The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Su, S. B.
Right arrow Articles by Caspi, R. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Su, S. B.
Right arrow Articles by Caspi, R. R.
The Journal of Immunology, 2003, 171: 2314-2319.
Copyright © 2003 by The American Association of Immunologists

Dissociating the Enhancing and Inhibitory Effects of Pertussis Toxin on Autoimmune Disease

Shao Bo Su, Phyllis B. Silver, Peng Wang, Chi-Chao Chan and Rachel R. Caspi1

Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pertussis toxin (PT) has both enhancing and inhibitory effects on experimental autoimmune disease, depending on its time of administration relative to immunization. The inhibitory effect is due to blocking of Gi-coupled receptors by the enzymatic A subunit. In this study, we attribute the enhancing effect of PT to the cell-binding B subunit (PT-B). C57BL/6 mice, a strain that requires PT to develop experimental uveitis, were immunized with a retinal Ag and were injected with whole PT, PT-B, or vehicle. Disease and associated immunological responses were evaluated. The results showed that PT-B, determined to be free of biologically significant contamination with whole PT or with endotoxin, was able to mimic all the effects of PT with respect to disease induction, enhancement of delayed-type hypersensitivity, enhancement of lymphocyte proliferation, induction of an innate IL-12 response, and promotion of an adaptive IFN-{gamma} response to the uveitogenic Ag. Our results suggest that PT-B is largely responsible for the disease-enhancing properties of PT.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pertussis toxin (PT), 2 a bacterial toxin elaborated by Bordetella pertussis, which is the causative agent of the disease whooping cough in human, is a noncovalently linked heterohexameric protein. It is structurally and functionally divided into A and B subunits (1, 2, 3). The A subunit (A protomer, PT-A) is composed of a single peptide (S1) with ADP-ribosyltransferase activity that modifies GTP-binding regulatory proteins (G proteins), thus interfering with G protein-dependent signal transduction in mammalian cells. The B subunit (B oligomer, PT-B), composed of a pentameric protein complex (S2, S3, two S4 and S5), confers the binding capacity of PT to cell surface glycoprotein receptors and delivers the A protomer into the cell (1, 2, 4, 5).

PT has been used for many years as an adjuvant to enhance induction of organ-specific autoimmune diseases elicited by immunization with tissue Ags. Such models include experimental autoimmune encephalomyelitis, experimental autoimmune orchitis, experimental autoimmune uveitis (EAU), and others (6, 7, 8, 9, 10, 11). Previous work by us and by others showed that enhancing effect of PT on autoimmunity is due at least in part to promotion of the Th1 response owing to effects on APCs (12, 13, 14).

In contrast, we recently showed in the EAU model that PT also has potent inhibitory effects on disease that are especially prominent when it is administered at a later stage in disease development. The disease-inhibitory activity required the PT-A subunit, and was attributed to inhibition of effector cell migration to target organ secondary to disruption of G protein-coupled signals by PT-A (15, 16).

The present study was designed to dissociate the inhibitory from the enhancing effects of PT on EAU by evaluating the effects of purified PT-B on the induction of disease. The effect of purified PT-A could not be evaluated, as PT-A can only enter into the cell as a complex with PT-B. Mice given PT-B or whole PT concurrently with immunization developed the disease with similar maximal scores. PT-B treatment also increased delayed-type hypersensitivity (DTH), enhanced lymphocyte proliferation, and elevated IFN-{gamma} production to the uveitogenic Ag. PT-B induced mRNA and protein expression of IL-12 p70 by splenocytes. These data indicate that PT-B mimics the activity of whole PT in triggering Th1-polarizing innate immunity responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Six- to 8-wk-old C57BL/6, C3H/HeNCr, and C3H/HeJ mice supplied by The Jackson Laboratory (Bar Harbor, ME) were kept under specific pathogen-free conditions and given standard laboratory chow and water ad libitum. Animal care and use were in compliance with institutional guidelines.

Ag and reagents

Whole bovine interphotoreceptor retinoid-binding protein (IRBP) was purified from retinas by Con A-Sepharose affinity chromatography and HPLC (17). Whole PT or PT-B isolated from whole PT was purchased from Sigma-Aldrich (St. Louis, MO).

SDS-PAGE

SDS-PAGE was performed in a standard Laemmli system using 10% gel. The bands were visualized by Coomassie blue staining and scanned. The ratio of contamination of PT-B with A protomer was analyzed with National Institutes of Health Image 1.62 software.

Chemotaxis assay

Cell migration was assessed by using a 48-well microchemotaxis chamber, as described previously (18, 19). The number of migrated cells in three high-powered fields (x400) was counted by light microscopy after coding the samples. Results are expressed as the mean (±SD) value of the migration in triplicate samples.

LPS measurement

The amount of LPS contamination in PT and PT-B preparations was measured using the Chromogenic Limulus Amebocyte Lysate Test Kit (Cambrex, Walkersville, MD), according to manufacturer’s instructions. The kit yields LPS levels in units. Levels of LPS in pg/ml were calculated from a calibration curve constructed from serial dilutions of a known concentration of LPS.

Treatment with inhibitors

PT (50 µg/ml), PT-B (50 µg/ml), or LPS (10 µg/ml) was pretreated with proteinase K (25 µg/ml) for 45 min at 37°C in Dulbecco’s PBS (containing Ca2+ and Mg2+). The final concentrations of PT, PT-B, LPS, and the LPS inhibitor polymyxin B added to the cultures were 100 ng/ml, 100 ng/ml, 10 ng/ml, and 25 µg/ml, respectively.

EAU induction and scoring

EAU was induced by immunization with 150 µg bovine IRBP in CFA, as described before (20). At the same time, graded doses of PT-B or PT were injected i.p. Eyes were harvested 21 days after immunization. H&E-stained tissue sections of the whole eye were prepared and were graded by a masked observer on a scale of 0–4 in half-point increments, using the criteria described previously (12).

Delayed-type hypersensitivity

A total of 10 µg IRBP in 10 µl PBS or 10 µl PBS was injected into the pinna of right or left ear. Ear thickness was measured 48 h later using a spring-loaded micrometer.

Lymphocyte proliferation assay

Draining (inguinal and iliac) lymph nodes from immunized mice were collected 21 days after immunization, and were pooled within the group. Lymphocyte proliferation was assessed by tritiated thymidine uptake, as described previously (20). The data are shown as cpm.

Cytokine assays

Lymph node cells from IRBP-immunized mice or splenocytes from naive mice were cultured in 24-well flat-bottom plates (5 x 106 cells/ml culture medium per well) with the specified stimulants. Supernatants were collected 48 h later and were kept frozen in small aliquots at -70°C. Cytokine production was measured by ELISA using Ab pairs from Endogen (Boston, MA) for IFN-{gamma}, as described previously (21). IL-12 p70, the bioactive heterodimer, was determined using a kit (Quantikine M) from R&D Systems (Minneapolis, MN).

IL-12 p40 mRNA determination

Total cellular RNA was extracted from splenocytes for IL-12 p40 mRNA amplification, as follows: splenocytes (5 x 106/ml) from naive C57BL/6 mice were suspended in DMEM supplemented with 1% normal mouse serum and incubated with the specified concentrations of PT, PT-B, or LPS at 37°C overnight. In other experiments, naive mice were injected with the specified amounts of LPS, PT, or PT-B. The spleens were removed 2 h later. Total cellular RNA was extracted from collected cells and spleens. Reverse transcription was performed as per instructions in the kit (Clontech, Palo Alto, CA). PCR amplifications were performed for 25 cycles on the resultant cDNA for the expression of IL-12 p 40 mRNA. The sequences of the specific primers used in the detection of IL-12 p40 are as follows: sense, 5'-ATCGTTTTGCTGGTGTCTCC-3'; antisense, 5'-AGTCCCTTTGGTCCAGTGTG-3'. PCR amplification products were electrophoresed and viewed on a 1% agarose gel and stained with ethidium bromide. Bands were scanned and were quantitated using National Institutes of Health Image.

Reproducibility and data presentation

Experiments were repeated at least twice and usually three or more times. Results were highly reproducible. Figures show representative experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The preparation of PT-B has no detectable contamination by intact PT

Bioactive PT ADP-ribosylates Gi-type G proteins and interferes with cell migration by blocking chemokine receptor signaling. This activity is mediated by the enzymatic A subunit. In the present study, we wished to evaluate the effects of PT-B, free of the A subunit, on induction of EAU. Because commercially available PT-B is purified after dissociation of the A and the B subunits of PT, it was necessary to first ascertain that the PT-B preparation was free of contamination with whole PT. Fig. 1, A and B, demonstrates that contamination with PT-A was virtually undetectable by SDS-PAGE. Because biological assays are in general more sensitive, we examined the PT-B preparation for inhibition of cell migration in a standard Boyden chamber chemotaxis assay. Unlike intact PT, the tested preparation of PT-B did not inhibit cell migration to the chemokine, monocyte chemoattractant protein-1, even at a concentration as high as 1000 ng/ml (Fig. 1C), indicating that the preparation does not contain whole bioactive PT.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 1. Purity assessment of PT-B preparation. A, SDS-PAGE analysis. PT or PT-B (2 µg) sample was run under denaturing conditions. The 28-kDa band is PT-A. B, Intensity ratio of scanned bands was analyzed by NIH Image 1.62 software, as described in Materials and Methods. C, Preparation PT-B does not inhibit chemotaxis to monocyte chemoattractant protein-1. Human PBMC suspension treated with 100 ng PT or with graded doses of PT-B at 37°C for 1 h was submitted to chemotactic assay using a Boyden chamber. The number of migrated cells in three high-powered fields was counted by light microscopy after coding the samples. Results shown are representative of three independent experiments.

 
PT-B subunit mimics the effects of whole PT on induction of EAU and associated immunological responses

PT administered at the time of immunization with the autoantigen promotes induction of EAU and strongly enhances its associated immunological responses, including lymphocyte proliferation and DTH (22, 23). To evaluate the effects of PT-B, C57BL/6 mice immunized with IRBP in CFA were given a concurrent i.p. injection of an optimal dose of whole PT (determined to be 0.5 µg; Fig. 2A), graded doses of PT-B, or vehicle. Evaluation of eyes collected for histopathology on day 21 showed that vehicle-treated mice failed to develop disease (Fig. 2B). In contrast, PT-B facilitated development of EAU in a dose-dependent manner, ultimately reaching the same maximal disease scores as mice treated with an optimal dose of PT. The same mice challenged 2 days before the termination of the experiment for DTH responses to IRBP exhibited strongly augmented DTH scores (Fig. 2C). Similarly to the DTH response, lymphocyte proliferation was markedly enhanced by inclusion of PT-B in the immunization regimen (Fig. 2D). Thus, PT-B was able to mimic the enhancing effect of the whole PT on EAU, DTH, and Ag-specific lymphocyte proliferation.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 2. Enhancement of EAU and associated immunological response by PT-B. C57BL/6 mice were immunized with 150 µg IRBP in CFA, and the indicated doses of PT or PT-B were injected i.p. The data in each panel are representative of three or more repeat experiments. A and B, EAU scores of eyes as evaluated by histopathology 21 days after immunization. Each point represents one mouse (average of both eyes). C, DTH scores of mice challenged with 10 µl of IRBP into the ear pinna 19 days after immunization. Scores represent ear thickness after 48 h following Ag or PBS injection. D, Ag-specific proliferation of draining lymph node cells 21 days after immunization. Shown are cpm of [3H]thymidine ± SE of triplicates.

 
PT-B promotes development of a Th1 response

Our previous data indicated that EAU is dependent on the presence of a type 1 response to the uveitogenic Ag. We therefore compared the IFN-{gamma} production in response to IRBP of mice immunized and treated, or not, with PT or PT-B. Draining lymph node cells were collected 21 days after immunization. Ag-stimulated supernatants were generated from the different groups and were assayed for content of IFN-{gamma} by ELISA. The results showed that the lymphocytes from mice treated with PT-B produced at least as much IFN-{gamma} as mice treated with an optimal dose of PT-B, and both groups made considerably more IFN-{gamma} than vehicle-treated mice (Fig. 3). Taken together, these data suggest that similarly to intact PT, PT-B enhances disease expression by promoting cellular responses and increasing Th1 polarization.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 3. PT-B up-regulates Ag-specific IFN-{gamma} production. Lymph node cells harvested 21 days after immunization were stimulated with IRBP. IFN-{gamma} titers in 48-h supernatants were assayed by ELISA. The data are representative of three experiments.

 
PT-B induces IL-12 expression

IL-12 is a pivotal cytokine of the innate immune response that promotes cellular immunity (24). In the EAU model, it is required to generate uveitogenic effector cells and to maintain their function (25). It was therefore important to examine the effect of PT and PT-B on induction of innate IL-12. Induction of IL-12 was assessed in vitro and in vivo by expression of IL-12 p40 message, and by secretion of the IL-12 p70 heterodimer. LPS served as positive control.

Splenocytes of naive mice stimulated in culture with PT or with PT-B overnight expressed IL-12 p40 mRNA, and supernatants collected from these cells at 48 h contained IL–12 p70 protein detectable by ELISA (Fig. 4, A and B). More importantly, IL-12 was also induced in vivo. Spleens collected from mice 2 h after in vivo challenge with PT or PT-B expressed IL-12 p40 message (Fig. 4C). Splenocytes from parallel mice, placed in culture for 48 h without further stimulation, secreted IL-12 p70 protein into the supernatants (Fig. 4D). These results indicate that PT-B can closely mimic the effect of whole PT in triggering an innate IL-12 response, which could in part explain the observed skewing of the adaptive anti-IRBP response toward the Th1 phenotype and the enhancement of cellular responses and disease scores.



View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 4. PT-B enhances IL-12 expression. A, mRNA expression by splenocytes: splenocytes (5 x 106/ml) were cultured with PT (100 ng/ml), PT-B (100 ng/ml), or LPS (10 ng/ml) at 37°C overnight. RT-PCR for IL-12 p40 gives rise to expected size band of IL-12 p40 with length of 514 bp in the gel. Bands were scanned and quantitated using National Institutes of Health Image. The fold increase over unstimulated control was calculated after normalization to {beta}-actin. B, Protein expression by parallel cultures of splenocytes incubated for 48 h. IL-12 p70 in the supernatants was analyzed by ELISA. C, mRNA expression in spleens of mice treated in vivo with LPS (100 µg), PT (2 µg), or PT-B (2 µg). The spleens were removed after 2 h, and RT-PCR was performed as in A. Bands were scanned and quantitated using National Institutes of Health Image. The fold increase over untreated control was calculated after normalization to {beta}-actin. D, IL-12 protein in 48-h supernatants of splenocytes from mice treated in vivo as in C and incubated without further stimulation. Shown is IL-12 p70 protein, as assayed by ELISA in 48-h supernatants. Detection limit in this assay was 7.8 pg/ml. The data are representative of three repeat experiments.

 
The biological effects of PT-B cannot be attributed to LPS contamination

LPS is a bacterial component with adjuvant effects, and is a common contaminant in biological preparations. To rigorously exclude the possibility that any of the results shown above might be caused, or contributed to, by LPS contamination of the PT-B preparation, a number of experiments were performed. First, we quantitated the amount of endotoxin in the PT-B preparation and compared it with the minimal amount of LPS needed to induce production of IL–12 from mouse splenocytes in culture. The amount of LPS in the stock solution (100 µg/ml) of PT-B preparation was less than 0.3 endotoxin units (= less than 0.1 ng), as measured by the Chromogenic Limulus Amebocyte Lysate Test. More than 5 ng/ml of LPS was needed to trigger IL-12 message and IL–12 protein production from splenocytes, which is 250-fold more than the amount of LPS present in the 2 µg/ml solution of PT-B used for the assay (Fig. 5, A and B). Second, induction of IL–12 by the PT-B preparation was abrogated completely by proteinase K, attesting to the protein nature of the IL-12-inducing factor, whereas LPS-induced IL-12 production was resistant to proteinase K treatment (Fig. 5C). Conversely, the LPS inhibitor polymyxin B did not lower IL-12 production induced by the PT-B preparation, whereas it completely blocked IL-12 induction by LPS (Fig. 5C). Third, PT-B triggered IL-12 production by splenocytes of C3H/HeJ mice, which do not respond to LPS due to a Toll-like receptor 4 defect (Fig. 5C). Fourth, LPS was administered to mice concurrently with IRBP immunization in graded doses up to several orders of magnitude more than would be present as contaminant in PT-B. LPS was unable at any dose to reproduce the enhancing effect of PT-B on EAU and its associated immunological responses, including DTH, Ag-specific lymphocyte proliferation, and Ag-specific IFN-{gamma} production (Fig. 5, D, E, F, and G). These experiments indicated that none of the observed effects of PT-B could be attributed to traces of LPS present in the preparation.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 5. Biological effects of LPS vs PT-B in the EAU model. Dose response of LPS on IL-12 mRNA (A) and protein induction (B) in vitro: splenocytes (5 x 106/ml) from naive mice were cultured with the indicated amounts of LPS at 37°C overnight for message expression or 48 h for protein induction. Determination of IL-12 p40 by RT-PCR and p70 protein concentration in culture supernatants by ELISA was performed, as described previously. Effects of PT-B on IL-12 expression in splenocytes of C3H/HeNCr and C3H/HeJ mice (C): splenocytes (5 x 106/ml) from naive C3H/HeNCr mice (LPS responsive) and C3H/HeJ mice (LPS unresponsive) were cultured with PT (100 ng/ml), PT-B (100 ng/ml), and LPS (10 ng/ml) at 37°C for 48 h. Reagents (PT, PT-B, LPS) were treated with proteinase K (PK) before being added to the culture, as described in Materials and Methods. IL-12 p70 in the supernatants was analyzed by ELISA. Effects of LPS on EAU and its associated immunological responses: C57BL/6 mice were immunized with 150 µg IRBP in CFA, and the indicated doses of PT or LPS were injected i.p. EAU scores (D), DTH (E), Ag-specific proliferation of draining lymph node cells (F), and the effects of LPS on Ag-specific IFN-{gamma} production (G) were determined, as described previously.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PT has been used routinely in conjunction with immunization with tissue-specific Ags as an adjuvant to enhance induction of autoimmune diseases (6, 7, 8, 9, 10, 11). However, it is not clear which subunit of PT elicits these disease-enhancing effects. The report of He et al. (14) showing enhancement of IL-12 production in mouse splenocytes secondary to blockade of Gi protein signaling would appear to implicate the enzymatic PT-A subunit in this effect. In contrast, we (15) previously demonstrated that PT-A is responsible for inhibitory effects of PT on EAU, secondary to blockade of Gi protein-coupled receptors and disruption of chemokine signaling, and a subsequent study by others pointed out similar inhibitory effects of PT also on a Th2-driven model of allergic lung inflammation (26). In the present study, we dissociate the inhibitory from the enhancing effect of PT, by demonstrating that purified PT-B, free of biologically significant contamination with whole PT or with LPS, is able to closely mimic the effects of whole PT on EAU induction, on enhancement of cellular responses, and on promotion of Th1 polarization as judged by autoantigen-induced production of IFN-{gamma}. Furthermore, PT-B mimicked the effect of whole PT in being able to induce IL-12 message and IL-12 protein synthesis in splenocytes both in vitro and in vivo. This demonstrates that there is another route for inducing IL-12 by PT than through inhibition of Gi protein signaling.

Ours is not the first study that examines various activities of the PT-B subunit independent of the enzymatic A subunit. PT-B binds to cell surface glycoproteins, such as fetuin, haptoglobin, and the Lewisx blood group determinant on the cell surface; triggers phospholipase C and tyrosine kinase-dependent signal transduction events; and inhibits HIV replication and virus expression in human macrophages (3, 4, 5, 27, 28). Tamura et al. (29) reported that the B subunit is directly mitogenic to lymphocytes in vitro. We too observed that PT as well as PT-B enhanced proliferation when added directly to lymphocyte cultures (data not shown). Notably, Ryan et al. (30) found that a mutated PT with an enzymatically inactive A subunit exhibited adjuvant activities, including Ag-specific lymphocyte proliferation, cytokine and Ab production in vivo, and enhanced expression of B7 molecules on APC in vitro. Our study confirms and extends these data to an autoimmune disease model in which tissue pathology is dependent on Th1 polarization (31). In contrast, our data are at variance with Ben-Nun et al. (32), who reported that pretreatment of animals with PT-B 2–3 wk before an encephalitogenic challenge protected them from experimental autoimmune encephalomyelitis. We believe that in this case protection may have been due to induction of Abs in response to the pretreatment, which neutralized or eliminated the PT that was administered 2–3 wk later as necessary component of the encephalitogenic immunization protocol.

To understand exactly how PT-B exerts its immunostimulatory effects, the specific receptor(s) for PT-B will need to be identified. Although PT-B delivers PT-A into the cell, enabling it to block ADP ribosylation, one cannot assume that PT-B interacts directly with Gi protein-coupled receptors. Additional experiments, which are beyond the scope of the present study, are needed to identify the cellular receptor(s) for PT-B involved in this process and the signaling pathway(s) they activate.

The dose response of the enhancing effect of PT-B demonstrated that on a molar basis it was less than that of whole PT: to obtain a maximal enhancing effect, 2 µg of PT-B was needed vs 0.5 µg of whole PT. Because contamination of the PT-B preparation was excluded as a possible reason for this, two nonmutually exclusive explanations could be invoked. If all the enhancing activity of PT resides in the B subunit, the weaker adjuvanticity of PT-B compared with PT might be caused by a subtle configurational change of the B subunit as a result of dissociation from the A subunit, conceivably resulting in reduction of biological activity. Our data do not negate the possibility that a part of the enhancing activity might reside in the A subunit, as suggested by the data of He et al. (14), showing that inhibition of Gi protein signaling up-regulates IL-12 and promotes the Th1 response. It should be kept in mind, however, that the same Gi protein inhibition by the A subunit also strongly suppresses disease expression due to inhibition of responses to chemokines and prevention of effector cell migration to the target organ (15, 16). All these considerations notwithstanding, an important conceptual advance that stems from the present study is that neither the ADP-ribosyltransferase activity of PT-A, nor the IL-12-enhancing effects of Gi protein blockade that it causes, are necessary for Th1 polarization of immunological responses and for disease induction in the EAU model.

In summary, the present data, in conjunction with our previous reports (15, 16), demonstrate that the enhancing and inhibitory effects of PT on experimental autoimmunity can be dissociated from each other. Although the A subunit is largely responsible for the inhibitory effects of PT on EAU secondary to inhibition of cell migration by Gi-coupled receptor inhibition, the enhancing effect can be duplicated by the B subunit that mimics the effects of whole bioactive PT on induction of innate IL-12, enhancement of adaptive IFN-{gamma}, potentiation of cellular immunity, and facilitation of autoimmune tissue pathology.


   Acknowledgments
 
We are grateful to Drs. Joost Oppenheim and Ji Ming Wang, Frederick Cancer Research Facility, National Cancer Institute, for helpful critique of the manuscript.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Rachel R. Caspi, Laboratory of Immunology, National Eye Institute, National Institutes of Health, 10 Center Drive, 10/10N222, Bethesda, MD 20892-1857. E-mail address: rcaspi{at}helix.nih.gov Back

2 Abbreviations used in this paper: PT, pertussis toxin; DTH, delayed-type hypersensitivity; EAU, experimental autoimmune uveitis; IRBP, interphotoreceptor retinoid-binding protein. Back

Received for publication April 24, 2003. Accepted for publication June 3, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Merritt, E. A., W. G. Hol. 1995. AB5 toxins. Curr. Opin. Struct. Biol. 5:165.[Medline]
  2. Kaslow, H. R., D. L. Burns. 1992. Pertussis toxin and target eukaryotic cells: binding, entry, and activation. FASEB J. 6:2684.[Abstract]
  3. Wong, W. S., P. M. Rosoff. 1996. Pharmacology of pertussis toxin B-oligomer. Can. J. Physiol. Pharmacol. 74:559.[Medline]
  4. Saukkonen, K., W. N. Burnette, V. L. Mar, H. R. Masure, E. I. Tuomanen. 1992. Pertussis toxin has eukaryotic-like carbohydrate recognition domains. Proc. Natl. Acad. Sci. USA 89:118.[Abstract/Free Full Text]
  5. Van’t Wout, J., W. N. Burnette, V. L. Mar, E. Rozdzinski, S. D. Wright, E. I. Tuomanen. 1992. Role of carbohydrate recognition domains of pertussis toxin in adherence of Bordetella pertussis to human macrophages. Infect. Immun. 60:3303.[Abstract/Free Full Text]
  6. Raine, C. S.. 1984. Biology of disease: analysis of autoimmune demyelination: its impact upon multiple sclerosis. Lab. Invest. 50:608.[Medline]
  7. Tuohy, V. K.. 1994. Peptide determinants of myelin proteolipid protein (PLP) in autoimmune demyelinating disease: a review. Neurochem. Res. 19:935.[Medline]
  8. Kerlero de Rosbo, N., I. Mendel, A. Ben-Nun. 1995. Chronic relapsing experimental autoimmune encephalomyelitis with a delayed onset and an atypical clinical course, induced in PL/J mice by myelin oligodendrocyte glycoprotein (MOG)-derived peptide: preliminary analysis of MOG T cell epitopes. Eur. J. Immunol. 25:985.[Medline]
  9. Mendel, I., N. Kerlero de Rosbo, A. Ben-Nun. 1995. A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H-2b mice: fine specificity and T cell receptor V{beta} expression of encephalitogenic T cells. Eur. J. Immunol. 25:1951.[Medline]
  10. Caspi, R. R., F. G. Roberge, C. C. Chan, B. Wiggert, G. J. Chader, L. A. Rozenszajn, Z. Lando, R. B. Nussenblatt. 1988. A new model of autoimmune disease: experimental autoimmune uveoretinitis induced in mice with two different retinal antigens. J. Immunol. 140:1490.[Abstract]
  11. Hart, M. N., D. S. Linthicum, M. M. Waldschmidt, S. K. Tassell, R. L. Schelper, R. A. Robinson. 1987. Experimental autoimmune inflammatory myopathy. J. Neuropathol. Exp. Neurol. 46:511.[Medline]
  12. Caspi, R.. 1997. Experimental autoimmune uveoretinitis (EAU) – mouse and rat. A. Kruisbeek, and D. Margulies, and E. Shevach, and W. Strober, eds. In Current Protocols in Immunology Vol. January:Unit 15.6. John Wiley & Sons, New York.
  13. Hofstetter, H. H., C. L. Shive, T. G. Forsthuber. 2002. Pertussis toxin modulates the immune response to neuroantigens injected in incomplete Freund’s adjuvant: induction of Th1 cells and experimental autoimmune encephalomyelitis in the presence of high frequencies of Th2 cells. J. Immunol. 169:117.[Abstract/Free Full Text]
  14. He, J., S. Gurunathan, A. Iwasaki, B. Ash-Shaheed, B. L. Kelsall. 2000. Primary role for Gi protein signaling in the regulation of interleukin 12 production and the induction of T helper cell type 1 responses. J. Exp. Med. 191:1605.[Abstract/Free Full Text]
  15. Su, S. B., P. B. Silver, M. Zhang, C. C. Chan, R. R. Caspi. 2001. Pertussis toxin inhibits induction of tissue-specific autoimmune disease by disrupting G protein-coupled signals. J. Immunol. 167:250.[Abstract/Free Full Text]
  16. Agarwal, R. K., S. H. Sun, S. B. Su, C. C. Chan, R. R. Caspi. 2002. Pertussis toxin alters the innate and the adaptive immune response in a pertussis-dependent model of autoimmunity. J Neuroimmunol. 129:133.[Medline]
  17. Pepperberg, D. R., T. L. Okajima, H. Ripps, G. J. Chader, B. Wiggert. 1991. Functional properties of interphotoreceptor retinoid-binding protein. Photochem. Photobiol. 54:1057.[Medline]
  18. Falk, W., R. H. Goodwin, Jr, E. J. Leonard. 1980. A 48-well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration. J. Immunol. Methods 33:239.[Medline]
  19. Su, S. B., H. Ueda, O. M. Howard, M. C. Grimm, W. Gong, F. W. Ruscetti, J. J. Oppenheim, J. M. Wang. 1999. Inhibition of the expression and function of chemokine receptors on human CD4+ leukocytes by HIV-1 envelope protein gp120. Chem. Immunol. 72:141.[Medline]
  20. Tarrant, T. K., P. B. Silver, J. L. Wahlsten, L. V. Rizzo, C. C. Chan, B. Wiggert, R. R. Caspi. 1999. Interleukin 12 protects from a T helper type 1-mediated autoimmune disease, experimental autoimmune uveitis, through a mechanism involving interferon {gamma}, nitric oxide, and apoptosis. J. Exp. Med. 189:219.[Abstract/Free Full Text]
  21. Xu, H., L. V. Rizzo, P. B. Silver, R. R. Caspi. 1997. Uveitogenicity is associated with a Th1-like lymphokine profile: cytokine-dependent modulation of early and committed effector T cells in experimental autoimmune uveitis. Cell. Immunol. 178:69.[Medline]
  22. Caspi, R. R., P. B. Silver, C. C. Chan, B. Sun, R. K. Agarwal, J. Wells, S. Oddo, Y. Fujino, F. Najafian, R. L. Wilder. 1996. Genetic susceptibility to experimental autoimmune uveoretinitis in the rat is associated with an elevated Th1 response. J. Immunol. 157:2668.[Abstract]
  23. Silver, P. B., C. C. Chan, B. Wiggert, R. R. Caspi. 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. Invest. Ophthalmol. Visual Sci. 40:2898.[Abstract/Free Full Text]
  24. Trinchieri, G.. 1998. Proinflammatory and immunoregulatory functions of interleukin-12. Int. Rev. Immunol. 16:365.[Medline]
  25. Tarrant, T. K., P. B. Silver, C. C. Chan, B. Wiggert, R. R. Caspi. 1998. Endogenous IL-12 is required for induction and expression of experimental autoimmune uveitis. J. Immunol. 161:122.[Abstract/Free Full Text]
  26. Mathew, A., B. D. Medoff, A. D. Carafone, A. D. Luster. 2002. Cutting edge: Th2 cell trafficking into the allergic lung is dependent on chemoattractant receptor signaling. J. Immunol. 169:651.[Abstract/Free Full Text]
  27. Alfano, M., G. Vallanti, P. Biswas, C. Bovolenta, E. Vicenzi, B. Mantelli, T. Pushkarsky, R. Rappuoli, A. Lazzarin, M. Bukrinsky, G. Poli. 2001. The binding subunit of pertussis toxin inhibits HIV replication in human macrophages and virus expression in chronically infected promonocytic U1 cells. J. Immunol. 166:1863.[Abstract/Free Full Text]
  28. Alfano, M., T. Pushkarsky, G. Poli, M. Bukrinsky. 2000. The B-oligomer of pertussis toxin inhibits human immunodeficiency virus type 1 replication at multiple stages. J. Virol. 74:8767.[Abstract/Free Full Text]
  29. Tamura, M., K. Nogimori, M. Yajima, K. Ase, M. Ui. 1983. A role of the B-oligomer moiety of islet-activating protein, pertussis toxin, in development of the biological effects on intact cells. J. Biol. Chem. 258:6756.[Abstract/Free Full Text]
  30. Ryan, M., L. McCarthy, R. Rappuoli, B. P. Mahon, K. H. Mills. 1998. Pertussis toxin potentiates Th1 and Th2 responses to co-injected antigen: adjuvant action is associated with enhanced regulatory cytokine production and expression of the co-stimulatory molecules B7-1, B7-2 and CD28. Int. Immunol. 10:651.[Abstract/Free Full Text]
  31. Brenner, T., O. Abramsky. 1993. Natural and experimental transfer of anti-pertussis antibodies confers resistance to experimental autoimmune encephalomyelitis. J. Neurol. Sci. 114:13.[Medline]
  32. Ben-Nun, A., I. Mendel, N. Kerlero de Rosbo. 1997. Immunomodulation of murine experimental autoimmune encephalomyelitis by pertussis toxin: the protective activity, but not the disease-enhancing activity, can be attributed to the nontoxic B-oligomer. Proc. Assoc. Am. Physicians 109:120.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. Bo Su, P. B. Silver, P. Wang, C.-C. Chan, and R. R. Caspi
Cholera Toxin Prevents Th1-Mediated Autoimmune Disease by Inducing Immune Deviation
J. Immunol., July 15, 2004; 173(2): 755 - 761.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
K. Darabi, A. Y. Karulin, B. O. Boehm, H. H. Hofstetter, Z. Fabry, J. C. LaManna, J. C. Chavez, M. Tary-Lehmann, and P. V. Lehmann
The Third Signal in T Cell-Mediated Autoimmune Disease?
J. Immunol., July 1, 2004; 173(1): 92 - 99.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Su, S. B.
Right arrow Articles by Caspi, R. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Su, S. B.
Right arrow Articles by Caspi, R. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS