Astrocytes and microglia express inducible nitric oxide synthase in mice with experimental allergic encephalomyelitis

https://doi.org/10.1016/S0165-5728(96)00215-9Get rights and content

Abstract

Nitric oxide (NO), produced by inducible NO synthase (iNOS), may play a role in inflammatory demyelinating diseases of the central nervous system (CNS). We show upregulation of iNOS mRNA in CNS of SJL/J mice with experimental allergic encephalomyelitis (EAE). Using antibodies against mouse iNOS, GFAP (a marker for astrocytes) and Mac-1/CD11b (a marker for macrophages/microglia), both astrocytes and macrophages/microglia were identified as iNOS-expressing cells in situ in EAE lesions. GFAP+ astrocytes not associated with inflammatory infiltrates were also found to express iNOS. Because microglia rather than astrocytes are implicated in demyelinating pathology, we propose that microglial NO may be cytopathic whereas astrocyte-derived NO may be protective in EAE.

Introduction

Experimental allergic encephalomyelitis (EAE) is an autoimmune inflammatory disease of the central nervous system (CNS) that can be induced by immunization with myelin proteins (e.g., myelin basic protein, MBP) or by adoptive transfer of myelin-reactive CD4+ T cells. In mice, the relapsing-remitting progression and pathology of EAE resemble those of the human disorder multiple sclerosis (MS) (Raine, 1984; Raine et al., 1984). Like MS, EAE is characterized by blood–brain barrier breakdown, infiltration of the CNS by active CD4+ T cells and macrophages, activation of adjacent microglial and astroglial cells, and demyelination (Raine, 1984; McFarlin and McFarland, 1982). Although precise pathogenetic mechanisms underlying EAE are still not fully understood, complex interactions between infiltrating and resident cells of the CNS are thought to result in the formation and release of mediators that contribute to tissue damage.

The free radical nitric oxide (NO) is implicated in neurotransmission, vasorelaxation and host defense against microbial infections of neural tissues (Paakkari and Lindsberg, 1995; Bi et al., 1995; Schmidt and Walter, 1994). NO has been suggested to mediate tissue injury in demyelinating diseases. Activated glial cells have been shown in vitro to express the enzyme inducible NO synthase (iNOS or type 2 NOS) that catalyzes a high output of NO to which cultured neurons and oligodendrocytes are vulnerable (Skaper et al., 1995; Dawson et al., 1994; Merrill et al., 1993; Boje and Arora, 1992; Chao et al., 1992). Leukocytes isolated from the CNS during hyperacute EAE secrete substantially increased amounts of reactive nitrogen intermediates (MacMicking et al., 1992). Furthermore, induction of iNOS mRNA occurs in spinal cord of mice (Okuda et al., 1995), in brain of rats (Koprowski et al., 1993) with EAE and in MS brain (Bö et al., 1994). In mice, this expression of iNOS mRNA correlates with severity of EAE (Okuda et al., 1995) and levels of NO rise significantly in spinal cord during an episode of EAE (Lin et al., 1993). Consistent with this, a selective inhibitor of iNOS can prevent adoptive transfer of EAE in mice (Cross et al., 1994).

There has been much interest in discerning the primary cell type(s) responsible for such in vivo overproduction. Astrocytes are the major glial population in the CNS. In both MS and EAE lesions, astrocytic activation is prominent. In MS lesions, astrocytes have been suggested as the main source of excess NO (Bö et al., 1994; Brosnan et al., 1994). Yet, comparative immunohistochemical analyses of adjacent tissue sections showed localization of the iNOS protein to be restricted to invading macrophages and/or activated microglial cells within inflammatory infiltrates in brain or spinal cord of rodents with EAE (Ruuls et al., 1996a, Ruuls et al., 1996b; Van Dam et al., 1995; Okuda et al., 1995). This apparent divergence in the identity of cellular sources of a potential cytopathic mediator, NO, between MS and EAE, could reflect different pathogenetic mechanisms underlying the two diseases.

We report here not only that astrocytes express iNOS in SJL/J mice with EAE, but that astrocytes distal from the inflammatory infiltrates do so, as well. These findings may offer new insights into intercellular communication in the CNS, as well as resolving an apparent paradox.

Section snippets

Induction of EAE and tissue collection

Animal maintenance and experimental protocols were in accordance with guidelines set forth by the McGill University Animal Care Committee. Pathogen-free 6–8-week-old female SJL/J mice (Harlan–Sprague Dawley) were immunized s.c. at the base of tail and boosted s.c. in the flanks 7 days later with 400 μg of bovine MBP (prepared as described by Cheifetz et al., 1984) emulsified in complete Freund's adjuvant containing 50 μg of Mycobacterium tuberculosis H37 RA (Difco). Symptoms were first observed

iNOS gene expression in CNS during EAE

We analyzed the brains and spinal cords of SJL/J mice by Northern blot and RT-PCR to confirm iNOS gene expression in EAE. As shown in Fig. 1A, upregulation of iNOS mRNA occurred at levels detectable by Northern blot in EAE CNS, as compared to normal controls. Indeed, EAE brains or spinal cords contained four- to fivefold (after normalization with β-actin signal) increases in iNOS transcripts. That expression was low in normal tissues was suggested by use of a more sensitive assay, RT-PCR (Fig. 1

Discussion

We have examined expression of iNOS mRNA and protein in SJL/J mice with EAE. We have confirmed expression of iNOS mRNA in both the brain and spinal cord during an episode of EAE, and we have unequivocally localized enhanced iNOS expression to reactive astrocytes (as judged by their characteristic morphology and increased GFAP immunoreactivity) in EAE CNS. Interestingly, we find astrocytes expressing iNOS not only in association with inflammatory lesions, but also in the parenchyma at varying

Acknowledgements

We thank Michelle Krakowski for review of this manuscript. We are also grateful to Dr. Danuta Radzioch (Montréal General Hospital) for the ANA-1 cell line, Dr. Wee Yong for advice on histology, Fiona Yong for her expertise in confocal microscope imaging, Grace Chan for MBP preparation, and Lawrence Le for technical assistance. This work was supported by operating grants (to T.O.) from Medical Research Council and Multiple Sclerosis Society of Canada. E.H.T. was supported by ``Fonds pour la

References (51)

  • Baker, D., O'Neill, J.K. and Turk, J.L. (1991) Cytokines in the central nervous system of mice during chronic relapsing...
  • Barres, B.A., Hart, I.K., Coles, H.S.R., Burne, J.F., Voyvodic, J.T., Richardson, W.D. and Raff, M.C. (1992) Cell death...
  • Bi, Z., Barna, M., Komatsu, T. and Reiss, C.S. (1995) Vesicular stomatitis virus infection of the central nervous...
  • Blasi, E., Mathieson, B.J., Varesio, L., Cleveland, J.L., Bochert, P.A. and Rapp, U.R. (1985) Selective immortalization...
  • Bö, L., Dawson, T.M., Wesselingh, S., Mörk, S., Choi, S., Kong, P.A., Hanley, D. and Trapp, B.D. (1994) Induction of...
  • Boje, K.M. and Arora, P.K. (1992) Microglial-produced NO and reactive nitrogen species mediate neuronal cell death....
  • Bredt, D.S., Hwang, P.M. and Snyder, S.H. (1990) Localization of nitric oxide synthase indicating a neural role for...
  • Bredt, D.S. and Snyder, S.H. (1990) Isolation of nitric oxide synthase, a calmodulin-requiring enzymes. Proc. Natl....
  • Brosnan, C.F., Attistini, L., Raine, C.S., Dickson, D.W., Casadevall, A. and Lee, S.C. (1994) Reactive nitrogen...
  • Chao, C.C., Hu, S., Molitor, T.W., Shaskan, E.G. and Peterson, P.K. (1992) Activated microglia mediate neuronal cell...
  • Cheifetz, S., Moscarello, M. and Deber, C. (1984) NMR investigations of the charge isomers of bovine myelin basic...
  • Cross, A.H., Misko, T.P., Lin, R.F., Hickey, W.F., Trotter, J.L. and Tilton, R.G. (1994) Aminoguanidine, an inhibitor...
  • Dawson, V.L., Brahmbhatt, H.P., Mong, J.A. and Dawson, T.M. (1994) Expression of inducible nitric oxide synthase causes...
  • Franklin, R.J., Crang, A.J. and Blakemore, W.F. (1991) Transplanted type-1 astrocytes facilitate repair of...
  • Galea, E., Reis, D.J. and Feinstein, D.L. (1994) Cloning and expression of inducible nitric oxide synthase from rat...
  • Hewett, S.J., Misko, T.P., Keeling, R.M., Behrens, M.M., Choi, D.W. and Cross, A.H. (1996) Murine encephalitogenic...
  • Jensen, A.M. and Chiu, S.-Y. (1993) Astrocyte networks. In: S.R. Murphy (Ed.), Astrocytes: Pharmacology and Function....
  • Koprowski, H., Zeng, Y.M., Heber-Katz, E., Frazer, N., Rorke, L., Fu, Z.F., Hanlon, C. and Dietzschold, B. (1993) In...
  • Kubes, P., Suzuki, M., Granger, D.N. (1991) Nitric oxide: An endogenous modulator of leukocyte adhesion. Proc. Natl....
  • Lee, S.C., Dickson, D.W., Liu, W. and Brosnan, C.F. (1993) Induction of nitric oxide synthase activity in human...
  • Lin, R.F., Lin, T.S., Tilton, R.G. and Cross, A.H. (1993) Nitric oxide localized to spinal cord of mice with...
  • Lipton, S.A., Choi, Y.B., Pan, Z.H., Lei, S.Z., Chen, H.S.V., Sucher, N.J., Loscalzo, J., Singel, D.J. and Stamler,...
  • Louis, J.-C., Magal, E., Takayama, S. and Varon, S. (1993) CNTF protection of oligodendrocytes against natural and...
  • Lowenstein, C.J., Glatt, C.S., Bredt, D.S. and Snyder, S.H. (1992) Cloned and expressed macrophage nitric oxide...
  • Lyons, C.R., Orloff, G.J. and Cunningham, J.M. (1992) Molecular cloning and functional expression of an inducible...
  • Cited by (0)

    View full text