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
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lassmann, S.
Right arrow Articles by Campbell, I. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lassmann, S.
Right arrow Articles by Campbell, I. L.
The Journal of Immunology, 2001, 167: 5485-5493.
Copyright © 2001 by The American Association of Immunologists

Induction of Type 1 Immune Pathology in the Brain Following Immunization Without Central Nervous System Autoantigen in Transgenic Mice With Astrocyte-Targeted Expression of IL-121

Silke Lassmann2, Carrie Kincaid, Valérie C. Asensio3 and Iain L. Campbell4

Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037 Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-12, a cytokine produced by microglia, may regulate cellular immunity at a localized level in the CNS. To investigate this further, we examined the consequences of peripheral immune stimulation without specific autoantigen in wild-type or transgenic (termed GF-IL12) mice with astrocyte production of the bioactive IL-12 p75 heterodimer. Active immunization with CFA and pertussis toxin, a procedure known to stimulate a robust type 1-biased immune response, produced CNS immune pathology from which GF-IL12 but not wild-type mice developed signs of clinical disease consisting of loss of activity, piloerection, mild tremor, and motor change. All immunized mice had some degree of mononuclear cell infiltration into the brain; however, the severity of this was markedly increased in GF-IL12 mice where leukocytes accumulated in perivascular and parenchymal locations. Accumulating cells consisted of CD4+ and CD8+ T cells and macrophage/microglia. Moreover, expression of cytokines (IFN-{gamma} and TNF), chemokines (IFN-inducible protein-10 and RANTES), the immune accessory molecules, MHC class II, B7.2, ICAM-1 and VCAM-1, and NO synthase-2 was induced in the CNS of the GF-IL12 mice. Therefore, peripheral immunization of GF-IL12 but not wild-type mice can provoke active type 1 immunity in the brain—a process that does not require CNS-specific immunizing autoantigen. These findings indicate that the cytokine milieu of a tissue can dramatically influence the development of intrinsic immune responses and associated pathology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Despite the enigmatic etiology of most human organ-specific autoimmune diseases, evidence strongly suggests that both genetic and/or environmental factors play a crucial role (1, 2, 3, 4). Whether the failure to maintain self-tolerance primarily occurs at the level of the target organ or systemically is still debatable. This is certainly true for the human demyelinating disease multiple sclerosis (MS)5 (5, 6), a presumed autoimmune disease of the CNS whose etiology is associated with both environmental (e.g., viral and bacterial infections; Ref. 7) and genetic factors (8, 9). CNS-specific, circulating autoreactive T cells are present in the periphery of patients with MS and are believed to mediate the disease (10, 11, 12, 13). However, such autoreactive T cells can also be found in the blood of healthy individuals, suggesting that the mere presence of autoreactive T cells in the periphery is not sufficient for disease and requires further triggers to activate a pathogenic CNS-targeted autoimmune response. The possibility that these triggers involve, in part, environmental factors is tantalizing and is supported by numerous clinical reports documenting an association between antecedent viral and bacterial infections with the development or exacerbation of MS (7, 14, 15, 16).

Insights to the pathological mechanisms of MS have been gained from studies of what is considered to be the prototypic animal model of this human disorder, experimental autoimmune encephalomyelitis (EAE) (17). In rodents, EAE induction is triggered in the periphery by actively immunizing with CNS-specific Ag(s), thereby initiating a type 1 immune response directed against the CNS by autoreactive CD4+ Th1 cells. These models of active EAE invariably require potent adjuvants, above all, CFA often combined with pertussis toxin (PTX) treatment. The primary constituent of CFA that makes it so effective is the presence of microbial products such as Mycobacterium or bacterial DNA. A likely role of these microbial agents is to stimulate production of the cytokine IL-12 by APC (18, 19, 20, 21, 22, 23). The presence of IL-12 during the crucial stages in the development of CNS-Ag reactive CD4+ T cells then drives these cells into Th1-cell lineage commitment and function (24, 25). PTX supports Ab (26) and delayed-type hypersensitivity (27) responses and potentiates polyclonal (28) and Ag-specific T cell (29, 30) activation as well as IFN-{gamma} production (31). Both CFA and PTX may also increase disruption of blood-tissue barriers so as to facilitate autoreactive T cell access to target organs (32, 33, 34). Significantly, the combination of CFA and PTX is superior for the stimulation of type I immunity (35) and likely accounts for the need for both agents to effectively induce EAE.

IL-12 has a dominant role in melding the innate and adaptive arms of the immune response (for reviews see Refs. 36, 37). The functionality of this cytokine relies on the formation of a heterodimer between the p35 and p40 subunits encoded by two separate genes. As noted above, IL-12 is a key positive regulator of Th1-type T cell development and it induces large amounts of IFN-{gamma} production by newly differentiated as well as by mature Th1 T cells. Additionally, IL-12 promotes the functional activation and IFN-{gamma} production by NK and CD8+ T cells. IL-12 is essential for the normal development of type 1 immune responses to microbial infection. This is well illustrated in mice lacking IL-12 due to targeted disruption of the IL-12 gene. The ability of these animals to generate Th1 immunity is severely impaired (38, 39). IL-12 is also central to the induction of a variety of experimental autoimmune diseases that involve autoaggressive CD4+ Th1 cells (40, 41). Consistent with this, normally susceptible mice are completely resistant to the development of EAE in the absence of IL-12 (42), while treatment with anti-IL-12 Abs also abated disease (43, 44).

Interestingly, IL-12 expression has been demonstrated in the brain in active MS (45, 46) and in EAE (47, 48). In the CNS, in addition to infiltrating leukocytes, resident glial cells can also produce IL-12. Thus, induction of IL-12 p40 gene expression by microglia and astrocytes in vivo occurs following repeated systemic injection of sublethal doses of LPS (49). Similar to other APC, studies in vitro show that secretion of the IL-12 heterodimer is stimulated from microglia by microbial products such as LPS in combination with IFN-{gamma} as well as by CD40-CD40L interaction (49, 50, 51, 52). In all, these experimental and clinical data highlight that IL-12 production can be elicited from cells intrinsic to the brain, thus raising the possibility that IL-12 may regulate, at a localized level, cellular immunity within the CNS. In support of this, recent studies by us showed that CNS-targeted expression of IL-12 in transgenic mice induces a spontaneous CNS-specific type 1 immune-mediated neurological disease and also accelerates EAE induction (53).

Accumulating evidence indicates that bystander activation of T cells, mediated by tissue damage and/or the cytokine environment of the target tissue, can contribute to pathogenic T cell responses independent of Ag and TCR signaling (54, 55, 56). Additionally, because cytokines such as IL-12 are potent activators of NK cells and phagocytes, the cytokine milieu of a tissue might also contribute to innate immune responses. However, whether and to what extent the inflammatory milieu of the brain can initiate and/or drive immune responses locally is unclear. Therefore, in the present study we used the GF-IL12 transgenic model to investigate the influence of the CNS milieu on the development of localized immune responses. Specifically, we wished to address the question as to whether the "cytokine-primed" CNS of GF-IL12 mice could promote the targeting and activation of cellular immunity following induction of type 1 immune responses in the periphery induced by immunization with CFA and PTX without CNS-specific immunizing Ag. The results indicated that an active type 1 immune response developed in the brain of the GF-IL12 but not wild-type mice soon after peripheral immune challenge. Therefore, glial production of IL-12 can act as a local adjuvant for the propagation in the brain of peripherally induced type 1 immune responses—a process that does not require CNS-specific immunizing autoantigen. These findings strongly suggest that the cytokine milieu of a tissue can dramatically influence the development of intrinsic immune responses and associated pathology.


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

Transgenic mice expressing the IL-12 p35 and p40 subunit genes in astrocytes were generated as recently described (53). Mice of the hemizygous GF-IL12 line expressing chronic low levels of bioactive IL-12 were used in all studies. All mice were of the C57BL/6 x SJL hybrid background. For controls, nontransgenic littermate mice were obtained from the breeding of the GF-IL12 line and were therefore of the same genetic background. All mice studied were between 2 and 3 mo of age, well before the onset of spontaneous CNS immunity in the GF-IL12 mice, which occurred in some mice from ~5 mo of age.

Immunization protocol

All experiments were performed with new sets of emulsifying syringes designated exclusively for the following specific immunization schedule. Mice were actively immunized into the hind flanks with 200 µl of a 1:1 emulsion of BSA (1 mg/ml; Sigma-Aldrich, St. Louis, MO) in PBS in CFA (containing 4 mg/ml Mycobacterium tuberculosis H37RA; Difco, Detroit, MI), and each mouse received an additional i.p. injection of 500 ng of PTX (Sigma-Aldrich) at days 0 and 2. After immunization, all mice were observed for the time periods indicated and were then killed for RNA and histological analysis. Disease scores were assessed as follows: each ruffled fur, hunched posture, general "sickness", and shaky or mild motor function loss was assigned a grade of 0.5, whereas severe forms of these symptoms were graded as 1. For each animal, the daily score was assessed by taking the sum of all these grades. The mean score was calculated by adding the maximal scores for each animal, irrespective of when this score was reached, and dividing by the number of sick mice.

RNA isolation

Organs were removed and immediately snap frozen in liquid nitrogen and stored at -80°C pending RNA extraction. Poly(A)+ RNA was isolated according to a previously published method (57). Total RNA was extracted with TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer’s protocol.

RNase protection assays (RPA)

RPA were performed as described previously (58). The RNA samples (5 µg of total RNA or 1.5 µg of poly(A)+ RNA) were hybridized with [32P]UTP labeled probe sets to the cytokines, IFN-{gamma} and TNF (59), the chemokines IP-10 and RANTES (60), leukocyte subset markers (61), and host-response (62) genes. For all probe sets, a fragment of the RPL32–4A gene (63) served as an internal loading control. For quantitation, autoradiographs were scanned and analyzed by densitometry using National Institutes of Health Image 1.47. The densitometric value for each transcript was expressed as a ratio to the L32 RNA, which served as a control for RNA loading.

Immunohistochemistry

For immunophenotyping and cellular adhesion molecule immunostaining, mice were killed, and their organs were removed and immediately snap frozen in liquid nitrogen and stored at -70°C until sectioning. Sagittal cryomicrotome cut serial sections (10 µm) were air-dried, fixed in cold (-20°C) acetone-methanol (1:1) for 45 s, and nonspecific binding was blocked by incubating the sections for 1 h in blocking buffer (PBS with 3% rabbit and 3% goat serum). Sections were then incubated for 1 h at room temperature with rat mAbs to identify leukocytes (CD45), lymphocytes (CD4, CD8, and B220; all from BD PharMingen, San Diego, CA), MHC class II, (clone M5/114; American Type Culture Collection, Manassas, VA), Mac-1, (clone TIB 126; American Type Culture Collection), and cellular adhesion molecules and VCAM-1 (BD PharMingen) and ICAM-1 (clone YN11.1 kindly provided by Dr. F. Takei, Toronto, Canada). All Abs were used at a final concentration of 5 µg/ml diluted in the blocking buffer. Bound Ab was detected using Vectastain ABC kits (Vector Laboratories, Burlingame, CA). Before mounting, sections were counterstained with Mayer’s hematoxylin, and were dehydrated in graded ethanols.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Peripheral immunization with CFA and PTX alone induced clinical signs in GF-IL12 mice

To investigate the effect of local tissue IL-12 production on the development of a peripherally induced immune response, GF-IL12 mice and matched nontransgenic littermate mice were immunized with CFA in the absence of a CNS-Ag-specific trigger and were boosted with PTX. In two independent experiments, 20 of 23 GF-IL12 mice (87%) immunized in this fashion developed clinical signs, whereas wild-type mice appeared to remain healthy (Fig. 1Go). In the GF-IL12 mice, the mean onset of clinical signs was 5.7 ± 2.6 days (mean ± SD; n = 20) postimmunization and included ruffled fur and hunched posture. These progressed to mild motor dysfunction around days 14–18 and remained until day 34 at which time the mice were euthanized. The maximal clinical score reached was 1.2 ± 0.7 (mean ± SD; n = 20). The disease signs observed in CFA/PTX-treated GF-IL12 mice were not due to spontaneous CNS immunity seen in old GF-IL12 mice, as all animals used were at an age younger than that seen for the onset of spontaneous disease, and nonimmunized littermate control GF-IL12 mice remained healthy throughout the experiment (not shown). The clinical signs of the GF-IL12 mice after peripheral CFA/PTX challenge were also clearly different to those observed in mice with myelin oligodendrocyte glycoprotein-EAE. Thus, immunization with myelin oligodendrocyte glycoprotein induced progressive signs of "classical" EAE with tail paraparesis and frank hind limb paralysis (not shown), whereas CFA/PTX-treated GF-IL12 mice exhibited signs of motor dysfunction, but failed to develop tail paraparesis and paralysis.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 1. The incidence and temporal appearance of clinical signs following CFA/PTX immunization in wild-type and GF-IL12 mice. Wild-type ({diamond}) or GF-IL12 ({diamondsuit}) mice were immunized on day 0 with CFA and boosted with PTX as described in Materials and Methods. Disease scores were assessed at the indicated time points as follows: each ruffled fur, hunched posture, general "sickness," and shaky or mild motor function loss was assigned a grade of 0.5, whereas severe forms of these symptoms were graded as 1. For each animal, the total daily score was sum of all grades. Values are mean total scores for the group at each time point.

 
CFA/PTX challenge induced marked immune cell accumulation in the brain of GF-IL12 mice

To determine the basis for the clinical phenotype of immunized GF-IL12 mice, CFA/PTX-treated and nontreated wild-type and GF-IL12 mice were killed at various time points following immunization (n = 3) and the cerebellum and spinal cord were removed and RNA isolated for immunophenotyping by RPA (Fig. 2Go). Previous work by us established that transgene-encoded IL-12 production was highest in the cerebellum and was not detectable in the spinal cord (53). In untreated (day 0) wild-type or GF-IL12 mice, CD4, CD8, and CD3 RNA transcripts, which would reflect the presence of T cells in the brain, were not detectable in either brain region. In cerebellum, by day 6 postimmunization, the RNA transcripts defining these T cell subsets were detectable at similar levels for both wild-type and GF-IL12 mice. However, although the level of these transcripts showed no further increase in wild-type mice, in GF-IL12 mice, significant increases in both CD4 and CD3-RNA transcripts and all three RNA transcripts was observed at day 12 and day 18, respectively. At day 34, CD4, CD8, and CD3 RNA transcripts were slightly reduced. The levels of RNA corresponding to the macrophage/microglial marker Mac-1 were also significantly increased at day 18 and 34 postimmunization in GF-IL12 mice. In contrast to the cerebellum, no detectable CD4, CD8, and CD3-RNA transcripts were found in spinal cord at any time following CFA/PTX immunization in wild-type or GF-IL12 mice.



View larger version (67K):
[in this window]
[in a new window]
  		FIGURE 2. Immunophenotypic marker gene expression in the CNS of CFA/PTX-immunized mice. Immunophenotypic marker RNA was detected by RPA using a multiprobe RPA set as shown (A) and described in Materials and Methods. Poly(A)+ RNA (5 µg per sample) or total RNA (10 µg per sample) was prepared at the times shown from cerebellum or spinal cord, respectively. Quantitative analysis (B) of immunophenotype marker RNA levels in cerebellum from A. Densitometric analysis of each lane was performed on scanned autoradiographs using National Institutes of Health Image 1.57 software. The density level for each RNA was normalized to the respective level of L32 RNA, and the mean plus standard error of the mean was calculated. The statistical significance (*, p < 0.05 or less) of any difference between the GF-IL12 and corresponding wild-type sample was determined using Student’s t test.

 
Further characterization of the immunoinflammatory lesion induced by CFA/PTX immunization was performed by immunohistochemical staining of brain sections (Fig. 3Go). Immunostaining for the pan-leukocyte marker CD45 showed a prominent inflammatory response in the brain of GF-IL12 (Fig. 3GoE) mice at day 18 following peripheral CFA/PTX immunization. Lesions that were most severe in the cerebellum were also observed in the brain stem, while increased numbers of CD45+ cells also accumulated in the lateral ventricle and choroids plexus, but were largely absent from spinal cord (not shown). In the cerebellum, lesions consisted of significant numbers of CD45+ leukocytes surrounding vessels (Fig. 3GoE, arrow) and in the parenchyma (Fig. 3GoE, arrowhead). Interestingly, accumulation of small numbers of CD45+ leukocytes was also seen in cerebellum of wild-type mice immunized with CFA/PTX, but this was restricted to small perivascular foci (Fig. 3GoA, arrow). In contrast, in age-matched nonimmunized GF-IL12 (Fig. 3GoI) and wild-type (not shown) mice, CD45+ staining was restricted to occasional perivascular microglia. Further immunophenotyping of the infiltrating lesions in the cerebellum of CFA/PTX-treated GF-IL12 mice showed these were composed predominantly of CD4 (Fig. 3GoF) and CD8 (Fig. 3GoG)-positive T cells and Mac-1-positive macrophages (Fig. 3GoH). The small perivascular infiltrates in the immunized wild-type mice also contained variable numbers of these different leukocyte subsets (Fig. 3Go, B–D). In contrast, with the exception of some Mac-1-positive microglia, there was little or no detectable CD4- or CD8-positive T cells in brain from nonimmunized GF-IL12 (Fig. 3Go, J–L) or wild-type mice (not shown).



View larger version (92K):
[in this window]
[in a new window]
  		FIGURE 3. Immunohistochemical detection of infiltrating leukocytes in brain from CFA/PTX-immunized wild-type and GF-IL12 mice. Immunohistochemistry was performed on brain sections from wild-type (A–D) and GF-IL12 (E–H) mice at day 18 after immunization, or on nonimmunized GF-IL12 (I–L) mice. Sections were immunostained for CD45 (A, E, and I), CD4 (B, F, and J), CD8 (C, G, and K), and Mac-1 (D, H, and L). Perivascular (arrows) and parenchymal (arrowheads) infiltration of leukocytes was particularly marked in the cerebellum from the immunized GF-IL12 mice. Original magnification for all panels, x250.

 
In all, these findings indicated that CFA/PTX immunization without CNS autoantigen can induce a mild inflammatory response in the brain of wild-type mice. However, both the magnitude and severity of this response was markedly increased in the brain of GF-IL12 mice and typically was composed of predominantly CD4- and CD8-positive T cells as well as macrophages.

CFA/PTX immunization of GF-IL12 mice induced type 1 cytokine and chemokine gene expression in the CNS

To determine whether the CNS immune pathology in CFA/PTX-immunized mice was associated with a functional response, we next analyzed by RPA the cerebral gene expression of the type 1 cytokines, TNF and IFN-{gamma}, and the chemokines, IP-10 and RANTES (Fig. 4Go). In nonimmunized (day 0) wild-type and GF-IL12 mice there was no detectable expression of any of these gene products. However, in immunized GF-IL12 but not wild-type mice, both TNF and IFN-{gamma} RNA transcripts were detectable by day 6 and increased further by day 18 postimmunization (Fig. 4Go, A and B). In contrast to the proinflammatory cytokines, the chemokines IP-10 and RANTES showed a minor elevation in wild-type brain following immunization (Fig. 4Go, C and D). This was particularly so for RANTES RNA, which increased to modest levels by day 12 postimmunization and coincided with cerebral infiltration by low numbers of T cells. However, the expression of these chemokine genes was increased greatly in brain from GF-IL12 mice immunized with CFA/PTX. The levels of these chemokine RNAs were maximal by day 6 and remained at similarly elevated levels out to day 34 postimmunization.



View larger version (59K):
[in this window]
[in a new window]
  		FIGURE 4. Analysis of proinflammatory cytokine and chemokine gene expression in the brain of CFA/PTX-immunized wild-type and GF-IL12 mice. TNF and IFN-{gamma} (A) and IP-10 and RANTES (B) mRNA levels were detected by RPA as described in Materials and Methods. Poly(A)+ RNA (5 µg per sample) from cerebellum was prepared at the times shown. For quantitative analysis (B and D), densitometric analysis of each lane was performed on scanned autoradiographs using National Institutes of Health Image 1.57 software. The density level for each RNA was normalized to the respective level of L32 RNA, and the mean plus SEM was calculated.

 
These experiments demonstrated that the CNS infiltration of mononuclear cells in CFA/PTX-immunized GF-IL12 but not wild-type mice was associated with the induction of significant type 1 cytokine and chemokine gene expression and reflected an active immunological process.

CFA/PTX immunization induced increased expression of a number of immune accessory and host response molecules in the brain

To further define the nature of the immunoinflammatory lesion in the GF-IL12 mice following immunization with CFA/PTX, we examined the expression of a number of immune accessory molecules. Immunohistochemical staining was performed on brain sections from GF-IL12 or wild-type mice at day 14 after CFA immunization. The levels of the cellular adhesion molecules ICAM-1 (Fig. 5Go) and VCAM-1 (Fig. 5Go) was increased on cerebrovascular endothelium and infiltrating leukocytes in brain from immunized wild-type mice compared with nonimmunized controls. However, the expression of these molecules was increased much more significantly in the immunized GF-IL12 mice where dense staining was evident (Fig. 5Go). Expression of the MHC class II molecules showed a similar qualitative and quantitative response as the cellular adhesion molecules and were dramatically increased in brain from the immunized GF-IL12 mice (Fig. 5Go). These results indicated that a number of key immune accessory molecules associated with leukocyte migration and Ag presentation were significantly increased in the brain following peripheral immune challenge in GF-IL12 mice.



View larger version (134K):
[in this window]
[in a new window]
  		FIGURE 5. Immunohistochemical detection of immune accessory molecule expression in brain of CFA/PTX-immunized wild-type and GF-IL12 mice. Immunohistochemistry was performed as described in Materials and Methods. Sections of brain from immunized wild-type control (A–C) or GF-IL12 mice (D–F) and nonimmunized GF-IL12 mice (G–I) were immunostained for ICAM-1 (A, D, and G), VCAM-1 (B, E, and H), or MHC class II (C, F, and I). Original magnifications in all panels, x200. In the CFA/PTX-treated GF-IL12 specimens, the increased expression of ICAM-1, VCAM-1, and MHC class II is widely distributed and on vascular endothelium, infiltrating immune cells (arrows), and ramified parenchymal cells (arrows), presumed to be microglia.

 
We next examined by RPA the cerebral expression of a number of host response genes, ICAM-1, NO synthase-2, and glial fibrillary acidic protein, commonly associated with immune pathology in the brain. As shown in Fig. 6Go, A and B, expression of ICAM-1 RNA paralleled that of the corresponding protein, showing a minor increase from days 6–18 postimmunization in wild-type mice, but being more significantly increased at these times in GF-IL12 mice. With the exception of a small increase at day 6 postimmunization, NOS-2 RNA was largely undetectable in brain from wild-type mice and in GF-IL12 animals at day 0. However, NOS2 mRNA transcripts increased significantly from days 6–34 postimmunization in the brain of GF-IL12 animals (Fig. 6Go, A and B). Finally, expression of the astrocyte gene glial fibrillary acidic protein increased from days 6–18 and 6–34 postimmunization in wild-type mice and GF-IL12 mice, respectively. These findings show that a number of genes associated with the host response to inflammation and injury were significantly elevated in the brain of GF-IL12 following peripheral immunization.



View larger version (67K):
[in this window]
[in a new window]
  		FIGURE 6. Analysis of host response gene expression in the brain of CFA/PTX-immunized wild-type and GF-IL12 mice. Host response gene mRNA levels was detected by RPA as described in Materials and Methods. Poly(A)+ RNA (0.5 µg per sample) from cerebellum was prepared at the times shown. For quantitative analysis (B), densitometric analysis of each lane was performed on scanned autoradiographs using National Institutes of Health Image 1.57 software. The density level for each RNA was normalized to the respective level of L32 RNA, and the mean plus SEM was calculated. GFAP, glial fibrillary acidic protein.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Through its ability to act on NK cells and CD4- and CD8+ T cells, IL-12 is a potent activator of type 1 innate and adaptive immunity (36, 37). Not surprisingly, IL-12 is critical for the efficient control of many different microbial infections and is linked to the pathogenesis of autoimmune diseases (40, 41, 64, 65) including MS (45, 46) and is required for the induction of EAE in rodents (42, 44, 66, 67). The availability of a transgenic mouse model in which IL-12 production was restricted to astrocytes in the brain (53) allowed us to examine the question of whether and to what extent the cytokine milieu of this organ could influence the development of immune responses locally. This is an important issue when one considers that microglia and astrocytes in vitro and in vivo can be induced to produce IL-12 (49, 50, 51, 52), and as noted above, this cytokine is present in the CNS in active MS and EAE. We used a peripheral immune challenge model in which mice were actively immunized with CFA/PTX but in the absence of any CNS targeting autoantigen. This immunization strategy is known to provoke a strong type 1 immune response activating both innate and adaptive cellular immunity (30, 35). The results indicated that CFA/PTX immunization could sustain in the normal mouse a low-grade, nonpathogenic inflammatory process in the brain. Remarkably, however, in similarly treated GF-IL12 mice, the extent of this process was significantly exacerbated and included functional activation typical of a type 1 immune response and was associated with signs of neurological disease. The severity of this inflammatory process was worst in the cerebellum and brain stem where it is known that the transgene-encoded IL-12 production is highest (53). Given the potent ability of IL-12 to stimulate type 1 immune responses, it is perhaps not surprising that the molecular and cellular characteristics of the inflammatory response in the GF-IL12 mice induced by CFA/PTX immunization are quite similar to the spontaneous neuroimmune pathology that develops in some older GF-IL12 mice (53). However, a number of considerations indicated that the immune response in CFA/PTX-immunized GF-IL12 mice was not simply the development of spontaneous immune pathology. First, the mice used for CFA/PTX immunization were 2–3 mo of age, considerably younger than the age at which spontaneous disease has been detected. Second, no age-matched nonimmunized GF-IL12 control mice exhibited either clinical or pathologic alterations that would suggest the presence of spontaneous disease. Finally, both the onset and incidence of immune pathology was very consistent between mice following immunization. In contrast, the spontaneous disease shows variable penetrance and the onset varies widely in different transgenic mice from 5 to >12 mo of age. Therefore, we conclude that glial production of IL-12 can act as a local adjuvant for the propagation in the brain of peripherally induced type 1 immune responses—a process that does not require CNS-specific immunizing autoantigen. These findings strongly suggest that the cytokine milieu of a tissue may be important in contributing to intrinsic immune responses and associated pathology.

In many respects, the CNS is somewhat immunoincompetent and presents the immune system with a salvo of hurdles that affect how immune cells traffic in and out and recognize foreign Ag (for reviews, see Refs. 68, 69, 70). These include the blood-brain barrier (BBB), restricted expression of MHC molecules, an absence of efficient Ag-presenting dendritic cells, and brain-derived factors that may suppress or counter-regulate T cell activation and proinflammatory processes. Despite these impediments, activated T cells do enter the CNS parenchyma after first migrating through the BBB (71). More recent studies using transgenic T cells with defined TCR specificities in rats (72) and mice (73, 74) document that Ag-specific T cells and T cells of irrelevant Ag specificity may also readily enter the brain environment without prior activation. However, prior activation and cognate Ag recognition increases the efficiency of the recruitment to and duration of residence of these cells in the CNS (71, 72). The T cell recruitment to the CNS observed by us in wild-type mice immunized with CFA/PTX is consistent with these previous findings. As noted above, this immunization protocol activates type 1 immunity in the periphery generating a repertoire of activated T cells with specificity for the immunizing Ags. In mice, T cells with specificity for CNS autoantigens are known to be present in healthy animals (75) and might conceivably also become activated. These activated T cells might have then entered the CNS, but presumably due to the lack of further TCR stimulation by specific Ag(s) or the appropriate cytokine "milieu," these cells remained few in number and inactive in wild-type mice and could not further propagate a pathologic immune response. The minor increase in the expression of MHC class II and adhesion molecules observed in the brain under these conditions may reflect a response to the infiltrating leukocytes or alternatively to circulating mediators generated by the immunization reaction. Rabchevsky et al. (33) showed that peripheral injections of CFA without PTX into wild-type C57BL/6 mice resulted in a permeabilization of the BBB to serum proteins, but did not lead to T cell infiltration. These findings suggest that cerebrovascular responses to peripheral immunization can occur in the absence of T cell infiltration. More recently, Winer and colleagues (76), in a study of the disease fidelity of autoreactive T cells, showed that PTX administration alone to nonobese diabetic mice induced autoimmune encephalitis. In preliminary work, we have confirmed the requirement for PTX for the development of the CNS inflammatory response in CFA-immunized wild-type and GF-IL12 mice (S. L. Lassmann and I. L. Campbell, unpublished observations). The coadministration of PTX, a potent inducer of T cell activation (28, 29, 30), IFN-{gamma} production (31), as well as BBB disruption (32) might therefore break the threshold for induction of T cell infiltration and functional activation of the recruited as well as local cell populations. It is possible that PTX administration alone can elicit CNS immune pathology in the GF-IL12 mice and this is the subject of ongoing investigations.

The salient cellular and molecular features of the CNS immune pathology evoked by CFA/PTX immunization in the GF-IL12 mice included infiltrating CD4+ and CD8+ T cells and macrophages, the presence of IFN-{gamma} and TNF cytokine gene expression and IP-10 and RANTES chemokine gene expression, together with the induction of a number of key immune accessory molecules such as MHC class II, ICAM-1, VCAM-1, and NOS-2. This pathologic process is typical of a type 1 immune response and is remarkably similar to the immune pathology associated with EAE, itself an IL-12-dependent disease (77, 78). However, in marked contrast to EAE, induction of neuroimmunity in the GF-IL12 mouse did not require immunization with CNS autoantigen. Thus, the presence of astrocyte-produced IL-12 is sufficient to propagate a functional encephalitogenic immune response after peripheral immune challenge. So how is such an encephalitogenic response initiated when IL-12 is sequestered in the brain? The ability of T cells and NK cells to respond to IL-12 is determined by their expression of the IL-12R. The IL-12R consists of two subunits, IL-12R{beta}1 and IL-12R{beta}2, with the latter subunit being the primary signal transduction domain of the receptor (79). Naive T cells and NK cells are unable to respond to IL-12 due to the absence of IL-12R{beta}2 on their surface (80, 81, 82, 83). However, activation of these cells by mitogen or exposure to Ag or cytokines such as IFN-{gamma} results in rapid induction/up-regulation of IL-12R expression and the acquisition of IL-12 responsiveness (84, 85). CFA immunization induces circulating IL-12R-positive T cells that respond to the immunizing Ags (78). It is also conceivable that the activation of T cells by PTX noted above would induce or up-regulate IL-12R expression. Therefore, in the case of the GF-IL12 mice, the net effect of CFA/PTX immunization might be to produce a coordinate increase in the overall numbers of IL-12R-positive T cells circulating in the periphery. This in turn would be expected to markedly increase the pool of activated-IL-12R-positive cells in the brain, which would therefore be competent to respond to IL-12. The appearance of IFN-{gamma} gene expression in the brain of the GF-IL12 mice, which occurred as a synchronous event within 6 days after immunization and paralleled the leukocyte infiltration, is consistent with this proposed mechanism. Such a mechanism also may explain the sporadic and delayed onset of spontaneous type 1 immunity seen in the brain of unmanipulated aged GF-IL12 mice. These animals would be expected to be exposed to only infrequent and nominal antigenic challenge and thus the numbers of IL-12R-bearing T cells in the circulation and the brain environment would be minimal. The CFA/PTX immunization protocol may therefore accelerate and synchronize the onset and increase the incidence of the spontaneous disease in GF-IL12 mice.

As we noted above, CFA/PTX immunization induces a polyclonal T cell population that responds to the immunizing Ags. Therefore, it is most probable that the initial IL-12R-positive T cells that entered the brain in immunized GF-IL12 mice were not reactive with CNS Ags. However, because the specificity and function of the infiltrating T cells in the immunized GF-IL12 mice was not evaluated in this study, we cannot rule out that the encephalitogenic response in these mice also involved autoreactive T cells. Circulating T cells with reactivity for CNS Ags occur in the periphery of healthy humans (10, 11, 12, 13) and rodents (75, 86), which in the case of the latter, may, under appropriate stimulation, become encephalitogenic (75). Certainly, the environment of the inflamed CNS together with the presence of IL-12 in the immunized GF-IL12 brain would be conducive to the local presentation of Ag and activation of autoreactive IL-12R-bearing T cells recruited to the brain as bystander cells. Significant recruitment of bystander T cells to the CNS is known to occur in EAE and in MS (87). That de novo activation of autoreactive T cells recruited initially as bystander cells can occur in the CNS is well documented in EAE and results when autoantigens are released following inflammatory tissue damage (88). Moreover, autoreactive T cells arising from this so-called epitope spreading process can play a major pathogenic role in disease progression (89). In ongoing studies we are evaluating the T cell specificities associated with CFA/PTX-induced immune pathology in the GF-IL12 brain.

Bystander activation of CD44high memory T cells may serve as an important mechanism for the long-term survival and proliferation of these cells (90). Cytokines produced at inflammatory sites following microbial infection appear to be central in this process. Thus, TCR-independent bystander activation of memory CD8+ T cells is mediated by IFN-{alpha} and by IFN-{alpha}-induced IL-15 (56). Interestingly, it has been more recently demonstrated that IL-12 alone or in combination with other type 1 cytokines including IFN-{gamma}, IL-15, and IL-18 can induce the proliferation and activation of NK cells and memory CD4+ and CD8+ T cells with concomitant stimulation of IFN-{gamma} production (91, 92, 93) Due to their lack of IL-12R expression, naive T cells are not susceptible to bystander activation. This would account, in part, for the lack of activation that was observed with naive T cells trafficking to the inflamed brain of mice with EAE (74). We suggest in this study that a plausible alternative mechanism for the immune pathology induced in the brain of GF-IL12 mice by CFA/PTX immunization is independent of any Ag-specific TCR-mediated T cell response, but rather involves Ag-independent bystander activation of T cells by IL-12. Under these circumstances, a self-perpetuating cycle might be expected where the chronic transgene-encoded IL-12 production drives the continued responsiveness and expansion of activated T cells. Although we did observe a monophasic and persistent neuroimmune response in the immunized GF-IL12 mice, this appeared to decrease by 34 days. Thus, whatever the nature of this immune pathology, counter-regulatory mechanisms may also be at work in the brain in an attempt to reduce the adverse consequences of an unmitigated immune response. One possible candidate in this context is TGF-{beta}1, whose expression is increased significantly in the brain of GF-IL12 mice with spontaneous neuroimmune disease (53).

Our findings are relevant to the pathogenesis of MS where the initial development or subsequent exacerbation of disease is often associated with viral or bacterial infections (7, 14, 15, 16). It is envisaged that infectious episodes could act to not only stimulate type 1 immune responses in the periphery, but also to induce the local glial production of IL-12 in the brain akin to what has been previously demonstrated in mice following LPS injections (49). As shown in the present study, the activation of peripheral type 1 immunity results in the increased migration of T cells to the brain and this can occur independent of sensitizing autoantigen or preexisting CNS disease. The intrinsic milieu of the CNS may then play a central role in determining the outcome of this initial CNS-immune encounter. In this context, the production of IL-12 by glial cells would act as a local adjuvant for the further expansion and perpetuation of type 1 encephalitogenic immunity. It will now be of interest to determine further the nature of this encephalitogenic immunity; in particular, the respective roles of innate vs adaptive as well targeted vs bystander immune responses in causing disease in this model.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 11. The incidence and temporal appearance of clinical signs following CFA/PTX immunization in wild-type and GF-IL12 mice. Wild-type ({diamond}) or GF-IL12 ({diamondsuit}) mice were immunized on day 0 with CFA and boosted with PTX as described in Materials and Methods. Disease scores were assessed at the indicated time points as follows: each ruffled fur, hunched posture, general "sickness," and shaky or mild motor function loss was assigned a grade of 0.5, whereas severe forms of these symptoms were graded as 1. For each animal, the total daily score was sum of all grades. Values are mean total scores for the group at each time point.

 


View larger version (67K):
[in this window]
[in a new window]
  		FIGURE 21. Immunophenotypic marker gene expression in the CNS of CFA/PTX-immunized mice. Immunophenotypic marker RNA was detected by RPA using a multiprobe RPA set as shown (A) and described in Materials and Methods. Poly(A)+ RNA (5 µg per sample) or total RNA (10 µg per sample) was prepared at the times shown from cerebellum or spinal cord, respectively. Quantitative analysis (B) of immunophenotype marker RNA levels in cerebellum from A. Densitometric analysis of each lane was performed on scanned autoradiographs using National Institutes of Health Image 1.57 software. The density level for each RNA was normalized to the respective level of L32 RNA, and the mean plus standard error of the mean was calculated. The statistical significance (*, p < 0.05 or less) of any difference between the GF-IL12 and corresponding wild-type sample was determined using Student’s t test.

 


View larger version (92K):
[in this window]
[in a new window]
  		FIGURE 31. Immunohistochemical detection of infiltrating leukocytes in brain from CFA/PTX-immunized wild-type and GF-IL12 mice. Immunohistochemistry was performed on brain sections from wild-type (A–D) and GF-IL12 (E–H) mice at day 18 after immunization, or on nonimmunized GF-IL12 (I–L) mice. Sections were immunostained for CD45 (A, E, and I), CD4 (B, F, and J), CD8 (C, G, and K), and Mac-1 (D, H, and L). Perivascular (arrows) and parenchymal (arrowheads) infiltration of leukocytes was particularly marked in the cerebellum from the immunized GF-IL12 mice. Original magnification for all panels, x250.

 


View larger version (59K):
[in this window]
[in a new window]
  		FIGURE 41. Analysis of proinflammatory cytokine and chemokine gene expression in the brain of CFA/PTX-immunized wild-type and GF-IL12 mice. TNF and IFN-{gamma} (A) and IP-10 and RANTES (B) mRNA levels were detected by RPA as described in Materials and Methods. Poly(A)+ RNA (5 µg per sample) from cerebellum was prepared at the times shown. For quantitative analysis (B and D), densitometric analysis of each lane was performed on scanned autoradiographs using National Institutes of Health Image 1.57 software. The density level for each RNA was normalized to the respective level of L32 RNA, and the mean plus SEM was calculated.

 


View larger version (134K):
[in this window]
[in a new window]
  		FIGURE 51. Immunohistochemical detection of immune accessory molecule expression in brain of CFA/PTX-immunized wild-type and GF-IL12 mice. Immunohistochemistry was performed as described in Materials and Methods. Sections of brain from immunized wild-type control (A–C) or GF-IL12 mice (D–F) and nonimmunized GF-IL12 mice (G–I) were immunostained for ICAM-1 (A, D, and G), VCAM-1 (B, E, and H), or MHC class II (C, F, and I). Original magnifications in all panels, x200. In the CFA/PTX-treated GF-IL12 specimens, the increased expression of ICAM-1, VCAM-1, and MHC class II is widely distributed and on vascular endothelium, infiltrating immune cells, and ramified parenchymal cells presumed to be microglia.

 


View larger version (67K):
[in this window]
[in a new window]
  		FIGURE 61. Analysis of host response gene expression in the brain of CFA/PTX-immunized wild-type and GF-IL12 mice. Host response gene mRNA levels was detected by RPA as described in Materials and Methods. Poly(A)+ RNA (0.5 µg per sample) from cerebellum was prepared at the times shown. For quantitative analysis (B), densitometric analysis of each lane was performed on scanned autoradiographs using National Institutes of Health Image 1.57 software. The density level for each RNA was normalized to the respective level of L32 RNA, and the mean plus SEM was calculated. GFAP, glial fibrillary acidic protein.

 

   Acknowledgments
 
We gratefully acknowledge the administrative support of Heather Kemlein.


   Footnotes
 
1 This work was supported by the U.S. Public Health Service (Grant NS 36979 to I.L.C.). S.L. was supported by Deutscher Akademischer Austauschdienst (North Atlantic Treaty Organization Postdoctoral Fellowship). V.C.A. was a postdoctoral fellow of the National Multiple Sclerosis Society. This is manuscript 14064-NP from the Scripps Research Institute. Back

2 Current address: Institut für Molekulare Pathologie, TU München, Klinikum r.d. Isar, Trogerstrasee 18, D-81675 München, Germany. Back

3 Current address: Digital Gene Technologies, La Jolla, CA 92037. Back

4 Address correspondence and reprint requests to Dr. Iain L. Campbell, Department of Neuropharmacology, SP-315, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: icamp{at}scripps.edu Back

5 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; PTX, pertussis toxin; RPA, RNase protection assays; NOS-2, NO synthase-2; BBB, blood-brain barrier. Back

Received for publication April 23, 2001. Accepted for publication September 4, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Baxter, A. G.. 1997. Immunogenetics and the cause of autoimmune disease. Autoimmunity 25:177.[Medline]
  2. Gianani, R., N. Sarvetnick. 1996. Viruses, cytokines, antigens, and autoimmunity. Proc. Natl. Acad. Sci. USA 93:2257.[Abstract/Free Full Text]
  3. Theofilopoulos, A. N.. 1995. The basis of autoimmunity. I. Mechanisms of aberrant self-recognition. Immunol. Today 16:90.[Medline]
  4. Theofilopoulos, A. N.. 1995. The basis of autoimmunity. II. Genetic predisposition. Immunol. Today 16:150.[Medline]
  5. Calder, V., S. Owen, C. Watson, M. Feldman, A. Davison. 1989. MS: a localized immune disease of the central nervous system. Immunol. Today 10:99.[Medline]
  6. Hafler, D. A., H. L. Weiner. 1989. MS: a CNS and systemic autoimmune disease. Immunol. Today 10:104.[Medline]
  7. Hafler, D. A.. 1999. The distinction blurs between an autoimmune versus microbial hypothesis in multiple sclerosis. J. Clin. Invest. 104:527.[Medline]
  8. Compston, D. A., H. Kellar Wood, N. Robertson, S. Sawcer, N. W. Wood. 1995. Genes and susceptibility to multiple sclerosis. Acta. Neurol. Scand. Suppl. 161:43.[Medline]
  9. Sawcer, S., H. B. Jones, R. Feakes, J. Gray, N. Smaldon, J. Chataway, N. Robertson, D. Clayton, P. N. Goodfellow, A. Compston. 1996. A genome screen in multiple sclerosis reveals susceptibility loci on chromosome 6p21 and 17q22. Nat. Genet. 13:464.[Medline]
  10. Jingwu, Z., R. Medaer, G. Hashim, Y. Chin, E. van den Berg-Loonen, J. C. Raus. 1992. Myelin basic protein-specific T lymphocytes in multiple sclerosis and controls: precursor frequency, fine specificity and cytotoxicity. Ann. Neurol. 32:330.[Medline]
  11. Meinl, E., F. Weber, K. Drexler, C. Morelle, M. Ott, G. Saruhan-Direskeneli, N. Goebels, B. Ertl, G. Jechart, G. Giegerich, et al 1993. Myelin basic protein-specific T lymphocyte repertoire in multiple sclerosis: complexity of the response and dominance of nested epitopes due to recruitment of multiple T-cell clones. J. Clin. Invest. 92:2633.
  12. Olsson, T., J. Sun, J. Hillert, B. Hojeberg, H. P. Ekre, G. Andersson, O. Olerup, H. Link. 1992. Increased numbers of T cells recognizing multiple myelin basic protein epitopes in multiple sclerosis. Eur. J. Immunol. 22:1083.[Medline]
  13. Ota, K., M. Matsui, E. L. Milford, G. A. Mackin, H. L. Weiner, D. A. Hafler. 1990. T-cell recognition of an immunodominant myelin basic protein in multiple sclerosis. Nature 346:183.[Medline]
  14. Compston, D. A., B. N. Vakarelis, E. Paul, W. I. McDonald, J. R. Batchelor, C. A. Mims. 1986. Viral infection in patients with multiple sclerosis and HLA-DR matched controls. Brain 109:325.[Abstract/Free Full Text]
  15. Panitch, H. S.. 1994. Influence of infection on exacerbations of multiple sclerosis. Ann. Neurol. 36:S25.
  16. Sibley, W. A., C. R. Bamford, K. Clark. 1985. Clinical viral infections and multiple sclerosis. Lancet 1:1313.[Medline]
  17. Wekerle, H., K. Kojima, J. Lannes-Vieira, H. Lassmann, C. Linington. 1994. Animal models. Ann. Neurol. 36:S47.
  18. Cooper, A. M., J. Magram, J. Ferrante, I. M. Orme. 1997. Interleukin 12 (IL-12) is crucial to the development of protective immunity in mice intravenously infected with Mycobacterium tuberculosis. J. Exp. Med. 186:39.[Abstract/Free Full Text]
  19. Cowdery, J. S., N. J. Boerth, L. A. Norian, P. S. Myung, G. A. Koretzky. 1999. Differential regulation of the IL-12-p40 promoter and of p40 secretion by CpG DNA and lipopolysaccharide. J. Immunol. 162:6770.[Abstract/Free Full Text]
  20. Fulton, S. A., J. M. Johnsen, S. F. Wolf, D. S. Sieburth, W. H. Boom. 1996. Interleukin-12 production by human monocytes infected with Mycobacterium tuberculosis: role of phagocytosis. Infect. Immunol. 64:2523.[Abstract]
  21. Segal, B. M., D. M. Klinman, E. M. Shevach. 1997. Microbial products induce autoimmune disease by an IL-12-dependent pathway. J. Immunol. 158:5087.[Abstract]
  22. Segal, B. M., J. T. Chang, E. M. Shevach. 2000. CpG oligonucleotides are potent adjuvants for the activation of autoreactive encephalitogenic T cells in vivo. J. Immunol. 164:5683.[Abstract/Free Full Text]
  23. Verhasselt, V., C. Buelens, F. Willems, D. De Groote, N. Haeffner-Cavaillon, M. Goldman. 1997. Bacterial lipopolysaccharide stimulates the production of cytokines and the expression of costimulatory molecules by human peripheral blood dendritic cells: evidence for a soluble CD14-dependent pathway. J. Immunol. 158:2919.[Abstract]
  24. Schmitt, E., P. Hoehn, C. Huels, S. Goedert, N. Palm, E. Rude, T. Germann. 1994. T helper type 1 development of naive CD4+ T cells requires the coordinate action of interleukin-12 and interferon-{gamma} and is inhibited by transforming growth factor-{beta}. Eur. J. Immunol. 24:793.[Medline]
  25. Seder, R. A., R. Gazzinelli, A. Sher, W. E. Paul. 1993. Interleukin 12 acts directly on CD4+ T cells to enhance priming for interferon-{gamma} production and diminishes interleukin 4 inhibition of such priming. Proc. Natl. Acad. Sci. USA 90:10188.[Abstract/Free Full Text]
  26. Munoz, J. J., M. G. Peacock. 1990. Action of pertussigen (pertussis toxin) on serum IgE and on Fc{epsilon} receptors on lymphocytes. Cell. Immunol. 127:327.[Medline]
  27. Sewell, W. A., P. A. de Moerloose, J. A. Hamilton, J. W. Schrader, I. R. Mackay, M. A. Vadas. 1987. Potentiation of delayed-type hypersensitivity by pertussigen or cyclophosphamide with release of different lymphokines. Immunology 61:483.[Medline]
  28. Lobet, Y., C. Feron, G. Dequesne, E. Simoen, P. Hauser, C. Locht. 1993. Site-specific alterations in the B oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin. J. Exp. Med. 177:79.[Abstract/Free Full Text]
  29. Kamradt, T., P. D. Soloway, D. L. Perkins, M. L. Gefter. 1991. Pertussis toxin prevents the induction of peripheral T cell anergy and enhances the T cell response to an encephalitogenic peptide of myelin basic protein. J. Immunol. 147:3296.[Abstract]
  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. Sewell, W. A., P. A. de Moerloose, J. L. McKimm-Breschkin, M. A. Vadas. 1986. Pertussigen enhances antigen-driven interferon-{gamma} production by sensitized lymphoid cells. Cell. Immunol. 97:238.[Medline]
  32. Linthicum, D. S., J. J. Munoz, A. Blaskett. 1982. Acute experimental autoimmune encephalomyelitis in mice: adjuvant action of Bordetella pertussis is due to vasoactive amine sensitization and increased vascular permeability of the central nervous system. Cell. Immunol. 73:299.[Medline]
  33. Rabchevsky, A. G., J. D. Degos, P. A. Dreyfus. 1999. Peripheral injections of Freund’s adjuvant in mice provoke leakage of serum proteins through the blood-brain barrier without inducing reactive gliosis. Brain Res. 832:84.[Medline]
  34. Reiber, H., A. J. Suckling, M. G. Rumsby. 1984. The effect of Freund’s adjuvants on blood-cerebrospinal fluid barrier permeability. J. Neurol. Sci. 63:55.[Medline]
  35. Shive, C. L., H. Hofstetter, L. Arredondo, C. Shaw, T. G. Forsthuber. 2000. The enhanced antigen-specific production of cytokines induced by pertussis toxin is due to clonal expansion of T cells and not to altered effector functions of long-term memory cells. Eur. J. Immunol. 30:2422.[Medline]
  36. Gately, M. K., L. M. Renzetti, J. Magram, A. S. Stern, L. Adorini, U. Gubler, D. H. Presky. 1998. The interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses. Annu. Rev. Immunol. 16:495.[Medline]
  37. Trinchieri, G.. 1998. Interleukin-12: a cytokine at the interface of inflammation and immunity. Adv. Immunol. 70:83.[Medline]
  38. Magram, J., J. Sfarra, S. Connaughton, D. Faherty, R. Warrier, D. Carvajal, C. Y. Wu, C. Stewart, U. Sarmiento, M. K. Gately. 1996. IL-12-deficient mice are defective but not devoid of type 1 cytokine responses. Ann. NY Acad. Sci. 795:60.[Medline]
  39. Magram, J., S. E. Connaughton, R. R. Warrier, D. M. Carvajal, C. Y. Wu, J. Ferrante, C. Stewart, U. Sarmiento, D. A. Faherty, M. K. Gately. 1996. IL-12-deficient mice are defective in IFN-{gamma} production and type 1 cytokine responses. Immunity 4:471.[Medline]
  40. Caspi, R. R.. 1998. IL-12 in autoimmunity. Clin. Immunol. Immunopathol. 88:4.[Medline]
  41. Seder, R. A., B. L. Kelsall, D. Jankovic. 1996. Differential roles for IL-12 in the maintenance of immune responses in infectious versus autoimmune disease. J. Immunol. 157:2745.[Abstract]
  42. Segal, B. M., B. K. Dwyer, E. M. Shevach. 1998. An interleukin (IL)-10/IL-12 immunoregulatory circuit controls susceptibility to autoimmune disease. J. Exp. Med. 187:537.[Abstract/Free Full Text]
  43. Constantinescu, C. S., M. Wysocka, B. Hilliard, E. S. Ventura, E. Lavi, G. Trinchieri, A. Rostami. 1998. Antibodies against IL-12 prevent superantigen-induced and spontaneous relapses of experimental autoimmune encephalomyelitis. J. Immunol. 161:5097.[Abstract/Free Full Text]
  44. Leonard, J. P., K. E. Waldburger, S. J. Goldman. 1995. Prevention of experimental autoimmune encephalomyelitis by antibodies against interleukin 12. J. Exp. Med. 181:381.[Abstract/Free Full Text]
  45. Balashov, K. E., D. R. Smith, S. J. Khoury, D. A. Hafler, H. L. Weiner. 1997. Increased interleukin 12 production in progressive multiple sclerosis: induction by activated CD4+ T cells via CD40 ligand. Proc. Natl. Acad. Sci. USA 94:599.[Abstract/Free Full Text]
  46. Windhagen, A., J. Newcombe, F. Dangond, C. Strand, M. N. Woodroofe, M. L. Cuzner, D. A. Hafler. 1995. Expression of costimulatory molecules B7-1 (CD80), B7-2 (CD86), and interleukin 12 cytokine in multiple sclerosis lesions. J. Exp. Med. 182:1985.[Abstract/Free Full Text]
  47. Bright, J. J., B. F. Musuro, C. Du, S. Sriram. 1998. Expression of IL-12 in CNS and lymphoid organs of mice with experimental allergic encephalitis. J. Neuroimmunol. 82:22.[Medline]
  48. Issazadeh, S., A. Ljungdahl, B. Hojeberg, M. Mustafa, T. Olsson. 1995. Cytokine production in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis: dynamics of mRNA expression for interleukin-10, interleukin-12, cytolysin, tumor necrosis factor-{alpha} and tumor necrosis factor-{beta}. J. Neuroimmunol. 61:205.[Medline]
  49. Stalder, A. K., A. Pagenstecher, N. C. Yu, C. Kincaid, C. S. Chiang, M. V. Hobbs, F. E. Bloom, I. L. Campbell. 1997. Lipopolysaccharide-induced IL-12 expression in the central nervous system and cultured astrocytes and microglia. J. Immunol. 159:1344.[Abstract]
  50. Aloisi, F., G. Penna, J. Cerase, B. Menendez Iglesias, L. Adorini. 1997. IL-12 production by central nervous system microglia is inhibited by astrocytes. J. Immunol. 159:1604.[Abstract]
  51. Becher, B., V. Dodelet, V. Fedorowicz, J. P. Antel. 1996. Soluble tumor necrosis factor receptor inhibits interleukin 12 production by stimulated human adult microglial cells in vitro. J. Clin. Invest. 98:1539.[Medline]
  52. Becher, B., M. Blain, J. P. Antel. 2000. CD40 engagement stimulates IL-12p70 production by human microglial cells: basis for Th1 polarization in the CNS. J. Neuroimmunol. 102:44.[Medline]
  53. Pagenstecher, A., S. Lassmann, M. J. Carson, C. L. Kincaid, A. K. Stalder, I. L. Campbell. 2000. Astrocyte-targeted expression of IL-12 induces active cellular immune responses in the central nervous system and modulates experimental allergic encephalomyelitis. J. Immunol. 164:4481.[Abstract/Free Full Text]
  54. Gangappa, S., J. S. Babu, J. Thomas, M. Daheshia, B. T. Rouse. 1998. Virus-induced immunoinflammatory lesions in the absence of viral antigen recognition. J. Immunol. 161:4289.[Abstract/Free Full Text]
  55. Horwitz, M. S., L. M. Bradley, J. Harbertson, T. Krahl, J. Lee, N. Sarvetnick. 1998. Diabetes induced by Coxsackie virus: initiation by bystander damage and not molecular mimicry. Nat. Med. 4:781.[Medline]
  56. Tough, D. F., P. Borrow, J. Sprent. 1996. Induction of bystander T cell proliferation by viruses and type I interferon in vivo. Science 272:1947.[Abstract]
  57. Badley, J. E., G. A. Bishop, T. St. John, J. A. Frelinger. 1988. A simple, rapid method for the purification of poly(A)+ RNA. BioTechniques 6:114.[Medline]
  58. Stalder, A., A. Pagenstecher, C. Kincaid, I. L. Campbell. 1999. Analysis of gene expression by multiprobe RNase protection assay. J. Harry, and H. A. Tilson, eds. Neurodegeneration Methods and Protocols 53. Human Press, Totowa.
  59. Hobbs, M. V., W. O. Weigle, D. J. Noonan, B. E. Torbett, R. J. McEvilly, R. J. Koch, G. J. Cardenas, D. N. Ernst. 1993. Patterns of cytokine gene expression by CD4+ T cells from young and old mice. J. Immunol. 150:3602.[Abstract]
  60. Asensio, V. C., I. L. Campbell. 1997. Chemokine gene expression in the brains of mice with lymphocytic choriomeningitis. J. Virol. 71:7832.[Abstract]
  61. Asensio, V. C., C. Kincaid, I. L. Campbell. 1999. Chemokines and the inflammatory response to viral infection in the central nervous system with a focus on lymphocytic choriomeningitis virus. J. Neurovirol. 5:65.[Medline]
  62. Chiang, C. S., A. Stalder, A. Samimi, I. L. Campbell. 1994. Reactive gliosis as a consequence of interleukin-6 expression in the brain: studies in transgenic mice. Dev. Neurosci. 16:212.[Medline]
  63. Dudov, K. P., R. P. Perry. 1984. The gene family encoding the mouse ribosomal protein L32 contains a uniquely expressed intron-containing gene and an unmutated processed gene. Cell 37:457.[Medline]
  64. Lamont, A. G., L. Adorini. 1996. IL-12: a key cytokine in immune regulation. Immunol. Today 17:214.[Medline]
  65. Trembleau, S., T. Germann, M. K. Gately, L. Adorini. 1995. The role of IL-12 in the induction of organ-specific autoimmune diseases. Immunol. Today 16:383.[Medline]
  66. Bright, J. J., C. Du, M. Coon, S. Sriram, S. J. Klaus. 1998. Prevention of experimental allergic encephalomyelitis via inhibition of IL-12 signaling and IL-12-mediated Th1 differentiation: an effect of the novel anti-inflammatory drug lisofylline. J. Immunol. 161:7015.[Abstract/Free Full Text]
  67. Waldburger, K. E., R. C. Hastings, R. G. Schaub, S. J. Goldman, J. P. Leonard. 1996. Adoptive transfer of experimental allergic encephalomyelitis after in vitro treatment with recombinant murine interleukin-12: preferential expansion of interferon-{gamma}-producing cells and increased expression of macrophage-associated inducible nitric oxide synthase as immunomodulatory mechanisms. Am. J. Pathol. 148:375.[Abstract]
  68. Sedgwick, J. D.. 1995. Immune surveillance and autoantigen recognition in the central nervous system. Aust. N. Z. J. Med. 25:784.[Medline]
  69. Shrikant, P., E. N. Benveniste. 1996. The central nervous system as an immunocompetent organ: role of glial cells in antigen presentation. J. Immunol. 157:1819.[Abstract]
  70. Williams, K. C., W. F. Hickey. 1995. Traffic of hematogenous cells through the central nervous system. Curr. Topics Microbiol. Immunol. 202:221.[Medline]
  71. Hickey, W. F., B. L. Hsu, H. Kimura. 1991. T-lymphocyte entry into the central nervous system. J. Neurosci. Res. 28:254.[Medline]
  72. Bauer, J., M. Bradl, W. F. Hickley, S. Forss-Petter, H. Breitschopf, C. Linington, H. Wekerle, H. Lassmann. 1998. T-cell apoptosis in inflammatory brain lesions: destruction of T cells does not depend on antigen recognition. Am. J. Pathol. 153:715.[Abstract/Free Full&n