International Immunology, Vol 4. No. 5, pp.
615-620
© 1992 Oxford University Press
Evidence of endogenous regulatory function of transforming growth factor-/ experimental allergic encephalomyelitis
in
Michael K. Racke, Barbara Cannella3, Paul Albert1, Michael Sporn2, Cedric S. Raine3, and Dale E. McFarlin The Neuroimmunology Branch and biometry and Field Studies Branch, National Institute of Neurological Diseases and Stroke and 2Laboratory of Chemoprevention, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA, 3Division of Neuropathology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
Abstract Experimental allergic encephalomyelitis (EAE) Is an autoimmune disease characterized by inflammation and demyellnation in the central nervous system (CNS). Administration of transforming growth factor-/? (TGF-/3) has been shown to inhibit EAE. In this study, the possible role of endogenous TGF-/3 in the regulation of relapsing EAE produced by the transfer of myelin basic protein-specific T cell lines was assessed. Although TGF-/3 Is not present In the normal CNS, this cytokine was detected by immunohistology In areas of central nervous system inflammation in both acute and chronic disease. The administration of anti-TGF-0 at the disease onset led to a worsening of the clinical course of EAE and more extensive pathological lesions. These findings provide direct evidence for a role of endogenous TGF-/9 In the remissions seen in chronic relapsing EAE. Introduction
Methods +
Transfer of MBP-specific CD4 class II MHC-restricted T cells into naive, syngeneic SJL/J mice produces chronic, relapsing EAE, a disorder that resembles the human disease multiple sclerosis both clinically and pathologically (1,2). Recently, it has been demonstrated that administration of TGF-/31 improved the clinical course of EAE, even when given during ongoing disease (3-5). TGF-/31 belongs to a family of molecules with profound immunosuppressive effects including inhibition of T cell activation and proliferation, decreased lymphokine production, inhibition of IFN-7-induced class II MHC expression, and decrease in the generation of cytotoxic lymphocytes (6-10). TGF-0 has been postulated to participate in the pathogenesis of other inflammatory disorders such as arthritis (4,11), hepatitis (12,13), and glomerulonephritis (14,15) and it has been suggested that the secretion of TGF-0 by T cells during recovery from EAE contributes to remissions of the disease (16). In this study, the endogenous role of TGF-/3 in the regulation of EAE was examined.
Mice Female SJL/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME) at 4 weeks of age. All mice used in the experiments described were 8 - 1 2 weeks of age. Production of EAE MBP was prepared from guinea pig spinal cords (Pel-Freez, Rogers, AR) (17). MBP-specific LNC were generated by immunizing with MBP (400 /*g) and harvesting the draining nodes after 10 days (3). After 4 days of incubation with MBP (25 /ig/ml), LNC were washed and injected (3x10 7 cells/0.2 ml) i.v. into nave mice. These mice were examined daily for signs of disease and graded on a 0 - 5 scale of increasing severity: 0, no abnormality; 1, a floppy tail with mild hindlimb weakness; 2, a floppy tail with moderate hindlimb weakness; 3, severe hind leg paresis but not complete paralysis; 4, total hindlimb paralysis, which may be associated with moderate forelimb weakness; 5, quadriplegia or premoribund state (2).
Corresponding author: M. K. Racke Transmitting editor: L. Sieinman
Received 15 January 1992, accepted 14 February 1992
Downloaded from http://intimm.oxfordjournals.org/ at University of Otago on July 10, 2015
Key words: experimental allergic encephalomyelitis, transforming growth factor-/3
616
Function for TGF-/3 in EAE
Anti-TGF-01 Antjserum to TGF-/31 was produced in turkeys (18). The potency of this antiserum was measured by blocking the binding of [12SI]TGF-/31 to A549 human lung carcinoma cells, which have TGF-/3 receptors (18). It was determined that 1 /d of antiserum blocked the binding of 4 ng TGF-01. Normal turkey serum (NTS) was used as a control. Treatment of EAE with anti-TGF-/31 Groups of five mice that received activated MBP-specific LNC were given either anti-TGF-/31 or NTS diluted in PBS via the tail vein on indicated days post-transfer of encephalitogenic cells. Control mice injected with the antiserum did not develop any signs of illness. Clinical observers of mice were blinded as to whether mice had received anti-TGF-/31 or placebo.
cells. Prior to the development of inflammation, TGF-/3 was not detected by immunostaining. This is consistent with observations that there is negligible expression of TGF-/31 in the CNS of normal adult mice (22). Immunohistochemical studies of the CNS during the first episode of disease and following the first and third episodes of disease consistently demonstrated TGF-/3 in the areas of perivascular and subpial inflammation characteristic of EAE (Fig. 1). Similar staining for TGF-/3 was observed in both control and anti-TGF-/31 treated animals. Astrocytes in the nearby white matter also showed positive staining for TGF-0. TGF-/3 was also present in inflamed areas of the CNS in animals during the chronic phase of disease. Enhancement of EAE by administering anti-TGF-/31 Because administration of TGF-01 suppressed clinical EAE, a series of experiments was performed to determine if anti-TGF-
Immunocytochemistry and pathology
4 *.* B
Statistical methods The average clinical scores for 8-day blocks of time were computed on each mouse in each experiment. Comparisons between anti-TGF-j31 and placebo groups for each block of time were made with two-sample f-tests (20). Treatment effects were also assessed by using a multivariate approach (Hotellings T2), which takes into account the correlation across time in comparing the resulting curves (21). Results Demonstration of TGF-f} in CNS inflammatory lesions in EAE The presence of TGF-/3 in the CNS was examined by immunohistology at various stages after the adoptive transfer of immune
Fig. 1. (A) Frozen section of lumbar spinal cord from a mouse treated with anti-TGF-01 for 5 days and sampled during the acute phase of EAE. The section is reacted with anti-TGF-/3 and shows TGF-0 + cells along a penetrating blood vessel and TGF-/3+ astrocytes in the white matter parenchyma x420 (B) Similar section from an anti-TGF-01 -treated mouse sampled during the first episode of disease. Several TGF-0+ cells are seen in the subpial region to the right and following the course of a penetrating vessel. x420.
Downloaded from http://intimm.oxfordjournals.org/ at University of Otago on July 10, 2015
CNS tissue was removed after whole body perfusion with PBS and embedded in OCT compound (Lab-Tek Division, Miles Laboratories, Naperville, IL) in a dry ice bath for immunocytochemistry or fixed by immersion in 2.5% glutaraldehyde for evaluation of pathology in 1 ^m thick epoxy sections. The expression of various molecules recognized by the antibodies was assessed by indirect staining with the avidin - biotin - peroxidase (ABC) technique (19). After fixation with acetone for 10 min, cryostat sections were treated with 0.03% H2O2 to quench endogenous peroxidase, followed by blocking with normal serum. Primary antibodies were applied overnight at 4°C at the following dilutions: anti-TGF-/3 (R&D Systems, Minneapolis, MN) 1:100; anti-l-As from MKS-4 hybridoma (ATCC, Rockville, MD) purified by affinity chromatography, at 1:25; anti-H-2 (Boehringer-Mannheim, Indianapolis, IN) at 1:20; anti-CD4 and anti-CD8 (BectonDickinson, Mountain View, CA) at 1:50. The anti-TGF-/3 used to stain tissues recognizes both TGF-/31 and TGF-/32. Sections were then incubated with the secondary antibody, followed by the ABC reagent and 3',3-diaminobenzidme. The immunostained preparations were evaluated by two 'blinded' observers and the levels of immunoreactivity and inflammation graded as follows: - , no inflammatory cells; + / - , a few positive cells; +, organization of inflammatory infiltrates around blood vessels; + +, extensive perivascular cuffing with extension into the adjacent subarachnoid space; + + + to + + + +, extensive perivascular cuffing and increasing degrees of subarachnoid inflammation (19).
Function for TGF-/3 in EAE 617 01 would exacerbate the disease. The studies showed that administration of anti-TGF-/31 after the adoptive transfer of encephalitogenic T cells led to a more severe disease course (Fig. 2). The daily administration of 10 /J of anti-TGF-/31, capable of neutralizing 40 ng of TGF-/31, for five consecutive days (days 6 - 1 0 post transfer of encephalitogenic cells) produced a marked increase in disease severity from days 1 5 - 2 3 (Fig. 2a). During this period, the anti-TGF-01 treated mice had a mean clinical score of 2.1 compared to 1.1 for the NTS treated group (P
615-620
© 1992 Oxford University Press
Evidence of endogenous regulatory function of transforming growth factor-/ experimental allergic encephalomyelitis
in
Michael K. Racke, Barbara Cannella3, Paul Albert1, Michael Sporn2, Cedric S. Raine3, and Dale E. McFarlin The Neuroimmunology Branch and biometry and Field Studies Branch, National Institute of Neurological Diseases and Stroke and 2Laboratory of Chemoprevention, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA, 3Division of Neuropathology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
Abstract Experimental allergic encephalomyelitis (EAE) Is an autoimmune disease characterized by inflammation and demyellnation in the central nervous system (CNS). Administration of transforming growth factor-/? (TGF-/3) has been shown to inhibit EAE. In this study, the possible role of endogenous TGF-/3 in the regulation of relapsing EAE produced by the transfer of myelin basic protein-specific T cell lines was assessed. Although TGF-/3 Is not present In the normal CNS, this cytokine was detected by immunohistology In areas of central nervous system inflammation in both acute and chronic disease. The administration of anti-TGF-0 at the disease onset led to a worsening of the clinical course of EAE and more extensive pathological lesions. These findings provide direct evidence for a role of endogenous TGF-/9 In the remissions seen in chronic relapsing EAE. Introduction
Methods +
Transfer of MBP-specific CD4 class II MHC-restricted T cells into naive, syngeneic SJL/J mice produces chronic, relapsing EAE, a disorder that resembles the human disease multiple sclerosis both clinically and pathologically (1,2). Recently, it has been demonstrated that administration of TGF-/31 improved the clinical course of EAE, even when given during ongoing disease (3-5). TGF-/31 belongs to a family of molecules with profound immunosuppressive effects including inhibition of T cell activation and proliferation, decreased lymphokine production, inhibition of IFN-7-induced class II MHC expression, and decrease in the generation of cytotoxic lymphocytes (6-10). TGF-0 has been postulated to participate in the pathogenesis of other inflammatory disorders such as arthritis (4,11), hepatitis (12,13), and glomerulonephritis (14,15) and it has been suggested that the secretion of TGF-0 by T cells during recovery from EAE contributes to remissions of the disease (16). In this study, the endogenous role of TGF-/3 in the regulation of EAE was examined.
Mice Female SJL/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME) at 4 weeks of age. All mice used in the experiments described were 8 - 1 2 weeks of age. Production of EAE MBP was prepared from guinea pig spinal cords (Pel-Freez, Rogers, AR) (17). MBP-specific LNC were generated by immunizing with MBP (400 /*g) and harvesting the draining nodes after 10 days (3). After 4 days of incubation with MBP (25 /ig/ml), LNC were washed and injected (3x10 7 cells/0.2 ml) i.v. into nave mice. These mice were examined daily for signs of disease and graded on a 0 - 5 scale of increasing severity: 0, no abnormality; 1, a floppy tail with mild hindlimb weakness; 2, a floppy tail with moderate hindlimb weakness; 3, severe hind leg paresis but not complete paralysis; 4, total hindlimb paralysis, which may be associated with moderate forelimb weakness; 5, quadriplegia or premoribund state (2).
Corresponding author: M. K. Racke Transmitting editor: L. Sieinman
Received 15 January 1992, accepted 14 February 1992
Downloaded from http://intimm.oxfordjournals.org/ at University of Otago on July 10, 2015
Key words: experimental allergic encephalomyelitis, transforming growth factor-/3
616
Function for TGF-/3 in EAE
Anti-TGF-01 Antjserum to TGF-/31 was produced in turkeys (18). The potency of this antiserum was measured by blocking the binding of [12SI]TGF-/31 to A549 human lung carcinoma cells, which have TGF-/3 receptors (18). It was determined that 1 /d of antiserum blocked the binding of 4 ng TGF-01. Normal turkey serum (NTS) was used as a control. Treatment of EAE with anti-TGF-/31 Groups of five mice that received activated MBP-specific LNC were given either anti-TGF-/31 or NTS diluted in PBS via the tail vein on indicated days post-transfer of encephalitogenic cells. Control mice injected with the antiserum did not develop any signs of illness. Clinical observers of mice were blinded as to whether mice had received anti-TGF-/31 or placebo.
cells. Prior to the development of inflammation, TGF-/3 was not detected by immunostaining. This is consistent with observations that there is negligible expression of TGF-/31 in the CNS of normal adult mice (22). Immunohistochemical studies of the CNS during the first episode of disease and following the first and third episodes of disease consistently demonstrated TGF-/3 in the areas of perivascular and subpial inflammation characteristic of EAE (Fig. 1). Similar staining for TGF-/3 was observed in both control and anti-TGF-/31 treated animals. Astrocytes in the nearby white matter also showed positive staining for TGF-0. TGF-/3 was also present in inflamed areas of the CNS in animals during the chronic phase of disease. Enhancement of EAE by administering anti-TGF-/31 Because administration of TGF-01 suppressed clinical EAE, a series of experiments was performed to determine if anti-TGF-
Immunocytochemistry and pathology
4 *.* B
Statistical methods The average clinical scores for 8-day blocks of time were computed on each mouse in each experiment. Comparisons between anti-TGF-j31 and placebo groups for each block of time were made with two-sample f-tests (20). Treatment effects were also assessed by using a multivariate approach (Hotellings T2), which takes into account the correlation across time in comparing the resulting curves (21). Results Demonstration of TGF-f} in CNS inflammatory lesions in EAE The presence of TGF-/3 in the CNS was examined by immunohistology at various stages after the adoptive transfer of immune
Fig. 1. (A) Frozen section of lumbar spinal cord from a mouse treated with anti-TGF-01 for 5 days and sampled during the acute phase of EAE. The section is reacted with anti-TGF-/3 and shows TGF-0 + cells along a penetrating blood vessel and TGF-/3+ astrocytes in the white matter parenchyma x420 (B) Similar section from an anti-TGF-01 -treated mouse sampled during the first episode of disease. Several TGF-0+ cells are seen in the subpial region to the right and following the course of a penetrating vessel. x420.
Downloaded from http://intimm.oxfordjournals.org/ at University of Otago on July 10, 2015
CNS tissue was removed after whole body perfusion with PBS and embedded in OCT compound (Lab-Tek Division, Miles Laboratories, Naperville, IL) in a dry ice bath for immunocytochemistry or fixed by immersion in 2.5% glutaraldehyde for evaluation of pathology in 1 ^m thick epoxy sections. The expression of various molecules recognized by the antibodies was assessed by indirect staining with the avidin - biotin - peroxidase (ABC) technique (19). After fixation with acetone for 10 min, cryostat sections were treated with 0.03% H2O2 to quench endogenous peroxidase, followed by blocking with normal serum. Primary antibodies were applied overnight at 4°C at the following dilutions: anti-TGF-/3 (R&D Systems, Minneapolis, MN) 1:100; anti-l-As from MKS-4 hybridoma (ATCC, Rockville, MD) purified by affinity chromatography, at 1:25; anti-H-2 (Boehringer-Mannheim, Indianapolis, IN) at 1:20; anti-CD4 and anti-CD8 (BectonDickinson, Mountain View, CA) at 1:50. The anti-TGF-/3 used to stain tissues recognizes both TGF-/31 and TGF-/32. Sections were then incubated with the secondary antibody, followed by the ABC reagent and 3',3-diaminobenzidme. The immunostained preparations were evaluated by two 'blinded' observers and the levels of immunoreactivity and inflammation graded as follows: - , no inflammatory cells; + / - , a few positive cells; +, organization of inflammatory infiltrates around blood vessels; + +, extensive perivascular cuffing with extension into the adjacent subarachnoid space; + + + to + + + +, extensive perivascular cuffing and increasing degrees of subarachnoid inflammation (19).
Function for TGF-/3 in EAE 617 01 would exacerbate the disease. The studies showed that administration of anti-TGF-/31 after the adoptive transfer of encephalitogenic T cells led to a more severe disease course (Fig. 2). The daily administration of 10 /J of anti-TGF-/31, capable of neutralizing 40 ng of TGF-/31, for five consecutive days (days 6 - 1 0 post transfer of encephalitogenic cells) produced a marked increase in disease severity from days 1 5 - 2 3 (Fig. 2a). During this period, the anti-TGF-01 treated mice had a mean clinical score of 2.1 compared to 1.1 for the NTS treated group (P
