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*
The Picower Institute for Medical Research, Manhasset, NY 11030;
Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461;
The Kenneth S. Warren Laboratories, Tarrytown, NY 10591; and
§
Laboratory of Cellular Immunology, North Shore University Hospital, Manhasset, NY 11030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Proinflammatory cytokines and other macrophage products occupy a
pivotal role in the pathogenesis of MS and its respective animal model
EAE (4, 5, 6). For example, local TNF production within the CNS can lead
to increased blood-brain barrier leakage, induction of adhesion
molecules on the CNS vasculature (7), direct damage to myelin and
oligodendrocytes (8), and induction of other proinflammatory cytokines
(9, 10). Activated macrophages are a significant source of TNF in
neuroinflammatory disorders, but infiltrating lymphocytes (11, 12) as
well as resident astrocytes (13), microglia (14, 15), and smooth muscle
cells may also contribute. In EAE, depletion of macrophages immediately
before the onset of clinical signs has been shown to significantly
reduce disease expression, supporting a key role for these cells in
disease pathogenesis (16, 17, 18). Macrophage activation is thought to be
mediated by Th1-type cytokines such as IFN-
, resulting in the
release of a range of proinflammatory cytokines and chemokines, and
other toxic factors such as the free radical nitric oxide (NO), which
contribute significantly to the expansion of the inflammatory response
and associated tissue damage (19). The central role of TNF in EAE
pathogenesis has been established using anti-TNF antisera (20, 21),
soluble TNF receptors (22, 23), and other natural and pharmacologic TNF
inhibitors. Similarly, TNF has been identified in MS lesions (24, 25),
while cerebrospinal fluid TNF levels predict MS progression (26).
Moreover, T cells isolated from cerebrospinal fluid of MS patients with
active disease have a greater capacity to secrete TNF compared with
controls (27, 28). Therefore, modulation of macrophage proinflammatory
activities offers a potential target for chemotherapeutic intervention,
and suppression and/or inhibition of TNF production has become a
central strategy in the design of novel selective approaches to the
treatment of MS.
We recently developed a family of experimental therapeutics designed as
inhibitors of cytokine-indubible macrophage arginine uptake (and
subsequent NO synthesis). One tetravalent guanylhydrazone member of
this class,
(N,N'-bis{3,5-bis[1-[(aminoiminomethyl)hydrazono]ethyl}phenyl]
decanediamide tetrahydrochloride) (CNI-1493) was found to reversibly
inhibit the phosphorylation of p38 mitogen-activated protein kinase,
which plays a key role in posttranscriptional regulation of
proinflammatory cytokine synthesis (29). When given at an estimated
therapeutic to toxic ratio of 40:1, CNI-1493 suppresses LPS-stimulated
production of macrophage TNF, IL-1, IL-6, MIP-1
, MIP-1ß, and NO
production. In contrast, no effect is found on the constitutive
synthesis of TGF-ß or MHC class II up-regulation by IFN- (30).
Furthermore, CNI-1493 retains its cytokine-suppressive activities even
in the presence of IFN-, in contrast to the immunosuppressive
activities of glucocorticoids such as dexamethasone (30). A beneficial
effect of CNI-1493 has been noted in several models of
macrophage-mediated toxicity including: reduced edema formation in
carrageenan-induced inflammation in the footpad; protection against the
toxic effects of LPS; a reduction in infarct volume in cerebral
ischemia; protection against adult respiratory distress syndrome by
cecal ligation and puncture; reduction in clinical signs in adjuvant
arthritis; and a prolongation of cardiac allograft survival (reviewed
in 31 . The efficacy of this compound in protecting against
diseases of macrophage-derived toxicity, combined with its low toxicity
and short elimination half-life, makes it an attractive new therapeutic
compound.
In this study we have addressed the potential efficacy of this potent inhibitor of macrophage activation in an inflammatory condition of the CNS in which proinflammatory cytokines have been shown to play a central role. The model system that we have chosen is EAE in the SJL/J mouse induced by the passive transfer of T cells sensitized to MBP. We show that i.p. administration of CNI-1493 successfully prevents clinical manifestations in mild EAE and significantly reduces severe EAE. The clinical improvement was accompanied by a significant decrease in related histopathology and a decrease in proinflammatory cytokine expression at the lesion site. In addition, treatment with CNI-1493 was found to significantly inhibit the chronic phase of EAE. Finally, we show that CNI-1493 has no effect on T cell proliferation at the therapeutic concentrations used in this study.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female SJL/J (H-2s) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and used between the ages of 6 and 8 wk. Mice were housed and maintained in a federally approved animal facility at the Albert Einstein College of Medicine (Bronx, NY) in accordance with National Institutes of Health guidelines. For induction of passive transfer EAE, donor mice were immunized s.c. with 0.5 mg MBP (Sigma Chemical, St. Louis, MO) emulsified with an equal volume of CFA (Difco Laboratories, Detroit, MI). Ten days later, the draining axial, brachial, and inguinal lymph nodes were removed and homogenized into a single cell suspension. Lymph node cells were washed and activated with 50 µg/ml MBP in RPMI 1640 supplemented with 10% heat-inactivated FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), glutamine (2 mM), 2-ME (50 µM), sodium pyruvate (1 mM), HEPES buffer (0.1 M), and nonessential amino acids (0.1 mM) (Sigma Chemicals, St. Louis, MO) at a density of 4 x 106 cells/ml in 24-well plates (2 ml/well) for 4 days at 37°C in humidified 8% CO2-air. For adoptive transfer, naive syngeneic animals were injected in the lateral tail vein with 5 x 107 to 1 x 108 MBP-sensitized lymph node cells in 0.2 ml. The lower number of cells resulted in a mild disease course, while the higher number resulted in the severe form of the disease. Before transfer, cells were washed extensively in HBSS. To obtain a clinical index (CI), mice were scored on a scale of 0 to 5 according to standard procedures as follows: 0, no clinical disease; 1, limp/flaccid tail; 2, hind limb weakness (ataxia) with incomplete paralysis; 3, complete paralysis of one or two hind limbs; 4, forelimb weakness or partial paralysis; 5, moribund. Intermediate scores were assigned if neurologic signs were of lower severity than typically observed. Onset of disease was determined by loss of tail tone. Recovery was defined as a clear clinical improvement of at least one grade following a paralytic incident. Relapses were defined as worsening of the clinical index by 1.0 grade for two consecutive days.
CNI-1493 and treatment protocol
CNI-1493 (Cas. Reg. No. 164301-51-3) was synthesized and purified as previously described (32). The purity was >99% as estimated by melting point, nuclear magnetic resonance, elution from HPLC, and elemental analysis. For each experiment, CNI-1493 was freshly prepared as a 1 mM working stock solution in sterile, endotoxin-free distilled water, and diluted to the desired concentration in sterile saline. Drug or vehicle was administered daily by the i.p. route at a dose of 1 or 5 mg/kg in a volume of 100 µl of sterile saline.
The statistical significance of differences in clinical index between groups was analyzed using Student’s two-tailed t test. Significance was accepted if p < 0.05.
Histopathology
Animals were anesthetized by ether inhalation and perfused intracardially with 20 ml ice-cold PBS. Sections of the brain and lumbar spinal cord were removed and either flash frozen in liquid nitrogen and stored at -80°C, or immersion-fixed in Trump’s fixative (4% paraformaldehyde, 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). For histopathologic analysis, fixed tissue was dehydrated through a graded series of ethanol, cleared in propylene oxide, and embedded in Epon 812 (EM Sciences, Fort Washington, PA). For each animal, four to six 1-µm epoxy sections were stained with 1% toluidine blue and examined by light microscopy. Coded slides were scored according to density of inflammatory cells in perivascular CNS compartments using the following scale: 1, perivascular inflammation of less than three cells thick; 2, perivascular inflammation of more than 3 cells thick; 3, parenchymal inflammation. The histopathologic score was then calculated for each animal by adding all the scores for the individual.
RNase protection assay
Three animals per group were sensitized as described above,
killed, and perfused intracardially with saline. The lumbar spinal cord
was dissected out and snap-frozen in liquid nitrogen. RNA was prepared
from the samples using Tri-Reagent per manufacturer’s instructions
(Molecular Research Center, Cincinnati, OH). Twenty micrograms of RNA
was subjected to RPA using Riboquant MCK-2 probe set (PharMingen, San
Diego, CA). The assay was performed using the Ambion RPA II kit
essentially as described by the manufacturer. Briefly, 50 ng of the
MCK-1 probe mixture was used as the template for transcription of
antisense RNA probes using T7 RNA polymerase and
[-32P]UTP. Labeling was quantitated by liquid
scintillation counting, and labeling efficiency was typically greater
than 90%. Twenty micrograms of total RNA were hybridized to 3.5
x 106 cpm of probe overnight at 45°C. Unhybridized probe
was digested with RNase A and T1, and hybrids were precipitated.
Protected fragments were resolved on a 5% polyacrylamide/8 M urea gel.
The gel was fixed, dried, and bands were visualized by autoradiography
on Fuji film RX. Messenger RNA levels for each of the cytokines was
quantitated by densitometry of autoradiographs using the NIH-Image
software package. Sample loading was normalized by comparison of L32
and glyceraldehyde-phosphate dehydrogenase mRNA levels.
T cell proliferation assays
Anti-CD3-mediated proliferation. Proliferative responses were assessed with T cells purified from naive spleens using high affinity negative selection mouse T cell enrichment columns (R&D Systems, Cambridge, MA). Ninety-six-well plates were coated for 3 h at 37°C in a humidified chamber with hamster anti-murine CD3 (PharMingen, San Diego, CA) at 2 µg/ml. Plates were then washed three times with water. T lymphocytes were added at a density of 0.5 to 5 x 106 cells/well in the presence or absence of CNI-1493 at various dilutions in 0.2 ml final volume. Plates were then incubated for 2 days at 37°C in a CO2 incubator and 8 h before harvest pulsed with 1 µCi of [3H]TLR (NEN-Dupont, Boston, MA) per well. Cells were harvested and incorporated radioactivity was quantified on a Top Count Microplate Scintillation Counter (Packard Instrument, Meriden, CT).
Ag-specific proliferation. BR6.9 is an I-Ab-restricted CD4 T cell clone responsive to the 146–162 peptide of the nicotinic acetyl choline receptor derived from C57BL/6 (B6) mice and was obtained from Dr. Anthony J. Infante of the University of Texas Southwestern Medical Center, San Antonio, TX. Nicotinic acetyl choline receptor peptide (1 µg/ml) was added to 2.5 x 104 responder cells and coincubated with 2.5 x 105 APCs (mitomycin C-treated syngeneic spleen cells) for 3 days. [3H]Thymidine (1 µCi) was added during the last 6 h of culture, and cells were harvested onto glass fiber mats and counted for radioactivity in a Betaplate counter.
Superantigen-specific proliferation. BR6.9 cells express Vß6 and are thus responsive to the endogenous murine mammary tumor retroviral superantigen Mls1a expressed by cells from DBA12 mice. To determine whether CNI-1493 affects superantigen responses, 2.5 x 104 BR6.9 cells were coincubated with 2.5 x 105 mitomycin C-treated DBA12 spleen cells for 3 days in the presence or absence of CNI-1493. [3H]thymidine (1 µCi) was added during the last 6 h of culture, cells were harvested onto glass fiber mats and counted for radioactivity in a Betaplate counter.
TNF ELISA. Immunlon II (Dynatech, Chantilly, VA) ELISA plates were coated with 5 µg/ml hamster anti-murine TNF mAb (Genzyme Diagnostics, Cambridge, MA). The plates were blocked with Superblock blocking buffer (Pierce, Rockland, IL) and washed in Tris-buffered saline (TBS)/0.05% Tween-20. Culture supernatants and TNF standards (rTNF, R&D Systems, Minneapolis, MN) were diluted in TBS/0.2% Tween-20 and incubated overnight at 4°C. After washing, rabbit anti-murine TNF antiserum (BAS 16.248) was added at a dilution of 1:800 using 1x TBS, 0.2% Tween-20, and 1% goat serum, and incubated for 2 h at room temperature. Alkaline phosphatase-conjugated goat anti-rabbit IgG (Boehringer Mannheim, Indianapolis, IN) was added at a 1:3000 dilution after washing the plate in TBS/0.05% Tween-20. After a 1-h incubation, the substrate (0.5 mg/ml of p-nitrophenyl-phosphate (pNPP) in 10 mM diethanolamine/0.5 mM MgCl2, pH 9.5) was added to the wells and incubated in the dark for 30 min at room temperature. After development, the plate was then read at 405 nm using a Dynatech ELISA reader. Unknown samples were compared against a standard curve between 1,000 and 60,000 pg/ml. The data were plotted with the Biolynx program (Dynatech) using a sigmoidal, log/linear scales with tails curve fitting.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals sensitized by the passive transfer of MBP-reactive T cells develop a relapsing-remitting disease course that is characterized clinically by an ascending paralysis and pathologically by focal areas of submeningeal and perivascular inflammation and demyelination, localized preferentially to the spinal cord. The acute phase of the disease usually starts 5 to 7 days posttransfer (dpt), peaks approximately 11 to 12 dpt, and is followed by a period of remission lasting 10 to 12 days. Most animals then relapse with peak clinical signs occurring approximately 28 to 40 dpt. The pattern of subsequent relapses and remissions is variable and is linked to the severity of early events in the disease process, with more severe episodes of the acute disease associated with more frequent relapses and a gradual progression into a chronic disease state.
To determine the efficacy of CNI-1493 to suppress disease, we initially
tested its ability to suppress mild EAE. Sensitized animals were
divided into coded groups and injected i.p. on a daily basis with 5
mg/kg CNI-1493 or vehicle alone commencing immediately before, and
continuing through, the peak of the disease. Clinical expression of
disease was assessed on a daily basis through day 40. Control animals
treated with saline developed a typical disease course (Fig. 1
A). In two separate
experiments, CNI-1493 treatment at 5 mg/kg from days 4 to 10 completely
suppressed both the acute and chronic phases of the disease. In the
representative experiment illustrated (Fig. 1A), the mean
clinical index (MCI) at peak of disease (9 dpt) was 1.6 ± 0.5 in
untreated and 0.0 ± 0.0 for the treated group (mean ± SD,
n = 5 per treatment group).
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Having ascertained that the compound inhibited mild EAE, we then
tested its effect on a more severe form of the disease. Animals were
again divided into coded groups and injected daily i.p. with two doses
(5 and 1 mg/kg) throughout the course of the acute phase (12 days). The
results of one representative experiment are shown in Figure 1
B. In animals treated with either saline or 1 mg/kg
CNI-1493 a typical disease course was observed with severe paralysis
noted during the acute phase of the disease (days 7–9), which was
followed by a remission (days 14–23) and subsequent relapse (day 25
on). In contrast, animals treated with 5 mg/kg CNI-1493 showed only
mild clinical signs during the acute phase of the response that was
followed by a delayed and more attenuated chronic phase of the disease
(Fig. 1B). The MCI at the peak of disease (7 dpt) was
4.3 ± 0.7 in controls animals as compared with 0.5 ± 0.3
for treated animals (mean ± SD, n = 4,
p < 0.05). This dose is well below the
LD50 of 90 mg/kg i.p. in mice (33). The onset of the
relapse, defined as an increase in MCI of 0.5 grade for more than 2
days, was delayed in animals receiving the 5 mg/kg dose as compared
with animals injected with vehicle alone (24–26 dpt vs 27–29 dpt,
p < 0.05). Similar results were obtained with a
treatment course of 6 days overlapping the onset of disease. Five
animals per group were treated for 6 days after the first sign of
clinical illness, as described above. The untreated group had a MCI at
peak disease of 3.5 ± 0.0, while the treated group had a MCI of
0.2 ± 0.2.
Effect of the treatment regimen on chronic-relapsing EAE
We then examined the effect of treatment with CNI-1493 at
different stages of the acute clinical episode (Table I). When given before disease onset
(defined as the first day of clinical signs) no clinical efficacy was
noted (Table I, Expt. 1). Similarly, when given after peak expression
of disease (defined as the height of clinical signs), no effect was
noted on either the clinical expression of the disease or on the
kinetics of the recovery process (Table I, Expt. 2). Interestingly,
however, when a single dose was given at the onset of disease, a 50%
decrease in clinical disease was noted (Table I, Expt. 3).
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Having ascertained that CNI-1493 suppressed the acute phase EAE,
we then tested the effect of drug treatment on the chronic phase of the
disease. EAE was induced by adoptive transfer as before and the animals
monitored for clinical signs. Following the acute phase of the disease,
the animals went into remission followed by a relapse. At the time of
relapse, 10 animals were paired by clinical index, coded, and one
animal from each pair was given 5 mg/kg/day for 6 days (25–31 dpt) and
the other animal vehicle alone. The cumulative data, with the MCI
expressed as a percentage of the control animal, for all pairs is shown
in Figure 2A. Representative
pairs from this experiment are shown in Figure 2B. As shown
in Figure 2, animals that received CNI-1493 had a significantly
improved disease course compared with those that received saline alone.
Figure 2 also shows that animals continued to improve clinically for an
extended period of time (25 days) after treatment was terminated.
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Histopathology of mice treated with CNI-1493
To determine the effect of CNI-1493 on pathologic expression of
EAE during the acute phase of the disease, two additional experiments
were performed for histopathologic analysis. In untreated,
control animals (CI = 3.5), focal submeningeal areas of
perivascular mononuclear cell inflammation and demyelination in
white matter were detected in the lumbar and cervical regions of the
cord. Edema, as determined by evidence of perivascular accumulations of
proteinaceous exudate, was also present particularly in association
with vessels in the anterior fissure. In contrast, in animals treated
with CNI-1493 (CI = 0.5), no demyelination was detected and only
minimal evidence of perivascular inflammation and edema formation was
observed. A histologic index determined from coded slides gave a value
of +3.0 (n = 3) for the control animals and +0.5
(n = 3) for the CNI-1493-treated animals.
Representative pathology from these animals is shown in Figure 3.
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To determine the effect of CNI-1493 on the inflammatory cytokine
expression in EAE lesions during the acute phase of the disease, two
additional experiments were performed for analysis of levels of
proinflammatory cytokine mRNA by RNAse protection assay. In untreated,
control animals, high levels of mRNA of the proinflammatory cytokines
IL-1, IL-6, IL-2, and IFN- were detected at the lesion site. Animals
that received CNI-1493 had reduced levels of proinflammatory cytokine
levels at the lumbar spinal cord (Fig. 4). RNA levels for IL-1, IL-2, and
IFN- were reduced 
50%, while IL-6 message was reduced by
about 75%.
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Since EAE is known to be caused by neuroantigenic-specific
CD4+ T cells, we sought to determine whether CNI-1493 had
any effect on T cell proliferative responses. In vivo pharmacologic
studies have determined that the maximal possible blood levels of
CNI-1493 after i.p. injection of 5 mg/kg in mice is 0.025 µg,
corresponding to a concentration of 30 nM (33). T cell proliferation
was induced by cross-linking of CD3 in the presence of various
concentrations of CNI-1493. As can be seen in Figure 5A, CNI-1493 concentrations
more than 10-fold higher than 30 nM had no effect on T cell
proliferation. We then tested the effects of CNI-1493 on specific T
cell responses to neuroantigen (nicotinic acetyl choline receptor,
NACR) and superantigen (murine mammary tumor retroviral superantigen,
Mls1a). Again, concentrations more than 10-fold higher than
30 nM had no effect on specific T cell proliferation to NACR peptide
(Fig. 5B) or to superantigen (Fig. 5C). Finally,
no suppression of TNF responses by T cells was observed in any of these
experiments (Fig. 5D). Similar results were obtained using
MBP as the antigenic stimulus and T cells derived from SJL mice (data
not shown). Therefore, our results suggest little, if any, effect of
CNI-1493 on infiltrating T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EAE can be successfully treated by a variety of immunotherapies directed at proinflammatory cytokine production. These include procedures that result in the down-regulation of proinflammatory cytokines TNF, lymphotoxin, and IL-1, as well as those that inhibit macrophage migration and activation (16, 17, 18, 20, 21, 22, 23). The most extensive and successful strategies have examined the role of the TNF family of proteins. In initial studies, a central role for TNF was demonstrated by the ameliorating effect of administering Abs to TNF or the soluble TNF receptor (21, 23). These promising results then prompted further investigations of pharmacologic TNF inhibitors. Several of these have shown efficacy in EAE, including matrix metalloprotease inhibitors that block processing of TNF; Rolipram, a selective inhibitor of phosphodiesterase type IV; other phosphodiesterase type IV inhibitors such as pentoxifylline, which inhibit TNF production by activated macrophages; thalidomide, which inhibits the stability of TNF mRNA without affecting the stability of mRNA for other cytokines, and phosphatidyl serine, a phospholipid of the cell membrane, which has been found to inhibit LPS-induced TNF production in vitro (reviewed in 34 . Pathologic analyses of the results obtained with these various anti-TNF treatment modalities suggest that these procedures principally interfere with the homing of inflammatory cells to the CNS and affect the development of EAE at a step subsequent to the generation of autoimmune cells, a situation relevant to MS where the putative autoimmune reactive cells have been generated before clinical presentation. However, although extremely effective in animal models, these specific immunotherapies are less likely to work in human disease in which the pathogenic mechanisms involved are expected to be more complex and to vary at different stages of the disease process.
The precise mechanism involved in suppression of EAE by CNI-1493 is not
known. Results presented here demonstrate a lack of inhibitory activity
of CNI-1493 on T cell proliferation in response to the CNS-specific Ags
PLP and MBP. A more extensive study of the effects of CNI-1493 on T
cells and macrophages has recently been published by Bjork et al. (35).
They quantitated proinflammatory cytokine synthesis at the single cell
level (using computerized image analysis) following different routes of
cell activation. Their results clearly demonstrated that the production
of IL-2, IFN, TNF-, and TNF-ß by activated T cells (via CD3 and
CD28 ligation or PMA/ionomycin treatment) was not affected by CNI-1493
treatment (35). On the other hand, similar treatment resulted in a
profound inhibition of LPS-induced TNF-, IL-1, IL-1ß, IL-6, and
IL-8 production by macrophages, independently of IFN- priming. These
results correspond well with previous published reports in which
CNI-1493 has been shown to inhibit the release from activated
macrophages of the cytokines TNF and IL-1, the chemokines MIP-1 and
MIP-1ß, as well as NO produced via the inducible form of nitric oxide
synthase (iNOS) (30). Each of these factors has been implicated in the
pathogenesis of EAE (11). Elevated levels of TNF, IL-1, MIP-1,
MIP-1ß and iNOS have been clearly demonstrated in affected CNS
tissues, with levels correlating with the kinetics and severity of
disease expression. Inhibition of TNF, IL-1, MIP-1, and iNOS have
been found to block disease expression. Peroxynitrates formed as a
major consequence of NO production via iNOS have been implicated in the
pathogenesis of EAE (36). Thus, CNI-1493 provides a potentially wider
range of inhibitory activities against these inflammatory mediators
than the specific inhibitors described above.
Since CNI-1493 does not affect T cell proliferation or proinflammatory
cytokine production, but profoundly inhibits the production of
potentially pathogenic macrophage cytokines, we hypothesize that the
mechanism of suppression of EAE by this drug is likely to involve
macrophage deactivation. We found a decrease in the expression of the
proinflammatory cytokines IL-6, IFN-, IL-1, and IL-2 at the lesion
site. We interpreted the decrease of T cell-derived cytokines (IL-2 and
IFN-) as an indirect effect due to macrophage deactivation and
subsequent generalized decrease in the proinflammatory status of the
lesion area, which is supported by the histologic data. Of particular
note, however, is the observation that treatment of activated
macrophages with CNI-1493 has not been shown to affect the production
of TGF-ß, a cytokine with known regulatory activity. Natural recovery
from the acute clinical episode of EAE has been associated with the
production of TGF-ß by spleen cells, and administration of this
cytokine to animals sensitized to develop EAE blocks progression of
disease (34). Furthermore, enhanced TGF-ß secretion has been proposed
to mediate the immunosuppression observed following the induction of
oral tolerance (37).
In addition to its effect in the acute clinical episode, treatment with CNI-1493 was found to significantly ameliorate the chronic phase of the disease. Although it is widely assumed that the mechanisms involved in the induction of a relapse resemble those found during the acute phase of the disease, this has by no means been formally tested (38, 39). Most of the studies that have addressed mechanisms of disease pathogenesis, as well as those that have tested various therapeutic regimes, have focused principally on the acute clinical episode in the mouse model or have used the monophasic disease that develops in the Lewis rat. The striking ability of CNI-1493 to inhibit chronic disease, even after the cessation of treatment, is in contrast to data obtained with either Abs to TNF or integrins, or with the soluble TNF receptor, where EAE usually develops rapidly following cessation of treatment (21, 22, 40). Since the in vivo anti-inflammatory effects of CNI-1493 are relatively short lived, these data suggest that the long-term beneficial effects of CNI-1493 result from the lack of effect on TGF-ß that could result in a shift in the cytokine profile of the autoreactive cells from one dominated by proinflammatory cytokines to one in which a Th2 or Th3 profile dominates. If this is indeed the case, then CNI-1493 may, like other drugs such as retinoids and phosphodiesterase inhibitors, exert part of its effect by altering the balance between Th1 and Th2 type responses and further studies are planned to specifically address this issue.
In conclusion, we have documented the efficacy of a new type of pharmacologic agent, a multivalent guanylhydrazone compound, in an inflammatory disease of the CNS. This compound has many potential benefits for diseases of a chronic inflammatory nature; it has broad inhibitory activity for proinflammatory factors released from activated macrophages while leaving immunoregulatory cytokines unaffected; the anti-inflammatory activities of CNI-1493 are short lived and thus should not result in long-term immunosuppression leaving the patient vulnerable to intercurrent infections and, like other successful pharmacologic agents, has a lower cost, higher specificity, and known biophysical characteristics compared with biologic immunotherapies. Finally, this compound provides a useful tool to study the effects of simultaneously removing multiple cytokines from an inflammatory disease model. In summary, these studies identify a prototype compound that should lead to the development of novel macrophage-modulating anti-inflammatory therapies that could have significant benefits for many diseases of a chronic inflammatory nature.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James A. Martiney, The Kenneth S. Warren Laboratories, 765 Old Saw Mill Road, Tarrytown, NY 10591. E-mail address:
3 Current address: Fish & Richardson P.C., 45 Rockefeller Plaza, Suite 2800, New York, NY 10111.
4 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; MBP, myelin basic protein; CNS, central nervous system; NO, nitric oxide; CI, clinical index; dpt, days post-transfer; MCI, mean clinical index; MIP-1, -1ß, macrophage inflammatory protein-1 , ß; iNOS, inducible nitric oxide synthase; NACR, nicotinic acetyl choline receptor; RPA, RNase protection assay.
Received for publication July 1, 1997. Accepted for publication January 30, 1998.
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N. Kawakami, S. Lassmann, Z. Li, F. Odoardi, T. Ritter, T. Ziemssen, W. E.F. Klinkert, J. W. Ellwart, M. Bradl, K. Krivacic, et al. The Activation Status of Neuroantigen-specific T Cells in the Target Organ Determines the Clinical Outcome of Autoimmune Encephalomyelitis J. Exp. Med., January 20, 2004; 199(2): 185 - 197. [Abstract] [Full Text] [PDF]
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 A. C. La Flamme, K. Ruddenklau, and B. T. Backstrom Schistosomiasis Decreases Central Nervous System Inflammation and Alters the Progression of Experimental Autoimmune Encephalomyelitis Infect. Immun., September 1, 2003; 71(9): 4996 - 5004. [Abstract] [Full Text] [PDF]
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 A. A. Babcock, W. A. Kuziel, S. Rivest, and T. Owens Chemokine Expression by Glial Cells Directs Leukocytes to Sites of Axonal Injury in the CNS J. Neurosci., August 27, 2003; 23(21): 7922 - 7930. [Abstract] [Full Text] [PDF]
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T. R. Bernik, S. G. Friedman, M. Ochani, R. DiRaimo, L. Ulloa, H. Yang, S. Sudan, C. J. Czura, S. M. Ivanova, and K. J. Tracey Pharmacological Stimulation of the Cholinergic Antiinflammatory Pathway J. Exp. Med., March 18, 2002; 195(6): 781 - 788. [Abstract] [Full Text] [PDF]
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J. L. McQualter, R. Darwiche, C. Ewing, M. Onuki, T. W. Kay, J. A. Hamilton, H. H. Reid, and C. C.A. Bernard Granulocyte Macrophage Colony-Stimulating Factor: A New Putative Therapeutic Target in Multiple Sclerosis J. Exp. Med., October 1, 2001; 194(7): 873 - 882. [Abstract] [Full Text] [PDF]
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A. E. Juedes and N. H. Ruddle Resident and Infiltrating Central Nervous System APCs Regulate the Emergence and Resolution of Experimental Autoimmune Encephalomyelitis J. Immunol., April 15, 2001; 166(8): 5168 - 5175. [Abstract] [Full Text] [PDF]
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 M. B. Atkins, B. Redman, J. Mier, J. Gollob, J. Weber, J. Sosman, B. L MacPherson, and T. Plasse A Phase I Study of CNI-1493, an Inhibitor of Cytokine Release, in Combination with High-Dose Interleukin-2 in Patients with Renal Cancer and Melanoma Clin. Cancer Res., March 1, 2001; 7(3): 486 - 492. [Abstract] [Full Text]
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A. E. Juedes, P. Hjelmstrom, C. M. Bergman, A. L. Neild, and N. H. Ruddle Kinetics and Cellular Origin of Cytokines in the Central Nervous System: Insight into Mechanisms of Myelin Oligodendrocyte Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis J. Immunol., January 1, 2000; 164(1): 419 - 426. [Abstract] [Full Text] [PDF]
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 S. Xue, N. Sun, N. Van Rooijen, and S. Perlman Depletion of Blood-Borne Macrophages Does Not Reduce Demyelination in Mice Infected with a Neurotropic Coronavirus J. Virol., August 1, 1999; 73(8): 6327 - 6334. [Abstract] [Full Text]
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