Rational design for the development of epidermal growth factor receptor antagonists

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) bind with similar high affinity to the human EGF receptor. Using a domain-exchange strategy we have shown that the C-terminal linear region of these molecules is involved in high affinity receptor binding. By further single amino acid substitution in this linear C-terminal region, a putative interaction site of these ligands with their receptor has been identified. This identification of a receptor binding domain in EGF/TGF alpha provides an important initial step in the development of EGF receptor antagonists with significant clinical potential.     

Decorin is a biological ligand for the epidermal growth factor receptor

Ectopic expression of decorin induces profound cytostatic effects in transformed cells with diverse histogenetic backgrounds. The mechanism of action has only recently begun to be elucidated. Exogenous decorin activates the epidermal growth factor (EGF) receptor, thereby triggering a signaling cascade that leads to phosphorylation of mitogen-activated protein (MAP) kinase, induction of p21, and growth suppression. In this study we demonstrate a direct interaction of decorin with the EGF receptor. Binding of decorin induces dimerization of the EGF receptor and rapid and sustained phosphorylation of MAP kinase in squamous carcinoma cells. In a cell-free system, decorin induces autophosphorylation of purified EGF receptor by activating the receptor tyrosine kinase and can also act as a substrate for the EGF receptor kinase itself. Using radioligand binding assays we show that both immobilized and soluble decorin bind to the EGF receptor ectodomain or to purified EGF receptor. The binding is mediated by the protein core and has relatively low affinity (Kd approximately 87 nM). Thus, decorin should be considered as a novel biological ligand for the EGF receptor, an interaction that could regulate cell growth during remodeling and cancer growth.     

The epidermal growth factor receptor is not required for tumor necrosis factor-alpha action in normal mammary epithelial cells

Our laboratory has shown that tumor necrosis factor-alpha (TNF alpha) can regulate normal mammary epithelial cell (MEC) growth, morphogenesis, and, under certain circumstances, functional differentiation in a manner similar to epidermal growth factor (EGF). As TNF alpha has been shown to up-regulate EGF receptor (EGFR) expression and function in other systems, the present studies were undertaken to determine whether TNF alpha action in MEC was indirect through stimulation of the EGFR. An inhibitor of EGFR tyrosine kinase activity, PD158780, failed to block proliferation induced by 40 ng/ml TNF alpha and only partially inhibited growth in response to 2 ng/ml TNF alpha. PD158780 was also unable to suppress the extensive morphological development induced by either TNF alpha concentration. In contrast, the effects of TNF alpha and PD158780 on functional differentiation (i.e. casein accumulation) were time dependent. When measured on day 7 after 48 h of treatment, casein accumulation was unaffected by either concentration of TNF alpha or by PD158780. When assessed on day 21 after 16 days of treatment, however, casein levels were decreased by 40 ng/ml TNF alpha and increased by PD158780. Significantly, this PD158780-induced increase in casein was not observed in MEC that had been treated with both PD158780 and TNF alpha. These results thus suggest that EGFR tyrosine kinase activity is not necessary for TNF alpha action in normal MEC.     

Intestinal adaptation is enhanced by epidermal growth factor independent of increased ileal epidermal growth factor receptor expression

BACKGROUND/PURPOSE: Intestinal adaptation after massive small bowel resection (SBR) is augmented by epidermal growth factor (EGF) via an unknown mechanism. We recently have observed that EGF increases the expression of EGF receptor mRNA and protein content in the remnant ileum after SBR. The purpose of this study was to determine whether the magnitude of EGF-induced receptor expression correlates with intestinal adaptation. METHODS: A 50% proximal SBR or sham operation (bowel transection with reanastomosis) was performed on male ICR mice. Animals from each group were then selected randomly to receive either human recombinant EGF (150 microg/kg/d) or saline by twice daily intraperitoneal injections. The remnant ileum was harvested at 1 week, and parameters of adaptation measured as changes in protein content. Ileal EGF receptor mRNA was quantitated using a ribonuclease protection assay. Changes in the expression ileal EGF receptor protein were determined by Western blot after immunoprecipitation. Comparisons of mean values between groups was performed using analysis of variance (ANOVA) and a P value of less than .05 was considered significant. Values are presented as mean +/- SEM. RESULTS: EGF was mitogenic to the ileum after sham operation as monitored by increases in ileal protein content (2.21 +/- 0.002 mg/cm Sham v 2.97 +/- 0.25 mg/cm Sham +/- EGF; P < .05). After SBR, adaptation resulted in increased ileal protein content (4.45 +/- 0.27 mg/cm), which was substantially boosted by EGF (5.98 +/- 0.39 mg/cm; P < .05). No differences were detected in ileal EGF receptor mRNA or protein expression between Sham or SBR groups that did not receive EGF. However, EGF significantly enhanced the expression of ileal EGF receptor mRNA to an equal extent after both sham and SBR (approximately threefold). The magnitude of this increase in EGF receptor protein (four- to sixfold) was similar in both EGF groups as shown by Western blotting. CONCLUSIONS: Changes in ileal EGF receptor expression are not mandatory for adaptation to occur. EGF upregulates the expression of mRNA and protein for its own intestinal receptor in vivo. Because EGF-induced receptor expression was comparable after both SBR and Sham operation, the beneficial effect of EGF during adaptation is likely caused by other factors in addition to increased receptor expression.     

Hormonal regulation of epidermal growth factor receptor content and signaling in bovine mammary tissue

Mammary tissue from midpregnant heifers was cultured with epidermal growth factor (EGF) or transforming growth factor alpha for 1-3 days. After 1 day, 10 nM EGF or transforming growth factor alpha doubled DNA synthesis, whereas lower concentrations (0.1 or 1 nM) increased DNA synthesis 2- to 3-fold after 2-3 days in culture. In other studies, bovine mammary tissue was transplanted to ovariectomized athymic mice and treated for 10 days with saline, estradiol (1 microg/day), progesterone (1 mg/day), or estradiol + progesterone. Subsequent explant culture of the bovine tissue indicated that estradiol + progesterone augmented the ability of EGF to stimulate DNA synthesis. The increased response to EGF was associated with increased EGF binding and with increased EGF-induced tyrosine kinase that paralleled the increased EGF binding. In other studies, athymic mice bearing xenografted bovine mammary tissue were primed for 10 days with estradiol and progesterone, followed by 2-day treatment with saline (control), hydrocortisone (200 microg/day), PRL (1 mg/day), or hydrocortisone + PRL. Hydrocortisone and PRL alone decreased, and PRL + hydrocortisone eliminated, EGF-induced DNA synthesis. EGF receptor content was unaffected by hydrocortisone but was reduced by PRL or hydrocortisone + PRL. Furthermore, the ability of EGF to induce tyrosine kinase activity was decreased by PRL and by hydrocortisone + PRL. The decreased kinase activity was greater than the decrease in receptor binding, suggesting a specific modulation of EGF receptor kinase activity in response to lactogenic hormones.     

Basic fibroblast growth factor is neither necessary nor sufficient for the development of retinal neovascularization

Basic fibroblast growth factor (FGF2) is constitutively expressed in the retina and its expression is increased by a number of insults, but its role in the retina is still uncertain. This study was designed to test the hypothesis that altered expression of FGF2 in the retina affects the development of retinal neovascularization. Mice with targeted disruption of the Fgf2 gene had no detectable expression of FGF2 in the retina by Western blot, but retinal vessels were not different in appearance or total area from wild-type mice. When FGF2-deficient mice were compared with wild-type mice in a murine model of oxygen-induced ischemic retinopathy, they developed the same amount of retinal neovascularization. Transgenic mice with a rhodopsin promoter/Fgf2 gene fusion expressed high levels of FGF2 in retinal photoreceptors but developed no retinal neovascularization or other abnormalities of retinal vessels; in the ischemic retinopathy model, they showed no significant difference in the amount of retinal neovascularization compared with wild-type mice. These data indicate that FGF2 expression is not necessary nor sufficient for the development of retinal neovascularization. This suggests that agents that specifically antagonize FGF2 are not likely to be useful adjuncts in the treatment of retinal neovascularization and therapies designed to increase FGF2 expression are not likely to be complicated by retinal neovascularization.     

Oncogenic Ha-Ras-dependent mitogen-activated protein kinase activity requires signaling through the epidermal growth factor receptor

C3H10T1/2 fibroblasts transformed by the minimal expression of oncogenic Ha-Ras (V12H10 cells) or N-Ras (K61N10 cells) have constitutive mitogen-activated protein kinase (MAPK) activity and proliferate in serum-free medium. The constitutive MAPK activity and serum-independent proliferation of V12H10 cells are sensitive to the growth factor antagonist, suramin (Hamilton, M., and Wolfman, A. (1998) Oncogene 16, 1417-1428), suggesting that Ha-Ras-mediated regulation of the MAPK cascade is dependent upon the action of an autocrine factor. Serum-free medium conditioned by V12H10 cells contains an activity that stimulates MAPK activity in quiescent fibroblasts. This MAPK stimulatory activity could be specifically blocked by the epidermal growth factor receptor (EGFR) inhibitors, PD153035 and PD158780. These inhibitors also blocked the serum-independent proliferation of V12H10 cells. Immunodepletion of conditioned medium with antibodies to transforming growth factor alpha and EGF significantly inhibited its ability to stimulate MAPK activity. Stable transfection of EGFR-negative NR6 and EGFR-positive Swiss3T3 cells with oncogenic (G12V)Ha-Ras demonstrated that only the Ha-Ras-transfected Swiss 3T3 cells possessed constitutive MAPK activity, and this activity was sensitive to PD153035. These data suggest that autocrine activation of the EGFR is required for the regulation of the MAPK cascade in cells minimally expressing oncogenic Ha-Ras.     

Activation and function of the epidermal growth factor receptor and erbB-2 during mammary gland morphogenesis

The hormonal stimulation of mammary gland morphogenesis is believed to occur through growth factor receptor signaling pathways. To determine the importance of the epidermal growth factor receptor (EGFR) pathway, we examined extracts of inguinal mammary glands from prepubertal and pubertal mice for tyrosine-phosphorylated EGFR and other erbB receptors. Tyrosine phosphorylation of both EGFR and erbB-2 was detected in normal female BALB/c mice at 5-6 weeks of age, but not during the prepubertal stage, e.g., 24 days of age. Treatment of mice with estradiol or epidermal growth factor also stimulated the formation of mammary EGFR/erbB-2 phosphotyrosine. Waved-2 mice, which have impaired EGFR kinase activity, exhibited less mammary development than did wild-type (wt) mice when both were evaluated at 36 days of age. Because EGFR knockout (KO) mice die shortly after birth, glands from the newborns were implanted under the renal capsules of female nude mice. Under these conditions, extensive ductal growth was observed in mammary glands from wt animals; in contrast, glands from EGFR KO mice failed to grow beyond rudimentary structures. Tissue recombinants revealed that the wt fat pad supported the morphogenesis of EGFR KO epithelium, whereas the EGFR KO fat pad did not. Taken together, these data suggest that EGFR is essential for morphogenesis of the mammary ducts and functions during this period of mammary development as a heterodimer with erbB-2 in the mammary stroma.     

Abnormal astrocyte development and neuronal death in mice lacking the epidermal growth factor receptor

Stimulation of the epidermal growth factor receptor (EGF-R) produces numerous effects on central nervous system (CNS) cells in vitro including neuronal survival and differentiation, astrocyte proliferation and the proliferation of multipotent progenitors. However, the in vivo role of EGF-R is less well understood. In the present study, we demonstrate that EGF-R null mice generated on a 129Sv/J Swiss Black background undergo focal but massive degeneration the olfactory bulb, piriform cortex, neocortex, and thalamus between postnatal days 5 and 8 which is due, at least in part, to apoptosis. Some of the neuronal populations that degenerate do not normally express EGF-R, indicating an indirect mechanism of neuronal death. There were also delays in GFAP expression within the glia limitans and within structures outside the germinal zones in early postnatal ages. At or just prior to the onset of the degeneration, however, there was an increase in GFAP expression in these areas. The brains of EGF-R (-/-) animals were smaller but cytoarchitecturally normal at birth and neuronal populations appeared to be intact, including striatal GABAergic and midbrain dopaminergic neurons which have previously been shown to express EGF-R. Multipotent progenitors and astrocytes derived from EGF-R (-/-) mice were capable of proliferating in response to FGF-2. These data demonstrate that EGF-R expression is critical for the maintenance of large portions of the postnatal mouse forebrain as well as the normal development of astrocytes.     

Insufficiency of self-phosphorylation for the activation of epidermal growth factor receptor

Polyclonal immunoglobulins were produced against the carboxy terminus, -SEFIGA, of the receptor for epidermal growth factor (EGF). The addition of these immunoglobulins to a solution containing EGF receptor resulted in the activation of its protein tyrosine kinase. The levels of activation were assessed by measuring the initial velocities of the phosphorylation of the tyrosine in angiotensin II. The enzymatic activity induced by the immunoglobulins was significant, usually 50-70% of the maximum activity induced by EGF, and the induction occurred over a narrow range of concentration of the immunoglobulins. In order to achieve the activation, the immunoglobulins had to be bivalent; the addition of monovalent Fab fragments to EGF receptor did not produce any activation of the protein tyrosine kinase. The activation produced by the immunoglobulins was found to be reversible upon the addition of the synthetic peptide SEFIGA against which the immunoglobulins had been produced. Self-phosphorylation of the EGF receptor also occurred as the enzyme was activated by the immunoglobulins. Tryptic peptide maps demonstrated that the self-phosphorylation caused by the immunoglobulins had the same signature as that produced by EGF. When the synthetic peptide that had been used as the hapten was added to EGF receptor that had been self-phosphorylated in the presence of the immunoglobulins, the stimulated enzymatic activity was lost even though the protein remained phosphorylated. It follows from the results of deletion mutation [Walton, G. M., Chen, W. S., Rosenfeld, M. G., & Gill, G. N. (1990) J. Biol. Chem. 265, 1750-1754] and the results reported here that self-phosphorylation is neither necessary nor sufficient for the activation of EGF receptor.     

Immunocytochemical localization of transforming growth factor alpha, epidermal growth factor and epidermal growth factor receptor in the cat endometrium and placenta

Hoxb-5 is one of the few homeobox genes strongly expressed in the developing mouse lung. To explore the hypothesis that Hoxb-5 acts to regulate epithelial cell fate and branching morphogenesis in the developing lung, we studied the temporal, spatial, and cell-specific expression of Hoxb-5 from gestational day (d) 13.5 to postnatal day (P) 2. Immunocytochemistry demonstrated regional localization of Hoxb-5 protein to developing conducting airways and surrounding mesenchyme. The cellular expression pattern changed from diffusely positive nuclei of mesenchymal cells on d13.5 to become more localized to nuclei of subepithelial fibroblasts and some adjacent columnar and cuboidal epithelial cells on d14.5. After d14.5, Hoxb-5 protein expression continued to decrease in mesenchymal cells distal from developing airways, but persisted in fibroblasts underlying conducting airways. Hoxb-5 protein expression persisted in nuclei of columnar and cuboidal epithelial cells on d16.5 and d17.5, with expression in low cuboidal epithelial cells as well from d17.5 to P2. Western blot analysis showed temporal and quantitative changes in Hoxb-5 protein expression with peak expression on d14.5-15.5. We conclude that Hoxb-5 protein is developmentally regulated in a temporal, spatial, and cell-specific manner throughout the pseudoglandular, canalicular, and terminal saccular periods of lung development in the mouse. This localization and expression pattern suggests that Hoxb-5 may influence branching morphogenesis, cell-cell communication, cell fate, and differentiation of conducting airway epithelia.     

pH sensitivity of epidermal growth factor receptor complexes

The association/dissociation binding kinetics of 125I-labeled mouse epidermal growth factor (EGF) to receptors on human fibroblast cells in monolayer culture have been measured at 4 degrees C as a function of extracellular pH from pH 5-9. At pH 8, steady-state total binding is maximal. As pH is lowered to 6.5, total binding monotonically decreases dramatically. It changes further only slightly between pH 6.5 and 5 to about 20% of the maximum binding value. Scatchard binding plots at pH 7.5 and above show the commonly observed concave-upward, non-linear curve; as pH is lowered, this plot becomes much more linear, indicating that the "high affinity" bound receptor population is greatly diminished. Application of our ternary complex binding model [Mayo et al., J Biol Chem 264:17838-17844, 1989], which hypothesizes complexation of the EGF-bound receptor with a cell surface interaction molecule, indicates that pH may have some direct effects on ternary complex formation, but the major effect is on EGF-receptor dissociation.     

Inhibition of parietal cell acid secretion is mediated by the classical epidermal growth factor receptor

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) inhibit gastric acid secretion both in vivo and in vitro. Previous studies have indicated that EGF and TGF-alpha bind to the same EGF/TGF-alpha receptor. Nevertheless, we and others have previously demonstrated that inhibition of acid secretion by these growth factors requires concentrations of the peptides that are 10-fold higher than those necessary for induction of mitogenesis. Therefore, we have sought to investigate whether gastric parietal cells may possess a second EGF/TGF-alpha receptor class. Two systems were studied: First, [125I]TGF-alpha was cross-linked to the receptor in isolated rabbit parietal cell membranes, and labeled species were resolved on SDS-PAGE. Second, acid secretion was evaluated in pylorus-ligated waved-2 mutant mice, which carry a disabling point mutation in their classical EGF/TGF-alpha receptor. In isolated parietal cells, [125I]TGF-alpha was cross-linked into a single species of 170 kDa. Cross-linking was inhibited in the presence of unlabeled TGF-alpha with an IC50 of 80 nM. In the pylorus-ligated mice, control littermate mice demonstrated a dose-dependent inhibition of acid secretion by EGF with an IC50 of 20 micrograms/kg. In contrast, EGF had no inhibitory effect on acid secretion in waved-2 mice at concentrations up to 100 micrograms/kg. No alterations in parietal cell or gastrin cell numbers were observed. These results in both isolated rabbit parietal cells and waved-2 mice support the existence of only a single class of EGF/TGF-alpha receptors in parietal cells. Differences in growth factor affinity are likely due to the modification of the receptor or one of its coordinate regulators.     

Androgen regulation of epidermal growth factor receptor binding activity during fetal rabbit lung development

The luteolytic response to a prostaglandin F2 alpha analogue, cloprostenol, was investigated in vivo and in vitro at defined stages of the luteal phase. In vivo administration of cloprostenol to female marmoset monkeys on day 3 after ovulation had no effect on plasma progesterone concentrations, whereas administration on day 14 after ovulation reduced plasma progesterone to preovulatory concentrations within 4 h. To identify the cellular basis for this luteolytic action, marmoset luteal tissue obtained on days 3, 6 and 14 after ovulation was incubated in vitro and progesterone production, cAMP accumulation and phosphoinositide (PI) turnover measured in response to cloprostenol, human chorionic gonadotrophin (hCG) with or without cloprostenol, or dibutyryl-cAMP with or without cloprostenol. Progesterone production was stimulated by both hCG and dbcAMP at all stages of the luteal phase. Although neither hCG nor dbcAMP had any significant effects on PI turnover, hCG also increased cAMP accumulation. In marmoset luteal tissue obtained on day 3 after ovulation, cloprostenol had no significant effect on basal or hCG/dbcAMP-stimulated progesterone production but significantly stimulated PI turnover. In contrast, on days 6 and 14 after ovulation, cloprostenol significantly inhibited hCG- and dbcAMP-stimulated progesterone production and the cAMP response to hCG, but had no significant effect on PI turnover. Since progesterone production by the marmoset corpus luteum depends on the luteotrophic support of luteinizing hormone (LH), these observations suggest that the luteolytic action of cloprostenol in vivo involves the inhibition of LH/hCG action at sites both prior and subsequent to cAMP accumulation. However, such luteolytic effects do not appear to require the generation of inositol phosphates by increased PI turnover.     

CrkII signals from epidermal growth factor receptor to Ras

A rat fibroblast mutant defective in oncogenic transformation and signaling from epidermal growth factor receptor to Ras has been isolated. The mutant contains dominant negative-type point mutations in the C-terminal SH3 domain of one crkII gene. Among the adapters tested, the mutant is complemented only by crkII cDNA. Expression of the mutated crkII in parent cells generates the phenotype indistinguishable from the mutant cell. Yet overexpression or reduced expression of Grb2 in the mutant before and after complementation with crkII have little effect on its phenotype. We conclude that adapter molecules are highly specific and that the oncogenic growth signal from epidermal growth factor receptor to Ras is predominantly mediated by CrkII in rat fibroblast.     

Signalling by the Drosophila epidermal growth factor receptor is required for the specification and diversification of embryonic muscle progenitors

Muscle development initiates in the Drosophila embryo with the segregation of single progenitor cells, from which a complete set of myofibres arises. Each progenitor is assigned a unique fate, characterized by the expression of particular identity genes. We now demonstrate that the Drosophila epidermal growth factor receptor provides an inductive signal for the specification of a large subset of muscle progenitors. In the absence of the receptor or its ligand, SPITZ, specific progenitors fail to segregate. The resulting unspecified mesodermal cells undergo programmed cell death. In contrast, receptor hyperactivation generates supernumerary progenitors, as well as the duplication of at least one SPITZ-dependent myofibre. The development of individual muscles is differentially sensitive to variations in the level of signalling by the epidermal growth factor receptor. Such graded myogenic effects can be influenced by alterations in the functions of Star and rhomboid. In addition, muscle patterning is dependent on the generation of a spatially restricted, activating SPITZ signal, a process that may rely on the localized mesodermal expression of RHOMBOID. Thus, the epidermal growth factor receptor contributes both to muscle progenitor specification and to the diversification of muscle identities.     

Disulfide bond structure of human epidermal growth factor receptor

The extracellular domain of the human epidermal growth factor receptor (sEGFR) consists of 621 amino acid residues, including 50 cysteines. The connections of the 25 disulfide bonds in the recombinant sEGFR protein, obtained from Chinese hamster ovary cells, have been determined using N-terminal sequencing and matrix-assisted laser desorption/ionization mass spectroscopy. We identified a basic repeat of eight cysteines with a 1-3, 2-4, 5-6, and 7-8 disulfide pairing pattern in the two cysteine-rich regions of sEGFR. By comparison to other cysteine-rich motifs, it was concluded that the cysteine-rich repeat of sEGFR belongs to the laminin-type EGR-like (LE) structural motif. Three-dimensional structure models of the two cysteine-rich regions have been built, based on the three-dimensional structures of the LE domains from the laminin gamma1 chain and secondary structure predictions for the EGF receptor.     

Expression of epidermal growth factors and epidermal growth factor receptor in normal cycling human ovaries

Immunolocalization of transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), cripto-1, amphiregulin and epidermal growth factor receptor (EGFR) was studied in 51 premenopausal human ovaries at various phases of the menstrual cycle. Localization of mRNA for TGF alpha and EGF was also studied by in-situ hybridization. Immunoreactive TGF alpha was observed predominantly in theca cells in 12 of 33 antral follicles in the follicular phase (6/14 dominant follicles, and 6/19 non-dominant) but not in any of the 18 follicles in the luteal phase or in primordial and pre-antral follicles. TGF alpha immunoreactivity was present predominantly in the luteinized granulosa cells in 13 of 15 corpora lutea in the luteal phase, which are considered to be active in steroidogenesis, but not in any of the regressed corpora lutea. Accumulation of TGF alpha mRNA hybridization signal was observed only in the theca cells in the follicles and luteinized theca cells in the ovaries that were immunohistochemically positive for TGF alpha. EGFR immunoreactivity was detected in 24 of 33 antral follicles in the follicular phase and in two of 18 follicles in the luteal phase but not in any of the corpora lutea. Immunoreactive EGF, cripto-1 and amphiregulin or EGF mRNA was not detected in any follicles, corpora lutea, or the stroma cells examined. These results indicate that, of the epidermal growth factors examined in this study, TGF alpha is locally synthesized in normal cycling human ovaries and TGF alpha may be synthesized in theca cells and act on the granulosa cells in a paracrine fashion through the EGFR in ovarian follicles.     

The epidermal growth factor receptor tyrosine kinase phosphorylates connexin32

To elucidate the role of excessive nitric oxide (NO) via the inducible nitric oxide synthase (iNOS) in experimental allergic encephalomyelitis (EAE), the effect of a selective iNOS inhibitor, aminoguanidine, was investigated using mice with actively induced EAE. Administration of aminoguanidine by intraperitoneal or intracisternal injection from day 2 to day 12 after immunization produced a significant delay in the onset of EAE. On the other hand, administration of aminoguanidine by intraperitoneal or intracisternal injection for 10 days after the onset of EAE enhanced the clinical severity and mortality rate and hastened the onset of relapse significantly. The histological study at day 11 after the onset revealed that more inflammatory cells were present in the central nervous system of mice treated with aminoguanidine as compared with mice without aminoguanidine treatment. These results suggested that NO via iNOS was a pathogenetic factor in the induction phase of EAE, but had an inhibitory role in the progression phase of EAE. Although the effect of NO synthase inhibitors on EAE has been controversial, the present study suggested that the timing of administration might be an important consideration and might explain the previous contradictory reports.