April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Diurnal-distribution-selective Down- Regulation Of Cytokines but Not Chemokines In Tears
Author Affiliations & Notes
  • Robert A. Sack
    Biological Sciences, SUNY-Opt, New York, New York
  • A Beaton
    Biological Sciences, SUNY-Opt, New York, New York
  • B. Cooper
    Biological Sciences, SUNY-Opt, New York, New York
  • S. Sathe
    Biological Sciences, SUNY-Opt, New York, New York
  • A Mezentsev
    Pharmacology, NYMC, Valhalla, New York
  • M Schwartzman
    Pharmacology, NYMC, Valhalla, New York
  • L Bellner
    Pharmacology, NYMC, Valhalla, New York
  • P. Istroavich
    Biological Sciences, SUNY-Opt, New York, New York
  • Footnotes
    Commercial Relationships  Robert A. Sack, patent application (P); A. Beaton, None; B. Cooper, None; S. Sathe, None; A. Mezentsev, None; M. Schwartzman, None; L. Bellner, None; P. Istroavich, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 1134. doi:
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      Robert A. Sack, A Beaton, B. Cooper, S. Sathe, A Mezentsev, M Schwartzman, L Bellner, P. Istroavich; Diurnal-distribution-selective Down- Regulation Of Cytokines but Not Chemokines In Tears. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1134.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Overnight eye closure results in a stagnant tear layer extremely enriched in pro-inflammatory and immune-modulating cytokines. This study was designed to further characterize this environment and to determine how cytokine bioactivity is down-regulated.

Methods: : ; Open (O) and Closed (C) tears from Ns were subjected to multiple micro well array assays to obtain quantitative data on >80 low abundance proteins. Samples were also separated by molecular sieve HPLC; the eluents were bioassayed for net angiogenic activity and the elution profiles of chemokines, cytokines and interactive proteins established. Cytokine-protein complexes and other protein-protein complexes were characterized using a novel laboratory-developed multiplex micro well assay.

Results: : Protein array analysis reveal statistically significant selective >10x increases in many pro-inflammatory and immune-modulating cytokines, chemokines and several integrins in C fluid, accompanied by a parallel build-up of soluble (s)-decoy receptors and other interacting proteins with the levels of the latter greatly exceeding that of the targeted cytokines (pg versus ng/ml range). HPLC separation followed by array analysis, bioassay, and protein-protein binding assays reveals that the vast majority of pro-inflammatory and immune-modulating cytokines in C are devoid of bioactivity and elute in a common high molecular weight fraction (>360 kDa) in which they can be shown to be bound to specific soluble cytokine-receptors as well as to a yet to be fully characterized complexing protein(s). The pattern of distribution suggests a 2-step binding process that inactivates and tags the cytokines for cleavage. In contrast, nearly all of the accumulating chemokines (IP-10, GROα, MCP-1 and most IL-8), which lack soluble receptors, elute as bioactive monomers or oligomers.

Conclusions: : Pro-inflammatory and immune- modulating cytokines accumulating in C fluid are inactivated by a novel 2-step co-operative binding process that maybe involved in controlling inflammation elsewhere. Formation of these complexes can also mask epitopes employed in cytokine sandwich ELISAs and array assays thereby becoming a source of error.

Keywords: cytokines/chemokines • cornea: tears/tear film/dry eye • immunomodulation/immunoregulation 

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