ELISpot analysis of lipopolysaccharide-stimulated leukocytes: human granulocytes selectively secrete IL-8, MIP-1beta and TNFalpha
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KeywordsPeripheral Blood Mononuclear Cell Secreting Cell ELISpot Assay Polymorphonuclear Cell Human Donor
Granulocytes or polymorphonuclear cells (PMN) represent the majority of leukocytes in peripheral blood. As terminally differentiated cells, they contain few ribosomes and assist innate immunity mainly through phagocytosis and degranulation. Whether or not they can release proinflammatory cytokines such as TNFα and IL-1β has been a controversial issue. To clarify the role of PMN in this aspect, lipopolysaccharide (LPS)-induced cytokine secretion from PMN was analyzed at the single-cell level with the ELISpot technique.
PMN and peripheral blood mononuclear cells (PBMC) from healthy human donors were prepared by gradient-based centrifugation to a purity >98%. ELISpot assays were used for detection of a large panel of inflammatory mediators. Cells were stimulated with endotoxin (LPS, 100 ng/ml) for 20 hours and the numbers of secreting cells were quantified with an ELISpot reader. For comparison, cytokine production was also analyzed by ELISA. In some experiments, PBMC were depleted of monocytes using anti-CD14 magnetic beads.
Purified PMN secreted IL-8 and MIP-1β and a sub-population also released TNFα after LPS stimulation. In contrast and different from some earlier reports, we were unable to detect secretion of IL-1β, IL-12, granulocyte–macrophage colony-stimulating factor, IL-6 or IFNγ. Furthermore, granulocytes did not secrete the cytotoxic molecules perforin or granzyme B in response to LPS. Compared with the limited cytokine production by PMN, PBMC secreted significant amounts of all substances investigated and were found to require a 100× lower concentration of LPS than granulocytes to obtain the maximum number of responding cells. In addition, CD14+ monocytes were found to be the primary source of production.
By use of the ELISpot method we could establish the cytokine profiles for both PBMC and PMN based on the frequency and pattern of cytokine secreting cells, rather than the amount of produced cytokine as by ELISA. This way, low levels of contaminating monocytes present in our PMN preparations could be discriminated from the granulocytes. Additionally, we could demonstrate that ELISpot, compared with ELISA, not only provides a more sensitive means of detection but potentially gives biologically more relevant information.
LPS-stimulated PMN were shown to secrete IL-8, MIP-1β and TNFα but not IL-1β, IL-6, IL-10, IL-12, granulocyte–macrophage colony-stimulating factor, IFNγ, perforin or granzyme B. Our findings suggest that the ELISpot assay may be a suitable tool in further studies of cellular signaling.
This article is published under license to BioMed Central Ltd.