Autoimmunity to a pathogenic retinal antigen begins as a balanced cytokine response that polarizes towards type 1 in a disease-susceptible and towards type 2 in a disease-resistant genotype

Bing Sun, Shu-Hui Sun, Chi-Chao Chan, Barbara Wiggert1 and Rachel R. Caspi

Laboratory of Immunology, and
1 Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, NIH, Bethesda, MD 20892, USA

Correspondence to: R. R. Caspi, Laboratory of Immunology, NEI/NIH, 10 Center Drive, MSC 1857, 10/10N222, Bethesda, MD 20892-1857, USA


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Susceptible, but not resistant, strains of rodents immunized for induction of experimental autoimmune uveitis (EAU) with the uveitogenic protein interphotoreceptor retinoid-binding protein (IRBP) exhibit a type 1 response at the time of disease expression. Here we investigate the evolution of this response using the prototypic EAU-susceptible and EAU-resistant mouse strains, B10.A and BALB/c. Disease severity and IRBP-specific responses (proliferation, cytokines and antibody isotypes) were evaluated 7, 14 and 21 days after uveitogenic immunization. B10.A mice initially exhibited an IgG1-dominated antibody response, and their lymph node cells produced IL-4 and IL-5 in addition to IFN-{gamma}. On day 14 and 21, however, the IgG2a isotype became predominant, and the primed lymph node cells produced mainly IFN-{gamma} and IL-12. B10.A mice developed EAU before day 14. BALB/c mice initially produced IL-12 and IFN-{gamma} in addition to IL-5, IL-4 and IL-10. At later time points IL-12 and IFN-{gamma} production diminished, and IL-4, IL-5 and IL-10 increased. An IgG1-dominated antibody response was maintained throughout. BALB/c mice failed to develop EAU even at day 21. Thus, both susceptible and resistant genotypes initially mount a balanced, type 0-like cytokine response to a uveitogenic challenge, that subsequently polarizes towards type 1 in the susceptible strain and towards type 2 in the resistant strain.

Keywords: autoimmunity, cytokines, rodent, Th1/Th2, uveitis


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Experimental autoimmune uveitis (EAU) is a T-cell-mediated autoimmune disease that can be induced in rodents and non-human primates by immunization with retinal antigens such as the interphotoreceptor retinoid-binding protein (IRBP) or its fragments. EAU leads to destruction of the neural retina and related tissues (1,2), and ultimately to blindness. EAU closely resembles human uveitic conditions of a putative autoimmune etiology and serves as a model for these sight-threatening diseases.

The balance between type 1 and type 2 immune responses plays a crucial role in determining the outcome of a uveitogenic challenge (36). While susceptible strains of mice and rats consistently exhibit a dominant type 1 response to the uveitogenic antigen at the time of disease expression, resistant strains exhibit a low type 1, or an overt type 2, response at the same time. Examples of such strains are the susceptible B10.A and the resistant BALB/c, which 21 days after a uveitogenic immunization with IRBP exhibit polarized type 1 and type 2 responses respectively (3). Although there have been significant advances in our understanding of the dynamic process leading to development of polarized Th1 and Th2 effectors at the single-cell level (7,8), the evolution of a type 1 or type 2 responses in vivo remains poorly documented.

We asked the question whether the response pattern typical of resistance or susceptibility is present from the outset or whether there is an evolution towards what is observed as the final response pattern. We therefore followed the responses of B10.A and BALB/c mice starting with immunization and until 1 week after EAU onset. The results demonstrated that both B10.A and BALB/c mice had a Th0-like response to IRBP 1 week after uveitogenic challenge. Subsequently, however, in B10.A mice the response became polarized towards Th1 and in BALB/c towards the Th2 profile. The shift to a type 1-dominated response correlated with onset of disease in the B10.A strain. Thus, the evolution of the response in vivo on a population level, that ultimately results in susceptibility or resistance, appears to recapitulate the same pattern as phenotype maturation thought to occur in individual T cells.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
B10.A and BALB/c female mice, 6–8 weeks old, were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals were kept in microisolator cages under specific pathogen-free conditions and were handled in compliance with National Institutes of Health and National Eye Institute guidelines for animal care and use.

Reagents
IRBP was isolated from bovine retinas, as previously described, by concanavalin A–Sepharose affinity chromatography and fast performance liquid chromatography (9). IRBP preparations were aliquoted and stored at –70°C. BSA, {alpha}-methyl mannopyranoside, pertussis toxin and complete Freund's adjuvant (CFA) were purchased from Sigma (St Louis, MO); Mycobacterium tuberculosis strain H37RA was from Difco (Detroit, MI).

EAU induction and scoring
Mice were immunized s.c. with 50 µg of IRBP in 0.2 ml emulsion 1/1 v/v with CFA that had been supplemented with M. tuberculosis to the final concentration of 2.5 mg/ml and were given 1 µg pertussis toxin in 0.1 ml i.p. as an additional adjuvant. Eyes were collected 7, 14 and 21 days after immunization. Freshly enucleated eyes were fixed for 1 h in 4% phosphate-buffered glutaraldehyde and transferred into 10% phosphate-buffered formaldehyde until processing. Fixed and dehydrated tissue was embedded in methacrylate, and 4–6 mm sections, cut through the pupillary–optic nerve plane, were stained by standard hematoxylin & eosin. Six sections cut at different levels were examined for each eye in a masked fashion by one of us (C.-C. C.), and the presence and extent of lesions were determined. Severity of EAU was scored on a scale of 0–4 in half-point increments, according to a semiquantitative system described previously (10). Briefly, the minimal criterion to score an animal as positive by histopathology was inflammatory cell infiltration of the iris, ciliary body, choroid, vitreous and retina. Progressively higher grades were assigned for presence of discrete to diffuse lesions in the tissue such as vasculitis, granuloma, retinal folding and/or detachment, photoreceptor damage, etc. The grading system takes into account lesion type, size and number.

Lymphocyte proliferation assay
Single-cell suspensions were prepared from inguinal and iliac lymph nodes of IRBP-immunized mice 7, 14 and 21 days after immunization with IRBP, and were pooled within each group. Triplicate 0.2 ml cultures were incubated in 96-well tissue culture plates (4x105 cells/well) with IRBP (30, 3 and 0.3 µg/ml) in RPMI 1640 medium containing 1% syngeneic normal mouse serum, 10–5 M 2-mercaptoethanol, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 mg/ml gentamycin, 20 mg/ml {alpha}-methyl mannopyranoside (to neutralize possible traces of concanavalin A which is used in the purification of IRBP) and 1 mM sodium pyruvate. The cultures were incubated for a total of 72 h at 37°C in 5% CO2 in air and were pulsed with 1 µCi [3H]thymidine per well during the last 18 h of incubation. The plates were harvested using a PHD cell harvester (Cambridge, MA) and isotope incorporation was determined using standard liquid scintillation.

Cytokine assays
Cytokines were measured in culture supernatants by ELISA. For assay of antigen-specific responses, draining lymph node cells collected 7, 14 and 21 days after immunization with IRBP were pooled within each group, and were cultured in 24-well Linbro plates (ICN/Flow, Costa Mesa, CA) at a concentration of 5x106 cells/well in 1 ml RPMI 1640 as described above for 48 h with IRBP (30 µg/ml). For assay of innate responses, lymph node cells of naive mice were cultured with phytohemagglutinin (PHA; 1 µg/ml). Supernatants were collected after 48 h for determination of secreted IL-4, IL-5, IL-10 and IFN-{gamma}, IL-12 and tumor necrosis factor (TNF)-{alpha} by ELISA using kits from Genzyme (Cambridge, MA).

Determination of IRBP-specific IgG antibody isotypes
Serum levels of anti-IRBP IgG2a and IgG1 subclasses were determined by ELISA as previously described (3). Briefly, 96-well microtiter plates (Costar, Cambridge, MA) were coated with IRBP (1 µg/ml). After blocking the plates with BSA and overnight incubation with samples of the tested sera, the plates were developed using horseradish peroxidaseconjugated goat anti-mouse IgG1 or goat anti-mouse IgG2a antibodies (Southern Biotechnology Associates, Birmingham, AL). The amount of each isotype bound to the IRBP-coated wells was estimated from standard curves constructed by coating wells with the same goat anti-mouse IgG1 or goat anti-mouse IgG2a antibodies and adding dilutions of Ig standards of the pertinent isotype.

Reproducibility and data presentation
Experiments were repeated at least twice. Response patterns were highly reproducible. Graphs depict representative experiments. Lymphokine titers shown are an average of two independent determinations performed on the same supernatant.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Kinetics of uveitic versus lymphocyte proliferative responses of B10.A and BALB/c mice
We have previously reported that 3 weeks after uveitogenic immunization B10.A mice (a type 1 responder) express disease, whereas BALB/c mice (a type 2 responder) do not, although both show lymphocyte proliferation to IRBP. We wished to test the kinetics of these responses, on the premise that the BALB/c mice might have a delayed response that would account for their not developing disease. Both strains were immunized with IRBP, and responses were evaluated 7, 14 and 21 days following immunization. On day 7 both strains were negative for disease, but B10.A mice already had full-blown EAU on day 14, whereas BALB/c mice remained disease-free through day 21 (Fig. 1Go). In experiments not shown here BALB/c mice were seen to remain disease-free even at 5 weeks after immunization (11). The IRBP-specific lymphocyte proliferation of both strains showed similar kinetics, but the response of B10.A mice was more vigorous (Fig. 2aGo). A dose–response curve on day 21 showed that the BALB/c lymphocytes required 10-fold more antigen in culture than B10.A to elicit the same proliferative response (Fig. 2bGo).



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Fig. 1. EAU scores in B10.A and BALB/c mice. EAU score was evaluated by histopathology 7, 14 and 21 days after immunization with IRBP. The horizontal bar denotes the average of each group.

 


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Fig. 2. IRBP-specific lymph node cell proliferation. Lymph node cells were harvested 7, 14 and 21 days after immunization, and were stimulated with IRBP. (a) Kinetics as a function of time after immunization, at 30 µg/ml IRBP in culture. (b) Dose–response to graded doses of IRBP of cells collected 21 days after immunization. The results are expressed as c.p.m.x10–3.

 
Evolution of the cytokine response in B10.A and BALB/c mice
We previously noted that 21 days after immunization, which is 9–11 days after onset of disease, B10.A mice express a dominant type 1 cytokine response. At the same time BALB/c expresses a dominant type 2 response. To examine whether the response patterns were fixed from the time of priming, the cytokine profiles were analyzed during the development of EAU in both strains. Draining lymph node cells of IRBP-primed mice were stimulated in culture with IRBP and supernatants collected after 48 h were analyzed for type 2 cytokines (IL-4, IL-5 and IL-10) and for type 1 cytokines (IL-12, IFN-{gamma} and TNF-{alpha}) by ELISA. Although IL-12 is made by non-T cells and TNF-{alpha} can be made by a variety of cells, they are referred to as type 1 because the synthesis of both is positively regulated by IFN-{gamma}. Furthermore, IL-12 drives T cell polarization towards the Th1 phenotype.

Unexpectedly, the EAU-susceptible B10.A mice exhibited a relatively high IL-4, IL-5 and IL-10 production on day 7, which decreased on days 14 and 21 (Fig. 3Go). In the EAU-resistant BALB/c, on the other hand, production of the same cytokines increased with time, so that by day 21 BALB/c produced more IL-4 and IL-5 than B10.A, but continued to produce less IL-10. The 21 day cytokine pattern is consistent with our previously reported observations in those two strains. Evolution of the type 1 cytokine response showed the opposite pattern. IL-12 and IFN-{gamma} rose from day 7 to day 21 in B10.A mice, but decreased in BALB/c mice (Fig. 4Go). While B10.A produced 10-fold higher amounts of IFN-{gamma} than BALB/c on day 7, the latter strain nevertheless produced a respectable 1 ng/ml of IFN-{gamma}, which fell to a quarter that by day 21. In contrast, the day 7 IFN-{gamma} production in B10.A almost tripled by day 21 (note that the IFN-{gamma} panels are plotted on a log scale). Thus, in both strains the response initially starts as a more balanced, type 0-like response and subsequently polarizes in the opposite direction, to yield the final response profile of a type 1-dominant response in B10.A and a type 2-dominant response in BALB/c.



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Fig. 3. Antigen-specific production of type 2 cytokines. Lymph node cells collected 7, 14 and 21 days after immunization were stimulated with IRBP. Supernatants were collected after 48 h and were assayed by ELISA. Cytokine protein in supernatants of unstimulated cells was undetectable.

 


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Fig. 4. Antigen-specific production of type 1 cytokines. Type 1 cytokines were measured by ELISA in the same supernatants as in Fig. 3Go. Cytokine protein was undetectable in supernatants of unstimulated cells.

 
Evolution of IRBP-specific antibody isotypes in the serum
Because IFN-{gamma} promotes isotype switching from IgM to IgG2a, and IL-4 promotes switching to IgG1, the relative amounts of these antibody isotypes in the serum reflect the (Th1 or Th2) response that develops to the antigen in vivo. On day 7, both strains exhibited an excess of IRBP-specific IgG1 isotype (Fig. 5Go). However, on days 14 and 21, B10.A mice switched to an IgG2a-predominant profile, whereas BALB/c mice retained an IgG1-dominated profile. These data are in line with the antigen-specific cytokine data described in the previous paragraph.



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Fig. 5. Isotype analysis of anti-IRBP serum antibodies. Anti-IRBP IgG subclasses were measured by isotype-specific ELISA in sera of individual mice at different time points following immunization. The horizontal bar denotes the average of each group.

 
The innate response in B10.A and BALB/c strains
The development of the adaptive immune response is influenced by the innate immune response (12). We therefore wished to assess the response to a mitogenic stimulus in the two strains as a measure of their innate response. The T cell specific mitogen, PHA, was used to stimulate naive lymph node cells from both strains. Representative type 1 cytokines (IL-12 and IFN-{gamma}) and representative type 2 cytokines (IL-4 and IL-5) were measured in supernatants collected after 48 h. The results indicated that the innate response of B10.A mice was biased towards production of type 1 cytokines, whereas the response of BALB/c mice was biased towards type 2 cytokines (Fig 6Go).



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Fig. 6. PHA-induced type 1 and type 2 cytokines. Lymph node cells collected from naive mice were stimulated with PHA. Supernatants were collected after 48 h and were assayed by ELISA. Cytokine protein in supernatants of unstimulated cells was undetectable.

 

    Discussion
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 Abstract
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 Methods
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 Discussion
 References
 
The present study examines the evolution of the antigen-specific response over time in an EAU-susceptible and an EAU-resistant mouse strain. Because B10.A had previously been seen to be a dominant type 1 responder and BALB/c a dominant type 2 responder to IRBP, we initially expected that a polarized response pattern would be apparent from the beginning. To our surprise, the early response as measured on day 7 in terms of cytokines and antibody isotypes was much more balanced in both strains. B10.A produced relatively high quantities of IL-4, IL-5 and IL-10 on day 7, and in fact produced more of those cytokines than did BALB/c. Both strains had an early IgG antibody response predominantly of the IgG1 isotype. The most striking difference between B10.A and BALB/c in their early cytokine profile was that B10.A produced 10-fold more antigen-specific IFN-{gamma} than BALB/c; however, at that time B10.A also produced more type 2 cytokines than BALB/c. It could therefore be argued that B10.A is a more vigorous cytokine producer overall and that the Th1/Th2 balance in this strain occurs at a higher quantity of all cytokines. At later time points all the assayed type 1 cytokines in B10.A increased and all type 2 cytokines decreased. Interestingly, the major change towards the Th1 profile in B10.A in terms of IL-4, IL-5 and the predominant antibody isotype appears to take place by day 14, roughly coincident with onset of disease. However, the cytokine and antibody responses continued to polarize even after EAU onset, which may be seen as being compatible with the course of disease, that reaches its peak by day 21. The evolution of the cytokine response pattern in BALB/c was exactly the opposite: in comparison to day 7 all type 1 cytokines decreased, and type 2 cytokines increased, over time. BALB/c mice remained free of EAU through day 21 when the experiments were harvested and in other experiments not shown here remained resistant even 5 weeks after immunization, the latest time point examined (11). Thus, both strains initially mounted a more balanced, type 0-like response to IRBP, that subsequently become polarized in opposite directions.

Although the mice chosen for this study are a prototypic Th2-dominant (resistant) and Th1-dominant (susceptible) strain, it is possible that the evolution of cytokine response in those two may not be representative of all strains. Specifically, the present study did not address resistant strains that at the time of disease expression display a low Th1 response without a dominant Th2 response, or minimally susceptible strains that display a Th0-like response (3,6). The present study also did not examine the possibility that evolution of the cytokine profile in lymphoid compartments other than the draining lymph node might differ. However, the shift in the antibody isotypes strongly suggests that the polarization of the response is a systemic, rather than a local phenomenon.

It is important to keep in mind that the cytokine responses measured in this study are not to be regarded as equivalent to the responses of individual T cells. Rather, they reflect the profile of the response at the population level and represent the sum total of the cytokines produced by different T cells (as well as by non-T cells) in the lymph node cell population. Nevertheless, due to their antigen-specific nature, these responses are T cell driven, if not entirely T cell derived. To illustrate this point, most of the IL-12 and at least some of the TNF-{alpha} would be produced by macrophages and dendritic cells, driven by the IFN-{gamma} produced by T cells as a result of antigenic stimulation.

A number of factors influence the direction of effector T cell differentiation towards Th1 or Th2. The major ones described to date are: signaling through the antigen receptor (receptor occupancy or avidity of interaction) (13,14), cytokine milieu (in part determined by innate immunity) (7,12) and identity of antigen-presenting cell (co-stimulation and cytokines/chemokines) (15). While the identity of antigen-presenting cells might be presumed in our study to be the same in both strains, since the method of antigen delivery is identical, there certainly are differences between the two strains in antigen recognition and cytokines produced as part of the innate response.

With regard to effect of antigen, BALB/c had lower proliferative responses than B10.A to a particular dose of IRBP at all time points after immunization. An antigen dose curve revealed that the response in BALB/c was shifted down by an order of magnitude compared to B10.A. The reasons behind this dose–response shift could not be addressed in the present study, because the B10.A (I-Ak) and the BALB/c (I-Ad) differ not only in the antigenic epitopes that they present, but also in multiple non-MHC genes. Nevertheless, it is tempting to speculate that one these reasons might be antigen-specific interactions of lower avidity in the BALB/c, which would effectively result in lower receptor occupancy, a situation that in some systems has been suggested to preferentially stimulate Th2 cells (14,16). In this context, it is relevant to mention that BALB/k, a MHC-congenic strain to BALB/c having I-Ak, that is EAU susceptible, exhibits a (day 21) response pattern to IRBP that is intermediate between the BALB/c and B10.A (3). This suggests that antigen-specific interactions can influence the direction of the adaptive response in this model.

To the extent that response of naive lymph node cells to the mitogen PHA is representative of the innate immune response, that response differed considerably between the two strains. B10.A made a type 1 response, whereas BALB/c made a type 2 response to the mitogen. A similar cytokine bias in mouse strains having this background was also noted by others in response to parasite and microbial antigens (reviewed in 12). It is interesting that despite the clear bias of the innate response to PHA, the adaptive response to IRBP did not reflect the same polarization from the beginning, but rather showed a gradual evolution. The present data cannot distinguish whether this evolution reflects a change at the single-cell level or at the population level, i.e. whether cells that have been primed early on are progressively differentiating towards a more polarized phenotype or whether newly recruited cells at later times after immunization are committing to a more polarized phenotype than cells recruited earlier. Thus, the polarization of the adaptive response in the two strains could represent the combined effect of the avidity of antigen specific interactions and the innate cytokine milieu in which the effector T cells are differentiating.

The question must be asked whether polarization of the cytokine response is causally related to resistance and susceptibility to EAU or whether it is merely an association. In a previous study, we showed that treatment of B10.A mice with a combination of recombinant murine IL-4 and IL-10 during the first 5 days after uveitogenic immunization resulted in a mixed antigen-specific cytokine response profile on day 21, similar to the response profile on day 7 in the present study, and resulted in strongly reduced disease scores (17). At that time our interpretation was that the treatment precipitated a response shift towards Th2. In light of the present data, we would modify this to state that it prevented a response shift towards Th1. Interestingly, the converse manipulation, early treatment of BALB/c with IL-12, does not result in EAU susceptibility and in fact aborts disease induction in susceptible genotypes (18). The mechanism behind this phenomenon appears to be inhibition of effector cell generation due to inhibitory effects of IFN-{gamma}, NO and induction of apoptosis, rather than a cytokine response shift, underscoring the unexpected complexities that may result when attempting to manipulate delicately balanced biological systems.

Recently Doncarli et al. (19) reported that a conversion from Th0/Th1 to a Th2-like response after immunization with collagen type II takes place in mice that develop collagen-induced arthritis. In this case, susceptibility was connected to a dominant type 2 response. Together with our present report, the data support the contention that a gradual polarization of an initially neutral response towards a pathogenic phenotype represents a general phenomenon, and is not unique only to the antigen IRBP and to uveitis. Thus, the in vivo response phenotype at the population level appears to be a dynamic process that, after a fashion, recapitulates the process of phenotype commitment thought to occur in individual T cells. As such, at least during early stages of its development, it appears be amenable to directed immunomodulation away from a phenotype that is pathogenic to the autoimmune disease in question. As a corollary, the time from onset of autoimmune symptoms may be a predictive factor whether an immunotherapeutic approach or a conventional immunosuppressive approach should be used in a given clinical situation.


    Abbreviations
 
EAUexperimental autoimmune uveoretinitis
CFAcomplete Freund's adjuvant
IRBPinterphotoreceptor retinoid-binding protein
PHAphytohemagglutinin
TNFtumor necrosis factor

    Notes
 
Transmitting editor: J. A. Berzofsky

Received 5 March 1999, accepted 28 April 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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