Proinflammatory and regulatory cytokine production associated with innate and adaptive immune responses in children with autism spectrum disorders and developmental regression☆
Introduction
Autism spectrum disorder (ASD) is a complex developmental disorder diagnosed on the basis of clinical characteristics (DSM-IV). Many parents of ASD children have reported that the onset of developmental regression and autistic behavior and/or further deterioration occurred following immunization and/or benign childhood infection. Many ASD children also appear to be intolerant to common dietary protein antigens (Ag), suffering from a variety of gastrointestinal (GI) symptoms including diarrhea, constipation, colic, gastroesophageal reflux, and GI discomfort. ASD children who have undergone GI workup were found to have ileocolonic lymphoid nodular hyperplasia and mucosal inflammation at high frequency (Wakefield et al., 2000). These observations may indicate that autistic children have aberrant immune responses against benign environmental factors at high frequency.
Recent progress in basic immunology has revealed an intricate immune defense network mounted by innate and adaptive immunity. Innate immunity provides initial immune responses by recognizing pathogen-associated molecular patterns (PAMPs) shared by groups of microbial pathogens (Medzhitov and Janeway, 2000). PAMPs are recognized by pattern recognition receptors (PRRs) expressed by Ag-presenting cells (APC; macrophages, monocytes, dendritic cells, etc.) and natural killer (NK) cells. One of the most extensively studied PAMPs is LPS Ulevitch, 2000, Wright, 1999. PAMPs recognized by PRRs activate these innate immune cells, resulting in production of proinflammatory (TNF-α, IL-1β, IL-6) and counter-regulatory (sTNFRI, sTNFRII, and IL-1ra) cytokines and phagocytosis/destruction of pathogens Medzhitov and Janeway, 2000, Wright, 1999. The proinflammatory cytokines produced by innate immunity are also known to cross the intact blood–brain barrier and affect hypothalamo–pituitary–adrenocortical (HPA) axis inducing stress responses Barkhudaran and Dunn, 1999, Dunn et al., 1999, Mohankumar et al., 1998, Mossner et al., 1998, Wang and Dunn, 1999.
Innate immunity plays a key role in development of subsequent adaptive immune responses by facilitating costimulatory/adhesion molecule expression and Ag processing of activated APC (Medzhitov and Janeway, 2000). Cytokine concentrations in the microenvironment also affect differentiation of T-helper (Th) and cytotoxic T (Tc) subsets; increased IL-12/IL-18 levels in the microenvironment facilitate type 1 T (T1) cell differentiation Flavell, 1999, Swain, 1999. Although innate immunity is crucial for subsequent adaptive immune responses, dysregulated innate immune responses could be harmful and may lead to broken tolerance and development of autoimmune phenomena (Medzhitov and Janeway, 2000).
Upon Ag presentation, resting T cells are activated into the effector-stage T cell subsets with distinguished patterns of cytokine production Flavell, 1999, Swain, 1999. T1 responses induce phagocytic cell-mediated immune responses by producing T1 cytokines (IFN-γ, IL-2, and TNF-β) and facilitating production of IgG1/IgG3 antibodies (Ab) that enhance opsonization. Type 2 T cell (T2) responses induce eosinophil-mediated inflammatory responses by production of T2 cytokines (IL-4, IL-5, and IL-13) and augmenting production of IgG4/IgE Ab Flavell, 1999, Swain, 1999. T1 and T2 cytokines counter-regulate each other and an imbalance of T1/T2 responses is implicated with pathogenesis of various disorders. For example, skewed T1 responses may be associated with organ-specific autoimmune disease (multiple sclerosis, etc.), while excessive T2 responses may lead to atopic disorders Romagnani, 1997, Swain, 1999. Skewed T1 or T2 responses were also indicated in ASD children Gupta et al., 1998, Singh, 1996. Autoimmunity against neuronal cells and neurotransmitters has also been implicated as part of potential etiology of ASD Singh et al., 1993, Singh et al., 1997.
On the basis of these findings, we designed this study to test our hypothesis that subtle but aberrant innate immune responses and resultant dysregulated adaptive immune responses may predispose ASD children to adverse reactions to benign environmental factors including childhood common infection, immunizations, and dietary proteins. Such adverse reactions could trigger and/or aggravate developmental regression and autistic behavior in ASD children. This study characterizes ASD children with developmental regression from the view of innate and adaptive immune responses as an initial step in testing our hypothesis.
Section snippets
Method
The study included children with developmental regression and autism spectrum disorders (ASD), healthy unrelated children, and developmentally normal siblings (Table 1). Reference values for cytokine production assays were obtained from normal adult volunteers (N=10). ASD children include autism (N=65) and PDD-NOS (N=6) as diagnosed by DSM-IV criteria (American Psychiatric Association, 1994). Five patients with autism have seizure disorders confirmed with EEG and have been treated with valproic
Innate immune responses
PBMCs from a significant number of ASD children produced >2 standard deviations (SD) above the control mean (CM) values of proinflammatory and counter-regulatory cytokines (Table 2). Specifically, in the absence of LPS, 27, 26, 27, and 19 out of 71 ASD PBMCs produced >2 SD above the CM values of TNF-α, IL-6, sTNFRI, and sTNFRII, respectively. With LPS (0.1 μg/ml), 40, 22, 30, 20, and 12 out of 71 ASD PBMCs produced >2 SD above the CM values of TNF-α, IL-6, IL-1β, sTNFRII, and sTNFRII,
Discussion
This study was undertaken to evaluate innate and adaptive immune responses in ASD children with developmental regression, secondary to frequent parental reports of adverse reactions to benign environmental factors including vaccination, childhood infection, and dietary proteins in these children. We observed that PBMCs from a number of ASD children produced excessive proinflammatory and regulatory cytokines associated with innate immunity compared to controls (Table 2). Adaptive immune
Acknowledgements
Authors are thankful to Ms. Maura Fisk for her fundraising efforts and general support for this project. We are also thankful to Mr. Robert Weyerhaeuser and Mr. and Mrs. Joseph and Jane Micallef for their interest in and generous support for this study.
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Research funded by Cherbec Advancement Foundation (Robert M Weyerhaeuser), St. Paul, MN, Carl A Weyerhaeuser Charitable Trust (Robert M Weyerhaeuser), St. Paul, MN, Autism Research Institute, San Diego, CA, US Davis M.I.N.D. Institute, Sacramento, CA and private donation (Joseph and Jane Micallef, St. Paul, MN).