, C-C., Caspi, R. R., Nussenblatt, R. B., and Whitcup, S., Expression of cell adhesion molecules in mice with experimental autoimmune uveitis. Invest. Ophthulmol. Vis. Sci. 33 (Suppl.), 796, 1992. Caspi, R. R., Basic mechanisms in immune mediated uveitic disease. In “Immunology of Eye Diseases” (S. Lightman, Ed.), pp. 61-86, Kluwer Academic, Boston, 1989.
