Eye ( 1989) 3,69-78
Immunomodulation of Experimental Autoimmune Uveitis Using a Rat Anti Retinal S-Antigen Specific Monoclonal Antibody: Evidence for a Species Difference.
H.S. DUA, J. LIVERSIDGE andJ.V. FORRESTER. Aberdeen
Summary Experimental Autoimmune Uveitis (EAU) induced by retinal antigens, particularly S-antigen, forms a useful model of human chronic intraocular inflammation particularly endogenous post erior uveitis. It provides a means of assessing the efficacy of various agents used in the control of such inflammation. We induced an autoimmune uveitis and its associated pinealitis in Dun kin-Hartley guinea pigs and Lewis rats by inoculating them with bovine retinal S-antigen. In rats, induction of EAU but not of Experimental Autoimmune Pinealitis (EAP) could be pre vented by the administration of S-antigen specific rat monoclonal antibody simultaneously with the S-antigen. Inhibition of EAU was accompanied by significantly raised levels of anti-S antibodies during the first two weeks post-immunisation. In contrast, the same monoclonal antibody failed to inhibit both EAU and EAP in guinea pigs. Immunocytochemical staining of rat tissues for lymphocyte subsets, monocytes and macrophages showed that eyes of monoc lonal antibody treated animals contained no immunocompetent inflammatory cells unless they also had clinical signs of inflammation. In contrast, the inflammatory exudate in the pineal glands of both treated and untreated animals contained equal numbers of infiltrating lympho cytes and monocytes in the same relative proportions. These results indicate that the inhibitory effect of the monoclonal antibody S2.4.CS is directed towards the effector arm of the immune mediated cytotoxic response. A possible mechanism by which the antibody may be preferen tially inhibiting the inflammatory response in the eyes but not in the pineal glands of rats, is suggested.
Chronic intraocular inflammation is an immunological disturbance indicating that important cause of ocular morbidity and vis immune mechanisms are involved in initiat ual loss particularly among young adults. ing and/or establishing the chronicity of the Several exogenous and endogenous factors disease and its tendency to recur. 5-9 Experi have been identified in the Correspondence to: J.V. Forrester, Department of Opthalmology, Medical School, Foresterhill, Aberdeen, AB92ZD. 70 H. S. DUA ET AL. EAU has been used extensively in studying subdivided into a test group of five animals the Table I Immunisation protocol Expt. Group No. S-Ag.* CFA** Pertussist S2.4.C5tt RatIgG@ (ug) (mls) (mls) (mls) (mls) Guinea 1 5 50 0.25 2 pigs 2 4 50 0.25 Rats 1 10 50 0.25 1 1 2 6 50 0.25 1 3 6 50 0.25 1 * Bovine S-antigen (200 micro grams/ml in phosphate buffered saline (PBS) pH 7.4). ** Complete Freunds Adjuvant, H37Ra (Difco). The S-antigen and adjuvant were mixed and injected into the foot pad. t B. pertussis organisms, suspension of 1010 cells/ml in PBS injected intraperitoneally. tt Rat anti-S (Bovine) monoclonal antibody, S2.4.C5., IgG1, 1 mg/ml in PBS given by intraperitoneal injection. @ Non-specific rat IgG (Sigma), 1 mg/ml in PBS given intraperitoneally. ANTI-S MONOCLONAL ANTIBODY IN EAU 71 further purified by affinity chromatography that did not show any clinical disease were on Protein-A Sepharose, and shown to con killed 28 to 30 days post-immunisation. tain over 99% IgG by SDS-PAGE (Phar Blood samples were collected and both eyes macia Phast system). and the pineal gland were obtained for examination. One eye from each animal and Immunisation protocol: Eyes of all animals half of the pineal glands from each group were examined by slit lamp biomicroscopy were snap frozen. The other eye and the prior to immunisation to exclude any pre remaining pineal glands were fixed in 3% existing pathology. The animals were then phosphate buffered glutaraldehyde and pro� immunised as described in Table I. Rats were cessed for electron and light microscopy. tail bled after inoculation, and serum was stored at -70°C. Immunocytochemistry: Lymphocyte sub populations were tested in inflammatory Clinical observation: All animals in both experiments were observed from day one lesions in rat eyes using a panel of monoc post-inoculation. Clinical examination of the lonal antibodies (Table II). Frozen sections eyes with the slit lamp and direct ophthal on chrome-alum gelatin coated glass slides moscope was carried out after dilation of were stained by hematoxylin and eosin and pupils with 1% cyclopentolate. The day of also by the immunocytochemical Alkaline onset of uveitis, its severity and its course Phosphatase Anti-Alkaline Phosphatase were noted. The severity of the inflammatory (AP AAP) technique. Rabbit anti-mouse immunoglobulin (Dakopatts) was used as the activity was graded on a scale of 0 - 4 as secondary antibody and APAAP (mouse) described by Forrester et al.l7 Rats were tail bled at weekly intervals until sacrificed and complexes (Dakopatts) as the tertiary anti serum stored at -70°C. body. Semi-thin sections of glutaraldehyde fixed tissues, stained with toluidine blue and Sample preparation: Guinea pigs were sac basic fuschin, were also used for his rificed on days 22123 post-inoculation. Both topathological study. eyes and the pineal gland were obtained for examination. One eye was snap frozen in Tis Antibodies to Retinal S-antigen: Antibody sue-TEK, OCT compound (Miles Scientific) titres to retinal S-antigen were estimated by immersing in liquid arcton near its freez using a standard ELISA technique24 mod ing point. The other eye was fixed in 3% ified for rat serum. Microtiter 96 well plates phosphate buffered glutaraldehyde and pro were coated with bovine S-antigen (1 microg cessed for electron and light microscopy. The ram/ml in 0.02M Tris/HCI buffer, pH9), pineal gland was fixed in acetic formal,saline blocked with 0.5% bovine serum albumin, and processed for light microscopy. then incubated with the test sera. Bound Rats were sacrificed at various time inter antibodies were detected using peroxidase vals, mostly around the peak of the inflam conjugated rabbit anti-rat immunogloblins matory activity or soon thereafter. Animals (Dakopatts). Table II Monoclonal markers used (Serotec): Monoclonal markers used (serotec): 1. W3 -25 (CD4) T-heJper, macrophages, thymocytes. 2. OX 8 (CD8) T-supressor/cytotoxic, N-K cells. 3. OX 19 (CDS) Pan-T cells. 4. OX 44 Pan-T cells. 5. MARD3 B cells, (anti-IgD heavy chain) . 6. OX39 Interleukin 2 receptor. 7. OX 6 DRila monomorphic, (MHC class II). 8. EDI Monocytes and macrophages. 72 H. S. DUA ET AL. Results but in most it was uniformly spread through Guinea Pigs out the substance of the gland (Fig. lc) dis Clinical: All five guinea pigs in Group 1 rupting the normal architecture (Fig. Id). In (monoclonal antibody plus S-antigen) had contrast to the ocular inflammation the signs of uveitis by day 10 and by day 12 the pineal inflammatory response was predomin uveitis had reached its peak (4+). All four antly lymphocytic with an absence of guinea pigs in Group 2 (S-antigen alone) had polymorphonuclear cells. signs of uveitis on day 12 and the severity of inflammationhad reached its maximum (4+) Rats by day 14 (Table III). At the peak of the Clinical: Five of the six control animals inflammatory response the eyes showed con (Group 2, immunised with S-antigen alone) gestion, corneal haze, cells and flare in the developed a severe early onset uveitis involv anterior chamber, hyperaemia of the iris, ing the anterior and posterior segments of haemorrhagic retro-iridial hypopyon and vit- both eyes. Clinically this was manifest as a reous exudate obscuring retinal details. hypopyon uveitis with obscuration of the fun Ocular pathology: Eyes from all guinea dal view at its peak of inflammation. Simi pigs, both treated and untreated with larly, five of the six rats immunised with S S2.4.C5 showed similar features on histol antigen and non-specific IgG (Group 3) ogy. The lesions within an eye ranged from developed a marked uveitic response early inflammatory infiltration in the choroid although this was slightly reduced in severity with some disruption of the subjacent photo with a later onset. In contrast, only two of ten receptor layer (Fig. la) to massive infiltration rats that were immunised with S-antigen and of the entire chorioretina, optic nerve, ciliary the rat monoclonal antibody S2.4.C5 body and iris (Fig. Ib). The choroid was thic developed uveitis, in each case uveitis was kened with inflammatory cells, chiefly lym also delayed in onset and of least severity. phocytes, monocytes, polymorphonuclear Ocular pathology: Eyes from S-antigen leukocytes and macrophages. The retinal pig immunised rats treated with the monoclonal ment epithelium was disrupted at places with antibody S2.4.C5 which were clinically not loss of the photoreceptor layer. The retina inflaIlled (80%) showed no evidence of showed serous detachment at places with a inflammatory cell infiltration (Fig. 2a). In predpminance of neutrophils and monocytes contrast, eyes from untreated rats or rats in the exudate. At other places the retinal treated with non-specific IgG demonstrated architecture was totally disorganised making multiple focal inflammatory lesions which it impossible to distinguish the various layers. varied in severity from perivasculitis with Pineal inflammation: The pineal glands of early round cell infiltration in the photo all animals in both groups showed massive receptor layer (Fig. 2b) to extensive mononuclear infiltration. In some glands it granulomatous infiltration of the retina and was largely confined to the subcapsular zone choroid with loss of photoreceptor cells (Fig. Table III Uveitic and pinealitic response to S-antigen Guinea pigs Rats Group* Group* I 2 1 2 3 Total number of animals 5 4 10 6 6 Number with uveitis 5 4 2 5 5 Number with pinealitis 5 4 10 5 5 Severity (mean)@ 4 4 1 3 2 Day of onset@ 10 12 19 14 19 * 1. S2.4.C5 treated, 2. Untreated, 3. Rat IgG treated. @Ofuveitis. ANTI-S MONOCLONAL ANTIBODY IN EAU 73 Fig.1. Ocular changes in guinea pigs (a) Early choroidal lesion (arrow) showing inflammatory cell infilt ration around vessels with thickening of the choroid, in an animal from the control group. (Toluidine blue and basic fuschin x220). (b) Advanced lesion showing a grossly thickened choroid packed with inflam matory cells, loss of photo receptors and an atrophic retina (arrow) in an animal from the group treated with S2. 4. C5. (Haematoxylin and eosin x85). Pineal changes in guinea pigs (c) Dense mononuclear cell infiltration in the pineal gland of an animal treated with S2. 4. C5 (Haematoxylin and eosin x 200). (d) Nor mal guinea pig pineal (Haematoxylin and Eosin x200). 2c) as described previously for Lewis rats.16 2d) and CD4 positive cells. CD8 positive cells In most cases, retinal damage was severe and were less common while there were few B was characterised by a cellular infiltrate con cells. The proportions of cells were the same sisting of monocytes, macrophages, lympho irrespective of the site of the lesion ego cytes and a few neutrophils. Similar cells around retinal vessels, in the photoreceptor infiltrated the choroidal layer. layer or in the choroid. Many cells in the Immunocytochemical staining of the cell focal lesions expressed Ia antigen but were infiltrate indicated that a high proportion of negative for interleukin 2 receptor. the cells were monocytes/macrophages (Fig. Pineal Inflammation: Pinealitis was pre- 74 H. S. DUA ET AL. a IPL - INL- OPL- ONL- ROS - RPE- c- S- c d Fig. 2. Ocular changes in rats. (a) Normal retina in an animal from Group 1 treated with anti-S monoc lonal antibody S2. 4. CS. The rod outer segments (ROS) are well preserved. IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium; C, choroid; S, sclera. (Toluidine blue and basic fuschin, x2BO). (b) Early lesion. Showing round cell infiltration in the photoreceptor layer (arrow) with vacuolation and disruption of the rod outer segments. (Toluidine blue and basic fuschin, x3S0). (c) Advanced lesion. Showing perivascular infiltra tion (thick arrow), loss of photoreceptor layer with subretinal infiltration (thin arrow) and a thickened choroid packed with inflammatorycells. (Toluidine blue and basic fuschin, X 200). (d) Subretianal exudate showing predominantly cells that are stained by the monocyte/macrophage monoclonal marker (EDI, arrow). Note absence of photoreceptor layer. (Alkaline phosphatase-Antialkaline phosphatase (APAAP) xIOO). sent in all three groups of S-antigen groups occurred as a mild to moderate sub immunised rats whether untreated or treated capsular focal granuloma, frequently close to with either rat IgG or the monoclonal anti the pineal stalk. Perivasculitis was a common body S2.4.C5 (Figs 3b and c). Two rats (one feature and the predominant cells were lym from each of the control group) that failed to phocytes and monocytes. Immunocytochemi develop EAU did not show any pineal cally, CD4 cells were scattered throughout inflammation either (Fig. 3a). Pinealitis in all the gland (Fig. 3d) with monocytes and CD8 ANTI-S MONOCLONAL ANTIBODY IN EAU 7S Fig. 3. Pineal changes in rats. (a) Normal rat pineal. (Haematoxylin and eosin x200). (b) Subcapsular focal granulomatous infiltration. (arrow, Ha�matoxylin and eosin x200). (c) Perivasculitis (arrow) with subcapsular infiltration. (Toluidine blue and basic fuschin x450). (d) Inflammatory infiltration in the pineal stalk region showing predominantly cells that are stained by the T-helper cell monoclonal marker CD4 (W3-27). (APAAP x350). cells next in frequency. A few B cells were ELISA between 7 and 21 days post immuni also noted. The relative proportions of cells sation (Fig. 4). Rats treated with monoclonal in inflammatory lesions was the same for all antibody S2.4.C5 consistently showed higher three groups of rats. Ia antigen expression levels of anti-S antibodies in their serum than was a common feature, as expected, but cells untreated or IgO treated controls (Fig. 4). expressing interleukin 2 receptor were not After 21 days antibody levels were maximal observed. for all three groups. Humoral response to S-antigen Discussion Untreated and IgO treated rats which were Modulation of the host immune response immunised with bovine S-antigen showed a using monoclonal antibodies, although still progressive increase in anti-S antibodies by experimental, has great potential value in the 76 H. S. DUA ET AL. ANTI-S ANTIBODY TITRES IN THE DIFFERENT GROUPS OF RATS epitopes of S-antigen may be identical. They synthesised 23 peptides corresponding to the 2.8 2.4 entire 404 amino acid sequence of the S-anti • MoAb 2.0 o SALINE gen molecule and demonstrated that one dRAT IgG 1.6 peptide nominated peptide M (18 amino A490 1.2 acids in length) was highly pathogenic and 0.8 consistently induced EAU and EAP in Lewis 0.4 rats. If the protection provided by the monoclonal antibody S2.4.C5, against EAU 7 14 21 28 DAYS was induced by idiotype anti-idiotype net work responses then it should also have inhi Fig. 4. Graph showing anti-S antibody titres in the various groups of rats measured by ELISA bited EAP. (A490) at weekly intervals from day 1 to day 28. It is also unlikely that the monoclonal anti body could have masked antigenic epitopes on the S-antigen molecule in the rod outer management of autoimmune diseases. The segments27 thereby interfering either with its aim of this approach is to induce selective presentation to the lymphoid cells or with its tolerance to certain antigens whilst retaining interaction with effector cells since target cell responsiveness to others. Benjamin and responses occurred normally in the pineal Waldman25 were able to induce tolerance in gland in S2.4.C5 treated rats. An alternative mice to immunogenic antigens by treatment explanation for the inhibitory effect of with rat monoclonal antibodies directed S2.4.C5 on EAU but not on EAP may lie in against the CD4 cells. the requirement for effective homing De Kozak et al19 succeeded in inhibiting mechanisms for sensitised cytotoxic lympho EAU in rats by the administration of mouse cytes. Whereas, in the eye a blood-retinal S-antigen specific moncolonal antibodies barrier is well defined, a blood pineal barrier simultaneously with the S-antigen. They does not exist.28 In EAU sensitised lympho suggested that the heterologous monoclonal cytes adhere to activated retinal vascular antibody assumed a protective role by induc endothelium and it has been suggested that ing the formation of anti-idiotypic this mechanism is essential for directing lym antibodies. The mouse monoclonal afforded phocyte traffic to sites of inflammation.29 It is protection from S-antigen induced uveitis in possible that S2.4.C5 inhibits this interaction rats probably because of the large number of between lymphocytes and endothelium at the antigenic determinants shared by these two blood-retinal barrier, but has no effect in the species of rodents. However, the heterolog barrier-free pineal. gland where the vascular ous system that we used demonstrated that endothelium is fenestrated.30,31 the protective role of the S-antigen specific The presence of relatively higher levels of monoclonal antibody, S2.4.C5, does not antibodies during the first two weeks of extend far across species. All the guinea pigs immunisation in S2.4.C5 treated animals is of that received S2.4.C5 along with S-antigen interest. Although the fate of the monoclonal developed a severe uveitis and pinealitis. The antibody after inoculation in the rats is not onset of uveitis was two days earlier than in known, some of this anti-S activity was prob the control group indicating that the monoc ably due to the inoculated monoclonal anti lonal may have in fact acted like an adjuvant. body in the early stage. After one week it is On the other hand, our homologous system probable that much of the injected monoc using a rat monoclonal antibody in rats, was lonal antibody had been degraded or proces extremely effective in inhibiting EAU. sed. In a heterologous system, de Kozak et Interestingly this contrasted with the obser apo inoculated rats with a mouse anti-S vation that S2.4.C5 was not effective in monoclonal antibody and failed to detect its inhibiting the pineal inflammation in rats. presence in the sera after one week, although Recent evidence presented by Donoso et aP6 in the present homologous system experi indicates that EAU and EAP inducing ment, some of the monoclonal antibody may ANTI-S MONOCLONAL ANTIBODY IN EAU 77 7 have persisted for longer periods. In sub Dumonde DC, Kasp-Grochowska E, Graham sequent weeks, particularly the third and E, Banga JP, Sanders MD, Stanford MA, fourth weeks post-immunisation, anti-S titres Faure JP, de Kozak Y, Tuyen VV: Autoim were equally raised in all groups. At this mune mechanisms in inflammatory eye dis stage the polyclonal host-response to S-anti ease. Trans Ophthalmol Soc UK 1985, 104: 232-8. gen would have predominated and was 8 Hallet JW, Wolkowicz MI, Leopold IH, clearly not related to induction of tolerance Canamucio C, Wijewski E: Autoimmune and disease prevention as it was similar in all complement fixation test in endogenous groups of animals. uveitis. Arch Ophthalmol1962,68: 168-71. Whatever the mechanism, it is clear that 9 Rahi AHS, Holborow El, Perkins ES, Gurgen monoclonal antibody S2.4.C5 is highly effec YY, Dinning WI: Immunological investiga tive in abrogating EAU and this result raises tions in uveitis. Trans Ophthalmol Soc UK the possibility of similar strategies being 1976,96: 113--22. 10 adopted in human disease. There are, how Kalsow CM and Wacker WB: Pineal reactivity ever, many problems which will have to be of anti-retina sera. Invest Ophthalmol Vis Sci 1977, 16: 181--4. overcome not least of which is identifying 11 Kalsow CM and Wacker WB: Rabbit ocular and patients with chronic intraocular inflamma pineal autoimmune response to retina anti tion who have clear cut evidence of autoim gens. Curr Eye Res 1986,5: 579-86. munity to retinal antigens. For this to be 12 Mochizuki M, Charley 1, Kuwabara T, Nus achieved we must await appropriately sensi senblatt RB, Gery I: Involvement of the tive tests which will differentiate 'natural' pineal gland in rats with experimental autoim autoimmunity from pathological autoim mune uveitis. 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