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The Journal of Immunology, 2006, 177: 6896-6903.
Copyright © 2006 by The American Association of Immunologists, Inc.

Pertussis Toxin Is Superior to TLR Ligands in Enhancing Pathogenic Autoimmunity, Targeted at a Neo-Self Antigen, by Triggering Robust Expansion of Th1 Cells and Their Cytokine Production1

Chiaki Fujimoto*, Cheng-Rong Yu2,*, Guangpu Shi2,*, Barbara P. Vistica*, Eric F. Wawrousek{dagger}, Dennis M. Klinman{ddagger}, Chi-Chao Chan*, Charles E. Egwuagu* and Igal Gery3,*

* Laboratory of Immunology and {dagger} Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892; and {ddagger} Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Microbial products are assumed to play a major role in triggering pathogenic autoimmunity. Recently accumulated data have shown that these products stimulate the immune system by interacting with TLRs, expressed on APCs. To examine the capacity of various TLR ligands to trigger pathogenic autoimmunity, we used a system in which naive CD4 cells, specific against hen egg lysozyme (HEL), are injected into recipient mice expressing HEL in their eyes. Only when stimulated, the naive cells acquire pathogenic capacity and induce ocular inflammation. Seven TLR ligands were tested in this system: lipoteichoic acid/peptidoglycan, zymosan, poly (I:C), LPS, pertussis toxin (PTX), flagellin, and CpG oligodeoxynucleotide. Treatment of recipient mice with HEL alone stimulated proliferation of the transferred cells, but no disease, whereas ocular inflammation did develop in recipient mice coinjected with HEL and any one of the seven TLR ligands. Inflammation induced by PTX surpassed by its severity those induced by all other tested TLR ligands and was accompanied by a dramatic increase in number of the transferred cells that acquired features of effector Th1 lymphocytes. Ocular inflammation and number of transferred cells in recipients injected with PTX and HEL were substantially reduced by treatment with Abs against IFN-{gamma} or IL-12, thus indicating the role of these cytokines in the PTX effect. Overall, our observations demonstrate that various TLR ligands are capable of triggering pathogenic autoimmunity and that PTX surpasses other microbial products in this activity, by stimulating excessive proliferation and polarization toward Th1 of naive T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The negative selection process in the thymus is incomplete, allowing T cells with specificity toward self Ags to escape deletion (1, 2, 3, 4). Such cells are present in healthy individuals in a "resting" state, but when activated, these resting cells acquire effector features and the capacity to invade the target tissue and initiate pathogenic autoimmune processes (3, 5, 6). The mechanisms whereby self-specific T cells are activated are not completely clear, but there is ample indirect evidence to suggest that microbial infection plays a major role in this process (3, 7, 8, 9). This notion has been supported by the well-known requirement for microbial components in adjuvants used to promote pathogenic autoimmunity in experimental animals. Moreover, the role played by microbial molecules in triggering autoimmunity has been clearly defined following the discovery of TLRs in vertebrates and the analysis of their crucial role in triggering the innate immune response (9, 10, 11, 12). Further, these data have shown that microbial molecules serve as ligands for TLRs on APC and activate these cells to reach maturity and acquire enhanced capacity to efficiently present specific Ags to naive T cells (9, 10, 11, 12). When the activated T cells are specific against self Ags, the outcome could be an autoimmune response, with subsequent potential pathogenicity.

Fourteen TLRs have been discovered so far in vertebrates, with a number of microbial components identified as the specific ligands for the majority of these TLRs. Major known TLR ligands include peptidoglycan (PGN)4 and lipoteichoic acid (LTA), from Gram-positive bacteria, that are ligands for TLR2; zymosan, a product of fungi, a ligand for TLRs 2 and 6; dsRNA of viruses, represented by polyriboinosinic polyribocytidylic acid (poly(I:C)), a ligand for TLR3; LPS from Gram-negative bacteria, a ligand for TLR4; bacterial flagellin, a ligand for TLR5 and CpG oligodeoxynucleotide (ODN), the ligand for TLR9 (for more detail, see reviews by Medzhitov (9) and Pulendran (12)). Several of these microbial TLR ligands have been known for their capacity to enhance immune responses and have been used in various forms of adjuvants for decades.

A microbial product of particular interest is pertussis toxin (PTX). This multifunctional molecule has been used for its capacity to promote pathogenic autoimmunity; PTX was found to enhance a variety of immune responses (13, 14, 15, 16, 17, 18, 19, 20) and to be essential for induction of experimental autoimmune diseases in the CNS (13, 21) and the eye (22, 23). The mode of action of PTX in promoting autoimmune diseases has been attributed to several different mechanisms, including its effect on blood-tissue barriers (5, 14), increased cytokine production (24, 25), or selective enhancement of Th1 immune response (26). More recently, PTX was found to also function as a ligand for TLR4 (7, 27) and its capacity to enhance experimental autoimmune encephalomyelitis was attributed in part to induction of adhesion molecules on brain endothelial cells (7, 27).

In this study, we present data of a study in which PTX was compared with six other TLR ligands for their capacity to trigger pathogenic autoimmunity by activating naive CD4 cells specific against a neo-self Ag. In the system we used, transgenic (Tg) mice expressing hen egg lysozyme (HEL) in their eyes are injected with naive CD4 cells that transgenically express a TCR specific against HEL (6, 28, 29). No pathological changes were detected in eyes of the recipient mice, even following injection with HEL. In contrast, treatment of these recipient mice with any of the tested TLR ligands resulted in development of inflammatory ocular changes. PTX activity was found in this experimental system to exceed by far that of all other tested TLR ligands. Analysis of the PTX effect revealed that treatment with this agent is characterized by a dramatic increase in number of donor cells, with unusual kinetics, as well as by profound production of Th1 cytokines. We propose that these unique activities of PTX are responsible, at least in part, for the capacity of this agent to enhance pathogenic autoimmunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

HEL-Tg mice expressing membrane-bound HEL in the lens under control of the {alpha}A-crystallin promoter, on the FVB/N background, were generated as described elsewhere (30). HEL-specific TCR Tg mice, designated 3A9, on the B10.BR background (31) were a gift from M. Davis (Stanford University, Stanford, CA). Tg mice from each of the two lines were mated to produce (FVB/N x B10.BR)F1 hybrids, expressing either HEL in their lens (HEL-Tg mice), or the HEL specific TCR on their T cells (3A9 mice). Only F1 hybrids expressing either one of the two transgenes were used in the present study. In all adoptive transfer experiments recorded here, the cells used were from 3A9 donors, whereas recipients were HEL-Tg mice. Mice were housed in a pathogen-free facility and maintained in a 12-h light-12-h dark cycle. All procedures involving animals were performed according to the guidelines of the National Institute of Health Resolution for the Care and Use of Laboratory Animals.

Reagents

HEL, PTX, PGN, LTA from Staphylococcus aureus, and poly(I:C) were provided by Sigma-Aldrich. LPS from Salmonella typhimurium was purchased from Difco. Flagellin from S. typhimurium was purchased from InvivoGen. Zymosan A from Saccharomyces cerevisiae was provided by Molecular Probes. CpG ODNs, GCTAGACGTTAGCGT and TCAACGTTGA, were synthesized at the Center for Biologics Evaluation and Research, Food and Drug Administration (CBER, FDA) core facility (Bethesda, MD) and were administered together. The concentration of contaminating endotoxin in the tested agents was <10 endotoxin units/ml as measured by a Limulus amebocyte assay (Cambrex). The doses used for injection into mice were: 100 µg each for PGN and LTA, which were administered together in all experiments recorded here, 150 µg for poly(I:C), 50 µg for LPS and zymosan, 10 µg for flagellin, 80 µg for CpG ODN, and 0.5 or 1.0 µg of PTX per mouse.

Adoptive transfer of CD4 cells and induction of disease

Naive CD4 cells were isolated from spleens and lymph nodes of 3A9 mice using T cell columns and MACS beads as described in detail elsewhere (28, 29). Isolated CD4 cells (5 x 106) were injected via the tail vein into naive HEL-Tg mice. On the following day, the recipient mice were injected i.v. with PBS or the TLR ligands, at the indicated doses, with or without HEL, at 100 µg/mouse. Recipients were euthanized on the indicated days following the transfer of CD4 cells. Eyes were fixed in 4% glutaraldehyde for 1 h, followed by 4% formaldehyde. Fixed eyes were embedded in methacrylate, sectioned via the pupillary-optic nerve head axis and stained with H&E. The severity of inflammation was scored as described elsewhere (28). Briefly, the level of inflammation was evaluated separately in the anterior segment, vitreous, and retina, on a scale of 0–3. The final score consisted of the sum of the three subscores on a final scale of 0–9.

CFSE dilution assay

Naive CD4 cells (5 x 107 cells/ml) were labeled with CFSE (Molecular Probes) as described by Bird et al. (32). CFSE-labeled cells were injected i.v. into HEL-Tg mice (5 x 106 cells in 200 µl). Recipient mice were euthanized on the indicated days postadoptive transfer, and splenocytes were collected and stained with allophycocyanin (ALPC)-conjugated anti-CD4 mAb (BD Pharmingen). Cell suspensions were analyzed on a FACScan Cytometer (BD Biosciences) and were gated for CD4+CFSE+ dye dilution peaks, with 106 events being acquired in a live cell gate.

Flow cytometry analysis

mAbs against murine CD4-ALPC (L3T4), CD49d-PE (R1–2), CD62L-PE (MEL-14), and isotype rat IgG controls-FITC, -PE, or -ALPC were purchased from BD Pharmingen. A clonotypic mAb specific for the Tg TCR of the 3A9 mice, designated 1G12, a gift from E. Unanue (Washington University, St. Louis, MO), was used conjugated with FITC. Anti-CD16/CD32 Abs (2.4G2; BD Pharmingen) was used to block FcRs in all stainings.

Spleen cells of recipient mice were collected at different time points following adoptive transfer of donor cells and single-cell suspensions were prepared by conventional methods. Collected cells were treated with ammonium chloride potassium ("ACK") buffer (Cambrex) and washed in staining buffer. For cell surface staining, incubation steps were performed for 30 min at 4°C. Flow cytometry analysis was performed on a FACSCalibur (BD Biosciences) using FlowJo (Tree Star).

Measurement of cytokine production

Spleen cells of recipient mice were cultured in 24-well plates at 5 x 106 cells/well in 1 ml of RPMI 1640 medium, supplemented with HL-1 serum replacement (Cambrex), antibiotics, and 2-ME (50 µM), with or without stimulants, as indicated. Supernatants were collected after incubation for 48 h and their cytokine levels were determined by the Pierce, using Multiplex SearchLight technology.

Real-time RT-PCR analysis

Total RNA was extracted with TRIzol from whole eyes of recipient mice on day 7 postadoptive transfer of CD4 cells. RNA (10 µg), SuperScript II Reverse Transcriptase (Invitrogen Life Technologies) and oligo(dT)12–16 were used for first-strand cDNA synthesis as previously described (33). Real-time 5'-nuclease fluorogenic RT-PCR analysis was performed on an ICycler iQ Real-Time PCR Sequence Detection System (Bio-Rad). PCR was conducted with the following primers: IFN-{gamma}, 5'-CAGCAACAGCAAGGCGAA-3' and 5'-CTGGACCTGTGGGTTGTTGAC-3'; IL-4, 5'-ACAGGAGAAGGGACGCCAT-3', 5'-CTGTGGTGTTCTTCGTTGCTG-3', β-actin, 5'-CGGTTCCGATGCCCTGAGGCTC-3', 5'-CAGCAACAGCAAGGCGAA-3'. Fluorescence-labeled probes used are: IFN-{gamma}, 5'-FAM-CCCAAGAAGGAAGGCTGG A-AHQ-13'; IL-4, 5'-FAM-AAGGATGCATTCATGAGTATTGCCAAGTTTGA-AHQ-13'; β-actin, 5'-TET-ACGGAGATGGATGTGCCAAACGTCCT-AHQ-13'.

Triplicate samples of 10-fold serial dilutions of cDNA were assayed and used to construct standard curves. PCR parameters were as recommended for the TaqMan Universal PCR master mix kit (Applied Biosystems). β-actin was used as an external copy number standard to enable the measurement of relative amounts of IFN-{gamma} and IL-4 mRNA. It should be emphasized that the standard curves generated from cDNA dilution series showed excellent linearity indicating precise quantitative relationship between cDNA copy number and fluorescence intensity within the dynamic range of the assay.

Treatment with anti-IL-12 Ab and anti-IFN-{gamma} Ab

Rat mAbs against murine IL-12, clone C17.8, a gift from G. Trinchieri (Schering-Plough Research Institute, Dardilly, France) and rat mAb against murine IFN-{gamma} (clone R4–6A2), purchased from American Type Culture Collection, were administered i.p., at 0.5 mg/mouse on days 1, 3, and 5 postadoptive transfer of naive CD4 cells. Control mice were injected with normal rat IgG2a according to the same schedule. Recipient mice were euthanized on day 7 and pathological changes in their eyes, as well as numbers of donor cells in their spleens were determined as described above.

Statistical analysis

Data are shown as the mean values ± SEM. For histological scores, each mouse (average of both eyes) is shown and treated as one event for the purpose of statistical analysis, using the Mann-Whitney rank sum test. Statistical significance of differences for all other assays was analyzed by independent Student’s t test. Differences of p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PTX is superior to TLR ligands in triggering pathogenicity by naive CD4 cells

To examine the capacity of PTX and other TLR ligands to trigger pathogenicity in naive T cells, we used an experimental system in which naive CD4 cells specific against HEL are transferred into Tg-recipient mice in which HEL is expressed in the lens. No inflammatory ocular changes are detected in recipients of naive CD4 cells, but severe inflammation develops in eyes of recipients injected with CD4 cells activated with Th1 cytokines in culture before transfer (6, 28). This system thus made it possible to assess the capacity of agents to trigger pathogenicity in CD4 cells in vivo, by treating recipients of naive CD4 cells with the tested agents and assessing the development of ocular inflammatory changes in these recipient mice. The tested agents included PTX and six TLR ligands, i.e., the combination of PGN and LTA, poly(I:C), LPS, flagellin, zymosan, and CpG ODN. Data accumulated in repeated experiments are summarized in Fig. 1A. All tested agents were injected with or without HEL.


Figure 1
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  		FIGURE 1. Enhancement of ocular inflammation by treatment with various TLR ligands. A, Recipient HEL-Tg mice were injected with 5 x 106 naive CD4 cells from 3A9 mice and 1 day later were treated with HEL, 100 µg, along with PBS, or one of the seven TLR ligands, at the doses cited in Materials and Methods. Eyes of the recipient mice were collected on day 7 postcell injection and the level of inflammation, on a scale of 0–9, was determined as described in Materials and Methods and in Ref. 28 . Circles represent individual mice. Inflammation severity in eyes of all treated groups was significantly higher than in the control group (PBS), with p < 0.05 for the zymosan (Zym) treated mice and p < 0.01 for all other groups. The inflammation severity in the PTX-treated mice was also significantly higher (p < 0.01) than in all other treated groups. B, Typical histological changes in eyes of recipient mice. There are no inflammatory changes in the eye of a recipient treated with HEL (100 µg) plus PBS, moderate levels of inflammation in the recipients treated with HEL plus LPS (50 µg), or CpG ODN (80 µg), and very severe changes in the eye of the mouse treated with HEL plus PTX (1 µg). Please note that the lens in all eyes presented here show distortion of the normal morphology, due to the expression of the transgene (30 ). Higher magnifications of the optic nerve head area of eyes from recipients treated with CpG ODN or PTX are shown below the corresponding eye sections. H&E, original magnifications: x10 or x40. Additional abbreviations, used for certain ligands in this and other Figures only: PGN, a combination of PGN and LTA; PIC, poly (I:C); Flg, flagellin; Zym, zymosan.

 
No ocular changes were seen in recipients treated with any of the agents without HEL (data not shown). Marginal or no inflammatory changes were detected in recipients injected with HEL alone, whereas ocular inflammation of different severity levels was observed in recipient mice injected with the combination of HEL and any of the tested agents (Fig. 1A). Treatment with PTX was superior to all six TLR ligands, with the majority of PTX-treated recipient mice developing remarkably more severe inflammatory changes than recipients treated with any of the tested TLR ligands.

Typical changes in eyes of mice treated with LPS, CpG, or PTX are depicted in Fig. 1B. The moderate ocular changes in the eyes of recipient mice treated with LPS or CpG included mainly infiltration of inflammatory cells, mostly lymphocytes. The infiltration characteristically localized in the entry sites of the cells, i.e., large vessels at the optic nerve head, limbus, and retinal blood vessels. Inflammatory cells were also seen in most cases in the vitreous as well. Dramatically more severe inflammation was seen, in contrast, in eyes of recipient mice treated with PTX. Typically, the major changes in these eyes included heavy cellular infiltration in most ocular tissues, severe retinal detachment, with serous and cellular exudates in the subretinal space, various degrees of destruction to the retina, edema of the cornea, and protein material in the vitreous.

PTX stimulates vigorous donor cell proliferation, with unique kinetics

The severity of ocular inflammation in recipient mice in the experimental system used here is determined to a large extent by the number of transferred effector cells (28). To examine the possibility that the superior activity of PTX in the transferred inflammation system is due to vigorous proliferation of donor cells in the recipient mouse, we monitored the division level of donor cells in recipient mice by the CFSE assay. Data collected in mice treated with CpG ODN were also included, for comparison. As seen in Fig. 2, essentially no division was observed on day 3 postcell transfer in donor cells in the spleen of mice treated with either PTX or CpG ODN alone, but active proliferation did develop in mice injected with these molecules in combination with HEL. Interestingly, the division rate in mice treated with PTX or CpG ODN and HEL did not differ much from that seen in mice injected with HEL alone (Fig. 2). The division rate in all mice injected with HEL was rapid and the CFSE was diluted beyond clear detection as soon as day 5 (data not shown). These results thus indicate that the division rate of donor cells at the early phase following cell transfer does not explain the unusual severity of ocular inflammation in mice treated with PTX.


Figure 2
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  		FIGURE 2. Donor cell division in recipient mice treated with HEL or TLR ligands, alone or together. Naive CD4 cells of 3A9 donors, 5 x 106, were labeled with CFSE (5 µM) before being adoptively transferred to HEL-Tg recipient mice, that were treated a day later as indicated. Spleens of recipients were collected on day 3 postcell injection and assessed for proliferation by CFSE dilution of CD4+ cells. A similar level of cell division is seen in all recipient mice treated with HEL (100 µg) with PBS, PTX (1 µg), or CpG ODN (80 µg), whereas no division was induced in recipients treated with PTX or CpG ODN alone. The result is representative of three independently performed experiments.

 
Another approach we used to monitor the effect of PTX and other microbial products on the number of donor cells in the recipient mice, at different time points, was to actually count these cells in the recipient spleen. This approach was made possible by the availability of a clonotypic Ab, designated "1G12," that is specific for the 3A9 mouse TCR (34, 35). Fig. 3A shows the flow cytometric data, on day 7 postcell injection, when the mouse eyes were collected for examination. The percent of 1G12+ cells in spleens of PTX-treated mice (6.4%) was ~13-fold higher in this experiment than that in CpG ODN-treated mice (0.5%) (Fig. 3A). The calculated actual numbers of 1G12+ cells in spleens of recipient mice treated with all tested agents, plus HEL, are shown in Fig. 3B. Treatment with all six TLR ligands initiated increases in donor cell numbers, as compared with PBS-injected controls, but the effect of treatment with PTX profoundly exceeded that of other ligands. Also of interest are the findings that 1) the effect of all stimuli was completely dependent on the accompanying injection with HEL (data not shown) and that 2) HEL alone induced only a marginal increase in donor cell number on day 7, despite its activating cell division immediately following injection, as shown in Fig. 2.


Figure 3
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  		FIGURE 3. Treatment with PTX exceeds by far that with other TLR ligands in stimulating increase in number of transferred donor cells. HEL-Tg mice were injected with 5 x 106 naive CD4 cells from 3A9 donor mice and a day later were treated with HEL plus PBS or the TLR ligands, as indicated. Spleens of recipients were collected on day 7 postcell transfer and analyzed by flow cytometry for the donor cells (1G12+). A, Data representative of five independent experiments, measuring the percent of 1G12+ cells (the recorded values) in pooled spleens of recipient mice treated with HEL (100 µg) plus PBS, PTX (1 µg), or CpG (80 µg). B, Actual number of donor cells in the spleen of recipient mice treated with HEL plus PBS or the different TLR ligands. The number of donor cells was determined in pools of three mice in each treatment group and the data are the calculated mean ± SEM of three to nine independently performed experiments. The difference between the number of donor cells in recipients treated with flagellin (Flg) or zymosan (Zym) and controls was insignificant, but reached significance in groups treated with the other ligands (p < 0.05 for LPS and p < 0.01 for all other groups). The number of donor cells in the PTX-treated mice was significantly higher than in all other groups (p < 0.01). Additional abbreviations used for certain ligands, are given in the legend for Fig. 1.

 
The uniqueness of PTX effect on the number of donor cells was further demonstrated when comparing its kinetics to that of the other stimuli. Fig. 4A records the number of donor cells in spleens of recipient mice at different time points following treatment with HEL alone, or in combination with PTX or CpG ODN. A marginal increase in donor cells was seen on days 3 and 5 in mice treated with HEL alone. A moderate increase in 1G12+ cells was recorded in mice treated with CpG ODN plus HEL, reaching a peak on day 5 and declining thereafter. In contrast, donor cell numbers in PTX-treated mice reached a high peak on day 7 and decreased only slightly on day 9. Examination for inflammation of eyes of the mice tested for donor cell number (Fig. 4B) revealed that the peak of severity was reached on day 7 in both groups, treated with PTX or CpG ODN, with a moderate decrease by day 9.


Figure 4
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  		FIGURE 4. Kinetics of increase in donor cell number in the recipient mouse spleen (A) and severity of ocular inflammation (B) in recipients treated with HEL plus PBS, PTX (1 µg), or CpG ODN (80 µg). Groups of HEL-Tg mice were injected with 5 x 106 naive CD4 cells from 3A9 donors, and treated a day later as indicated. Subgroups of three recipients were euthanized at the indicated time points, their spleens were pooled and the number of donor cells (1G12+) were determined by flow cytometry, while the levels of ocular inflammation were scored as described in Materials and Methods. The presented values are means ± SEM, collected in three independent experiments.

 
Donor cells in PTX-treated mice efficiently acquire adhesion molecule profile characteristic for tissue invasion

The inflammation-inducing capacity of T cells is determined to a large extent by the profile of molecules on their surface. Of particular importance are the adhesion molecules CD49d ("VLA4") and CD62L ("L-selectin"); studies of our group and others have shown that tissue invading T lymphocytes are characterized by high expression of CD49d and low expression of CD62L (6, 36, 37). To examine the effect of treatment with PTX and the TLR ligands on the profile of surface molecules on donor cells, we determined the expression of CD49d and CD62L on 1G12+ cells in spleen of recipient mice. Fig. 5 summarizes the flow cytometric data collected in repeated experiments. Spleen cells of recipients treated with most ligands exhibited increased expression of CD49d and reduction in CD62L, but these changes in mice treated with PTX surpassed those induced by all other agents.


Figure 5
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  		FIGURE 5. Expression levels of cell surface adhesion molecules on donor cells in recipient mice treated with HEL plus PBS, or the various TLR ligands. HEL-Tg mice were injected with 5 x 106 naive CD4 cells from 3A9 donors and treated a day later as indicated. Recipient spleens were collected 7 days postcell transfer and their donor cells (1G12+) were analyzed by flow cytometry for expression of CD49d ("VLA4") (A), or CD62L ("L-selectin") (B). Donor cells from the PTX-treated mice exhibited the highest levels of CD49d and lowest levels of CD62L, with the differences reaching significance (p < 0.01) when compared with most other groups. The data are mean values ± SEM of three to nine independent experiments. Additional abbreviations, used for certain ligands, are spelled out in the legend for Fig. 1.

 
PTX treatment promotes Th1 cytokine production by donor cells

Naive CD4 cells undergo polarization toward Th1 or Th2 immune types following antigenic stimulation. To examine the type and level of polarization of donor cells in recipient mice of the different groups, we cultured spleen cells of these recipient mice and measured the level of type-specific cytokines following stimulation with HEL (Fig. 6). Levels of IL-12 and IFN-{gamma}, Th1-specific cytokines, were dramatically higher in cultures from PTX-treated mice than in cultures of all tested TLR ligands. In contrast, production of IL-10 in cultures from the PTX-treated mice was one of the lowest among the seven groups. Spleen cells from PTX-treated mice also produced IL-1β, IL-2, and IL-6 in levels significantly higher than splenocytes from other mouse groups (Fig. 6), suggesting that enhanced production of these cytokines played a role in the disease induction process in the recipient mice. It is of note that, unlike the enhanced production of cytokines in response to the Ag (HEL) by spleen cells from PTX-treated mice, no enhancement was observed in the response of these cells to the non-Ag-specific stimulation by Con A (data not shown).


Figure 6
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  		FIGURE 6. A unique cytokine production profile of donor cells from recipient mice treated with PTX. HEL-Tg mice were injected with 5 x 106 naive CD4 cells from 3A9 donors and treated a day later with HEL plus PBS, or the tested TLR ligands. Recipient spleens were collected 7 days postcell transfer, their cells were cultured with HEL for 48 h, and cytokine levels in supernatants of these cultures were measured as described in Materials and Methods. The data are means ± SEM of three independent experiments. The levels of IL-12, IFN-{gamma}, IL-6, IL-1β, and IL-2 were significantly higher in cultures of PTX-treated mice than in those of the other groups (p < 0.05 for IL-6 and p < 0.01 for all other mentioned cytokines). Abbreviations for certain ligands are spelled out in the legend for Fig. 1.

 
To further assess the role of polarization toward Th1 in the PTX effect, we analyzed the profile of cytokine mRNA transcripts in the recipient mouse eye. We extracted RNA from eyes of recipient mice of the different treatment groups and determined the level of IFN-{gamma} and IL-4 transcripts, using real-time RT-PCR. The results of two experiments are summarized in Fig. 7. Eyes of PTX-treated mice expressed high levels of IFN-{gamma} transcript, but low levels of the IL-4 transcript. It is also of interest that a high level IFN-{gamma} but low level of IL-4 transcripts were also found in eyes of recipients treated with CpG ODN, while relatively higher levels of IL-4 transcript were found in eyes of mice treated with PGN/LTA (PGN), poly (I:C), and LPS.


Figure 7
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  		FIGURE 7. Expression of IFN-{gamma} and IL-4 transcripts in inflamed eyes of recipient mice following treatment with HEL plus PTX or the other TLR ligands. HEL-Tg mice were injected with 5 x 106 naive CD4 cells from 3A9 donors and treated a day later with HEL plus PBS or the tested TLR ligands. Recipient mouse eyes were collected on day 7 postcell transfer and whole RNA extracts were tested by quantitative real-time PCR for the expression of IFN-{gamma} or IL-4, as detailed in Materials and Methods. The data are mean values ± SEM of five or six independent samples. Expression of IFN-{gamma} transcript in eyes of recipients treated with PTX was significantly higher than that in all other groups (p < 0.01). Abbreviations for certain ligands are given in the legend for Fig. 1.

 
The PTX effect is neutralized by Abs against IFN-{gamma} and IL-12

In view of the vigorous production of IL-12 and IFN-{gamma} in mice treated with PTX, we examined the role of these Th1 cytokines in the pathogenic process by treating PTX-injected recipient mice with Abs against these cytokines. As shown in Fig. 8, treatment with either one of these Abs reduced both the level of ocular inflammation (Fig. 8A) and the number of donor cells in the recipients’ spleen (Fig. 8, B and C). These observations thus suggest that both IL-12 and IFN-{gamma} play active roles in the PTX-induced pathogenic process.


Figure 8
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  		FIGURE 8. Treatment with Abs against IFN-{gamma} or IL-12 reduced the severity of ocular inflammation and the increase in number of donor cells in recipients treated with PTX. HEL-Tg mice were injected with 5 x 106 naive CD4 cells from 3A9 donors and treated on day 1 with the combination of HEL (100 µg) and PTX (0.5 µg). Groups of three mice were then injected on days 1, 3, and 5, i.p., with rat Abs against IFN-{gamma} or IL-12, 0.5 mg/mouse. Control mice were similarly injected with the corresponding normal rat Ig isotype (IgG). A, Severity of ocular inflammation. B, Flow cytometric analysis of the recipient spleen, determining the percent of donor cells in the whole spleen population of mice of each group; C, actual numbers of donor cells (1G12+) in the spleen of recipient mice of each group. Data recorded in A and C are mean ± SEM of three independent experiments, while B depicts representative data of these experiments. The treatment effect reached significance (p < 0.05) only when IFN-{gamma} Ab was used and the severity of ocular changes were assessed.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study compared, for the first time, several major known TLR ligands for their capacity to trigger CD4-mediated pathogenic autoimmune processes. The ligands’ activities were assessed by an experimental system we developed, in which naive CD4 cells, specific against HEL, are adoptively transferred into recipient mice that express HEL in their eyes (28, 29). The transferred naive cells do not induce disease in untreated recipients, but do so in mice treated with agents capable of stimulating the naive CD4 cells to become effector cells (6, 28). Our system thus provides an experimental model that imitates the presumable process by which pathogenic autoimmunity is initiated by infection. Our system has several advantages over other systems that have been used in recently published studies (3, 8, 38), in which the capacity of TLR ligands to induce pathogenic autoimmunity has been shown: 1) the population of adoptively transferred naive CD4 cells in our system is homogeneous, 2) the number of cells used can be modified at will, 3) the transferred cells can be identified by a clonotypic Ab and any changes in their number or surface markers can be readily monitored, and 4) the immune response of the transferred T cells can be differentiated from that of the host cells, since only the former cells recognize HEL.

All tested TLR ligands were found capable of triggering pathogenic autoimmune processes in our system, thus indicating that autoimmunity may be stimulated by a wide spectrum of microbial products. Whereas moderate levels of ocular inflammation were found in mice treated with six of the seven tested TLR ligands, exceptionally severe changes were seen in eyes of recipients treated with PTX (Fig. 1A), reaching severity levels comparable only to those observed in mice injected with 106 or more in vitro-stimulated Th1 cells (28). Data we collected in the present study suggest that the strikingly severe ocular changes in PTX-treated recipient mice could be attributed to the dramatic increase in number of transferred cells, which acquired features of "activated" Th1 cells and produced very high levels of Th1 type cytokines.

The observation concerning the increase in number of transferred cells in the PTX-treated mice sheds new light on the activity of this microbial product in triggering pathogenic autoimmunity. Our observation was made possible by the availability of the clonotypic Ab, 1G12, that identifies the donor cells and differentiates them from the recipient host cells. It is noteworthy that PTX was found to stimulate proliferation of T cells in vitro, when added without any other antigenic stimulus (39). In contrast, however, PTX had no effect in our in vivo system unless the Ag, HEL, was administered as well.

Of particular interest is the unique kinetics by which donor cells increased in number in the spleen of PTX-treated recipient mice. The increase in donor cells in PTX-treated mice resembled that in recipients treated with another TLR ligand, CpG ODN, but only during the first 5 days following cell transfer. Later, the number of donor cells declined in the spleen of recipient mice treated with CpG ODN, but dramatically increased in recipients treated with PTX, with their numbers remaining high at least until day 9 postcell transfer (Fig. 4A).

In all experiments recorded in this study, the treatment with PTX was given 1 day following the adoptive transfer of the naive CD4 cells. To examine the PTX activity when administered before the cells, we treated groups of mice with PTX and HEL on days –1 or –3. We found that the typical severe ocular changes (as seen in Fig. 1B) developed in these experimental mice, but in smaller proportions than that observed in mice treated on day +1. In addition, the number of donor cells in recipient spleens, as well as these cells’ capacity to release inflammatory cytokines (e.g., IFN-{gamma}) in response to HEL stimulation in vitro, was lower in recipients treated with PTX before the adoptive transfer of the cells, as compared with mice treated with PTX on day +1 (data not shown). These results indicate that the PTX effect is retained in the recipient mouse, but is slowly diminishing. We also examined the PTX effect when administered again, on days +7 or +10, following the routine treatment on day +1. The repeated treatment with PTX had essentially no effect when the recipient mice were examined on day +14 for ocular inflammation and number and cytokine release of donor cells in the recipient spleen (data not shown).

In addition to the profound increase in the number of donor cells, development of severe inflammatory changes in eyes of recipients treated with PTX could be attributed in part to changes in the adhesion molecule profile on the donor cells, i.e., increase in CD49d and decrease in CD62L (Fig. 5). This profile characterizes tissue-invading cells (6, 36, 37) and was also observed on 3A9 Th1 cells following their activation in culture with HEL (6).

Another feature that characterizes donor cells in PTX-treated recipients, which is assumed to contribute to the potent disease-inducing capacity of these cells, is their vigorous production of IFN-{gamma} when stimulated in culture with HEL (Fig. 6). In addition, it is assumed that IFN-{gamma} released by the Th1 cells stimulated the recipient’s macrophages and dendritic cells to produce the high levels of IL-12 measured in these whole spleen cultures (Fig. 6). These two cytokines play major roles in shifting the immune response toward the Th1 pole, and their participation in the pathogenic process is indicated by the reduced ocular inflammation seen in recipient mice treated with Abs against IFN-{gamma} or IL-12 (Fig. 8). Furthermore, the actual involvement of IFN-{gamma} in the inflammatory response promoted by PTX was indicated by the finding of high expression of the IFN-{gamma} transcript in eyes of PTX-treated recipient mice (Fig. 7). Our finding concerning the selective stimulation of Th1 cytokine production in PTX-treated mice is in line with data reported by Hou et al. (26). It should be mentioned, however, that IL-12 shares a major component, p40, with another cytokine, IL-23 (40) and therefore, treatment with the IL-12 Ab could neutralize IL-23 as well. Because IL-23 drives the subset of Th17 lymphocytes (40, 41), this Ab treatment could also have affected the immunopathogenic Th17 population. This possibility is currently under investigation.

Analysis of the Th1/Th2 profiles of the infiltrating cells in inflamed eyes of recipient mice treated with the different TLR ligands (Fig. 7) revealed remarkable selectivity in the effect of the different ligands. Whereas PTX and CpG ODN promoted "Th1 response" (high IFN-{gamma}, low IL-4), treatment with PGN, poly(I:C), and LPS skewed the response toward the Th2 type (low IFN-{gamma}, high IL-4). Our data are thus in line with recent observations by the groups of Pulendran (42, 43) and Raz (44), showing that different TLR ligands differentially regulate the Th1/Th2 balance.

Recent publications indicated that the enhancing effect of PTX on the immune response is mediated mainly via the activation of TLR4 (7, 27). Data collected in the present study suggest, however, that the effect of PTX in our system could not be entirely attributed to this mode of action. LPS, the "hallmark" ligand for TLR4, was included in our study and its stimulatory activity differed remarkably from that of PTX by all tested parameters, i.e., severity of ocular inflammation, increase in number of donor cells, and, in particular, the profile of cytokines produced by the donor cells in culture. It is conceivable, therefore, that the unique activity of PTX in the system we used in this study is mediated mainly via a mechanism other than the "conventional" activation of TLR4. It is also of note that TLR4 was found not to be essential for another immunopathogenic process in the eye in which PTX plays an essential role, i.e., induction of experimental autoimmune uveitis (EAU). Su et al. (45) reported that mice deficient in TLR4 developed EAU similarly to their wild-type control, whereas no disease could be induced in mice deficient in IL-1R.

It is noteworthy that the PTX effect in our experimental system depended on cotreatment of the recipient mice with HEL, because no inflammation was detected in recipients of naive CD4 cells treated with PTX alone (data not shown). This observation suggests that PTX does not directly affect the naive CD4 cells, but rather, this molecule possibly enhances the pathogenic process by promoting the antigenic stimulation of these cells by a unique mechanism. It is also of interest that marginal or no ocular inflammation was seen in recipient mice injected with HEL alone, with no additional stimulus, despite the proliferation of donor cells observed in these mice (Fig. 2). This finding indicates that the process of Ag-induced cell division by itself does not elicit pathogenic capacity in naive T cells specific against self Ags. It is also of note that Thompson et al. (46) have recently found in a similar cell transfer system that T cells stimulated by the Ag alone divided well, but exhibited poor survival, as compared with cells stimulated with the combination of the Ag and a TLR ligand.

Our finding that PTX triggers a powerful pathogenic autoimmune process by stimulating naive CD4 cells to vigorously proliferate and acquire effector Th1 cell features sheds new light on the mode of action of this microbial product as an adjuvant for induction of pathogenic autoimmunity. Treatment with PTX is essential for induction of experimental autoimmune diseases such as experimental autoimmune encephalomyelitis (13, 21) or EAU (22, 23) and its mode of action has been attributed to several mechanisms, in particular increasing vascular permeability (5, 14), activation of the innate immunity via TLR4 (7, 27), reduction in number of Treg cells (47, 48), and enhancement of Th1 responses by stimulation of dendritic cells (26). Our observations suggest that enhanced proliferation of Th1 cells and, perhaps, of Th17 cells, specific against the target self Ag combines with the other effects of PTX to produce the unique adjuvant activity of this molecule.

In summary, using an experimental system in vivo that detects the conversion of naive CD4 cells into pathogenic effector Th cells, we found that all tested TLR ligands exhibited this capacity, thus supporting the notion that infection plays a major role in triggering autoimmunity. PTX, also a TLR ligand, surpassed by far all other ligands in triggering severe inflammation, following vigorous cell proliferation and Th1 cytokine production. Data collected here thus extend our knowledge about the capacity of PTX to promote pathogenic autoimmunity.


   Acknowledgments
 
We thank Robert S. Lee for tail DNA analysis, Ricardo Dreyfuss for digital microphotography, and the National Eye Institute Histology Core Facility for tissue section preparations.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Intramural Research Program of the National Eye Institute, National Institutes of Health. Back

2 C.-R.Y. and G.S. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Igal Gery, Laboratory of Immunology, National Eye Institute, National Institutes of Health, Building 10, Room 10N208, Bethesda, MD 20892-1857. E-mail address: geryi{at}nei.nih.gov Back

4 Abbreviations used in this paper: PGN, peptidoglycan; LTA, lipoteichoic acid; ODN, oligodeoxynucleotide; PTX, pertussis toxin; poly(I:C), polyriboinosinic polyribocytidylic acid; Tg, transgenic; ALPC, allophycocyanin; HEL, hen egg lysozyme; EAU, experimental autoimmune uveitis. Back

Received for publication December 29, 2005. Accepted for publication August 24, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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