| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the Neurosciences Research Unit,* The Canberra Hospital, Canberra, Australia; the Faculty of Medicine, University of Utrecht, Utrecht, The Netherlands; The John Curtin School of Medical Research, Australian National University, Canberra; and The Australian National University School of Medicine, Canberra, Australia
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Results from much of the early work on NO and EAE,6-9 including our own,10 suggested a deleterious effect of NO in this CNS inflammation. Later studies, however, began to shift the focus from the idea of NO as a "bad" molecule to one in which it plays an important role in down-regulating the disease process.5 We11 and others12,13 showed that inhibition of NO production by treatment of Lewis rats with nitric oxide synthase (NOS) inhibitors enhances actively induced EAE. We further demonstrated that the EAE-resistant PVG rat produces up to four times higher serum reactive nitrogen intermediates (RNI) levels than Lewis rats following immunization with myelin basic protein with complete Freund’s adjuvant (MBP-CFA) and that treatment of immunized PVG rats with the potent NOS inhibitor, N-methyl-L-arginine (L-NMA), rendered them highly susceptible to disease induction.11 We also showed that the major source of NO in the PVG rat was most likely the spleen in that splenectomy completely abrogated the increased serum RNI levels following immunization of the PVG rat, and again rendered them highly susceptible to EAE.14
Recently, other studies have demonstrated that the acute monophasic nature of EAE in the Lewis rat as well as the Lewis rat’s resistance to re-induction of disease following recovery can also be accounted for, at least in part, by the increased production of NO.15,16 Rats recovering from MBP-CFA-induced EAE have significantly increased levels of serum RNI, indicative of increased NO production. These levels remain elevated after the recovery period and increase even further early after a re-challenge with MBP-CFA. These animals are totally refractory to a second episode of disease.17 Oral treatment of rats with L-NMA beginning at peak disease prolongs the disease episode until the drug is stopped. Treatment of fully recovered rats with L-NMA beginning 24 hours before a re-challenge with MBP-CFA leads to decreased serum RNI levels and results in a second episode of EAE in 100% of animals. In point of fact, treatment of recovered Lewis rats with L-NMA even in the absence of re-challenge with antigen results in a secondary episode of disease.16 These data point to NO as a potent regulator of this autoimmune response.
The Brown Norway (BN) rat has long been regarded as "resistant" to EAE18-20 and this resistance has been mapped to both MHC and non-MHC regions of the genome.18,21,22 Recently, studies on the BN rat have described the BN as mounting a Th2 type of immune response and a number of non-MHC regulated mechanisms have been suggested, such as endogenous corticosteroid levels,23 enhanced TGFß production,24,25 and CD8 bias.26 Here we re-examine the resistance of the BN rat to EAE in light of our current knowledge of the role NO can play in regulation of this disease. We report experiments in which we demonstrate both in vitro and in vivo that the BN rat produces significantly more NO (as reactive nitrogen intermediates, RNI) following stimulation or immunization with neuroantigen and complete Freund’s adjuvant (CFA) than either the PVG or Lewis rat. Using the unique adjuvant carbonyl iron (CI), as originally described by Levine and Sowinski,20 we are able to induce EAE in BN rats and we further demonstrate that with this adjuvant these rats do not increase their NO production following immunization. If complete Freund’s adjuvant is administered simultaneously with carbonyl iron and neuroantigen, the BN rats produce high levels of NO and fail to develop EAE. The pathology in the BN rat immunized using CI is unique in that the lesions show an extensive infiltration of macrophages/activated microglia. The cytokine profiles of both inflamed spinal cord and draining lymph nodes do not differ between Lewis rats immunized with neuroantigen in CFA and BN rats immunized with neuroantigen plus CI, suggesting both responses are Th1 responses. These results once more point to the important role of the innate immune system, and in particular NO, in immune regulation.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Lewis and PVG rats were bred by the Animal Breeding Establishment of the John Curtin School of Medical Research, Australian National University. Breeding pairs of BN rats were obtained from the Animal Resource Centre, Canning Vale, Western Australia, and bred on the premises at the Canberra Hospital. The use of animals and all procedures performed were approved by the Animal Experimentation Ethics Committee of the Australian National University following the guidelines of the National Health and Medical Research Council of Australia.
Immunizations
MBP was purified from frozen guinea pig spinal cord according to the method of Eylar et al.27 MBP in saline was emulsified in an equal volume of incomplete Freund’s adjuvant with added Mycobacterium butyricum (Bacto Laboratories, Liverpool, Australia) (4 mg/ml). Rats received 100-µl emulsion in each hind footpad. When using rat spinal cord homogenates (SCH) the original procedure of Levine and Sowinski20 was followed. Frozen spinal cords were passed through a mesh and 40% homogenates in saline were left in a 60°C water bath for 1 hour. SCHs were either emulsified with equal volume of CFA (containing 4 mg/ml Mycobacterium tuberculosis) and injected at a total of 100 µl per rat (50 µl in each hind footpad) or mixed with CI (Sigma, 100 mg CI:1 ml SCH) and 50 µl injected into one hind footpad.
Evaluation of EAE Clinical Signs
Rats were examined on a daily basis and clinical scores recorded from day 5 to termination of the experiment. Clinical disease severity was assessed and scored as previously described17 using a scale from 1 to 5 as follows: 0, asymptomatic; 1, flaccid distal half of tail; 2, entire tail flaccid; 3, ataxia, difficulty in righting; 4, hind limb weakness; 5, hind limb paralysis. Body weight was also recorded in some experiments.
Measurement of Nitric Oxide Production
The levels of nitrate and nitrite in serum, urine, and cell culture samples were determined as an indirect measurement of NO production (described in detail by Cowden et al).11 Briefly, 30-µl aliquots were added in duplicate to a V-bottomed plates (Nunc). Standard curves were generated using culture media or dialysed rat serum to which sodium nitrite or sodium nitrate had been added at concentrations ranging from 1 mmol/L to 1 µmol/L. To measure nitrate, the addition of nitrate reductase and NADPH (Boehringer Mannheim, Mannheim, Germany) for 30 minutes is required for conversion to nitrite. Nitrite was measured by addition of Griess reagent. Trichloroacetic acid was added to precipitate protein, the plates were centifuged and the optical density of each supernatant was read at 540 nm with a reference wavelength of 650 nm using a Multiscan Platereader (Thermo Labsystems). Nitrate and nitrite levels were quantified by reading against the appropriate standard curves. The results were expressed as micromolar (µM) concentrations of reactive nitrogen intermediates (RNI) ie, the sum of nitrate and nitrite concentrations.
In Vitro Cytokine Stimulation of Spleen Cells
Spleen cell suspensions were cultured at a concentration of 2 x 105 per well in 96-well plates in DMEM medium with 10% fetal calf serum (FCS), 2ME, and L-glutamine. Cultures were set up in the absence or presence of 10 U/ml rat recombinant IFN
(GIBCO BRL) for 48 hours.
Immunopathology and Immunohistochemistry
Two centimeters of spinal cord encompassing the lumbar enlargement were taken from each of three animals and sectioned longitudinally at three different levels through the cord. Sets of six serial sections were cut and three sections (every other one) were mounted on each of two slides. This was repeated three times leaving six sections between sets. This was repeated at three levels through the cord. Half the slides were stained with H&E and the other used for immunohistochemistry (see below). A total of 27 sections were examined. Lesions were assessed semi-quantitatively and were scored 0 to +++, with 0, no lesions; +, one lesion in an occasional low-power field, ++, one lesion in almost every field; and +++, several lesions in many fields.
Serial paraffin sections (7 µm) of lumbar spinal cord (as above), lymph nodes, and spleens were subjected to a 20-minute antigen retrieval in citrate buffer (pH 6.0) at 95°C. Peritoneal exudate cells were harvested 3 days after an i.p. injection of 100 µl SCH-CFA or 50 µl SCH-CI. Slides of peritoneal exudate cells were prepared by cytospin and fixed with acetone. The endogenous peroxidase activity and the non-specific antibody binding were blocked by incubation with peroxide block and power block (InnoGenex Immunohistochemistry Kit, San Remon, CA). Biotinylated mouse anti-rat ED1 monoclonal antibody (Serotec, Oxford, England) and rabbit anti-rat iNOS polyclonal antiserum (Sapphire Bioscience, Australia) were used as primary antibodies. The secondary antibodies and the developing were according to the manufacturer’s instructions (InnoGenex). The sections were counterstained with Mayer’s hematoxylin and mounted in SuperMount (Sapphire Bioscience).
PCR Analysis of Cytokines
On different days after immunization with SCH-CFA or SCH-CI Lewis and BN rats were anesthetized, perfused with cold phosphate-buffered saline (PBS), and the popliteal lymph nodes and lumbar spinal cords were stored in RNAzol at –70°C. mRNA was extracted and transcribed into cDNA using reverse transcriptase (Qiagen, Victoria, Australia). Specific cytokine cDNA fragments were amplified with Taq polymerase (Epicenter, Madison, WI) in FailSafe buffer E (for IL2 and IFN), and buffer D (for TNF
, IL4, and IL10). Amplified PCR products were run in 2.3% agarose gel. The sequences of primer pairs (synthesized by GeneWorks, Australia) were as follows:
ß-actin: sense 5'-CTATCGGCAATGAGCGGTTC-3' and
antisense 5'-CTTAGGAGTTGGGGGTGGCT-3';
IL-2: sense 5'-CCACTTCAAGCCCTGCAAAGGA-3' and
antisense 5'-GACAGATGGCTATCCATCTCCTCAG-3';
IFN: sense 5'-TGAACGCTACACACTGCATCTTGG-3' and
antisense 5'-CGACTCCTTTTCCGCTTCCTGAG-3';
TNF: sense 5'-TCGAGTGACAAGCCCGTAG-3' and
antisense 5'-GGCAGAGAGGAGGCTGACTT-3';
IL4: sense 5'-TCCATGCACCGAGATGTTTGTACC-3' and
antisense 5'-CTTTCAGTGTTGTGAGCGTGGACT-3';
IL10: sense 5'-TAAGGTTACCTTGGGTTGCCAAG-CC-3' and
antisense 5'-AGGGGAGAAATCGATGACAGCGCC-3'.
The intensities of the bands of the amplified PCR products were determined by densitometry and values have been normalized to that of ß-actin and expressed as percentage.
Statistics
The analysis was done with Student’s t-test or analysis of variance with Dunnett’s Post-Hoc Test.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Spleen cells from groups of non-immunized age-matched female Lewis, PVG, and BN rats were cultured in vitro with or without IFN. Supernatants were assayed for RNI 48 hours later. The results (Table 1)
demonstrate that PVG splenocytes produce approximately 2.5 times the amount of RNI as the Lewis splenocytes and the BN splenocytes twice that of the PVG and five times that of the Lewis cells. Thus, with respect to the in vitro RNI production by splenocytes: BN>PVG>Lewis.
|
The in vivo levels of serum RNI seen in the three rat strains following immunization with MBP-CFA do not in fact parallel the in vitro levels of RNI produced by spleen cells following IFN stimulation. The PVG rat is relatively resistant to EAE induction compared to the Lewis rat and following immunization produces up to three times higher serum RNI levels than the Lewis rat; this being similar to the in vitro situation. The BN rat, however, is strongly resistant to MBP-CFA-induced EAE, their spleen cells produce twice the amount of RNI as PVG cells, yet in vivo, following immunization, the BN serum RNI levels are only equal to or even slightly lower than the PVG (Figure 1A)
. Furthermore, oral treatment of immunized PVG rats with the NOS inhibitor N-methyl-L-arginine (L-NMA) has been shown to reduce the RNI levels and render the rats highly susceptible to EAE induction,11
but interestingly, we have been unable to lower the serum RNI levels of the immunized BN rat with non-toxic doses of NOS inhibitors or to convert the BN to EAE susceptibility with such NOS inhibitors (data not shown). The puzzle of the BN splenocytes making significantly more RNI in culture than the PVG splenocytes, yet this difference not being reflected in the serum RNI levels following in vivo immunization, and our inability to lower serum levels with drugs, suggested to us the possibility that the BN rat may in fact be making more RNI in vivo than the PVG rat but excreting them in the urine at a faster rate. As seen in Figure 1B
, this would appear to be the case as the BN rats excreted up to three times more RNI in the urine than the PVG on a background of equal serum levels.
|
The increased production and maintenance of high levels of RNI in the BN rat prevented our assessing the role of NO in the resistance of the BN to EAE by the use of NOS inhibitors. We therefore revisited the original experiments of Levine and Sowinski,20
who showed that the BN rat could develop severe EAE if carbonyl iron was used as adjuvant. In doing so we asked if one possible mechanism for the efficacy of carbonyl iron was that it did not induce NO production in the BN rat following immunization. Soluble MBP could not be used with carbonyl iron as the two dissociate too rapidly and therefore we used spinal cord homogenate (SCH) as did Levine and Sowinski.20
The EAE susceptibility profile of the two rat strains is the same with SCH-CFA as with MBP-CFA, ie, Lewis>BN (Table 2)
. Carbonyl iron as adjuvant in the Lewis rat had the effect of increasing the severity and duration of disease when compared with CFA as adjuvant, though the increase in duration was not significant. Carbonyl iron usage in the BN rat significantly increased the incidence of disease and the disease produced was quite severe. Interestingly, the duration of disease in the BN immunized with CI was significantly longer than the CI-induced disease in the Lewis rat (Table 2)
. In another experiment (Figure 2A)
, we demonstrate that there is no disease and no weight loss in BN rats immunized with SCH-CFA and this on a background of elevated serum RNI levels. Rats immunized with SCH-carbonyl iron (Figure 2B)
show no increase of serum RNI levels and develop severe disease with significant weight loss.
|
|
We also examined the difference in NO production by various BN rat tissues following immunization with either CFA or with carbonyl iron to determine whether increased tissue iNOS might reflect serum RNI levels following CFA. To do this we performed immunohistochemistry for iNOS expression in draining lymph nodes and spleen taken 7 days after immunization in the foot pads with SCH and either adjuvant. As shown in Figure 3, A and B
, CFA immunization led to intense expression of iNOS in draining lymph nodes and spleen whereas immunization with carbonyl iron (Figure 3, D and E)
showed much less staining for iNOS in these tissues. We also injected SCH-CFA or SCH-CI directly into the peritoneal cavity and exudate was harvested 3 days later. Cells from animals stimulated with CFA (Figure 3C)
exhibited much greater iNOS staining than cells from animals stimulated with CI (Figure 3F)
. Carbonyl iron particles can be seen in the latter photo (arrows). Thus, CFA drives the production of iNOS whereas carbonyl iron does not.
|
To further demonstrate the role for NO in resistance of the BN rat we increased NO production by immunizing with CFA at the same time as SCH-CI. BN rats were immunized with SCH-CI plus CFA in one hind footpad. Positive-control rats received only SCH-CI in one footpad and negative-control rats received only SCH-CFA in one footpad. The animals were observed for clinical disease and serum RNI levels were measured at a single time point (day 14 post-immunization). As shown in Table 3
, SCH-CFA-immunized rats did not develop clinical EAE whereas the SCH-CI animals did. As before, serum RNI levels were two to three times higher (32 to 46 µmol/L) in CFA-immunized rats than in carbonyl iron-immunized rats (11 to 14 µmol/L). Rats given both CFA and carbonyl iron had serum RNI levels similar to those given CFA alone, failed to develop clinical EAE, and showed only marginal weight loss. Similar results were obtained when the CFA was given in one hind footpad and SCH-carbonyl iron in the other (data not shown).
|
When immunized with SCH-CFA, only the Lewis rats developed clinical EAE and Figure 4A
shows a typical spinal cord meningeal lesion from a rat with clinical score of 3. Figure 4E
shows a similar area of spinal cord meninges of a BN rat immunized with SCH-CFA for comparison and demonstrates that there are no lesions in these clinically disease-free animals. SCH-CI induced EAE in both Lewis and BN rats as demonstrated above. At one and the same clinical score the extent and the intensity of the inflammatory lesions in the lumbar spinal cord were essentially the same in the Lewis rats immunized with SCH-CFA and SCH-CI and in the BN rats immunized with SCH-CI. The lesion load at three levels of the lower lumbar spinal cord in three animals of each of the above three groups was: ++ in the meninges; +++ in the white matter; + to ++ in the gray matter. There were no differences between the groups. Typical pictures after H&E staining are shown in Figure 4, B to D
-Lewis and 4, F to H-BN.
|
for various phenotypic cell markers and the striking result observed was with respect to ED-1+ cells. As shown in Figure 5
, a large proportion of the inflammatory cells seen in the meninges (Figure 5F)
, white matter (Figure 5G)
, and gray matter (Figure 5H)
of the BN rats are ED-1+ ie, macrophages/microglia. The inflammatory lesions in the Lewis rats, however, showed only minimal involvement of such cells (Figure 5, B to D)
. We made no attempt to quantify the proportion of ED-1+ cells but the areas shown are representative of random fields taken from three sections at three different levels of the spinal cord. Quite interestingly, despite this intense macrophage/microglial infiltration and activation in the BN rat there are few cells expressing iNOS. Figure 4
has also been stained immunohistochemically for iNOS; note the reddish brown staining indicated by arrows.
|
In an attempt to determine whether the BN rat was responding to immunization with SCH-carbonyl iron by developing a Th1 or Th2 response, we compared cytokine mRNA expression in draining lymph node cells from Lewis and BN rats after immunization with either SCH-CFA or SCH-carbonyl iron. At day 7 after immunization with either adjuvant, the draining lymph nodes of both Lewis and BN rats showed message for the five cytokines studied: IL2, IFN, TNF, IL4, and IL10 (Table 4)
. The Lewis rat showed higher expression of IL2 than the BN rat irrespective of the adjuvant used. Also with respect to the type of adjuvant, there was lower expression of IFN after immunization with SCH-carbonyl iron in both rat strains. The other difference in the cytokine profile in the draining lymph nodes was that the BN expressed higher levels of IL10 after immunization with SCH-CFA than after SCH-CI. The same differences seen at day 7 were also observed at day 12 (not shown). In the lumbar spinal cord of animals with clinical EAE at day 14, Lewis rats immunized with SCH-CFA expressed TNF, IFN, IL-4, and IL-10 whereas the BN rats, taken at the same time yet having no disease when immunized with SCH-CFA, had only background levels of these cytokines. Both Lewis and BN rats immunized with SCH-CI have the same profile as the Lewis with CFA except for a very low expression of IFN (data not shown).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
stimulation than do PVG splenocytes and the PVG splenocytes make more than Lewis splenocytes. This observation is in agreement with previous data for splenocytes incubated with ConA or IFN.28-30
In vivo, following immunization with neuroantigen and CFA, the BN and PVG rats show equal amounts of serum NO, both of which are three to four times higher than the Lewis, and the two former are resistant to EAE induction. We have previously shown11
that the PVG rat could be converted to high susceptibility by inhibiting NO production with the NOS inhibitor L-NMA, however, such treatment of the BN rat failed to reduce serum NO levels or render the rats EAE susceptible. It now appears that the BN rat may in fact produce significantly more NO than the PVG but the RNI are rapidly excreted in the urine. This is most likely a homeostatic mechanism to prevent the build up of toxic concentrations of nitrate and nitrite in the blood. This apparent increased production may account for our inability to pharmacologically reduce RNI levels in the BN rat sufficiently to convert them to susceptibility. Where the high levels of NO are produced is not known but we have shown here that, following immunization with CFA, the draining lymph nodes, spleen, and induced peritoneal exudate cells all express high levels of iNOS which are not present following CI immunization. A previous study from this lab showed that the increased serum NO level in the PVG compared to the Lewis rat when immunized with MBP-CFA was most likely produced by the spleen as splenectomy of the PVG rats lowered their NO levels following immunization and converted these relatively EAE-resistant rats to high susceptibility.14
The increased NO production by BN spleen cells in vitro suggests the spleen may be over-producing NO in vivo as well. Here we used an adjuvant, carbonyl iron, first described almost 30 years ago by Levine and Sowinski20 to induce EAE in the BN rat. We demonstrate that SCH-CI immunization does not induce an increase in NO production as SCH-CFA does and the BN rats subsequently develop severe disease. If CFA is administered simultaneously with SCH-CI, serum RNI levels increase significantly and the rats again become resistant to development of disease. Thus, lowering NO levels enhances disease while replacing increased NO levels again restores resistance, strongly suggesting that NO contributes significantly to the resistance of the BN rat to EAE.
A number of mechanisms have previously been proposed to explain the resistance of the BN rat to EAE. First, of course, is the demonstration that susceptibility or resistance is partially controlled by genes encoded by a region within the MHC locus. However, the fact that congenic strains with the Lewis MHC expressed on the BN background are still resistant to EAE indicates clearly that genes outside the MHC also play a significant role.18,19,21,31,32 Steroids have been examined as possible regulating molecules and indeed the BN rat has been shown to have increased basal levels of corticosterone compared to Lewis and other EAE susceptible strains.33 However, treatment of BN rats with the steroid antagonist RU486 (Mifepristone) failed to influence the resistance to EAE. Similarly, adrenalectomy before sensitization did not allow the development of clinical EAE.23 Furthermore, even though DA and BN rats both produce significantly more ACTH and corticosterone than the Lewis following environmental stress, the DA are highly susceptible to EAE while the BN rats remain resistant.34 The consensus would seem to be, therefore, that the hypothalamic-pituitary-adrenocortical axis characteristics of a rat strain are not a predictor of disease susceptibility.
Other studies suggest that the Th1/Th2 bias of the CD4+ T cell population in the various strains determines the type of autoimmunity that develops. Thus the Lewis mounts a Th1 response and is susceptible to EAE and autoimmune uveoretinitis, both Th1 mediated, while the BN is susceptible to mercuric chloride-induced glomerulopathy35
and gold salts autoimmunity,36
both Th2 responses. Cautain et al25
suggested that the natural resistance of the BN to EAE is associated with a defect in the ability to mount a Th1 response. Thus, BN lymph nodes produce more IL-4 than Lewis lymph nodes and the serum of BN rats have high amounts of TGFß following MBP immunization. Neutralization of TGFß rendered BN rats susceptible to EAE. These investigators examined the origin of the TGFß and excluded CD8 T cell involvement in its production. Interestingly, in another paper by the same group26
they report that the CD8 compartment of the BN rat plays a dominant role in their inability to mount a Th1 type response. In this latter in vitro study it was shown that the CD8 compartment of the BN rat was only one-third that of the Lewis rat and produced only 5% of the IFN produced by Lewis rats. This study also reported that even though BN T cells proliferate less than Lewis T cells to Con A, (Lew X BN) F1 T cells proliferated to Con A stimulation equally as well with either BN or Lewis splenocytes as APC, suggesting that the defect is in the T cell compartment and not the APC. Also in an APC-free stimulation culture using anti-TCR and anti-CD28, BN T cells proliferated slightly less than Lewis cells. This effect was, however, quite dose dependent in that when a lower dose of anti-CD28 was used proliferation of the two cell types was the same.
In the present work our attempt to determine the Th phenotype of the BN rat with EAE induced with SCH-CI was inconclusive. We did not do quantitative PCR and looked simply for large differences. What can be said is that, in general, the BN rat does make more IL-10 than the Lewis rat, which is in agreement with the work of others.37
Also in both the lymph nodes and spinal cord of the BN rat immunized with CI there was less of the Th1 cytokine IFN. This does not, however, necessarily suggest that CI is inducing a Th2 disease. Work from our lab38,39
and others40
has demonstrated in the mouse that even though EAE is a Th1-mediated disease, IFN is not essential for its expression and in fact appears to be essential for recovery from disease. If the mouse work can be extrapolated to the rat then the fact that CI-immunized BN rats responded with IL-2 and TNF production suggests a Th1 response.
Whether SCH-CI-induced EAE is a Th1 or Th2 response may in fact be a moot point. We do not doubt that the BN and the Lewis rat preferentially produce different cytokines on immunization with the same antigen. What we suggest, however, is that this Th bias may be due to a differential ability to produce NO. NO has been shown to influence immune reactivity in a number of ways. NO inhibits lymphocyte proliferation probably by preventing the activation of Janus kinases,41 molecules critical in cytokine receptor signal transduction.42 Other studies have described a NO-induced bias of cytokine gene expression in which NO preferentially down-regulates Th1-type cytokines specifically while up-regulating Th2-associated molecules, resulting in a Th2 bias.43 Such bias would prevent expansion of Th1 cells essential for EAE pathology. A similar regulatory effect might be exerted by NO at the level of antigen presentation as it has been shown that NO induces transcription of the IL-12 p40 gene in macrophages but not the p35 gene. Because the p40 homodimer acts as an antagonist for IL-12,44 this might lead to less Th1 reactivity in the presence of NO.
Yet another possible limiting effect of NO on immune reactivity is its ability to inhibit T cell migration. We have shown, at least in vitro, that NO inhibits migration of encephalitogenic T cells through CNS endothelial monolayers in a dose-dependent manner.45 This migration inhibition is thought to be due to NO’s ability to polarize T lymphocytes resulting in a morphology suggestive of migrating cells, ie, ones with a leading edge and trailing uropod. We suggested that these NO-induced morphological changes result in T cells with predefined migratory directionality, thus limiting the options these cells have to respond to other migratory signals. The genetic loci outside the MHC described as contributing to susceptibility to autoimmune disease may contain elements regulating iNOS gene expression.
Complete Freund’s adjuvant can be used in the BN rat to induce EAE if myelin oligodendroglial glycoprotein (MOG) is used as antigen.46 MOG-induced EAE requires both T cells and antibody for its full expression and the resultant CNS pathology in the BN rat is characterized by the deposition of C9 and IgG and a cellular infiltrate comprised of large numbers of neutrophils as well as eosinophils. Interestingly, in the MOG model, there were fewer macrophages in the infiltrates of the BN rat than the Lewis rat. These investigators concluded that the disease in the BN rat induced with MOG was independent of a substantial Th2 T cell response and was mediated by Th1 CD4+ T cells with clinical expression dependent on anti-MOG antibody.
The pathology seen in the BN rat immunized with carbonyl iron as adjuvant is of considerable interest. Both the Lewis and the BN rats immunized with CI had extensive inflammatory lesions in the meninges, white and gray matter which, based on H&E staining, were of equal intensity as well as similar distribution. The lesions were, however, qualitatively different between the strains. Thus, lesions in the BN rat had extensive involvement of ED-1+ cells whereas there was considerably less involvement of these cells in lesions seen in the Lewis rat. This was true in all three of each type of rat examined. The reason for this difference is not known. It should be remembered that the antigen used was whole spinal cord homogenate and not a defined antigen. The different strains of rat may be responding to different neuroantigens within the homogenate leading to different cellular responses. Conversely, the rats may be responding to the same antigen but with a different cellular response. ED-1 defines both peripheral macrophages and resident activated microglia and therefore either of these cells may be selectively up-regulated by the BN and not the Lewis. We are currently investigating whether the ED-1+ cells in the BN are predominately macrophages or microglia.
This difference in pathology between strains of rats in this study certainly has parallels with the human disease multiple sclerosis (MS). MS is extremely heterogeneous in its clinical presentation, response to therapy, radiological findings, structural pathology, and pathogenesis. There is no way of determining which patients will develop which type of disease. One form of disease is acute MS, or Marburg’s variant. It is a rare event but runs an exceptionally severe course, which typically ends in death within a year of diagnosis. Interestingly the pathology of Marburg’s is characterized by extensive macrophage infiltration rather than the more usual lymphocytic response seen in other forms of MS. Marburg’s may also be associated with an altered myelin basic protein structure and the deposition of immunoglobulins.47,48
Finally, another important question raised by this work is what is the mechanism of adjuvanticity of carbonyl iron? It was interesting to note that the footpads of CI-immunized rats showed virtually no inflammation or swelling when compared to immunization with CFA. Also the draining lymph nodes of the CI immunized rats had substantially fewer macrophages, as determined by ED-1 staining, yet the BN rat developed severe EAE with extensive macrophage infiltration into the CNS. The mechanism(s) by which carbonyl iron exerts its adjuvant activity is under investigation.
|
Acknowledgements |
|---|
primers, and Mark Fenning, Karen Edwards, and Julie Macklin from the photolab of JCSMR. |
Footnotes |
|---|
Supported by grants from Multiple Sclerosis Australia and the National Health and Medical Research Council (NH&MRC) of Australia as well as the Private Practice Fund of The Canberra Hospital. D.O.W. is a Fellow of the NH&MRC.
Accepted for publication August 9, 2004.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  K. Rejdak, A. Petzold, Z. Stelmasiak, and G. Giovannoni Cerebrospinal fluid brain specific proteins in relation to nitric oxide metabolites during relapse of multiple sclerosis Multiple Sclerosis, January 1, 2008; 14(1): 59 - 66. [Abstract] [PDF]
|
|
|
|
 |
|
 B. Zhu, Y. Bando, S. Xiao, K. Yang, A. C. Anderson, V. K. Kuchroo, and S. J. Khoury CD11b+Ly-6Chi Suppressive Monocytes in Experimental Autoimmune Encephalomyelitis J. Immunol., October 15, 2007; 179(8): 5228 - 5237. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. A. Dietlin, F. M. Hofman, B. T. Lund, W. Gilmore, S. A. Stohlman, and R. C. van der Veen Mycobacteria-induced Gr-1+ subsets from distinct myeloid lineages have opposite effects on T cell expansion J. Leukoc. Biol., May 1, 2007; 81(5): 1205 - 1212. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Giordano, D. M. Magaletti, and E. A. Clark Nitric oxide and cGMP protein kinase (cGK) regulate dendritic-cell migration toward the lymph-node-directing chemokine CCL19 Blood, February 15, 2006; 107(4): 1537 - 1545. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
