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The Journal of Immunology, 1998, 160: 5955-5962.
Copyright © 1998 by The American Association of Immunologists

The Effect of {gamma}{delta} T Cell Depletion on Cytokine Gene Expression in Experimental Allergic Encephalomyelitis1

Alice J. Rajan2, Jonathan D. S. Klein and Celia F. Brosnan

Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, we showed previously that depletion of {gamma}{delta} T cells using the mAb GL3 immediately before disease onset, or during the chronic phase, significantly ameliorated clinical severity. We now report on the effect of {gamma}{delta} T cell depletion on expression of five cytokine genes, IL-1, IL-6, TNF, lymphotoxin, and IFN-{gamma} in spinal cords of mice during the pre-onset, onset, height, and recovery phases of EAE, and on expression of type II nitric oxide synthase. In control animals, the mRNAs for IL-1 and IL-6 rose dramatically at disease onset and peaked before disease height, whereas the mRNAs for TNF, lymphotoxin, and IFN-{gamma} rose more slowly and peaked with peak of disease. In GL3-treated animals, a dramatic reduction in all five cytokines was noted at disease onset, but only IFN-{gamma} remained significantly reduced at a time point equivalent to height of disease in control animals. ELISA data confirmed the reduced levels of IL-1 and IL-6 at disease onset in GL3-treated animals, and pathologic analysis demonstrated a marked reduction in meningeal infiltrates at the same time point. Studies of type II NOS also demonstrated a significant reduction in both mRNA and protein expression at the height of disease in GL3-treated animals. These results suggest that {gamma}{delta} T cells contribute to the pathogenesis of EAE by regulating the influx of inflammatory cells into the spinal cord and by augmenting the proinflammatory cytokine profile of the inflammatory infiltrates.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Experimental allergic encephalomyelitis (EAE)3 is an inflammatory demyelinating disease of the central nervous system (CNS) that is the most commonly used animal model for multiple sclerosis. It can be initiated in susceptible species by sensitization with CNS myelin Ags or by the adoptive transfer of CNS myelin Ag-specific CD4+ T cells. Depending on the species of animals tested, the age at the time of sensitization, and the nature of the Ag(s) used, EAE may present as an acute monophasic disease from which the animals recover without subsequent relapse, or may display periods of exacerbation and remission, usually evolving into a chronic disease state. Analysis of the pathologic expression of EAE has shown that disease onset is usually well correlated with the abrupt development of inflammation in the CNS. Although the exact mechanism of disease induction remains to be defined, most studies support the conclusion that in normal immunocompetent animals, activation of a Th1-type cytokine cascade that results in the recruitment to the CNS and activation in situ of large numbers of lymphocytes and macrophages plays a key role in disease pathogenesis (reviewed in 1 .

Several studies now support the conclusion that T cells expressing the {gamma}{delta} TCR are also present in this inflammatory infiltrate, as well as in multiple sclerosis lesions (2, 3, 4, 5, 6, 7). Although the exact function of these cells remains unknown, studies in EAE have shown that their numbers fluctuate in association with disease activity and that they are principally localized to the edge of lesioned areas of the CNS, particularly at sites in which altered expression of heat shock proteins has been documented (3). {gamma}{delta} T cells have been shown to display in vitro cytotoxicity toward oligodendrocytes, and may thus contribute to demyelinating activity within the lesion (8), and may also be a source of Th1-type cytokines that could contribute to the induction and/or maintenance of the proinflammatory activity of cells within the lesion (9). Recently, three studies have attempted to define a role for {gamma}{delta} T cells in EAE either by depleting {gamma}{delta} T cells using Abs raised against the {gamma}{delta} TCR, or by immunizing with a peptide specific for V{gamma}6, a V region gene that has been shown to predominate in the CNS of animals with EAE. Although the results of these studies gave conflicting results, since disease amelioration was noted in two (10, 11), and disease exacerbation in the other (12), they collectively support the conclusion that {gamma}{delta} T cells contribute to the inflammatory process in EAE.

In our own studies, we showed that in a chronic-relapsing mouse model of EAE, induced by the passive transfer of myelin basic protein (MBP)-activated lymph node cells, treatment with the mAb GL3 (10), which transiently depletes {gamma}{delta} T cells from the circulation, significantly ameliorated disease expression when given either immediately before the acute clinical episode, or during the more chronic stages of the disease. FACS analysis showed that treatment with this Ab resulted in a significant depletion of {gamma}{delta} T cells both in the spleen and in the CNS of affected animals, persisting for approximately 15 days. In an attempt to define more precisely the mechanism by which {gamma}{delta} T cells contribute to the encephalitogenic process during the acute phase of the disease, we have now studied cytokine gene expression at varying stages of the disease in GL3-treated and control animals using multiprobe ribonuclease protection assays (RPA). The results obtained have been further verified for some cytokines by studying protein levels in the spinal cord, and by assessment of the activation of type II nitric oxide synthase (iNOS), an enzyme that would be expected to be regulated by proinflammatory cytokines in the lesion. The results support the conclusion that {gamma}{delta} T cells participate in the encephalitogenic process by regulating the influx of inflammatory cells into the spinal cord and by augmenting the proinflammatory cytokine profile of the inflammatory infiltrates in the CNS.


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

Six- to eight-week-old female SJL/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were housed and maintained in a federally approved animal facility, and all protocols were approved by Institutional Animal Care and Use Committee of Albert Einstein College of Medicine (Bronx, NY).

Immunization and induction of EAE

Mice were immunized with MBP (Sigma, St. Louis, MO), as described previously (10). Draining axial, brachial, and inguinal lymph nodes were removed after 10 days, and a single cell suspension was prepared in RPMI 1640 medium containing 10% FCS, 100 µg/ml penicillin/streptomycin, 2 mM glutamine, 5 x 10-5 M 2-ME, 1 mM sodium pyruvate, 0.1 M HEPES, and nonessential amino acids (Sigma). Cells were plated at a density of 4 x 106 cells/ml in 24-well plates and activated in vitro with 50 µg/ml MBP at 37°C in 8% CO2 for 4 days. For adoptive transfer, 5 x 107 cells in 0.1 to 0.2 ml were injected into naive syngeneic recipients via the lateral tail vein. Clinical expression of disease was graded on a clinical index (CI) scale of 0 to 5, as follows: grade 1, limp tail; grade 2, hind limb weakness; grade 3, plegia of both hind limbs; 4, plegia of three or four limbs; grade 5, moribund. Recipient mice first showed signs of EAE 6 to 8 days posttransfer. Animals in recovery were defined as a clear clinical improvement of at least one grade persisting for 24 to 48 h following a paralytic incident. Animals were studied from 0 to 20 days posttransfer.

In vivo depletion of {gamma}{delta} T cells

Culture supernatants of the hamster IgG mAb against pan TCR-{gamma}{delta} (GL3) (gift of Dr. Lefrancois, University of Connecticut, Farmington, CT) were collected, and the IgG was affinity purified using an Affigel protein A MAPS II Kit (Bio-Rad, Hercules, CA). Mice were injected i.p. on 2 consecutive days with 1 mg of purified GL3 Ab (13) given in two equal doses twice daily. Control group mice were injected with an equivalent dose and amount of normal hamster IgG (NHIgG; Accurate Chemicals, Westbury, NY).

Neuropathology

For pathologic analysis of the tissue, mice were anesthetized by ether inhalation and perfused through the left ventricle with 20 ml cold PBS. The spinal cord was removed, and sections of the lumbar cord were immersion fixed in Trump’s fixative (4% paraformaldehyde, 1% glutaraldehyde, in 0.1 M phosphate buffer, pH 7.4), or snap frozen in optimal cooling compound. For light microscopy, the tissue fixed in Trump’s solution was dehydrated through a graded series of ethanol, cleared in propylene oxide, and embedded in Epon 812 (Electron Microscopy Sciences, Fort Washington, PA). One-micrometer epoxy sections were placed on glass slides and stained with 1% toluidine blue.

Quantitation of cells in the CNS by FACS analysis

Sensitized mice (n = 4 per group) that had been treated with NHIgG or GL3 as above were perfused through the ascending aorta with 30 ml ice-cold PBS on day 6 or day 7 posttransfer of MBP-reactive T cells. The spinal cord was dissected free from the vertebral column and dissociated by passing through a stainless steel mesh grid. Leukocytes were isolated by Percoll density-gradient centrifugation, and single cell suspensions were stained for identification of the total leukocyte population using Abs to CD45 and CD3 (PharMingen, San Diego, CA), as described previously (10). FACS analysis was performed using a Becton Dickinson FACScan (Becton Dickinson, Mountain View, CA). A total cell count was obtained following gating of the appropriately labeled cell populations.

Immunohistochemistry

For immunohistochemistry, 10-µm sections were prepared from the snap-frozen tissue. Sections were air dried for 30 min and fixed in ice-cold acetone for 10 min at -20°C. Slides were rinsed in PBS, followed by incubation in 0.3% H2O2 in methanol for 15 min. After washing with PBS three times, the sections were blocked with 10% normal goat serum for 1 h. For staining of IL-1, slides were then incubated with an Ab to IL-1ß (Genzyme, Cambridge, MA) overnight at 4°C, washed three times in PBS, and incubated with biotinylated secondary Ab to hamster IgG (PharMingen) for 2 h at room temperature. Control tissues were incubated with hamster NHIgG and processed as above. Slides were washed three times in PBS and incubated with an avidin-biotin horseradish peroxidase complex (Vector Laboratories, Burlingame, CA) diluted 1/100, mixed, and incubated for 1 h at room temperature. Diaminobenzidine tetrahydrochloride (3,3') (Vector Laboratories) was used for chromogenic detection of peroxidase staining. After the final wash with PBS, sections were dehydrated and mounted in Permount. For analysis of type II NOS, slides were blocked as above and incubated with a polyclonal Ab to type II NOS (Transduction Laboratories, Lexington, KT) diluted 1/200 overnight at +4°C. They were then washed three times with PBS and incubated with a Cy3-coupled anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature in the dark. Control slides were incubated with PBS. Slides were then washed, mounted in aqua-mount (Lerner Laboratories, Pittsburgh, PA), and examined by fluorescence microscopy.

Preparation of total RNA

Following administration of a lethal dose of sodium pentobarbital, mice were perfused through the ascending aorta with 20 ml ice-cold PBS. The spinal cord was then dissected free from the spinal column, and total RNA was extracted using TRIREAGENT (Molecular Research Center, Cincinnati, OH).

Ribonuclease protection assay

RPA using either the ML-11 multiprobe template set (kindly provided by Dr. Monte Hobbs) (14) or the mCK-1 template set (PharMingen) was performed essentially as described (14). Briefly, [{alpha}-32P]UTP-labeled antisense RNA transcripts for TNF-{alpha}, lymphotoxin (LT), IL-1{alpha} and ß, IL-6, IFN-{gamma}, and L-32 (large ribosomal subunit protein 32) were generated using the template sets and T7 RNA polymerase. Ten to twenty micrograms of total RNA from each sample were allowed to hybridize to the labeled probe for 20 h at 45°C. ssRNA was digested with an RNase A/T1 mixture (Ambion, Austin, TX), and the hybrids were analyzed on denaturing urea/polyacrylamide gels. Protected fragments were visualized by autoradiography and quantified using Image Quant (Molecular Dynamics, Sunnyvale, CA). The ML-11 probe set was used to measure levels of IL-1, IL-6, TNF-{alpha}, and LT, and the mCK1 for IFN-{gamma}. For each sample, a ratio of the intensity of the cytokine band was obtained using the band for ml32.

Competitive RT-PCR/MIMIC PCR

mRNA for iNOS was quantified by competitive RT-PCR or MIMIC PCR, according to the manufacturer’s instructions (Clontech Laboratories, Palo Alto, CA). Briefly, total RNA from spleen and spinal cord was isolated as above, and 5 µg of RNA was reverse transcribed using Ready-to-go you-prime first-strand beads (Pharmacia Biotech, Piscataway, NJ). A series of 10-fold dilutions of known iNOS MIMIC cDNA, a commercial DNA that is amplified with similar efficiency as target cDNA for iNOS by the specific primers, but produces a product of smaller size, was added to a constant amount (0.75 µg) of target cDNA containing a final concentration of 2 U/50 µl Amplitaq DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT), 0.4 µM 5' primer, 0.4 µM 3' primer (Clontech Laboratories), 0.2 mM of each dNTP, 1.5 mM magnesium chloride, and 10 mM PCR buffer (Perkin-Elmer/Cetus). PCR amplifications were performed for 35 cycles, each cycle consisting of 94°C for 45 s, 65°C for 45 s, and 72°C for 2 min, followed by a 10-min extension at 72°C. PCR products were separated by electrophoresis on a 1.6% agarose gel and visualized by UV light after staining with ethidium bromide. A fine-tuned competitive PCR was performed using twofold serial dilutions after determination of the 10-fold dilution in which the PCR MIMIC and target cDNA template gave bands of equal intensity. PCR amplifications were done as before, except that one of the dNTP was [{alpha}-32P]dCTP. The PCR products were then resolved on a 1.6% ethidium bromide agarose gel, and the bands corresponding to the iNOS target and MIMIC were excised from the agarose gel and the amount of radioactivity was determined by scintillation counting. The log of the ratio of the radioactivity of the target and MIMIC DNA was plotted against the log of the MIMIC molecules added to the PCR. Results were derived from a linear regression of the data. Statistical analysis was performed using Student’s t test.

ELISA

The ELISAs for IL-1ß and IL-6 were performed using kits purchased from R&D Systems (Minneapolis, MN). Affinity-purified polyclonal Abs for IL-1ß or IL-6 were precoated onto microtiter plates. Animals were perfused with ice-cold PBS, as above, and the spinal cord and spleen were homogenized with 1 ml PBS containing 0.1 M PMSF. Duplicate 50 µl of standards, controls, and spinal cord or spleen homogenates were added to the wells and incubated at room temperature for 2 h. After washing, enzyme-linked polyclonal Abs specific for mouse IL-1ß, or IL-6 were added and incubated for 2 h at room temperature. The plates were then washed and reacted with substrate solution for 30 min, stop solution was added, and the plates were read at an OD of 450 nm. The sample values were then calculated from the standard curve.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cytokine gene expression in {gamma}{delta} T cell-depleted mice

To determine the effect of {gamma}{delta} T cell depletion on cytokine gene expression in the CNS of animals with EAE, mice were sensitized by the passive transfer of MBP-reactive lymph node cells and treated with the GL3 Ab, or with NHIgG as a control, on days 4 and 5 posttransfer. At varying times in the disease process, animals were perfused with PBS, the spinal cords were removed, total RNA was extracted, and cytokine gene expression was assessed using the multiprobe template RPA system. Since preliminary data indicated that changes occurring at the early stages of the disease process were critical to this analysis, we took tissue immediately before (day 6) and at the time of (day 7) disease onset, as well as at the height (days 10/11) and during the recovery phase (day 20) of the disease. A total of four animals from two separate experiments was analyzed for each time point. The mean CI for the NHIgG animals taken on day 6 was 0, day 7 was 1.3 ± 0.6, days 10/11 was 2.8 ± 0.8, and day 20 was 1.3 ± 0.7. For the animals treated with GL3, the mean CI for day 6 was 0, day 7 was 0, days 10/11 was 0.75 ± 0.6, and day 20 was 0.8 ± 0.2. Using the ML-11 RPA multiprobe template set (14), we were able to distinguish signals for IL-1, IL-6, TNF-{alpha}, and LT. The results are shown in Figure 1Go and are expressed as a ratio of cytokine mRNA to mL-32. In the NHIgG animals, levels of mRNA for IL-1 rose abruptly at the time of disease onset, then fell gradually over the course of the disease. Treatment with GL3 dramatically reduced the levels of IL-1 mRNA at the pre-onset and onset phases, but had essentially no effect on levels detected at the height and recovery phases. A similar pattern of cytokine mRNA expression was observed for IL-6 in the NHIgG-treated animals, with peak levels noted at the time of disease onset. Treatment with GL3 also dramatically changed the IL-6 mRNA pattern of expression, with low levels noted at the expected time of disease onset, but with levels that were actually higher than the controls during the recovery phase. In contrast to the data for IL-1 and IL-6, cytokine mRNA levels for TNF-{alpha} and LT peaked at the height of disease. Treatment with GL3 led to significantly lower levels of mRNA for these cytokines at disease onset, but at later stages of the disease no differences were noted between treated and control animals. The levels of mRNA for IFN-{gamma} in these same samples were determined using a separate RPA kit (Fig. 1GoE). The results show that, like TNF and LT, IFN-{gamma} levels peaked with the height of disease. However, in contrast to the results obtained with TNF-{alpha} and LT, treatment with GL3 led to reduced expression of mRNA for IFN-{gamma} at all stages of the disease process.



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  		FIGURE 1. Analysis of cytokine gene expression in the spinal cords of EAE mice using RPA. Animals were sensitized by the passive transfer of MBP-reactive T cells and treated with GL3 or NHIgG on days 4 and 5. On days 6 (pre-onset), 7 (onset), height (days 10–11) and remission (remiss, days 20) phases of the disease, the animals were perfused with saline, the spinal cords were removed, and mRNA for the different cytokine genes was determined using the multiprobe RPA assay. The data are expressed as a ratio of the band of the cytokine gene to the band for mL-32, as described in Materials and Methods. Each value shown represents the mean ± SD of a total of four animals from two separate experiments for IL-1 (A), IL-6 (B), TNF (C), LT (D), and IFN-{gamma} (E).

 
Cytokine protein expression in the CNS of {gamma}{delta} T cell-depleted mice

To determine whether the changes noted in the mRNA levels for IL-1 and IL-6 in the {gamma}{delta} T cell-depleted animals were reflected in the amounts of cytokine present in the CNS, we used IL-1ß- and IL-6-specific ELISAs to assess protein levels present in the spinal cord on day 6 and day 7 postsensitization. The results are shown in Table IGo. On day 6, only low levels of IL-1 and IL-6 were detected in spinal cord homogenates from both the NHIgG- and GL3-treated animals. However, on day 7, coincident with disease onset, there was a dramatic increase in the levels of both of these cytokines in the NHIgG animals that was not detected in the GL3-treated animals. No differences were detected in IL-1 or IL-6 levels in the spleen cell samples harvested from the same animals (data not shown). These data indicate, therefore, that the results obtained with the RPA were reflected in the total levels of protein present in the CNS, and indicate that depletion of {gamma}{delta} T cells led to significantly reduced levels of the proinflammatory cytokines IL-1 and IL-6 at the time of disease onset.


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  		Table I. ELISA for IL-1ß and IL-6 production by NHIgG and GL3-treated mice1

 
Pathology of early onset phase in {gamma}{delta} T cell-depleted mice

It is now well recognized that the commencement of clinical expression of EAE in susceptible animals is marked by an abrupt onset of inflammation in the CNS, particularly localized to the lumbar spinal cord. In our previous studies, we showed that {gamma}{delta} T cell depletion led to a significant reduction in the extent of inflammation in the CNS detectable at the height of the acute clinical episode (10) but, in those experiments, we did not study the early phases of the disease process. Therefore, we repeated the {gamma}{delta} T cell depletion experiments and took tissue for pathology from the day 6 and day 7 time points. The results are shown in Figure 2Go. They indicated that in the animals treated with NHIgG on day 6, immediately before disease onset, a low level of inflammation could be observed in the lumbar spinal cord sections that was restricted to the meninges (Fig. 2GoA). On day 7, the extent of this inflammation had increased considerably, although the bulk of the inflammatory infiltrate was still restricted to the meningeal vessels (Fig. 2GoB). In contrast, in animals treated with GL3, only small foci of inflammation could be detected in the meninges on day 6 (Fig. 2GoC) that was not measurably increased by day 7 (Fig. 2GoD). To provide a more quantitative analysis of these events, an additional set of animals was sensitized and treated as before. On days 6 (n = 4 per group) and 7 (n = 4 per group), the spinal cord was removed after transcardial perfusion with PBS, and a total leukocyte count was performed using FACS analysis. In animals treated with NHIgG, the total number of leukocytes isolated from the spinal cord on day 6 was 6,308 ± 1,959, and on day 7 was 622,990 ± 8,290. In animals treated with GL3, the total number of leukocytes isolated on day 6 was 2,426 ± 762, and on day 7 was 9,476 ± 3,027. Thus, {gamma}{delta} T cell depletion led to a striking decrease in the extent of infiltration during the early phases of the inflammatory episode.



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  		FIGURE 2. One-micron toluidine-blue-stained epoxy cross-sections from the L6 region of the lumbar spinal cord of mice sensitized to develop EAE by the adoptive transfer of MBP-reactive lymph node cells 6 or 7 days previously. A, High power view of the lateral columns of an animal sensitized 6 days previously and treated with NHIgG (CI +0). Note the mononuclear cell infiltrate associated with the leptomeninges and a penetrating vessel. B, Identical region to A from an animal sensitized 7 days previously and treated with NHIgG (CI +1). C, Identical region to A from an animal sensitized 6 days previously and treated with GL3 (CI +0). D, Identical region to A from an animal sensitized 7 days previously and treated with GL3 (CI +0). Magnification x165.

 
We next took frozen sections from the same day 6 and day 7 time points to determine the sites of IL-1 production in the spinal cord. The results showed that the infiltrating cells themselves were the major sites of IL-1 production at this time point (Fig. 3Go).



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  		FIGURE 3. Immunohistochemical analysis of IL-1ß in spinal cord lesions. A, Frozen sections of spinal cord from a mouse sensitized 7 days previously by the passive transfer of MBP-reactive T cells and reacted with Abs to IL-1ß. Note the intense immunoreactivity for IL-1ß in the leptomeninges corresponding to the inflammatory infiltrates. B, Serial section to A reacted with an irrelevant isotype-matched control Ab. The meninges lie above. Magnification x165.

 
The effect of {gamma}{delta} T cell depletion on type II NOS expression

The most persistent effects of {gamma}{delta} T cell depletion on cytokine gene expression in EAE were noted for IFN-{gamma} in which reduced levels of mRNA for this cytokine were detected at each of the time points tested. IFN-{gamma} is thought to contribute to the pathology of EAE by activating macrophages to produce a range of toxic factors involved in the disruption of the blood-brain barrier, and the loss of myelin from the axon. One of the factors that has been strongly implicated in this process is the activation of type II NOS, which, once activated, leads to the prolonged release of large amounts of nitric oxide (15). In EAE, both activated macrophages and reactive astrocytes have been implicated as the major sources of type II NOS (16). To study the effects of {gamma}{delta} T cell depletion on type II NOS expression in EAE, we treated animals as before with NHIgG and GL3 and, at the height of the acute clinical episode, harvested spleens and spinal cords and performed a semiquantitative analysis for type II NOS using a MIMIC PCR-based assay (see Materials and Methods). No PCR products for type II NOS were detected in any of the spleen samples. However, in spinal cord tissues, a strong signal for type II NOS was obtained. As shown in Figure 4Go, all of the animals (n = 5) treated with NHIgG had detectable product for type II NOS with a mean level of 1.3 ± 0.7 attomoles/spinal cord, ranging from 0.94 (CI +0.5) to 2.5 (CI +3.5) in individual animals, consistent with the data obtained by Cross et al. (17). In contrast, in the animals treated with GL3, the mean level of activity was 0.36 ± 0.5 attomoles activity (n = 5), ranging from 0 (CI +0.5) to 1.25 (CI +1), indicating a significant (p < 0.02) reduction in the levels of type II NOS activity associated with the depletion of {gamma}{delta} T cells.



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  		FIGURE 4. Semiquantitative RT-PCR analysis of iNOS mRNA in mouse spinal cord tissue. At the height of disease (day 9), total RNA was prepared from spinal cord tissue of control (NHIgG, n = 5) and {gamma}{delta} T cell-depleted (GL3, n = 5) mice, and the presence of mRNA for iNOS was determined using a MIMIC-based RT-PCR technique, as described in Materials and Methods. Results are expressed as attomoles mRNA per spinal cord. The mean value for the NHIgG-treated animals was 1.3 ± 0.7, and for the GL3-treated animals was 0.36 ± 0.5 (p < 0.02).

 
We then stained frozen sections from these same animals to determine the extent of immunoreactivity for type II NOS in the tissues. As shown in Figure 5GoA, animals treated with NHIgG showed prominent staining for type II NOS associated with inflammatory foci and glia that was considerably reduced in the animals treated with GL3 (Fig. 5GoB). Thus, both mRNA and protein for type II NOS were found to be reduced in the animals depleted of {gamma}{delta} T cells. These data would be consistent with the low levels of IFN-{gamma} detected in these animals, since this cytokine has been strongly implicated in synergizing with other cytokines in the production of high levels of type II NOS.



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  		FIGURE 5. Immunohistochemical analysis of iNOS expression in mouse spinal cord. A, Frozen section of the lumbar spinal cord reacted with a polyclonal Ab to type II NOS from a mouse that had been sensitized 9 days previously and treated with NHIgG on days 4 and 5 (CI +3.5). Note the intense immunoreactivity for type II NOS in inflammatory cells present in the anterior median fissure. B, An identical region to A from an animal that had been treated with GL3 on days 4 and 5 (CI +0.5). Note that only a faint immunoreactive signal could be detected in this tissue. Magnification x82.5.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have addressed the effect of {gamma}{delta} T cell depletion on cytokine gene expression in the spinal cords of mice in which an acute attack of EAE was induced by the passive transfer of MBP-reactive T cells. Two different patterns of response were observed. For IL-1, IL-6, TNF, and LT, {gamma}{delta} T cell depletion markedly reduced the levels of mRNA for these cytokines at disease onset but, at a time point that corresponded to the height of disease in control animals, no differences were detected in the mRNA levels between depleted and control animals. In contrast, mRNA levels for IFN-{gamma} were reduced in {gamma}{delta} T cell-depleted animals at all stages of the disease process. The differences noted in the levels of IL-1 and IL-6 at disease onset were confirmed by ELISA, and the reduction in the levels of mRNA for IFN-{gamma} was supported by reduced expression of type II NOS, an enzyme for which IFN-{gamma} functions as a major inducing cytokine. The fact that each of these proinflammatory cytokines is up-regulated in EAE tissues has been amply documented in various different models (18, 19, 20, 21, 22, 23, 24, 25). The results presented in this work are substantially in agreement with these other studies, but also suggest that the timing and sequence of release of these cytokines may be as important as their relative levels, since {gamma}{delta} T cell-depleted mice had clearly elevated levels of IL-1, IL-6, TNF, and LT at a time when they expressed little to no clinical evidence of disease (mean CI 0.75 ± 0.6), although pathologic evidence of inflammation could be readily detected (10).

The results for IL-1, IL-6, TNF, and LT suggest a regulatory role for {gamma}{delta} T cells at disease onset, a concept that was supported by reduced levels of inflammation at this time point in the cord meninges in depleted animals. Several studies have demonstrated that the onset of EAE is accompanied by an abrupt onset of inflammation in the CNS, and clinical disease has been shown to correlate with the extent of this inflammatory infiltrate (26, 27). A critical role for TNF in the initiation of the CNS inflammatory process has been shown using anti-TNF Abs as well as soluble forms of the TNF receptor (28, 29, 30). Similarly, a role for IL-1 in the initiation of inflammation is supported by the exacerbating effect of administration of IL-1, as well as by the ameliorating effect of the IL-1R antagonist (31, 32), in EAE. Recently, several studies have documented a role for {gamma}{delta} T cells in the regulation of inflammation at various tissue sites. Thus, it has been shown that {gamma}{delta} T cells control inflammatory reactivity and prevent excessive liver damage in mice infected with Listeria monocytogenes by regulating the influx of neutrophils into the liver (33). {gamma}{delta} T cells have also been shown to be required for the accumulation of eosinophils in the pleural cavity of mice following challenge with LPS, an effect that was postulated to occur via amplification of the role of macrophages in this response (34). In mice with acquired immunity to Mycobacterium tuberculosis, {gamma}{delta} T cells have also been shown to regulate cellular traffic into the pleural cavity, promoting the influx of lymphocytes and monocytes and inhibiting the trafficking of neutrophils, possibly through the secretion of specific chemokines (35).

The role of {gamma}{delta} T cells as a source of chemokines has not been documented extensively, but intraepithelial {gamma}{delta} T cells can be induced to express MIP-1{alpha}, MIP-1ß, RANTES, and lymphotactin, but not monocyte-chemotactic protein-1, with lymphotactin being the most abundantly produced (36). In these cells, the expression of RANTES remained elevated following activation in contrast to the kinetics of RANTES expression in {alpha}ß T cells, suggesting that {gamma}{delta} T cells may be a potent source of this chemokine during chronic inflammatory episodes (36). In early Listeria infection, {gamma}{delta} T cells have been found to be a major source of a monocyte-chemotactic factor, a cytokine/chemokine known to be crucial for protection against Listeria infection (37). Chemokines are expressed abundantly in EAE lesions (38), and ongoing studies of chemokine expression in our {gamma}{delta} T cell-depleted mice have shown a dramatic reduction in the levels of monocyte-chemotactic protein-1, MIP-1{alpha}, MIP-1ß, and RANTES in the spinal cord, particularly in the early phases of the disease process (Rajan et al., manuscript in preparation). These data would be consistent with an important role for {gamma}{delta} T cells in orchestrating the influx of inflammatory cells into the CNS lesions during the early stages of lesion formation.

The results for IFN-{gamma} suggest that {gamma}{delta} T cells themselves are either a major source of this cytokine or regulate its expression in other cells in inflamed tissues. Both T cells and NK cells are known to be major sources of IFN-{gamma} in vivo, and our results would be consistent with the observations of others that {gamma}{delta} T cells share with NK cells a propensity toward high IFN-{gamma} release (39, 40, 41, 42, 43). The role of IFN-{gamma} in EAE, however, has been shown to be complex with both pro- and antiinflammatory effects noted (44, 45, 46). Although the reasons for these discrepancies remain unclear, they most likely reflect the fact that IFN-{gamma} plays a key regulatory role in both the inductive and protective phases of the disease process.

A major effect of IFN-{gamma} is thought to be its role in activation of cells of the monocyte/macrophage series. As a measure of this in our experiments, we chose to investigate the expression of type II NOS, an enzyme that is induced in many cell types in response to combinations of cytokines, but for which IFN-{gamma} has been recognized as the major regulatory cytokine. The fact that {gamma}{delta} T cell-depleted mice showed lower levels of type II NOS would be consistent with observations in experimental mucosal candidiasis, in which {gamma}{delta} T cells have been shown to enhance macrophage nitric oxide production in vitro via IFN-{gamma} secretion, as well as in vivo, in which it was shown that depletion of {gamma}{delta} T cells abrogated NOS expression in affected mucosal sites (47). However, again like IFN-{gamma}, a role for this enzyme in EAE remains controversial, with both exacerbating and beneficial effects of inhibitors of NOS having been documented extensively (reviewed in 1 .

That {gamma}{delta} T cells play an important role in immunity toward bacterial and viral infections has been documented extensively, but their potential role in autoimmune conditions has not been so widely studied. However, there is a growing body of evidence supporting a role for {gamma}{delta} T cells as immunoregulatory cells. {gamma}{delta} T cells have been shown to be involved in the control of {alpha}ß T cell activation (48), and to both propagate and regulate murine lupus in the MRL/Mp+/+ strain (49). In addition, {gamma}{delta} T cells have been shown to be critical mediators of tolerance induction in a number of different model systems, including those induced by either aerosolized or ingested soluble Ags and involving humoral and cell-mediated immune responses (41, 42, 50, 51). Although the mechanisms involved in these complex regulatory pathways have not yet been defined, they have been shown to be mediated by remarkably low numbers of {gamma}{delta} T cells, ranging from ~2 x 105 cells/animal in the studies of McMenamin et al. (41), to ~2 x 103 cells/animal in the studies of Szczepanik et al. (50), which would be within the range of {gamma}{delta} T cells found in the spinal cord of animals with EAE.

The results presented in this work could be interpreted in one of two ways: either {gamma}{delta} T cells promote the inflammatory process providing additional cytokines that facilitate the activation and migration of myelin-reactive T cells across the blood-brain barrier, or {gamma}{delta} T cells function to down-regulate a regulatory cell that blocks the activation of these autoimmune cells. Studies in TCR-transgenic mice specific for MBP have clearly shown that only a few mice spontaneously express disease, unless they are challenged with Ag, pertussis toxin, agents present in a nonsterile environment (52), or are crossed with Rag-/- animals (53). A critical step in this process appears to be transfer into the CNS, which is known to require cell activation and expression of certain sets of adhesion molecules (27). We propose, therefore, that {gamma}{delta} T cells facilitate the activation and transfer of myelin-reactive T cells into the CNS by providing appropriate proinflammatory cytokines and chemokines. As noted above, these effects may not require large numbers of cells and suggest that interpretations made from the relative numbers of different cell populations may not necessarily reflect the role of these cells in the disease process (10). We further propose that when autoantigen-reactive T cell lines and/or clones are driven in vitro by repeated stimulation with Ag and cytokines, they become sufficiently activated that they function independently of factors supplied by {gamma}{delta} T cells, and under these conditions are able to transfer disease in the absence of {gamma}{delta} T cells, as has been shown in the TCR-{gamma}{delta} knockout mouse (54). An alternative possibility is that {gamma}{delta} T cells function to regulate a regulatory cell, but the results obtained in the TCR-{gamma}{delta} knockout mouse would tend to argue against this.

In conclusion, the results of this study suggest that {gamma}{delta} T cells participate in the development of EAE by functioning to regulate leukocyte transfer across the blood-brain barrier and acting as a source of proinflammatory cytokines. As such, these data form part of a growing awareness of the role of {gamma}{delta} T cells in regulating a number of inflammatory events. However, in many of these conditions, the effect of {gamma}{delta} T cell depletion has led to conflicting results, with amelioration or exacerbation of disease noted in often closely related animal model systems. Although some of these differences may be attributable to differences in the various strategies used, the role of {gamma}{delta} T cells in these conditions remains confusing. As such, these data are reminiscent of the plethora of effects attributable to CD4+ {alpha}ß TCR+ T cells before the establishment of the Th1 vs Th2 paradigm (55). Whether or not {gamma}{delta} T cells can be subdivided into different functional subsets remains to be determined, but it is clear that the regulatory pathways involved in {gamma}{delta} T cell activation differ from that found in {alpha}ß T cells. However, given the growing number of Ag-specific immune responses that can now be attributed to {gamma}{delta} T cells, it is to be expected that progress in our understanding of the biology of this subset of T cells will proceed more rapidly.


   Acknowledgments
 
We thank Hong Zhang for help in the sensitization of the animals and the preparation of tissues for immunohistochemistry. We also thank Clemens Cayetano for preparation of the semithin sections.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants NS31919, NS11920, and NS07098. J.D.S.K. participated in this study as a Roth Scholar in the Roth Institute Summer Undergraduate Honors Research Program at Albert Einstein College of Medicine of Yeshiva University. Back

2 Address correspondence and reprint requests to Dr. Alice J. Rajan, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Back

3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; CI, clinical index; CNS, central nervous system; iNOS, inducible nitric oxide synthase; LT, lymphotoxin; MBP, myelin basic protein; MIP, macrophage-inflammatory protein; NHIgG, normal hamster immunoglobulin G; NOS, nitric oxide synthase; RANTES, regulated upon activation, normal T cell expressed and secreted; RPA, ribonuclease protection assay; RT-PCR, reverse-transcriptase polymerase chain reaction. Back

Received for publication September 30, 1997. Accepted for publication February 18, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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