β-interferon regulates the immunomodulatory activity of neonatal rodent microglia
Introduction
Multiple sclerosis (MS) is the commonest cause of acquired neurological disability in young adults. The precise mechanisms of immunologically mediated tissue damage are not fully understood, but there is a consensus that activated T lymphocytes cross the blood–brain barrier and initiate an inflammatory response within the central nervous system (CNS). Soluble factors are important in myelin and oligodendrocyte injury [1] and infiltrating macrophages and activated resident microglia subsequently phagocytose myelin sheaths. Non-specific immunosuppressive and cytotoxic treatment has been relatively unsuccessful in the treatment of MS, but more recently, β-IFN has been assessed as a potential treatment. There is accumulating evidence that it may suppress some aspects of disease activity [1,2] and alter the natural history of MS. However, the exact mechanism of action of β-IFN in patients with MS has not yet been elucidated.
Although first described on the basis of its antiviral properties, β-IFN has now been documented to have immunomodulatory functions on a wide range of target cells. β-IFN has a direct antiproliferative action on some cells including macrophages and counteracts the mitogenic stimulus of certain cytokines [3,4]. It acts on large granular lymphocytes to increase killing, augments natural killer cell activity [5] and leads to increased generation of cytotoxic T lymphocytes [6]. B cell proliferation is augmented as is secretion of Ig M, G and A. The effects of β-IFN on accessory cells such as the peripheral macrophage are well characterised. β-IFN prevents proliferation and causes terminal differentiation and activation [7]. The resultant morphological changes include cell enlargement, spreading, pseudopod formation and vacuolation. Adherence is increased, as is the number and density of Fc receptors (FcR) [8] leading to increased phagocytosis. Destruction and detoxification of internalised pathogens is maximised by induction of lysosomal enzymes. Class I MHC expression is upregulated leading to increased class I restricted antigen presentation to CD8 cells. By contrast, in certain other accessory cells, namely the murine macrophage and the human astrocyte, not only does β-IFN not induce class II MHC, the expression of which is required for antigen-specific activation of CD4 + T cells via the T cell receptor (TCR), but it actually inhibits upregulation of class II surface expresson mediated by γ-IFN [9], [10], [11].
In MS, T cells are thought to be activated within the periphery and enter the CNS where maintenance of activation occurs after encountering autoantigen. Immunoglobulin also contributes to the disease process; B lymphocytes are identified in the perivascular infiltrate [12] and oligo-clonal bands in the cerebrospinal fluid and, furthermore, in the experimental allergic encephalomyelitis model, passive transfer of myelin oligodendrocyte glycoprotein-specific antibody results in a more severe disease with pathology resembling the primary demyelination seen in MS [13]. Central FcR expressing cells could directly phagocytose opsonised targets and immune complexes. Specific peptide within the immune complex would be processed and presented in the context of class II MHC, and hence activate CD4-positive T cells.
In the CNS, microglia fulfill the criteria of immunologically active accessory cells; on activation in vitro they increase their expression of class II MHC [14,15], and FcR [15]; they are phagocytic and can process and present antigen in an MHC restricted fashion to both resting and naive T cells [16], [17], [18] and can upregulate constitutive expression of the Th1 co-stimulatory molecule B7 [19]. In addition, microglia have important direct cytotoxic effector functions involving adherence to, and phagocytosis of, opsonised targets, the ability to mount a respiratory burst in response to appropriate stimulation [15] and the production of potentially harmful cytokines such as TNFα [20].
We have systematically studied the effects of recombinant rat β-IFN on a comprehensive range of immunological accessory and cytotoxic effector functions using cultured rodent microglia and have examined the interaction of β-IFN with γ-IFN, which has been proposed as the main pro-inflammatory cytokine in the pathophysiology of MS. Neonatal rodent microglial morphology, class II MHC expression, proliferation, FcR expression, ability to mount a respiratory burst and TNFα secretion have each been examined. We identified both antagonistic and synergistic effects of these cytokines, re-emphasising the complexity of immune interactions between immunocompetent cells, cytokines and potentially beneficial immunotherapeutic agents.
Section snippets
Cytokines
Rat recombinant γ-IFN and β-IFN were obtained from Biosource International (Camarillo, CA, USA). γ-IFN was used in all experiments at a concentration of 100 units/ml. β-IFN was used at 200 units/ml.
Tissue culture
Microglia were isolated and cultured from 1–2-da-old Sprague– Dawley rats using the method of Giulian and Baker [21] with modifications as described by Zajicek et al. [20]. This protocol was further modified such that, briefly, mixed brain cultures were shaken on day 8 for 2 h to remove microglia from
Microglial proliferation
To study proliferation, microglia were cultured for 48 h, alone or in the presence of IFNs, and pulsed with [3H] thymidine; cells were harvested and radioactivity counted (Fig. 1). In the presence of β-IFN, there was approximately a 60% reduction in proliferation, whereas γ-IFN alone, or in combination with β-IFN, completely inhibited proliferation.
Class II MHC induction
Class II MHC expression was examined by indirect immunocytochemistry. Microglia were incubated for 48 h in the presence of 10% FCS without cytokines
Discussion
Our study shows that the effects of β-IFN and γ-IFN on neonatal rodent microglia are both complex and interactive. The results do not produce a complete explanation for the clinical effects of β-IFN, but highlight the potential benefits and complications of cytokine treatment. Both β-IFN and γ-IFN are antiproliferative for microglia alone or in combination and they augment TNFα secretion induced by sub-optimal concentrations of LPS. In contrast, β-IFN antagonises both γ-IFN-induced upregulation
Acknowledgements
This work was supported by the Wellcome Trust. We would like to thank Miss Sarah Stevens for expert technical support.
References (42)
- et al.
Macrophage activation by interferon α and β is associated with a loss of proliferative capacity
Cell Immunol.
(1987) - et al.
Interferon-β impairs induction of HLA-DR antigen expression in cultured human astrocytes
J. Neuroimmunol.
(1989) - et al.
Microglia present myelin antigens to T cells after phagocytosis of oligodendrocytes
Cellular Immunology
(1993) - et al.
Functional antagonism between type I and type II interferons on human macrophages
Biochem. Biophys. Res. Commun.
(1986) - et al.
Interferon-β specifically inhibits interferon-γ-induced class II major histocompatability complex gene transcription in a human astrocytoma cell line
J. Neuroimmunol.
(1991) - et al.
Does interferon beta cause initial exacerbations of multiple sclerosis?
The Lancet
(1995) Interferon beta-1b is effective in relapsing-remitting multiple sclerosis. I. Clinical results of a multicenter, randomized, double-blind, placebo-controlled trial
Neurology
(1984)- et al.
The UBC MS/MRI study group, the IFN-β multiple sclerosis study group Interferon beta-1b is effective in relapsing-remitting multiple sclerosis. II MRI analysis results of a multicenter randomized, double-blind, placebo-controlled trial
Neurology
(1993) - et al.
Endogenous regulation of macrophage proliferative expansion by colony stimulating factor-induced interferon
Science
(1984) - et al.
Interferon effects on PDGF action