518 Journal of Leukocyte Biology Volume 59, April 1996

Dissection of the pathologies induced by transmembraneand wild-type tumor necrosis factor in transgenic mice

Lesley Probert, Katerina Akassoglou, Lena Alexopoulou, Eleni Douni, Sylva Haralambous,Sally Hill, George Kassiotis, Dimitris Kontoyiannis, Manolis Pasparakis, David Plows, andGeorge KolliasDepartment of Molecular Genetics, Hellenic Pasteur Institute, Athens, Greece

Abstract: With increasing awareness that seemingly

diverse immune-mediated diseases involve similar

pathogenetic mechanisms, and the identification of a

growing number of key effector molecules, it is

becoming possible to design and generate effective

transgenic models for such diseases. Tumor necrosis

factor (TNF) plays a prominent role in immune and

host defense responses and there is strong evidence

that abnormal TNF production contributes to disease

initiation and progression in rheumatoid arthritis,

systemic inflammatory response syndrome, diabetes,

multiple sclerosis, and many other immune-mediated

disorders. The generation of TNF transgenic mice,

in which TNF production is deregulated, has pro-

vided us with direct evidence that, in vivo, this

cytokine can indeed trigger the development of such

complex disease phenotypes. Transgenic mice that

have been engineered to overexpress human or mur-

me TNF molecules in peripheral joints, T cells, or

neurons of the central nervous system represent

important animal models for human rheumatoid ar-

thritis, systemic inflammation, and multiple sclerosis,respectively. In addition to establishing a central role

for TNF in such diseases, these animal models have

already proved valuable for identifying additional

important disease-effector molecules, and for gain-

ing an insight into the complex in vivo mechanisms

that are involved in disease pathogenesis. For exam-

ple, in the case of arthritis, TNF has been found to

transmit its pathogenic effects entirely through inter-

leukin-1, which may therefore represent an addi-

tional important target for therapeutic interventionin the human disease. In summary, TNF transgenic

models of human disease are expected to serve as

important in vivo tools for defining details of disease

pathogenesis, potential targets for therapeutic inter-

vention, and for evaluating the possible involvement

of additional genetic and environmental factors on

the disease state. J. Leukoc. Riot. 59: 518-525;

1996.

Key Words: autoin�muniuy . disease models . inflammation

neuroimmunology arthritis cytokine

INTRODUCTION

Tumor necrosis factor a (TNF-a) is a proinflammatory

cytokine produced mainly by activated macrophages, but

also by many other cell types including T cells, pre-B

cells, natural killer cells, mast cells, neutrophils, fi-

broblasts, keratinocytes, and microglial cells. It is pro-

duced during immune and host defense responses and,

under physiological conditions, acts to preserve architec-

tural and biochemical homeostasis [1]. Thus, TNF-a is

involved in tissue remodeling, defense against infection,

and inflammation. In contrast to this, overproduction of

TNF-a can cause severe pathological changes leading to

tissue injury, shock, and even death. TNF-a has been

directly implicated in the pathogenesis of several disease

states such as the systemic inflammatory response syn-

drome, graft-versus-host reactions, and autoimmune dis-

eases including diabetes, rheumatoid arthritis, and

multiple sclerosis.

The pleiotropism of TN F-a action is a result of the mul-

tilevel mechanisms that operate to regulate its expression

and functional potency. In addition to its diverse cellular

source, TNF-a occurs naturally in two biologically active

molecular forms, a 26-kDa membrane-anchored precursor

protein (pro-TNF-a) acting locally through cell-to-cell

contact, and a secreted, mature 17-kDa form that is

cleaved from pro-TNF-a by proteolytic enzymes and is

capable of acting on distant targets [2]. Evidence is accu-

mulating that the two molecular forms of TNF-a can medi-

ate distinct biological functions. For example,

transmembrane TNF-a has been associated with contact-

dependent lymphocyte and monocyte-mediated cytotoxic-

ity [3] and may play a role in B cell activation [4, 5].

Diversity of TNF-a action is further acheived at the recep-

tor level. TNF-a signaling is mediated by two specific

high-affinity receptors, the p55 TNF-a-R (CD12Oa) and

p75 TNF-a-R (CD12Ob) [see ref. 6]. The two receptors are

co-expressed in most tissues but contain very different

Abbreviations: TNF-a, tumor necrosis factor-a; IL, interleukin; CNS, central

nervous system; MS. multiple sclerosis; RAE, experimental autoimmune encepha-lomyelitis.

Reprint requests: Dr. George Kollias, Department of Molecular Cenetics, Hel-lenic Pasteur Institute, 127 Vass Sofias Avenue, 11521 Athens, Hellas.

Received November29, 1995; revised January 19, 1996; accepted January 22,1996.

Probert et al. Tranamembrane and wild-type TNF tranagenic mice 519

intracellular domains associated with distinct cytoplasmic

proteins [7, 8], and thus mediate distinct cellular re-

sponses in vivo. The murine p55 TNF-a-R appears to mo-

nopolize TNF-a-mediated signaling and can signal almost

all reported TNF-a activities including cytotoxicity [9, 10],

apoptosis [11], and proliferation [12]. In contrast, p75

TNF-a-R signaling is limited to few cell systems, and pro-

motes proliferation of thymocytes, T lymphocytes [13], and

other cells of hemopoietic origin, as well as apoptotic sig-

nals in mature, activated CD8� T cells [14]. Recent evi-

dence indicates that the 26-kDa transmembrane TNF-a

molecule is superior to soluble TNF-cx in activating the

p’75 TNF-ct-R in various systems such as T cell activation

and thymocyte proliferation [15] and implies an important

physiological role for this receptor in local inflammatory

responses. Both the p55 and p7S TNF-a-R exist as trans-

membrane proteins that are inducibly sheddable. The re-

sulting soluble receptors can compete for TNF-ct and thus

modulate its bioavailability either inhibiting or enhancing

its action depending upon the microenvironment [16].

Considering the multiple and potent actions of TNF-a it is

not surprising that the biological availability of this protein

is strictly regulated. As with other pro-inflammatory cytok-

ines, TNF-a mRNA contains a 3’ UTR region that is

thought to be critical in the regulation of both mRNA sta-

bility [17] and translational efficiency [18, 19].

Although we are now beginning to gain insight into sev-

eral aspects of TNF-cx biology, for example TNF-a-R sig-

naling mechanisms, the regulation of TNF-a production,

and the relative roles of membrane-associated and soluble

TNF-a and TNF-a-R proteins, we know little of their bio-

logical significance in the in vivo context during physi-

ological and pathophysiological processes. Our laboratory

has been using transgenic technology to investigate the in

vivo biological potential of TNF-a during health and dis-

ease, and to analyze the multiple mechanisms that regulate

its expression and functional potency. It is possible, for

example, to differentiate between the two TNF-a receptors

in vivo, by expressing huTNF-a transgenes in transgenic

mice. In this way, the function of the p55 TNF-a-R is

specifically probed by virtue of its wide species cross-re-

activity, while the species-specific mouse p75 TNF-a-R is

unable to mediate signals from huTNF-a [20]. In addition,

analysis of the role of 26-kDa transmembrane huTNF-a in

vivo can be examined by use of a mutant huTNF-a trans-

gene in which the triplets encoding the first to twelfth

amino acids containing the putative cleavage site for ma-

ture soluble TNF-a, have been deleted (iM-12 TNF-a).

This i�1-12 mutant TNF-a gene has been shown to pro-

duce a transmembrane huTNF-a protein that remains

bioactive against cellular targets in vitro [21].

A TRANSGENIC MOUSE MODEL OF CHRONICINFLAMMATORY ARTHRITIS

Rheumatoid arthritis is an autoimmune disease involving

localized inflammation of the synovial joint. Immunoge-

Fig. 1. Sections taken through the knee joint of an arthritic Tg197(huTNF-a-globin) mouse. Immunocytochemical demonstration of largenumbers of 132 integrin (CD18)-immunoreactive cells adhering to vascular

endothelium and infiltrating the proliferated synovium. The anti-CD18

antibody was kindly provided by Dr. Leslie Moloney, Tanabe Research

Laboratories, San Diego, CA. Scale bar, 200 Ltm.

netic susceptibility, physical stress or injury, infectious

agents, and endogenous substances have been implicated

in triggering the primary immune response in arthritis. The

subsequent amplification and perpetuation of joint inflam-

mation involves a complex interplay between pro-inflam-

matory and immunomodulatory cytokines, neuropeptides,

metalloproteinases, adhesion molecules, and other inflam-

matory mediators. Elevated levels of pro-inflammatory cy-

tokines such as TNF-cz, interleukin-1 (IL-i), IL-6, IL-8,

and granulocyte/macrophage colony stimulating factor are

present in the synovial fluid and serum of patients with

rheumatoid arthritis [22-24] and TNF-a and its receptors

are expressed by rheumatoid synoviocytes [25-28].

TNF-a shows many biological activities that are consis-

tent with it playing a central role in the development of

chronic arthritic disease. TNF-a triggers the activation and

proliferation of synoviocytes in vitro [29, 30] and may

interfere with cartilage and bone metabolism through in-

duction of collagenase production by synovial cells [31,

32], inhibition of proteoglycan synthesis by articular chon-

drocytes [33, 34], and stimulation of bone resorption in

vitro [35, 36]. Other indirect activities include the induc-

tion of additional cytokines [37, 38], and the triggering of

adhesion molecule expression on endothelial cells, which

enables lymphocytes to home to the site of inflammation

[39]. These mechanisms are considered to be decisive in

the development of arthritis.

To investigate the role of TNF-a in vivo in the develop-

ment of arthritis, our laboratory has generated transgenic

mice that overexpress TNF-a. Our early work showed that

a human TNF-a transgene could be expressed efficiently

in peripheral joints of transgenic mice when its 3’ UTR and

3’ flanking sequences were replaced with corresponding

sequences from the human �3-globin gene to stabilize the

mRNA and enhance its translational efficiency. In these

mice exogenous huTNF-a was produced in both trans-

membrane (26 kDa) and soluble (17 kDa) molecular forms.

Mice expressing the huTNF-a-globin construct develop

Fig. 2. Histological analysis of sections taken through the cerebellum of a 3-month-old, ataxic Tg6074 (muTNF-a-globin) mouse. Scale bars, 200 �tm.(A) In situ hybridization localization of transgene expression (using a �P-labeled 5’ muTNF-a antisense riboprobe) in neurons of the granule layer.

(B) GFAP-immunoataining of a serial section to that shown in A, showing marked astrocytosis in the granule and molecular layers. (C) Oil

red-haematoxylin staining demonstrating numerous lipid-laden macrophages and myelin debris in the molecular layer.

520 Journal of Leukocyte Biology Volume 59, April 1996

chronic progressive inflammatory polyarthritis with 100%

phenotypic penetrance. For example, in the Tg197

(huTNF-a-globin) line, arthritis develops timely with mac-

roscopic signs of disease detectable by 4-5 weeks of age

and histologically evident synovial hyperplasia and inflam-

matory infiltration from 3 weeks of age [40]. The arthritis

that develops in these mice shows histological features

characteristic of human rheumatoid arthritis [41] including

hyperproliferation of synovial cells, inflammatory infil-

trates of the synovial space, fibrous tissue and pannus

formation, articular cartilage destruction, and bone resorp-

tion. Development of arthritis in these transgenic mice can

be completely suppressed by the administration of neutral-

izing anti-human TNF-a monoclonal antibodies (kindly

provided by Drs. Sue Stephens and Roly Foulkes, Celitech,

UK) confirming that human TNF-a has a specific and

direct in vivo role in the pathogenesis of this disease.

The mode of action of huTNF-a in arthritis development

is unclear but may be direct, for example by driving prolif-

eration of the synovial cells [29, 30]. TNF-a may also act

indirectly, for example by the induction of other pro-in-

flammatory cytokines [37, 38] and by interfering with im-

mune homeostasis in the joint space. TNF-a induces the

expression of adhesion molecules on vascular endothelal

cells, thereby increasing leukocyte adhesion. In Tg197

mice, numerous cells present within the arthritic synovium

are immunoreactive for CD18 (the a subunit of the �

integrin adhesion molecule, and a marker for T cells,

monocytes, and neutrophils; Fig. 1). Moreover, TNF-a is

chemotactic for monocytes in vitro [42] and may be di-

rectly responsible for their recruitment into the arthritic

joint. Interestingly, high levels of nerve growth factor have

also been demonstrated in Tgi97 synovium [43]. Nerve

growth factor increases in the synovium of rheumatoid ar-

thritis patients and in experimentally induced arthritis,

and may modulate the inflammatory state by inducing the

release of biologically active compounds from nerve termi-

nals, mast cells, and/or immune cells [44].

A prominent action of TNF-a is the induction of IL-i

expression [45]. Uke TNF-a, IL-i is also strongly impli-

cated in the pathogenesis of arthritic disease. The involve-

ment of this cytokine in the development of

collagen-induced arthritis in mice has been demonstrated

by neutralization of the disease by use of antibodies to IL-i

[46, 47] or an endogenous inhibitor of IL-i, the IL-i re-

ceptor antagonist [48]. However, the relative contributions

of IL-i and TNF-a in the development and progression of

arthritis remains questionable. We have used our TNF-a

transgenic mouse model of arthritis to investigate the func-

tional hierarchy between TNF-a and IL-i in the patho-

genesis of joint disease. A neutralizing monoclonal

antibody to the murine type 1 IL-i receptor (kindly pro-

vided by Dr. John Simms at Immunex), administered in-

traperitoneally from birth to 9 weeks of age, completely

neutralized the development of arthritis. Moreover, serum

levels of circulating TNF-a were significantly lower in

treated than in untreated arthritic controls before or after

onset of arthritis [49]. Taken together, these results show

that in TNF-a-transgenic mice the IL-i receptor accepts

the whole pathogenic load of TNF-ct, thereby acting as a

potent downstream mediator in the pathogenesis of chronic

arthritis. In addition, our results suggest that IL-i signaling

blockade exerts a direct negative feedback effect on TNF-

a production.

It is important to note that in transgenic mice which

express huTNF-a, the arthritogenic potential of huTNF-a

Fig. 3. Section taken through the cervical spinal cord of an 8-month-old

Tg6074 mouse that had kyphosis and severe ataxia. Normal myelin is

absent and is replaced by lipid-filled macrophages and myelin debris as

shown by oil red-haematoxylin staining. Scale bar, 200 l,tm.

Probert et al. Tranamembrane and wild-type TNF tranagenic mice 521

can be mediated exclusively through the pSS TNF-a-R. As

already mentioned, the murine p75 TNF-a-R is species

specific [20]. Here it may be argued that, under physi-

ological conditions, the availability of a functional p7S

TNF-a-R may act to control disease development. To test

this possibility we have generated transgenic mice that

express an equivalent murine TNF-a-globin transgene so

that overexpressed TNF-a protein would interact with both

of the TNF-a receptors. Development of arthritis, with a

disease profile and histological features similar to that seen

in huTNF-a transgenic counterparts, was also induced in

these mice, suggesting that the availability of functional

p’ZS TNF-ct-R is not able to counteract the arthritogenic

effects of TNF-a overproduction in vivo.

A further question concerns the differential actions of

the soluble and transmembrane molecular forms of TNF-a

in disease pathogenesis. To investigate whether transmem-

brane TNF-a is sufficient to function as a trigger in the

pathogenesis of arthritis we have generated transgenic

mice that express the mutant uncleavable hu z�i-i2 TNF-

cz-globin transgene in their joints [49a]. Consistent with

previous data from in vitro studies using a similar construct

[2, 21], the high levels of huTNF-cz detectable in trans-

genic mouse sera, or supematants from CHO cells trans-

fected with the Ai-12 TNF-a construct, were biologically

inactive by cytotoxicity assay on L929 cells. Contact cyto-

toxicity assays further confirmed that this mutant transgene

encodes biologically active TNF-a only as a membrane-as-

sociated protein [49a]. Interestingly, transgenic mice that

express the mutant transgene develop typical chronic in-

flammatory polyarthritis, similar to the arthritis triggered

by wild-type huTNF-a transgenes. In the established

Ig5453 (A1-i2 huTNF-ct-globin) line arthritis develops

with 100% phenotypic penetrance and is evident as mac-

roscopic swelling of the hind ankle joints at around 5-6

weeks of age. Histologically, synovial hyperplasia and in-

flammatory infiltration by polymorphonuclear and lympho-

cytic cells, pannus formation, and articular cartilage

destruction are all common features in these mice. As in

mice expressing wild-type huTNF-a-globin, arthritis in the

�1-i2 huTNF-ct-globin mice is also neutralized by the

administration of anti-huTNF-a antibodies. These results

establish transmembrane TNF-a as a potent effector of

joint inflammation.

T CELL TARGETED TNF-a TRANSGENES;LOCAL AND SYSTEMIC TNF-a-MEDIATEDDISEASE

To analyze the biological potential of T cell-specific TNF-

a production and to further evaluate the role of the p55

TNF-cZ-R, in vivo, we generated transgenic mice that ex-

press constitutively huTNF-a or �1-i2 huTNF-a in their

T cells [49a, 50]. This was achieved by placing the TNF-a

transgenes under the control of the T cell-specific, locus

control region of the human CD2 gene. As already men-

tioned, the use of huTNF-a transgenes ensures that trans-

gene signaling is mediated exclusively by the p55

TNF-a-R. High level, T cell-specific expression of the

wild-type and M-12 huTNF-a transgenes was seen to

parallel expression of the endogenous CD2 gene with high

levels of huTNF-a mRNA found in thymus, lymph nodes,

and spleen. Immunochemical analysis showed that both

constructs produce the high- molecular-weight transmem-

brane TNF-a, and that wild-type transgenes also produce

i7-kDa TNF-a. Phenotypically, mice developed marked

histological and cellular changes locally in their lymphoid

organs. Thymuses were hypoplastic with a markedly re-

duced cortex, whereas lymph nodes were enlarged and

lacked distinct B and T cell regions. Histological changes

were accompanied by reduced numbers of thymocytes and

peripheral T cells. TNF-a is known to induce thymocyte

proliferation, in vitro, via the p75TNF-a-R [51]. However,

our results provide in vivo evidence that TNF-a may also

induce cytotoxicity in murine thymocytes by signaling

through the p55TNF-a-R, and that 26-kDa transmem-

brane TNF-a is sufficient to induce this effect by a con-

tact-dependent mechanism [49a]. Cytotoxicity of

thymocytes and peripheral T cells may be a direct action

of TNF-a. The p55 TNF-a-R shares sequence homology

with Fas including the area identified as the apoptotic or

death domain, which is sufficient for producing cytotoxic

signals [ii, 52]. Alternatively, T cell cytotoxicity may oc-

cur indirectly, for example by affecting the normal func-

tioning of thymic stromal cells, which play an important

supportive role for thymocytes in the thymic microenviron-

ment. Consistent with the latter hypothesis, pSS TNF-a-Rs

are abundant on epithelial cells, and in lymphoid organs

are limited to the germinal center dendritic reticulum cells

[53], whereas the p75 TNF-a-Rs are the major ligand

binding receptors on lymphocytes [51].

522 Journal of Leukocyte Biology Volume 59, April 1996

High-level T cell-specific expression of the wild-type

TNF-a transgene resulted in spillover of soluble TNF-a

protein into the circulation. In the established Tg2ii

(CD2-huTNF-a-globin) line, for example, serum levels of

huTNF-a ranged between 100 and 1300 pg/mI. The pres-

ence of circulating TNF-cx protein was correlated with de-

velopment of a lethal wasting syndrome characterized by

widespread vascular necrosis and ischemic tissue necrosis

in peripheral organs, particularly the liver, pancreas, and

lymph nodes. As for the lymphoid organ abnormalities, the

wasting disease could be completely neutralized by anti-

huTNF-a antibodies [50, 54]. TNF-a is known to exert a

variety of in vitro effects on vascular endothelial cells,

altering morphology and growth rates [55], and stimulating

adhesion molecule expression [56, 57]. In addition, TNF-a

acts to induce IL-i production by vascular endothelial

cells [58, 59] and both cytokines act to produce procoagu-

lant activity [60-62]. As a consequence, TNF-a may cre-

ate a situation in which both leukocytes and fibrin

accumulate along the walls of vessels and hemostasis is

induced. Confirmation that the systemic pathology was ef-

fected by soluble circulating TNF-a, rather than by trans-

membrane TNF-a present on the surface of circulating T

cells, was obtained in transgenic mice expressing only the

uncleavable huTNF-a protein. These animals, despite

similar overall levels of TNF-a mRNA production, do not

develop systemic pathology or wasting syndrome [49a].

The development of wasting disease in TNF-a trans-

genic mice fits well with some of the documented effects of

TNF-a in vivo. TNF-a has been shown to be directly in-

volved in the pathogenesis of endotoxic shock [63, 64] and

cachexia induced in mice bearing tumors that secrete

TNF-a [65]. Similarly, the chronic administration of

sublethal doses of TNF-a to experimental animals leads to

wasting accompanied by specific inflammatory changes in

several tissues [66], whereas single lethal doses induce

intravascular thrombosis with hemorrhagic necrosis of tis-

sues [67]. Our finding that the overexpression of huTNF-a

in transgenic mice leads to vascular obstruction, hemosta-

sis, and resulting ishemic tissue necrosis and wasting dis-

ease, demonstrates that such effects can be mediated by

the pSS TNF-a-R and that specifically blocking the action

of this receptor might find therapeutical application to aid

the management of systemic inflammation and its conse-

quences.

CNS-SPECIFIC EXPRESSION OF TNF-aINDUCES INFLAMMATORY DEMYELINATINGDISEASE

In addition to their classical role in peripheral immune

function and host defense, cytokines are now emerging as

important mediators of central nervous system (CNS)

physiology and pathophysiology. TNF-a is produced byneurons in the normal murine brain [68] and exerts neuro-

modulatory effects [69]. The expression of TNF-a is up-

regulated in a wide range of CNS disorders, including mul-

tiple sclerosis (MS) [70, 71], AIDS dementia complex [72],

bacterial meningitis [73], Parkinson’s disease [74], and

human and murine cerebral malaria [75]. This increased

expression might derive from activated infiltrating T cells

or macrophages [1], activated astrocytes [76], and micro-

glial cells [77]. The contribution to disease pathogenesis of

increased TNF-a production in the CNS and its mecha-

nism of action are at present ill-defined. However, there is

strong in vitro evidence that in the CNS, TNF-a acts as a

potent pro-inflammatory cytokine and a major effector of

immune-mediated demyelination. TNF-a can induce oh-

godendrocyte degeneration and myelin vesiculation in

murine CNS explants [78, 79] and causes selective cyto-

toxicity to primary oligodendrocytes [80]. Moreover, stud-

ies in experimental autoimmune encephalomyelitis (EAE),

which is often used as a model for human MS, have pro-

vided additional in vivo evidence supporting the impor-

tance of TNF-a in disease pathogenesis. The adoptive

transfer of this disease by MBP-reactive T cells can be

prevented by the administration of neutralizing antibody to

TNF-a [81] or by treatment with soluble pSS TNF-a R

[82].

To investigate the in vivo role of TNF-a overproduction

in the CNS, we have generated transgenic mice that ex-

press specifically in neurons of their CNS (see Fig. 2A), a

3’-untranslated region (UTR)-modified murine TNF-a

transgene under the control of its own promoter, (Tg6074

line, muTNF-cx-globin) [83]. From between 3 and 8 weeks

of age, mice spontaneously develop chronic inflammatory

demyelinating disease with 100% phenotypic penetrance.

Mice die prematurely within -8 months after developing

progressively severe neurological symptoms ranging from

mild tremors and ataxia to severe imbalances and seizures.

Other symptoms include loss of the limb flexion reflex,

hind limb paresis, and kyphosis. Histopathological fea-

tures were reactive astrocytosis (Fig. 2B) and microgliosis,

infiltration of the meninges and CNS parenchyma with

macrophages (Fig. 2C) and CD4� and CD8� T lympho-

cytes, and focal demyelination (Fig. 3). The direct action

of TNF-a in the pathogenesis of this disease was confirmed

by peripheral administration of a neutralizing anti-murine

TNF-a antibody. This treatment completely prevented the

development of neurological symptoms, T cell infiltration

into the CNS parenchyma, astrocytosis, and demyeiination,

and greatly reduced the severity of reactive microgliosis.

One of the earliest histopathological changes in the CNS

of Tg6074 mice, and one that is prominent by 3 weeks of

age, is the accumulation of T lymphocytes at the meningeal

surface and their subsequent infiltration into the CNS pa-

renchyma. T cell presence in the CNS is low under normal

conditions, but is markedly increased in inflammatory dis-

eases such as MS [84] and EAE [85] and is thought to be

central to disease pathogenesis [84, 86]. The prevailing

belief is that MS is primarily autoimmune in nature, in-

volving an integrated attack by T cells, B cells, and macro-

phages on the myelin sheath. In both MS and EAE,

Probert et al. Transmembrane and wild-type TNF transgenic mice 523

transgression of the blood-brain barrier by inflammatory

cells is a key initial event in disease initiation [86, 87],

and a similar pathogenetic mechanism may take place in

1g6074 mice. Antibody neutralization experiments show

that disease in Tg6074 mice can be completely prevented

if infiltration of the CNS by inflammatory cells is blocked.

TNF-a overexpression in the CNS of Tg6074 transgenic

mice may directly influence inflammatory cell trafficking

at the blood-brain barrier. TNF-a is known to induce the

expression of adhesion molecules on vascular endothe-

hum, thereby selectively enhancing their adhesiveness for

inflammatory cells [see ref. i]. Moreover, TNF-a is chemo-

tactic for monocytes [42]. It remains to be determined

whether the T cells that occupy the CNS in Tg6074 mice

show specificity for neuronal antigens and whether they

have a pathogenic or immunomodulatory role during the

development of disease.

The demyehination that occurs in Tg6074 mice substan-

tiates a wealth of previous in vitro evidence that has impli-

cated TNF-a as an effector of immune-mediated

demyehination. In addition to its selective cytotoxic effect

on oligodendrocytes [79, 80], TNF-cx-immunoreactive in-

flammatory cells and astrocytes are present in MS plaques

[70], and disease progression in MS has been correlated

with high cerebrospinal fluid levels of TNF-a [88]. There-

fore, demyehination in Tg6074 mice might occur directly

from transgene-expressed TNF-a or as a bystander effect

of locally activated astrocytes, macrophages/microglia, and

activated infiltrating T cells. Alternatively, TNF-a may act

through the induction of further inflammatory molecules

such as nitric oxide, which in turn induces oligodendrocyte

death [89, 90] and affects the normal differentiation and

growth of neuronal precursors [90].

The evidence for a major involvement of TNF-a in the

pathogenesis of inflammatory demyelinating diseases such

as MS and EAE is now strong, and many therapeutical

approaches target the action of this cytokine. EAE can be

inhibited by several anti-TNF-a strategies [81, 82, 91], 1

cell class switching from Thi (TNF-a-producing) to immu-

nomodulatory Th2 cells [92], and the anti-depressant

rohipram, which inhibits T cell activation and TNF-a se-

cretion [93]. Transgenic mice that express TNF-cz in their

CNS, such as the Tg6074 mice described here, represent

important model systems for inflammatory and/or demyeli-

nating diseases in humans, and are expected to aid patho-

genetic studies and the identification of potential targets

for therapeutic intervention.

ACKNOWLEDGMENTS

We thank Drs. Sue Stephens and Roly Foulkes (Celltech

Ltd. UK) for providing us with the anti-human and anti-

murine TNF-a antibodies we use to maintain our trans-

genic lines and Dr. John Simms (Immunex, USA) for the

anti-murine type I IL-i receptor antibody. This work was

supported by the Greek Secretariat for Research and Tech-

nology and European Commission Grants, BIO-CT94-2092

and ERBCHRX-CT930182.

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