November 1999
Volume 40, Issue 12
Free
Immunology and Microbiology  |   November 1999
The Requirement for Pertussis to Induce EAU Is Strain-Dependent: B10.RIII, but Not B10.A Mice, Develop EAU and Th1 Responses to IRBP without Pertussis Treatment
Author Affiliations
  • Phyllis B. Silver
    From the Laboratory of Immunology and the
  • Chi-Chao Chan
    From the Laboratory of Immunology and the
  • Barbara Wiggert
    Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland.
  • Rachel R. Caspi
    From the Laboratory of Immunology and the
Investigative Ophthalmology & Visual Science November 1999, Vol.40, 2898-2905. doi:
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Phyllis B. Silver, Chi-Chao Chan, Barbara Wiggert, Rachel R. Caspi; The Requirement for Pertussis to Induce EAU Is Strain-Dependent: B10.RIII, but Not B10.A Mice, Develop EAU and Th1 Responses to IRBP without Pertussis Treatment. Invest. Ophthalmol. Vis. Sci. 1999;40(12):2898-2905.

      Download citation file:


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

      ×
  • Supplements
Abstract

purpose. Experimental autoimmune uveoretinitis (EAU) in mice is an important model for elucidating basic mechanisms in autoimmune eye disease. The need for pertussis toxin (PTX) as an additional adjuvant to elicit EAU has limited the usefulness of this model in some types of studies by introducing a pleiotropic factor with confounding effects on the immune response.

methods. In the present study the authors examined the ability of B10.RIII mice, the most susceptible strain known so far, to develop EAU in response to the retinal antigen, interphotoreceptor retinoid-binding protein (IRBP), and to a major uveitogenic epitope of IRBP, peptide (p)161-180, in the absence of PTX treatment.

results. The data indicate that high disease scores in response to IRBP and p161-180 were found in B10.RIII mice, without the need for PTX as part of the immunization protocol. Unlike the B10.A strain in which appreciable disease did not develop without PTX, B10.RIII mice mounted a high IFN-γ response to IRBP in the absence of PTX treatment. Interestingly, and unlike the effect with IRBP, in vitro recall response to p161-180 was low in IFN-γ, despite good EAU scores.

conclusions. The data indicate that an important mechanism through which PTX facilitates induction of cell-mediated autoimmunity is by promoting a Th1 polarization of the immune response. The propensity of B10.RIII mice to mount a more polarized Th1 response to IRBP than other strains may contribute to their ability to develop EAU without pertussis adjuvant. Nevertheless, the induction of EAU by p161-180 in the context of a relatively limited IFN-γ production indicates that non–Th1- and Th-related mechanisms are likely to act in concert to determine the outcome of disease.

Experimental autoimmune uveoretinitis (EAU) is an important model for human uveitis. Over the years it has helped to elucidate basic mechanisms involved in pathogenesis and immune regulation of the disease process, has provided an important tool in studying the genetic factors predisposing to disease, and has served as a template for development of new therapies. The mouse model has been particularly useful in these studies because of the availability of specific reagents such as monoclonal antibodies and cytokines, as well as the availability of genetically defined congenic mouse strains and genetically modified transgenic and knockout strains. 
EAU in mice can be induced by immunization with the interphotoreceptor retinoid-binding protein (IRBP); the retinal S-Ag is poorly uveitogenic in mice. 1 Most mouse strains are relatively resistant to induction of EAU, and immunization protocols have required the use of pertussis adjuvant to elicit disease. 1 2 The most potent and most useful form of pertussis adjuvant is purified pertussis toxin (PTX), and our previous work has shown that there is little or no disease in the susceptible B10.A strain if PTX is not used as part of the immunization regimen. 2  
The need for PTX in the induction protocol confounds the picture by adding a potent adjuvant with pleiotropic activities to the immunization protocol. PTX has been used for many years to enhance induction of autoimmune disease and overcome resistance in low-responder strains. 3 It is known to have multifaceted effects on the immune response, including enhancement of vascular permeability, histamine sensitization, lymphocyte recirculation, mitogenic effects on T and B cells, and other effects. 4 5 6 7 8 9 10 11 12 13 This can make interpretation of experimental data difficult, and limits the usefulness of the model for some types of studies. 
In the present study we re-examined the possibility of inducing EAU in mice without pertussis toxin, using the most susceptible strain known thus far, B10.RIII, immunized with IRBP or with a major pathogenic epitope of IRBP. The results show that EAU in B10.RIII mice, unlike in B10.A mice, can be induced by IRBP without pertussis treatment. Furthermore, EAU can be induced in B10.RIII mice by the pathogenic peptide (p)161-180 without pertussis. Delayed hypersensitivity and lymphocyte proliferation were strongly enhanced by PTX treatment in all cases. Antigen-specific production of IFN-γ indicated that the cellular response of B10.RIII mice to IRBP in the absence of PTX was more polarized toward type 1 than that of B10.A, and that PTX treatment elevated IFN-γ production in B10.A mice in response to IRBP to the level found in B10.RIII mice. We propose that this explains in part the tendency toward development of EAU in the B10.RIII mouse after IRBP immunization without PTX treatment. We further propose that one of the mechanisms that contribute to the well-documented property of PTX to promote cell-mediated autoimmunity is its ability to polarize the immune response toward the Th1 pathway. 
Materials and Methods
Animals
Six- to 8-week-old B10.A and B10.RIII mice were supplied by Frederick Cancer Research Facility (Frederick, MD) and by Jackson Laboratories (Bar Harbor, ME), respectively. Animal care and use were in compliance with institutional guidelines and with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Antigens
Whole bovine IRBP was purified from retinas by concanavalin A-Sepharose–affinity chromatography and high-performance liquid chromatography. 14 Human p161-180 (sequence SGIPYIISYLHPGNTILHVD) was synthesized on a peptide synthesizer (model 461; Applied Biosystems; Foster City, CA) using Fmoc chemistry. 
EAU Induction and Scoring
EAU was induced by active immunization with graded doses of IRBP or p161-180 in phosphate-buffered saline (PBS) emulsified 1:1 vol/vol in complete Freund’s adjuvant (CFA) that had been supplemented with Mycobacterium tuberculosis strain H37RA (Sigma, St. Louis, MO) to 2.5 mg/ml. A total of 200 μl emulsion was injected subcutaneously, divided among three sites: base of tail and both thighs. In some groups, 0.5 μg of Bordetella pertussis toxin (PTX) (Sigma) was injected intraperitoneally at the same time. EAU by adoptive transfer was induced by intraperitoneal injection of pooled spleen and lymph node cells obtained from primed donors and stimulated in culture with 20 μM p161-180 in the presence or absence of 50 ng/ml interleukin (IL)-12, essentially as previously described. 15 In some adoptive transfer recipients, 1 μg PTX was administered intravenously just before the adoptive transfer. Clinical EAU was evaluated by fundoscopy under a binocular microscope after dilation of the pupil and graded on a scale of 0 to 4 using criteria described in detail elsewhere. 16 Eyes harvested 21 days after immunization or 10 days after adoptive transfer were prefixed in 4% phosphate-buffered glutaraldehyde for 1 hour (to prevent artifactual detachment of the retina), and then transferred to 10% phosphate-buffered formaldehyde until processing. Fixed and dehydrated tissue was embedded in methacrylate, and 4- to 6-μm sections were stained with standard hematoxylin and eosin. Eye sections cut at different planes were scored in a masked fashion. Incidence and severity of EAU were graded on a scale of 0 to 4 in half-point increments, using the criteria described previously, 16 which are based on the type, number, and size of lesions present. Incidence was shown as the number of positive animals of all animals in the group. Severity of disease was the average score of eyes from those animals in which disease developed (if disease was unilateral, both eyes were averaged). 
Delayed-Type Hypersensitivity
To assess delayed-type hypersensitivity (DTH), 10 μg IRBP or peptide in 10 μl PBS was injected into the ear pinna. Ear-thickness increment was measured 48 hours later using a spring-loaded micrometer. The response was calculated as the difference between ear thickness before and after challenge. 
Lymphocyte Proliferation Assay
Draining (inguinal and iliac) lymph nodes were collected after 21 days, and were pooled within the group. Triplicate 0.2-ml cultures containing 5 × 105 cells were seeded in round-bottomed 96-well microtiter plates. The RPMI medium (Biowhittaker, Walkersville, MD) was supplemented with mouse serum, mercaptoethanol, antibiotics, glutamine, and nonessential amino acids, as described, 1 and contained 30 μg/ml IRBP or 20 μM p161-180 as stimulants. The cultures were incubated for a total of 60 hours. Tritiated thymidine (1 μCi/well) was added during the last 18 hours. The data are shown as Δcpm (Δcpm = mean cpm in cultures with antigen, minus the mean cpm in control cultures without antigen). 
Cytokine Assays
Lymph node and spleen cells were cultured in 96-well flat-bottomed plates (1 × 106 cells/0.2 ml culture medium per well) either alone or with stimulants at the concentrations mentioned earlier. Supernatants were collected after 48 to 72 hours and were kept frozen in small aliquots at −70°C. Cytokine production was measured by enzyme-linked immunosorbent assay (ELISA) using antibody pairs from Pharmingen (La Jolla, CA) for IL-4, or from Endogen (Boston, MA) for IL-5 and IFN-γ, or from R&D (Minneapolis, MN) for TNF-α, as described previously. 17 18  
Adoptive Transfer of EAU
Donor B10.RIII mice were immunized with 50 μg p161-180. Lymph node cells and spleen cells collected on day 14 after immunization were pooled. The cell suspension was adjusted to 107 cells/ml in RPMI medium supplemented as for the proliferation assay, and the cultures were stimulated in 75-cm2 flasks for 72 hours with 20 μM p161-180 in the presence or absence of 50 ng/ml IL-12. To remove excess adherent cells (macrophages), the stimulating cultures were transferred into new flasks after 24 hours and again after 48 hours. After 3 days, the lymphocytes were separated from erythrocytes and debris by discontinuous density gradient centrifugation on Ficoll (Lympholyte M; Accurate, Westbury, NY) and counted. Each recipient mouse was injected intraperitoneally with the specified number of cells. Some recipient mice were injected with 1μ g PTX intravenously just before the adoptive transfer. Eyes were collected from the recipients after 10 days and were evaluated for EAU by histopathology. 
Reproducibility and Data Presentation
Experiments were repeated at least twice. Results were highly reproducible. Figures show pooled data from repeat experiments, or representative experiments, as indicated. 
Results
Induction of EAU with IRBP in B10.RIII, but Not in B10.A Mice, without Pertussis Treatment
To induce EAU, B10.A and B10.RIII mice were immunized with graded doses of IRBP in CFA, with or without a concomitant intraperitoneal injection of 0.5 μg PTX (Fig. 1) . Although both strains had high disease scores after the immunization regimen that included PTX, only B10.RIII mice had high disease scores without pertussis treatment, which at 25 μg IRBP per mouse were almost equal to those of the PTX-treated group. At the lower and higher limits of the dose–response curve, pertussis appeared to lower the threshold dose required for EAU induction and to forestall reduction of disease scores at higher IRBP doses. B10.A mice showed trace disease at best, which consisted of mild vitritis without retinal damage, even at the high antigen dose of 200 μg IRBP per mouse,. That appreciable disease did not develop in B10.A mice without PTX treatment largely confirms data in our previously published and unpublished data. 2  
To pinpoint the time of disease onset, groups of four mice were immunized with 50 μg IRBP, with or without 0.5 μg PTX, and were observed daily by fundoscopy. Kinetics of disease development are shown in Table 1 . B10.RIII mice showed clinical signs as early as 7 days after immunization if treated with PTX, or 8 days if untreated with PTX. Onset of disease in PTX-treated B10.A mice lagged by 2 days behind the corresponding B10.RIII group. Untreated B10.A mice began to show clinical signs only after day 12, and full-blown histologic disease did not develop even on day 21 (as shown in Fig. 1 ). It should be pointed out that fundoscopy scores did not translate directly to histology scores, but in general there was good correlation between both types of evaluations when performed in parallel. 
Induction of EAU in B10.RIII Mice with P161-180 without Pertussis Treatment
We have previously defined the human sequence of IRBP p161-180 as a major pathogenic epitope for B10.RIII mice. Animals immunized with peptide using a regimen that included PTX had disease scores that approached scores obtained after immunization with IRBP and PTX. Because experiments described in the previous section showed that EAU in B10.RIII could be elicited with IRBP without PTX, we wanted to test whether the same was true in response to the peptide. 
B10.RIII mice were immunized with graded doses of p161-180 in CFA, with or without concomitant administration of 0.5 μg PTX. Mice immunized without PTX had good EAU scores that at peptide doses between 10 and 25μ g peptide per mouse were only slightly lower than those in mice immunized with PTX (Fig. 2) . However, use of PTX appeared to lower the threshold of antigen dose required for EAU induction and to eliminate the plateau of maximal achievable disease score observed at the highest doses of immunizing peptide. Thus, similar to EAU induced by whole IRBP, EAU induced by p161-180 in B10.RIII mice appears relatively independent of PTX as an additional adjuvant, except at extremely low or high antigen doses. 
Kinetics of disease development in response to 25 μg p161-180 in CFA, with or without 0.5 μg PTX, are shown in Table 1
Dependence on PTX Treatment of DTH Scores and Lymphocyte Proliferation in Response to IRBP, Irrespective of Disease Development
The same mice that were immunized for development of EAU were challenged 2 days before the end of the experiment for DTH responses to the immunizing antigen (IRBP or p161-180). The ear-swelling responses 48 hours after challenge showed that in all cases DTH scores were highly dependent on inclusion of PTX in the immunization regimen (Fig. 3) . This was irrespective of the antigen, mouse strain, and disease scores resulting from the immunization. 
Lymphocyte proliferation to the immunizing antigen was tested on draining lymph node cells that were pooled within each group. Similarly to the DTH responses, lymphocyte proliferation was strongly enhanced by inclusion of PTX in the immunization regimen, whether or not it was needed for elicitation of EAU (Fig. 4)
Thus the pattern of DTH and lymphocyte proliferation showed an apparent dissociation from EAU development in PTX-untreated B10.RIII mice; even mice that had good disease scores without PTX had relatively low DTH and proliferative responses. A strong enhancing effect of PTX on DTH responses has previously been noted by others in various experimental situations 9 10 and could be influenced by combined effects of PTX on IFN-γ production, lymphocyte migration, and vascular permeability, that may tend to keep IFN-γ–producing, antigen-specific lymphocytes in the circulation for a longer than normal period, while facilitating their egress into inflammatory sites. 9 10 12 13 Similarly, effects on recirculation may inhibit emigration of primed lymphocytes from the draining lymph node, accounting for higher proliferative responses in vitro. 
Development of a More Polarized Type 1 Response to IRBP in B10.RIII Mice Than in B10.A Mice in the Absence of PTX Treatment
Our previous data indicate that EAU is strongly dependent on the presence of a type 1 response to the uveitogenic antigen. 19 20 We therefore examined the cytokine profile of the response to IRBP and to p161-180 in mice immunized in the presence and absence of PTX. Draining lymph node cells were collected 21 days after immunization, antigen-stimulated supernatants were generated from the different groups and were assayed for content of IFN-γ, IL-4, IL-5, and TNF-α by ELISA. The results showed that neither strain produced detectable IL-4 titers in response to IRBP and both had low antigen-specific IL-5 responses, indicating absence of a significant type 2 response. This response pattern was previously observed by us to be typical of the B6 and B10 genetic backgrounds. 18 20 Interestingly, however, B10.RIII mice produced 10 times more IFN-γ and almost 3 times as much TNF-α in response to IRBP than did B10.A mice (Table 2) . PTX treatment of B10.A mice elevated the production of IFN-γ and TNF-α to the levels produced by untreated B10.RIII mice. 
The Paradox of p161-180: Uveitogenicity without a High IFN-γ Response
Interestingly, in the case of the peptide the relationship between IFN-γ response in culture and disease development did not hold up. Although this peptide was uveitogenic even without PTX treatment, the in vitro response to the peptide was low in IFN-γ, indicating a low type 1 response (Table 3) . It was also low in IL-4 and IL-5, indicating absence of an appreciable type 2 response (Table 3) . Such a null cytokine pattern was previously observed by us to be associated with genetic resistance to EAU 19 20 and contrasts with the usual response to uveitogenic antigen in the susceptible strains. Nevertheless, culturing primed lymph node cells of (PTX-untreated) p161-180–immunized mice in the presence of IL-12 strongly enhanced their ability to produce IFN-γ as well as their ability to transfer disease adoptively to naive recipients (Table 4 and Fig. 5 ). The enhancement of uveitogenicity on adoptive transfer was particularly apparent at limiting cell numbers (Fig. 5) . Thus, 5 million cells from cultures treated in vitro with IL-12 and showing a predominant Th1 cytokine profile induced close to maximal disease, whereas the same number of cells without IL-12 treatment and showing the IFN-γ low cytokine profile were unable to transfer disease. Thus, although an effector response low in IFN-γ production induced EAU disease in the case of this peptide, polarization of the response toward Th1 enhanced disease expression. 
Effects of PTX on Adoptively Transferred EAU
Because PTX is a bacterial product that stimulates innate immunity, we treated some recipient mice with PTX just before infusion of peptide-primed cells from PTX-untreated donors, on the assumption that PTX would stimulate production of endogenous IL-12 in vivo and thus promote uveitogenicity of the p161-180–primed cells, similar to the effect of IL-12 treatment in vitro. Interestingly, pretreatment of recipient mice with PTX 8 hours before infusing 20 or 40 million primed cells, which caused severe disease in recipients that did not receive PTX, completely prevented development of EAU. The PTX-treated adoptive transfer recipients were still free of disease 10 days after the adoptive transfer, corresponding to 5 to 6 days after EAU onset in PTX-untreated recipients, at which point the experiment was terminated (Fig. 5) . A delay in onset of adoptively transferred EAU in rats after pretreatment of recipients with pertussis adjuvant (bacteria) has previously been reported by others. 21 Thus, PTX has dramatically different effects when administered at the induction, rather than at the expression stage of disease. 
Discussion
The present article reports that B10.RIII mice can develop EAU without PTX as an additional adjuvant. Our previous studies indicated that the development of EAU in rats and mice is closely correlated with their ability to mount a Th1-dominated response to IRBP. 19 20 We therefore believe that the development of EAU in the B10.RIII strain without PTX as additional adjuvant is at least in part because of its propensity to mount a Th1-dominated response to IRBP in the absence of such treatment. PTX-induced enhancement of the Th1 response in association with abrogation of resistance to disease has been seen in the rat EAU model. 19 The present study also points to a role for influences other than the Th1–Th2 balance in susceptibility, such as an ability to produce high levels of TNF-α. 22 The observation that EAU can be induced in B10.RIII mice without PTX treatment increases the usefulness of the mouse model for basic studies of ocular autoimmunity, by eliminating a pleiotropic adjuvant substance with many known and unknown effects on the immune system. 
Our results shed light on the long-debated effects of pertussis adjuvant on cell-mediated autoimmunity. Although it has long been known that pertussis administered concurrently with immunization promotes induction of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and EAU, the mechanism by which this comes about is controversial. Some investigators have attributed enhancement of EAE by pertussis to histamine sensitization of vascular endothelial cells and increase of vascular permeability, whereas others have proposed effects on the sensitization phase of EAE. 4 5 6 12 Because pertussis is administered at the time of immunization, it should be gone long before effector cell infiltration into the target organ occurs 7 to 9 days later. We therefore believe that the effects are more likely to be exerted on early events that coincide temporally with presence of PTX in the system, such as priming of effector T cells and their commitment to the Th1 pathway. 
Support for this interpretation is also provided by the observation that PTX completely abrogated disease when administered with adoptive transfer of exogenously generated uveitogenic effector cells. If effects on vascular permeability were a primary mechanism, enhancement of disease would be expected. We propose that inhibition of adoptively transferred EAU is related to the documented inhibitory effects of PTX on recirculation and homing of lymphocytes. 7 13 Because PTX is a known uncoupler of G-proteins, we hypothesize that these effects may be secondary to blocking of chemokine signaling through G-protein–coupled receptors, which is necessary for migration and extravasation of effector lymphocytes and recruited leukocytes into the target organ. 23 24 This hypothesis is currently under investigation in a separate study. 
Interestingly, B10.RIII mice appeared to produce less IFN-γ in response to p161-180 of IRBP than to the whole IRBP molecule. Furthermore, a Th1-dominant response to this epitope did not appear to be a prerequisite for its ability to induce disease, except under conditions of suboptimal uveitogenic challenge (insufficient or excessive immunization dose, or a limiting number of adoptively transferred effector cells). One reason for this apparent paradox may be the identity of the antigenic epitopes involved. It is known that the type of response evoked is in part dependent on the epitope itself, and is affected by the affinity of the interaction with T cell receptor and major histocompatibility complex (MHC). High-affinity (or high-avidity) interactions in several antigen systems have been seen to promote development of type 1 responses, whereas low-affinity interactions may encourage type 2 responses. 25 26 27 28 We hypothesize that the response to p161-180 involves mostly the self-specific repertoire from which high-affinity cells have been deleted, and therefore results in an IFN-γ–poor response. In contrast, the bovine IRBP molecule contains also multiple nonconserved, bovine-specific epitopes that can interact with the T-cell receptor with high affinity and cause abundant production of IFN-γ. Although these arguments may explain the difference in response phenotype to the peptide and the whole IRBP molecule, they do not explain the dissociation between the type of response to the peptide in vitro (low Th1) and the response to the same peptide in vivo (uveitis). The ability of p161-180 to induce disease in the context of a response relatively low in IFN-γ supports the interpretation that factors besides Th1–Th2 balance influence pathogenicity. 
In summary, EAU induction in the B10.RIII mouse strain by active immunization with IRBP or with its immunodominant epitope 161-180 was found not to require the use of pertussis adjuvant. Unlike B10.A, B10.RIII mice showed strong Th1 response and high TNF-α levels to IRBP without PTX treatment, although even in this strain the disease was enhanced by PTX under suboptimal or supraoptimal conditions of induction. Although in the past it has been proposed that PTX promotes induction of cell-mediated autoimmunity because of its effects on vascular permeability, the present data, as well as data published by us previously, 19 indicate that PTX-driven Th1 polarization may play an important role. Nevertheless, the ability of p161-180 to elicit EAU, apparently without inducing a strong Th1 response, remains a paradox and points to the involvement of additional factors besides Th1–Th2 balance in pathogenicity. The present investigation broadens the usefulness of the mouse EAU model and permits its use in studies in which pertussis treatment is undesirable or would confound the conclusions. 
 
Figure 1.
 
EAU scores of B10.RIII and B10.A mice immunized with IRBP, with or without concomitant pertussis treatment. (A) B10.RIII; (B) B10.A. The score at each dose is the average of all the mice in the group ± SE. EAU incidence (positive/total) is shown next to each point. The data are a composite of two experiments.
Figure 1.
 
EAU scores of B10.RIII and B10.A mice immunized with IRBP, with or without concomitant pertussis treatment. (A) B10.RIII; (B) B10.A. The score at each dose is the average of all the mice in the group ± SE. EAU incidence (positive/total) is shown next to each point. The data are a composite of two experiments.
Table 1.
 
Kinetics of Disease Onset in B10.RIII and B10.A Mice Immunized with or without PTX
Table 1.
 
Kinetics of Disease Onset in B10.RIII and B10.A Mice Immunized with or without PTX
Days after Immunization Strain and Immunization Regimen*
B10.RIII IRBP B10.RIII IRBP, PTX B10.RIII Peptide B10.RIII Peptide, PTX B10.A IRBP B10.A IRBP, PTX
6 0, 0, 0, 0, † 0, 0, 0, 0 0, 0, 0, 0 0, 0, 0, 0 0, 0, 0, 0 0, 0, 0, 0
7 0, 0, 0, 0.25 0.25, 0.5, 0.5, 0.5 0, 0, 0, 0 0, 0, 0.5, 0.5 0, 0, 0, 0 0, 0, 0, 0
8 0, 0.5, 0.75, 0.75 1.5, 1.25, 1.75, 2.5 0, 0.5, 1, 1 0.25, 1, 2, 3 0, 0, 0, 0 0, 0, 0, 0.75
9 3, 3, 3, 3 3, 3, 4, 4 0, 2, 3, 3 3, 3, 4, 4 0, 0, 0, 0 0, 0.25, 0.75, 2
10 ND ND ND ND 0, 0, 0, 0 0, 0.25, 0.75, 2.5
11 ND ND ND ND 0, 0, 0, 0 0, 0.25, 3, 3.5
12 ND ND ND ND 0, 0, 0.25, 0.5 0, 1, 3, 3.5
13 ND ND ND ND 0, 0, 0.5, 0.75 0, 3, 3, 3.5
14 ND ND ND ND 0, 0, 0.5, 0.75 0, 3, 3, 3.5
Figure 2.
 
EAU scores of B10.RIII mice immunized with p161-180 with or without concomitant pertussis treatment. The score at each dose is the average of all the mice in the group ± SE. EAU incidence (positive/total) is shown next to each point. The data are a composite of two experiments.
Figure 2.
 
EAU scores of B10.RIII mice immunized with p161-180 with or without concomitant pertussis treatment. The score at each dose is the average of all the mice in the group ± SE. EAU incidence (positive/total) is shown next to each point. The data are a composite of two experiments.
Figure 3.
 
Delayed-type hypersensitivity scores of B10.RIII and B10.A mice immunized with or without concomitant pertussis treatment. Mice were challenged by injection of the immunizing antigen into the ear pinna 19 days after immunization. DTH scores represent the increment in ear thickness after 48 hours. (A) B10.RIII immunized with IRBP; (B) B10.RIII immunized with peptide; (C) B10.A immunized with IRBP. EAU scores of these mice and the number of mice per group are shown in Figures 1 and 2 .
Figure 3.
 
Delayed-type hypersensitivity scores of B10.RIII and B10.A mice immunized with or without concomitant pertussis treatment. Mice were challenged by injection of the immunizing antigen into the ear pinna 19 days after immunization. DTH scores represent the increment in ear thickness after 48 hours. (A) B10.RIII immunized with IRBP; (B) B10.RIII immunized with peptide; (C) B10.A immunized with IRBP. EAU scores of these mice and the number of mice per group are shown in Figures 1 and 2 .
Figure 4.
 
Lymphocyte proliferation of B10.RIII and B10.A mice immunized with or without concomitant pertussis treatment. Lymph nodes harvested 21 days after immunization were stimulated in culture with the immunizing antigen. Proliferation is shown as cpm × 10−3 of[ 3H]thymidine after subtraction of background counts (500 to 5.5 × 103 cpm, depending on the group). (A) B10.RIII immunized with IRBP; (B) B10.RIII immunized with peptide; (C) B10.A immunized with IRBP. EAU scores of these mice and the number of mice per group are shown in Figures 1 and 2 .
Figure 4.
 
Lymphocyte proliferation of B10.RIII and B10.A mice immunized with or without concomitant pertussis treatment. Lymph nodes harvested 21 days after immunization were stimulated in culture with the immunizing antigen. Proliferation is shown as cpm × 10−3 of[ 3H]thymidine after subtraction of background counts (500 to 5.5 × 103 cpm, depending on the group). (A) B10.RIII immunized with IRBP; (B) B10.RIII immunized with peptide; (C) B10.A immunized with IRBP. EAU scores of these mice and the number of mice per group are shown in Figures 1 and 2 .
Table 2.
 
Antigen-Specific Cytokine Production by Lymph Node Cells of B10.RIII and B10.A Mice Immunized with IRBP with or without PTX
Table 2.
 
Antigen-Specific Cytokine Production by Lymph Node Cells of B10.RIII and B10.A Mice Immunized with IRBP with or without PTX
Strain PTX Treatment IFN-γ (ng/ml) TNF-α (pg/ml) IL-4 (ng/ml) IL-5 (ng/ml)
B10.RIII 56.4 585 <0.078 <0.156
+ 174.0 624 <0.078 0.232
B10.A 4.8 213 <0.078 <0.156
+ 52.9 682 <0.078 0.322
Table 3.
 
Antigen-Specific Cytokine Production by Lymph Node Cells of B10.RIII Mice Immunized with p161-180 with or without PTX
Table 3.
 
Antigen-Specific Cytokine Production by Lymph Node Cells of B10.RIII Mice Immunized with p161-180 with or without PTX
PTX Treatment IFN-γ (ng/ml) TNF-α (pg/ml) IL-4 (ng/ml) IL-5 (ng/ml)
1.1 391 <0.078 <0.156
+ 8.5 566 <0.078 <0.156
Table 4.
 
Antigen-Specific Production of IFN-γ in the Presence or Absence of IL-12 in Culture by Pooled Spleen and Lymph Node Cells of B10.R.III Mice Immunized with p161-180
Table 4.
 
Antigen-Specific Production of IFN-γ in the Presence or Absence of IL-12 in Culture by Pooled Spleen and Lymph Node Cells of B10.R.III Mice Immunized with p161-180
Time in culture* IL-12 IFN-γ (ng/ml)
Experiment 1 Experiment 2
48 hours 0.419 0.752
+ 9.340 9.738
72 hours 1.056 1.401
+ 9.456 10.327
Figure 5.
 
Effects of IL-12 and PTX on adoptive transfer of EAU by peptide-specific cells. B10.RIII were immunized with p161-180. Their pooled spleen and lymph node cells were cultured with the immunizing antigen, with or without IL-12, and graded doses of cells were infused into naive recipients. Some recipients of cells cultured without IL-12 were treated with PTX. Shown are average EAU scores of the recipients on day 10 after adoptive transfer. Incidence of disease in each group is indicated (positive/total). The data are a composite of two experiments. IFN-γ production by the transferred cells is shown in Table 3 .
Figure 5.
 
Effects of IL-12 and PTX on adoptive transfer of EAU by peptide-specific cells. B10.RIII were immunized with p161-180. Their pooled spleen and lymph node cells were cultured with the immunizing antigen, with or without IL-12, and graded doses of cells were infused into naive recipients. Some recipients of cells cultured without IL-12 were treated with PTX. Shown are average EAU scores of the recipients on day 10 after adoptive transfer. Incidence of disease in each group is indicated (positive/total). The data are a composite of two experiments. IFN-γ production by the transferred cells is shown in Table 3 .
Caspi RR, Roberge FG, Chan CC, et al. A new model of autoimmune disease: experimental autoimmune uveoretinitis induced in mice with two different retinal antigens. J Immuno. 1988;140:1490–1495.
Caspi RR, Chan CC, Leake WC, Higuchi M, Wiggert B, Chader GJ. Experimental autoimmune uveoretinitis in mice: induction by a single eliciting event and dependence on quantitative parameters of immunization. J Autoimmu. 1990;3:237–246. [CrossRef]
Lando Z, Teitelbaum D, Arnon R. Induction of experimental allergic encephalomyelitis in genetically resistant strains of mic. Natur. 1980;287:551–552. [CrossRef]
Linthicum DS, Frelinger JA. Acute autoimmune encephalomyelitis in mice, II: susceptibility is controlled by the combination of H-2 and histamine sensitization genes. J Exp Me. 1982;156:31–40. [CrossRef]
Linthicum DS, Munoz JJ, Blaskett A. Acute experimental autoimmune encephalomyelitis in mice, I: adjuvant action of Bordetella pertussis is due to vasoactive amine sensitization and increased vascular permeability of the central nervous system. Cell Immuno. 1982;73:299–310. [CrossRef]
Lando Z, Ben-Nun A. Experimental autoimmune encephalomyelitis mediated by T-cell line: II, specific requirements and the role of pertussis vaccine for the in vitro activation of the cells and induction of disease. Clin Immunol Immunopatho. 1984;30:290–303. [CrossRef]
Spangrude GJ, Araneo BA, Daynes RA. Site-selective homing of antigen-primed lymphocyte populations can play a crucial role in the efferent limb of cell-mediated immune responses in viv. J Immuno. 1985;134:2900–2907.
Sewell WA, Andrews P. Inhibition of lymphocyte circulation in mice by pertussis toxi. Immunol Cell Bio. 1989;67:291–296. [CrossRef]
de Moerloose PA, Hamilton JA, Sewell WA, Vadas MA, Mackay IR. Pertussigen in vivo enhances antigen-specific production in vitro of lymphokine that stimulates macrophage procoagulant activity and plasminogen activato. J Immuno. 1986;137:3528–3533.
Sewell WA, de Moerloose PA, McKimm–Breschkin JL, Vadas MA. Pertussigen enhances antigen-driven interferon-gamma production by sensitized lymphoid cell. Cell Immuno. 1986;97:238–247. [CrossRef]
Fish F, Cowell JL, Manclark CR. Proliferative response of immune mouse T-lymphocytes to the lymphocytosis-promoting factor of Bordetella pertussi. Infect Immu. 1984;44:1–6.
Sudweeks JD, Todd JA, Blankenhorn EP, et al. Locus controlling Bordetella pertussis-induced histamine sensitization (Bphs), an autoimmune disease-susceptibility gene, maps distal to T- cell receptor beta-chain gene on mouse chromosome . Proc Natl Acad Sci US. 1993;90:3700–3704. [CrossRef]
Lyons AB. Pertussis toxin pretreatment alters the in vivo cell division behaviour and survival of B lymphocytes after intravenous transfe. Immunol Cell Bio. 1997;75:7–12. [CrossRef]
Pepperberg DR, Okajima TL, Ripps H, Chader GJ, Wiggert B. Functional properties of interphotoreceptor retinoid-binding protei. Photochem Photobio. 1991;54:1057–1060. [CrossRef]
Tarrant TK, Silver PB, Chan C–C, Wiggert B, Caspi RR. Endogenous IL-12 is required for induction and expression of experimental autoimmune uveiti. J Immunol. 1998;161:122–127. [PubMed]
Caspi R. Experimental autoimmune uveoretinitis (EAU): mouse and ra. Curr Protocols Immunol. 1997;5:16.
Xu H, Rizzo LV, Silver PB, Caspi RR. Uveitogenicity is associated with a Th1-like lymphokine profile: cytokine-dependent modulation of primary and committed T cells in EAU. Cell Immuno. 1997;178:69–78. [CrossRef]
Jones LS, Rizzo LV, Agarwal RK, et al. IFN-gamma-deficient mice develop experimental autoimmune uveitis in the context of a deviant effector respons. J Immuno. 1997;158:5997–6005.
Caspi RR, Silver PB, Chan CC, et al. Genetic susceptibility to experimental autoimmune uveoretinitis in the rat is associated with an elevated Th1 respons. J Immuno. 1996;157:2668–2675.
Sun B, Rizzo LV, Sun SH, et al. Genetic susceptibility to experimental autoimmune uveitis involves more than a predisposition to generate a T helper-1-like or a T helper-2- like respons. J Immuno. 1997;159:1004–1011.
McAllister CG, Vistica BP, Sekura R, Kuwabara T, Gery I. The effects of pertussis toxin on the induction and transfer of experimental autoimmune uveoretiniti. Clin Immunol Immunopatho. 1986;39:329–336. [CrossRef]
de Kozak Y, Naud MC, Bellot J, Faure JP, Hicks D. Differential tumor necrosis factor expression by resident retinal cells from experimental uveitis-susceptible and -resistant rat strain. J Neuroimmuno. 1994;55:1–9. [CrossRef]
Loetscher P, Seitz M, Clark–Lewis I, Baggiolini M, Moser B. Monocyte chemotactic proteins MCP-1, MCP-2, and MCP-3 are major attractants for human CD4+ and CD8+ T lymphocyte. FASEB . 1994;8:1055–1060.
Cyster JG, Goodnow CC. Pertussis toxin inhibits migration of B and T lymphocytes into splenic white pulp cord. J Exp Me. 1995;182:581–586. [CrossRef]
Tao X, Constant S, Jorritsma P, Bottomly K. Strength of TCR signal determines the costimulatory requirements for Th1 and Th2 CD4+ T cell differentiatio. J Immuno. 1997;159:5956–5963.
Murray JS. How the MHC selects Th1/Th2 immunit. Immunol Toda. 1998;19:157–163. [CrossRef]
Chaturvedi P, Yu Q, Southwood S, Sette A, Singh B. Peptide analogs with different affinities for MHC alter the cytokine profile of T helper cell. Int Immuno. 1996;8:745–755. [CrossRef]
Tao X, Grant C, Constant S, Bottomly K. Induction of IL-4-producing CD4+ T cells by antigenic peptides altered for TCR bindin. J Immuno. 1997;158:4237–4244.
Figure 1.
 
EAU scores of B10.RIII and B10.A mice immunized with IRBP, with or without concomitant pertussis treatment. (A) B10.RIII; (B) B10.A. The score at each dose is the average of all the mice in the group ± SE. EAU incidence (positive/total) is shown next to each point. The data are a composite of two experiments.
Figure 1.
 
EAU scores of B10.RIII and B10.A mice immunized with IRBP, with or without concomitant pertussis treatment. (A) B10.RIII; (B) B10.A. The score at each dose is the average of all the mice in the group ± SE. EAU incidence (positive/total) is shown next to each point. The data are a composite of two experiments.
Figure 2.
 
EAU scores of B10.RIII mice immunized with p161-180 with or without concomitant pertussis treatment. The score at each dose is the average of all the mice in the group ± SE. EAU incidence (positive/total) is shown next to each point. The data are a composite of two experiments.
Figure 2.
 
EAU scores of B10.RIII mice immunized with p161-180 with or without concomitant pertussis treatment. The score at each dose is the average of all the mice in the group ± SE. EAU incidence (positive/total) is shown next to each point. The data are a composite of two experiments.
Figure 3.
 
Delayed-type hypersensitivity scores of B10.RIII and B10.A mice immunized with or without concomitant pertussis treatment. Mice were challenged by injection of the immunizing antigen into the ear pinna 19 days after immunization. DTH scores represent the increment in ear thickness after 48 hours. (A) B10.RIII immunized with IRBP; (B) B10.RIII immunized with peptide; (C) B10.A immunized with IRBP. EAU scores of these mice and the number of mice per group are shown in Figures 1 and 2 .
Figure 3.
 
Delayed-type hypersensitivity scores of B10.RIII and B10.A mice immunized with or without concomitant pertussis treatment. Mice were challenged by injection of the immunizing antigen into the ear pinna 19 days after immunization. DTH scores represent the increment in ear thickness after 48 hours. (A) B10.RIII immunized with IRBP; (B) B10.RIII immunized with peptide; (C) B10.A immunized with IRBP. EAU scores of these mice and the number of mice per group are shown in Figures 1 and 2 .
Figure 4.
 
Lymphocyte proliferation of B10.RIII and B10.A mice immunized with or without concomitant pertussis treatment. Lymph nodes harvested 21 days after immunization were stimulated in culture with the immunizing antigen. Proliferation is shown as cpm × 10−3 of[ 3H]thymidine after subtraction of background counts (500 to 5.5 × 103 cpm, depending on the group). (A) B10.RIII immunized with IRBP; (B) B10.RIII immunized with peptide; (C) B10.A immunized with IRBP. EAU scores of these mice and the number of mice per group are shown in Figures 1 and 2 .
Figure 4.
 
Lymphocyte proliferation of B10.RIII and B10.A mice immunized with or without concomitant pertussis treatment. Lymph nodes harvested 21 days after immunization were stimulated in culture with the immunizing antigen. Proliferation is shown as cpm × 10−3 of[ 3H]thymidine after subtraction of background counts (500 to 5.5 × 103 cpm, depending on the group). (A) B10.RIII immunized with IRBP; (B) B10.RIII immunized with peptide; (C) B10.A immunized with IRBP. EAU scores of these mice and the number of mice per group are shown in Figures 1 and 2 .
Figure 5.
 
Effects of IL-12 and PTX on adoptive transfer of EAU by peptide-specific cells. B10.RIII were immunized with p161-180. Their pooled spleen and lymph node cells were cultured with the immunizing antigen, with or without IL-12, and graded doses of cells were infused into naive recipients. Some recipients of cells cultured without IL-12 were treated with PTX. Shown are average EAU scores of the recipients on day 10 after adoptive transfer. Incidence of disease in each group is indicated (positive/total). The data are a composite of two experiments. IFN-γ production by the transferred cells is shown in Table 3 .
Figure 5.
 
Effects of IL-12 and PTX on adoptive transfer of EAU by peptide-specific cells. B10.RIII were immunized with p161-180. Their pooled spleen and lymph node cells were cultured with the immunizing antigen, with or without IL-12, and graded doses of cells were infused into naive recipients. Some recipients of cells cultured without IL-12 were treated with PTX. Shown are average EAU scores of the recipients on day 10 after adoptive transfer. Incidence of disease in each group is indicated (positive/total). The data are a composite of two experiments. IFN-γ production by the transferred cells is shown in Table 3 .
Table 1.
 
Kinetics of Disease Onset in B10.RIII and B10.A Mice Immunized with or without PTX
Table 1.
 
Kinetics of Disease Onset in B10.RIII and B10.A Mice Immunized with or without PTX
Days after Immunization Strain and Immunization Regimen*
B10.RIII IRBP B10.RIII IRBP, PTX B10.RIII Peptide B10.RIII Peptide, PTX B10.A IRBP B10.A IRBP, PTX
6 0, 0, 0, 0, † 0, 0, 0, 0 0, 0, 0, 0 0, 0, 0, 0 0, 0, 0, 0 0, 0, 0, 0
7 0, 0, 0, 0.25 0.25, 0.5, 0.5, 0.5 0, 0, 0, 0 0, 0, 0.5, 0.5 0, 0, 0, 0 0, 0, 0, 0
8 0, 0.5, 0.75, 0.75 1.5, 1.25, 1.75, 2.5 0, 0.5, 1, 1 0.25, 1, 2, 3 0, 0, 0, 0 0, 0, 0, 0.75
9 3, 3, 3, 3 3, 3, 4, 4 0, 2, 3, 3 3, 3, 4, 4 0, 0, 0, 0 0, 0.25, 0.75, 2
10 ND ND ND ND 0, 0, 0, 0 0, 0.25, 0.75, 2.5
11 ND ND ND ND 0, 0, 0, 0 0, 0.25, 3, 3.5
12 ND ND ND ND 0, 0, 0.25, 0.5 0, 1, 3, 3.5
13 ND ND ND ND 0, 0, 0.5, 0.75 0, 3, 3, 3.5
14 ND ND ND ND 0, 0, 0.5, 0.75 0, 3, 3, 3.5
Table 2.
 
Antigen-Specific Cytokine Production by Lymph Node Cells of B10.RIII and B10.A Mice Immunized with IRBP with or without PTX
Table 2.
 
Antigen-Specific Cytokine Production by Lymph Node Cells of B10.RIII and B10.A Mice Immunized with IRBP with or without PTX
Strain PTX Treatment IFN-γ (ng/ml) TNF-α (pg/ml) IL-4 (ng/ml) IL-5 (ng/ml)
B10.RIII 56.4 585 <0.078 <0.156
+ 174.0 624 <0.078 0.232
B10.A 4.8 213 <0.078 <0.156
+ 52.9 682 <0.078 0.322
Table 3.
 
Antigen-Specific Cytokine Production by Lymph Node Cells of B10.RIII Mice Immunized with p161-180 with or without PTX
Table 3.
 
Antigen-Specific Cytokine Production by Lymph Node Cells of B10.RIII Mice Immunized with p161-180 with or without PTX
PTX Treatment IFN-γ (ng/ml) TNF-α (pg/ml) IL-4 (ng/ml) IL-5 (ng/ml)
1.1 391 <0.078 <0.156
+ 8.5 566 <0.078 <0.156
Table 4.
 
Antigen-Specific Production of IFN-γ in the Presence or Absence of IL-12 in Culture by Pooled Spleen and Lymph Node Cells of B10.R.III Mice Immunized with p161-180
Table 4.
 
Antigen-Specific Production of IFN-γ in the Presence or Absence of IL-12 in Culture by Pooled Spleen and Lymph Node Cells of B10.R.III Mice Immunized with p161-180
Time in culture* IL-12 IFN-γ (ng/ml)
Experiment 1 Experiment 2
48 hours 0.419 0.752
+ 9.340 9.738
72 hours 1.056 1.401
+ 9.456 10.327
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×