April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Lumican Regulates Innate Immune Response And Phagocytosis In Macrophages
Author Affiliations & Notes
  • Seakwoo Lee
    School of Medicine, Johns Hopkins University, Baltimore, Maryland
  • Kristin Lohr
    School of Medicine, Johns Hopkins University, Baltimore, Maryland
  • Shukti Chakravarti
    School of Medicine, Johns Hopkins University, Baltimore, Maryland
  • Footnotes
    Commercial Relationships  Seakwoo Lee, None; Kristin Lohr, None; Shukti Chakravarti, None
  • Footnotes
    Support  NIH EY11654
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 1118. doi:
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      Seakwoo Lee, Kristin Lohr, Shukti Chakravarti; Lumican Regulates Innate Immune Response And Phagocytosis In Macrophages. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1118.

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

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Purpose: : Lumican is a leucine-rich repeat proteoglycan of the corneal stroma. We showed that lumican interacts with CD14 and CD18 on macrophage and neutrophil cell surfaces to promote innate immune response and cell migration. Here we show that lumican regulates phagocytosis of gram-negative bacteria through specific interactions with CD14 and CD18.

Methods: : Innate immune response to lipopolysaccharides (LPS) was determined in Lum+/+ and Lum-/- primary peritoneal macrophages by ELISA measurements of TNF-α. Phagocytosis of fluorophore-tagged E. coli and Pseudomonas aeruginosa by Lum+/+ and Lum-/- macrophages were determined by confocal microscopy. Recombinant lumican (rLum) and a mutant form (rLum-Y20A) were produced in HEK293 cells. Binding affinities between CD14 and rLum or rLum-Y20A were measured by Surface Plasmon Resonance (SPR) analysis.

Results: : Induction of TNF-α was delayed in Lum-/- macrophages. Treatment of Lum-/- macrophages with exogenous rLum restored TNF-α induction to wild type levels, while rLum-Y20A showed 30% recovery. CD14-/- and Lum-/-CD14-/- macrophages, also severely impaired in LPS-mediated TNF-α induction, did not respond to rLum restoration, which indicating lumican-LPS-mediated TNF-α induction to be entirely CD14 dependent. Lum-/- macrophages showed 40% decrease of bacterial phagocytosis compared to Lum+/+, and could be restored by exogenous rLum and not rLum-Y20A. Further confocal analysis showed colocalization of lumican, CD14 and CD18 on areas of the cell surface associated with phagocytosed bacteria. SPR analysis showed specific binding of rLum to CD14 (KA = 2.15 x 106 M-1). The mutated rLum-Y20A showed weaker binding to CD 14(KA = 3.50 x 104 M-1).

Conclusions: : Lumican promotes CD14 mediated innate immune response to LPS and phagocytosis of gram-negative bacteria via molecular interactions of its N-terminal end with CD14.

Keywords: cornea: basic science • inflammation • extracellular matrix 

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