Low natural killer cell cytotoxic activity in autism: The role of glutathione, IL-2 and IL-15

https://doi.org/10.1016/j.jneuroim.2008.09.005Get rights and content

Abstract

Although many articles have reported immune abnormalities in autism, NK cell activity has only been examined in one study of 31 patients, of whom 12 were found to have reduced NK activity. The mechanism behind this low NK cell activity was not explored. For this reason, we explored the measurement of NK cell activity in 1027 blood samples from autistic children obtained from ten clinics and compared the results to 113 healthy controls. This counting of NK cells and the measurement of their lytic activity enabled us to express the NK cell activity/100 cells. At the cutoff of 15–50 LU we found that NK cell activity was low in 41–81% of the patients from the different clinics. This NK cell activity below 15 LU was found in only 8% of healthy subjects (p < 0.001). Low NK cell activity in both groups did not correlate with percentage and absolute number of CD16+/CD56+ cells. When the NK cytotoxic activity was expressed based on activity/100 CD16+/CD56+ cells, several patients who had displayed NK cell activity below 15 LU exhibited normal NK cell activity. Overall, after this correction factor, 45% of the children with autism still exhibited low NK cell activity, correlating with the intracellular level of glutathione. Finally, we cultured lymphocytes of patients with low or high NK cell activity/cell with or without glutathione, IL-2 and IL-15. The induction of NK cell activity by IL-2, IL-15 and glutathione was more pronounced in a subgroup with very low NK cell activity. We conclude that that 45% of a subgroup of children with autism suffers from low NK cell activity, and that low intracellular levels of glutathione, IL-2 and IL-15 may be responsible.

Introduction

Autism is a developmental disorder of unknown etiology which occurs in childhood (Baird et al., 2003). As with many complex autoimmune diseases, genetic and environmental factors including infection, diet and xenobiotics, along with the resultant immune and neurological abnormalities, are thought to play a role in the development of autism (Edelson and Cantor, 2000, Vojdani et al., 2002, Vojdani et al., 2003, Vojdani et al., 2004, Wiznitzer, 2004, Rutter, 2000, Cohen et al., 2005, Fatemi et al., 2002, Fatemi et al., 2005, Sebat et al., 2007; Freitag, 2007, Weiss et al., 2008, Kim et al., 2008, Shi et al., 2003). Studies have shown several genetic links, including chromosomal abnormalities, each in a very small percentage of cases (Wiznitzer, 2004, Rutter, 2000, Cohen et al., 2005, Fatemi et al., 2005, Sebat et al., 2007, Freitag, 2007, Weiss et al., 2008, Kim et al., 2008).

Immune abnormalities in autism include changes in the numbers and activities of macrophages, T cells, B cells and natural killer (NK) cell activity (Ashwood et al., 2004, Ashwood et al., 2006 Ashwood and Wakefield, 2006, Stubbs and Crawford, 1977, Warren et al., 1986, Warren et al., 1987). In addition, it has been shown that children with ASD had increased activation of both Th1 and Th2 arms of the adaptive immune response, with a Th2 predominance, and without the compensatory increase in the regulatory cytokine IL-10 (Molloy et al., 2006). Another study reported that the innate and adaptive immune responses in children with autism were associated with elevations in TNF-α, IL-1β, and/or IL-6 (Jyonouchi et al., 2001).

In the mid-1970s NK cells were originally identified on a functional basis because of their ability to lyse certain tumor cells in vitro without the requirement for prior immune sensitization of the host (Kiessling et al., 1975, Herberman et al., 1975). Since then, much knowledge has been accumulated regarding their origin, differentiation, receptor repertoire and effector functions, as well as their ability to shape adaptive immune responses (Colucci et al., 2003, Raulet, 2004, Farag and Caligiuri, 2006). NK cells represent a unique subset of lymphocytes, distinct from T and B cells, that contribute to host anti-microbial and anti-tumor defense reactions (Biron et al., 1999, Wu and Lanier, 2001, Mueller et al., 2008). In humans, NK cells are broadly defined as CD3CD56+ lymphocytes. They can be further subdivided into two main subsets on the basis of their surface expression levels of CD56. CD56bright NK cells have predominantly immunoregulatory properties mediated by a potent cytokine producing capacity, while CD56dim NK cells have a marked cytotoxic function (Cooper et al., 2001). The latter cell subset also expresses high levels of the low-affinity Fc receptor for IgG (FcγRIII; also known as CD16) allowing them to mediate antibody-dependent cell-mediated cytotoxicity (ADCC).

NK cells can also be stimulated by several types of cytokines, in particular IL-2 and IL-15. Once activated by cytokines, NK cells proliferate and show increased secretory and cytolytic functions. Activated NK cells secrete interferon-γ (IFNγ), tumor necrosis factor (TNF) and granulocyte/macrophage colony-stimulating factor (GM-CSF). These cytokines, together with secreted chemokines, stimulate inflammatory responses. Cytokine secretion by NK cells also influences adaptive immune responses by modulating the growth and differentiation of monocytes, dendritic cells and granulocytes (Biron et al., 1999, Raulet, 2004). The exocytosis of lytic granules that contain membrane-disrupting proteins such as perforin and a family of structurally related serine proteases known as granzymes is considered to be the main mechanism used by NK cells to kill target cells. NK cells also express molecules of the TNF superfamily that can engage cell-death receptors on target cells, which results in the induction of apoptosis (Bryceson et al., 2006, Hayakawa and Smyth, 2006). Furthermore, there is a correlation between increased NK cell function and anti-tumor responses in individuals treated with IL-2 and IL-15 (Fehniger et al., 2002, Ljunggren and Malmberg, 2007).

In children with autism and the related disorder Rett syndrome, lower levels of circulating numbers of NK cells are noted compared with controls (Raulet, 2004, Farag and Caligiuri, 2006). Additionally, in children with ASD, decreased NK cell activity demonstrated by target lysis has been shown (Warren et al., 1987). This measurement of NK cell activity was carried out on the PBMCs of 31 patients with autism; 12 of the 31 (39%) were found to have reduced levels of cytotoxicity.

Therefore, this multi-center study was conducted on more than one thousand specimens obtained from 10 different clinics in different states. Low NK cell activity was demonstrated in children with autism in all 10 clinics. This low NK cell activity correlated with low intracellular levels of glutathione, a known potentiator of immunological function (Droge et al., 1994), and cytokines such as IL-2 and IL-15 (Ljunggren and Malmberg, 2007).

It seems that a decrease in the cellular cysteine supply decreases the intracellular glutathione (GSH) level. This, along with low levels of IL-2 and IL-15 production, may contribute to lower NK cell activity in autism.

Section snippets

Study population

Blood samples from 1027 (3:1 ratio of male to female), 2 to 15 years of age, with a diagnosis of autism were sent by different clinicians to our laboratory for immune system evaluation, including NK cytotoxic activity examination. The clinical diagnosis of autism was made according to the DSM IV and/or ICD-10 criteria established by the American Psychiatric Association (Washington, D.C.), as well as by a developmental pediatrician, a pediatric neurologist, and/or a licensed psychologist.

NK cell cytotoxic activity in autism

An investigation of the natural killer cytotoxic activity of peripheral blood mononuclear cells from 1027 patients with autism and 113 controls was carried out using Cr51 release assay with K562 tumor cells as the target. PMBCs from patients with autism exhibited significantly lower cytotoxic activity than that of the children in the control group. The distribution of the NK cell activity of all groups along with the mean values are presented in Fig. 1. The mean cytotoxic activity for controls

Discussion

Natural killer cells mediate early non-adaptive responses against viruses, intracellular bacteria, parasite-infected cells and malignancies (Deniz et al., 2008, Orange and Ballas, 2006). They mediate these effects through the production of cytokines and the direct killing of a transformed or infected cell by granule release (Kozlowski et al., 1999, Fortier and Kornbluth, 2006, Zhou et al., 2002, Huang et al., 2006).

In addition, these cells are involved in regulating immune response through

Disclosures

Doreen Granpeesheh, Ph.D., B.C.B.A., is the founder and executive director of The Center for Autism and Related Disorders (CARD). She is not on CARD's IRB and took no part in the review process for this study.

Acknowledgments

We would like to thank Dr. Mohsen Bazargan for his contributions in the statistical analysis of the data for this project, and Mr. Joel Bautista for the preparation of the manuscript.

References (65)

  • JyonouchiH. et al.

    Proinflammatory and regulatory cytokine production associated with innate and adaptive immune responses in children with autism spectrum disorders and developmental regression

    J. Neuroimmunol.

    (2001)
  • JohanssonS. et al.

    NK cells: elusive players in autoimmunity

    Trend Immunol.

    (2005)
  • KimH.-G. et al.

    Disruption of neurexin 1 (NRXN1) associated with autism spectrum disorder

    Am. J. Hum. Genet.

    (2008)
  • LiangC.-M. et al.

    Glutathione regulates interleukin-2 activity on cytotoxic T-cells

    J. Biol. Chem.

    (1989)
  • MolloyC.A. et al.

    Elevated cytokine levels in children with autism spectrum disorder

    J. Neuroimmunol.

    (2006)
  • OrangeJ.S. et al.

    Natural killer cells in human health and disease

    Clin. Immunol.

    (2006)
  • SegalB.M.

    The role of natural killer cells in curbing neuroinflammation

    J. Neuroimmunol.

    (2007)
  • VojdaniA. et al.

    Antibodies to neuron-specific antigens in children with autism: possible cross-reaction with encephalitogenic proteins from milk, Chlamydia pneumoniae and Streptococcus Group A

    J. Neuroimmunol.

    (2002)
  • WarrenR.P. et al.

    Reduced natural killer cell activity in autism

    J. Am. Acad. Child Adolesc. Psych.

    (1987)
  • AshwoodP. et al.

    Spontaneous mucosal lymphocyte cytokine profiles in children with autism and gastrointestinal symptoms: mucosal immune activation and reduced counter regulatory interleukin-10

    J. Clin. Immunol.

    (2004)
  • AshwoodP. et al.

    The immune response in autism: a new frontier for autism research

    J. Leukoc. Biol.

    (2006)
  • BairdG. et al.

    Diagnosis of autism

    Br. Med. J.

    (2003)
  • BironC.A. et al.

    Natural killer cells in antiviral defense: function and regulation by innate cytokines

    Annu. Rev. Immunol.

    (1999)
  • BrycesonY.T. et al.

    Activation, coactivation and costimulation of resting human natural killer cells

    Immunol. Rev.

    (2006)
  • CarsonW.E. et al.

    A potential role for interleukin-15 in the regulation of human natural killer cell survival

    J. Clin. Invest.

    (1997)
  • CohenD. et al.

    Specific genetic disorders and autism: clinical contribution towards their identification

    J. Autism Dev. Disord.

    (2005)
  • ColucciF. et al.

    What does it take to make a natural killer?

    Nature Rev. Immunology

    (2003)
  • DenizG. et al.

    Regulatory NK cells suppress antigen-specific T cell responses

    J. Immunol.

    (2008)
  • DrogeW. et al.

    Functions of glutathione and glutathione disulfide in immunology and immunopathology

    FASEB J.

    (1994)
  • EdelsonS.B. et al.

    The neurotoxic etiology of the autistic spectrum disorder: a replicative study

    Toxicol. Ind. Health

    (2000)
  • FatemiS.H. et al.

    Prenatal viral infection leads to pyramidal cell atrophy and macrocephaly in adulthood: implications for genesis of autism and schizophrenia

    Cell. Mol. Neurobiol.

    (2002)
  • FortierJ.M. et al.

    NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase

    J. Immunol.

    (2006)
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