The pathogenesis of lesions in the central nervous system (CNS) of patients with multiple sclerosis (MS) represents the end product of several immune processes. Secretion of cytokines by infiltrating inflammatory cells as well as by endogenous glia contributes directly and indirectly to the pathogenesis of the MS lesions [1
]. Whether the initial lesions in CNS white matter affect oligodendrocytes [3
] or activated microglia in normal appearing white matter (NAWM) [4
] is still not clear, but inflammatory cells are part of active and chronic active lesions, the type I and II lesions of Luchinnetti and Lassmann, as well as the type III and type IV lesions, which are considered primarily degenerative or toxic lesions of oligodendrocytes [5
]. It is also increasingly clear that cytokines are directly and indirectly involved in inhibition of lesion formation.
There has been renewed interest in the pathologic changes in axons in the white matter [6
] as well as lesions in the gray matter in patients with MS [8
]. These changes, which can be seen in the earliest lesions, have lead to the view of MS as being, at least in part, a degenerative disease of the CNS. Activated endogenous glia, particularly microglia, and perhaps infiltrating inflammatory cells may also contribute to the pathogenesis of lesions in other more classically degenerative diseases of the CNS such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis (ALS). [10
]. Since MS has a neurodegenerative component and because much of the permanent disability in MS results from axonal and neuronal dysfunction, irreversible damage and cell loss [14
], there is increasing interest in treatments that are able to attenuate neuronal damage and perhaps allow for regeneration, so called "neuroprotection" [16
]. Several studies have emphasized the potential for the immune system to provide neuroprotection and encourage repair in experimental immunopathogenic disorders of the CNS [19
] and peripheral nervous system (PNS) [22
] as well as in traumatic [23
] and degenerative diseases [25
]. While much of the work on neuroprotection is in animal models, there is some indirect evidence for neuroprotection provided by immunomodulatory therapy in patients with MS [27
]. In addition to protection of axons and neurons and stimulation of regeneration of damaged axons, it is naive to consider neurons and axons in isolation from glial cells. Thus there is also a need to identify factors that could inhibit demyelination and/or protect oligodendrocytes and enhance oligodendrocyte precursor maturation. Additionally the effects of cytokines, chemokines and growth factors on astrocytes and microglia, which undoubtedly play several roles in neuroprotection, axonal outgrowth and synaptogenesis as well as development of neurons, need consideration and study.
The development of MS lesions, as well as inhibition of lesion formation and reparative processes, involves complex interactions among mixtures of CNS cells. In addition single infiltrating inflammatory cells and glial cells do not secrete single cytokines, and glial cells do not respond by producing single growth factors or modifying only one of their many cell functions. MS is a highly complex disease with regards to basic immune and inflammatory mechanisms involving the systemic immune system as well the CNS. As such, complex techniques need to be applied in MS research [32
] so as to avoid oversimplification. For these reasons we have carried out a series of experiments to examine the effects of different cytokine mixtures, typical of Th1 and Th2 lymphocytes, as well as monocyte/macrophages (M/M) on early gene expression in mixed glial cell cultures. This approach also eliminates the confounding effects of infiltrating inflammatory cells. We chose to employ microarray technology, which allows one to simultaneously examine the regulatory effect of these mixtures of cytokines on a vast number of genes. Thus one may not only see effects on a large number of genes but may find regulatory effects on unanticipated genes. To identify genes more likely to be directly affected by the cytokine mixtures, we chose to start by examining the effect on gene expression at 6 hours in vitro
(early gene expression).
Our rationale is to use genomics to identify the significant early changes in gene expression in the interactive mixed glial cell environment, with subsequent experiments focused on identifying the specific cell types responsible for those changes. Because of the large number of genes regulated by these cytokine mixtures and issues of manuscript length we reported the effects on genes of immune system related molecules in an earlier separate report [34
] and divided other data into this paper with neurotrophins, other growth factors and structural proteins, and a third paper looking at ion channels, neurotransmitters and their receptors, mitochondria, signaling, transcription factors, and molecules involved in apoptosis, among others. In the first report we noted that expression of at least 814 genes of 7,985 analyzed were changed by a minimum of 2 fold by one or more of the cytokine mixtures at 6 hours (early gene response) in vitro
when compared to control cultures. In this report and another paper in progress, we present results of regulation of genes for molecules more classically related to glial and axonal/neuronal cells. We are aware that the assignment of molecules to one or the other such broad categories is arbitrary since many of these molecules act in the immune system as well as in the CNS. In addition, some molecules could be classified under any one of several categories in this report. As examples, BIG-2 could be considered a neuronal protein but is also an adhesion molecule; deleted in colon cancer (DCC) is a cancer-related protein but also a neuronal protein.