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Published online before print August 3, 2007

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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.1106683

Received for publication November 20, 2006.
Revised June 7, 2007.
Accepted for publication June 28, 2007.

Article

A 5.8-kDa component of manuka honey stimulates immune cells via TLR4

A. J. Tonks ^*@, E. Dudley , N. G. Porter , J. Parton ^*, J. Brazier ^*, E. L. Smith ^*, and A. Tonks

Departments of *Medical Microbiology and Haematology, Cardiff University, Cardiff, United Kingdom; Biochemistry Research Group, School of Biological Sciences, School of Environment and Society, University of Wales Swansea, United Kingdom; and Crop and Food Research Ltd., Christchurch, New Zealand

^@ To whom correspondence should be addressed. E-mail: tonksaj{at}cf.ac.uk.

	Abstract

Honey is used as a therapy to aid wound healing. Previous data indicate that honey can stimulate cytokine production from human monocytes. The present study further examines this phenomenon in manuka honey. As inflammatory cytokine production in innate immune cells is classically mediated by pattern recognition receptors in response to microorganisms, bacterial contamination of honey and the effect of blocking TLR2 and -4 on stimulatory activity were assessed. No vegetative bacteria were isolated from honey; however, bacterial spores were cultured from one-third of samples, and low levels of LPS were detected. Blocking TLR4 but not TLR2 inhibited honey-stimulated cytokine production significantly. Cytokine production did not correlate with LPS levels in honey and was not inhibited by polymyxin B. Further, the activity was reduced significantly following heat treatment, indicating that component(s) other than LPS are responsible for the stimulatory activity of manuka honey. To identify the component responsible for inducing cytokine production, honey was separated by molecular weight using microcon centrifugal filtration and fractions assessed for stimulatory activity. The active fraction was analyzed by MALDI-TOF mass spectroscopy, which demonstrated the presence of a number of components of varying molecular weights. Additional fractionation using miniaturized, reverse-phase solid-phase extraction resulted in the isolation of a 5.8-kDa component, which stimulated production of TNF- via TLR4. These findings reveal mechanisms and components involved in honey stimulation of cytokine induction and could potentially lead to the development of novel therapeutics to improve wound healing for patients with acute and chronic wounds.

Key Words: cytokines • antibacterial • separation • identification

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