Journal of Autism and Developmental Disorders

, Volume 43, Issue 11, pp 2686–2695 | Cite as

Cannabinoid Receptor Type 2, but not Type 1, is Up-Regulated in Peripheral Blood Mononuclear Cells of Children Affected by Autistic Disorders

  • Dario SiniscalcoEmail author
  • Anna Sapone
  • Catia Giordano
  • Alessandra Cirillo
  • Laura de Magistris
  • Francesco Rossi
  • Alessio Fasano
  • James Jeffrey Bradstreet
  • Sabatino Maione
  • Nicola Antonucci
Original Paper


Autistic disorders (ADs) are heterogeneous neurodevelopmental disorders arised by the interaction of genes and environmental factors. Dysfunctions in social interaction and communication skills, repetitive and stereotypic verbal and non-verbal behaviours are common features of ADs. There are no defined mechanisms of pathogenesis, rendering curative therapy very difficult. Indeed, the treatments for autism presently available can be divided into behavioural, nutritional and medical approaches, although no defined standard approach exists. Autistic children display immune system dysregulation and show an altered immune response of peripheral blood mononuclear cells (PBMCs). In this study, we investigated the involvement of cannabinoid system in PBMCs from autistic children compared to age-matched normal healthy developing controls (age ranging 3–9 years; mean age: 6.06 ± 1.52 vs. 6.14 ± 1.39 in autistic children and healthy subjects, respectively). The mRNA level for cannabinoid receptor type 2 (CB2) was significantly increased in AD-PBMCs as compared to healthy subjects (mean ± SE of arbitrary units: 0.34 ± 0.03 vs. 0.23 ± 0.02 in autistic children and healthy subjects, respectively), whereas CB1 and fatty acid amide hydrolase mRNA levels were unchanged. mRNA levels of N-acylphosphatidylethanolamine-hydrolyzing phospholipase D gene were slightly decreased. Protein levels of CB-2 were also significantly increased in autistic children (mean ± SE of arbitrary units: 33.5 ± 1.32 vs. 6.70 ± 1.25 in autistic children and healthy subjects, respectively). Our data indicate CB2 receptor as potential therapeutic target for the pharmacological management of the autism care.


Autistic disorders Cannabinoid system Gene expression PBMCs 



First and foremost, we thank the many autism families who volunteered as participants in this research study. The authors gratefully thank Mr. Enzo Abate, Ms. Giovanna Gallone and the no-profit organizations “La Forza del Silenzio” and “Cancellautismo”—Italy for their useful assistance. We thank the Autism Research Institute, USA (ARI grant “Research that makes a difference” 2010) for financial support of this study. The authors would like to thank Dr. Sarah Costantino, Second University of Naples, for her useful assistance in image analysis.


  1. Agudelo, M., Newton, C., Widen, R., Sherwood, T., Nong, L., Friedman, H., et al. (2008). Cannabinoid receptor 2 (CB2) mediates immunoglobulin class switching from IgM to IgE in cultures of murine-purified B lymphocytes. Journal of Neuroimmune Pharmacology, 3(1), 35–42.PubMedCrossRefGoogle Scholar
  2. Alessio, N., Squillaro, T., Cipollaro, M., Bagella, L., Giordano, A., & Galderisi, U. (2010). The BRG1 ATPase of chromatin remodeling complexes is involved in modulation of mesenchymal stem cell senescence through RB-P53 pathways. Oncogene, 29(40), 5452–5463.PubMedCrossRefGoogle Scholar
  3. Algeciras-Schimnich, A., Barnhart, B. C., & Peter, M. E. (2002). Apoptosis independent functions of killer caspases. Current Opinion in Cell Biology, 14, 721–726.PubMedCrossRefGoogle Scholar
  4. American Psychiatric Association. (2000). Diagnostic and statistical manual of mental disorders (4th ed.). Text Revision. Washington, DC: American Psychiatric Press.Google Scholar
  5. Ardura, J. A., & Friedman, P. A. (2011). Regulation of g protein-coupled receptor function by na +/h + exchange regulatory factors. Pharmacological Reviews, 63(4), 882–900.PubMedCrossRefGoogle Scholar
  6. Ashwood, P., Krakowiak, P., Hertz-Picciotto, I., Hansen, R., Pessah, I., & Van de Water, J. (2011). Elevated plasma cytokines in autism spectrum disorders provide evidence of immune dysfunction and are associated with impaired behavioral outcome. Brain, Behavior, and Immunity, 25(1), 40–45.PubMedCrossRefGoogle Scholar
  7. Ashwood, P., Wills, S., & Van de Water, J. (2006). The immune response in autism: A new frontier for autism research. Journal of Leukocyte Biology, 80(1), 1–15.PubMedCrossRefGoogle Scholar
  8. Barna, I., & Zelena, D. (2012). The biochemical complexity of the endocannabinoid system with some remarks on stress and related disorders: A minireview. Endocrine Regulations, 46(2), 107–124.PubMedCrossRefGoogle Scholar
  9. Basu, S., & Dittel, B. N. (2011). Unraveling the complexities of cannabinoid receptor 2 (CB2) immune regulation in health and disease. Immunologic Research, 51(1), 26–38.PubMedCrossRefGoogle Scholar
  10. Bradford, M. M. (1976). A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.PubMedCrossRefGoogle Scholar
  11. Cencioni, M. T., Chiurchiù, V., Catanzaro, G., Borsellino, G., Bernardi, G., Battistini, L., et al. (2010). Anandamide suppresses proliferation and cytokine release from primary human T-lymphocytes mainly via CB2 receptors. PLoS ONE, 5(1), e8688.PubMedCrossRefGoogle Scholar
  12. de Magistris, L., Familiari, V., Pascotto, A., Sapone, A., Frolli, A., Iardino, P., et al. (2010). Alterations of the intestinal barrier in patients with autism spectrum disorders and in their first-degree relatives. Journal of Pediatric Gastroenterology and Nutrition, 51(4), 418–424.PubMedCrossRefGoogle Scholar
  13. Di Filippo, C., Rossi, F., Rossi, S., & D’Amico, M. (2004). Cannabinoid CB2 receptor activation reduces mouse myocardial ischemia-reperfusion injury: Involvement of cytokine/chemokines and PMN. Journal of Leukocyte Biology, 75(3), 453–459.PubMedCrossRefGoogle Scholar
  14. Di Marzo, V., & Petrosino, S. (2007). Endocannabinoids and the regulation of their levels in health and disease. Current Opinion in Lipidology, 18(2), 129–140.PubMedCrossRefGoogle Scholar
  15. Dong, R., Dong, K., Wang, X., Chen, G., Shen, C., & Zheng, S. (2012). Interleukin-33 overexpression is associated with gamma-glutamyl transferase in biliary atresia. Cytokine, S1043–4666(12), 00771–00775.Google Scholar
  16. Enstrom, A. M., Onore, C. E., Van de Water, J. A., & Ashwood, P. (2010). Differential monocyte responses to TLR ligands in children with autism spectrum disorders. Brain, Behavior, and Immunity, 24(1), 64–71.PubMedCrossRefGoogle Scholar
  17. Galiègue, S., Mary, S., Marchand, J., Dussossoy, D., Carrière, D., Carayon, P., et al. (1995). Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. European Journal of Biochemistry, 232(1), 54–61.PubMedCrossRefGoogle Scholar
  18. Garcia-Gutierrez, M. S., & Manzanares, J. (2011). Overexpression of CB2 cannabinoid receptors decreased vulnerability to anxiety and impaired anxiolytic action of alprazolam in mice. Journal of Psychopharmacology, 25(1), 111–120.PubMedCrossRefGoogle Scholar
  19. Giordano, C., Siniscalco, D., Melisi, D., Luongo, L., Curcio, A., Soukupova, M., et al. (2011). The galactosylation of N(ω)-nitro-l-arginine enhances its anti-nocifensive or anti-allodynic effects by targeting glia in healthy and neuropathic mice. European Journal of Pharmacology, 656(1–3), 52–62.PubMedCrossRefGoogle Scholar
  20. Guo, Y., Xiao, P., Lei, S., Deng, F., Xiao, G. G., Liu, Y., et al. (2008). How is mRNA expression predictive for protein expression? A correlation study on human circulating monocytes. Acta Biochimica et Biophysica Sinica (Shanghai), 40(5), 426–436.CrossRefGoogle Scholar
  21. Gupta, S., Samra, D., & Agrawal, S. (2010). Adaptive and innate immune responses in autism: rationale for therapeutic use of intravenous immunoglobulin. Journal of Clinical Immunology, 30, S90–S96.CrossRefGoogle Scholar
  22. Gygi, S. P., Rochon, Y., Franza, B. R., & Aebersold, R. (1999). Correlation between protein and mRNA abundance in yeast. Molecular and Cellular Biology, 19(3), 1720–1730.PubMedGoogle Scholar
  23. Hegde, V. L., Nagarkatti, M., & Nagarkatti, P. S. (2010). Cannabinoid receptor activation leads to massive mobilization of myeloid-derived suppressor cells with potent immunosuppressive properties. European Journal of Immunology, 40(12), 3358–33571.PubMedCrossRefGoogle Scholar
  24. Högestätt, E. D., Jönsson, B. A., Ermund, A., Andersson, D. A., Björk, H., Alexander, J. P., et al. (2005). Conversion of acetaminophen to the bioactive N-acylphenolamine AM404 via fatty acid amide hydrolase-dependent arachidonic acid conjugation in the nervous system. Journal of Biological Chemistry, 280(36), 31405–31412.PubMedCrossRefGoogle Scholar
  25. Ishiguro, H., Horiuchi, Y., Ishikawa, M., Koga, M., Imai, K., Suzuki, Y., et al. (2010). Brain cannabinoid CB2 receptor in schizophrenia. Biological Psychiatry, 67(10), 974–982.PubMedCrossRefGoogle Scholar
  26. Jean-Gilles, L., Gran, B., & Constantinescu, C. S. (2010). Interaction between cytokines, cannabinoids and the nervous system. Immunobiology, 215(8), 606–610.PubMedCrossRefGoogle Scholar
  27. Kenny, P. J. (2011). Macrophage cannabinoid receptor goes up in smoke. Nature Neuroscience, 14, 1100–1102.PubMedCrossRefGoogle Scholar
  28. Klein, T. W., & Cabral, G. A. (2006). Cannabinoid-induced immune suppression and modulation of antigen-presenting cells. Journal of Neuroimmune Pharmacology, 1(1), 50–64.PubMedCrossRefGoogle Scholar
  29. Klein, T. W., Newton, C., Larsen, K., Lu, L., Perkins, I., Nong, L., et al. (2003). The cannabinoid system and immune modulation. Journal of Leukocyte Biology, 74(4), 486–496.PubMedCrossRefGoogle Scholar
  30. Lamkanfi, M., Declercq, W., Kalai, M., Saelens, X., & Vandenabeele, P. (2002). Alice in caspase land. A phylogenetic analysis of caspases from worm to man. Cell Death and Differentiation, 9, 358–361.PubMedCrossRefGoogle Scholar
  31. Levy, S. E., Mandell, D. S., & Schultz, R. T. (2009). Autism. Lancet, 374(9701), 1627–1638.PubMedCrossRefGoogle Scholar
  32. Li, X., Chauhan, A., Sheikh, A. M., Patil, S., Chauhan, V., Li, X. M., et al. (2009). Elevated immune response in the brain of autistic patients. Journal of Neuroimmunology, 207(1–2), 111–116.PubMedCrossRefGoogle Scholar
  33. Li, C., Jones, P. M., & Persaud, S. J. (2011). Role of the endocannabinoid system in food intake, energy homeostasis and regulation of the endocrine pancreas. Pharmacology & Therapeutics, 129(3), 307–320.CrossRefGoogle Scholar
  34. Lord, C., Risi, S., Lambrecht, L., Cook, E. H., Jr, Leventhal, B. L., DiLavore, P. C., et al. (2000). The autism diagnostic observation schedule-generic: A standard measure of social and communication deficits associated with the spectrum of autism. Journal of Autism and Developmental Disorders, 30(3), 205–223.PubMedCrossRefGoogle Scholar
  35. Lord, C., Rutter, M., & Le Couteur, A. (1994). Autism diagnostic interview-revised: A revised version of a diagnostic interview for caregivers of individuals with possible Pervasive Developmental Disorders. Journal of Autism and Developmental Disorders, 24(5), 659–685.PubMedCrossRefGoogle Scholar
  36. Maccarrone, M., De Petrocellis, L., Bari, M., Fezza, F., Salvati, S., Di Marzo, V., et al. (2001). Lipopolysaccharide downregulates fatty acid amide hydrolase expression and increases anandamide levels in human peripheral lymphocytes. Archives of Biochemistry and Biophysics, 393(2), 321–328.PubMedCrossRefGoogle Scholar
  37. Maier, T., Güell, M., & Serrano, L. (2009). Correlation of mRNA and protein in complex biological samples. FEBS Letters, 583(24), 3966–3973.PubMedCrossRefGoogle Scholar
  38. Meyer, S., Temme, C., & Wahle, E. (2004). Messenger RNA turnover in eukaryotes: Pathways and enzymes. Critical Reviews in Biochemistry and Molecular Biology, 39(4), 197–216.PubMedCrossRefGoogle Scholar
  39. Minocci, D., Massei, J., Martino, A., Milianti, M., Piz, L., Di Bello, D., et al. (2011). Genetic association between bipolar disorder and 524A > C (Leu133Ile) polymorphism of CNR2 gene, encoding for CB2 cannabinoid receptor. Journal of Affective Disorders, 134(1–3), 427–430.PubMedCrossRefGoogle Scholar
  40. Molloy, C. A., Morrow, A. L., Meinzen-Derr, J., Schleifer, K., Dienger, K., Manning-Courtney, P., et al. (2006). Elevated cytokine levels in children with autism spectrum disorder. Journal of Neuroimmunology, 172(1–2), 198–205.PubMedCrossRefGoogle Scholar
  41. Mouslech, Z., & Valla, V. (2009). Endocannabinoid system: An overview of its potential in current medical practice. Neuro Endocrinology Letters, 30(2), 153–179.PubMedGoogle Scholar
  42. Nagarkatti, P., Pandey, R., Rieder, S. A., Hegde, V. L., & Nagarkatti, M. (2009). Cannabinoids as novel anti-inflammatory drugs. Future Medicinal Chemistry, 1(7), 1333–1349.PubMedCrossRefGoogle Scholar
  43. Nong, L., Newton, C., Cheng, Q., Friedman, H., Roth, M. D., & Klein, T. W. (2002). Altered cannabinoid receptor mRNA expression in peripheral blood mononuclear cells from marijuana smokers. Journal of Neuroimmunology, 127(1–2), 169–176.PubMedCrossRefGoogle Scholar
  44. Onore, C., Enstrom, A., Krakowiak, P., Hertz-Picciotto, I., Hansen, R., Van de Water, J., et al. (2009). Decreased cellular IL-23 but not IL-17 production in children with autism spectrum disorders. Journal of Neuroimmunology, 216(1–2), 126–129.PubMedCrossRefGoogle Scholar
  45. Pacher, P., & Mechoulam, R. (2011). Is lipid signaling through cannabinoid 2 receptors part of a protective system? Progress in Lipid Research, 50(2), 193–211.PubMedCrossRefGoogle Scholar
  46. Pascal, L. E., True, L. D., Campbell, D. S., Deutsch, E. W., Risk, M., Coleman, I. M., et al. (2008). Correlation of mRNA and protein levels: Cell type-specific gene expression of cluster designation antigens in the prostate. BMC Genomics, 9, 246.PubMedCrossRefGoogle Scholar
  47. Peralta, L., Agirregoitia, E., Mendoza, R., Expósito, A., Casis, L., Matorras, R., et al. (2011). Expression and localization of cannabinoid receptors in human immature oocytes and unfertilized metaphase-II oocytes. Reprod Biomed Online, 23(3), 372–379.PubMedCrossRefGoogle Scholar
  48. Pertwee, R. G., Howlett, A. C., Abood, M. E., Alexander, S. P., Di Marzo, V., Elphick, M. R., et al. (2010). Cannabinoid receptors and their ligands: Beyond CB1 and CB2. Pharmacological Reviews, 62(4), 588–631.PubMedCrossRefGoogle Scholar
  49. Rajagopalan, L. E., & Malter, J. S. (1997). Regulation of eukaryotic messenger RNA turnover. Progress in Nucleic Acid Research and Molecular Biology, 56, 257–286.PubMedCrossRefGoogle Scholar
  50. Robinson, S. A., Loiacono, R. E., Christopoulos, A., Sexton, P. M., & Malone, D. T. (2010). The effect of social isolation on rat brain expression of genes associated with endocannabinoid signaling. Brain Research, 1343, 153–167.PubMedCrossRefGoogle Scholar
  51. Romero-Calvo, I., Ocón, B., Martínez-Moya, P., Suárez, M. D., Zarzuelo, A., Martínez-Augustin, O., et al. (2010). Reversible Ponceau staining as a loading control alternative to actin in Western blots. Analytical Biochemistry, 401(2), 318–320.PubMedCrossRefGoogle Scholar
  52. Ross, J. (1996). Control of messenger RNA stability in higher eukaryotes. Trends in Genetics, 12(5), 171–175.PubMedCrossRefGoogle Scholar
  53. Schneider, M., & Koch, M. (2005). Deficient social and play behavior in juvenile and adult rats after neonatal cortical lesion: Effects of chronic pubertal cannabinoid treatment. Neuropsychopharmacology, 30(5), 944–957.PubMedCrossRefGoogle Scholar
  54. Schopler, E., Reichler, R. J., & Renner, B. R. (1993). The childhood autism rating scale (CARS). Los Angeles, CA: Western Psychological Services.Google Scholar
  55. Schultz, S. T. (2010). Can autism be triggered by acetaminophen activation of the endocannabinoid system? Acta Neurobiologiae Experimentalis, 70(2), 227–231.PubMedGoogle Scholar
  56. Siniscalco, D., Sapone, A., Giordano, C., Cirillo, A., de Novellis, V., de Magistris, L., et al. (2012). The expression of caspases is enhanced in peripheral blood mononuclear cells of autism spectrum disorder patients. Journal of Autism and Developmental Disorders, 42(7), 1403–1410.PubMedCrossRefGoogle Scholar
  57. Soukupová, M., Palazzo, E., De Chiaro, M., Gatta, L., Migliozzi, A. L., Guida, F., et al. (2010). Effects of URB597, an inhibitor of fatty acid amide hydrolase (FAAH), on analgesic activity of paracetamol. Neuro Endocrinology Letters, 31(4), 507–511.PubMedGoogle Scholar
  58. Stamova, B. S., Apperson, M., Walker, W. L., Tian, Y., Xu, H., Adamczy, P., et al. (2009). Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood. BMC Medical Genomics, 2, 49.PubMedCrossRefGoogle Scholar
  59. Suzuki, K., Matsuzaki, H., Iwata, K., Kameno, Y., Shimmura, C., Kawai, S., et al. (2011). Plasma cytokine profiles in subjects with high-functioning autism spectrum disorders. PLoS ONE, 6(5), e20470.PubMedCrossRefGoogle Scholar
  60. Tanikawa, T., Kurohane, K., & Imai, Y. (2011). Regulatory effect of cannabinoid receptor agonist on chemokine-induced lymphocyte chemotaxis. Biological and Pharmaceutical Bulletin, 34(7), 1090–1093.PubMedCrossRefGoogle Scholar
  61. Tománková, H., & Myslivecek, J. (2012). Regulation of receptors coupled to G proteins (GPCRs). Ceskoslovenska Fysiologie, 61(1), 15–23.PubMedGoogle Scholar
  62. Wilusz, C. J., Wormington, M., & Peltz, S. W. (2001). The cap-to-tail guide to mRNA turnover. Nature Reviews Molecular Cell Biology, 2(4), 237–246.PubMedCrossRefGoogle Scholar
  63. Yang, M., Liu, Y., Lu, S., Wang, Z., Wang, R., Zi, Y., et al. (2013). Analysis of the expression levels of survivin and VEGF in patients with acute lymphoblastic leukemia. Experimental and Therapeutic Medicine, 5(1), 305–307.PubMedGoogle Scholar
  64. Zanichelli, F., Capasso, S., Cipollaro, M., Pagnotta, E., Cartenì, M., Casale, F., et al. (2012). Dose-dependent effects of R-sulforaphane isothiocyanate on the biology of human mesenchymal stem cells, at dietary amounts, it promotes cell proliferation and reduces senescence and apoptosis, while at anti-cancer drug doses, it has a cytotoxic effect. Age (Dordr), 34(2), 281–293.CrossRefGoogle Scholar
  65. Zhu, C., Solorzano, C., Sahar, S., Realini, N., Fung, E., Sassone-Corsi, P., et al. (2011). Proinflammatory stimuli control N-acylphosphatidylethanolamine-specific phospholipase D expression in macrophages. Molecular Pharmacology, 79(4), 786–792.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Dario Siniscalco
    • 1
    • 2
    Email author
  • Anna Sapone
    • 3
    • 4
  • Catia Giordano
    • 1
  • Alessandra Cirillo
    • 5
  • Laura de Magistris
    • 3
  • Francesco Rossi
    • 1
  • Alessio Fasano
    • 4
    • 6
  • James Jeffrey Bradstreet
    • 7
  • Sabatino Maione
    • 1
  • Nicola Antonucci
    • 8
  1. 1.Division of Pharmacology, Department of Experimental MedicineSecond University of NaplesNaplesItaly
  2. 2.Centre for AutismCasertaItaly
  3. 3.Department of Internal and Experimental Medicine “Magrassi-Lanzara”Second University of NaplesNaplesItaly
  4. 4.Center for Celiac Research and Mucosal Immunology and Biology Research CenterMassachusetts General Hospital EastCharlestownUSA
  5. 5.Division of Biotechnology and Molecular Biology “A. Cascino”, Department of Experimental MedicineSecond University of NaplesNaplesItaly
  6. 6.MassGeneral Hospital for ChildrenBostonUSA
  7. 7.International Child Development Resource CenterCummingUSA
  8. 8.Biomedical Centre for Autism Research and TreatmentBariItaly

Personalised recommendations