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Astroglial overproduction of TGF-β1 enhances inflammatory central nervous system disease in transgenic mice
Journal of Neuroimmunology 77 Ž1997. 45–50
Astroglial overproduction of TGF-b 1 enhances inflammatory central nervous system disease in transgenic mice Tony Wyss-Coray a
a,)
, Persephone Borrow b, Madelyne J. Brooker b, Lennart Mucke
a
Gladstone Molecular Neurobiology Program and Department of Neurology, UniÕersity of California San Francisco, CA 94141, USA b Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037, USA Received 27 November 1996; revised 3 February 1997; accepted 5 February 1997
Abstract Cerebral expression of the injury response cytokine transforming growth factor-b 1 ŽTGF-b 1. has been found to be increased in several neurological diseases but it remains unclear whether its function is primarily beneficial or detrimental. Here we show that transgenic Žtg. mice that overexpress bioactive TGF-b 1 in the central nervous system ŽCNS. and show no overt phenotype in the unmanipulated state, are more susceptible to the immune-mediated CNS disease experimental autoimmune encephalomyelitis ŽEAE.. TGF-b 1 tg mice with EAE showed an earlier onset of clinical symptoms, more severe disease and increased mononuclear cell infiltration in their spinal cords compared with non-tg littermate controls with EAE. Whereas previous observations indicated that increased peripheral levels of TGF-b 1 can suppress EAE, our findings demonstrate that local expression of TGF-b 1 within the CNS parenchyma can enhance immune cell infiltration and intensify the CNS impairment resulting from peripherally triggered autoimmune responses. q 1997 Elsevier Science B.V. Keywords: Cytokine; Multiple sclerosis; Gliosis; Autoimmunity
1. Introduction TGF-b 1 is a multifunctional cytokine that has been implicated in the control of inflammation and immune responses, cellular proliferation and differentiation, extracellular matrix production and degradation, angiogenesis, cell adhesion and tissue repair Žfor review see Roberts and Sporn, 1988; Massague, ´ 1990; Border and Ruoslahti, 1992; McCartney-Francis and Wahl, 1994.. TGF-b 1-deficient mice develop a multifocal autoimmune-type inflammatory disease ŽShull et al., 1992; Kulkarni et al., 1993.. Given systemically, TGF-b 1 inhibits autoimmune diseases such as experimental autoimmune encephalomyelitis ŽEAE., whereas neutralizing antibodies against TGF-b 1 enhance EAE ŽRacke et al., 1991; Kuruvilla et al., 1991; Santambrogio et al., 1993; Johns and Sriram, 1993.. TGF-b 1 seems to play a significant role in the induction of oral tolerance against autoantigens, which is currently being studied as a treatment for EAE and multiple sclerosis Abbreviations: CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; MSCH, mouse spinal cord homogenate ) Corresponding author. Tel.: q1-415-6953835; fax: q1-415-8266541; e-mail: [email protected] 0165-5728r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 7 . 0 0 0 4 9 - 0
ŽFukaura et al., 1996.. In addition, TGF-b 1 has also been reported to mediate immune enhancement in some situations ŽAllen et al., 1990; Wahl et al., 1991; Weinberg et al., 1992; Sharma et al., 1996; Feldmann et al., 1996.. The role of TGF-b 1 in the CNS is not well understood Žfor review see Finch et al., 1993; Mattson et al., 1997.. TGF-b 1 has been detected in the normal murine CNS by immunohistochemical studies, mRNA analysis and TGFb 1 bioassays ŽHeine et al., 1987; Unsicker et al., 1991; Wyss-Coray et al., 1995. and TGF-b 1 protein has been detected in the cerebrospinal fluid of healthy human subjects ŽChao et al., 1994.. TGF-b 1 is expressed in most CNS cell types and is rapidly upmodulated, mainly in astrocytes and microglia, after neural injury ŽLindholm et al., 1992; Logan et al., 1992; Da Cunha et al., 1993; Morgan et al., 1993.. Increased levels of TGF-b 1 have been found in brains of patients with AIDS dementia ŽWahl et al., 1991; Da Cunha et al., 1993., Alzheimer’s disease ŽVan der Wal et al., 1993; Chao et al., 1994. and multiple sclerosis ŽLink et al., 1994.. Although these reports suggest a role for TGF-b 1 in CNS injury, it is still unclear whether overproduction of TGF-b 1 in these conditions has primarily beneficial or detrimental effects. Previously, we described a tg mouse model in which a
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bioactive form of TGF-b 1 was expressed in astrocytes ŽWyss-Coray et al., 1995.. High-level overexpression of TGF-b 1 resulted in the development of a communicating hydrocephalus, seizures, motor incoordination and runting, whereas low-level expression of the transgene induced no such alterations in tg mice. However, in all tg lines analyzed, astroglial overexpression of TGF-b 1 consistently induced a strong upmodulation of extracellular matrix proteins such as laminin, fibronectin and perlecan in the CNS, particularly in the vicinity of TGF-b 1-expressing perivascular astrocytes. None of our TGF-b 1 tg lines showed spontaneous CNS infiltration by hematogenous cells. While injections of TGF-b 1 into the rabbit retina increased vascular permeability ŽCuff et al., 1996., i.v. injections of Evan’s blue have revealed no disruption of the blood–brain barrier in unmanipulated TGF-b 1 tg mice Žunpublished observations.. To characterize more directly the effects of increased TGF-b 1 expression within the CNS on the course of immune-mediated neurological diseases, we challenged TGF-b 1 tg mice with EAE. Acute EAE in non-tg mice is characterized by focal areas of inflammation in brain and spinal cord and by the development of paralysis within 12–14 days after immunization with CNS antigens in adjuvant. This experimental model has been widely used to investigate the pathogenesis of multiple sclerosis Žfor review see Martin and McFarland, 1995.. TGF-b 1 tg mice showed a faster onset and significantly more severe disease than non-tg littermate controls. In addition, the extent of mononuclear cell infiltration of the lumbar spinal cord was substantially greater in immunized tg mice than in immunized non-tg controls.
showed no signs of hydrocephalus before or after experimental manipulations. 2.2. Preparation of mixed mouse spinal cord homogenate (MSCH) and induction of EAE Murine spinal cords were extracted as described ŽKennedy et al., 1992. from SJLrJ and Balbrc ByJ mice. Equal numbers of spinal cords from each strain were combined, homogenized with a tissue homogenizer ŽVirtis Co., Gardiner, NY. and kept at y708C until use. Mice were immunized with this mixed mouse spinal cord homogenate ŽMSCH. emulsified in an equal volume of CFA containing Mycobacterium tuberculosis H37 Ra ŽDifco Laboratories, Detroit, MI.. Each mouse received a total of 200 m l of MSCHrCFA emulsion injected subcutaneously in the base of the tail at two different sites. Immediately after antigen injection and 48 h later, 250 ng of pertussis toxin ŽList Biological Laboratories, Campbell, CA. in 200 m l of PBS was injected into a tail vein to promote the induction of EAE ŽMunoz and Mackay, 1984.. The mice were coded so that subsequent clinical and histological assessments could be performed in a blinded fashion. 2.3. Clinical eÕaluation
2. Materials and methods
From day 5 after immunization, all mice were weighed and examined daily for clinical signs of disease. Clinical signs of disease were scored as follows by an investigator blinded with respect to the genotype of the mice: Grade 0, no signs of disease: Grade 1, limp tail and ruffled fur; Grade 2, Grade 1 findings plus hind limb weakness; Grade 3, Grade 1 findings plus total hind limb paralysis and Grade 4, severe paralysis and moribund. Half grades were used for intermediate clinical signs.
2.1. Mice and DNA analysis
2.4. Histology and immunocytochemistry
Non-tg SJLrJ and Balbrc ByJ mice were purchased from Jackson Laboratories ŽBar Harbor, ME.. Heterozygous tg mice were from the glial fibrillary acidic protein ŽGFAP.-pTGF-b 1 expresser line 64 described previously ŽWyss-Coray et al., 1995.. These mice express in astrocytes a porcine TGF-b 1 cDNA mutated to result in the production of bioactive TGF-b 1. The construct was originally injected into Balbrc ByJ = SJLrJ F2 hybrid one-cell embryos. The mice used in this study had been backcrossed for at least eight generations onto the SJLrJ background or for at least four generations onto the Balbrc ByJ background. Tg mice were identified by slot blot analysis of tail DNA with an SV40-specific probe as described ŽWyss-Coray et al., 1995.. Non-tg littermates served as controls in all experiments. Similar numbers of female and male mice, 8–12 weeks old were used. Altogether, 30 heterozygous TGF-b 1 tg mice and 29 non-tg littermate controls were used in four independent experiments. The low-expresser mice used in the current study
Fourteen days after immunization, mice were anesthetized by intraperitoneal injection of 0.5 ml of 3.8% chloral hydrate solution and then perfused transcardially with 0.9% saline solution for 1–2 min. After dissection, hemibrains and lumbar spinal cords were postfixed in Bouin’s fixative for 5 h at 48C and then stored overnight in 70% ethanol at 48C, embedded in paraffin and sectioned. Opposite hemibrains and parts of the lower spinal cords were embedded in OCT compound ŽMiles Diagnostics Division, Elkhart, IN., snap-frozen in dry icerisopentane and stored at y708C before cryosectioning. Two to three paraffin-embedded sagittal brain sections and two to three longitudinal spinal cord sections from each mouse were stained with hematoxylin and eosin and were analyzed to assess mononuclear cell infiltrates. Infiltrates in spinal cords were scored as follows: Grade 0, no infiltrates; Grade 1, single infiltrating cells along meninges andror ventricular spaces; Grade 2, clear cellular infiltrates, some in small clusters; Grade 3, large numbers of
T. Wyss-Coray et al.r Journal of Neuroimmunology 77 (1997) 45–50
cellular clusters along meninges and blood vessels, clusters not connected in spinal cord and Grade 4, clear intraparenchymal infiltrates, large areas of infiltrating cells occupying up to one fifth of the width of the spinal cord. Scores obtained from spinal cords by two investigators blinded as to the genotypes of mice were averaged. Scores obtained from the investigators were identical in 11 of 20 cases; in six cases the scores differed by 0.5 grade and in three by 1 grade. Immunostaining for TGF-b 1 was performed essentially as described ŽWyss-Coray et al., 1995.. Briefly, sections were deparaffinized, blocked for 30 min with 2% goat serum in Tris-buffered saline ŽTBS. Ž50 mM Tris–HCl ŽpH 7.4., 150 mM NaCl. followed by sequential 10 min incubations with avidin and biotin solutions ŽVector Laboratories, Burlingame, CA.. Sections were then rinsed in TBS and incubated overnight at 48C with the rabbit antiTGF-b 1 antiserum G4 Ž1:400 in TBS.. Immunostaining was revealed using the rabbit peroxidase ABC kit from Vector Laboratories and development in hydrogen peroxide and 3,3X-diaminobenzidine ŽDAB. or 3-amino-9-ethyl carbazole ŽAEC. ŽBiogenex, San Ramon, CA.. Immunohistochemical staining for immune markers was performed on 10 m m cryosections as described ŽBorrow et al., 1995.. Briefly, sections were fixed with acetone, blocked with avidin and biotin solutions ŽVector Laboratories. and incubated first with primary antibodies diluted in PBS Žsee below. and then with species-matched biotinylated secondary antibodies ŽBioGenex, San Ramon, CA. diluted 1:50 in PBS. Biotinylated antibodies were detected with streptavidin-peroxidase ŽJackson Immunoresearch L a b o ra to rie s , W e s t G ro v e , P A . a n d diaminobenzidinerH 2 O 2 substrate. The following primary antibodies were used: rat IgG2a anti-mouse CD4 monoclonal antibody Ždiluted 1:100.; a combination of rat IgG2a anti-mouse CD8a monoclonal antibodies Ž1:50. and rat IgG1 anti-mouse CD8b monoclonal antibody Ž1:100. Žantibodies from Pharmingen, San Diego, CA.; F4r80 Ž1:100., a rat IgG2b monoclonal antibody that recognizes mature cells of the mouse monocytermacrophage lineage Žtissue culture supernatant used at 1:10.; M1r42 Ž1:100., a rat IgG2a monoclonal antibody against MHC class I ŽBoehringer Mannheim Biochemicals, Indianapolis, IN. and MK-S4 Ž1:200., a mouse IgG2b monoclonal antibody that recognizes MHC antigen I-As but not I-Ad ŽKappler et al., 1981.. Two-color immunocytochemical staining for simultaneous detection of macrophagermicroglia and MHC class II antigen was performed as described ŽSurh et al., 1992.. Briefly, 10 m m cryosections were fixed with acetone, blocked with avidin and biotin solutions ŽVector Laboratories. and incubated sequentially with F4r80, a biotinylated anti-rat secondary antibody ŽBioGenex. and alkaline phosphatase-conjugated streptavidin ŽJackson Immunoresearch Laboratories. diluted at 1:100 in PBS. They were then blocked again with avidin and biotin solutions, incubated
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with biotinylated MK-S4 and followed by peroxidase-conjugated streptavidin ŽJackson Immunoresearch Laboratories.. Two-color reactions were then carried out, the first using fast blue BB as a substrate for alkaline phosphatase and the second using AEC as a substrate for peroxidase. Cells of the macrophage lineage were thus identified by a blue product and MHC class II by a red product.
3. Results To test the effect of astroglial overproduction of TGF-b 1 on inflammatory CNS disease in vivo, we immunized TGF-b 1 tg mice and non-tg littermate controls with MSCHrCFA to induce EAE. TGF-b 1 tg mice showed an earlier onset and a more severe disease than non-tg controls. By 10 days after immunization, 67% of tg and 27% of non-tg mice showed some evidence of disease and at 12 and 14 days, clinical signs were significantly more severe in TGF-b 1 tg than in non-tg control mice ŽFig. 1.. No clear disease manifestations were observed in any of the immunized mice between days 5 and 10 after immunization, indicating that the enhanced disease in TGF-b 1 tg mice was not due to an increased susceptibility to the
Fig. 1. Development of experimental autoimmune encephalomyelitis ŽEAE. in TGF-b 1 tg mice and non-tg controls after peripheral immunization with mouse spinal cord homogenate and CFA. Beginning 5 days after immunization, groups of 15 heterozygous TGF-b 1 tg and 11 non-tg mice were assessed daily for clinical signs of disease by an investigator blinded with respect to their genotypes. Clinical symptoms were scored from 0–4 as outlined in Section 2. Results shown represent clinical scores 10, 12 and 14 days after immunization of TGF-b 1 tg mice and non-tg controls from two independent experiments. Each circle represents one animal; columns and error bars represent means and SEM, respectively. Clinical signs were more severe in TGF-b 1 tg mice than in non-tg controls at days 12 and 14 Ž ) ps 0.017; ) ) ps 0.0018, by two-tailed, unpaired Student’s t-test..
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pertussis toxin used in all mice to promote the development of acute EAE Žsee Section 2.. To evaluate further the disease-promoting effect of CNS-derived TGF-b 1 on EAE, we back-crossed GFAPTGF-b 1 tg mice from the EAE-sensitive SJLrJ genetic background to the more EAE-resistant Balbrc ByJ background. After four to six generations of back crossing onto Balbrc ByJ, mice were challenged with MSCHrCFA and
Fig. 3. Immunocytochemical analysis of TGF-b 1 expression and mononuclear cell infiltrates in spinal cords from TGF-b 1 tg mice with EAE. ŽA. TGF-b 1 immunolabeling of parenchymal astrocytes. ŽB, C. Large numbers of infiltrating cells positive for MHC class II Žred. and F4r80 Žblue. were found along the central canal ŽB., as well as in perivascular and meningeal infiltrates ŽC.. Original magnification for photomicrographs was =64.
Fig. 2. Analysis of mononuclear cell infiltrates in spinal cords of heterozygous TGF-b 1 tg and non-tg control mice 14 days after induction of EAE. ŽA, B. Hematoxylin and eosin-stained longitudinal sections from the lower lumbar spinal cord Žclose to the pial surface. from two mice with EAE, a non-tg control ŽA. and a TGF-b 1 tg mouse ŽB.. Original magnification for photomicrographs was =64. ŽC. The extent of mononuclear cell infiltration of spinal cords in TGF-b 1 tg mice Ž ns11. and non-tg controls Ž ns9. was scored as described in Section 2. Columns and error bars represent means and SEM, respectively. TGF-b 1 tg mice had significantly higher scores than non-tg mice; ) ps 0.013 by two-tailed, unpaired Student’s t-test.
analyzed. Signs of EAE were significantly more severe in TGF-b 1 tg Balbrc ByJ mice than in non-tg littermate controls Žmean clinical disease score, 2.4 " 0.22 Žtg. versus 1.5 " 0.2 Žnon-tg.; p s 0.008; two-tailed, unpaired Student’s t-test, n s 7 per groups.. These findings indicate that TGF-b 1 significantly increased the susceptibility of Balbrc ByJ mice to EAE. To determine why EAE developed more quickly and was more severe in TGF-b 1 tg mice, we analyzed EAE spinal cords from TGF-b 1 tg Ž n s 11. and non-tg Ž n s 9. mice of the SJLrJ background. Unmanipulated TGF-b 1 tg
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mice showed no immune infiltrates in their brains ŽWyssCoray et al., 1995. or spinal cords Ždata not shown.. However, at the peak of clinical disease manifestations Ž14 days after induction of EAE., the degree of mononuclear cell infiltration in the spinal cord ŽFig. 2. and brain Žnot shown. was significantly greater in TGF-b 1 tg mice than in non-tg controls. Transgene-derived TGF-b 1 was expressed widely by astrocytes in spinal cords of TGF-b 1 tg mice ŽFig. 3A.. Immunohistochemical analysis of brains and spinal cords with mAbs specific for immune cell surface markers confirmed the greater increase in mononuclear cell infiltrates in the spinal cords of TGF-b 1 tg mice compared with non-tg controls. Large numbers of MHC class II positive, and thus activated cells of the macrophage lineage were detected in TGF-b 1 tg spinal cords along the central canal ŽFig. 3B. as well as in perivascular and meningeal infiltrates ŽFig. 3C.. However, no clear differences in the cellular composition of these infiltrates were observed between the two groups of mice Ždata not shown..
4. Discussion TGF-b 1 protein levels in postmortem tissue or cerebrospinal fluid have been found to be elevated in patients with AIDS dementia, multiple sclerosis and Alzheimer’s disease ŽWahl et al., 1991; Da Cunha et al., 1993; Link et al., 1994; Chao et al., 1994.. However, it remains unclear whether the expression of TGF-b 1 in these conditions represents a strictly protective compensatory response or somehow contributes to the disease process ŽFinch et al., 1993; Mattson et al., 1997.. Several studies have demonstrated that systemically administered TGF-b 1 inhibits EAE ŽRacke et al., 1991; Kuruvilla et al., 1991; Santambrogio et al., 1993. and that TGF-b 1 is produced by myelin-specific T-lymphocytes isolated from peripheral blood and cerebrospinal fluid of patients with multiple sclerosis ŽLink et al., 1994.. Tlymphocytes that produce TGF-b 1 are thought to be antiinflammatory because they are specifically induced in oral immunization protocols that lead to attenuation of EAE and possibly multiple sclerosis ŽFukaura et al., 1996.. In contrast, the results presented here demonstrate that, at least in certain pathological conditions, overexpression of TGF-b 1 by resident CNS cells can promote inflammation and immune-mediated CNS disease. Our findings are consistent with recent studies in rheumatoid arthritis and kidney fibrosis, where TGF-b 1 enhanced local inflammation ŽFeldmann et al., 1996; Sharma et al., 1996.. These data emphasize how difficult it is to predict the in vivo effects of cytokines in specific pathophysiological situations without rigorous experimental assessment in the living organism. As demonstrated here, tg models can be helpful in this regard as they allow for the reliable expression of select proteins Ždevoid of any impurities. in specific organs and cells.
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It is possible that the EAE-enhancing effect of local TGF-b 1 expression relates to the chemoattractant properties of this cytokine ŽWahl et al., 1991.. The absence of inflammatory infiltrates in unmanipulated TGF-b 1 tg mice indicates that astroglial overexpression of TGF-b 1 per se is not sufficient to lead to CNS inflammation. However, a leaky blood–brain barrier and a CNS antigen-specific immune response may generate the appropriate scenario for TGF-b 1-induced chemotaxis to exert a significant immunopathological effect in the CNS environment. Chemokines may play an important role in this process. For example, chemokines are upmodulated in the CNS of animals with EAE and their expression levels correlate with disease severity ŽRansohoff et al., 1993; Godiska et al., 1995.. Notably, TGF-b 1 has been shown to induce the production of the chemokine monocyte chemoattractant protein-1 by astrocytes in vitro ŽHurwitz et al., 1995.. The ability of TGF-b 1 to induce proteoglycans ŽWyss-Coray et al., 1995. and integrins ŽIgnotz and Massague, 1988. may further increase the effectiveness of chemokines and facilitate increased transcytosis of inflammatory cells. We conclude that increased glial production of TGF-b 1 can have a detrimental effect on immune-mediated CNS disease and, hence, that overexpression of TGF-b 1 in the CNS parenchyma in such conditions could be harmful. It is possible that genetic traits or CNS disorders that promote upmodulation of TGF-b 1 levels in the CNS enhance an individual’s susceptibility to immune-mediated CNS damage.
Acknowledgements This work was supported in part by the National Institute on Aging ŽAG-11385; L.M... T.W.-C. was supported by a Fellowship Award from the Swiss National Science Foundation and L.M. by a Harry Weaver Neuroscience Scholarship Award from the National Multiple Sclerosis Society. P.B. was supported in part by the NIAID, Divison of AIDS ŽAI37430-02..
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Astroglial overproduction of TGF-b 1 enhances inflammatory central nervous system disease in transgenic mice Tony Wyss-Coray a
a,)
, Persephone Borrow b, Madelyne J. Brooker b, Lennart Mucke
a
Gladstone Molecular Neurobiology Program and Department of Neurology, UniÕersity of California San Francisco, CA 94141, USA b Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037, USA Received 27 November 1996; revised 3 February 1997; accepted 5 February 1997
Abstract Cerebral expression of the injury response cytokine transforming growth factor-b 1 ŽTGF-b 1. has been found to be increased in several neurological diseases but it remains unclear whether its function is primarily beneficial or detrimental. Here we show that transgenic Žtg. mice that overexpress bioactive TGF-b 1 in the central nervous system ŽCNS. and show no overt phenotype in the unmanipulated state, are more susceptible to the immune-mediated CNS disease experimental autoimmune encephalomyelitis ŽEAE.. TGF-b 1 tg mice with EAE showed an earlier onset of clinical symptoms, more severe disease and increased mononuclear cell infiltration in their spinal cords compared with non-tg littermate controls with EAE. Whereas previous observations indicated that increased peripheral levels of TGF-b 1 can suppress EAE, our findings demonstrate that local expression of TGF-b 1 within the CNS parenchyma can enhance immune cell infiltration and intensify the CNS impairment resulting from peripherally triggered autoimmune responses. q 1997 Elsevier Science B.V. Keywords: Cytokine; Multiple sclerosis; Gliosis; Autoimmunity
1. Introduction TGF-b 1 is a multifunctional cytokine that has been implicated in the control of inflammation and immune responses, cellular proliferation and differentiation, extracellular matrix production and degradation, angiogenesis, cell adhesion and tissue repair Žfor review see Roberts and Sporn, 1988; Massague, ´ 1990; Border and Ruoslahti, 1992; McCartney-Francis and Wahl, 1994.. TGF-b 1-deficient mice develop a multifocal autoimmune-type inflammatory disease ŽShull et al., 1992; Kulkarni et al., 1993.. Given systemically, TGF-b 1 inhibits autoimmune diseases such as experimental autoimmune encephalomyelitis ŽEAE., whereas neutralizing antibodies against TGF-b 1 enhance EAE ŽRacke et al., 1991; Kuruvilla et al., 1991; Santambrogio et al., 1993; Johns and Sriram, 1993.. TGF-b 1 seems to play a significant role in the induction of oral tolerance against autoantigens, which is currently being studied as a treatment for EAE and multiple sclerosis Abbreviations: CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; MSCH, mouse spinal cord homogenate ) Corresponding author. Tel.: q1-415-6953835; fax: q1-415-8266541; e-mail: [email protected] 0165-5728r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 7 . 0 0 0 4 9 - 0
ŽFukaura et al., 1996.. In addition, TGF-b 1 has also been reported to mediate immune enhancement in some situations ŽAllen et al., 1990; Wahl et al., 1991; Weinberg et al., 1992; Sharma et al., 1996; Feldmann et al., 1996.. The role of TGF-b 1 in the CNS is not well understood Žfor review see Finch et al., 1993; Mattson et al., 1997.. TGF-b 1 has been detected in the normal murine CNS by immunohistochemical studies, mRNA analysis and TGFb 1 bioassays ŽHeine et al., 1987; Unsicker et al., 1991; Wyss-Coray et al., 1995. and TGF-b 1 protein has been detected in the cerebrospinal fluid of healthy human subjects ŽChao et al., 1994.. TGF-b 1 is expressed in most CNS cell types and is rapidly upmodulated, mainly in astrocytes and microglia, after neural injury ŽLindholm et al., 1992; Logan et al., 1992; Da Cunha et al., 1993; Morgan et al., 1993.. Increased levels of TGF-b 1 have been found in brains of patients with AIDS dementia ŽWahl et al., 1991; Da Cunha et al., 1993., Alzheimer’s disease ŽVan der Wal et al., 1993; Chao et al., 1994. and multiple sclerosis ŽLink et al., 1994.. Although these reports suggest a role for TGF-b 1 in CNS injury, it is still unclear whether overproduction of TGF-b 1 in these conditions has primarily beneficial or detrimental effects. Previously, we described a tg mouse model in which a
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bioactive form of TGF-b 1 was expressed in astrocytes ŽWyss-Coray et al., 1995.. High-level overexpression of TGF-b 1 resulted in the development of a communicating hydrocephalus, seizures, motor incoordination and runting, whereas low-level expression of the transgene induced no such alterations in tg mice. However, in all tg lines analyzed, astroglial overexpression of TGF-b 1 consistently induced a strong upmodulation of extracellular matrix proteins such as laminin, fibronectin and perlecan in the CNS, particularly in the vicinity of TGF-b 1-expressing perivascular astrocytes. None of our TGF-b 1 tg lines showed spontaneous CNS infiltration by hematogenous cells. While injections of TGF-b 1 into the rabbit retina increased vascular permeability ŽCuff et al., 1996., i.v. injections of Evan’s blue have revealed no disruption of the blood–brain barrier in unmanipulated TGF-b 1 tg mice Žunpublished observations.. To characterize more directly the effects of increased TGF-b 1 expression within the CNS on the course of immune-mediated neurological diseases, we challenged TGF-b 1 tg mice with EAE. Acute EAE in non-tg mice is characterized by focal areas of inflammation in brain and spinal cord and by the development of paralysis within 12–14 days after immunization with CNS antigens in adjuvant. This experimental model has been widely used to investigate the pathogenesis of multiple sclerosis Žfor review see Martin and McFarland, 1995.. TGF-b 1 tg mice showed a faster onset and significantly more severe disease than non-tg littermate controls. In addition, the extent of mononuclear cell infiltration of the lumbar spinal cord was substantially greater in immunized tg mice than in immunized non-tg controls.
showed no signs of hydrocephalus before or after experimental manipulations. 2.2. Preparation of mixed mouse spinal cord homogenate (MSCH) and induction of EAE Murine spinal cords were extracted as described ŽKennedy et al., 1992. from SJLrJ and Balbrc ByJ mice. Equal numbers of spinal cords from each strain were combined, homogenized with a tissue homogenizer ŽVirtis Co., Gardiner, NY. and kept at y708C until use. Mice were immunized with this mixed mouse spinal cord homogenate ŽMSCH. emulsified in an equal volume of CFA containing Mycobacterium tuberculosis H37 Ra ŽDifco Laboratories, Detroit, MI.. Each mouse received a total of 200 m l of MSCHrCFA emulsion injected subcutaneously in the base of the tail at two different sites. Immediately after antigen injection and 48 h later, 250 ng of pertussis toxin ŽList Biological Laboratories, Campbell, CA. in 200 m l of PBS was injected into a tail vein to promote the induction of EAE ŽMunoz and Mackay, 1984.. The mice were coded so that subsequent clinical and histological assessments could be performed in a blinded fashion. 2.3. Clinical eÕaluation
2. Materials and methods
From day 5 after immunization, all mice were weighed and examined daily for clinical signs of disease. Clinical signs of disease were scored as follows by an investigator blinded with respect to the genotype of the mice: Grade 0, no signs of disease: Grade 1, limp tail and ruffled fur; Grade 2, Grade 1 findings plus hind limb weakness; Grade 3, Grade 1 findings plus total hind limb paralysis and Grade 4, severe paralysis and moribund. Half grades were used for intermediate clinical signs.
2.1. Mice and DNA analysis
2.4. Histology and immunocytochemistry
Non-tg SJLrJ and Balbrc ByJ mice were purchased from Jackson Laboratories ŽBar Harbor, ME.. Heterozygous tg mice were from the glial fibrillary acidic protein ŽGFAP.-pTGF-b 1 expresser line 64 described previously ŽWyss-Coray et al., 1995.. These mice express in astrocytes a porcine TGF-b 1 cDNA mutated to result in the production of bioactive TGF-b 1. The construct was originally injected into Balbrc ByJ = SJLrJ F2 hybrid one-cell embryos. The mice used in this study had been backcrossed for at least eight generations onto the SJLrJ background or for at least four generations onto the Balbrc ByJ background. Tg mice were identified by slot blot analysis of tail DNA with an SV40-specific probe as described ŽWyss-Coray et al., 1995.. Non-tg littermates served as controls in all experiments. Similar numbers of female and male mice, 8–12 weeks old were used. Altogether, 30 heterozygous TGF-b 1 tg mice and 29 non-tg littermate controls were used in four independent experiments. The low-expresser mice used in the current study
Fourteen days after immunization, mice were anesthetized by intraperitoneal injection of 0.5 ml of 3.8% chloral hydrate solution and then perfused transcardially with 0.9% saline solution for 1–2 min. After dissection, hemibrains and lumbar spinal cords were postfixed in Bouin’s fixative for 5 h at 48C and then stored overnight in 70% ethanol at 48C, embedded in paraffin and sectioned. Opposite hemibrains and parts of the lower spinal cords were embedded in OCT compound ŽMiles Diagnostics Division, Elkhart, IN., snap-frozen in dry icerisopentane and stored at y708C before cryosectioning. Two to three paraffin-embedded sagittal brain sections and two to three longitudinal spinal cord sections from each mouse were stained with hematoxylin and eosin and were analyzed to assess mononuclear cell infiltrates. Infiltrates in spinal cords were scored as follows: Grade 0, no infiltrates; Grade 1, single infiltrating cells along meninges andror ventricular spaces; Grade 2, clear cellular infiltrates, some in small clusters; Grade 3, large numbers of
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cellular clusters along meninges and blood vessels, clusters not connected in spinal cord and Grade 4, clear intraparenchymal infiltrates, large areas of infiltrating cells occupying up to one fifth of the width of the spinal cord. Scores obtained from spinal cords by two investigators blinded as to the genotypes of mice were averaged. Scores obtained from the investigators were identical in 11 of 20 cases; in six cases the scores differed by 0.5 grade and in three by 1 grade. Immunostaining for TGF-b 1 was performed essentially as described ŽWyss-Coray et al., 1995.. Briefly, sections were deparaffinized, blocked for 30 min with 2% goat serum in Tris-buffered saline ŽTBS. Ž50 mM Tris–HCl ŽpH 7.4., 150 mM NaCl. followed by sequential 10 min incubations with avidin and biotin solutions ŽVector Laboratories, Burlingame, CA.. Sections were then rinsed in TBS and incubated overnight at 48C with the rabbit antiTGF-b 1 antiserum G4 Ž1:400 in TBS.. Immunostaining was revealed using the rabbit peroxidase ABC kit from Vector Laboratories and development in hydrogen peroxide and 3,3X-diaminobenzidine ŽDAB. or 3-amino-9-ethyl carbazole ŽAEC. ŽBiogenex, San Ramon, CA.. Immunohistochemical staining for immune markers was performed on 10 m m cryosections as described ŽBorrow et al., 1995.. Briefly, sections were fixed with acetone, blocked with avidin and biotin solutions ŽVector Laboratories. and incubated first with primary antibodies diluted in PBS Žsee below. and then with species-matched biotinylated secondary antibodies ŽBioGenex, San Ramon, CA. diluted 1:50 in PBS. Biotinylated antibodies were detected with streptavidin-peroxidase ŽJackson Immunoresearch L a b o ra to rie s , W e s t G ro v e , P A . a n d diaminobenzidinerH 2 O 2 substrate. The following primary antibodies were used: rat IgG2a anti-mouse CD4 monoclonal antibody Ždiluted 1:100.; a combination of rat IgG2a anti-mouse CD8a monoclonal antibodies Ž1:50. and rat IgG1 anti-mouse CD8b monoclonal antibody Ž1:100. Žantibodies from Pharmingen, San Diego, CA.; F4r80 Ž1:100., a rat IgG2b monoclonal antibody that recognizes mature cells of the mouse monocytermacrophage lineage Žtissue culture supernatant used at 1:10.; M1r42 Ž1:100., a rat IgG2a monoclonal antibody against MHC class I ŽBoehringer Mannheim Biochemicals, Indianapolis, IN. and MK-S4 Ž1:200., a mouse IgG2b monoclonal antibody that recognizes MHC antigen I-As but not I-Ad ŽKappler et al., 1981.. Two-color immunocytochemical staining for simultaneous detection of macrophagermicroglia and MHC class II antigen was performed as described ŽSurh et al., 1992.. Briefly, 10 m m cryosections were fixed with acetone, blocked with avidin and biotin solutions ŽVector Laboratories. and incubated sequentially with F4r80, a biotinylated anti-rat secondary antibody ŽBioGenex. and alkaline phosphatase-conjugated streptavidin ŽJackson Immunoresearch Laboratories. diluted at 1:100 in PBS. They were then blocked again with avidin and biotin solutions, incubated
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with biotinylated MK-S4 and followed by peroxidase-conjugated streptavidin ŽJackson Immunoresearch Laboratories.. Two-color reactions were then carried out, the first using fast blue BB as a substrate for alkaline phosphatase and the second using AEC as a substrate for peroxidase. Cells of the macrophage lineage were thus identified by a blue product and MHC class II by a red product.
3. Results To test the effect of astroglial overproduction of TGF-b 1 on inflammatory CNS disease in vivo, we immunized TGF-b 1 tg mice and non-tg littermate controls with MSCHrCFA to induce EAE. TGF-b 1 tg mice showed an earlier onset and a more severe disease than non-tg controls. By 10 days after immunization, 67% of tg and 27% of non-tg mice showed some evidence of disease and at 12 and 14 days, clinical signs were significantly more severe in TGF-b 1 tg than in non-tg control mice ŽFig. 1.. No clear disease manifestations were observed in any of the immunized mice between days 5 and 10 after immunization, indicating that the enhanced disease in TGF-b 1 tg mice was not due to an increased susceptibility to the
Fig. 1. Development of experimental autoimmune encephalomyelitis ŽEAE. in TGF-b 1 tg mice and non-tg controls after peripheral immunization with mouse spinal cord homogenate and CFA. Beginning 5 days after immunization, groups of 15 heterozygous TGF-b 1 tg and 11 non-tg mice were assessed daily for clinical signs of disease by an investigator blinded with respect to their genotypes. Clinical symptoms were scored from 0–4 as outlined in Section 2. Results shown represent clinical scores 10, 12 and 14 days after immunization of TGF-b 1 tg mice and non-tg controls from two independent experiments. Each circle represents one animal; columns and error bars represent means and SEM, respectively. Clinical signs were more severe in TGF-b 1 tg mice than in non-tg controls at days 12 and 14 Ž ) ps 0.017; ) ) ps 0.0018, by two-tailed, unpaired Student’s t-test..
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pertussis toxin used in all mice to promote the development of acute EAE Žsee Section 2.. To evaluate further the disease-promoting effect of CNS-derived TGF-b 1 on EAE, we back-crossed GFAPTGF-b 1 tg mice from the EAE-sensitive SJLrJ genetic background to the more EAE-resistant Balbrc ByJ background. After four to six generations of back crossing onto Balbrc ByJ, mice were challenged with MSCHrCFA and
Fig. 3. Immunocytochemical analysis of TGF-b 1 expression and mononuclear cell infiltrates in spinal cords from TGF-b 1 tg mice with EAE. ŽA. TGF-b 1 immunolabeling of parenchymal astrocytes. ŽB, C. Large numbers of infiltrating cells positive for MHC class II Žred. and F4r80 Žblue. were found along the central canal ŽB., as well as in perivascular and meningeal infiltrates ŽC.. Original magnification for photomicrographs was =64.
Fig. 2. Analysis of mononuclear cell infiltrates in spinal cords of heterozygous TGF-b 1 tg and non-tg control mice 14 days after induction of EAE. ŽA, B. Hematoxylin and eosin-stained longitudinal sections from the lower lumbar spinal cord Žclose to the pial surface. from two mice with EAE, a non-tg control ŽA. and a TGF-b 1 tg mouse ŽB.. Original magnification for photomicrographs was =64. ŽC. The extent of mononuclear cell infiltration of spinal cords in TGF-b 1 tg mice Ž ns11. and non-tg controls Ž ns9. was scored as described in Section 2. Columns and error bars represent means and SEM, respectively. TGF-b 1 tg mice had significantly higher scores than non-tg mice; ) ps 0.013 by two-tailed, unpaired Student’s t-test.
analyzed. Signs of EAE were significantly more severe in TGF-b 1 tg Balbrc ByJ mice than in non-tg littermate controls Žmean clinical disease score, 2.4 " 0.22 Žtg. versus 1.5 " 0.2 Žnon-tg.; p s 0.008; two-tailed, unpaired Student’s t-test, n s 7 per groups.. These findings indicate that TGF-b 1 significantly increased the susceptibility of Balbrc ByJ mice to EAE. To determine why EAE developed more quickly and was more severe in TGF-b 1 tg mice, we analyzed EAE spinal cords from TGF-b 1 tg Ž n s 11. and non-tg Ž n s 9. mice of the SJLrJ background. Unmanipulated TGF-b 1 tg
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mice showed no immune infiltrates in their brains ŽWyssCoray et al., 1995. or spinal cords Ždata not shown.. However, at the peak of clinical disease manifestations Ž14 days after induction of EAE., the degree of mononuclear cell infiltration in the spinal cord ŽFig. 2. and brain Žnot shown. was significantly greater in TGF-b 1 tg mice than in non-tg controls. Transgene-derived TGF-b 1 was expressed widely by astrocytes in spinal cords of TGF-b 1 tg mice ŽFig. 3A.. Immunohistochemical analysis of brains and spinal cords with mAbs specific for immune cell surface markers confirmed the greater increase in mononuclear cell infiltrates in the spinal cords of TGF-b 1 tg mice compared with non-tg controls. Large numbers of MHC class II positive, and thus activated cells of the macrophage lineage were detected in TGF-b 1 tg spinal cords along the central canal ŽFig. 3B. as well as in perivascular and meningeal infiltrates ŽFig. 3C.. However, no clear differences in the cellular composition of these infiltrates were observed between the two groups of mice Ždata not shown..
4. Discussion TGF-b 1 protein levels in postmortem tissue or cerebrospinal fluid have been found to be elevated in patients with AIDS dementia, multiple sclerosis and Alzheimer’s disease ŽWahl et al., 1991; Da Cunha et al., 1993; Link et al., 1994; Chao et al., 1994.. However, it remains unclear whether the expression of TGF-b 1 in these conditions represents a strictly protective compensatory response or somehow contributes to the disease process ŽFinch et al., 1993; Mattson et al., 1997.. Several studies have demonstrated that systemically administered TGF-b 1 inhibits EAE ŽRacke et al., 1991; Kuruvilla et al., 1991; Santambrogio et al., 1993. and that TGF-b 1 is produced by myelin-specific T-lymphocytes isolated from peripheral blood and cerebrospinal fluid of patients with multiple sclerosis ŽLink et al., 1994.. Tlymphocytes that produce TGF-b 1 are thought to be antiinflammatory because they are specifically induced in oral immunization protocols that lead to attenuation of EAE and possibly multiple sclerosis ŽFukaura et al., 1996.. In contrast, the results presented here demonstrate that, at least in certain pathological conditions, overexpression of TGF-b 1 by resident CNS cells can promote inflammation and immune-mediated CNS disease. Our findings are consistent with recent studies in rheumatoid arthritis and kidney fibrosis, where TGF-b 1 enhanced local inflammation ŽFeldmann et al., 1996; Sharma et al., 1996.. These data emphasize how difficult it is to predict the in vivo effects of cytokines in specific pathophysiological situations without rigorous experimental assessment in the living organism. As demonstrated here, tg models can be helpful in this regard as they allow for the reliable expression of select proteins Ždevoid of any impurities. in specific organs and cells.
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It is possible that the EAE-enhancing effect of local TGF-b 1 expression relates to the chemoattractant properties of this cytokine ŽWahl et al., 1991.. The absence of inflammatory infiltrates in unmanipulated TGF-b 1 tg mice indicates that astroglial overexpression of TGF-b 1 per se is not sufficient to lead to CNS inflammation. However, a leaky blood–brain barrier and a CNS antigen-specific immune response may generate the appropriate scenario for TGF-b 1-induced chemotaxis to exert a significant immunopathological effect in the CNS environment. Chemokines may play an important role in this process. For example, chemokines are upmodulated in the CNS of animals with EAE and their expression levels correlate with disease severity ŽRansohoff et al., 1993; Godiska et al., 1995.. Notably, TGF-b 1 has been shown to induce the production of the chemokine monocyte chemoattractant protein-1 by astrocytes in vitro ŽHurwitz et al., 1995.. The ability of TGF-b 1 to induce proteoglycans ŽWyss-Coray et al., 1995. and integrins ŽIgnotz and Massague, 1988. may further increase the effectiveness of chemokines and facilitate increased transcytosis of inflammatory cells. We conclude that increased glial production of TGF-b 1 can have a detrimental effect on immune-mediated CNS disease and, hence, that overexpression of TGF-b 1 in the CNS parenchyma in such conditions could be harmful. It is possible that genetic traits or CNS disorders that promote upmodulation of TGF-b 1 levels in the CNS enhance an individual’s susceptibility to immune-mediated CNS damage.
Acknowledgements This work was supported in part by the National Institute on Aging ŽAG-11385; L.M... T.W.-C. was supported by a Fellowship Award from the Swiss National Science Foundation and L.M. by a Harry Weaver Neuroscience Scholarship Award from the National Multiple Sclerosis Society. P.B. was supported in part by the NIAID, Divison of AIDS ŽAI37430-02..
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