Interleukin 6 production in autoimmune encephalomyelitis
Eur. J. Immunol. 1990. 20: 233-235
233
Short paper Koen Gijbelson, Jo Van Dammeov, Paul ProostO, Willy PutO, Herwig Carton+ and Alfons BilliauO
Interleukin 6 production in the central nervous system during experimental autoimmune encephalomyelitis*
Departments of Microbiologyo, Rega Institute and Brain and Behaviour Research , University of Leuven Medical School, Leuven +
Interleukin 6 (IL 6) is one of the major inflammation-associated cytokines. Elevated serum or tissue levels of I L 6 have been reported to occur in several human diseases, including infections of the central nervous system (CNS), but not in non-infectious CNS inflammation, e.g. multiple sclerosis. While studying experimental autoimmune encephalomyelitis (EAE) as an animal model for autoimmune inflammation of the CNS, we found increased IL 6 levels in the CNS of mice suffering from a lethal form of the disease. IL6 levels in the spleens and sera were not significantly increased. These findings are indicative of local production of I L 6 in the CNS during EAE, and represent the first demonstration of IL 6 production in non-infectious CNS inflammatory disease.
1 Introduction Interleukin 6 (IL6), also called B cell stimulatory factor-2 (BSF-2),hepatocyte stimulating factor or hybridomdplasmacytoma growth factor (HPGF), is a cytokine that can be produced by various cell types, including monocytes, endothelial cells, activated T cells and fibroblasts [l].The production of IL 6 is induced by various infectious and inflammatory stimuli (e.g. IL 1, bacterial LPS, virus infection).The numerous biological activities of IL 6 include the stimulation of B cell growth and differentiation, the stimulation of T cell proliferation and differentiation, the induction of differentiation of hemopoietic cells and the regulation of production of acute phase proteins by hepatocytes. I L 6 also induces fever by a direct or indirect effect on the hypothalamus [2], and may play a role as a neuro-endocrine mediator, as is suggested by the fact that the folliculostellate cells of the anterior pituitary gland produce IL6 [3].
nervous system (CNS), but not in non-infectious inflammatory diseases of the CNS, such as multiple sclerosis (MS) [4, 51. Yet I L 6 is found in patients with other diseases in which autoimmunity may play a role, such as rheumatic arthritis [6, 71 and lupus erythematosus [8]. In order to explore further the apparent discrepancy between IL 6 production in the CNS and elsewhere in the body during autoimmune diseases and to assess the possible role of IL6 in autoimmune disorders of the CNS, we have used experimental autoimmune encephalomyelitis (EAE) as an animal model. E AE is an autoimmune T cellmediated, experimentally induced inflammation of the CNS. We determined I L 6 levels in sera, CSF and tissue homogenates of mice developing EAE.
2 Materials and methods 2.1 Mice and EAE induction
When considering the circumstances in which IL6 is induced and the possible effects exerted by this cytokine, one can expect it to play an important role in inflammatory reactions. In humans, I L 6 has been detected in the cerebrospinal fluid (CSF) during infections of the central [I 79051
* A
This work was supported by grants from the Belgian Ministry of Science Policy (GOA Programme) and the WOMS (Wetenschappelijk Onderzoek Multiple Sclerose). Research Assistant of the Belgian National Fund for Scientific Research. Research Associate of the Belgian National Fund for Scientific Research.
SJLN mice, 8-9 weeks old, were obtained from the Animal Breeding Centre of the University of Leuven. Animals were kept in a conventional animal house. An emulsion was prepared consisting of 100 mg/ml of NMRI spinal cord (homogenized, lyophilized) in phosphate buffer and 4 mg/ml Mycobacterium tuberculosis (strain H37-RA) in CFA (bothreagentsfrom Difco, Detroit, MI). On day 0 the mice were injected in each of the two hind footpads with 50 p1 of this emulsion. Immediately thereafter and on day 2, 50 p1 of a Bordetella pertussis suspension (containing 1O1O cells; kind gift of Dr. R. H. Tiesjema, Rijksinstituut voor de Volksgezondheid, RIV, Bilthoven, The Netherlands) was given i.v. Control animals did not receive any treatment.
Correspondence: Koen Gijbels, Laboratory of Immunobiology, Rega Institute, Minderbroederstraat 10, B-3000 Leuven, Belgium
2.2 Sampling
Abbreviations: CSF Cerebrospinal fluid CNS: Central nervous system EAE: Experimental autoimmune encephalomyelitis MS: Multiple sclerosis
Animals were examined daily for disease symptoms. When the clinical status reached a critical stage, the mice were killed by ether anesthesia. Blood was collected by cardiac
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
0014-2980/90/0101-0233$02.50/0
234
K. Gijbels, J.Van Damme, €? Proost et al.
Eur. J. Immunol. 1990. 20: 233-235
puncture and allowed to clot overnight at 4 "C. After centrifugation serum was decanted. Cerebrospinal fluid was taken by suboccipital puncture as described elsewhere [9] and pooled for each group. The brains, spinal cords and spleens were removed in a sterile fashion and homogenized in 10 volumes of Eagle's minimum essential medium containing 10% FCS. During this procedure the samples were kept on ice. The resulting suspension was clarified by centrifugation at 5 0 0 x g for 10min. All samples were stored at -20°C until assay for I L 6 activity.
Table 2. IL6 levels in the CSF of mice with EAE as compared to those in controls at time of death
Exp. no. 1 2 3
Group
Control
< 1.2 (4)Q
Eur. J. Immunol. 1990. 20: 233-235
233
Short paper Koen Gijbelson, Jo Van Dammeov, Paul ProostO, Willy PutO, Herwig Carton+ and Alfons BilliauO
Interleukin 6 production in the central nervous system during experimental autoimmune encephalomyelitis*
Departments of Microbiologyo, Rega Institute and Brain and Behaviour Research , University of Leuven Medical School, Leuven +
Interleukin 6 (IL 6) is one of the major inflammation-associated cytokines. Elevated serum or tissue levels of I L 6 have been reported to occur in several human diseases, including infections of the central nervous system (CNS), but not in non-infectious CNS inflammation, e.g. multiple sclerosis. While studying experimental autoimmune encephalomyelitis (EAE) as an animal model for autoimmune inflammation of the CNS, we found increased IL 6 levels in the CNS of mice suffering from a lethal form of the disease. IL6 levels in the spleens and sera were not significantly increased. These findings are indicative of local production of I L 6 in the CNS during EAE, and represent the first demonstration of IL 6 production in non-infectious CNS inflammatory disease.
1 Introduction Interleukin 6 (IL6), also called B cell stimulatory factor-2 (BSF-2),hepatocyte stimulating factor or hybridomdplasmacytoma growth factor (HPGF), is a cytokine that can be produced by various cell types, including monocytes, endothelial cells, activated T cells and fibroblasts [l].The production of IL 6 is induced by various infectious and inflammatory stimuli (e.g. IL 1, bacterial LPS, virus infection).The numerous biological activities of IL 6 include the stimulation of B cell growth and differentiation, the stimulation of T cell proliferation and differentiation, the induction of differentiation of hemopoietic cells and the regulation of production of acute phase proteins by hepatocytes. I L 6 also induces fever by a direct or indirect effect on the hypothalamus [2], and may play a role as a neuro-endocrine mediator, as is suggested by the fact that the folliculostellate cells of the anterior pituitary gland produce IL6 [3].
nervous system (CNS), but not in non-infectious inflammatory diseases of the CNS, such as multiple sclerosis (MS) [4, 51. Yet I L 6 is found in patients with other diseases in which autoimmunity may play a role, such as rheumatic arthritis [6, 71 and lupus erythematosus [8]. In order to explore further the apparent discrepancy between IL 6 production in the CNS and elsewhere in the body during autoimmune diseases and to assess the possible role of IL6 in autoimmune disorders of the CNS, we have used experimental autoimmune encephalomyelitis (EAE) as an animal model. E AE is an autoimmune T cellmediated, experimentally induced inflammation of the CNS. We determined I L 6 levels in sera, CSF and tissue homogenates of mice developing EAE.
2 Materials and methods 2.1 Mice and EAE induction
When considering the circumstances in which IL6 is induced and the possible effects exerted by this cytokine, one can expect it to play an important role in inflammatory reactions. In humans, I L 6 has been detected in the cerebrospinal fluid (CSF) during infections of the central [I 79051
* A
This work was supported by grants from the Belgian Ministry of Science Policy (GOA Programme) and the WOMS (Wetenschappelijk Onderzoek Multiple Sclerose). Research Assistant of the Belgian National Fund for Scientific Research. Research Associate of the Belgian National Fund for Scientific Research.
SJLN mice, 8-9 weeks old, were obtained from the Animal Breeding Centre of the University of Leuven. Animals were kept in a conventional animal house. An emulsion was prepared consisting of 100 mg/ml of NMRI spinal cord (homogenized, lyophilized) in phosphate buffer and 4 mg/ml Mycobacterium tuberculosis (strain H37-RA) in CFA (bothreagentsfrom Difco, Detroit, MI). On day 0 the mice were injected in each of the two hind footpads with 50 p1 of this emulsion. Immediately thereafter and on day 2, 50 p1 of a Bordetella pertussis suspension (containing 1O1O cells; kind gift of Dr. R. H. Tiesjema, Rijksinstituut voor de Volksgezondheid, RIV, Bilthoven, The Netherlands) was given i.v. Control animals did not receive any treatment.
Correspondence: Koen Gijbels, Laboratory of Immunobiology, Rega Institute, Minderbroederstraat 10, B-3000 Leuven, Belgium
2.2 Sampling
Abbreviations: CSF Cerebrospinal fluid CNS: Central nervous system EAE: Experimental autoimmune encephalomyelitis MS: Multiple sclerosis
Animals were examined daily for disease symptoms. When the clinical status reached a critical stage, the mice were killed by ether anesthesia. Blood was collected by cardiac
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
0014-2980/90/0101-0233$02.50/0
234
K. Gijbels, J.Van Damme, €? Proost et al.
Eur. J. Immunol. 1990. 20: 233-235
puncture and allowed to clot overnight at 4 "C. After centrifugation serum was decanted. Cerebrospinal fluid was taken by suboccipital puncture as described elsewhere [9] and pooled for each group. The brains, spinal cords and spleens were removed in a sterile fashion and homogenized in 10 volumes of Eagle's minimum essential medium containing 10% FCS. During this procedure the samples were kept on ice. The resulting suspension was clarified by centrifugation at 5 0 0 x g for 10min. All samples were stored at -20°C until assay for I L 6 activity.
Table 2. IL6 levels in the CSF of mice with EAE as compared to those in controls at time of death
Exp. no. 1 2 3
Group
Control
< 1.2 (4)Q
