ELISpot analysis of LPS-stimulated leukocytes: human granulocytes selectively secrete IL-8, MIP-1beta and TNF-alpha.
Granulocytes and monocytes/macrophages represent key effector cells of the innate immune system. While human monocytes have been recognized as capable of secreting a broad spectrum of cytokines, the situation has been less clear in granulocytes with studies often showing conflicting results. In this study, lipopolysaccharide (LPS)-induced cytokine secretion from polymorphonuclear cells (PMN) and peripheral blood mononuclear cells (PBMC) was analyzed at the single cell level with the enzyme-linked immunospot (ELISpot) assay. This method allowed us to establish the cytokine profiles for both PBMC and PMN based on the frequency and pattern of cytokine secreting cells, rather than on the amount of produced cytokine detectable in solution by ELISA. As a result, low levels of contaminating mononuclear cells present in our PMN preparations could be discriminated from granulocytes. Using this technique, neutrophils were found to secrete the two chemokines, IL-8 and MIP-1beta in response to LPS. Also TNF-alpha was secreted but in lower amounts and by significantly fewer cells. However, and as opposed to several other reports, we were unable to detect secretion of IL-1beta, IL-6, IL-10, IL-12 and GM-CSF. In contrast to the limited cytokine production by PMN, PBMC secreted considerably larger amounts of the investigated cytokines with CD14(+) monocytes being the primary source of production. Finally, we believe that the cytokine ELISpot technique may provide a powerful tool by which cells of the innate immune system can be studied from a functional perspective at the single cell level.
Division of Infectious Diseases, Department of Medicine, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden. christian.smedman@ki.se
Christian
C
Smedman
Bengt
B
Gårdlund
Kopek
K
Nihlmark
Patrik
P
Gille-Johnson
Jan
J
Andersson
Staffan
S
Paulie
D002478:Cells, Cultured; D054407:Chemokine CCL4 / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007113:Immunity, Innate / Q000187:drug effects; D016209:Interleukin-8 / Q000378:metabolism*; D007958:Leukocyte Count; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000276:immunology; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D008070:Lipopolysaccharides / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects* / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
C066507:CCL4 protein, human; C508606:CXCL8 protein, human; D054407:Chemokine CCL4; D016209:Interleukin-8; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha