By
From the Department of Neurological Sciences, Beckman Center for Molecular and Genetic Medicine, Stanford University, Stanford, California 94305; and Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
Experimental autoimmune encephalomyelitis (EAE) has
served as a prototypic model of T cell-mediated, organspecific autoimmune disease, and as a useful model for the
human disease, multiple sclerosis (MS) (1). Frei et al. (2)
demonstrate that in two strains of mice with a double
knockout, where both TNF- Contradiction: TNF- There is abundant evidence that TNF- Reconciliation of the Data On the Role of TNF Cytokines may exert several, even opposing effects at
distinct points, both anatomically or temporally during an
immune response. Produced and released naturally during
an immune response, cytokines will be regulated, and their
action will usually only target cells at the site of production.
Conversely, systemic administration of cytokines inevitably
cannot be under such control, and thus adverse rather than
physiologic effects may ensue. Thus, cytokines often produce paradoxical effects when delivered systemically, rather
than via effector cells at the site of pathology in situ: TNF- There are numerous effector molecules in EAE. It is
clear that IL-6 (27), nitric oxide (28, 29), and TNF- The remarkable redundancy in cytokine function is elegantly demonstrated in mice with a transgene for a TCR
for myelin basic protein and an inactivated RAG-1 gene.
In these mice EAE still develops without Th1 T cells. Examination of their brains reveals IL-4 production without
evidence for TNF- Given these intricacies and redundancies in cytokine
pathways, it may not be accurate to claim that TNF- There are major abnormalities in many of the new strains
of mice with disrupted (knocked out) genes using the contemporary technology. Often, the genes under study are
inactivated during the entire life of the organism, and are
inactivated throughout the organism. In these TNF- Perhaps the next generation of knockout animals with
spatial and temporal control of the inactivated gene will be
more useful (32). Wilson and Tonegawa write: "The major
drawback of the current gene-knockout technology, as applied to the brain, is the lack of regional and temporal specificity." (32). Identical problems also confound studies on
immune function with the current knockouts for cytokine
genes, now being used. Work with the current generation
of knockouts in EAE show that disease develops well when
gamma interferon is inactivated (33), when IL-4 is inactivated (34), and when TNF- and LT-
are inactivated,
that EAE may develop. The disease in these double knockout mice progresses in an apparently typical fashion, concordant with what is observed in the usual inbred strains of mice where EAE is induced: there is clinical paralysis, and
histopathology reveals intense perivascular and parenchymal infiltration with CD4+ T cells and demyelination.
They conclude, and I agree, that the results are surprising,
given the large body of information suggesting that TNF-
and LT-
are important in the pathogenesis of EAE and
MS. However, before accepting their ultimate conclusion that, "these results indicate that TNF
and LT-
are not essential for the development of EAE," it is worthwhile to
consider the limitations of the first-generation knockouts
that have been employed in their study. Certain misconceptions have arisen concerning the interpretation of experiments with these contemporary knockouts. This is especially true when trying to understand the role of critical
effector molecules like cytokines, in the development of
complex phenotypes, like the paralysis and inflammation
seen in EAE. Many of these cytokine molecules have diverse biological activities, and many of the functions of
these molecules can be duplicated by other cytokines.
Thus, in animals with disrupted or "knocked out" cytokine
genes, one may expect many diverse changes in several
physiological processes, and one might find that after all is
done, that another gene and its product can replace the function of the gene that was disrupted.
and LT-
Are Critical in
the Development of EAE, Yet Disease Occurs in the
TNF-
-LT-
Double Knockout
and LT-
are
critical in the development of EAE, and in the human disease, MS (3). Both TNF-
and LT-
mRNA and protein are in the central nervous system in acute EAE (3).
T cell clones, reactive to myelin basic protein, are more capable of mediating EAE, when they produce higher amounts
of TNF-
and LT-
(7). Blockade of clinical paralysis in
EAE has been successful with anti-TNF antibodies (8, 9) or
soluble TNF type I receptors (10, 11). Reversal of EAE is
seen with altered peptide ligands of myelin basic protein that reduce production of TNF-
(12, 13). Reduction of
TNF-
with type I phosphodiesterase inhibitors like the
antidepressant, Rolipram, also leads to the reversal of EAE
(14, 15). Relapsing attacks of paralysis in EAE, which can
be induced with superantigens, are blocked with anti-TNF
(16). TNF is produced in high amounts by glial cells in
strains that are susceptible to EAE, but not in resistant
strains (17). Demyelination is mediated in vitro in oligodendroglial cultures by TNF-
and LT-
(18). Overexpression of TNF-
in the central nervous system leads to
demyelination (19). This experiment in a transgenic mouse
with the TNF-
transgene expressed in the central nervous
system, stands in contrast to the double knockout mice
used here, where the LT-
and TNF-
genes are disrupted
throughout the animal, not only in the central nervous system. Injection of TNF-
can trigger relapses of EAE (20, 21). All these experiments in EAE, reinforce the findings
indicating that TNF-
and LT-
play a pathogenic role in
MS: TNF-
and LT-
are found in demyelinating lesions
in the brains of MS patients, and increases in TNF can be
seen in the spinal fluid before relapses (22).
in
Demyelinating Disease
may inhibit demyelinating disease, for instance, in Theiler's virus induced demyelination (23). Likewise in EAE, delivery of TNF-
by a recombinant vaccinia virus inhibited
EAE (24). Similarly with Th2 cytokines, systemic delivery
of IL-4 worsens EAE (25), while local delivery of IL-4 via
T cell clones ameliorates disease (26).
may
all play a role in the immunopathology of EAE and MS.
Given the redundant function of these molecules, it is not
surprising that inactivating one or more of them, may still
not influence a change in phenotype.
. Thus even without Th1 cytokines,
EAE can still develop with the appearance of Th2 cytokines in active lesions (Lafaille J, J. Van de Keere, J. Baron, W. Haas, and S. Tonegawa, manuscript submitted for publication).
and
LT-
are not essential for EAE in normal animals. As Frei
and colleagues write, "Alternatively, in their absence other
cytokines may compensate for the defect." (27). I would
support this interpretation, and I would inquire about what
other cytokines were expressed in these inflamed brains.
Were other Th1 cytokines expressed, or were Th2 cytokines
present? Indeed gamma interferon transcripts were found in
the brains of animals showing EAE with TNF-
and LT-
inactivated. Paradoxically perhaps in inbred mice without disrupted genes, administration of gamma interferon inhibits EAE, while antibody to gamma interferon enhances EAE
(27, 30).
- and
LT-
-deficient mice there is abnormal spleen architecture,
blood lymphocytosis, absence of lymph nodes, and functional defects in T cell physiology (31). Are these appropriate conditions to study gene function and make conclusions about the role of these genes in autoimmunity?
and LT-
are inactivated (2).
Obviously in these knockout mice other cytokines and
mediators may then assume the functions of the deleted
products of the inactivated genes. Future studies involving
knockouts need to take these issues into consideration.
Given new technologies involving microarrays, it is now
possible to screen for a wide variety of cytokines, chemokines, metalloproteases, adhesion molecules, and other critical mediators on a single sample of RNA from the brain of
a mouse with EAE (35). Comparison of the array of mediators transcribed in such knockout mice with EAE to other
mice without the disrupted genes, who nevertheless develop
EAE, will give further clues to how alternative pathways
are utilized to achieve a classical phenotype. The concept
that there is a single mediator ultimately causing pathology
in a phenotype as complex as EAE, is probably flawed. The
culmination of a complex sequence of pathological events
results in disease. Studies with the current generation of
knockouts reveal the redundancy of certain mediators, like
TNF-
and LT-
, in complex pathophysiological events.
Address correspondence to Lawrence Steinman, Department of Neurological Sciences, Beckman Center for Molecular and Genetic Medicine, B002, Stanford University, Stanford, CA 94305-5429.
Received for publication 17 April 1997.
1. | Steinman, L.. 1996. Multiple sclerosis: a coordinated immunological attack against myelin in the central nervous system. Cell. 85: 299-302 [Medline]. |
2. | Frei, K., H.P. Eugster, M. Popst, C. Constantinescu, E. Lavi,
and A. Fontana. 1997. Tumor necrosis factor and lymphotoxin-![]() |
3. |
Issazadeh, S.,
A. Ljungdahl,
B. Hojeberg,
M. Mustafa, and
T. Olsson.
1995.
Cytokine production in the central nervous
system of Lewis rats with experimental autoimmune encephalomyelitis: dynamics of mRNA expression for interleukin10, interleukin-12, cytolysin, tumor necrosis factor ![]() ![]() |
4. |
Renno, T.,
M. Krakowski,
C. Piccirillo,
J. Lin, and
T. Owens.
1995.
TNF-![]() |
5. | Baker, D., J.K. O'Nell, and J.L. Turk. 1991. Cytokines in the central nervous system of mice during chronic relapsing experimental allergic encephalomyelitis. Cell. Immunol. 134: 505-510 [Medline]. |
6. |
Held, W.,
R. Meyermann,
Y. Qin, and
C. Mueller.
1993.
Perforin and tumor necrosis factor ![]() |
7. | Powell, M.B., D. Mitchell, J. Lederman, J. Buckmeier, S.S. Zamvil, M. Graham, N.H. Ruddle, and L. Steinman. 1990. Lymphotoxin and tumor necrosis factor-alpha production by myelin basic protein-specific T cell clones correlates with encephalitogenicity. Intern. Immunol. 2: 539-544 [Medline]. |
8. | Ruddle, N.H., C.M. Bergman, K.M. McGrath, E.G. Lingenheld, M.L. Grunnet, S.J. Padula, and R.B. Clark. 1990. An antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis. J. Exp. Med. 172: 1193-1200 [Abstract]. |
9. | Selmaj, K., C.S. Raine, and A.H. Cross. 1991. Anti-tumor necrosis factor therapy abrogates autoimmune demyelination. Ann. Neurol 30: 694-700 [Medline]. |
10. | Selmaj, K., W. Paplerz, A. Glabinski, and T. Kohno. 1995. Prevention of chronic relapsing experimental autoimmune encephalomyelitis by soluble tumor necrossis factor receptor I. J. Neuroimmunol. 56: 135-141 [Medline]. |
11. |
Klinkert, W.E.F.,
K. Kojima,
W. Lesslauer,
W. Rinner,
H. Lassmann, and
H. Wekerle.
1997.
TNF-![]() |
12. |
Karin, N.,
D. Mitchell,
N. Ling,
S. Brocke, and
L. Steinman.
1994.
Reversal of experimental autoimmune encephalomyelitis by a soluble variant of a myelin basic protein epitope: T
cell receptor antagonism and reduction of Interferon-![]() ![]() |
13. | Brocke, S., K. Gijbels, M. Allegretta, I. Ferber, C. Piercy, T. Blankenstein, R. Martin, U. Utz, N. Karin, D. Mitchell, et al . 1996. Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein. Nature (Lond.). 379: 343-345 [Medline]. |
14. | Sommers, N., P.A. Loschmann, G.H. Northoff, M. Weller, A. Steinbrecher, J.P. Steinbach, R. Lichtenfels, R. Meyermann, A. Rietmuller, A. Fontana, et al . 1995. The anti-depressant rolipram suppresses cytokine production and prevents autoimmune encephalomyelitis. Nat. Med 1: 244-248 [Medline]. |
15. | Genain, C.P., T. Roberts, R.L. Davis, M. Nguyen, A. Uccelli, D. Faulds, Y. Li, J. Hedgpeth, and S.L. Hauser. 1995. Prevention of autoimmune damage in non-human primates by a cAMP specific phosphodiesterase inhibitor. Proc. Natl. Acad. Sci. USA 92: 3602-3605 . |
16. | Brocke, S., A. Gaur, C. Piercy, A. Gautam, K. Gijbels, C.G. Fathman, and L. Steinman. 1993. Induction of relapsing paralysis in experimental autoimmune encephalomyelitis by bacterial superantigen. Nature (Lond.). 365: 642-644 [Medline]. |
17. |
Chung, I.Y.,
J.G. Norris, and
E.N. Benveniste.
1991.
Differential tumor necrosis factor ![]() |
18. | Selmaj, K.W., and C.S. Raine. 1988. Tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro. Ann. Neurol. 23: 339-346 [Medline]. |
19. |
Probert, L.,
K. Akassoglou,
M. Pasparakis,
G. Kontogeorgos, and
G. Kollias.
1995.
Spontaneous inflammatory demyelinating disease in transgenic mice showing central nervous systemspecific expression of tumor necrosis factor ![]() |
20. |
Kuroda, Y., and
Y. Shimamoto.
1991.
Human tumor necrosis factor-![]() |
21. |
Crisi, G.M.,
L. Santambrogio,
G.M. Hochwald,
S.R. Smith,
J.A. Carlino, and
G.J. Thorbecke.
1995.
Staphylococcal enterotoxin B and tumor necrosis factor ![]() ![]() |
22. |
Sharief, M.K., and
R. Hentges.
1991.
Association between
tumor necrosis factor-![]() |
23. | Paya, C.V., P.J. Leibson, A.K. Patick, and M. Rodriguez. 1990. Inhibition of Theiler's virus-induced demyelination in vivo by tumor necrosis factor alpha. Int. Immunol. 2: 909-913 [Medline]. |
24. | Willenborg, D.O., S.A. Fordham, W.B. Cowden, and I.A. Ramshaw. 1995. Cytokines and murine autoimmune encephalomyelitis: inhibition or enhancement of disease with antibodies to select cytokines, or by delivery of exogenous cytokines using a recombinant vaccinia virus system. Scand. J. Immunol. 41: 31-41 [Medline]. |
25. |
Steinman, L..
1996.
A few autoreactive cells in an autoimmune infiltrate control a vast population of nonspecific cells:
A tale of smart bombs and the infantry.
Proc. Natl. Acad. Sci.
USA.
93:
2253-2256
|
26. | Shaw, M.K., J.B. Lorens, A. Dhawan, R. DalCanto, H.Y. Tse, A.B. Tran, C. Bonpane, S.L. Eswaran, S. Brocke, N. Sarvetnick et al. 1997. Local delivery of interleukin-4 by retrovirus-transduced T lymphocytes ameliorates experimental autoimmune encephalomyelitis. J. Exp. Med. In press. |
27. | Gijbels, K., S. Brocke, J. Abrams, and L. Steinman. 1995. Administration of neutralizing antibodies to interleukin-6 (IL-6) reduces experimental autoimmune encephalomyelitis and is associated with elevated levels of IL-6 bioactivity in central nervous system and circulation. Mol. Med. 1: 795-805 [Medline]. |
28. | Brenner, T., S. Brocke, F. Szafer, R. Sobel, J.F. Parkinson, D.H. Perez, and L. Steinman. 1997. Inhibition of nitric oxide synthase for treatment of experimental autoimmune encephalomyelitis. J. Immunol. 158: 2940-2946 [Abstract]. |
29. | Bo, L., T.M. Dawson, S. Wesselingh, S. Mork, S. Choi, P.A. Kong, D. Hanley, and B.D. Trapp. 1994. Induction of nitric oxide synthase in demyelinating regions of multiple sclerosis brains. Ann. Neurol. 36: 778-784 [Medline]. |
30. |
Krakowski, M., and
T. Owens.
1996.
Interferon-![]() |
31. |
Eugster, H.P.,
M. Muller,
U. Karrer,
B.D. Car,
B. Schnyder,
V.M. Eng,
G. Woerly,
M. Le,
M. Aguet,
R.M. Zinkernagel, et al
.
1996.
Multiple immune abnormalities in tumor necrosis
factor and lymphotoxin ![]() |
32. | Wilson, T., and S. Tonegawa. 1997. Synaptic plasticity, place cells and spatial memory: study with second generation knockouts. Trends Neurosci 20: 102-106 [Medline]. |
33. |
Ferber, I.A.,
S. Brocke,
C. Taylor-Edwards,
W. Ridgway,
C. Dinisco,
L. Steinman,
D. Dalton, and
C.G. Fathman.
1996.
Mice with a disrupted interferon-![]() |
34. | Liblau, R., L. Steinman, and S. Brocke. Experimental autoimmune encephalomyelitis in IL-4 deficient mice. Int. Immunol. In press. |
35. |
Heller, R.A.,
M. Schena,
A. Chai,
D. Shalon,
T. Bedilion,
J. Gilmore,
D.E. Woolley, and
R.W. Davis.
1997.
Discovery
and analysis of inflammatory disease-related genes using
cDNA microarrays.
Proc. Natl. Acad. Sci. USA.
94:
2150-2155
|