Although the pathogenesis of ASD is not well understood, recent studies have suggested that localized inflammation of the central nervous system may contribute to the pathogenesis of ASD. A number of studies have shown that TNFα, IFNγ, IL-1β and IL-12 were increased in the peripheral blood of ASD patients (Zimmerman et al., 2005
; Molloy et al, 2006
; Ashwood et al, 2006
). TNFα was also shown to be increased in the cerebral spinal fluid of autistic patients (Vargas et al, 2005
; Chez MG, 2007). However, there is only one study that has been conducted to investigate the inflammatory cytokine profile in the brain of autistic patients using protein arrays (Vargas et al, 2005
). To further characterize the nature of the inflammatory responses in autistic brains, we used a recently developed flow cytometry method (multiplexed bead analysis) to measure cytokine levels in the brain (cerebral cortex) extracts. This method provides many advantages over the traditional ELISA methods, and with better results for characterization of cytokines (Khan et al., 2007; DuPont et al., 2005
). This method also makes it possible to measure several cytokines in a single sample to establish a profile of the inflammatory reaction. Our results showed that pro-inflammatory cytokines (TNFα, IL-6 and GM-CSF) were significantly increased in the brains of ASD patients in comparison with the control subjects. TNF-α and IL-6 are cytokines involved in cell-mediated immune response and their production has been shown to be associated with tissue inflammation and necrosis (Beutler and Cerami, 1989
). The role of TNF-α as a neuromodulating agent has also been described in brain development, and it may play a role in neurons and neuroglial cells modulating glutaminergic transmission (Pickering et al., 2005
). Excessive glutamate excitotoxic effects acting on NMDA receptors could occur in the presence of excess TNF-α (Pickering et al, 2005
). This occurrence can lead to effects on microglial activation, as well as on nuclear factor kappa-B (NF-kB). Such changes have also been seen in models of inflammation inducing epileptic activity in which neuroglial inflammation has caused epileptic spikes (Pickering et al, 2005
). Thus elevations of TNF-α, IL-6 and GM-CSF in the brain of ASD patients suggest that children with ASD have a heightened immune response that may be associated with localized brain inflammation and tissue necrosis.
Immune responses that are stimulated by exposure to specific antigens are considered adaptive responses and are often driven by CD4+ T helper (Th) cells. Th cell clones have been classified into distinct functional types on the basis of the cytokines they secrete (Abbas et al, 1996
). The most clearly defined of these subsets are Th1, which is characterized by the production of interferon (IFN)-γ and IL-2, and Th2, which is characterized by the production of IL-4, IL-5 and IL-10. The main effect or function of Th1 cytokines is phagocyte mediated defense, especially against intracellular microbes, while Th2 cytokines function to affect IgE and eosinophil/mast cell-mediated immune responses. An imbalance of these cytokines, skewed toward Th2, is seen in allergic responses (Ngoc et al, 2005
) and some systemic autoimmune responses such as systemic lupus erythematosus (Spadaro et al, 2003
). A skewing toward Th1 cytokines is seen in some organ specific autoimmune disorders such as insulin-dependent diabetes mellitus and multiple sclerosis (Liblau et al, 1995
). In our study, we detected significant elevations in IFN-γ (Th1 origin), but not in IL-4, IL-5 and IL-10 (Th2 origin), suggesting that the brain of ASD patients have an excess adaptive response through activation of the Th1 pathway, rather than activation of the Th2 pathway. The increased activation of Th1 cytokines, but not Th2 cytokines in the ASD brain also suggests that an autoimmune disorder may be involved in the pathogenesis of ASD, but not an allergic response. Moore et al (2001)
reported that children with ASD had increased activation of both Th2 and Th1 immune response in blood mononuclear cells, with Th2 predominance. This finding is different from our results, suggesting that immune responses in the brain and blood mononuclear cells in autistic patients could be different. In addition, the ratio of IFN-γ to IL-10 was significantly higher in ASD patients than in controls, showing an absence of a compensatory increase in IL-10. This supports the concept of a dysregulation of the adaptive immune responses in ASD patients. These results are also consistent with those reported by Jyonouchi et al (2002)
involving heightened and dysregulated innate immune responses in children with ASD as evidenced by elevated proinflammatory cytokine production from their peripheral blood mononuclear cells.
In addition, our study demonstrated that chemokine IL-8 was also significantly increased in the brain of ASD patients in comparison to the control group. IL-8 has powerful chemotactic effects on T cells and neutrophils. Elaboration of IL-8 by resident tissues is an important mechanism for directing leukocytes to migrate, especially through tissues without blood vessels. In recent years, increasing attention has been focused on chemokines as inflammatory mediators in the CNS. The limited number of studies that have investigated chemokine and chemokine receptor expression in Alzheimer's disease (AD) brains and in cell culture models seem to support a role for inflammation in AD pathogenesis (Grammas et al, 2006
). Studies have also suggested a possible role of chemokines as communication molecules between neurons and microglia (Wolfang J, 2001). The significant increase of IL-8 in the brain of ASD patients implicated a chemokine mediated inflammation and this excess activation of IL-8 may be a potent signal to recruit T lymphocytes that leads to the damage in the brain of ASD patients.
There are certainly limitations in this investigation. The sample sizes are relatively small. In addition, only 10 cytokines were analyzed. Despite these limitations, this is the first study to our knowledge to investigate various inflammatory cytokine expression levels in the brain of ASD patients using the most recently developed cytometry method.
In summary, we found that ASD is associated with change in expression levels of various pro-inflammatory and anti-inflammatory cytokines in the brain of ASD patients. These findings serve as further evidence that inflammation may be an important part of the pathogenesis of ASD. Based on these findings, further investigation directed at cytokine modulation as a therapeutic approach to ASD is a logical next step.