Granulocyte-activating mediators (GRAM)

In the present study we investigated the capability of human
epidermal cells to generate granulocyte-activating mediators
(GRAM). It could be shown that human epidermal cells as well as an
epidermoid carcinoma cell line (A431) produce an epidermal
cell-derived granulocyte-activating mediator (EC-GRAM) which
stimulates human granulocytes to release significant levels of
toxic oxygen radicals as measured by a lucigenin-dependent
chemiluminescence (CL). For further characterization of EC-GRAM the
A431 cell line was used. Supernatants of A431 cells usually
contained maximal EC-GRAM levels within 24 h of incubation. Factor
production was enhanced by bacterial lipopolysaccharide (LPS), but
not by silica particles and PHA. Moreover, freeze-thaw lysates of
A431 cells and extracts of heat-separated human epidermis contained
significant levels of EC-GRAM. Preincubation of granulocytes with
EC-GRAM resulted in an enhanced response to subsequent stimulation
with the chemotactic peptide f-met-phe. In contrast EC-GRAM did not
affect the response to PMA or zymosan particles. However, EC-GRAM
treated granulocytes were unresponsive to restimulation with
EC-GRAM. Upon high performance liquid chromatography (HPLC) gel
filtration EC-GRAM eluted within two major peaks exhibiting a
molecular weight of 17 kD and 44 kD. According to its biochemical
and biological properties EC-GRAM can be separated from other
cytokines such as ETAF/-interleukin 1, interleukin 2, interferons,
granulocyte colony-stimulating factor (G-CSF) and tumor necrosis
factor (TNF). However, an antibody to human GM-CSF neutralized
about 75% of the activity. These results indicate that EC-GRAM
activity stimulating the generation of reactive oxygen species by
granulocytes is probably due to GM-CSF.