September 2002
Volume 43, Issue 9
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Immunology and Microbiology  |   September 2002
Spontaneous Induction of Immunoregulation by an Endogenous Retinal Antigen
Author Affiliations
  • Dale S. Gregerson
    From the Department of Ophthalmology, University of Minnesota, Minneapolis, Minnesota.
  • Chunzhi Dou
    From the Department of Ophthalmology, University of Minnesota, Minneapolis, Minnesota.
Investigative Ophthalmology & Visual Science September 2002, Vol.43, 2984-2991. doi:
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      Dale S. Gregerson, Chunzhi Dou; Spontaneous Induction of Immunoregulation by an Endogenous Retinal Antigen. Invest. Ophthalmol. Vis. Sci. 2002;43(9):2984-2991.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Ocular immune deviation studies have relied on intraocular injections of antigen to induce altered immune responses. Contributions of the injection process itself have complicated study of the mechanisms and interpretations of biological significance. In the current study, transgenic mice were used to determine the presence of immune deviation or other evidence of immunoregulation, elicited by endogenous Escherichia coli β-galactosidase (β-gal) expressed through a retinal arrestin promoter.

methods. Mice that express β-gal in the retina and various control mice were immunized with β-gal and tested for immune responsiveness by the ear-swelling test for delayed-type hypersensitivity (DTH), in vitro proliferation assays, and cytokine assays. Spleen cells from transgenic mice were also transferred to normal recipients to test for transfer of immune deviation.

results. Endogenous retinal β-gal expression depressed the DTH response and proliferation assays after β-gal immunization. The ability to depress DTH was transferred by naïve spleen cells from transgenic mice to nontransgenic mice. Use of an immunization protocol that included high-dose Mycobacterium tuberculosis (M tb) H37Ra adjuvant and concurrent administration of pertussis toxin reversed the inhibition of DTH.

conclusions. An endogenous neo self-retinal antigen alters subsequent immune responses without intraocular injection, suggesting that normal retinal proteins in quiet eyes induce immunoregulatory responses. Based on cytokine assays, there were similarities to the immune deviation induced by intraocular inoculation in the IL-4 response, but the IL-10, IFN-γ, and TGF-β1 results were not similar, indicating that the mechanisms differ. The ability of supplemented adjuvant to overcome endogenous tolerance may be related to the requirement for supplemented adjuvants in the induction of experimental autoimmune uveoretinitis.

Immune deviation resulting from injection of antigen into the anterior chamber (AC) of the eye is now a well-described outcome, the mechanisms of which continue to be identified. 1 Recent studies have extended immune deviation-promoting properties beyond the AC to the vitreous cavity 2 and subretinal space. 3 4 A source of uncertainty in these studies is the role played by the antigen injection process itself. These are threefold: first, concern for the physiological relevance of administering a bolus of antigen; second, concerns that tissue trauma resulting from the mechanical injection plays a role in induction of immune deviation; and third, the possibility that antigen leaking from an ocular injection site may reach other tissues, especially external mucosal surfaces or the lymphatic drainage associated with external structures, leading to peripheral tolerance. Although efforts have been made by laboratories studying ocular immune deviation to refine the injection techniques, there has been no conclusive demonstration that these are not significant concerns. 
Our laboratory used several types of transgenic mice that express β-galactosidase (β-gal) on various promoters to achieve expression in selected tissues, including retinal photoreceptor cells. These mice provided the opportunity to test for the presence of immunoregulation induced by an endogenous protein, because no injection is needed to place antigen into the eye. Using these transgenic mice and negative control animals, we showed that endogenous β-gal expression in the retina leads to altered responses that differ from immune deviation elicited by ocular inoculation of antigen. 
Methods
Mice
B10.A mice carrying β-gal transgenes driven by various promoters were used. These include hi-arr-β-gal mice (photoreceptor cell expression), which were derived as previously described. 5 Mice expressing β-gal on a GFAP promoter (line 16) were obtained from Lennart Mucke, 6 and were backcrossed to B10.A for a minimum of 10 generations. The hi-arr-β-gal and GFAP mice are backcrossed to homozygosity for the β-gal transgene. B6-ROSA26 mice, which give widespread expression of β-gal, were purchased from Jackson Laboratories (Bar Harbor, ME) and backcrossed to B10.A for a minimum of 10 generations. The ROSA26 mice are hemizygous for the β-gal transgene. The ROSA26 mice were used as the positive control for the presence of peripheral tolerance to β-gal as a self-antigen. Their widespread expression of antigen, which is predominantly nonocular, leads to differences that are detected by a variety of immunoassays. Use of mice was in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Mice were housed under specific pathogen-free (SPF) conditions and maintained on lactose-free chow. 
Antigens and Immunization
β-Gal was purchased from Prozyme (San Leandro, CA). Several other sources were found to be contaminated with other proteins. Mice were immunized subcutaneously on a hind thigh with β-gal in CFA emulsions at the indicated doses. Two CFA preparations were used in two different immunization regimens. Low-dose CFA contained 1 mg/mL Mycobacterium tuberculosis (M tb) H37Ra in oil (Sigma, St. Louis, MO). Mice receiving low-dose CFA were not given pertussis toxin (PTx); experimental autoimmune uveoretinitis (EAU) did not develop in these mice. High-dose CFA-PTx is the combination of high-dose M tb in oil and concurrent inoculation of PTx that is commonly used to induce EAU with retinal proteins, including β-gal, in rodents. It was prepared from the conventional low-dose formulation by the addition of 4 mg/mL M tb H37Ra. Mice receiving the high-dose CFA received a single intraperitoneal injection of 0.5 μg PTx (Sigma) at the time of immunization. 
Xgal Staining
Except for the pineal gland, 5-bromo-4-chloro-3-indoyl-β-galactosidase (Xgal) staining was performed as previously described. 5 To test for β-gal in the pineal gland, mice were killed by CO2 inhalation and fixed with a 1-hour transcardiac perfusion of 2% paraformaldehyde, 0.1% glutaraldehyde, 0.02% NP-40, and 0.01% deoxycholate in PBS (pH 7.4). The skull was removed in the area of the lambda reference point. The attached dura and pineal gland were detached and postfixed for 1 hour. Xgal staining was performed at room temperature overnight. The stained tissue was washed with PBS, postfixed for 1 hour, and embedded in optimal temperature cutting compound (OTC). Serial cryosections were mounted and counterstained with nuclear fast red. 
Measurement of Delayed-Type Hypersensitivity
With a 30-gauge needle, mice were given an intrapinna injection of 100 μg β-gal in 10 μL saline in the left ear and saline only in the right ear. After 24 and 48 hours, ear thickness was measured with a micrometer (22-111; Mitutoyo, Kawasaki, Japan). The difference in ear thickness due to a delayed-type hypersensitivity (DTH) response to antigen was determined by measuring the thickness of the left ear and subtracting the thickness of the right ear and calculating the average difference in the group. Erythema resulting from the DTH reaction was determined by measuring the diameter of the reaction on two axes at a right angle. The average diameter was calculated and used to determine the area of the reaction, assuming a circular area. 
AC Inoculations
The cornea was penetrated near the limbus at a shallow angle with an 18-g microvitreo-retinal (MVR) knife, releasing a small amount of aqueous. A 33-g cannula was inserted into this slit, and 1.1 μL saline or saline containing β-gal was injected slowly into the AC. The cannula was withdrawn, and the eye was copiously flushed with saline to wash away antigen that may have leaked from the injection site. 
Lymphocyte Proliferation Assays
Spleen cells were prepared and stimulated with the indicated concentrations of β-gal in 96-well plates, as previously described. 5  
ONPG Assay
For the o-nitrophenyl-β-d-galactopyranoside (ONPG) assay, eyes and brain from Tg and control mice were first minced with scissors and then homogenized on ice in assay buffer (100 mM Na phosphate [pH 7.0], 10 mM KCl, 1 mM MgSO4, 35 μM β-mercaptoethanol) containing 50 μg/mL DNase I. Brain samples weighing approximately 0.4 g were obtained roughly as a cube from above the interaural line. Pineal glands were removed with a small piece of dura, and stalk material was left attached. Debris was removed by centrifugation, and supernatants were collected for assay of β-gal activity. Samples of aqueous humor were also collected by tapping the AC. Assays for β-gal content in the samples were performed with the ONPG substrate, as previously reported, with the use of purified Escherichia coli β-gal (Prozyme) as the standard. 7  
Cytokine Assays
The cytokine content of culture supernatants taken from lymphocyte proliferation assays was determined by ELISA, according to the manufacturer’s suggestions, as previously reported. 5 The antibody pairs and the murine cytokine standards for IL-4, IL-10, and IFN-γ were purchased from PharMingen (San Diego, CA). Reagents for the TGF-β1 assay were purchased from R&D Systems (Minneapolis, MN). 
Results
Xgal Staining for Expression of β-Gal in the Tg Mice
The tissue expression patterns and relative staining intensity of β-gal are shown in Figure 1 . Although some aspects of the expression have been reported elsewhere, 5 6 8 9 10 the mice have been backcrossed and bred for many generations, raising the possibility that expression has changed. As a result, we confirmed that the expected expression had been retained. Expression of β-gal in B10.A-GFAP-β-gal mice was indistinguishable from that of the line 16 mice from which they were derived (Figs. 1A 1B 1C 1D 1E) , showing expression in astrocytes of brain and retina, optic nerve, and the epithelium of lens and cornea. There was no thymic expression in GFAP mice by RNase protection assay, 6 or by Xgal or ONPG assays. The B10.A-ROSA26 mice expressed β-gal in a wide variety of tissues, including the eye, brain, bone marrow, kidney, thymus, and spleen (not shown), at levels ranging from low to high (Figs. 1F 1G 1H 1I 1J 1K 1L) . The hi-arr-β-gal mice continued to express at a high level in the retinal rod photoreceptor cells, at a much lower level in the pineal gland, and at a trace level in a small number of brain cells (Figs. 1M 1N 1O) , as previously reported. 5 9 11 We did not find expression elsewhere, including the spleen, bone marrow, and thymus, which was also tested by RT-PCR. 
Quantitation of β-Gal in Eyes and Brain of the Tg Mice
The amounts of β-gal expressed in tissues are shown in Table 1 . The homozygous hi-arr-β-gal mice expressed β-gal in the retina at levels consistent with those in our previous assays. 9 Expression in the hi-arr-β-gal pineal gland was less than 0.5 ng per pineal sample, but was detected by Xgal staining (Fig. 1N) . Similarly, expression of β-gal expression in brain was below detection by ONPG, but was found in a few cells by Xgal staining (Fig. 1O) . Because of the prominent expression of β-gal in the optic nerve of GFAP mice, a significant portion of the ocular β-gal in these mice is due to that part of the optic nerve that remains with the globe, even though the nerve is cut flush with the surface. To determine the levels of β-gal in the aqueous humor, samples were taken from transgenic eyes for assay. β-Gal was undetectable (<0.19 ng/3–5 μL aqueous humor) using samples from the GFAP-β-gal and hi-arr-β-gal transgenic mice. The 3- to 5-μL samples obtained from these eyes constitute a substantial portion of the total aqueous fluid from the anterior and posterior chambers. 
Induction of Immune Deviation by AC Inoculation with β-Gal
To establish that β-gal can induce both DTH and immune deviation, B10.A mice were inoculated in the AC with 35 μg β-gal in 1.1 μL normal unbuffered saline, or with saline only. After low-dose CFA immunization with β-gal, the mice were inoculated in the ears for DTH testing. Ear thickness and erythema were measured in all mice. Nonimmunized control mice showed no ear swelling due to intrapinna inoculation of β-gal, whereas immunized mice gave strong DTH responses (Fig. 2A) . Inoculation of β-gal into the AC inhibited subsequent development of DTH, as assessed by ear swelling, as found in studies of anterior-chamber–associated immune deviation (ACAID). Assessment of erythema produced similar results (Fig. 2B)
Development of DTH in β-Gal Transgenic Mice after Immunization
Because the transgenic mice express β-gal as an endogenous self-antigen under the control of different promoters, these mice were tested for evidence of immune deviation resulting from expression of endogenous antigen. DTH was strongly inhibited in all the transgenic mice, whether assessed by ear swelling (Fig. 3A) or erythema (Fig. 3B) . As expected, EAU did not develop in the hi-arr-β-gal mice when the low-dose CFA immunization protocol was used. 
Because DTH can be absent even in the presence of a substantial immune response, additional evidence of responses in these mice was sought. Antigen-specific proliferation of spleen cells was largely inhibited in cells harvested from the hi-arr-β-gal and ROSA26 transgenic mice immunized with β-gal, compared with β-gal–negative control animals (Fig. 4) . Spleen cells from the GFAP-β-gal mice gave a modest proliferative response that was reduced compared with the response in the control mice. These results show that endogenous expression of β-gal in the photoreceptor cells, as well as elsewhere, is capable of modulating the systemic response to immunization with β-gal in CFA. 
Transfer of Immunoregulation by Spleen Cells from Naive β-Gal Transgenic Mice
One of the hallmarks of ACAID is that the regulatory activity can be transferred to recipient mice by cells. 12 Spleen cells from naïve β-gal–negative and transgenic mice were collected, washed, and transferred intraperitoneally to β-gal–negative recipients. After 1 day, the recipients were immunized with β-gal in low-dose CFA and the ear test performed 7 days later. Spleen cells from all the transgenic donors, but not β-gal-negative donors depressed the DTH response (Fig. 5) . The recipient mice were also tested for responsiveness in vitro by proliferation assay. Although DTH was inhibited, β-gal–induced proliferation of spleen cells from the recipient mice was not significantly inhibited (Fig. 6)
Effect of EAU-Inducing Adjuvants on Endogenous Immunoregulation
The use of CFA supplemented with high doses of M tb, especially the H37Ra strain, together with intraperitoneal or intravenous inoculation of PTx has been in wide use in many models of experimental autoimmune disease. Its use in the murine retinal antigen EAU model, including the B10.A strain, was introduced and shown to be required by Caspi et al. 13 If the results of the previous figures using β-gal apply to the retinal proteins of non-Tg mice, including interphotoreceptor matrix retinoid-binding protein (IRBP) and arrestin, it is reasonable to ask why mice are susceptible to retinal antigen-induced EAU. At least part of the answer is shown in Figure 7 , which shows that the low-dose CFA and high-dose CFA-PTx immunization protocols produced different outcomes. DTH was strongly inhibited in the hi-arr-β-gal mice immunized with the low-dose protocol, whether assessed by ear swelling (Fig. 7A) or erythema (Fig. 7B) . Conversely, there was no difference in magnitude between the DTH responses of the non-Tg versus the hi-arr-β-gal mice if the high-dose CFA-PTx immunization protocol was used. The DTH response of the high-dose mice appeared lower than that of the low-dose control mice in two of the three repeats of this experiment, but the difference was not statistically significant. 
Cytokine Production
Within 3 days of completion of the ear-swelling tests, mice were killed and spleen cells cultured in the presence or absence of antigen. After 48 hours, supernatants were collected for cytokine analysis (Fig. 8) . The IFN-γ production by the Tg and control mice that received low-dose CFA was low, and not different from each other, even though the DTH responses of these mice were very different (Fig. 7) . IFN-γ production by the high-dose CFA-PTx primed mice was substantial, but did not differ between the Tg and control mice. The highest IL-4 response, which was elicited by antigen, was found in the low-dose CFA-Tg mice whose DTH response was inhibited (Fig. 7) . The IL-10 and TGF-β1 responses did not seem to correlate with the Tg status, use of CFA-PTx, or the DTH response. Only the IL-4 results are consistent with ACAID-like immune deviation. 
Discussion
Our findings provide evidence that extends knowledge of the means by which the eye and the retina alter responses to antigens. The results show that the presence of antigen in the retina can alter responses without the manipulations or conditions associated with inoculation of antigen and that the altered response can be transferred to normal mice with naïve spleen cells. 
The ocular dependence of the deviation is a significant consideration. The expression pattern is representative of photoreceptor cell proteins in general, because the photoreceptor conserved element (PCE)-1 promoter drives the pattern of expression. 14 We looked for β-gal in sites where arrestin expression has been reported, because the hi-arr-β-gal mice possess an arrestin-derived promoter. The reported sites are numerous and based on a variety of techniques, including Tg mice prepared to test various elements of the arrestin promoter. These sites include retina, 11 pineal gland, 11 brain, 11 thymus, 15 choroid plexus, 16 ciliary epithelium, 17 iris, 18 lens, 19 and brain tumors. 20 The accuracy of some observations is uncertain, because of the high degree of homology between the various arrestins, which tests the fidelity of the probes. Furthermore, the promoter construct used in the hi-arr-β-gal mice is not the complete, endogenous promoter, and may not have regulatory sites that would give expression elsewhere, such as thymus. The only expression we find in these hi-arr-β-gal mice is in retina (≈300 ng/mouse), pineal gland (positive by Xgal staining, but <0.5 ng/pineal gland), and a very small number of brain cells found in a small, discrete cluster in the amygdala. Consequently, the retinal expression is almost certainly responsible for the degree of immune deviation we found, but the pineal or brain expression could make a small contribution. The converse notion—that is, that pineal–brain expression is responsible for the deviation rather than the 300 ng of retinal expression—would be difficult to defend. 
The mechanism of endogenous immunoregulation in hi-arr-β-gal mice is not clear at this time. The increased IL-4 production by spleen cells from the hi-arr-β-gal mice immunized with the low-dose CFA protocol is consistent with the depressed DTH of this group and is consistent with findings in ACAID studies. 21 The IFN-γ results are not consistent with ACAID results, in that they do not correlate with the magnitude of the ear-swelling test findings in which decreased IFN-γ is associated with decreased DTH. 21 However, the IFN-γ production correlates with the use of high-dose CFA-PTx. The IL-10 results are not consistent with the ACAID model, which predicts elevated IL-10 in mice with immune deviation. 21 22 The production of TGF-β1 was not increased by inclusion of antigen in the cultures and did not correlate with the Tg status of the mice, use of CFA, or the DTH response. Elevated TGF-β production is associated with ACAID. 21  
The immunoregulation found in the hi-arr-β-gal mice does not appear to involve conventional central tolerance by deletion of β-gal–specific T cells in the thymus, for several reasons. First, we previously reported that the repertoire of immunodominant epitopes recognized by CD4 T cells after immunization (using high-dose CFA with PTx) of hi-arr-β-gal mice compared with β-gal–negative mice was unchanged. 5 Second, the use of the high-dose immunization protocol restored the DTH response of hi-arr-β-gal mice. If there were a substantial reduction in the number of mature β-gal–specific T cells exiting the thymus, reversal would not be the predicted response. Third, thymic expression of β-gal in hi-arr-β-gal mice was not detected by several assays. Because the thymic expression in the ROSA26 mice is obvious by Xgal staining, and these mice retain a significant CD4 T cell response to β-gal, 8 it is unlikely that negative selection could account for the results in the hi-arr-β-gal mice, which gave no evidence of thymic expression. Furthermore, the ROSA26 mice generated transferable regulation, even though they had detectable thymic expression. Fourth, by several in vitro assays, none of our CD4 T-cell lines and clones show evidence of activation due to endogenous β-gal after incubation with hi-arr-β-gal thymus, spleen, or lymph node (Gregerson Lab, unpublished observations, 1997–2000). Finally, the inhibition was transferable by spleen cells from naïve hi-arr-β-gal mice. 
The ability to transfer the inhibition with spleen cells from naïve hi-arr-β-gal mice suggests that cells carry the activity, consistent with the findings for ACAID, which is well known to be transferable with spleen cells. 12 23 However, the endogenous regulatory response may well differ from ACAID because of antigen dose and location. Because little antigen, compared with that used in ACAID inoculation protocols, is available to induce the response found in the current study and the antigen is not detectable in the aqueous of the hi-arr-β-gal mice, it is possible that the mechanisms leading to the endogenous regulatory response differ from those induced by inoculation of the AC with antigen. 
Another difference is that inoculation with a bolus of antigen has been used in studies of ACAID and similar studies, whereas the transgenic mice express a much smaller amount of antigen continuously and intracellularly. In studies of ACAID, it has been shown that antigen is gathered by antigen-presenting cells (APCs) in the AC after inoculation and delivered to the spleen, where the interactions of several cells, some of which are thymus dependent, 24 25 are required for expression of immune deviation. 26 27 28  
Although the β-gal is intracellular, there is the possibility that it leaks out of the photoreceptor cells of the retina, possibly during recycling of the rod photoreceptor cell outer segments. Because the level of β-gal that is present in the aqueous humor of hi-arr-β-gal mice is undetectable, we think it more likely that the antigen is instead gathered near the source in the parenchyma of the retina or the subretinal space, presumably by microglia, recently shown to migrate to that space, 29 or by other macrophage-like cells. Whether and how these cells leave the retina and whether they have a regulatory function is unknown at this time. 
The inhibition found in the GFAP-β-gal mice may occur by entirely different mechanisms, because in these mice β-gal is expressed not only in brain, where brain-associated immune deviation (BRAID) has been shown to exist, 30 31 but also in optic nerve and the epithelium of the cornea and lens. Normal turnover of β-gal–positive corneal epithelium in GFAP-β-gal mice provides the potential for continuous contact with the local mucosal immune system, known to induce peripheral tolerance. 32 There is also the potential for Langerhans cells to acquire and present the antigen from these corneal epithelial cells. 33 34 As a result, there may be several mechanisms contributing to immunoregulation in these mice. 
The immunoregulatory response of the ROSA26 mice is likely to be different from that found in hi-arr-β-gal mice. The ROSA26 mice express β-gal at comparatively low levels in many tissues, including thymus, spleen and bone marrow. Despite widespread expression, tolerance induction is incomplete because these mice respond to β-gal at reduced levels in lymphocyte proliferation assays when immunized with the high-dose CFA-PTx protocol used for EAU induction. 8 They also produce lower levels of circulating antibody to β-gal. 8  
These results call for a revised paradigm that integrates both this new finding and existing, reliable data that do not, at first glance, fit with these results. 9 The single most significant factor may be related to our finding that the high-dose CFA-PTx reversed the inhibition of DTH found in the hi-arr-β-gal mice. The finding of immune regulation to β-gal when present as an endogenous retinal antigen may explain in part why EAU induction protocols for retinal autoantigens, including β-gal in the hi-arr-β-gal mice, require high-dose adjuvants. In all our previous studies, we have used the high-dose protocol. Inhibition of proliferative responses to β-gal found in the transgenic mice is less effective when the CFA is supplemented in the high-dose manner required for induction of autoimmune disease. 5 9  
 
Figure 1.
 
Xgal staining of tissues from the Tg mice. (AE) GFAP-β-gal mice; (FL) B10.A-ROSA26 mice; and (MO) hi-arr-β-gal mice. (A) retina; (B) optic nerve; (C) low-power collage of a horizontal section through the brain showing staining in multiple structures; (D) lens epithelium; (E) corneal epithelium; (F) retina; (G) corneal epithelium; (H) brain, cerebellum; (I) brain, hippocampus; (J) bone marrow; (K) kidney; (L) thymus; (M) retina; (N) pineal gland; and (O) brain. Arrows: regions of Xgal staining. Magnification: (A, D, E, F, KM) ×20; (B, G, J, N, O) ×40; (H, I) ×4.
Figure 1.
 
Xgal staining of tissues from the Tg mice. (AE) GFAP-β-gal mice; (FL) B10.A-ROSA26 mice; and (MO) hi-arr-β-gal mice. (A) retina; (B) optic nerve; (C) low-power collage of a horizontal section through the brain showing staining in multiple structures; (D) lens epithelium; (E) corneal epithelium; (F) retina; (G) corneal epithelium; (H) brain, cerebellum; (I) brain, hippocampus; (J) bone marrow; (K) kidney; (L) thymus; (M) retina; (N) pineal gland; and (O) brain. Arrows: regions of Xgal staining. Magnification: (A, D, E, F, KM) ×20; (B, G, J, N, O) ×40; (H, I) ×4.
Table 1.
 
Quantitation of β-Gal in Eyes and Brain from Tg and Control Mice
Table 1.
 
Quantitation of β-Gal in Eyes and Brain from Tg and Control Mice
Mice Whole Eye (ng) Brain (ng/g Wet Weight)
Hi-arr-β-gal 164 ± 36 BD, †
GFAP-β-gal 62.3 ± 8.7 299 ± 77
B10.A-ROSA26 6.8 ± 1.2 81 ± 27
β-gal BD* BD, †
Figure 2.
 
DTH develops in B10.A mice immunized with β-gal in CFA, and ACAID develops when β-gal is injected into the AC before immunization. β-gal–negative B10.A mice were inoculated in the AC of one eye with 35 μg β-gal in 1.1 μL saline or with saline only. Eight days later, the mice were immunized subcutaneously (low-dose protocol) with or without β-gal (200 μg in 200 μL CFA emulsion). Control mice were similarly immunized with β-gal or not immunized. After 7 days, intrapinna injections of β-gal (100 μg in 10 μL saline) were performed in the left ear and saline only in the right ear. After 24 and 48 hours, the ears were measured for swelling (A) and erythema (B). Probabilities were calculated by t-tests of the data in the indicated groups. Results are from 48-hour readings.
Figure 2.
 
DTH develops in B10.A mice immunized with β-gal in CFA, and ACAID develops when β-gal is injected into the AC before immunization. β-gal–negative B10.A mice were inoculated in the AC of one eye with 35 μg β-gal in 1.1 μL saline or with saline only. Eight days later, the mice were immunized subcutaneously (low-dose protocol) with or without β-gal (200 μg in 200 μL CFA emulsion). Control mice were similarly immunized with β-gal or not immunized. After 7 days, intrapinna injections of β-gal (100 μg in 10 μL saline) were performed in the left ear and saline only in the right ear. After 24 and 48 hours, the ears were measured for swelling (A) and erythema (B). Probabilities were calculated by t-tests of the data in the indicated groups. Results are from 48-hour readings.
Figure 3.
 
Development of DTH after immunization with β-gal was inhibited in β-gal–expressing transgenic mice. Transgenic and control mice were immunized subcutaneously with 200 μg in 100 μL CFA (low-dose protocol). One group of control mice was not immunized. Seven days later, ears were inoculated with 100 μg β-gal or saline only. After 24 and 48 hours, the ears were measured for swelling (A) and erythema (B). Probabilities were calculated by t-tests of the data in the indicated groups compared with the nonimmune B10.A group. None of the results in the transgenic mice were significantly different from those in the nonimmune control animals. Results are from 48-hour readings.
Figure 3.
 
Development of DTH after immunization with β-gal was inhibited in β-gal–expressing transgenic mice. Transgenic and control mice were immunized subcutaneously with 200 μg in 100 μL CFA (low-dose protocol). One group of control mice was not immunized. Seven days later, ears were inoculated with 100 μg β-gal or saline only. After 24 and 48 hours, the ears were measured for swelling (A) and erythema (B). Probabilities were calculated by t-tests of the data in the indicated groups compared with the nonimmune B10.A group. None of the results in the transgenic mice were significantly different from those in the nonimmune control animals. Results are from 48-hour readings.
Figure 4.
 
Proliferation assays of spleen cells collected after the ear-swelling assays. Spleen cells from the indicated control and transgenic mice were stimulated in vitro with β-gal to assess proliferative responses. All mice were immunized as described in Figure 2 , except the nonimmune control mice. Assays included β-gal–negative B10.A (▪); hi-arr-β-gal (□); GFAP-β-gal (○); ROSA26 (⋄); and nonimmune B10.A (•).
Figure 4.
 
Proliferation assays of spleen cells collected after the ear-swelling assays. Spleen cells from the indicated control and transgenic mice were stimulated in vitro with β-gal to assess proliferative responses. All mice were immunized as described in Figure 2 , except the nonimmune control mice. Assays included β-gal–negative B10.A (▪); hi-arr-β-gal (□); GFAP-β-gal (○); ROSA26 (⋄); and nonimmune B10.A (•).
Figure 5.
 
Spleen cells from β-gal transgenic mice transferred inhibition of DTH to normal β-gal recipients. Spleen cells were collected from naïve transgenic and nontransgenic mice. Erythrocytes were lysed, and 30 × 106 cells transferred to β-gal–negative recipients. The next day, all recipient mice were immunized with 200 μg β-gal (low-dose protocol), followed by an ear-swelling test 7 days later, as described in Figure 2 . Probabilities are for comparisons with the recipients of spleen cells from β-gal–negative donors. Results are from 48-hour readings.
Figure 5.
 
Spleen cells from β-gal transgenic mice transferred inhibition of DTH to normal β-gal recipients. Spleen cells were collected from naïve transgenic and nontransgenic mice. Erythrocytes were lysed, and 30 × 106 cells transferred to β-gal–negative recipients. The next day, all recipient mice were immunized with 200 μg β-gal (low-dose protocol), followed by an ear-swelling test 7 days later, as described in Figure 2 . Probabilities are for comparisons with the recipients of spleen cells from β-gal–negative donors. Results are from 48-hour readings.
Figure 6.
 
Proliferation assays of spleen cells collected after the ear-swelling assays. Spleen cells from the recipient mice were stimulated in vitro with β-gal to assess proliferative responses. All mice were immunized. Assays included β-gal–negative B10.A (▪); hi-arr-β-gal (□); GFAP-β-gal (○); and ROSA26 (⋄).
Figure 6.
 
Proliferation assays of spleen cells collected after the ear-swelling assays. Spleen cells from the recipient mice were stimulated in vitro with β-gal to assess proliferative responses. All mice were immunized. Assays included β-gal–negative B10.A (▪); hi-arr-β-gal (□); GFAP-β-gal (○); and ROSA26 (⋄).
Figure 7.
 
Effect of adjuvant on the development of DTH in hi-arr-β-gal and control mice. Groups of mice were immunized with either the low-dose or high-dose protocols. All mice underwent an ear-swelling test for DTH 7 days later, as described in Figure 2 . Probabilities are for comparisons between data in the bracketed groups. Results are from 48-hour readings of ear swelling (A) and erythema (B). This experiment was performed three times with similar results.
Figure 7.
 
Effect of adjuvant on the development of DTH in hi-arr-β-gal and control mice. Groups of mice were immunized with either the low-dose or high-dose protocols. All mice underwent an ear-swelling test for DTH 7 days later, as described in Figure 2 . Probabilities are for comparisons between data in the bracketed groups. Results are from 48-hour readings of ear swelling (A) and erythema (B). This experiment was performed three times with similar results.
Figure 8.
 
Production of cytokines by spleen cells harvested from the B10.A and hi-arr-β-gal mice described in Figure 7 . Spleens were collected 3 days after completion of ear-swelling tests. Samples were taken from wells with and without antigen (150 μg/mL β-gal) stimulation. Results are expressed as the mean ± SD, and the number of animals in each assay is indicated below each panel. All experiments were performed at least twice.
Figure 8.
 
Production of cytokines by spleen cells harvested from the B10.A and hi-arr-β-gal mice described in Figure 7 . Spleens were collected 3 days after completion of ear-swelling tests. Samples were taken from wells with and without antigen (150 μg/mL β-gal) stimulation. Results are expressed as the mean ± SD, and the number of animals in each assay is indicated below each panel. All experiments were performed at least twice.
The authors thank Jing Yang for assistance with producing the transgenic mice used in these studies. 
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Figure 1.
 
Xgal staining of tissues from the Tg mice. (AE) GFAP-β-gal mice; (FL) B10.A-ROSA26 mice; and (MO) hi-arr-β-gal mice. (A) retina; (B) optic nerve; (C) low-power collage of a horizontal section through the brain showing staining in multiple structures; (D) lens epithelium; (E) corneal epithelium; (F) retina; (G) corneal epithelium; (H) brain, cerebellum; (I) brain, hippocampus; (J) bone marrow; (K) kidney; (L) thymus; (M) retina; (N) pineal gland; and (O) brain. Arrows: regions of Xgal staining. Magnification: (A, D, E, F, KM) ×20; (B, G, J, N, O) ×40; (H, I) ×4.
Figure 1.
 
Xgal staining of tissues from the Tg mice. (AE) GFAP-β-gal mice; (FL) B10.A-ROSA26 mice; and (MO) hi-arr-β-gal mice. (A) retina; (B) optic nerve; (C) low-power collage of a horizontal section through the brain showing staining in multiple structures; (D) lens epithelium; (E) corneal epithelium; (F) retina; (G) corneal epithelium; (H) brain, cerebellum; (I) brain, hippocampus; (J) bone marrow; (K) kidney; (L) thymus; (M) retina; (N) pineal gland; and (O) brain. Arrows: regions of Xgal staining. Magnification: (A, D, E, F, KM) ×20; (B, G, J, N, O) ×40; (H, I) ×4.
Figure 2.
 
DTH develops in B10.A mice immunized with β-gal in CFA, and ACAID develops when β-gal is injected into the AC before immunization. β-gal–negative B10.A mice were inoculated in the AC of one eye with 35 μg β-gal in 1.1 μL saline or with saline only. Eight days later, the mice were immunized subcutaneously (low-dose protocol) with or without β-gal (200 μg in 200 μL CFA emulsion). Control mice were similarly immunized with β-gal or not immunized. After 7 days, intrapinna injections of β-gal (100 μg in 10 μL saline) were performed in the left ear and saline only in the right ear. After 24 and 48 hours, the ears were measured for swelling (A) and erythema (B). Probabilities were calculated by t-tests of the data in the indicated groups. Results are from 48-hour readings.
Figure 2.
 
DTH develops in B10.A mice immunized with β-gal in CFA, and ACAID develops when β-gal is injected into the AC before immunization. β-gal–negative B10.A mice were inoculated in the AC of one eye with 35 μg β-gal in 1.1 μL saline or with saline only. Eight days later, the mice were immunized subcutaneously (low-dose protocol) with or without β-gal (200 μg in 200 μL CFA emulsion). Control mice were similarly immunized with β-gal or not immunized. After 7 days, intrapinna injections of β-gal (100 μg in 10 μL saline) were performed in the left ear and saline only in the right ear. After 24 and 48 hours, the ears were measured for swelling (A) and erythema (B). Probabilities were calculated by t-tests of the data in the indicated groups. Results are from 48-hour readings.
Figure 3.
 
Development of DTH after immunization with β-gal was inhibited in β-gal–expressing transgenic mice. Transgenic and control mice were immunized subcutaneously with 200 μg in 100 μL CFA (low-dose protocol). One group of control mice was not immunized. Seven days later, ears were inoculated with 100 μg β-gal or saline only. After 24 and 48 hours, the ears were measured for swelling (A) and erythema (B). Probabilities were calculated by t-tests of the data in the indicated groups compared with the nonimmune B10.A group. None of the results in the transgenic mice were significantly different from those in the nonimmune control animals. Results are from 48-hour readings.
Figure 3.
 
Development of DTH after immunization with β-gal was inhibited in β-gal–expressing transgenic mice. Transgenic and control mice were immunized subcutaneously with 200 μg in 100 μL CFA (low-dose protocol). One group of control mice was not immunized. Seven days later, ears were inoculated with 100 μg β-gal or saline only. After 24 and 48 hours, the ears were measured for swelling (A) and erythema (B). Probabilities were calculated by t-tests of the data in the indicated groups compared with the nonimmune B10.A group. None of the results in the transgenic mice were significantly different from those in the nonimmune control animals. Results are from 48-hour readings.
Figure 4.
 
Proliferation assays of spleen cells collected after the ear-swelling assays. Spleen cells from the indicated control and transgenic mice were stimulated in vitro with β-gal to assess proliferative responses. All mice were immunized as described in Figure 2 , except the nonimmune control mice. Assays included β-gal–negative B10.A (▪); hi-arr-β-gal (□); GFAP-β-gal (○); ROSA26 (⋄); and nonimmune B10.A (•).
Figure 4.
 
Proliferation assays of spleen cells collected after the ear-swelling assays. Spleen cells from the indicated control and transgenic mice were stimulated in vitro with β-gal to assess proliferative responses. All mice were immunized as described in Figure 2 , except the nonimmune control mice. Assays included β-gal–negative B10.A (▪); hi-arr-β-gal (□); GFAP-β-gal (○); ROSA26 (⋄); and nonimmune B10.A (•).
Figure 5.
 
Spleen cells from β-gal transgenic mice transferred inhibition of DTH to normal β-gal recipients. Spleen cells were collected from naïve transgenic and nontransgenic mice. Erythrocytes were lysed, and 30 × 106 cells transferred to β-gal–negative recipients. The next day, all recipient mice were immunized with 200 μg β-gal (low-dose protocol), followed by an ear-swelling test 7 days later, as described in Figure 2 . Probabilities are for comparisons with the recipients of spleen cells from β-gal–negative donors. Results are from 48-hour readings.
Figure 5.
 
Spleen cells from β-gal transgenic mice transferred inhibition of DTH to normal β-gal recipients. Spleen cells were collected from naïve transgenic and nontransgenic mice. Erythrocytes were lysed, and 30 × 106 cells transferred to β-gal–negative recipients. The next day, all recipient mice were immunized with 200 μg β-gal (low-dose protocol), followed by an ear-swelling test 7 days later, as described in Figure 2 . Probabilities are for comparisons with the recipients of spleen cells from β-gal–negative donors. Results are from 48-hour readings.
Figure 6.
 
Proliferation assays of spleen cells collected after the ear-swelling assays. Spleen cells from the recipient mice were stimulated in vitro with β-gal to assess proliferative responses. All mice were immunized. Assays included β-gal–negative B10.A (▪); hi-arr-β-gal (□); GFAP-β-gal (○); and ROSA26 (⋄).
Figure 6.
 
Proliferation assays of spleen cells collected after the ear-swelling assays. Spleen cells from the recipient mice were stimulated in vitro with β-gal to assess proliferative responses. All mice were immunized. Assays included β-gal–negative B10.A (▪); hi-arr-β-gal (□); GFAP-β-gal (○); and ROSA26 (⋄).
Figure 7.
 
Effect of adjuvant on the development of DTH in hi-arr-β-gal and control mice. Groups of mice were immunized with either the low-dose or high-dose protocols. All mice underwent an ear-swelling test for DTH 7 days later, as described in Figure 2 . Probabilities are for comparisons between data in the bracketed groups. Results are from 48-hour readings of ear swelling (A) and erythema (B). This experiment was performed three times with similar results.
Figure 7.
 
Effect of adjuvant on the development of DTH in hi-arr-β-gal and control mice. Groups of mice were immunized with either the low-dose or high-dose protocols. All mice underwent an ear-swelling test for DTH 7 days later, as described in Figure 2 . Probabilities are for comparisons between data in the bracketed groups. Results are from 48-hour readings of ear swelling (A) and erythema (B). This experiment was performed three times with similar results.
Figure 8.
 
Production of cytokines by spleen cells harvested from the B10.A and hi-arr-β-gal mice described in Figure 7 . Spleens were collected 3 days after completion of ear-swelling tests. Samples were taken from wells with and without antigen (150 μg/mL β-gal) stimulation. Results are expressed as the mean ± SD, and the number of animals in each assay is indicated below each panel. All experiments were performed at least twice.
Figure 8.
 
Production of cytokines by spleen cells harvested from the B10.A and hi-arr-β-gal mice described in Figure 7 . Spleens were collected 3 days after completion of ear-swelling tests. Samples were taken from wells with and without antigen (150 μg/mL β-gal) stimulation. Results are expressed as the mean ± SD, and the number of animals in each assay is indicated below each panel. All experiments were performed at least twice.
Table 1.
 
Quantitation of β-Gal in Eyes and Brain from Tg and Control Mice
Table 1.
 
Quantitation of β-Gal in Eyes and Brain from Tg and Control Mice
Mice Whole Eye (ng) Brain (ng/g Wet Weight)
Hi-arr-β-gal 164 ± 36 BD, †
GFAP-β-gal 62.3 ± 8.7 299 ± 77
B10.A-ROSA26 6.8 ± 1.2 81 ± 27
β-gal BD* BD, †
×
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