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LNFPIII/LeX-Stimulated Macrophages Activate Natural Killer Cells via CD40-CD40L Interaction

Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 02115

Received 13 April 2005/ Returned for modification 27 May 2005/ Accepted 15 June 2005

Lacto-N-fucopentaose III (LNFPIII) is a human milk sugar containing the biologically active Lewis X (LeX) trisaccharide. LNFPIII/LeX is also expressed by immunosuppressive helminth parasites, by bacteria, and on a number of tumor/cancer cells. In this report, we first demonstrate that LNFPIII activates macrophages in vitro as indicated by upregulation of Gr-1 expression on F4/80⁺ cells. Further, we investigated the effect of LNFPIII-activated macrophages on NK cell activity. We found that LNFPIII-stimulated F4/80⁺ cells were able to activate NK cells, inducing upregulation of CD69 expression and gamma interferon (IFN-) production. The experiments show that NK cell activation is macrophage dependent, since NK cells alone did not secrete IFN- in response to LNFPIII. Furthermore, we found that activation of NK cells by glycan-stimulated macrophages required cell-cell contact. As part of the cell-cell contact mechanism, we determined that CD40-CD40L interaction was critical for IFN- secretion by NK cells, as the addition of anti-CD40L antibodies to the coculture blocked IFN- production. We also demonstrated that LNFPIII-stimulated macrophages secrete prostaglandin E₂, interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-) but a very low level of IL-12. Interestingly, addition of anti-TNF-, anti-IL-10, or anti-IL-12 monoclonal antibodies did not significantly alter NK cell activity. Our data show that these soluble mediators are not critical for LNFPIII-stimulated macrophage activation of NK cells and provide further evidence for the importance of cell-cell contact and CD40-CD40L interactions between macrophages and NK cells.